CONFERENCE ABSTRACTS & POSTER PRESENTATIONS

CONFERENCE ABSTRACTS& POSTER PRESENTATIONSTHE 8 THZEBRAFISH DISEASEMODELS CONFERENCEJOSEPH B. MARTIN CONFERENCE CENTER // BOSTON // USA

ZDM8Boston | USA 2015ZDM8 ABSTRACTSPaper # Title Presenter Day Page #Keynote Presentations1Fly Fishing: A Model Organism Approach to Developing CancerTherapeuticsRoss Cagan Monday 172 Mark Fishman Wednesday3Programming and reprogramming muscle stem cells for therapy inmuscle degenerative diseaseAmy Wagers Thursday 18Oral Presentations4Role of the bZIP Oncogenic Transcription Factor JDP2 in High-RiskT-ALLTom Look Monday 195 The CXCR3-CXCL11 signaling axis mediates macrophage Annemarie Meijer Monday 206 The study of amygdalar and hippocampal functions in zebrafish Koichi Kowakami Monday 2178Dissecting the mechanistic basis for congenital muscular dystrophypathology in zebrafish modelsGenetic compensation induced by deleterious mutations but not geneknockdownsPeter Currie Monday 22Didier Stainier Monday 239 Approaches to elucidate cancer metabolism using zebrafish models Maria Caterina Mione Monday 2410111213The tyrosine phosphatase PRL3 as a novel drug target in T-cell acutelymphoblastic leukemiaA novel tissue-specific CRISPR technique establishes arid2 as a tumorsuppressorgene in melanomaProstate metastasis in zebrafish is attenuated by targeting cancer stemcells and overexpression of novel tumor suppressor miR-25An epigenetic strategy for inhibition of MITF function in malignantmelanomaJessica Blackburn Monday 25Julien Ablain Monday 26Ewa Snaar-Jagalska Monday 27Angelo Casa Monday 2814 Drug-Leads and New Targets in the Melanocyte Lineage and Melanoma Elizabeth Patton Monday 2915Reinforcement of cell fate decisions is crucial for maintaining cardiacchamber integrityDebbie Yelon Tuesday 3016 Epigenetic Mechanisms Regulating Heart Regeneration in Zebrafish Caroline Burns Tuesday 31171819Shp2 variant signaling in zebrafish models of Noonan and LEOPARDsyndromeThe AP-1 transcription factor component Fosl2 potentiates the rate ofmyocardial differentiation from the zebrafish second heart fieldThe Key Role of Collagen-Repeat Domains in Hennekam SyndromePatientsJeroen den Hertog Tuesday 32Geoffrey Burns Tuesday 33Stefan Schulte-Merker Tuesday 3428 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts

ZDM8 ABSTRACTSZDM7Madison | USA 2015ZDM8Boston | USA 2015Paper # Title Presenter Day Page #202122232425262728293031Fish models for lysosomal diseases: how a new tool enables tochallenge an old paradigm of disease pathogenesisCentroacinar cells are pancreatic progenitors that contribute toregeneration of insulin producing β-cellsIdentification of a new PPARa/PPARg dual agonist for the treatment offatty liver diseaseYap reprograms glutamine metabolism to support growth during liverdevelopment and tumorigenesisDNA hypomethylation induces interferon response and macrophageactivation in uhrf1 mutant zebrafish embryosThe pro-apoptotic factor Bax regulates necrosis during MycobacterialinfectionDisruption to the basal transcription machinery causes intestinaldysplasiaLeukotriene signaling during host defense responses to bacterialinfectionZebrafish models of Staphylococcus aureus pathogenesis: Seeing isbelievingA new immunometabolic mechanism driving macrophage activationduring gouty arthritisTrim9 Mediates Zebrafish Macrophage Chemotaxis in a RINGdependentMannerGenetic basis of intramembranous bone vascularization in a rarevascular syndromeEnrico Moro Tuesday 35Mike Parsons Tuesday 36Henry Krause Tuesday 37Andrew Cox Tuesday 38Yelena Chernyavskaya Tuesday 39Francisco Roca Tuesday 40Joan Heath Tuesday 41Anna Huttenlocher Tuesday 42Stephen Renshaw Tuesday 43Chris Hall Tuesday 44Jeff Yoder Tuesday 45Jingwei Rachel Xiong Tuesday 4632 The lymphatic vasculature in health and disease Jonathan Astin Tuesday 47333435Exploiting the regenerative biology of zebrafish tendons to build aroadmap for human regenerative medicine applicationsChemical and Genetic Approaches to Identify Therapeutic Targets forEwing SarcomaEstablishing a zebrafish model of craniosynostosis; P236R fgfr1amissense mutation introduced by CRISPR/Cas9 mediated homologousrecombinationJenna Galloway Wednesday 48Joanie Neumann Wednesday 49Jolanta Topezewska Wednesday 5036 RAP1-mediated MEK-ERK pathway defects in Kabuki syndrome I-Chun Tsai Wednesday 513738NAD+ Biosynthesis Improves Muscle Structure and Function in aSecondary DystroglycanopathyMuscle cell dynamics and regeneration in zebrafish muscular dystrophymodelsClarisa Henry Wednesday 52Sharon Amacher Wednesday 5339 A novel role for Wnt9a in hematopoietic stem cell development. David Traver Wednesday 548 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts3

ZDM8Boston | USA 2015ZDM8 ABSTRACTSPaper # Title Presenter Day Page #40 Characterization of zebrafish erythroid burst cell line Petr Bartunek Wednesday 5541 Fam210b regulates cellular iron uptake and heme synthesis Barry Paw Wednesday 5642 Vitamin D3 modulates vertebrate hematopoietic stem cell homeostasis Mauricio Cortes Wednesday 574344454647Thymocyte selection-associated HMG box protein (TOX) inducesgenomic instability in T-cell Acute Lymphoblastic LeukemiaPerphenazine induces apoptosis in T-cell acute lymphoblastic leukemiaby direct activation of PP2A.Identification and Characterization of a Novel Cancer Susceptibility Genethat Regulates Ras ActivityExpanding psychiatric genetics: 16p11.2 gene interactions affectmultiple whole body phenotypesAcceleration of hepatic steatosis to carcinogenesis transition byneutrophils and macrophages in kras-induced hepatocellular carcinomamodel in zebrafishRiadh Lobbardi Wednesday 58Alejandro Guiterriez Wednesday 59Sahar Nissim Wednesday 60Jasmine McCammon Wednesday 61Chuan Yan Wednesday 6248 Molecular control of Schwann cell development by Fbxw7 Breanne Harty Wednesday 6349In vivo imaging of host-pathogen interactions in Staphylococcus aureusinfectionJustyna Serba Wednesday 6450 Severe ossification defects in Medaka mutants for Osterix/Sp7 Tingsheng Yu Wednesday 655152535455Investigating the Pathobiology of Myofibrillar Myopathies and PotentialTherapies using ZebrafishInvestigation of the role of the HACE1 tumor suppressor in high-riskpediatric neuroblastomaGlucocorticoid anti-inflammatory agents activate hypoxia induciblesignallingZebrafish Caudal Hematopoietic Embryonic Stromal Trunk (CHEST) CellsSupport Hematopoietic Stem and Progenitor Cell (HSPC) ExpansionThe Serotonergic Nervous System Regulates HSC Production Via theHypothalamic-Pituitary-Interrenal Axis In ZebrafishAvnika Ruparelia Wednesday 66Babak Razaghi Wednesday 67Freek VanEeden Thursday 68David Stachura Thursday 69Wanda Kwan Thursday 7056 R-spondin-1 is a novel regulator of hematopoietic stem cell specification Jamie Genthe Thursday 7157Calmodulin inhibition rescues the effects of ribosomal protein deficiencyin Diamond Blackfan anemia modelsElizabeth Macari Thursday 7258Key determinants at the cross roads of innate immunity and metaboliccontrolHerman SpainkThursday59 Meeting challenges in psychiatric and neurodevelopmental disorders Hazel Sive Thursday 7348 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts

ZDM8 ABSTRACTS & POSTERSZDM7Madison | USA 2015ZDM8Boston | USA 2015Paper # Title Presenter Day Page #606162A systems biology approach to understanding hearing regeneration inzebrafishEpithelial damage and MMP-13 overexpression contribute to paclitaxelinducedperipheral neuropathy in zebrafishZebrafish Mutants of the Autism Risk Gene Cntnap2 Identify GABAergicDefects and Uncover Estrogens as Phenotypic SuppressorsShawn Burgess Thursday 74Sandra Rieger Thursday 75Ellen Hoffman Thursday 7663 Mechanisms of glial bridging during spinal cord regeneration Mayssa Mokalled Thursday 7764 SHP2 activation synergizes with MYCN in neuroblastoma pathogenesis Shizhen (Jane) Zhu Thursday 7865 Studies of blood stem cells and cancer in the zebrafish Len Zon Thursday 7966 Premature ageing anticipates cancer in tert-deficient zebrafish Miguel Godinho Ferrerira Thursday 8067686970p53 isoform Δ113p53/Δ133p53 promotes DNA double-strand breakrepair to protect cell from death and senescence in response to DNAdamageLive imaging reveals NFkB signaling to be a key regulator for a TrophicInflammatory Response during tumour initiationZebrafish Chemical Screen Identifies Prolyl Isomerase PIN1 as aTherapeutic Target in Radiation-Resistant CancerCD4-transgenic zebrafish: a resource for interrogating helper T cellbiologyJinrong Peng Thursday 81Yi Feng Thursday 82Samuel Sidi Thursday 83Adam Hurlstone Thursday 84Poster # Title Presenter Day Page #Poster PresentationsPoster Session 1 - Tuesday 24th August (even numbers)Poster Session 2- Wednesday 25th August (odd numbers)* indicates a Lightning TalkCancer100* Oncogenic Roles of TCF3 and ID3 in T Lymphoblast Cancers J. Kimble FrazerPosterSession 185101Role of Notch-signaling in tumor progression in a zebrafish model ofgliomaMarie MayrhoferPosterSession 286102Reemergence of neural crest progenitor identity is a key event inmelanomainitiationCharles KaufmanPosterSession 187103Cell type-specific effects of caffeic acid phenethyl ester in the neuralcrestand melanomaChristie CiarloPosterSession 288104Zebrafish Model for Anti-Intrahepatic Cholangiocarcinoma in vitro and invivo Drug Screening PlatformJen-Leih WuPosterSession 1898 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts5

ZDM8Boston | USA 2015ZDM8 POSTERSPoster # Title Presenter Day Page #105 Hallmarks of cancer in the human-zebrafish xenograft model Rita Fior106 Modeling cytotoxic T cells recruitment to zebrafish melanoma Aya LudinPosterSession 2PosterSession 19091107The tumor suppressor adenomatous polyposis coli (apc) regulatesmitochondrial pyruvate carrier 1 (mpc1): a novel role for apc in pyruvatemetabolismImelda SandovalPosterSession 292108Myogenic Regulatory Factors and their role in EmbryonalRhabdomyosarcomaInes M. TenentePosterSession 193109Notch Signaling Increases the Number of Relapse-driving TumorPropagating Cells in Embryonal RhabdomyosarcomaMyron IgnatiusPosterSession 294110 Circadian Effect of MTX in Zebrafish Toxicity and Proliferation Veronica AklePosterSession 195111*The GAP-related Domain of NF1 is required for tumor-suppression ofMYCN-induced neuroblastoma in vivo.Shuning HePosterSession 296112A role for Wnt Signaling in self-renewal and differentiation of EmbryonalRhabdomyosarcomaMadeline HayesPosterSession 197113Loss of function of ARID1A synergizes with MYCN in neuroblastomapathogenesisTing TaoPosterSession 298114DEF plays a role in pre-rRNA processing that is upregulated to synergizewith MYCN in neuroblastoma pathogenesisHui ShiPosterSession 199115 Modeling thyroid cancer in zebrafish Ana AlmeidaPosterSession 2100116Arsenic and DNA methylation: Using zebrafish to uncover themechanism of arsenic induced toxicity and methylome reprogrammingKathryn BambinoPosterSession 1101117Central Nervous System Infiltration by Zebrafish T LymphoblastMalignanciesChiara BorgaPosterSession 2102118Using zebrafish to study the significance of tumour cell apoptosis andtumour-associated macrophages in aggressive B-cell lymphomaZiyuan ChangPosterSession 1103119Identification of a zebrafish germline mutation that causes T cell cancerand apoptosis resistanceBarbara SquibanPosterSession 2104120 SCID zebrafish as a universal transplant platform John Moore121 Optical Control of Cancer Initiation from Single Cells in Zebrafish Zhiping FengPosterSession 1PosterSession 2105106122*Hemodynamic profiles tune the arrest and extravasation of circulatingtumor cellsSofia AzevedoPosterSession 1107123Continuous low frequency in vivo mutagenesis: a novel system fortumour inductionLara SicouriPosterSession 210868 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts

ZDM8 POSTERSZDM7Madison | USA 2015ZDM8Boston | USA 2015Poster # Title Presenter Day Page #124The BRIP1/FANCJ DNA helicase as novel 17q driver oncogene inneuroblastomaSuzanne VanhauwaertPosterSession 1109125Exploring the TLX1-PHF6 cooperative interrelationship in T-cell acutelymphoblastic leukemia through zebrafish modelingSiebe LoontiensPosterSession 2110126Identifying novel combination therapies in BRAFV600E-driven melanomain the zebrafish model.Michelle DangPosterSession 1111127An Orthotopic Model of Glioblastoma in Zebrafish Exhibits KeyHallmarks of Brain Tumors.Alessandra WelkerPosterSession 2112128Using Zebrafish to Investigate the Synergistic Inhibition of FocalAdhesion Kinase and Aurora Kinases on Ewing Sarcoma Proliferationand MetastasisNicole MelongPosterSession 1113129 Glypican4 modulates collective cell migration non-cell autonomously Marina Venero GalanternikPosterSession 2114130Investigating the Role of polk in Melanoma Heterogeneity and DrugResistanceKelsey TemprinePosterSession 1115131SATB2 accelerates melanoma progression and invasion by altering cellfate and activating actin remodelingEllen Van RooijenPosterSession 2116132 Uncovering the function of Polycomb proteins in malignant melanoma Sara DiNapoliPosterSession 1117133Histone deacetylase inhibitors antagonize distinct pathways to suppresstumorigenesis of embryonal rhabdomyosarcomaTerra Vleeshouwer-NeumannPosterSession 2118134A novel model of thyroid cancer in zebrafish reveals early morphologicand developmental abnormalities induced by oncogenic BRAFV600EViviana AnelliPosterSession 1119135Investigation of MYC Collaborating Oncogenes in T-cell AcuteLymphoblastic Leukemia Progression and RelapseElaine GarciaPosterSession 2120136Establishment of a zebrafish model for the study of metastatic processof human cancer cellsWen-Wei ChangPosterSession 1121137 Melanoma regression and recurrence in zebrafish Sonia Wojciechowska138 A Novel Function for the PRL3 Phosphatase in the Melanocyte Lineage Judith ZichPosterSession 2PosterSession 1122123139Role of oncogenic glutamate receptor signaling in melanocyte andmelanoma developmentAna NetoPosterSession 2124140kit inhibits BRAFV600E-driven melanoma formation by facilitatingcompetition with wild-type RAFJames NeiswenderPosterSession 1125141 Use of oral gavage to treat adult zebrafish with small molecule drugs. Raul Martinez-McFalinePosterSession 2126142A Genome-Editing Approach for the Stepwise Establishment ofZebrafish Models of Pediatric High-Grade Gliomas and MPNSTsFelix OppelPosterSession 11278 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts7

ZDM8Boston | USA 2015ZDM8 POSTERSPoster # Title Presenter Day Page #143UFD1: a novel contributor to MYC-driven T-cell acute lymphoblasticleukemogenesisLeah HuitingPosterSession 2128144 Understanding the Role of DLST in MYCN-driven Neuroblastoma Nicole Anderson145 Zebrafish Modeling of PAX3-FOXO1 Driven Rhabdomyosarcoma Genevieve KendallPosterSession 1PosterSession 2129130146Loss of chd5-mediated tumor suppression accelerates MYCN-drivenneuroblastoma tumorigenesis in zebrafishMark ZimmermanPosterSession 1131147Overexpression of platelet-derived growth factor receptor alpha (PDGFRA)synergizes with loss of NF1 in the molecular pathogenesis of malignantperipheral nerve sheath tumor (MPNST)Dong Hyuk KiPosterSession 2132148Modelling cancer Inflammation and treatments including surgery andradiotherapyPaul MartinPosterSession 1133149 Investigating the role of tetraploid intermediates in melanoma progression Revati DarpPosterSession 2134150In vivo Modeling of a Single Nucleotide Polymorphism within LMO1Associated with High Risk Neuroblastoma Using TALEN Genome Editingin ZebrafishNina WeichertPosterSession 1135151A Novel Missense Mutation in the Extracellular Domain of PDGFRaInduces Functional Consequences in vivoAmanda TempletonPosterSession 2136152Tumor Suppression Mechanism of CCR4-NOT transcription complexsubunit 3 (CNOT3) in T-cell Acute Lymphoblastic LeukemiaZhaodong LiPosterSession 1137153* Identifying GDF6 as a novel pro-survival melanoma oncogene Arvind VenkatesanPosterSession 2138154Building personalized genetic models of human cancer in zebrafish topredict response to therapy and characterize disease mechanismsJacques VillefrancPosterSession 1139155Characterize the Role of Protein Kinase CK2 in MYCN-drivenNeuroblastomaHaiwei LianPosterSession 2140156Tumor diversity and evolution revealed through restriction-site associatedDNA sequencingElizabeth PerryPosterSession 1141157 Bace2 in normal and malignant melanocytes Yan Zhang158 Bace2 in zebrafish melanocyte development Milena Zimmer159 Quantifying Metastatic Spread In A Zebra-fish Melanoma Model Silja Heilmann160 The Role of Adipocytes in Melanoma Metastasis Xiang ZhangPosterSession 2PosterSession 1PosterSession 2PosterSession 114214314414588 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts

ZDM8 POSTERSZDM7Madison | USA 2015ZDM8Boston | USA 2015Poster # Title Presenter Day Page #161Cell of origin studies of malignant melanoma using both transgeniczebrafish and human pluripotent stem cell-derived melanocytesScott CallahanPosterSession 2146162 Nuclear Hormone receptors regulate transcriptional pausing in neural crest Cristina SantorielloPosterSession 1147Cardio-Vascular Diseases and Disorders163The Apelin receptor and its ligand Elabela modulate Nodal/TGFβsignalling differently for proper cardiac developmentSerene ChngPosterSession 2148164Unravelling the function of a novel unannotated gene responsible forLeft-Right Asymmetry defects in HumansEmmanuelle SzenkerPosterSession 1149165Distinct Pathophysiologies of BCL2-associated Athanogene 3 –basedCardiomyopathy Models in Adult ZebrafishYonghe DingPosterSession 2150166Study of Developmental and Molecular Processes Regulated by Sorbs1using a Combination of in vitro and in vivo modelsAlexandra VelosoPosterSession 1151167Missense Mutation in COL22A1 Is Associated With IntracranialAneurysmsQuynh TonPosterSession 2152168Wnt signaling balances cardiac and pharyngeal muscle progenitorspecification in zebrafishAmrita MandalPosterSession 1153169Heart development is severely affected by an impairment of theHedgehog pathway in type II mucopolysaccharidosis (MPSII) animalmodels.Roberto CostaPosterSession 2154170The role of inflammatory cell signalling during cardiac scar formation andregression in adult zebrafishRebecca RichardsonPosterSession 1155171Genetic studies of histone demethylases kdm6ba and kdm6bb duringzebrafish cardiogenesis and regenerationAlexander AkerbergPosterSession 2156172High Throughput Forward and Reverse Screen for Cardiotoxicology ofAnticancer Drugs in ZebrafishToshio TanakaPosterSession 1157173Interactions between a pair of cell adhesion molecules control theproduction of outflow tract progenitor cells from the second heart fieldXin-Xin ZengPosterSession 2158Gaestrointestinal And Liver Disease174Transgenic and Patient Derived Xenograft Zebrafish Model forPersonalized Medicine of Hepatocellular CarcinomaChiou-Hwa YuhPosterSession 1159175Overfeeding accelerates thioacetamide-induced liver damage inzebrafish.Elena TurolaPosterSession 21608 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts9

ZDM8 POSTERSZDM7Madison | USA 2015ZDM8Boston | USA 2015Poster # Title Presenter Day Page #194 The Role of Calreticulin (Calr) in Vertebrate Hematopoiesis Ki Fong ManPosterSession 1179195A screen for epigenetic regulators of hematopoiesis reveals arequirement for Ing4 in HSC specificationKatie KathreinPosterSession 2180196Investigating the Role of Wnt16 Signaling during HSC NicheSpecificationNikki GlennPosterSession 1181197* Decreased leukemia initiating cells in hdac1 haploinsufficient T-ALL Margaret Pruitt198 Using oncogene combinations to model myeloid leukaemia in zebrafish Elisa AlghisiPosterSession 2PosterSession 1182183199Development of Multi-lineage Hematopoiesis in Two Novel Zebrafishrunx1 MutantsErica BrescianiPosterSession 2184200Distinct roles for matrix metalloproteinases 2 and 9 in zebrafishhematopoietic stem cell emergenceLindsay TheodorePosterSession 1185201Identification of approved drugs that are selectively active againsthematopoietic stem and progenitor cells with inactivating mutations ofTET2Chang-Bin JingPosterSession 2186202 The role of transcription pausing in hematopoietic stem cell development Xiaoying BaiPosterSession 1187203PDGFRB signaling acts downstream of metabolic alteration to affecthematopoietic stem cell induction in the zebrafish embryoSung-Eun LimPosterSession 2188204*Phenotypic Assays using Zebrafish Hematopoesis Models forElucidation of Hematopoietic ToxicityAnna LenardPosterSession 1189205Transgenic zebrafish expressing mCherry under the control of thehemogen promoter recapitulate the erythropoietic program andknockdown of hemogen causes||pronounced embryonic anemiaMichael PetersPosterSession 2190206 Role of Sclerotome in Hematopoietic Stem Cell Development Clair KelleyPosterSession 1191207Lightsheet live imaging reveals the earliest stages of zebrafish kidneymarrow colonization by hematopoietic stem cellsOwen TamplinPosterSession 2192208 A Novel Phytosterol increases Hematopoietic Stem Cell Homing Troy LundPosterSession 1193Infection And Immunity209Zebrafish: A novel tool to study 3-O sulfated heparan sulfate and HSV-1infectionVaibhav TiwariPosterSession 2194210T lymphocytes control microbial composition by actively suppressingovergrowth of pathogenic bacteria in the zebrafish gutSylvia BrugmanPosterSession 11958 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts11

ZDM8Boston | USA 2015ZDM8 POSTERSPoster # Title Presenter Day Page #211A zebrafish embryonic model for comparative studies of Aspergillusinfection.Bjørn KochPosterSession 2196212 System-wide analysis of the T-cell response Merja JaronenPosterSession 1197213Analysis and manipulation of phagocyte calcium dynamics in infectionand inflammationMolly MattyPosterSession 2198214*XPR1 is specifically required for the differentiation of tissue-residentmacrophagesAna Meireles De SousaPosterSession 1199215Establishment and characterization of a trypanosome infection inzebrafishEva DoroPosterSession 2200216 Shuttling spores between leukocytes in zebrafish fungal infection models Vahid Pazhakh217 The role of vascular dysfunction during mycobacterial infection Stefan OehlersPosterSession 1PosterSession 2201202218Ancient collection of antigen processing and presentation genesuncovered by genomic sequencing of clonal zebrafishSean McConnellPosterSession 1203Inflammation and Hemostasis219Identification of benzopyrone as a common structural feature incompounds with anti-inflammatory activity in a zebrafish phenotypicscreen.Anne RobertsonPosterSession 2204220Tracking PGE2 effect on HSPC progenitor populations via thetranscription factor CREBEva FastPosterSession 1205221 The role of mir-223 in neutrophil reverse migration Wenqing ZhouPosterSession 2206222The role of lipid mediators in inflammation resolution elucidated by anovel ptges reporter line.Catherine LoynesPosterSession 1207223Integrating human and zebrafish studies to find new drug targets in theneutrophil kinomeKatherine HenryPosterSession 2208224 Interleukin 1 induction in sterile tissue injury Nikolay OgryzkoPosterSession 1209225Cigarette smoke-induced inflammation and tissue remodelling ofzebrafish gillsFränze ProgatzkyPosterSession 2210226TNBS-induced enterocolitis as an inflammatory model for large- scalescreening of environmental factors.Jessica KenisonPosterSession 1211227Inflammatory regulation of hematopoietic stem cell formation andfunction in the normal and hyperglycemic niche.Virginie EsainPosterSession 2212128 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts

ZDM8 POSTERSZDM7Madison | USA 2015ZDM8Boston | USA 2015Poster # Title Presenter Day Page #228The molecular mechanism underlying glucocorticoid resistance ofinflammatory||leukocyte migrationMarcel SchaafPosterSession 1213229Genome editing of factor X reveals unexpected early survival and lateonset||hemorrhageJordan ShavitPosterSession 2214Musculo-Skeletal Disorders230 Idiopathic Scoliosis development in zebrafish and humans. Simone LaupheimerPosterSession 1215231*Alterations in actin capping protein Capzb causes craniofacialmalformation and hypotonia in zebrafishKusumika MukherjeePosterSession 2216232*Neuromuscular regulation in zebrafish by a large AAA+ ATPase/ubiquitinligase, mysterin/RNF213Yuri KotaniPosterSession 1217233Successful treatment with inhibitors of the mitochondrial permeabilitytransition pore in a zebrafish model of collagen VI muscular dystrophyMarco SchiavonePosterSession 2218234 A zebrafish model for human FOP Melissa LaBontyPosterSession 1219235Integration of Hedgehog signaling into networks controlling zebrafishregenerative osteogenesisBenjamin ArmstrongPosterSession 2220236Intra-ray mesenchymal cells generate the Wnt-producing progenitorniche that orchestrates caudal fin regenerationGabriel YettePosterSession 1221237Investigating potential therapeutics for merosin-deficient congenitalmuscular dystrophySarah SmithPosterSession 2222238 Connexin 34.4 may play important role in the myogenesis of zebrafish Jiann-Jou Yang239 Linking motile ciliary dysfunction to adolescent idiopathic scoliosis Curtis BoswellPosterSession 1PosterSession 2223224240Zebrafish Protein-Trap Gene-Break Transposon System to ExploreVertebrate Proteome of Skeletal Muscle RemodelingNoriko UmemotoPosterSession 1225241Modeling human mitochondrial diseases caused by mutations in nucleargenomeHirotaka AtaPosterSession 2226242Mutation of zebrafish kif5B genes causes craniofacial malformation andmyopathyFlorence MarlowPosterSession 1227243Nervous System Abnormalities in a Zebrafish Model of DuchenneMuscular DystrophySarah KucenasPosterSession 2228244 Zebrafish models for human craniofacial and skeletal diseases. Pamela YelickPosterSession 1229245Extraocular Muscle Regeneration in Adult Zebrafish. A ParadigmChange?Alfonso Saera-VilaPosterSession 22308 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts13

ZDM8Boston | USA 2015ZDM8 POSTERSPoster # Title Presenter Day Page #246Study of the koliber mutant points to the novel role of cholesterolsynthesis gene in skeletogenesisJacek TopczewskiPosterSession 1231Neurological Disorders247Human Amyloid Protein Expression and Self Assembly in vivo in theZebrafish LensOwen LawrencePosterSession 2232248 Feeling the sleep pressure in health and disease Sabine ReichertPosterSession 1233249Domoic acid targets developing oligodendrocytes and causesdevelopmental neurotoxicityJennifer PanlilioPosterSession 2234250Behavioural, Neuroanatomical and Pharmacological Profiling in ZebrafishModels of AutismMarcus GhoshPosterSession 1235251A Novel Zebrafish Model of Neurodegeneration-Induced AstrocyteActivationFrancisco QuintanaPosterSession 2236252Novel genes critical for hypoxic preconditioning in zebrafish areregulators of insulin and glucose metabolismFarhad ImamPosterSession 1237253Screening natural products for neuroprotective potential in the zebrafishlarvae PD modelSuzita NoorPosterSession 2238254Modeling the Chemical Pathways of Fast-acting Antidepressants inZebrafishAndrew RennekampPosterSession 1239255*Characterizing vertebrate stress response with mutant zebrafish strainsin the hypothalamic-pituitary-adrenal axisHan LeePosterSession 2240256Expression of the transcription factor gs homeobox 1 defines neuronsthat are required for prepulse inhibitionSadie BergeronPosterSession 1241257 Using zebrafish to model the role of GEMIN5 in neurological disease Katherine SchafferPosterSession 2242258A new model for Multiple Sclerosis: Using zebrafish to directly investigatedemyelination and remyelination in vivoAngela MorrisPosterSession 1243259 Effects of cocaine on visual processing in zebrafish Elizabeth RileyPosterSession 2244Techniques and Technology260Tools for zebrafish research, from activity analysis to detailed observationin embryos and larvaeBrooke AprilPosterSession 1245261Imaging the dynamics of tumor cell heterogeneity followingtransplantation in optically clear, immune compromised zebrafishQin TangPosterSession 2246148 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts

ZDM8 POSTERSZDM7Madison | USA 2015ZDM8Boston | USA 2015Poster # Title Presenter Day Page #262CRISPR MultiTargeter: A Web Tool to Find Common and UniqueCRISPR Single Guide RNA Targets in the ZebrafishSergey PrykhozhijPosterSession 1247263Zebrabow clonal analysis with fate-mapping defines the number of stemcells arising during hematopoietic developmentJonathan HenningerPosterSession 2248264Zebrafish Transgenic Embryos Specifically Respond to EndoplasmicReticulum StressHuai-Jen TsaiPosterSession 1249265Dissection of normal, aberrant, and malignant hematopoiesis at singlecell resolution in zebrafishFinola MoorePosterSession 2250266High-Throughput Gene Targeting and Phenotyping using CRISPR/Cas9for modeling Human diseasesGaurav VarshneyPosterSession 1251Other/ Various267Wnt and BMP signaling pathways alterations are a hallmark of bonedefects in a fish model for Gaucher diseaseIlaria ZancanPosterSession 2252268FGF signaling impairment substantially contribute to bone pathologicalmanifestations in animal models for Mucopolysaccharidosis Type II(MPSII)Stefania BellessoPosterSession 1253269 A Pak1/Erk signaling module as a therapeutic target in Rasopathies Daniela Araiza-Olivera270 Autophagy confers the therapeutic effects of mTOR inhibition in PKD Ping Zhu271 BioEYES: A Partnership to Advance K-12 Science Valerie ButlerPosterSession 2PosterSession 1PosterSession 2254255256272Generation of a MLIV zebrafish disease model using both ZFN andCRISPR/Cas9 genome editingHuiqing LiPosterSession 1257273 Healthier communities through Prescription Education and InSciEd Out Chris Pierret274 Resource Construction at the China Zebrafish Resource Center Luyuan Pan275 The T Protein: Vertebrae Fit to a T Michael Pickart276 The Regulation of Endoderm Development by Nuclear Receptor Nr5a2 Olivia Weeks277 Adapting zebrafish disease assays to the K-12 education space Madeleine HammerlundPosterSession 2PosterSession 1PosterSession 2PosterSession 1PosterSession 2258259260261262278In vivo imaging of mammalian HSCs engraftment and colonization intothe CHT of zebrafish embryosMargarita Parada KuszPosterSession 12638 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts15

ZDM8Boston | USA 2015ZDM8 POSTERSPoster # Title Presenter Day Page #279 Low cost technology for science education partnerships Michael P. EkkerPosterSession 2264280HIF-Independent Hypoxia-Induced Genes are Required for HypoxicPreconditioning in ZebrafishDanhua ZhangPosterSession 1265281 Low cost, low power, mobile genotyping Austin S. McCoy282 Supporting Zebrafish and Human Disease Research at ZFIN. Sabrina ToroPosterSession 2PosterSession 1266267283Crush injury in zebrafish bmp1 mutant as a model to study humanOsteogenesis Imperfecta fracture repairMonika Jagoda TomeckaPosterSession 2268284Ldb2 Regulates Vegf-A-dependent Arterial Specification via Expressionof Delta-like 4 in ZebrafishSeung-Sik RhoPosterSession 1269285A signal through the noise: quantitative evaluation of the scienceeducation intervention InSciEd OutLinnea R. ArcherPosterSession 2270286Live intracellular calcium dynamics during podocyte development andinjury in zebrafishRitu TomarPosterSession 1271287Annotation of the Expression and Function of Mitochondrial GenesThrough Zebrafish Insertional MutantsRoberto Lopez CerveraPosterSession 2272288Prostaglandin Signalling Regulates Ciliogenesis By ModulatingIntraflagellar TransportDaqing JinPosterSession 1273289BBS1 localization and functional changes due to a disease-associatedmutationAnthony ScottPosterSession 2274290Regeneration of zebrafish melanocytes involves direct differentiation andconcurrent replenishment of progenitor cellsSharanya IyengarPosterSession 1275291 Developmental Control of DNA Replication Christopher SansamPosterSession 2276292CRISPR mediated targeted mutagenesis at the NHGRI Zebrafish Core:Efficient protocols to generate mutant fishBlake CarringtonPosterSession 2277168 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts

ZDM8 ABSTRACTSZDM7Madison | USA 2015ZDM8Boston | USA 2015Fly Fishing: A Model Organism Approach to Developing CancerTherapeuticsRoss L. CaganDept. of Developmental and Regenerative BiologyIcahn School of Medicine at Mount SinaiCancer has proven a difficult disease to control long-term with drug therapies: genomic and wholebody complexity as well as emergence of various mechanisms of resistance continue to prove achallenge. Model organisms are beginning to play a key role in exploring this complexity, reminiscentof their roles at the forefront of genetics, cell biology, and development. I will discuss our effortswith Drosophila to capture disease complexity and to leverage the use of fly genetics to develop newtherapeutics.Building fly tumor models with increasing genetic complexity often led to progressive resistanceagainst targeted therapies. In general, therapeutics that suppressed tumor progression in flies andmice with genetically simple tumors failed as complexity increased. We find that complex drugcocktails often better address this level of complexity. I will discuss our efforts—in collaboration withchemists including Kevan Shokat, Arvin Dar, and Avner Schlessinger—to develop a new generation oflead compounds that emphasize 'rational, balanced polypharmacology'. Combining fly genetics withmedicinal and computational chemistry, our goal is to determine whether these lead compounds aremore successful at addressing tumor progression, resistance, and heterogeneity in both flies andpatients.8 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts17

ZDM8Boston | USA 2015ZDM8 ABSTRACTSProgramming and reprogramming muscle stem cells for therapyin muscle degenerative diseaseAuthors: Amy WagersDuchenne Muscular Dystrophy (DMD) is a progressive muscle wasting disease causedby mutations in the DYSTROPHIN gene. Gene correction and autologous transplantationof muscle stem cells (i.e., satellite cells) presents a promising therapeutic approach forrestoring DYSTROPHIN expression in muscle fibers of these patients; however the lowfrequency of satellite cells in adult muscle is an obstacle for obtaining sufficient numbersof cells for therapeutic engraftment. To address this issue, we have sought cultureconditions that would support the ex-vivo expansion of satellite cells and potentiallyprovide more cells for transplantation. We found that forskolin, an adenylyl cyclaseactivator, dramatically expanded mouse satellite cells in culture. Forskolin-treated culturedcells retained the immunophenotypic characteristics of engraftable satellite cells, andtransplantation of compound-treated cells into dystrophic muscle yielded a significantlyhigher level of engraftment compared to control cells. Forskolin also dramatically expandssatellite cells from the mdx mouse model of DMD in culture, and together with basicfibroblast growth factor (bFGF) and a GSK3b antagonist can drive myogenic specificationof human induced pluripotent stem cells (iPSCs). As proof of concept for a combined geneand cell therapy approach for treating DMD, we targeted the mutated DYSTROPHIN locuspatient-derived iPSCs and in ex-vivo expanded dystrophic satellite cells from mice, usingCRISPR/Cas9 gene editing technology. Efficient gene targeting and expansion of genemodified cells provides new opportunities for applying combined gene- and cell-basedtherapies for DMD and related neuromuscular disorders.188 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts

ZDM8 ABSTRACTSZDM7Madison | USA 2015ZDM8Boston | USA 2015Role of the bZIP Oncogenic Transcription Factor JDP2 in High-Risk T-ALLMarc R. Mansour 1,2 , Shuning He 1 , Zhaodong Li 1 , Riadh M. Lobbardi 3 ,Clemens Hug 1 , Evisa Gjini 1 , Alejandro Gutierrez 4 , Song Yang 5 , You-YiKuang 5,6 , Yi Zhou 5 , Leonard I. Zon 5 , Takaomi Sanda 7 , David M. Langenau 3and A. Thomas Look 11 Department of Pediatric Oncology, Dana-Farber Cancer Institute, Harvard Medical School,Boston MA, 02115, USA.2 Department of Haematology, UCL Cancer Institute, University College London, WC1E 6BT, UK.3 [1] Molecular Pathology Unit, Massachusetts General Hospital, Boston, Massachusetts, USA. [2]Cancer Center, Massachusetts General Hospital, Boston, Massachusetts, USA. [3] Center forRegenerative Medicine, Massachusetts General Hospital, Boston, Massachusetts, USA. [4]Harvard Stem Cell Institute, Cambridge, Massachusetts, USA4 Division of Hematology/Oncology, Boston Children's Hospital, Dana-Farber Cancer Institute andHarvard Medical School, Boston, MA 02115, USA5 Stem Cell Program and Division of Hematology/Oncology, Children's Hospital and Dana FarberCancer Institute, and Harvard Stem Cell Institute, Harvard Medical School, 1 Blackfan Cir., Karp 7,Boston, MA 02115, USA.6 Heilongjiang River Fisheries Research Institute, Chinese Academy of Fishery Sciences, No. 232,Hesong Avenue, Daoli District Harbin, Heilongjiang, 150070; P.R. China.7 Cancer Science Institute of Singapore, National University of Singapore, and Department ofMedicine, Yong Loo Lin School of Medicine, 117599, Singapore.The outcome of acute lymphoblastic leukemia (ALL), the most common malignancy ofchildhood, has improved dramatically over the last 20 years, with upwards of 80% ofpatients cured with current approaches. However, a subset of patients with mutations ofmembers of the PRC2 complex (EZH2, SUZ12 and EED) has an extremely poorprognosis. In this study, we identified a basic-leucine-zipper (bZIP) transcription factor,Jun dimerization protein 2 (JDP2), as significantly upregulated in thymocytes from ezh2+/- zebrafish. Chemical inhibition of EZH2 in human T-ALL cell lines led to upregulationof JDP2 in vitro. Furthermore, we established that JDP2 is required for T-ALL cellsurvival, given shRNA knockdown of JDP2 led to rapid apoptosis, establishing JDP2 asan important onco-requisite gene in human T-ALL. To investigate its role in tumorinitiation, we coinjected single cell zebrafish embryos with rag2-Myc and rag2-jdp2.Strikingly, jdp2 coinjection markedly increased tumor penetrance compared to rag2-Mycalone, indicating synergy between jdp2 and Myc in thymocyte transformation. We alsoestablished a stable transgenic zebrafish line overexpressing rag2-jdp2/rag2-mCherry.Overexpression of jdp2 in our transgenic fish resulted in early thymic enlargement,followed by a long-latency aggressive T-ALL with high penetrance, making it the fourthoncogene with this capacity (along with Myc, Notch and activated Akt). These studiesestablish JDP2 as a novel oncogene in T-ALL, required for maintenance of themalignant cell state and with the capacity to initiate tumors in vivo. Our jdp2 transgeniczebrafish line provides the ideal model to elucidate the mechanisms through whichoverexpression of jdp2 initiates T-ALL, as well as its potential role in mediating drugresistance downstream of the PRC2 complex.8 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts19

ZDM8Boston | USA 2015ZDM8 ABSTRACTSThe CXCR3-CXCL11 signaling axis mediates macrophagerecruitment and dissemination of mycobacterial infectionVincenzo Torraca, Julien Rougeot, Annemarie H. MeijerInstitute of Biology, Leiden University, Leiden, The NetherlandsMycobacteria are the causative agents of life-threatening infectious diseases such astuberculosis and leprosy. These pathogens can parasitize macrophages, in order to surviveand disseminate inside the host. Recruitment of macrophages relies strongly on theproduction of chemokines by the infected host and CXC chemokine receptor 3 (CXCR3) isan important node in the chemokine signaling network. However, its role in mycobacterialdisease remains poorly explored. We used the optically accessible zebrafish embryo modelto investigate the function of the CXCR3-CXCL11 axis in macrophage recruitment and foundthat disruption of this signaling pathway increases the resistance to mycobacterial infection.In a mutant of the zebrafish ortholog of CXCR3 (cxcr3.2), macrophage chemotaxis tobacterial infections was attenuated. Notably, this phenotype could be mimicked also bypharmacological treatment with the NBI74330, a high-affinity antagonist for the humanreceptor CXCR3. We also identified two zebrafish CXCL11-like chemokines as the functionalligands of Cxcr3.2, showing that the recombinant proteins exerted a Cxcr3.2-dependentchemoattraction when administrated in vivo. During infection of zebrafish embryos withMycobacterium marinum, a well-established model for tuberculosis, we found that Cxcr3.2deficiency could also limit the macrophage-mediated dissemination of mycobacteria andattenuated the formation of granulomatous lesions, the typical histopathological features oftuberculosis. Prevention of mycobacterial dissemination by targeting the CXCR3 pathwayshould drive further research into the CXCR3-CXCL11 axis as a potential target for hostdirectedtherapy against tuberculosis and we are currently exploring this possibility in thezebrafish model.208 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts

ZDM8 ABSTRACTSZDM7Madison | USA 2015ZDM8Boston | USA 2015The study of amygdalar and hippocampal functions in zebrafishKoichi Kawakami, Lal Pradeep, Hideyuki TanabeDivision of Molecular and Developmental Biology, National Institute of Genetics, Mishima, JapanThe amygdala and hippocampus are two major components of the mammalian brainsand perform crucial roles in the processing of emotional memory and episodic andspatial memory, respectively. In teleost, the medial and lateral zone of the dorsaltelencephalon (Dm and Dl) have been postulated to be homologs of the mammalianamygdala and hippocampus based on neuroanatomical and functional studies.However, Dm and Dl are broad areas in the dorsal telencephalon and the neuralcircuitry mediating the amygdalar and hippocampal functions has yet to be explored.Here we identify the neuronal circuitry that is essential for emotional learning andepisodic and spatial learning by a genetic approach in zebrafish. We performedlarge-scale gene trap and enhancer trap screens and generated transgenic fish thatexpressed Gal4FF, a synthetic Gal4 transcription activator, in specific regions andneuronal circuits in the brain. Then we crossed these brain-specific Gal4FF transgenicfish lines with UAS-neurotoxin lines to inhibit the activity of the Gal4FF-expressingneurons, and analyzed behaviors of the double transgenic fish. We found that, when theactivity of a subpopulation of neurons in the Dm or Dl was inhibited, the fish showeddeficits in emotional learning (fear conditioning) or episodic (trace fear conditioning) andspatial learning paradigms. Thus, we think that these neuronal populations arefunctional equivalents of the mammalian amygdala and hippocampus, respectively. Thisfinding provides a basis for understanding essential neuronal circuits mediatingevolutionarily conserved behaviors in vertebrates and investigating neurologicaldiseases associated with amygdala and hippocampus pathology, such as anxietydisorders, epilepsy and Alzheimer’s disease.8 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts21

ZDM8Boston | USA 2015ZDM8 ABSTRACTSDissecting the mechanistic basis for congenital musculardystrophy pathology in zebrafish modelsTomHall 1, 2 , Ophelia V Ehrlich 1 , Rob Parton 2 and Peter D. Currie 1 .1. Australian Regenerative Medicine Institute, Level 1, 15 Innovation Walk Monash University,Wellington Road Clayton VIC 3800 Australia.2. Institute for Molecular Bioscience, The University of Queensland, St Lucia, Brisbane,Australia, 4072We have generated a zebrafish model of the most common form of CongenitalMuscular Dystrophy, merosin–deficient or Lamina alpha2-deficient congenitalmuscular dystrophy. Our studies have implicated extracellular matrix (ECM) failureas causative of the pathology evident in the zebrafish model of CMD and havesurprisingly revealed that detached muscle fibres can remain viable for considerableperiods after their detachment, and given the appropriate ECM environment, recontributeto muscle stability. Consequently, we have undertaken directedapproaches to stabilise the ECM environment using functionalised biomaterials togenerate artificial basement membrane structures for muscle attachment in diseasecontexts in vivo. Such approaches lead to fibre reattachment and a rescue of musclepathology. In this study we also reveal a deficit in the muscle stem cell compartmentof CMD animals, which can also be rescues by stabilising the ECM. We will discussa novel hypothesis for disease pathology for this group of disorders arising fromthese observations.228 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts

ZDM8 ABSTRACTSZDM7Madison | USA 2015ZDM8Boston | USA 2015Genetic compensation induced by deleterious mutations but notgene knockdownsAndrea Rossi1,*, Zacharias Kontarakis1,*, Claudia Gerri1, Hendrik Nolte1,2, SorayaHölper1, Marcus Krüger1,2 and Didier Y.R. Stainier1* Equal contributions1 Max Planck Institute for Heart and Lung Research, Bad Nauheim, Germany2 current address: Institute for Genetics and CECAD, University of Cologne, Cologne, GermanyCells sense their environment and adapt to it by fine-tuning their transcriptome. Wired intothis network of gene expression control are mechanisms to compensate for genedosage. The increasing use of reverse genetics in zebrafish, and other model systems, hasrevealed profound differences between the phenotypes caused by genetic mutations andthose caused by gene knockdowns at many loci, an observation previously reported inmouse and Arabidopsis. To identify the reasons underlying the phenotypic differencesbetween mutants and knockdowns, we generated mutations in zebrafish egfl7, anendothelial extracellular matrix gene of therapeutic interest, as well asin vegfaa. egfl7 morpholino-injected animals (morphants) exhibit severe vascular defectsbut eglf7 mutants do not show any obvious phenotypes. We further observedthat egfl7 mutants were less sensitive than their wild-type siblings to Egfl7 knockdown,arguing against residual protein function in the mutants or significant off-target effects of themorpholinos when used at a moderate dose. Comparing eglf7 mutant and morphantproteomes and transcriptomes, we identified a set of proteins and genes that wereupregulated in mutants but not in morphants. Among them were extracellular matrix genesthat can rescue egfl7 morphants, indicating that they could be compensating for the loss ofEgfl7 function in the phenotypically wild-type egfl7 mutants. Moreover, egfl7 CRISPRinterference, which obstructs transcript elongation and causes severe vascular defects, didnot cause the upregulation of these genes. Similarly, vegfaa mutants but not morphantsshow an upregulation of vegfab. Taken together, these data reveal the activation of acompensatory network to buffer against deleterious mutations, which was not observed aftertranslational or transcriptional knockdown.8 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts23

ZDM8Boston | USA 2015ZDM8 ABSTRACTSApproaches to elucidate cancer metabolism using zebrafishmodels.Marina Mione 1 , Andreas Kalkbrenner 2 , Victor Gourain 1 , Marie Mayrhofer 1 , SabrinaBurkart 1 , Svenja Meierjohann 4 , Claudia Muhle-Goll 2 , Burkhard Luy 2,31. Karlsruhe Institute of Technology, Institute for Toxicology and Genetics, Eggenstein-Leopoldshafen,76344, Germany2. Karlsruhe Institute of Technology, Institute of Organic Chemistry, 76131 Karlsruhe, Germany3. Karlsruhe Institute of Technology, Institute for Biological Interfaces 4 - Magnetic Resonance, 763444. University of Wurzburg, Dept. of Physiological Chemistry, 97074 Wurzburg, GermanyMetabolic reprogramming is a hallmark of cancer, as proliferating cells require a largeamount of nutrients, energy, and biosynthetic activity to produce the macromolecularcomponents required by the growing tumour cells. This leads to a switch from oxidativemetabolism to glycolysis (Warburg 1925; 1956) in order to utilize metabolites, such as lactateand glutamine, as building blocks for macromolecule biosynthesis. Previously, changes inmetabolism were mostly attributed to opportunistic utilization of alternative biochemicalpathways; however, the systematic analysis of cancer genomes and their expressionprofiling have revealed a surprising abundance of genetic alterations of key metabolicenzymes as early events in cancer. IDH1/2 dominant mutations are just one example(Parson et al., 2008). These are particularly interesting as enzymatic activities can betargeted with great specificity and success. To understand the consequences of alteredenzymatic activities on metabolism in cancer, analytical methods for quantifying metabolites,and system approaches to analyse the genetic and metabolome data in model systems arerequired. We have started to develop integrated maps of metabolic alterations at differentstages of melanoma and glioma development using our zebrafish cancer models (Santorielloet al., 2010; Spitzner et al., 2014). By combining metabolome analysis (through NMR) andgene expression data (through RNA-Seq) we identified metabolic reprogramming in thetrans-sulphuration pathway in melanoma, and increased glutamine /acetate production inglioma due to extensive reprogramming of the TCA cycle. Changes in metabolites andenzyme expression leading to these metabolic reprogramming events were alreadydetectable in transgenic larvae, thus suggesting that zebrafish cancer models may serve tostudy the evolution of metabolic reprogramming in cancer and provide an in vivo platform toscreen for metabolic vulnerability in different tumor types.248 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts

ZDM8 ABSTRACTSZDM7Madison | USA 2015ZDM8Boston | USA 2015The tyrosine phosphatase PRL3 as a novel drug target in T-cell acutelymphoblastic leukemiaJessica Blackburn 1 , Elaine Garcia 1 , Riadh Lobbardi 1 , Filip Malthijssens 2 , Aleksey Molodtsov 1 ,Pieter Van Vlierberghe 2 , Wilhelm Haas 1 , and David Langenau 11. Massachusetts General Hospital, Boston, MA2. Ghent University, BelgiumT-cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignancy of thymocytes.Despite significant improvements in treating primary leukemia, chemotherapies are oftenineffective against relapsed T-ALL, with 6,000 adult syngeneic zebrafish identified 6 of 47 clones thatspontaneously evolved increased leukemia propagating frequency and growth rate.Comparative genomic hybridization arrays determined that Protein Tyrosine Phosphatase 4A3(PRL3) was genomically amplified in 30% of clones with high relapse potential. Real-timequantitative PCR showed that 90% of all zebrafish T-ALL clones with high LPC frequency andgrowth expressed high levels of PRL-3, suggesting additional genetic pathways likely activatePRL3. PRL3 is also genomically amplified in 11% of human T-ALL and highly expressed in 58%of primary T-ALL patient samples, suggesting that this phosphatase is an important andpreviously undefined driver of human T-ALL. Indeed, PRL3 knock-down in human T-ALLsignificantly reduced the viability of cell lines in vitro and in xenograft models (p

ZDM8Boston | USA 2015ZDM8 ABSTRACTSA novel tissue-specific CRISPR technique establishes arid2 as atumor-suppressor gene in melanomaJulien Ablain 1 , Rachel Fogley 1 , Leonard Zon 1,2,3 .1 Stem Cell Program and Division of Hematology/Oncology, Boston Children's Hospital and DanaFarber Cancer Institute, Boston, MA 02115, USA.2Harvard Stem Cell Institute, Harvard University, Cambridge, MA 02138, USA.3Howard Hughes Medical Institute, Boston, MA 02115, USAGenome-wide analyses of recurrent mutations and copy-number alterations in cancers haveidentified a wealth of potential determinants of tumor initiation. Functional studies arerequired to assess the oncogenic or tumor-suppressive capacity of candidate genes. ARID2has been reported as one of the most frequently mutated epigenetic factors in melanoma. Itencodes a subunit of PBAF, a chromatin remodeling complex implicated in gene expressionand migration of neural crest progenitors, from which melanocytes arise. Here, we tested thehypothesis that arid2 acts as a tumor-suppressor gene in melanoma. We recently developeda vector system based on the CRISPR/Cas9 technology that enables tissue-specific genedisruption in zebrafish embryos (Ablain et al., Dev Cell 2015) and have here extended thiswork to study tumor-suppressors. In this study, we engineered this vector to target genesspecifically in melanocytes by expressing Cas9 under the control of the melanocyte-specificmitfa promoter. Injection of a vector targeting p53 into 50 Tg(mitfa:BRAF V600E ) zebrafishembryos initiated melanoma formation in every fish (median onset: 17 weeks) while a nontargetingvector never did. All tumors displayed biallelic frame-shift mutations at the p53target locus. We then disrupted known melanoma-suppressor genes like pten and rb1 inTg(mitfa:BRAF V600E ), p53 -/- zebrafish, which both resulted in a significant acceleration oftumor onset (medians: 10 and 16 weeks respectively) compared to the targeting of p53 usedas a negative control (median: 20 weeks). Importantly, we showed that the inactivation ofarid2 also leads to earlier tumors (median: 16 weeks). Our results identify ARID2 as acooperating tumor-suppressor gene of melanoma. This method allows for the systematicassessment of tumor-suppressive potential in vivo.268 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts

ZDM8 ABSTRACTSZDM7Madison | USA 2015ZDM8Boston | USA 2015Prostate metastasis in zebrafish is attenuated by targetingcancer stem cells and overexpression of novel tumorsuppressor miR-25Lanpeng Chen 1 , Eugenio Zoni 2 , Jeroen T. Buijs 2 , Arwin Groenewoud 1 , HermanSpaink 1 , Gabri van der Pluijm 2 and B. Ewa Snaar-Jagalska 11 Institute of Biology, Leiden University, Leiden, the Netherlands2Dept. of Urology, Leiden University Medical Centre, Leiden, The NetherlandsProstate cancer, once it has moved beyond the organ-confined state, is lethaldue to widespread, therapy resistant bone metastasis. Accumulating evidence showsthat prostate epithelial cancer stem/progenitor-like cells (PCSCs) play key roles in tumorinitiation, local and distant relapse, metastasis, and castration- and chemotherapyresistance.In this study, a zebrafish xenograft model has been established to verify the roleof cancer stem cells (CSCs) in the formation of metastasis.To isolate prostate cancer stem cells from human osteotropic PC-3M-Pro4 linewe used the ALDEFLUOR assay, which involves viable CSCs sorting based on ALDHenzyme activity. After sorting ALDH high and ALDH low subpopulations were injected intothe circulatory system of zebrafish embryos and dissemination, extravasation and distantmetastasis formation were examined. We observed that prostate cancer stem cellsidentified by high ALDH activity played a key role in the EMT-like driven invasion andformation of micro-metastasis controlled by cell proliferation. In addition, pretreatment ofALDH high cells with PCSCs inhibitors reduced metastatic progression confirming theirrole in the formation of prostate metastasis in zebrafish embryos.We also found that miR-25 expression in PCSCs was low/absent and steadilyincreased during their differentiation into cells with a luminal epithelial phenotype.Functional studies revealed that overexpression of miR-25 in prostate cancer stem cellsstrongly affected the invasive cytoskeleton causing reduced migration in vitro andmetastasis via attenuation of extravasation in vivo. We demonstrated, for the first time,that miR-25 can act as a tumor suppressor in highly metastatic PCSCs by directfunctional interaction with the pro-invasive αv- and α6 integrins. Our experimentalmetastasis data in zebrafish model support the findings in vitro and show that miR-25negatively regulates the acquisition of an invasive, metastatic phenotype in humanprostate cancer cells.8 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts27

ZDM8Boston | USA 2015ZDM8 ABSTRACTSAn epigenetic strategy for inhibition of MITF function inmalignant melanomaAngelo Casa 1 , Caitlin Bourque 1 , Sara DiNapoli 1 , Sagar Chhangawala 1 , Gil Blum 2 ,Liudmila Dzhekieva 2 , Glorymar Ibanez 2 , Minkui Luo 2 , and Yariv Houvras 1, 31. Dept. of Surgery, Weill Cornell Medical College, New York, NY 100652. Molecular Pharmacology and Chemistry Program, Memorial Sloan-Kettering Cancer Center,New York, NY 100653. Dept. of Medicine, Weill Cornell Medical College, New York, NY 10065MITF is a lineage-restricted transcription factor that is amplified and overexpressed in asubset of patients with malignant melanoma. Using a chemical-genetic approach inzebrafish, we have identified a novel requirement for SET8, the sole histonemethyltransferase that catalyzes mono-methylation of histone H4 lysine 20 (H4K20me1),in MITF function. We characterized a novel, small-molecule SET8 inhibitor and showedthat it impairs melanocyte differentiation and decreases levels of H4K20me1 in zebrafishembryos. Compared to wild-type embryos, mitfa heterozygotes are sensitized to SET8inhibition. Transgenic expression of human SET8 in zebrafish melanocytes in aBRAF V600E/V600E ; p53 M214K/M214K background gives rise to tumors that tend to be morepigmented compared to tumors arising in GFP controls. Additionally, SET8 inhibition inboth cultured human epidermal melanocytes and human melanoma cell lines results indecreased expression of MITF and its target genes. SET8 inhibition also has antiproliferativeeffects on several human melanoma cell lines. Gene ontology analysis ofglobal transcriptional changes following SET8 inhibition in the human melanoma cell lineMALME-3M identified several pathways involved in melanocyte differentiation andpigmentation. Analysis of this same RNA-seq data set showed that genes repressed bySET8 inhibition were positively correlated with MITF expression across human tumors inthe TCGA melanoma data set. Using chromatin immunoprecipitation and DNAsequencing (ChIP-seq), we find a broad distribution of H4K20me1 across several genebodies, including MITF target genes. For a number of these genes, these peaks aredecreased following SET8 inhibition. Finally, overall survival in the TCGA melanomadataset shows significant stratification by SET8 in MITF high melanomas when SET8-high/MITF-high was compared to SET8-low/MITF-high for overall survival. These studiesestablish SET8 as a key component for MITF transcription during melanocytedevelopment and in melanoma. We propose that pharmacologic inhibition of SET8 maybe a viable strategy for inhibition of MITF function in melanoma.288 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts

ZDM8 ABSTRACTSZDM7Madison | USA 2015ZDM8Boston | USA 2015Drug-Leads and New Targets in the Melanocyte Lineage andMelanomaE. Elizabeth PattonInstitute of Genetics and Molecular Medicine, MRC Human Genetics Unit and University ofEdinburgh Cancer Research Centre, Edinburgh, UK. e.patton@igmm.ed.ac.ukMelanoma (cancer of the melanocyte) kills over 20,000 Europeans each year andincidence continues to rise rapidly. BRAF V600E inhibitors have led to clinicallysignificant improvements in outcomes for melanoma patients, yet many patients withmetastatic melanoma rapidly succumb to the disease due to eventualchemoresistance, or insensitivity to the drug. Thus, it is critical to identify newtherapies that can act alone, or be combined with available treatments for enhancedefficacy and/or to overcome drug resistance.An important and new therapeutic concept for melanoma is to target the melanocytelineage. Recent evidence reveals that a melanocyte lineage specific programmemaintains melanoma survival, and we have engineered the first animal model inzebrafish to demonstrate that targeting the master melanocyte lineage transcriptionfactor MITF leads to rapid melanoma regression. Thus, understanding and targetingthe melanocyte lineage is directly relevant to melanoma, and reveals therapeuticallytargetable processes.We are using live-imaging of the melanocyte lineage as the basis for phenotypicchemical screens in zebrafish to find drugs/leads and identify targetable processesthat might elucidate pathways for cancer therapy. Screening for targets of themelanocyte lineage is highly relevant to melanoma because melanocytes are themelanoma cell of origin, and genes that specify the melanocyte stem cells and thelineage during embryogenesis are the same genes that play fundamental roles incancer. We use innovative chemical-biology to capture and validate targets in vivo,and perform chemo-preventative and -therapeutic trials in zebrafish melanomamodels using known and novel drug-delivery methods. Ultimately, we aim totranslate our most promising drug/leads and targets into the mammalian system, toestablish the basis for patent applications and clinical trials.8 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts29

ZDM8Boston | USA 2015ZDM8 ABSTRACTSReinforcement of cell fate decisions is crucial for maintainingcardiac chamber integrityArjana Pradhan 1 , Xin-Xin Zeng 1 , Sara Marques 2 , Vanessa George 3 , Kimara Targoff 3 ,and Deborah Yelon 11Division of Biological Sciences, University of California, San Diego, La Jolla, CA, 920932Developmental Genetics Program, Skirball Institute of Biomolecular Medicine, New YorkUniversity School of Medicine, New York, NY, 100163Division of Pediatric Cardiology, Department of Pediatrics, College of Physicians & Surgeons,Columbia University, New York, NY, 10032The atrial and ventricular chambers of the heart behave as distinct subunits with uniquemorphological, electrophysiological, and contractile properties. Proper maintenance ofatrial and ventricular features is therefore essential for the development andhomeostasis of a functional heart. However, chamber fate assignments seem relativelyplastic: for example, work in zebrafish has demonstrated that cells retain the potential toalter their chamber-specific traits, even after initiating differentiation. Our recent studiesof zebrafish nkx2.5 and nkx2.7 mutants has shown that these transcription factors playkey roles in enforcing chamber-specific gene expression programs, but the signalingpathways that function upstream of these factors are less well understood. Here, weshow that the FGF signaling pathway, which facilitates ventricular specification, isrequired to preserve ventricular identity. We find that inhibition of FGF signaling with theFGFR antagonist SU5402 can generate ectopic atrial cardiomyocytes within the alreadydifferentiated ventricle; similar phenotypes are evident in fgf8 mutants and in embryosoverexpressing a dominant-negative FGF receptor. Additionally, temporally restrictedmanipulation of the FGF pathway reveals a specific time window during which ventricularchamber identity is vulnerable to the loss of FGF signaling. Analysis usingphotoconvertible reporter transgenes indicates that the ectopic cells in SU5402-treatedembryos are not derived from the atrium. Instead, examination of the dynamiclocalization of atrial and ventricular proteins suggests that ectopic atrial cells areproduced through transformation of ventricular cardiomyocytes. Furthermore, we findthat FGF signaling is required for normal maintenance of nkx2.5 and nkx2.7 expression.Overexpression of nkx2.5 can improve the preservation of ventricular character inSU5402-treated embryos, suggesting that FGF signaling functions upstream of Nkxfactors to promote retention of ventricular identity. Together, our data suggest a modelin which continuous FGF signaling acts to suppress the plasticity of differentiatedcardiomyocytes and to preserve the integrity of the ventricular chamber.308 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts

ZDM8 ABSTRACTSZDM7Madison | USA 2015ZDM8Boston | USA 2015Epigenetic Mechanisms Regulating Heart Regeneration inZebrafishRaz Ben-Yair 1,2 , Vincent L. Butty 3 , Laurie A. Boyer 3 , Alon Goren 4 , *C. GeoffreyBurns 1,2 , *Caroline E. Burns 1,2,31Cardiovascular Research Center, Massachusetts General Hospital, Charlestown, MA 02129,2Harvard Medical School, Boston, MA 02115, 3 Department of Biology, Massachusetts Institute ofTechnology, Cambridge, MA 02139, 4 Broad Technology Labs (BTL), The Broad Institute ofHarvard and MIT, Cambridge, Massachusetts 02142, 5 Harvard Stem Cell Institute, Cambridge,MA 02138, *Equal contribution to this work.Mammalian hearts fail to regenerate following experimental (mice) or naturally occurring(human) acute myocardial infarction (MI). Instead, damaged muscle is replaced with anon-contractile scar that maintains ventricular wall integrity but also undermines pumpfunction, often to the point of overt congestive heart failure. Remarkably, adult zebrafishcan achieve near-perfect heart regeneration following several forms of injury throughrobust cardiomyocyte proliferation. Understanding the determinants of myocardialproliferation in zebrafish will identify barriers that exist in higher vertebrates and open thedoor for the development of innovative therapeutic strategies that stimulate regenerationof the infarcted heart in humans. I will discuss our unpublished work regarding the role ofepigenetic influences on cardiomyocyte proliferation and heart regeneration in zebrafish.8 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts31

ZDM8Boston | USA 2015ZDM8 ABSTRACTSShp2 variant signaling in zebrafish models of Noonan and LEOPARDsyndromeJeroen den Hertog 1,21. Hubrecht Institute-KNAW and University Medical Center Utrecht, Utrecht, the Netherlands2. Institute Biology Leiden, Leiden, the NetherlandsSHP2 is a modular protein with a protein-tyrosine phosphatase domain and two N-terminal SH2domains that has a central role in cell signaling. Mutations have been identified in humanPTPN11, encoding SHP2, that cause Noonan syndrome (NS) and LEOPARD syndrome (LS).We are using zebrafish to study the molecular-genetic and cell biological basis of NS and LS.Transient expression of mutant Shp2 with mutations that were identified in human NS and LSpatients, induced developmental defects that are reminiscent of the symptoms observed inpatients, including reduced length of the body axis, craniofacial defects and cardiac defects.Detailed analysis of cardiac development in zebrafish embryos expressing NS or LS variants ofShp2 indicated impaired heart displacement, resulting in defective left-right asymmetry. Wefound that defective ciliogenesis in Kupffer’s vesicle was at the basis of these laterality defects.We investigated signaling in these NS and LS embryos by mass spectrometry-basedphosphoproteomics. This led to the identification of candidate proteins whose tyrosinephosphorylation was different in NS or LS embryos, compared to wild type Shp2 expressingembryos. The involvement of two of these candidate proteins, the adaptor protein, PZR, and thetyrosine kinase, Fer, in the developmental defects in NS and LS embryos will be discussed.328 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts

ZDM8 ABSTRACTSZDM7Madison | USA 2015ZDM8Boston | USA 2015The AP-1 transcription factor component Fosl2 potentiates therate of myocardial differentiation from the zebrafish secondheart fieldLeila Jahangiri 1,2 , Meghan Adams 1,2 , Noelle-Paffett Lugassy 1,2 , Long Zhao 1,2 ,Kathleen Nevis 1,2 Burcu Guner-Ataman 1,2 , Caroline E Burns 1,2,3 , and C. GeoffreyBurns 1,21 Cardiovascular Research Center, Massachusetts General Hospital, Charlestown, MA 021292 Harvard Medical School, Boston, MA 021153 Harvard Stem Cell Institute, Cambridge, MA 02138In vertebrates, the muscle layer of the embryonic heart forms through two successivephases of cardiomyocyte differentiation. Initially, cardiomyocytes derived from first heartfield (FHF) progenitors assemble into the linear heart tube. Thereafter, second heart field(SHF) progenitors differentiate into cardiomyocytes that elongate the heart tube throughaccretion to the poles over a well-defined developmental window. Although deficienciesin heart tube extension can lead to life-threatening congenital heart defects, thevariables controlling the initiation, rate, and duration of SHF-mediated myocardialaccretion remain obscure. Here, we demonstrate that a component of the dimeric AP-1transcription factor, Fos-related antigen 2 (Fosl2), potentiates the rate of myocardialaccretion from the zebrafish SHF. Fosl2 null embryos initiate myocardial accretionappropriately, but the production of SHF-derived ventricular cardiomyocytes is sluggish,resulting in a ventricular myocardial deficit coupled with an accumulation ofundifferentiated progenitors. These findings demonstrate that Fosl2 promotes theprogenitor to cardiomyocyte transition. Surprisingly, mutant embryos eventually correctthe myocardial deficit by extending the differentiation window. Lastly, overexpression ofFosl2 results in precocious differentiation of SHF progenitors, demonstrating thesufficiency of this factor for driving myocardial differentiation. Taken together, our dataimplicate the AP-1 transcription factor in maximizing the rate of cardiomyocyteproduction from the SHF and uncover the existence of regulatory mechanisms to ensurethat a precise number of SHF-derived cardiomyocytes are produced irrespective ofembryonic stage.8 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts33

ZDM8Boston | USA 2015ZDM8 ABSTRACTSThe Key Role of Collagen-Repeat Domains in Hennekam SyndromePatientsRoukens, G. 1 , Peterson-Maduro 1 , J., Padberg, Y. 1,2,3 , Schulte, D. 1,3 , Schulte-Merker, S. 1,2,3(1) WWU Münster, Institute Cardiovascular Organogenesis and Regeneration, Mendelstraße 7, 48149Münster, Germany; (2) Cluster of Excellence ‘Cells in Motion’ - EXC 1003; (3) Hubrecht Institute, KNAW –UMC Utrecht, 3584CT Utrecht, the NL.Lymphatic vessels play vital roles during embryonic and adult fluid homeostasis and immunesurveillance. A few genes are known to be absolutely essential for lymphangiogenesis to occur,among them vascular endothelial growth factor-C (VEGFC), its receptor VEGF receptor 3(VEGFR3), and Collagen- and calcium-binding EGF domain-containing protein 1 (CCBE1). Allof these genes, in human, have been associated with clinical symptoms displaying lymphedema- e.g., mutations in CCBE1 have been connected to Hennekam syndrome. CCBE1 is a secretedprotein characterized by two EGF domains and two collagen repeats. The functional role of thedifferent CCBE1 protein domains is completely enigmatic, but very recently, CCBE1 hasemerged as a crucial regulator of vascular endothelial growth factor-C (VEGFC) signaling.We analyzed the functionality of several CCBE1 deletion mutants by generating knock-in miceexpressing these mutant protein forms, by analyzing their ability to enhance Vegfc signaling invivo in zebrafish, and by testing their ability to induce VEGFC processing in vitro. We found thatdeleting the collagen domains of CCBE1 has a much stronger effect on CCBE1 activity thandeleting the EGF domains. First, although CCBE1ΔCollagen mice fully phenocopy CCBE1knock-out mice, CCBE1ΔEGF knock-in embryos still form rudimentary lymphatics. Second,Ccbe1ΔEGF, but not Ccbe1ΔCollagen, could partially substitute for Ccbe1 to enhance Vegfcsignaling in zebrafish. Third, CCBE1ΔEGF, similarly to CCBE1, but not CCBE1ΔCollagen couldactivate VEGFC processing in vitro. Furthermore, a Hennekam syndrome mutation within thecollagen domain has a stronger effect than a Hennekam syndrome mutation within the EGFdomain.We propose that the collagen domains of CCBE1 are crucial for the activation of VEGFC in vivo.The EGF domains of CCBE1 are dispensable for regulation of VEGFC processing in vitro,however, they are necessary for full lymphangiogenic activity of CCBE1 in vivo.348 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts

ZDM8 ABSTRACTSZDM7Madison | USA 2015ZDM8Boston | USA 2015Fish models for lysosomal diseases: how a new tool enables tochallenge an old paradigm of disease pathogenesisMirella Filocamo 1 , Ilaria Zancan 2 Stefania Bellesso 3 , Roberto Costa 2 , and Enrico Moro 2 .1 Lab. Diagnosi Pre-Postnatale Malattie Metaboliche - IRCCS G. Gaslini, Genova, Italy.2Department of Molecular Medicine, University of Padova, Italy.3 Department of Women’s and Child’s Health, University of Padova, Italy.In the past decades a growing attention has been paid to the understanding of lysosomal functionin cell biology. The discovery of disrupted lysosomal activity in the etiology of neurodegenerativediseases has led to an increase in the spectrum of disorders, in which impaired lysosomal activityplays a key pathogenic role. Much effort has been spent to generate appropriate animal models toreproduce lysosomal storage diseases (LSDs), but in some cases murine models have beenpreviously shown to suffer from some limitations, including low viability and partial recapitulation ofdisease symptoms. In the last four years our group has been deeply involved in the generation andcharacterization of fish models for type II Mucopolyshaccharidosis (MPSII, OMIM #309900) andtype I Gaucher disease (OMIM#230800). In particular, we have focused our investigation on theearly molecular alterations responsible for bone and heart defects in MPSII and skeletalabnormalities in Gaucher disease. Our results challenge the current perspective of thepathogenesis of these LSDs based on the progressive lysosomal accumulation of undegradedsubstrates. We, indeed, found that in both disorders the precocious failure of lysosomal functionaffect cell differentiation and tissue homeostasis by altering target cell signaling pathways. Ourresults support a novel concept in which LSDs may be viewed as disorders, in which a primaryimpairment of specific cell signaling pathways is responsible for well-defined organ defects.8 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts35

ZDM8Boston | USA 2015ZDM8 ABSTRACTSCentroacinar cells are pancreatic progenitors that contribute toregeneration of insulin producing β-cellsMike ParsonsDiabetes is associated with the loss of insulin-producing β cells in the endocrine pancreas. Withthe aim of finding therapeutic routes to treat diabetes, we wish to find molecular and cellularmechanisms involved in β-cell neogenesis and regeneration. To expedite discovery of suchmechanisms we utilize the zebrafish model system where pancreatic cells readily regenerate.We have previously shown that larval zebrafish pancreas contains Notch-responsiveprogenitors that during development give rise to adult ductal, endocrine, and centroacinar cells(CACs). CACs in adults are also Notch-responsive and resemble their larval predecessors inmorphology. To assess if adult CACs are also progenitors we took two complementaryapproaches: 1) Using RNA-seq we established the transcriptome for adult CACs. Using geneontology, transgenic lines, and in situ hybridization we verified that the CAC transcriptome isenriched for progenitor markers. 2) Using our lineage tracing methods, we demonstrated thatCACs contribute to new endocrine cells following β-cell ablation or partial pancreatectomy. Wehave concluded, therefore, that CACs and their larval predecessors are the same cell type andrepresent an opportune model to study both β-cell neogenesis and β-cell regeneration.Furthermore, we present data from a mutant zebrafish line where β-cell regeneration isaccelerated. Studying the mechanism behind this heightened recovery may point to therapeuticapproaches to treat diabetes in humans.368 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts

ZDM8 ABSTRACTSZDM7Madison | USA 2015ZDM8Boston | USA 2015Identification of a new PPARa/PPARg dual agonist for the treatment offatty liver diseaseJens Tiefenbach 1 , Lilia Magdalonova 2 , Norrapat Shih 1 , Carolyn Cummins 2 and Henry M.Krause 11Donnelly Ctr, University of Toronto; 2 Dept of Pharmacology, University of TorontoWe are developing transgenic fish lines that respond to small molecules that modulate humannuclear receptors and their epigenetic regulating cofactors. These proteins, and the smallhormones and drugs that control their activities, are potential regulators of the vast majority oftoday’s most serious and costly diseases. The fish lines that we have developed indicate thepresence of active hormones and drugs via localized GFP production. We can then co-purify theactive ligands and specifically bound cofactors from responding tissues using triple affinitypurification of the activated NR protein, and then identify them via mass spectrometry. Using twoof our transgenic lines that contain human PPAR receptors, we have isolated dozens of bothknown and novel small molecule ligands. One of the new hits discovered is an existing drug withexcellent repurposing potential for the treatment of fatty liver diseases such as Non-alcoholicsteatohepatitus (NASH). Using affinity chromatography, we have also identified Coenzyme Q10as an endogenous ligand for both receptors. The ~50 transgenic fish lines expected shouldhave enormous potential for the understanding and prevention of other metabolic disorders andrelated diseases such as cancer, depression, memory, inflammatory and immune disorders.8 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts37

ZDM8Boston | USA 2015ZDM8 ABSTRACTSYap reprograms glutamine metabolism to support growth duringliver development and tumorigenesis.Andrew G. Cox 1,* , Katie L. Hwang 1,2* , Kimberley Evason 3 , Kristin K. Brown 4 ,Sebastian Beltz 1 , Keelin O’Connor 1 , Giorgio G. Galli 5 , Allison Tsomides 1 , DeanYimlamai 5 , Sagar Chhangawala 6 , Min Yuan 4 , Evan Lien 4 , Julia Wucherpfennig 1 ,Sahar Nissim 1 , Akihiro Minami 1 , David E. Cohen 1 , Fernando D. Camargo 5,6 , JohnAsara 4 , Yariv Houvras 7 , Didier Y. Stainier 3,8 , Wolfram Goessling 1,61. Brigham and Women’s Hospital, Harvard Medical School, Boston, MA2. Harvard/MIT MD-PhD Program, Harvard Medical School, Boston, MA3. University of California San Francisco, San Francisco, CA4. Beth Israel Deaconess Medical Centre, Harvard Medical School, Boston, MA5. Boston Children’s Hospital, Harvard Medical School, Boston, MA6. Harvard Stem Cell Institute, Cambridge, MA7. Weill Cornell Medical College and New York Presbyterian Hospital, NY8. Max Planck Institute, Bad Nauheim, Germany* equal contributionThe Hippo pathway has emerged as a master regulator of organ size and tumorigenesis.It is unclear, however, how the Hippo pathway affects cellular metabolism to provide theenergy and cellular building blocks required for rapid cell growth. Here, we utilizetransgenic zebrafish with liver-specific expression of the Hippo pathway effector Yap todemonstrate that Yap promotes hepatomegaly, liver dysplasia and increasedsusceptibility to chemical hepatocarcinogenesis. Transcriptomic and metabolomicprofiling identified glutamine metabolism as a predominant target of Yap. Yap activationenhances glutamine synthetase (Glul) expression and activity. This results in elevatedsteady-state levels of glutamine and enhanced nitrogen flux into de novo purine andpyrimidine biosynthesis. In vitro studies in human liver cancer cells identify Glul as adirect target of Yap. Yap-mediated activation of Glul is required for rapid cell and organgrowth, as Glul knockdown or intervention studies with the Glul inhibitor methioninesulfoximine diminish Glul activity, inhibit nitrogen flux into nucleotide biosynthesis andsuppress liver growth observed in Yap transgenics. We conclude that Yap regulates Glulexpression and reprograms nitrogen metabolism to enhance liver growth duringdevelopment and tumorigenesis. Our findings demonstrate that Yap integrates theanabolic demands of rapid cell proliferation by increasing the flux of glutamine intonucleotide biosynthesis.388 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts

ZDM8 ABSTRACTSZDM7Madison | USA 2015ZDM8Boston | USA 2015DNA hypomethylation induces interferon response andmacrophage activation in uhrf1 mutant zebrafish embryosYelena Chernyavskaya, Raksha Mudbhary, Debra Tokarz, Vinitha Jacob, SmitaGopinath, Xiaochen Sun, Jeffrey A. Yoder , Yujin Hoshida and Kirsten C. Sadler1. Division of Liver Diseases, Department of Medicine, Department of Developmental andRegenerative Biology.2. Division of Liver Diseases, Department of Medicine, Department of Developmental andRegenerative Biology, Graduate School of Biomedical Sciences.3. Department of Molecular Biomedical Sciences, Center for Comparative Medicine andTranslational Research, North Carolina State University4. Division of Liver Diseases, Department of Medicine, Department of Developmental andRegenerative Biology, Graduate School of Biomedical Sciences.5. Division of Liver Diseases, Department of Medicine, Department of Developmental andRegenerative Biology.6. Division of Liver Diseases, Department of Medicine, Liver Cancer Program, Tisch CancerInstitute, Icahn School of Medicine at Mount Sinai7. Department of Molecular Biomedical Sciences, Center for Comparative Medicine andTranslational Research, North Carolina State University.8. Division of Liver Diseases, Department of Medicine, Liver Cancer Program, Tisch CancerInstitute, Graduate School of Biomedical Sciences, Icahn School of Medicine at Mount Sinai.9. Division of Liver Diseases, Department of Medicine, Department of Developmental andRegenerative Biology, Graduate School of Biomedical Sciences.DNA methylation is a fundamental epigenetic modification that has been extensivelystudied in cultured cells however less is known about the consequences of globalmethylome remodeling in a whole organism. Using zebrafish mutants in two of the genesthat are required for DNA methylation - Ubiquitin-like, Containing PHD and RING FingerDomains 1 (UHRF1) and DNA methyltransferase 1 (DNMT1) – we have made thesurprising discovery that there is a dramatic induction of the innate immune system inresponse to the global DNA hypomethylation in these mutants. We found that 70% of thetop upregulated genes in uhrf1 mutants are involved in the immune system, with 8.5% ofall upregulated genes associated with the interferon response. We found that both uhrf1mutants and fish injected with unmethylated DNA have an expanded population ofactivated macrophages, suggesting that excess hypomethylated DNA mimics theimmune response to infection. In situ hybridization showed that interferon target genesare upregulated in those tissues most severely affected by uhrf1 mutation (head andliver). In addition, reduction of irf8 blocked macrophage development without repressingthe interferon response in uhrf1 mutants. The same immune genes are upregulated indnmt1 mutants, demonstrating that DNA hypomethylation induces the interferonresponse. We conclude that global DNA hypomethylation both induces the interferonresponse in non-immune cells and also stimulates macrophage activation andrecruitment to clear debris of cells that die due to methylome disruption. These findingsimplicate UHRF1 as a potential agent in immune-associated DNA hypomethylation.8 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts39

ZDM8Boston | USA 2015ZDM8 ABSTRACTSThe pro-apoptotic factor Bax regulates necrosis duringMycobacterial infectionF.J. Roca, S. Redmond, L. Ramakrishnan.Department of Medicine, University of Cambridge, MRC-LMB, Cambridge, UKIn the zebrafish-Mycobacterium Marinum model for tuberculosis infection, excessTNF triggers necrosis of infected macrophages which leads to exuberant extracellularbacterial growth and host susceptibility (Tobin et al.,2012; Roca and Ramakrishnan,2013). It has been shown that in infected macrophages, excess TNF induces productionof reactive oxygen species (ROS) which activate two death pathways one of whichinvolves the mitochondrial transition pore, and the other leads to ceramide productionand lysosomal permeabilization.In exploring the events downstream of ceramide production, we found thatceramide acts in a Cathepsin D-dependent manner to induce cell death via pro-apoptoticfactors including Bax. We engineered Bax mutants that lack its different functionaldomains and studied the ability of each mutant protein to regulate apoptosis andnecrosis. Then we infected zebrafish larvae expressing the Bax mutants that wereunable to induce apoptosis. We found the BH3 domain, which is required for Baxoligomerization during apoptosis, not to be required for necrosis under excess TNFconditions. Rather, an N-terminal transmembrane helix was found to be required fornecrosis.408 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts

ZDM8 ABSTRACTSZDM7Madison | USA 2015ZDM8Boston | USA 2015Disruption to the basal transcription machinery causes intestinaldysplasiaElizabeth L Christie 1 , Qian Du 1,2,3 , Naomi Mitchell 4 , Christopher Love 1,2,3 , Karen Doggett 2,3 ,Graham J. Lieschke 5 , Robert G Ramsay 6 , Leonie M Quinn 4 , Joan K Heath 1,2,31 Ludwig Institute for Cancer Research, Parkville, Victoria 3050, Australia; 2 Walter and Eliza Hall Instituteof Medical Research and 3 Department of Medical Biology, University of Melbourne, Parkville, Victoria3052, Australia; 4 Department of Anatomy and Neuroscience, University of Melbourne, Parkville, Victoria3010, Australia; 5 Australian Regenerative Medicine Institute, Monash University, Clayton, Victoria 3800,Australia; 6 Peter MacCallum Cancer Centre, East Melbourne, Victoria 3002, AustraliaCharacterisation of our collection of zebrafish intestinal mutants has uncovered novel andunexpected roles for ribosomal RNA processing proteins, general transcription factors (GTFs)and the minor class splicing machinery in cellular growth and division during development.GTFs are conserved multi-subunit complexes required for RNA Polymerase (Pol) II-dependenttranscription. Of these, TFIIH is the last to assemble on gene promoters and it plays a key rolein the initiation of transcription and RNA Pol II release.A relatively understudied component of TFIIH is gtf2h4/p52, which is mutated in sycorax s845 , ouronly mutant to exhibit an over-proliferation phenotype. sycorax larvae at 120hpf exhibit amultilayered, dysplastic intestinal epithelium, reminiscent of a pre-neoplastic lesion. To gaininsights into the transcriptional changes governing this increase in cell division, we conductedmicroarray analysis of micro-dissected wild type (WT) and sycorax intestines at 120hpf.sycorax intestines exhibited up-regulation of several key cell proliferation genes, including myca,mycb and myb.Transcriptional initiation and promoter escape are driven by two other TFIIH subunits, CDK7and cyclin H, which phosphorylate serine residues (Ser5 and Ser7) in the C-terminal domain(CTD) of POLR2A, the largest sub-unit of RNA Pol II. Subsequent to this, phosphorylation ofthe Ser2 residue of the CTD by CDK9/P-TEFb is required for productive elongation. Todetermine whether these events are dysregulated in sycorax, we conducted ChIP usingphosphor-specific antibodies to Ser5 and Ser2 in the RNA Pol II CTD along the zebrafish myca,mycb and myb genes. We found strikingly elevated levels of Ser5 phosphorylated RNA Pol IIon the myca, mycb and myb promoters of sycorax larvae (120hpf) compared to WT, but nochange on promoters of ubiquitously expressed genes, for example actin. Together these datasuggest that loss of the gtf2h4/p52 subunit of TFIIH specifically disrupts the mechanisms thatnormally regulate RNA Pol II activity, thereby promoting transcription of these important protooncogenes.8 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts41

ZDM8Boston | USA 2015ZDM8 ABSTRACTSleukotriene signaling during host defense responses to bacterialinfectionAnna HuttenlocherThe focus of discussion will be on leukotriene signaling during host defense responses tobacterial infection.The role of neutrophil-macrophage cross talk will be discussed.428 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts

ZDM8 ABSTRACTSZDM7Madison | USA 2015ZDM8Boston | USA 2015Zebrafish models of Staphylococcus aureus pathogenesis: Seeingis believing.Stephen RenshawStaphylococcus aureus (SA) is an important human pathogen, which has evolved a broadrange of strategies for incapacitating the cellular innate immune response. As antibioticresistant strains become more widespread, there is a pressing need to understand thepathogenesis of SA during infection in vivo. Zebrafish models allow visualisation of hostpathogeninteraction in realtime, and simultaneous assessment of pathogen dynamics. Bothof these approaches have accelerated our understanding of staphylococcal pathogenesis.Host phagocytes play a crucial role in controlling S. aureus infection, which can lead to apopulation ‘‘bottleneck’’ whereby clonal expansion of a small fraction of the initial inoculumfounds a systemic infection. The population dynamics of S. aureus pathogenesis were studiedin vivo using antibiotic-resistant strains constructed in an isogenic background, coupled withsystemic models of infection in zebrafish. Subsequent murine experiments revealedunexpected and complex bacterial population kinetics arising from clonal expansion duringinfection in particular organs.As part of studies to identify host factors important in regulating immune responses to SA, wefound that the cystine-knot protein, nerve growth factor b (NGFb), plays an unexpectedSpaetzle-like role in immunity to Staphylococcus aureus infection. Deleterious mutations ofeither human NGFb or its high-affinity receptor tropomyosin-related kinase receptor A (TRKA)were associated with severe S. aureus infections. NGFb was released by macrophages inresponse to S. aureus exoproteins and enhanced phagocytosis and killing, stimulated cytokineproduction, and promoted neutrophil recruitment. TrkA knockdown in zebrafish increasedsusceptibility to S. aureus infection, confirming an evolutionarily conserved role for NGFb-TRKA signaling in pathogen-specific host immunity.8 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts43

ZDM8Boston | USA 2015ZDM8 ABSTRACTSA new immunometabolic mechanism driving macrophageactivation during gouty arthritisChris Hall 1 , Maarten van der Kroef 1 , Leslie Sanderson 1 , Lisa Lawrence 1 ,Bregina Pool 2 , Nicola Dalbeth 2 , Kathy Crosier 1 and Phil Crosier 11 Department of Molecular Medicine and Pathology, Faculty of Medical and Health Sciences,University of Auckland, Auckland, New Zealand.2 Department of Medicine, Faculty of Medical and Health Sciences, University of Auckland,Auckland, New Zealand.The causative agent in gout is monosodium urate (MSU) crystals that accumulate in thejoint and stimulate an inflammatory response. MSU crystal-induced inflammation isdriven by mononuclear phagocytes that drive recruitment of neutrophils following releaseof pro-inflammatory cytokines, including IL-1β and TNF-α. Despite MSU crystals beingrecognised as the causative agent of gout, patients with deposits of MSU crystals do notalways experience gout attacks suggesting other contributing factors are required.Evidence suggests that fatty acids can act synergistically with MSU crystals to regulatemacrophage-driven inflammation. Understanding how fatty acids contribute tomacrophage activation, in response to MSU crystals, may reveal new therapeuticpossibilities in alleviating inflammation associated with gouty arthritis.We have established a zebrafish model of MSU crystal-driven inflammation that closelyresembles gouty arthritis. Injection of MSU crystals into Tg(mpeg1:EGFP) larvae wasfollowed by their rapid phagocytosis by macrophage-lineage cells that express proinflammatorymarkers, including il1b and tnfa, and immunoresponsive gene 1 (irg1). Wehave previously shown Irg1 (which we demonstrate is also expressed by mammalianmacrophages stimulated with MSU crystals) functions as an immunometabolic regulatorof macrophage activity by coupling fatty acid metabolism and mitochondrial reactiveoxygen species (mROS) production. Mitochondria-derived ROS is known to regulateaspects of macrophage function, including transcription of pro-inflammatory genes. Weshow the recruitment of neutrophils to injected MSU crystals is dependent on fatty acidβ-oxidation, macrophage Irg1 activity/mROS production, NF-κB and inflammasomeactivation. We propose a new immunometabolic mechanism controlling goutyinflammation where fatty acid β-oxidation fuels the production of Irg1-dependent mROSproduction within macrophages that contributes to NF-κB-driven expression of il1b andtnfa and neutrophil recruitment. Furthermore, by performing a drug repurposing screenwe have identified several drugs the potently suppress macrophage irg1 expression andinflammation in response to MSU crystals, supporting the ability to therapeutically targetthis immunometabolic pathway.448 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts

ZDM8 ABSTRACTSZDM7Madison | USA 2015ZDM8Boston | USA 2015Trim9 Mediates Zebrafish Macrophage Chemotaxis in a RINGdependentMannerDebra A. Tokarz 1 and Jeffrey A. Yoder 1,21Department of Molecular Biomedical Sciences, North Carolina State University, Raleigh, NC, USA2Center for Comparative Medicine and Translational Research, North Carolina State University, Raleigh,NC, USATissue infiltration by macrophages and neutrophils is an early and critical step in the innateimmune response, yet excessive infiltration by these leukocytes can result in tissue damage andperpetuate inflammation, contributing to a wide variety of disease conditions. Chemotacticfactors and cell surface receptors mediating leukocyte migration have been extensively studied,but the intracellular mediators of this process are less well-defined. Defining intracellularmediators of neutrophil and macrophage chemotaxis will reveal novel targets for modulatinginflammation. The tripartite motif (TRIM) proteins are a family of E3 ubiquitin ligases of whichseveral members have defined roles in innate immune responses. Although previouslyconsidered to be brain-specific, we have identified trim9 as a gene with increased transcriptlevels within macrophages and neutrophils of larval zebrafish following immune stimulation.Trim9 is highly conserved among vertebrate species, and we find that TRIM9 transcript levelsalso increase in human macrophages and neutrophils following stimulation with multiple toll-likereceptor agonists. Prior to our studies, Trim9 had no reported immune function, however, itspublished role in control of axon growth guidance suggests it is important in cell migration. Toinvestigate the role of Trim9 in leukocyte chemotaxis, we disrupted Trim9 function in zebrafishmacrophages through expression of a truncated form of Trim9 that lacks the RING domainrequired for ubiquitin ligase activity (ΔRING-Trim9). We have demonstrated that expression ofΔRING-Trim9 results in significantly reduced in vivo chemotaxis of zebrafish macrophages,while expression of full-length Trim9 under the same conditions does not. We propose thatTrim9, through its ubiquitin ligase activity, acts as a novel intracellular mediator of macrophagechemotaxis. Identifying Trim9 substrates and binding partners within macrophages may providenew therapeutic strategies for modulating inflammation.8 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts45

ZDM8Boston | USA 2015ZDM8 ABSTRACTSGenetic basis of intramembranous bone vascularization in arare vascular syndromeJingwei R. Xiong, Arda Cetinkaya, A. Nurten Akarsu, Bruno Reversade1. Institute of Medical Biology, A*STAR, Singapore2. Department of Medical Genetics, Hacettepe University Medical Faculty, Ankara, Turkey3. Department of Medical Genetics, Hacettepe University Medical Faculty, Ankara, Turkey4. Institute of Medical Biology, A*STAR, Singapore; Institute of Molecular and Cell Biology,A*STAR, SingaporeVascularization of bone is necessary for skeletal development, repair and lifelongmaintenance. As such, engineered bone implants which often lack adequatevascular networks fail to integrate successfully within the host tissue. To overcomethis problem, a deeper understanding of the molecular mechanisms regulating bonevascularization is needed.Here, we uncover the genetic basis of a rare Mendelian bone vascularizationdisorder known as Hereditary Intraosseous Vascular Malformation of the CraniofacialRegion (VMOS, MIM 606893). VMOS-associated pathology is defined by malformedblood vessel architecture within the jaw and skull, resulting in dramatic remodeling ofintramembranous bone tissue, but not of endochondral bone tissue. Homozygositymapping and exome sequencing of five affected families identify the causative gene,which is known to signal through a small GTPase.To model VMOS, we generated several maternal-zygotic knockout zebrafish of thecorresponding orthologue. Much to our dismay, mutant fish showed no discerniblebone or blood vessel phenotype, suggestive of species-specific gene function orgenetic redundancy between paralogues. Nonetheless, results from our humangenetic studies suggest that this gene is exclusive to vascularization ofintramembranous but not endochondral bones, a distinction which had no geneticbasis to date.468 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts

ZDM8 ABSTRACTSZDM7Madison | USA 2015ZDM8Boston | USA 2015The lymphatic vasculature in health and diseaseJonathan Astin 1 , Pauline Misa 1 , Kazuhide Okuda 1 , Stefan Oehlers 2 , ChristopherHall 1 , Felix Ellett 3 , Sultan Alasmari 3 , Graham Lieschke 3 , Kathryn Crosier 1 andPhil Crosier 11. Department of Molecular Medicine and Pathology, School of Medical Sciences, Universityof Auckland, Auckland, New Zealand.2. Department of Molecular Genetics and Microbiology, Duke University Medical Center,Durham 27710, USA3. Australian Regenerative Medicine Institute, Monash University, Clayton 3800 Victoria,AustraliaThe lymphatic vasculature plays an essential role in diverse biological processessuch as the maintenance of tissue fluid balance, immunosurveillance and thetransport of dietary fats. We are interested in understanding the development of thelymphatic vasculature within selected organ systems, the molecular regulation ofthese processes and how these systems may be compromised in disease settings.Solid tumours and many inflammatory-based diseases are associated with thegrowth of lymphatic vessels (lymphangiogenesis). In solid tumours,lymphangiogenesis is an integral step in the metastatic spread of tumour cells andthere is an important potential clinical niche for novel anti-lymphangiogenic agents.Using a zebrafish phenotype-based chemical screen, we have identified fourcompounds that have anti-lymphatic activity: kaempferol, a natural product found inplants, leflunomide, an inhibitor of pyrimidine biosynthesis, and cinnarizine andflunarizine, members of the type IV class of calcium channel antagonists. Mammaliananti-lymphatic activity was confirmed in Matrigel plug assays and in tumourxenografts. This screen provides important proof-of-principle that mammalian antilymphaticagents can be identified and developed in the zebrafish.Recent studies have shown that increased lymphangiogenesis may contribute to theresolution of chronic inflammation in diseases such as in inflammatory bowel disease(IBD), however the mechanisms underlying inflammatory lymphangiogenesis arelargely unknown. We have established a novel inflammatory lymphangiogenesismodel in zebrafish larvae involving colitogenic challenge stimulated by exposure to2,4,6-trinitrobenzenesulfonic acid (TNBS) or dextran sodium sulphate (DSS). We findthat zebrafish macrophages express vascular growth factors vegfaa, vegfc and vegfdand chemical ablation of these cells inhibits TNBS-induced lymphangiogenesis,suggesting that the recruitment of macrophages to the intestine upon colitogenicchallenge is required for the formation of IBD-associated lymphatic vessels. Webelieve this model can be used to investigate the role and mechanism oflymphangiogenesis in inflammatory diseases such as IBD.8 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts47

ZDM8Boston | USA 2015ZDM8 ABSTRACTSExploiting the regenerative biology of zebrafish tendons to build a roadmap forhuman regenerative medicine applicationsJenna L. GallowayCenter for Regenerative Medicine, Department of Orthopaedic Surgery, Massachusetts GeneralHospital, Harvard Medical School, Harvard Stem Cell InstituteTendons connect and transfer force between the muscles and bones of the body,making them highly prone to injury. These injuries affect millions of people each yearand incur significant healthcare costs. With inadequate long-term treatment options,lasting repair of the injured tissue remains a challenge. The reasons for this stem froman incomplete understanding of the fundamental cellular and molecular events thatgovern tendon repair. We have shown that zebrafish tendons share multiple hallmarkcharacteristics with mammalian tendons, including ultrastructure, gene expression,developmental regulation, and mechanical properties. To gain a better understanding ofthe pathways regulating the injury response, we have established acute tendon injuryand cell ablation models in the zebrafish. For this purpose, we have generatedfluorescent tendon transgenic zebrafish lines that allow us to visualize the tendon cellsfrom developmental to adult stages and during the repair process. We have exploitedthe optical properties of the tendon matrix and developed methods using SecondHarmonic Generation (SHG) with multiphoton microscopy to visualize collagen fiberorganization in the zebrafish tendon. For our acute injury studies, we have focused on asuperficially positioned cranial tendon that allows easy visualization of the injury repairprocess. In this model, we have found that adult zebrafish tendons re-align their collagenfibers in four weeks without residual scarring. Their initial injury response resembles thatfound in mammals with inflammatory, proliferative and remodeling phases. Moreover,this quick regenerative response can be used to determine the mechanisms underlyingcell and molecular pathway function in the context of vertebrate tendon repair. In ourtendon cell ablation model, we have engineered transgenic animals for inducing spatialand temporal specific tendon cell death. Using these genetic methods, we can ablatetendon cells during embryonic, juvenile, and adult stages, and observe cell recovery. Weare now using tendon transgenic lineage tracing and cell ablation lines to understand thecontribution and function of specific tendon cells in the injury repair process, and themolecular pathways regulating their activities. Together, these studies will expand ourunderstanding of the cellular and molecular pathways regulating tendon healing in aregenerative model system, impacting how we approach human tendon injury repair.488 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts

ZDM8 ABSTRACTSZDM7Madison | USA 2015ZDM8Boston | USA 2015Chemical and Genetic Approaches to Identify Therapeutic Targets forEwing SarcomaJoanie Neumann 1,2 and James F. Amatruda 1,2,31. Department of Pediatrics, UT Southwestern Medical Center, Dallas, TX, USA2. Department of Molecular Biology, UT Southwestern Medical Center, Dallas, TX, USA3. Department of Internal Medicine, UT Southwestern Medical Center, Dallas, TX, USAEwing Sarcoma is a malignant bone and soft tissue tumor of children and young adults causedby chromosomal translocations that create novel fusions of the EWS gene with an ETS familytranscription factor (most commonly FLI1). While chemotherapy can be curative, more than halfof adolescents with Ewing sarcoma die of disease within five years. The cellular origin andmechanisms of EWS-FLI1 mediated tumorigenesis are not known, impeding the development ofalternative therapies. Previously, we showed that somatic expression of human EWS-FLI1 inzebrafish causes Ewing sarcoma-like tumors (Leacock et al., DMM 2012). Given that humanEwing Sarcoma have neuroectodermal features we created a tissue specific model using themitfa promoter to drive the expression of hEWS-FLI1 specifically in neural crest-derived cells.Strikingly, EWS-FLI1 expression in melanocytes results in 1) increased number of early larvalmelanocytes in the head and 2) an almost a complete absence of pigmented melanocytes inadults, suggesting that EWS-FLI1 specifically interferes with the development and/or survival ofmelanocytes. Furthermore, a subset of tg(mitfa:hEWS-FLI1);p53 -/- fish develop tumorsresembling Ewing Sarcoma. To identify candidate compounds for targeted therapy of EwingSarcoma, we conducted a chemical suppressor screen on the Tg(mitfa:hEWS-FLI1) line testing~1200 compounds from libraries of known bioactive and FDA-approved compounds. Wecounterscreened against wildtype larvae to remove compounds affecting the growth andsurvival of melanocytes. We identified 70 compounds that suppressed the melanocytephenotype. Many of these compounds can be categorized together based on their role inseveral biological functions including the regulation of sex steroid signaling, inhibition of boneresorption, or regulation of prostaglandin production. We are further investigating thesecompounds and pathways to determine their potency and specificity as treatments for EwingSarcoma. These zebrafish models will provide molecular understanding of Ewing Sarcoma andwill foster the development of improved, targeted therapies.8 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts49

ZDM8Boston | USA 2015ZDM8 ABSTRACTSEstablishing a zebrafish model of craniosynostosis; P236R fgfr1amissense mutation introduced by CRISPR/Cas9 mediatedhomologous recombinationJolanta M. Topczewska 1 , Joanna P. Tomaszewski 1 , Jacek Topczewski 2 , Arun K. Gosain 1Northwestern University Feinberg School of Medicine, Ann & Robert H. Lurie Children’s Hospitalof Chicago, 1 Department of Surgery, 2 Department of PediatricsCraniosynostosis results from premature fusion of cranial bones, with a prevalence of1:2500 live births. Approximately 20% of all craniosynostosis cases are caused by mutations inFGF receptors. The majority of these mutations result in missense amino acid substitution thatconfers either ligand-dependent or ligand-independent activation of FGF signaling. Togenetically engineer P236R substitution into fgfr1a locus, we adopted a CRISPR targetedhomologous recombination. The CRISPR-fgfr1a construct was created in vitro, theoligonucleotide containing the mutated sequence, flanked by arms designed for homologousrecombination was manufactured; all RNAs were injected into zebrafish embryos. To confirmpresence of the mutation, specific primers were designed to amplify the modified sequence anda diagnostic restriction site was used to test for successful recombination. The high frequency ofCRISPR/Cas9 mediated mutagenesis in injected embryos allowed us to recover 9 adultfounders from a single experiment. As expected for a dominant mutation, the craniofacialphenotype was observed in G0, injected fish. Histological evaluation of adult fish revealedfusion of frontal bones underlying the interfrontal synostosis. Moreover, increased expression ofrunx2b in the suture was observed when compared with wild type siblings. A similar phenotype,and increased expression of Runx2 has been observed in a mouse model with the homologicalmutation P250R in Fgfr1. The increased expression was attributed to the P250R mutation andRunx2 being a target of Fgf/Fgfr1 signaling in the sutural tissue.In conclusion, zebrafish P236R fgfr1a mutation results in interfrontal suture fusion andskull malformation, providing the first evidence that modified Fgf/Fgfr signaling can inducecraniosynostosis in zebrafish, despite natural lifelong suture patency.508 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts

ZDM8 ABSTRACTSZDM7Madison | USA 2015ZDM8Boston | USA 2015RAP1-mediated MEK-ERK pathway defects in Kabuki syndromeNina Bögershausen 1,2,3* , I-Chun Tsai 4* , Esther Pohl 1,2,3 , Pelin Özlem Simsek Kiper 5 , FilippoBeleggia 1,2,3 , E. Ferda Percin 6 , Katharina Keupp 1,2,3 , Angela Matchan 7 , Esther Milz 1,2,3 ,Yasemin Alanay 5,8 , Hülya Kayserili 9 , Yicheng Liu 1,2,3 , Siddharth Banka 10 , Andrea Kranz 11 ,Martin Zenker 12 , Dagmar Wieczorek 13 , Nursel Elcioglu 14 , Paolo Prontera 15 , StanislasLyonnet 16 , Thomas Meitinger 17 , Francis Stewart 11 , Dian Donnai 10 , Tim M. Strom 17,18 , KorayBoduroglu 5 , Gökhan Yigit 1,2,3 , Yun Li 1,2,3 , Nicholas Katsanis 4,† , and Bernd Wollnik 1,2,3,†1 Institute of Human Genetics, University of Cologne, Cologne, Germany; 2 Center for MolecularMedicine Cologne (CMMC), University of Cologne, Cologne, Germany; 3 Cologne ExcellenceCluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University ofCologne, Cologne, Germany; 4 Center for Human Disease Modeling and Department of CellBiology, Duke University, Durham, North Carolina, USA; 5 Pediatric Genetics Unit, Department ofPediatrics, Hacettepe University Medical Faculty, Ankara, Turkey; 6 Department of MedicalGenetics, Gazi University Faculty of Medicine, Ankara, Turkey; 7 Oxford Gene Technology(OGT), Begbroke Science Park, Begbroke, Oxfordshire, UK; 8 Pediatric Genetics, Department ofPediatrics, Acibadem University, School of Medicine, Istanbul, Turkey; 9 Department of MedicalGenetics, Istanbul Medical Faculty, Istanbul University, Istanbul, Turkey; 10 Department ofGenetic Medicine, St Mary's Hospital, Manchester Academic Health Sciences Centre (MAHSC),University of Manchester, Manchester, UK; 11 Genomics, BioInnovationsZentrum, DresdenUniversity of Technology, Dresden, Germany; 12 Institute of Human Genetics, University HospitalMagdeburg, Magdeburg, Germany; 13 Institut für Humangenetik, Universitätsklinikum Essen,Essen, Germany; 14 Department of Pediatric Genetics, Marmara University Medical Faculty,Istanbul, Turkey; 15 Medical Genetics Unit, Hospital and University of Perugia, Perugia, Italy;16 Université Paris Descartes-Sorbonne Paris Cité, Institut Imagine, INSERM U781, HôpitalNecker-Enfants Malades, Paris, France; 17 Institute of Human Genetics, Helmholtz ZentrumMünchen, Neuherberg, Germany; 18 Institute of Human Genetics, Technische UniversitätMünchen, Munich, Germany.*These authors contributed equally to this workKabuki syndrome (KS) is a genetic disorder characterized by developmental delay andcongenital anomalies that include a distinctive facial appearance, loss of hearing, short stature,persistent fingerpads, and urogenital tract anomalies. Although mutations in chromatinregulators KMT2D (MLL2) and KDM6A have been recognized as a major cause of KS, thephysiological systems regulated by them are largely unknown. Using whole exome sequencingwe found a mutation converted to homozygosity by uniparental isodisomy (UPD) in RAP1A in apatient with a KS phenotype, and a de novo mutation in RAP1B in a second patient. To explorethe candidacy of this mutation and to gain insight into pathomechanism, we evaluated Rap1aand Rap1b in vivo and in vitro. These molecules belong to the Ras-like small GTPase family,regulate the MAPK pathway and are required for convergent extension (CE) movements duringdevelopment. Functional tests in vivo and in vitro showed that the Rap1a and Rap1b mutantsdisplay deficient CE movements and cause disregulated activation of MAPK signaling in bothzebrafish embryos and patient fibroblasts. We further show that Rap1 interacts genetically withKmt2d and that Rap1b expression is downregulated in both zebrafish kmt2d morphant andKMT2D deficient patient cells. Given that a distinctive jaw appearance is often seen in KSpatients, we show that depletion of Rap1 and Kmt2d affect the layout of pharyngeal skeleton byinhibiting cell intercalation, an essential process of CE movements. Importantly, thesephenotypes could be rescued by rebalancing MEK-ERK signaling via administration of smallmolecule inhibitors of MEK, offering a potential treatment paradigm for KS. Taken together, ourstudies have identified new genetic contributors of Kabuki syndrome and uncover the molecularmechanism of pathogenic features in KS patients, thereby providing a novel potential avenue fortreatment design in the future.8 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts51

ZDM8Boston | USA 2015ZDM8 ABSTRACTSNAD+ Biosynthesis Improves Muscle Structure and Function in a SecondaryDystroglycanopathyErin V. Carter, Joseph J. Belanger, Margaret E. Pasquarella, Linda S. Archambault, Clarissa A. HenrySchool of Biology and Ecology, University of Maine, Orono, ME 04469Muscular dystrophies are common, currently incurable diseases. A subset of dystrophies result fromgenetic disruptions in complexes that attach muscle fibers to their surrounding extracellular matrixmicroenvironment. We previously found that nicotinamide riboside kinase 2b-mediated NAD+biosynthesis, which functions as a small molecule agonist of muscle fiber-extracellular matrixadhesion, corrects dystrophic phenotypes in zebrafish lacking either a primary component of thedystrophin-glycoprotein complex, dystroglycan, or integrin alpha7. Mutations in at least 6 genesidentified thus far result in abnormal glycosylation of alpha-Dystroglycan. The resultant diseases arecalled secondary dystroglycanopathies. Our current goal is to determine whether NAD+supplementation ameliorates muscle degeneration and improves muscle function in secondarydystroglycanopathies. Our hypothesis is that because NAD+ benefited model of primarydystroglycanopathy where alpha and beta dystroglycan are absent, we expect this treatment tobenefit models of secondary dystroglycanopathy where alpha dystroglycan glycosylation is disrupted.We have used CRISPR-Cas9-mediated mutagenesis to generate zebrafish models for secondarydystroglycanopathies caused by mutations in pomgnt1, pomgnt2, large, large2, ispd, pomt1, pomt2,dpm2, dpm3, dolk, gmppb, b3galnt2, and fktn. These mutant lines are currently growing and PCR andDNA sequencing will be used to detect heritable mutations. In the mean time, we are testing ourhypothesis using previously published morpholino models of secondary dystroglycanopathies.Preliminary data indicate that FKRP-deficient embryos have thinner and disorganized muscle fibers,disrupted MTJs, reduced locomotion, and muscle degeneration. Supplementation with exogenousNAD+ improved muscle structure and function. In the future, we plan to characterize how FKRP andNAD+ contribute to neuromuscular junction integrity and analyze these same processes in otherdystroglycanopathy models. Overall, our results indicate that NAD+ supplementation may havetherapeutic implications for a broad spectrum of muscular dystrophies.528 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts

ZDM8 ABSTRACTSZDM7Madison | USA 2015ZDM8Boston | USA 2015Muscle cell dynamics and regeneration in zebrafish musculardystrophy modelsJared C. Talbot, Michael A. Berberoglu, Kimberly J. Hromowyk, and Sharon L. AmacherThe Ohio State University, Department of Molecular Genetics, Columbus, OH 43210Skeletal muscle is comprised of different muscle cell types that have different activities,physiology, and metabolism. Slow- and fast-twitch muscle fiber types can be differentiallyaffected in individuals suffering from muscle disease. For example, in Duchenne MuscularDystrophy (DMD) patients and animal models, fast-twitch fibers are more susceptible than slowtwitchfibers. To implement effective therapies and reliably target the susceptible muscle cellpopulation, it is critical to understand regulatory mechanisms that underlie the specification andmaintenance of the two cell types. We have zebrafish transgenic reporter lines that robustlyidentify slow and fast muscle cell membranes, nuclei, and Dystrophin protein dynamics andhave generated 10+ zebrafish knockouts in genes suspected to regulate fast muscle cellresponses, such as cell elongation and fusion, triggered by slow muscle cells. Using timelapseconfocal imaging, we are investigating slow and fast muscle cell interactions in live embryos toidentify cell behaviors tightly associated with fast muscle specification, differentiation, andmaintenance, and examine these behaviors in muscular dystrophy mutants and our geneknockouts, focusing on mutants that confer dystrophy-like phenotypes. To investigate whetheradult muscle stem cells recapitulate the embryonic program during muscle regeneration, we areanalyzing regenerative capacity in adults, in muscular dystrophy mutants, and in new mutantswe have generated that display dystrophic phenotypes. We aim to uncover regulatorymechanisms and interactions that explain why fast fibers are differentially lost in DMD patientsand to uncover cell interactions and behaviors that when abnormal, are early predictors of futurefiber loss. The long-term goal is to identify pathways that influence fiber type choice andenhance regenerative capacity and to generate robust zebrafish models for future drug screens.8 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts53

ZDM8Boston | USA 2015ZDM8 ABSTRACTSA novel role for Wnt9a in hematopoietic stem cell development.Stephanie Grainger* 1 , Jenna Richter* 1 , Claire Pouget 1 , Sara Wirth 2 , Kathrin S.Grassme 2 , Matthew Swift 3 , Brant M. Weinstein 4 , Wiebke Herzog 3,5,6 , David Traver ¥,1and Karl Willert ¥,1*These authors contributed equally to this work¥co-corresponding authors1Department of Cellular and Molecular Medicine, University of California, San Diego, La Jolla, California,92037, USA. 2University of Münster, Münster, Germany 3Department of Oncology, LombardiComprehensive Cancer Center, Georgetown University Medical Center, Washington, DC 20057 USA4Section on Vertebrate Organogenesis, Laboratory of Molecular Genetics, NICHD, NIH, Bethesda,Maryland, 20892, USA 5 Max-Planck-Institute for Molecular Biomedicine, Münster, Germany 6 Cells-in-Motion Cluster of Excellence (EXC 1003 – CiM), University of Münster, GermanyWnt signaling is critical in the formation and function of stem cell populations across many organsystems and organisms. Whereas Wnt signaling has been demonstrated to regulate thehomeostasis of hematopoietic stem cells (HSCs) in the adult mouse bone marrow, a role in thedevelopmental emergence of HSCs has not been fully addressed. We have uncovered atemporal requirement for Wnt signaling in the vasculature specifically mediated by Wnt9a.Genetic and small molecule manipulation of Wnt signaling affects the number of HSCs thatemerge in the dorsal aorta. Loss of Wnt9a function led to loss of HSCs, a phenotype that wasreversed only by Wnt9a overexpression but not by the closely related paralogue Wnt9b or bythe prototypical canonical Wnt3a ligand, indicating a unique requirement for Wnt9a in HSCemergence. These findings may inform the development of protocols to derive HSCs in vitrofrom human pluripotent precursors.548 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts

ZDM8 ABSTRACTSZDM7Madison | USA 2015ZDM8Boston | USA 2015Characterization of zebrafish erythroid burst cell lineOndrej Svoboda, Olga Machonova and Petr BartunekDepartment of Cell Differentiation, Institute of Molecular Genetics AS CR v.v.i., 142 20Prague 4, Czech Republic.Erythroid cells develop through several stages of committed progenitors, termed burstforming units erythroid (BFU-E) and colony forming units erythroid (CFU-E). Previously, wewere able to identify and characterize these progenitors by utilizing clonal assays in semisolidmedia and recombinant zebrafish erythropoietin (Epo).In addition to Epo, we also generated recombinant zebrafish Kit ligand a and b (Kitla/b orScfa/b). Here we show that addition of Kitla, but not Kitlb, act in synergy together with Epo toincrease self-renewal of BFU-Es. We established liquid cultures that enabled us to studyhematopoietic cells in a suspension. Addition of Epo, Kitla and dexamethasone (Dex) tozebrafish progenitors synergistically stimulated expansion of erythroid progenitors for up to14 days.By employing these conditions, we were able to spontaneously establish the zebrafisherythroid burst cell line (ZEB). The ZEB cells critically require Epo for their proliferation andlost their dependence on Kitla. In addition, it seems that the terminal differentiation of theZEB cells is blocked. The biochemical characterization of these cells revealed strong tyrosinephosphorylation upon stimulation with Epo, Kitla, IGF1 and Insulin, extensive proliferationassays however showed only mild response to IGF1 and Insulin in comparison to Epo.To understand the mechanisms underlying the self-renewal of ZEB cells, we analyzed theirtranscriptome by RNAseq. This analysis position them into the compartment of earlycommitted BFU-E progenitors. Finally, we were able to transplant the ZEBs into the zebrafishembryos and adults to study their engraftment.In summary, we describe the first zebrafish immortalized hematopoietic cell line. It will be ofmajor interest to identify a mechanisms underlying the immortalization of these cells. ZEBcells will serve as a valuable and unique tool in numerous studies, including thecharacterization of zebrafish erythroid signaling at a molecular level or cell engraftment aftertransplantation.8 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts55

ZDM8Boston | USA 2015ZDM8 ABSTRACTSFam210b regulates cellular iron uptake and heme synthesisBarry H. Paw 1,2,3 , Yvette Y. Yien 1 , Caiyong Chen 1 , Jiahai Shi 4 , Daniel E. Bauer 2,3 ,Carla M. Koehler 5 , James Palis 6 , Jerry Kaplan 7 , Stuart H. Orkin 2,3 , Harvey F.Lodish 41 Division of Hematology, Brigham and Women’s Hospital, Harvard Medical School, Boston,Massachusetts, USA.2 Division of Hematology-Oncology, Boston Children’s Hospital, and3 Department of Pediatric Oncology, Dana-Farber Cancer Institute, Harvard Medical School,Boston, Massachusetts, USA4 Whitehead Institute and Department of Biology, Massachusetts Institute of Technology,Cambridge, Massachusetts, USA.5 Department of Chemistry and Biochemistry, UCLA, Los Angeles, California, USA.6 Department of Pediatrics, University of Rochester School of Medicine and Dentistry, Rochester,New York, USA7 Department of Pathology, University of Utah School of Medicine, Salt Lake City, Utah, USA.While iron is crucial for many essential cellular processes, such as heme-mediatedoxygen-transport, oxidative phosphorylation, and DNA synthesis to name a few, it ishighly reactive and can participate in redox reactions that are detrimental to cells.Organisms have therefore evolved transport mechanisms to tightly regulate cellular andmitochondrial iron levels. To date, these critical mechanisms are poorly understood.Model organisms, such as the zebrafish, mouse and yeasts, have collectivelycontributed to our recent understanding of novel transporters and regulators ofintracellular iron and heme trafficking. We have identified Fam210b, which encodes amitochondrial transmembrane protein, as a novel regulator of cellular and mitochondrialiron levels. In zebrafish and mice, Fam210b is enriched in differentiating erythroid cellsand liver, which are tissues that require large amounts of iron for heme synthesis.Fam210b deficiency results in erythroid differentiation and heme synthetic defects as aresult of decreased mitochondrial and cellular iron levels. Supplementation of Fam210b-/- murine erythroleukemia cells and fam210b zebrafish morphants with non-transferriniron chelates restores erythroid differentiation and hemoglobin synthesis. Our findingsreveal that FAM210B facilitates mitochondrial iron import and is hence essential forheme synthesis in vertebrates. FAM210b may therefore function as a genetic modifierfor mitochondriopathies, anemias or porphyrias in humans.568 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts

ZDM8 ABSTRACTSZDM7Madison | USA 2015ZDM8Boston | USA 2015Vitamin D3 modulates vertebrate hematopoietic stem cellhomeostasisMauricio Cortes 1 , Michael J. Chen 2 , Sarah Y. Liu 1 , Wanda Kwan 1 David L. Stachura 3 ,Thorsten M. Schlaeger 2 , Wolfram Goessling 4 , George Q. Daley 2 , Trista E. North 11. Beth Israel Deaconess Medical Center, Harvard Medical School Boston, MA.2. Children’s Hospital, Harvard Medical School, Boston MA3. California State University, Chico, CA.4. Brigham and Women’s Hospital, Harvard Medical School, Boston, MAVitamin D was identified via a chemical screen in zebrafish as a modulator of expressionof the hematopoietic stem cell (HSC) markers runx1/cmyb in the aorta-gonadmesonephros, the site of definitive hematopoiesis. Treatment with the vitamin Dprecursor choleclaciferol (D3) during HSC niche formation resulted in decreasedrunx1/cmyb expression; however, D3 treatment during HSC induction and expansion,had no effect, suggesting D3 affects vascular niche formation. In contrast, treatment withactive vitamin D (1,25OH-D3) during either time window resulted in increasedrunx1/cmyb expression. FACS analysis of hematopoietic stem and progenitor cells(HSPCs) quantified and confirmed our findings. Hedgehog (Hh) signaling, anddownstream Notch activity, is essential for hemogenic endothelium specification.Treatment with D3 resulted in decreased Hh signaling as determined by FACS using aHh reporter line. Similarly, inhibition of CYP2R1, the enzyme required for D3 25-hydroxylation, via a mutation modeling humans with Vitamin D deficiency decreased Hhactivity and reduced HSPCs. Consistent with defects in Hh signaling, D3 treatedembryos had diminished Notch activity and abnormal specification of hemogenicendothelium. In contrast, HSPCs exposed to 1,25OH-D3 showed a significant increasein cell number, with 2-fold elevation in cell proliferation. Knockdown of the vitamin Dreceptor (VDRA) decreased runx1 expression and reduced HSPC number by FACS,indicating a receptor-mediated requirement in HSPC production. Colony forming (CFU)assays of FACS-sorted adult HSCs treated with 1,25OH-D3 showed a significantincrease in total CFUs indicating direct action on HSCs. Similarly, CFU-C assays ofCD34+ human umbilical cord blood cells (hUBCs) exposed to 1,25OH-D3 elicited a 2-fold increase in total CFUs. In sum, our studies have characterized two mechanisms bywhich vitamin D can modulate hematopoiesis. In addition, we have shown conservationof effect in hUBCs that indicates active vitamin D may have clinical utility for theexpansion of human HSCs for transplantation.8 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts57

ZDM8Boston | USA 2015ZDM8 ABSTRACTSThymocyte selection-associated HMG box protein (TOX) inducesgenomic instability in T-cell Acute Lymphoblastic LeukemiaRiadh Lobbardi 1,2 , Jordan Pinder 3,4§ , Barbara Martinez-Pastor 5§ , Jessica S.Blackburn 1,2§ , Nouran S. Abdelfattah 1,2 , Aleksey Molodtsov 1,2 , Marc R. Mansour 6,7 ,Brian J. Abraham 8 , Debra Toiber 5,9 , Manon de Waard 10 , Esha Jain 11 , Richard A.Young 8 , A. Thomas Look 6,7 , Ruslan I. Sadreyev 11 , John M. Asara 12 , RaulMostoslavsky 5 , Graham Dellaire 3,4 , David M. Langenau 1,21. Molecular Pathology and Cancer Center, Massachusetts General Hospital, Boston, MA 02129,USA.2. Harvard Stem Cell Institute, Cambridge MA 02139, USA.3. Departments of Pathology and Biochemistry and Molecular Biology; Dalhousie University;Halifax, NS Canada.4. Beatrice Hunter Cancer Research Institute; Halifax, NS Canada.5. Massachusetts General Hospital Cancer Center, Harvard Medical School, Boston, MA 02114,USA.6. Department of Pediatric Oncology, Dana-Farber Cancer Institute, Harvard Medical School,Boston MA, 02115, USA.7. Department of Haematology, UCL Cancer Institute, University College London, WC1E 6BT, UK.8. Whitehead Institute for Biomedical Research, 9 Cambridge Center, Cambridge, MA 02142,USA.9. Department of Life Sciences, Ben-Gurion University of the Negev, 84105 Beer Sheva, Israel.10. University of Leiden, Netherlands.11. Department of Molecular Biology, Massachusetts General Hospital, Harvard Medical School,Boston, United States.12. Division of Signal Transduction, Beth Israel Deaconess Medical Center and Department ofMedicine, Harvard Medical School, 330 Brookline Ave, Boston, Massachusetts 02215, USA.§. These authors contributed equally to this work.MYC and NOTCH are the major oncogenic drivers in T-ALL, yet additional collaboratinggenetic lesions likely collaborate to induce malignancy. To identify these factors, a largescaletransgenic screen was completed where 28 amplified and over-expressed genesfound in human T-ALL were assessed for accelerating leukemia onset in transgeniczebrafish models of disease. TOX synergized with both MYC and NOTCH to induce T-ALL. TOX is dynamically regulated in T-cell development with peak expression occurringwhen thymocytes are actively undergoing T-cell receptor (TCR) recombination. Despitebeing genomically amplified in a subset of human and mouse T-ALL, beingoverexpressed nearly all human T-ALL, and being regulated by the TAL1/LMO2/MYBsuper-enhancer, a role for TOX in inducing T-ALL has not been reported.Characterization of zebrafish T-ALLs revealed that TOX expands the overall number ofmalignant T-ALL clones and promoted genomic instability as assessed by changes inDNA content and Whole Genome Sequencing. Moreover, xenograft transplantationexperiments showed TOX is important for continued tumor growth and maintenance inhuman T-ALL. Proteomic experiments in human T-ALL identified that KU70/KU80 bindto TOX. Given that TOX elevated genomic instability in the zebrafish model and boundto KU70/KU80, we hypothesized that TOX is a negative regulator of Non-HomologousEnd Joining (NHEJ) pathway. Fluorescent repair assays confirmed that TOX inhibitsNHEJ, while dynamic real-time imaging studies showed that TOX suppressesrecruitment of fluorescent-tagged KU80 to DNA breaks. Importantly, TOX loss offunction increased NHEJ in human T-ALL cells and reduced time to DNA repair asassessed by fluorescent Traffic Light Reporter assays and quantitative assessment of53BP1 and γH2A.X foci resolution following irradiation. Thus, TOX has prominent rolesin initiating T-ALL by suppressing KU70/KU80 function to promote genomic instabilityand ultimately elevating rates at which acquired mutations and rearrangements areamassed in developing pre-malignant T cells.588 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts

ZDM8 ABSTRACTSZDM7Madison | USA 2015ZDM8Boston | USA 2015Perphenazine induces apoptosis in T-cell acute lymphoblasticleukemia by direct activation of PP2AAlejandro GuiterriezCurrent therapies for T-cell acute lymphoblastic leukemia (T-ALL) are toxic and fail in asignificant subset of patients. To identify novel therapies, we performed a small moleculescreen in our zebrafish model of MYC-induced T-ALL. This screen revealed thatperphenazine, an FDA-approved antipsychotic, effectively induces apoptosis in zebrafishand human T-ALL. Perphenazine is best known as an inhibitor of dopaminergic signaling,but we found that its activity was unrelated to any of its known molecular targets. Our datainstead suggested that its anti-leukemic activity was mediated by an unknown, low-affinitymolecular target. Identifying biologically relevant low-affinity targets of drugs that have otherhigh-affinity targets has been a major roadblock in drug target discovery. To address this,we developed an approach termed affinity correlation proteomics, which is based on affinitychromatography and mass spectrometry proteomics, and used it to identify proteinphosphatase 2a (PP2A) as a novel target of perphenazine. Perphenazine directly binds andactivates the enzymatic activity of PP2A, which dephosphorylates and inactivates many ofthe best-known oncoproteins in human cancer, including AKT, ERK and MYC. Moreover,PP2A is required for the antineoplastic activity of perphenazine. These findings reveal thepotential of direct pharmacologic activation of the PP2A tumor suppressor for treatment of T-ALL and other cancers driven by hyper-phosphorylated PP2A substrates. Moreover, activitycorrelation proteomics represents a promising approach to identify unknown moleculartargets of biologically active drugs.8 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts59

ZDM8Boston | USA 2015ZDM8 ABSTRACTSIdentification and Characterization of a Novel Cancer SusceptibilityGene that Regulates Ras Activity.Sahar Nissim 1,2,4 , Ignaty Leshchiner 3 , Joe Mancias 1,2,4 , Ophélia Maertens 1 , John Hedgepeth 1 ,Andrew J. Kim 1 , Julia Wücherpfennig 1 , Xiao-xu Wang 1,2 , Wade Harper 4 , Karen Cichowski 1 , AlecKimmelman 1,2 , Sapna Syngal 1,2 , Wolfram Goessling 1,2,3,4 .1. Brigham and Women’s Hospital, Boston, MA2. Dana-Farber Cancer Institute, Boston, MA3. Broad Institute, Boston, MA4. Harvard Medical School, Boston, MAPancreatic ductal adenocarcinoma (PDAC) remains one of the most lethal cancers today.Approximately 10% of PDAC cases are thought to have a hereditary basis, arising fromgermline mutations with high penetrance. A culprit gene has not been identified in the vastmajority of these families. In order to discover novel cancer-causing gene mutations, weperformed whole-genome sequencing in one family with a clustered incidence of PDAC. Thisrevealed a novel early nonsense mutation in the gene RABL3 that strongly co-segregates withcancer occurrence in all family members. Using Crispr-CAS, we generated zebrafish mutantswith a corresponding early stop codon in the rabl3 gene. Adult rabl3 heterozygous mutant fish ina p53 null background have dramatically accelerated spontaneous cancer rates with evidenceof loss-of-heterozygosity, validating the impact of this mutation on cancer risk. Unbiasedproteomic studies suggest that RABL3 may regulate the intracellular trafficking and localizationof RAS proteins. Confirming the proteomic data, loss of function of RABL3 in both zebrafish andin vitro assays with human pancreatic ductal epithelial cells results in abnormal RAS activity.Furthermore, assessment of the homozygous rabl3 mutant embyros reveals morphologicabnormalities resembling RASopathy syndromes, including dysmorphic craniofacial structures.While zebrafish heterozygous mutants of rabl3 appear morphologically normal, they aresusceptible to oxidative stress, revealing a proximal cancer-relevant phenotype in embryos thatcan be used to query modulators of rabl3 function. These results demonstrate the identificationof a mutation in RABL3 by whole genome sequencing and its subsequent validation as a novelPDAC susceptibility gene in zebrafish. Our data posit a novel mechanism by which RAS activitycan be dysregulated in cancer formation.608 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts

ZDM8 ABSTRACTSZDM7Madison | USA 2015ZDM8Boston | USA 2015Expanding psychiatric genetics: 16p11.2 gene interactions affectmultiple whole body phenotypesJasmine McCammon 1 , Alicia Blaker-Lee 1 , Xiao Chen 2 , Hazel Sive 1,21. Whitehead Institute for Biomedical Research2. Massachusetts Institute of TechnologyOur goal is to identify critical genes in the 16p11.2 copy number variant region (CNV), a~600 kb region of the genome where duplication or deletion is associated withpsychiatric disorders including autism, schizophrenia, bipolar disorder, and majordepression. Comorbid pathologies such as hypotonia, epilepsy, macrocephaly, obesity,gastrointestinal problems, and brain birth defects are also connected to this CNV. Thelinkage of these issues with variable 16p11.2 copy number is likely due to changes ingene dosage within the region. Our focus is to associate molecular, cellular, andbehavioral phenotypes with changes in gene copy number, a strategy that will be crucialin patients for unbiased diagnosis of each disorder. Because no variants of single genesfrom the core 25 genes affected by 16p11.2 CNVs have emerged from extensive patientsequencing analysis, we hypothesize that two or more genes from this interval interact toconfer copy number sensitivity. We have analyzed 162 pair-wise interactions of 16p11.2homologs to determine whether they impact brain development in the zebrafish, and 15pairs have emerged as being dosage-sensitive in a synergistic manner. All interactionsdiscovered are novel, nor could they have been predicted based on known genefunction. Genes that interact to alter brain development include: asphd1, fam57b, hirip3,kctd13, and kif22. A subset of these interactions also affect neuronal specification,cranial motoneuron morphology, muscle fiber patterning, and enteric neuron density.Each gene pair displays a unique phenotypic signature indicating that the variousphenotypes are under complex genetic control that can be defined in the zebrafish. Weare currently examining genetic interactions impacting other patient-relevantphenotypes, including obesity, seizure susceptibility, locomotion, and explicit memory.8 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts61

ZDM8Boston | USA 2015ZDM8 ABSTRACTSAcceleration of hepatic steatosis to carcinogenesis transitionby neutrophils and macrophages in Kras-inducedhepatocellular carcinoma model in zebrafishC. Yan 1,2 , X. Huo 2 , Z. Gong 1,21NUS Graduate School for Integrative sciences and engineering; 2 Biological Sciences,National University of SingaporeHepatocellular carcinoma (HCC), the third most deadliest cancer in the world, oftendevelops from nonalcoholic steatohepatits (NASH). However, the mechanisminitiating the transition from NASH to HCC remains poorly characterised. NASH ismarked with an increased infiltration of immune cells and an inflammatorymicroenvironment. In our kras-induced HCC zebrafish model, we observed hepaticsteatosis, together with increased infiltration of neutrophils and macrophages withinthe first 24 hours of induction of oncogenic kras expression in transgenic larvae,where early HCC phenotype was developed after 96 hours of oncogenic krasexpression. The hepatic steatosis aforementioned was caused by a MEK/ERKsignaling pathway-mediated elevation of pparγ in oncogenic hepatocytes. From Highcholesterol/High sugar feeding experiments, we showed that the extent of hepaticsteatosis correlated with leptin expression in oncogenic hepatocytes. As leptin is notonly a satiety hormone secreted by adipocytes to curb hunger but also an importantinflammatory mediator, we found that both hepatocytes and immune cells includingneutrophils and macrophages expressed leptin receptor and were susceptible toleptin influence. Leptin level was correlated with the infiltration level of neutrophilsand macrophages in a Tgfβ−dependent manner. Since Tgfβ is a prominent mediatorof the tumor microenvironment, in particular, to polarize tumor infilrated neutrophilsand macrophages to a pro-tumor role, we depleted neutrophils and macrophagesand demonstrated that tumor associated neutrophils and macrophages had roles inpromoting liver carcinogenesis with increased proliferation and decreased apoptosisin oncogenic hepatocyte. In sum, we propose a mechanism where hepatic steatosisinducedleptin expression initiates an inflammatory response in the liver, attractingneutrophils and macrophages to aid in the possible transition of NASH to HCC.628 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts

ZDM8 ABSTRACTSZDM7Madison | USA 2015ZDM8Boston | USA 2015Molecular control of Schwann cell development by Fbxw7Breanne L. Harty 1 , Melanie Holmgren 1 , Sarah D. Ackerman 1 , Amy L. Herbert 1 , Charleen L. Johnson 1 , Kelly R.Monk 1,21Washington University School of Medicine2Hope Center for Neurological DisordersMyelin is a multilamellar sheath generated by specialized glial cells that iteratively spiral their plasmamembranes around axon segments. Myelination enables rapid propagation of action potentials and providestrophic support essential for neuronal survival. In the peripheral nervous system (PNS), myelin is made bySchwann cells (SCs). Disruptions in SC development and myelination lead to devastating symptoms in severalneurological disorders and can result in both benign and malignant peripheral nerve tumors. To developeffective therapies for these patients, we must first understand the mechanisms that govern SC development,myelination, and myelin maintenance.In a forward genetic screen in zebrafish to define new regulators of myelinating glial cell development,we recovered stl64 mutants, which display increased expression of myelin-related genes and hypermyelinationin both the CNS and PNS. Using whole genome sequencing, we determined that stl64 disrupts fbxw7, whichencodes the substrate recognition component of E3 ubiquitin ligase complexes. Notable Fbxw7 targets aremaster regulators of transcription and cell cycle including: mTOR, Notch, and cyclin E. Thus, Fbxw7 is requiredfor critical cellular processes such as proliferation and differentiation.Fbxw7 can be regulated in part by differential expression of three splice isoforms – α, β, γ – whichdictate subcellular localization. Our preliminary data suggests that SCs only express the fbxw7α isoform, andour work to define isoform-specific Fbxw7 functions in SCs will be discussed. Furthermore, ultrastructuralanalyses of stl64 mutants revealed significantly thicker myelin in the PNS. Given that mTOR levels must betightly regulated to achieve proper myelin thickness, current work to test if loss of Fbxw7 regulation of mTOR isthe primary cause of the SC defects observed in stl64 mutants will also be discussed. Importantly, ourpreliminary analyses of fbxw7 stl64 zebrafish mutants provide the first evidence that Fbxw7 is an importantmodulator of Schwann cell development and myelination.8 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts63

ZDM8Boston | USA 2015ZDM8 ABSTRACTSIn vivo imaging of host-pathogen interactions in Staphylococcusaureus infectionJ. J. Serba 1,3 , T. K. Prajsnar 5 , S. J. Foster 2,4 , S. A. Renshaw 1,3,41. Bateson Centre, The University of Sheffield, Sheffield, United Kingdom2. Molecular Biology and Biotechnology Department, The University of Sheffield, Sheffield, UnitedKingdom3. Department of Infection and Immunity, The University of Sheffield, United Kingdom4. Krebs Institute, The University of Sheffield, United Kingdom5. Institute of Biology, Leiden University, Leiden, The NetherlandsS. aureus has evolved strategies to manipulate host-pathogen interactions to its own endsand avoid killing by professional phagocytes. It has been suggested that S. aureus can surviveinside professional phagocytes – mainly neutrophils and macrophages. Moreover, theintracellular environment, instead of being disruptive, could constitute a beneficial “niche”. Ourhypothesis is that S. aureus evade elimination by subverting one or more uptake andintracellular processing pathways. Internalisation and cellular trafficking are host responseprocesses already reported to potentially favour pathogenesis of other bacteria. Lightfluorescence and confocal microscopy has been used to track pathogens inside of thetransparent zebrafish embryo. Whole body quantification of bacteria and phagocytes has beenperformed to determine the number of phagocytes in infected embryos. S. aureus strains werestained before injection with pH-sensitive and insensitive fluorescent dyes to assess the stageof endosome maturation by pH decrease. We have used fluorescently labelled membranetransgenic zebrafish lines to visualise dynamics of intracellular compartments. Nomarski DICcombined with high power fluorescence microscopy enabled imaging of pathogen uptake,phagocyte-to-phagocyte interactions and bacterial acidification in vivo. Results of whole bodycounts of bacteria internalised by phagocytes showed more intake into macrophages than intoneutrophils. Quantification of bacteria labelled with pH sensitive bacterial cell wall dyesindicated different acidic conditions in bacteria-containing endosomes and lower acidificationrates in neutrophils. We demonstrated that bacterial cells co-localise with vesicles of differentsize in professional phagocytes. Complex quantification experiments combined with real timein vivo imaging have the potential to help increase our understanding of host-pathogeninteractions at the different stages of infection.648 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts

ZDM8 ABSTRACTSZDM7Madison | USA 2015ZDM8Boston | USA 2015Severe ossification defects in Medaka mutants forOsterix/Sp7Yu Tingsheng 1 , Dasyani M 1 , Sundaram S 1 , Tan Wen Hui 1 , Buettner A 1 ,Witten PE 2 , and Winkler C 11 Department of Biological Sciences and NUS Centre for Bioimaging Sciences(CBIS), National University of Singapore;2 Biology Department, Ghent University, BelgiumOsterix/Sp7 (Osx) is a zinc finger transcription factor that is expressed inpremature and mature osteoblasts of vertebrates. In mammals, Osx is akey regulator of osteoblast differentiation and is required for themaintenance of adult bone. In teleost fish, however, the role of Osx remainscontroversial as no early mutant bone phenotypes were reported in fish sofar. We have previously shown that a transient Morpholino-mediatedknock-down of osx in the Japanese medaka (Oryzias latipes) results in aloss or delay of mineralization in early cranial intramembranous bones,vertebrae and otoliths. To confirm these Morpholino data and study thelong-term effect of a constitutive osx deficiency, we generated osxknock-out mutants in medaka using CrispR/Cas9. Medaka osx mutantsexhibit severe bone defects from embryonic stages onwards. Mineralizationof cranial bones is strongly reduced while cartilage forms normally. Usinglive imaging in transgenic reporter lines, we find that formation of cranialcol10a1 positive osteoblast progenitors proceeds normally but theirmaturation into differentiated osteoblasts is impaired. Interestingly, whilemineralization of arches and fins is stronly disrupted in osx mutants,mineralization of the centrae appears intact. This coincides with thepresence of col10a1 positive progenitors around the prospective centrasuggesting that immature osteoblast progenitors are sufficient formineralization in the centra region. Together, our data show that medakaosx plays a crucial role in osteoblast maturation and skeletogenesis, andtherefore shares important functional features with its mammalian ortholog.In a second project, we studied the role of osteoblast progenitors andmature osteoblasts for bone formation in the regenerating medaka fin.Using conditional cell ablation, we show that col10a1 positive osteoblastprogenitors are essential for fin regeneration. In conclusion, our studies inmedaka provide novel insight into the role of osx during bone formation andregeneration in teleost fish.This work is supported by funds from the Singapore Ministry of(MOE2013-T2-2-126).Education8 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts65

ZDM8Boston | USA 2015ZDM8 ABSTRACTSInvestigating the Pathobiology of Myofibrillar Myopathies andPotential Therapies using ZebrafishAvnika A. Ruparelia 1 , Viola Oorschot 2 , Georg Ramm 2 , Robert J. Bryson-Richardson 11School of Biological Sciences, Monash University, Melbourne 3800, Australia2 Monash Micro Imaging, Monash University, Melbourne 3800, AustraliaMyofibrillar myopathies exhibit progressive muscle weakness and are characterisedby structural failure of the muscle at the Z-disk and the formation of cytoplasmicprotein aggregates. Mutations in the Actin binding protein Filamin C (FLNC) areknown to cause myofibrillar myopathy, with the most prevalent mutation beingFLNC W2710X . To investigate the mechanism of disease in FLNC W2710X myofibrillarmyopathy we overexpressed fluorescently tagged full-length human wildtype orFLNC W2710X in zebrafish and characterised the resulting muscle phenotype. Ourstudies reveal that expression of FLNC W2710X results in the formation of proteinaggregates, recapitulating a key feature of the disease. Remarkably, FLNC W2710Xlocalized correctly to the Z-disk and was capable of rescuing the fibre failurephenotype resulting from loss of FLNC, suggesting it retained functions necessary formuscle integrity. For the first time, utilizing the advantages of the zebrafish modelsystem, we performed fluorescent recovery after photobleaching experiments, invivo, to compare wildtype FLNC and FLNC W2710X , identifying an alteration in proteindynamics at the Z-disk. Furthermore, using a combination of knockdown experimentsand drug treatments, we investigated if impaired autophagy has a role in theformation of FLNC W2710X containing protein aggregates and examined theupregulation of autophagy as a potential therapy for FLNC-related myofibrillarmyopathy. We will present this data, that combined with detailed characterization byimmunolabelling and confocal and electron microscopy, has allowed us to determinethe mechanistic basis of disease and suggest potential therapeutic approaches.668 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts

ZDM8 ABSTRACTSZDM7Madison | USA 2015ZDM8Boston | USA 2015Investigation of the role of the HACE1 tumor suppressor in high-riskpediatric neuroblastomaBabak Razaghi, 1 Jaime N. Wertman, 2 Graham Dellaire, 3 Poul H.B. Sorensen, 4,5 Meredith Irwin, 6& Jason N. Berman 1,2,31. Pediatrics, Dalhousie University/IWK Health Centre, Halifax, NS, Canada2. Microbiology & Immunology, Dalhousie University, Halifax, NS, Canada3. Pathology, Dalhousie University, Halifax, NS, Canada4. Molecular Oncology, British Columbia Cancer Research Centre, Vancouver, BC, Canada5. Pathology and Laboratory Medicine, University of British Columbia Vancouver, BC, Canada6. Pediatrics, Hospital for Sick Children/University of Toronto, Toronto, ON, CanadaNeuroblastoma (NBL) is the most common extra-cranial solid tumor in children. Metastases arepresent at diagnosis in 50% of children and 5-year overall survival rates are 6 cells were inthe tail. Wild type SK-N-AS cells appear to migrate within 3 days post-injection. We used azebrafish model to reveal that HACE1 loss results in increased reactive oxygen species (ROS)and DNA damage (Daugaard et al, Nat. Commun 2013), suggesting that antioxidant ROSscavenging agents may provide previously unrecognized therapeutic benefits in NBL. A hace1CRISPR fish line was generated, which will be monitored for tumor incidence and examined forROS, DNA damage and apoptosis. Tumor bearing fish will be treated with the most effectivedrug combinations found in xenograft studies. This innovative zebrafish project has potential tocontribute new insights and provide an applicable adjuvant therapy for NBL.8 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts67

ZDM8Boston | USA 2015ZDM8 ABSTRACTSGlucocorticoid anti-inflammatory agents activate hypoxiainducible signalling”David Greenald*, Andrea Vettori*, Garrick Wilson*, Margherita Peron, LuisaDalla Valle, Mathavan Sinnakaruppan, Jane McKeating*, Francesco Argenton*,Freek van Eeden*Inflammation and hypoxia occur together in many disease states. Thus crosstalkbetween inflammatory regulators and hypoxia inducible factor (HIF) signaling is to beexpected, and has indeed been reported (reviewed in (Eltzschig and Carmeliet,2011; Palazon et al., 2014)). The majority of studies report synergistic interactionsbetween HIF and pro-inflammatory mediators. In contrast, the potential crosstalkbetween anti-inflammatory glucocorticoid (GC) and HIF signaling pathways is poorlyunderstood. We hypothesise that such cross talk occurs and may provide newtargets for therapeutic intervention. We present results from a 2000-compoundunbiased in vivo chemical screen for activators of HIF signalling in zebrafish, and,surprisingly, identify GC agonists as potent and specific HIF activators in the liver.GCs activate hypoxia response elements (HREs), rather than glucocorticoidresponse elements (GREs). Importantly, GC treatment of zebrafish expressing aglucocorticoid receptor (GR) mutant lacking a functional DNA binding domain couldstill enhance HRE-dependent activity. This shows that the GR uses a non-canonicalroute for GC-HIF cross talk. Importantly, we confirm the relevance of our zebrafishfindings in human Huh-7 hepatoma cells and in a unique liver slice culture, showingthat GCs stabilize HIF. We show that anti-inflammatory GCs activate HIF signallingpathways that may explain the differential capacity of GCs to trans-activate or transrepressgene expression in different cell types. The crosstalk between these majortranscriptional factors associated with inflammation may have relevance for thetreatment of several diseases with anti-inflammatory steroids.ReferencesEltzschig, H.K., Carmeliet, P., 2011. Hypoxia and inflammation. The New England journal ofmedicine 364, 656-665.Palazon, A., Goldrath, A.W., Nizet, V., Johnson, R.S., 2014. HIF transcription factors,inflammation, and immunity. Immunity 41, 518-528.*) equal contributions688 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts

ZDM8 ABSTRACTSZDM7Madison | USA 2015ZDM8Boston | USA 2015Zebrafish Caudal Hematopoietic Embryonic Stromal Trunk(CHEST) Cells Support Hematopoietic Stem and Progenitor Cell(HSPC) Expansion.David L Stachura 1 , Clyde A Campbell 2 , Jesse S Smith 1 , Julian Aggio 1 , DavidTraver 2 .1. California State University, Chico, Chico, CA, 959292. University of California, San Diego, La Jolla, CA, 92093Zebrafish are an excellent model system for studying hematopoiesis due to their externaldevelopment, high fecundity, and optical transparency. They are also geneticallyamenable, allowing large-scale mutagenesis and chemical screens for elucidating genesand molecular pathways involved in hematopoiesis. While forward genetic screens havebeen utilized to identify genes essential for the generation of primitive blood and theemergence of hematopoietic stem cells (HSCs), they have not elucidated genesessential for hematopoietic stem and progenitor cell (HSPC) proliferation anddifferentiation, due to a lack of methodologies to functionally assess HSPCs.We previously described techniques to test the developmental potential of HSPCs byculturing them on zebrafish kidney stromal (ZKS) cells, derived from the main site ofhematopoiesis in adult teleosts. We have also derived zebrafish embryonic stromal trunk(ZEST) cells, isolated from tissue surrounding the embryonic dorsal aorta, the site ofHSC emergence in developing fish. In addition to these cell lines, we now report theisolation and generation of caudal hematopoietic embryonic stromal trunk (CHEST) cellsfrom 48-hour post fertilization (hpf) caudal hematopoietic tissue, the site of embryonicHSC expansion. CHEST cells expand two-fold more hematopoietic cells than ZKS orZEST cells in vitro, and morphological and qRT-PCR analysis of these cultures indicatedlymphoid, myeloid, and erythroid differentiation. Transcriptional comparison of ZKS,ZEST, and CHEST cells will allow the discovery of molecular pathways expressed bythese cell lines that support HSPC proliferation and differentiation, but are temporallyand ontologically distinct. These cells should allow for the first time a comparison ofmolecular signals critical for HSPC proliferation and differentiation in the zebrafish, andcomparison with mammalian-supportive cell lines should further an evolutionaryunderstanding of vertebrate hematopoiesis. Importantly, they allow the testing andvalidation of bona fide HSPCs in zebrafish, cells commonly dysregulated in hematologicdiseases.8 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts69

ZDM8Boston | USA 2015ZDM8 ABSTRACTSThe Serotonergic Nervous System Regulates HSC ProductionVia the Hypothalamic-Pituitary-Interrenal Axis In ZebrafishWanda Kwan1, Mauricio Cortes1, Sarah Liu1, Wolfram Goessling2,3, Trista E. North1,31. Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA2. Brigham and Women’s Hospital and Dana-Farber Cancer Institute, Harvard Medical School,Boston, MA3. Harvard Stem Cell Institute, Cambridge, MAThere is increasing evidence that neuronal signals contribute to hematopoietic stem cell(HSC) production and maintenance; however, the mechanism by which the brainregulates HSCs and/or the HSC niche is unclear. Serotonin (5HT), a neurotransmitter inthe brain and a hormone in the peripheral system, was identified in a screen for HSCmodulators in zebrafish. Exposure to 5HT or fluoxetine (5HT reuptake inhibitor)increased hematopoietic stem and progenitor cell (HSPC) formation in the zebrafishaorta-gonad-mesonephros region as shown by runx1/cmyb in situ hybridization, FACS,and qRT-PCR analysis (p

ZDM8Boston | USA 2015ZDM8 ABSTRACTSCalmodulin inhibition rescues the effects of ribosomal proteindeficiency in Diamond Blackfan anemia modelsElizabeth R. Macari 1,2 , Alison M. Taylor 1,2 , David M. Raiser 2,3 , Kavitha Siva 4 , KatherineMcGrath 1 , Jessica M. Humphries 1 , Johan Flygare 4 , Benjamin L. Ebert 2,3,5 , and Leonard I.Zon 1,2,61 Stem Cell Program and Division of Hematology/Oncology, Boston Children's Hospital and Dana FarberCancer Institute, Harvard Stem Cell Institute, Boston, MA, USA2 Harvard Medical School, Boston, MA, USA3 Division of Hematology, Brigham and Women's Hospital, Boston, MA, USA.4 Stem Cell Center, Lund University, Lund, Sweden5 The Broad Institute of MIT and Harvard, Cambridge, MA, USA.6 Howard Hughes Medical InstituteRibosomal protein (RP) mutations are found in many diseases, including Diamond Blackfananemia (DBA), where defective erythropoiesis, craniofacial abnormalities and increased cancerrisk are major complications. RP mutations cause p53 activation through accumulation of freeRPs that bind and sequester MDM2. We characterized a zebrafish mutant in rps29. Rps29 -/-embryos have hematopoietic and endothelial defects, including decreased flk1 and mybexpression and defects in hemoglobinization. Consistent with other animal models of RPdysfunction, p53 knockdown in rps29 -/- embryos rescued these defects. To uncover novelcompounds that correct the phenotypes of DBA, we performed a chemical screen in rps29 -/-embryos. Several structurally distinct calmodulin (CaM) inhibitors successfully rescued flk1intersegmental vessel staining and hemoglobin (Hb) levels in the mutant embryo. Treatinghuman cord blood-derived CD34+ cells deficient in RPS19 with the CaM inhibitor,trifluoperazine (TFP), relieved the erythroid differentiation block. Injection of TFP in a DBAmurine model significantly increased red blood cell number and Hb levels. Mechanistic studiesin A549 cells infected with shRNA against RPS19 demonstrated that TFP blocks p53 nuclearaccumulation and induction of multiple p53 transcriptional target genes (p

ZDM8 ABSTRACTSZDM7Madison | USA 2015ZDM8Boston | USA 2015Meeting challenges in psychiatric and neurodevelopmentaldisordersHazel Sive 1,2 , Jasmine McCammon 1 , Xiao Chen 1,2 , Brad Carter 1 , Alicia Blaker-Lee 1 .1) Whitehead Institute and 2) MIT, Cambridge MA.The goals of understanding the genetic basis for psychiatric and neurodevelopmentaldisorders are compounded by two key challenges: 1. the multigenic nature of mostdisorders and 2. the multiple symptoms (co-morbidities) associated with each disorder.We are addressing two disorders, Pitt-Hopkins Syndrome (PTHS) that is associated withthe basic-helix-loop helix protein TCF4, and 16p11.2 Syndrome, that is associated withdeletion or duplication of the 16p11.2 copy number variant region (CNV) containingtwenty-five protein coding genes. PTHS is associated with loss of one copy of TCF4 andpresents with multiple symptoms, including intellectual disability, failure to thrive, facialdysmorphology, seizures and chronic constipation. We have developed a tcf4 mutantfish, and present a new general method to confirm the loss of Tcf4 protein in this line. Akey question for PTHS is how one gene regulates so many symptoms. A reciprocalpuzzle is seen in 16p11.2 Syndrome. Patients may present with autism, schizophrenia,seizures, intellectual disability, anxiety, hypotonia and obesity. Genetic data indicatethere is no single gene responsible for any symptom, and a key question is which16p11.2 genes interact to produce some or all symptoms. We have taken an antisenseand genetic approach in zebrafish to identify 16p11.2 interacting genes. Our data identifyfunctional interactions that could not have been predicted computationally. Theseapproaches highlight the outstanding capacity of the zebrafish to address complexhuman neural disorders.8 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts73

ZDM8Boston | USA 2015ZDM8 ABSTRACTSA systems biology approach to understanding hearing regeneration in zebrafishTissue regeneration is the result of a complex integration of injury signals, stemcell activation, inflammation responses, reactivation of developmental programsand regeneration-specific processes. We have developed a systematic approachto dissect and analyze the various pathways involved in hearing regeneration byusing zebrafish as a model organism. By using a high-throughput “guided”genetics and chemical genomics screen to identify the key genes and pathwaysinvolved in hearing regeneration, we have significantly enriched our success ratefor identifying regeneration-specific genes. From our previous work, we hadcollected 2000 candidate genes involved in hearing regeneration bytranscriptionally profiling regenerating zebrafish adult inner ears after sounddamage. We’ve built an efficient gene knockout pipeline, first using retroviralmutagenesis but now using CRISPR/Cas9 targeting and we are systematicallyinactivating the 2000 candidate genes and testing their role in both normal haircell development and hair cell regeneration. In addition, we have screened abroad number of well-characterized pharmacological inhibitors to identify genesthat cannot be easily tested by KO because of their important roles in earlydevelopment. From our first one hundred and fifty genes and twenty chemicalinhibitors tested, we have identified four genes that reduce the number of haircells in a normal embryo, and we have identified an additional seven genes andthree chemical inhibitors that specifically disrupt regeneration of the hair cellswithout affecting normal development. We will present data on the geneticstrategy used to generate and screen hundreds of zebrafish gene knockouts forhearing regeneration defects, the pathways emerging from our genetic andchemical genomic analysis, and the deeper phenotypic characterization of asample regeneration-specific mutant we have identified.748 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts

ZDM8 ABSTRACTSZDM7Madison | USA 2015ZDM8Boston | USA 2015Epithelial damage and MMP-13 overexpression contribute to paclitaxel-inducedperipheral neuropathy in zebrafishThomas S. Lisse, Leah Middleton, Paige Martin, Emily Spaulding, Olivia Lopes, Erin Carter, Elizabeth A.Brochu, Ashley Waldron, Sandra RiegerDavis Center for Regenerative Biology and Medicine, MDI Biological Laboratory, Kathryn W. Davis Building 227, Old BarHarbor Road, Salisbury Cove, ME 04672Paclitaxel is a microtubule-stabilizing drug that is most beneficial in the treatment of breast, lung, and ovariancancer. Paclitaxel is also part of a number of curative and palliative regimens. Despite its acknowledgedbenefits, however, it is also widely recognized that paclitaxel induces peripheral sensory neuropathy(paclitaxel-induced peripheral neuropathy, PIPN). PIPN is marked by symptoms such as tingling, numbnessand acute or chronic pain. In severe cases, chemotherapy must be terminated, depriving cancer patients of thefull benefits of paclitaxel treatment. Most studies have focused on the effects of paclitaxel directly in sensoryneurons but there is still no consensus about the underlying causes of this condition. This could relate to apossible function of other cell types, such as keratinocytes, in PIPN etiology. It is well established thatepidermal keratinocytes interact with cutaneous axons similarly to Schwann cells and signs of axondegeneration are initially visible in the epidermis. Thus keratinocytes are primary candidates for contributing toPIPN. To test this hypothesis, we have developed a unique PIPN model using zebrafish to study theinteractions between keratinocytes and cutaneous axons during paclitaxel treatment in real-time. Our studiesreveal that keratinocytes undergo morphological and molecular changes in the presence of paclitaxel prior toany visible cutaneous axon damage. Paclitaxel-treated zebrafish larvae show a defective skin barrier,indicating adhesion defects, and the skin is vulnerable to mechanical stress and injury. We found thatpaclitaxel induces the expression of matrix-degrading metalloproteinase 13 (MMP-13, collagenase 3) andMMP-13 inhibition prevents axon damage. Our studies therefore, for the first time, identify a possibly drugcandidate in the treatment of PIPN, which needs to be further tested in mammals.8 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts75

ZDM8Boston | USA 2015ZDM8 ABSTRACTSZebrafish Mutants of the Autism Risk Gene Cntnap2 IdentifyGABAergic Defects and Uncover Estrogens asPhenotypic SuppressorsEllen J. Hoffman 1,2 , Katherine J. Turner 3 , Joseph M. Fernandez 1,2 , Daniel Cifuentes 4 , MarcusGhosh 3 , Michael J. F. Barresi 5 , Stephen W. Wilson 3 , Jason Rihel 3, , Matthew W. State 1, 2, 4, 6 ,Antonio J. Giraldez 41 Child Study Center, Yale School of Medicine, New Haven, CT 06510, USA2 Program on Neurogenetics, Yale School of Medicine, New Haven, CT 06510, USA3 Department of Cell and Developmental Biology, University College London, Gower Street, LondonWC1E 6BT, UK4 Department of Genetics, Yale University School of Medicine, New Haven, CT 06510, USA5 Biological Sciences, Smith College, Northampton, MA 01063, USA6 Department of Psychiatry, University of California San Francisco, San Francisco, CA 94143, USAAutism spectrum disorders (ASD) are a group of devastating neurodevelopmentalsyndromes that affect up to 1 in 68 children. Despite advances in the identification of ASD riskgenes, the mechanisms underlying ASD remain unknown, limiting the development of targetedtreatments. Homozygous loss-of-function mutations in the Contactin Associated Protein-like 2(CNTNAP2) gene, which encodes a member of the neurexin family of cell adhesion molecules,are strongly linked to ASD and epilepsy in humans. Here we utilize a zebrafish model toinvestigate the function of Cntnap2 and to undertake pharmacological screens to identifyphenotypic suppressors. Using zinc finger nucleases, we generated deleterious mutations inboth zebrafish paralogs of Cntnap2. We find that Cntnap2 double mutants display GABAergicdeficits particularly in the telencephalon and hypersensitivity to drug-induced seizures. Highthroughputbehavioral profiling identifies nighttime hyperactivity as a prominent phenotype, whilepharmacological testing reveals dysregulation of both GABAergic and glutamatergic systems inCntnap2 mutants. Interestingly, we find that estrogen receptor agonists elicit a behavioralfingerprint that anti-correlates with the behavioral phenotype of Cntnap2 mutants. Finally, weshow that the phytoestrogen Biochanin A specifically reverses nighttime hyperactivity inCntnap2 mutants. These results identify estrogenic compounds as phenotypic suppressors andilluminate novel phenotypic and pharmacological pathways with relevance to autism.768 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts

ZDM8 ABSTRACTSZDM7Madison | USA 2015ZDM8Boston | USA 2015Mechanisms of glial bridging during spinal cord regeneration.Mayssa H. Mokalled and Kenneth D. Poss.Department of Cell Biology, Duke University Medical Center, USA.Injury to the mammalian spinal cord (SC) results in irreversible impairment of sensoryand motor functions. In contrast, adult zebrafish are capable of efficient, spontaneousrecovery after SC damage. Following SC transection, zebrafish will first initiate a glialbridge across the injury area. Axons then regenerate along the bridging glia, andneuronal connections are re-established to achieve full recovery. Although glial bridgingis a key step in SC repair, the subpopulation of glial cells that construct the bridge isunknown, and the signals that induce bridge formation are only partially understood. Tointerrogate these questions, we used transcriptome profiling followed by in situhybridization screening to identify factors that are expressed in the glial bridge during SCregeneration. We found that connective tissue growth factor (ctgf) is expressed in asubpopulation of glial cells that initiate the earliest bridging events. These ctgf +/ gfap +‘pioneer’ glial cells occupy the site of injury and stretch between the transected parts ofthe SC to establish the bridge. In addition, fish that overexpressed ctgf showedimproved glial bridging and SC regeneration, while ctgf mutant fish showed impairedregeneration. Our study has uncovered a novel cell population, the pioneer glia, thatdrives glial bridging and SC repair. We have also identified ctgf signaling as an essentialcomponent of SC regeneration. Uncovering the mechanisms of glial bridging inzebrafish opens new avenues to induce glial bridging as a means to improve SC repairin mammals.8 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts77

ZDM8Boston | USA 2015ZDM8 ABSTRACTSSHP2 activation synergizes with MYCN in neuroblastomapathogenesisXiaoling Zhang 1 , Zhiwei Dong 1 , A. Thomas Look 2 , Benjamin G. Neel 3 , and (Jane)Shizhen Zhu 1 .1. Mayo Clinic, Rochester, MN 55902, USA.2. Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA 02115, USA.3. Laura and Isaac Perlmutter Cancer Center, NYU Langone Medical Center, New York, NY 10016Neuroblastoma, a highly malignant embryonic tumor of the peripheral sympathetic nervoussystem (PSNS), arises from the sympathoadrenal lineage of neural crest cells and accounts for~10% of all cancer deaths in children. Recent next-generation genomic resequencing effortsrevealed that PTPN11 (encoding SHP2) is the second most frequently mutated gene in high-riskneuroblastoma, and is often accompanied by amplification of MYCN. Wild-type SHP2 and itsupstream activator, GAB2, are overexpressed in most neuroblastoma cases, suggesting thatthe SHP2 pathway might be activated by overexpression of upstream regulator(s) in cases thatlack PTPN11 mutations. To determine whether activation of the SHP2 pathway cooperates withMYCN in neuroblastoma pathogenesis, we developed two transgenic lines in which ptpn11 E69Kor wild-type Gab2 is overexpressed in the PSNS under control of the dopamine-betahydroxylase(dβh) promoter. Overexpression of either ptpn11 E69K or wild-type Gab2 with MYCNaccelerated the onset of neuroblastoma and increased the disease penetrance. Immunoblotanalysis of the neuroblastomas from these transgenic fish showed an increase of Erkphosphorylation in tumors overexpressing ptpn11 E69K or wild-type Gab2 with MYCN, comparedwith those overexpressing MYCN alone, suggesting that activated SHP2 can collaborate withMYCN in neuroblastoma tumorigenesis via activation of the RAS-ERK pathway. In addition, coexpressionof ptpn11 E69K and MYCN increased the proliferative rate of hyperplastic neuroblastcells in the interrenal gland, the zebrafish analogue of adrenal gland, and the sympatheticganglion, leading to an earlier onset and increased penetrance of neuroblastoma in bothregions, which faithfully recapitulate the sites of neuroblastoma in patients. Thus, zebrafishappears to provide a robust model system for functional genomic analysis and for theinvestigation of the mechanisms and pathways underlying mutations identified in ongoing tumorresequencing studies.788 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts

ZDM8 ABSTRACTSZDM7Madison | USA 2015ZDM8Boston | USA 2015Studies of blood stem cells and cancer in the zebrafishLeonard I. ZonStem Cell Program and Hematology/Oncology, Children’s Hospital and Dana-Farber Cancer Institute,HHMI, Harvard Stem Cell Institute, Harvard Medical School, Boston, MAThe zebrafish has emerged as an excellent system to study disease. We have beenstudying the hematopoietic system. The blood stem cells arise from the aorta duringembryogenesis, and sequentially colonize new sites of hematopoiesis. Using chemicalgenetics we have found a number of chemicals that modify the induction of stem cellsand their migration. PGE2 was found to be required for stem cell birth, and a stabilizedversion leads to potent numbers of stem cells. dmPGE2 can stimulate marrowengraftment of murine or human hematopoietic cells when transplanted. A clinical trialwith dmPGE2 for cord blood transplantation showed some activity. We have alsoundertaken a chemical screen to find small molecules that block neural crestdevelopment and that could be used for melanoma treatment. We found thatleflunomide, a chemical that inhibits pyrimidine synthesis, reduces neural crest and alsomelanoma. A clinical trial is underway in combination with BRAF and MEK inhibitors.We also have undertaken disease suppressor screens in the zebrafish. Some of thechemicals found point to mechanisms of disease rescue, and may become newtherapeutics. Our studies illustrate that new therapies can be found using the zebrafishsystem.8 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts79

ZDM8Boston | USA 2015ZDM8 ABSTRACTSPremature ageing anticipates cancer in tert-deficient zebrafishMadalena Carneiro and Miguel Godinho FerreiraInstituto Gulbenkian Ciência, Oeiras, PortugalWhy, and how, organisms age and ultimately die is one of the key questionsof modern biology. Telomeres are considered molecular timekeepers determiningcellular lifespans. Disparate studies showed that telomeres shorten with age, DNAdamage markers accumulate at telomeres and that animals lacking telomerasedisplay compromised tissue function. An integrated organism-based detailedanalysis is so far lacking.We show, for the first time, that short telomeres of specific tissues in naturallyaged zebrafish precede DNA damage, decline in cell proliferation and age-specificorgan decline. We use the telomere length of telomerase mutants to predict thecritical threshold at which tissue dysfunction should arise in old individuals. Criticallyshort telomeres accumulate specifically in the gut and muscle with aging, leading tocellular damage that culminates in local disruption of organ homeostasis.Strikingly, telomere shortening in these key tissues appears to be sufficient totrigger damage in others and precedes the onset of age-associated diseases,namely cancer. Thus, tissue-specific telomere length is limiting for local andsystemic physiological integrity, leading to tissue degeneration and disease in aging.808 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts

ZDM8 ABSTRACTSZDM7Madison | USA 2015ZDM8Boston | USA 2015p53 isoform ∆113p53/∆133p53 promotes DNA double-strandbreak repair to protect cell from death and senescence inresponse to DNA damageLu Gong 1 , Hongjian Gong 1 , Ting Tao 2 , Jinrong Peng 2 , Jun Chen 11 Key laboratory for Molecular Animal Nutrition, Ministry of Education, Innovation Center forSignaling Network, College of Life Sciences, 2 College of Animal Sciences, Zhejiang University,866 Yu Hang Tang Road, Hangzhou, Zhenjiang 310058, ChinaThe inhibitory role of p53 in DNA double-strand break (DSB) repair seemscontradictory to its tumor-suppressing property. The p53 isoform∆113p53/∆133p53 is a p53 target gene that antagonizes p53 apoptotic activity.However, information on its functions in DNA damage repair is lacking. Herewe report that ∆113p53 expression is strongly induced by γ-irradiation, but notby UV-irradiation or heat shock treatment. Strikingly, ∆113p53 promotes DNADSB repair pathways, including homologous recombination, non-homologousend joining and single-strand annealing. To study the biological significance of∆113p53 in promoting DNA DSB repair, we generated a zebrafish∆113p53M/M mutant via the transcription activator-like effector nucleasetechnique and found that the mutant is more sensitive to γ-irradiation. Thehuman ortholog, ∆133p53, is also only induced by γ-irradiation and functionsto promote DNA DSB repair. ∆133p53-knockdown cells were arrested at theG2 phase at the later stage in response to γ-irradiation due to a high level ofunrepaired DNA DSBs, which finally led to cell senescence. Furthermore,∆113p53/∆133p53 promotes DNA DSB repair via upregulating thetranscription of repair genes rad51, lig4 and rad52 by binding to a novel type ofp53-responsive element in their promoters. Our results demonstrate that∆113p53/∆133p53 is an evolutionally conserved pro-survival factor for DNAdamage stress by preventing apoptosis and promoting DNA DSB repair toinhibit cell senescence. Our data also suggest that the induction of ∆133p53expression in normal cells or tissues provides an important tolerance markerfor cancer patients to radiotherapy.8 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts81

ZDM8Boston | USA 2015ZDM8 ABSTRACTSLive imaging reveals NFkB signaling to be a key regulator fora Trophic Inflammatory Response during tumour initiationDerek W. Laux 1 , Isabel Ribeiro Bravo 1 , Thomas Ramezani 1 , Yi Feng 11. MRC Centre for Inflammation Research, Queen’s Medical Research Institute, University ofEdinburgh, Edinburgh, UKOur earlier work using a zebrafish cancer model to visualize the pre-neoplastic cell(PNC) at its inception, has revealed that the PNC elicits a Trophic InflammatoryResponse, in which recruited leukocytes promote its growth. Here, we use aninducible HRASG12V mediated zebrafish cancer model to unpick signals critical inestablishing the leukocyte Trophic Phenotype. Using a transgenic NFκB reporter wesee that both PNCs and recruited innate immune cells show elevated NFκB activity.Combining the inducible PNC model and NFκB reporter, we find that up-regulation ofNFκB in PNCs is an early event down stream of HRAS G12V induction before the upregulationof MyD88, an adaptor protein thought to be up stream of NFkB activation.More importantly, that this activation is involved in regulating immune cell recruitmenttoward PNCs. Our live imaging movies show that sustained contact with PNCs leadsto progressive increase in neutrophil NFκB levels. Furthermore, recruited neutrophilswith high NFκB activity show different behaviour compared with NFκB low neutrophils,suggesting that NFκB activity might regulate their Trophic phenotype. Blocking NFκBactivity with chemical inhibitors lead to a reduced PNC number. Similarly, transgenicover expression of NFκB super-repressor in PNC alone, suppresses PNCprogression. We are trying to identify signals that lead to NFκB activation inneutrophils in our model and also we are testing how inhibition of the NF-κB pathwayaffects leukocyte phenotype and trophism.828 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts

ZDM8 ABSTRACTSZDM7Madison | USA 2015ZDM8Boston | USA 2015Zebrafish Chemical Screen Identifies Prolyl Isomerase PIN1 as aTherapeutic Target in Radiation-Resistant CancerPeter H. Liu, Vincent Brechin, Jack M. Barbaro, Patrick Bradley and Samuel SidiDivision of Hematology/Oncology and Department of Developmental and Regenerative Biology,The Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA.Tumor cell resistance to radiation therapy (R-RT) remains a central obstacle to effectivecancer treatment. In head and neck squamous cell carcinoma and many other cancers,R-RT arises through mutational inactivation of p53, which blocks DNA damage-inducedtransactivation of proapoptotic genes. No effective therapies are currently available thatrestore radiosensitivity in the mutant tumors. To identify such compounds, we screenedfor small molecules that suppress R-RT in p53 mutant zebrafish embryos while havingno effect in non-irradiated wild-type siblings. These screens identified the FDA-approvedbenzimidazole anthelmintic, oxfendazole, acting independently of its ascribed target,tubulin. Through combinations of SAR, computational target prediction, in vitroenzymatic assays, and genetic and pharmacological validation in vivo, we identify thepeptidyl prolyl cis/trans isomerase, PIN1, as the key target of oxfendazole whoseinhibition is responsible for restoring radiosensitivity in p53 mutant fish. This wasvalidated in a panel of TP53 mutant human cancer cell lines. PIN1 was recentlyimplicated in the regulation of DNA end resection, a key step in double-strand DNAbreak (DSB) repair by homologous recombination. Consistent with this observation, wefind that PIN1 inhibition impairs radiation-induced DSB repair in vivo, leading toexcessive levels of DNA damage, activation of a p53-independent apoptotic pathway,and effective suppression of R-RT. As PIN1 is amplified or overexpressed in a widerange of human tumors, the data unveil a new, potentially broad strategy for overcomingR-RT in cancer patients.8 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts83

ZDM8Boston | USA 2015ZDM8 ABSTRACTSCD4-transgenic zebrafish: a resource for interrogating helper T cellbiologyChristopher Dee 1 , Raghavendar Nagaraju 1 , Christopher Secombes 2 , and AdamHurlstone 11Faculty of Life Sciences, The University of Manchester, Manchester, United Kingdom2Scottish Fish Immunology Research Centre, School of Biological Sciences, University of Aberdeen,Aberdeen, United KingdomHelper T (T H) lymphocytes are at the nexus of the innate and adaptive arms of the immunesystem. Best understood for mammals, T H cells integrate signals from professional antigenpresenting cells as well as other host and innate immune cells and then through theelaboration of cytokines elicit or bolster the action of appropriate innate and adaptiveimmune effector cells to neutralize a threat from pathogens. Ineffective engagement of T Hcells results in susceptibility to infectious disease, chronic inflammation and the escape ofmalignancies from immuosurveillance. Moreover, inappropriate T H cell activity results inpotentially life-threatening allergic reactions and autoimmunity. From their birth inlymphopoietic tissues, through education in the thymus, to activation in the periphery, thebehaviour of T H cells is complex and dynamic, entailing migration through diverse physicalbarriers, immune synapse formation with a range of cell types, regulated survival,proliferation and differentiation into subtypes with specialist functions. Here, using BACrecombineering and transgenesis to target mcherry expression to CD4+ cells, we havecreated zebrafish with vibrantly labelled T H cells and macrophages. We reveal that zebrafishCD4+ T cells are heterogeneous, responding to diverse immunological challenge asmammalian T H cells do through adopting particular transcription factor and cytokineexpression profiles. Moreover, we demonstrate the utility of this zebrafish resource forinterrogating at cellular resolution the complex behaviour of T H cells. Ultimately, this uniqueresource will generate insight into the evolution of adaptive immunity and may serve as avaluable tool for medical research.848 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts

ZDM8 POSTERSZDM7Madison | USA 2015ZDM8Boston | USA 2015Oncogenic Roles of TCF3 and ID3 in T Lymphoblast CancersBarbara Squiban 1 , Chiara Borga 1 , Matteo Marchesin 1 , Lance Batchelor 1 , J. Kimble Frazer 11 Pediatric Hematology-Oncology Section, University of Oklahoma Health Sciences Center, Oklahoma City, USACancer is the most lethal disease of U.S. children, and T-cell lymphoblastic leukemia (T-ALL) andlymphoma (T-LBL) are common pediatric malignancies. Diverse genetic lesions induce T-lymphoblastneoplasia. We study the etiology of T-cell oncogenesis using zebrafish T-lymphoblast cancer modelscreated by us and others. By profiling zebrafish T-cell cancers, we discovered silencing of id3, acandidate tumor suppressor. id3 repression is potent (~30-fold mRNA reduction) and coincides with id3promoter methylation. Since human ID3 inhibits E47 (an isoform of the TCF3 transcription factor), wehypothesized that id3 silencing fosters T-ALL/T-LBL by promoting E47 hyperactivity. Findings inmammals support this, as aberrant Id3 or Tcf3 function causes T-cell cancer in murine models.Therefore, TCF3 and ID3 have roles in both fish and mammalian T-lymphoblast oncogenesis. Furtherimplicating both genes, TCF3-activating and ID3-inhibiting mutations frequently enact E47 hyperactivityin human Burkitt lymphoma (BL). Thus, E47 and ID3 appear to be oncogenic or tumor suppressive,respectively, in human, murine, and zebrafish lymphocytes. To test our hypothesis that Tcf3hyperactivity induces zebrafish T-cell cancer, we are building transgenics with T-cell-specific overexpressionof zebrafish id3 or e47, human ID3 or E47, mutant ID3 and E47 from BL, and “ET2,” an IDproteininhibitor. Transgenes will be expressed in a WT background and in lines predisposed to T-cellcancer. Together, these will define if E47 hyperactivity induces cancer in WT fish, and if E47 augmentsand ID3 inhibits oncogenesis in cancer-prone lines. We will also make double-transgenics to testinteractions between human and zebrafish E47 and ID3, to elucidate their T-cell cancer-promoting and-suppressing properties in vivo. Coupled with in vitro studies to over-express or silence ID3 and E47 inhuman T-ALL lines, this project will establish if TCF3 is oncogenic and ID3 is tumor suppressive in bothzebrafish and human T-lymphoblasts.8 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts85

ZDM8Boston | USA 2015ZDM8 POSTERSRole of Notch-signaling in tumor progression in a zebrafish modelof glioma.Marie Mayrhofer 1 , Sabrina Burkart 1 , Victor Gourain 1 , Markus Reischl 2 , Pierre Affaticati 3 ,Arnim Jenett 3 , Jean-Stéphane Joly 3 , Marina Mione 11 Karlsruher Institute of Technology, Institute for Toxicology and Genetics, Hermann-von-Helmholtz-Platz 1, 76344 Eggenstein-Leopoldshafen, Germany2 Karlsruher Institute of Technology, Institute for Applied Informatics, Hermann-von-Helmholtz-Platz 1,76344 Eggenstein-Leopoldshafen, Germany3 Tefor Core Facility, Neuropsi Institute, CNRS, 1 Avenue de la Terrasse, 91 198 Gif-sur-Yvette,FranceGliomas are a diverse group of human brain cancers with the most common typeglioblastoma multiforme (GBM) being also the most aggressive form. Little is known aboutthe early stages of glioma development. We developed a zebrafish glioma model using theGal4-UAS system to express GFP tagged oncogenic Harvey RAS (HRAS V12 ) under a zic4enhancer (Spitzner, 2014, Eur J Cancer).Germline expression of GFP-HRAS V12 in the zic4:Gal4 line allows for efficient screeningapproaches as it induces hyperproliferation of dorsal neural progenitor cells, significantovergrowth of the telencephalon by 3 days post fertilization (dpf), increased number ofproliferating cells, but is accompanied by early death (98% by 10dpf).Somatic expression of the oncogene reduced lethality (75% by 30dpf) and thus allowedanalysis of tumors in adults. Tumors develop in the telencephalon and overexpress thehuman glioma markers GFAP and S100.Using zebrafish transgenic lines expressing specific neural markers to develop the tumors,we discovered cell fate alterations as an early effect of the oncogene. GFP-HRAS V12expression in neural progenitors promotes nestin and GFAP expression and repressesneuronal markers HU-C and neurogenin1 throughout brain tumor development. While Notchsignaling has been shown to play a similar role in normal brain development, it is notupregulated in oncogenic lesions in embryos and inhibition of the Notch pathway does notrescue the phenotypic alterations induced by HRAS V12 . However, tumors in juveniles (1-2months) and adult fish (≥ 3 months) selectively express increased levels of Notch targetgenes while non-proliferative lesions do not. This implies a specific role for the onset ofNotch-signaling in the establishment of malignant lesions versus benign tumors in this model.This model offers a powerful tool to study glioma development at cellular, molecular andgenetic levels in a living vertebrate and provides novel insights into the development ofsecondary GBM.868 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts

ZDM8 POSTERSZDM7Madison | USA 2015ZDM8Boston | USA 2015Understanding the initiating events that produce a fully competent cancer cell from afield of cancer-prone cells has remained challenging. To address this essential issue in cancerbiology, we focused on a zebrafish gene, crestin, that marks the embryonic neural crest from itsinitial formation through 72 hours post fertilization when it normally turns off. We previouslyfound that crestin mRNA is reexpressed selectively in melanoma tumors in adult zebrafish, thusreading out the presence of a neural crest progenitor (NCP) state. We cloned the crestinpromoter/enhancer and developed an EGFP reporter that recapitulates the embryonicexpression pattern of crestin mRNA and, crucially, marks grossly visible melanomas in ourBRAFV600E/p53 mutant melanoma zebrafish model. Remarkably, we also found thatcrestin:EGFP becomes active when lesions are only a few cells in number, potentially in the firstcell of the melanoma. We isolated small patches of crestin:EGFP positive cells and show thatthese precursor lesions are enriched for expression of the neural crest progenitor transcriptionfactor sox10, among other melanoma- and NCP-associated genes, by qPCR. In order to favora NCP-like state, we forced misexpression of sox10 in melanocytes of BRAFV600E/p53melanoma-prone zebrafish, and this led to enhanced melanoma formation. Through extensiveChIP-Seq and ATAC-Seq analysis, we identified super-enhancer elements in both human andzebrafish melanoma cells at the sox10 locus and an overall chromatin signature consistent withthe NCP state. These data support a model in which 1) BRAFV600E/p53 mutated melanocytesstochastically reactivate an NCP program through engagement of super-enhancer controlelements as read out by crestin reactivation at the initiation of melanoma formation and 2)reemergence of this progenitor state is important for melanoma initiation. We anticipate thatreemergence of progenitor identity will prove to be a broadly utilized mechanism during cancerinitiation.8 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts87

ZDM8Boston | USA 2015ZDM8 POSTERSCell type-specific effects of caffeic acid phenethyl ester in the neural crest andmelanomaChristie A. Ciarlo 1 , Charles K. Kaufman 1,2 , Beste Kinikoglu 3 , Eric C. Liao 3,4 and Leonard I. Zon 1,41 Stem Cell Program and Hematology/Oncology, Children’s Hospital Boston, Howard Hughes Medical Institute, HarvardMedical School, Boston, Massachusetts2 Dana Farber Cancer Institute, Dept. of Medical Oncology, Boston, Massachusetts3 Center for Regenerative Medicine and Division of Plastic and Reconstructive Surgery, Massachusetts General Hospital,Harvard Medical School, Boston, Massachusetts4 Harvard Stem Cell Institute, Cambridge, MassachusettsMetastatic melanoma, a tumor of pigment-producing cells of the skin, is a highly aggressive disease withlimited therapeutic options. Several lines of evidence indicate that melanoma cells revert to a neural crest stemcell fate, suggesting an avenue for therapeutic intervention. Using cultured cells from crestin:GFP transgeniczebrafish embryos, we have conducted a screen to identify chemicals that specifically decrease neural crestcell number. Crestin:GFP+ cells in this culture system are proliferative, migratory, and express neural crestspecifiers. This primary cell culture system is ideal for identifying chemicals that specifically affect neural crestdifferentiation and maintenance, as it is rapid, automatable, and preserves cell type heterogeneity. Compoundswere evaluated for their ability to specifically decrease the ratio of crestin:GFP+ to ubi:mCherry+ cells over 24hours. A library of 3400 compounds was screened at two dilutions, resulting in a 1.8% hit rate. Sixteen hitchemicals decrease crestin:GFP in vivo, including caffeic acid phenethyl ester (CAPE). CAPE rapidlydecreases crestin expression in zebrafish embryos and results in decreased pigmentation. Furthermore, CAPEselectively decreases the viability of melanoma cell lines. Eight melanoma and eight non-melanoma humancell lines were treated with CAPE for 48 hours, resulting in an average 3.3 fold difference in IC50 asdetermined by ATP content. Our studies of CAPE activity in the neural crest will provide valuable insight intoboth development and melanoma tumorigenesis.888 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts

ZDM8 POSTERSZDM7Madison | USA 2015ZDM8Boston | USA 2015Zebrafish Model for Anti-Intrahepatic Cholangiocarcinoma invitro and in vivo Drug Screening Platform1Jen-Leih Wu, 1, 2 Wei-Lin Teng, 3 Wangta Liu, 1 Ching-Yu Haung and 1 Chi-Hsueh Lin1. Institute of Cellular and Organismic Biology, Academia Sinica, Taiwan2. Department of Bioscience and Biotechnology, National Taiwan Ocean University, Taiwan3. Department of Biotechnology, Kaohsiung Medical University, TaiwanAbstractIntrahepatic Cholangiocarcinoma (ICC) is the second most common type ofprimary liver cancer. It is a rare malignant tumor that hard to discover at early stageand the treatment is not effectively. Therefore, to establish a platform for drugscreening is extremely important. To develop high throughput drug screening modelof ICC therapy, we establish two zebrafish model.One is transplanted fluorescently labeled human bile duct cancer cells into theyolk sac of 2-day-old zebrafish embryos to study invasion and dissemination behavior.The second is coexpressed hepatitis B virus X (HBx) and hepatitis C virus core (HCP)proteins in zebrafish livers can induce ICC formation. This phenomenon associatedsome signaling pathway like human, and the histopathologic characteristics are highlyidentical to human ICC.We choose 7 anti-cancer drugs and inhibitor, Paclitaxel(Taxol), Sorafenib(Nexavar), XAV-939, Vismodegib, DAPT, LY2109761 and SB431542 to treat bile ductcancer cell line, xenograft model and ICC zebrafish. The results show thatLY2109761(TGFBRI and TGFBRII inhibitor) , SB431542(TGFBRI inhibitor) canreduce the ICC cancer cell dissemination rate in xenograft model. In addition,LY2109761 can inhibit ICC formation rate (dysplasia rate) in adult zebrafish, and theTGFB1 downstream signaling were be suppressed.These results revealed the applicability of the zebrafish embryo as an in vivomodel for the pharmacodynamics in the metastatic behavior of human bile ductcancer cells. And ICC zebrafish model to be a platform for ICC mechanism-baseddrug screening.8 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts89

ZDM8Boston | USA 2015ZDM8 POSTERSHallmarks of cancer in the human-zebrafish xenograft modelRita Fior 1 , Vanda Póvoa 1 and Miguel Godinho-Ferreira 1,21.Champalimaud Foundation, Centre for the Unknown, Av. De Brasilia, 1400-038 Lisbon, Portugal2.Instituto Gulbenkian Ciência, Rua da Quinta Grande, 6 2780-156 Oeiras, PortugalThe human-zebrafish xenotransplant model not only is a powerful model system to studyhuman cancer but also may provide an in vivo functional platform for personalized cancertreatment in useful-time for patients. Larvae zebrafish xenotransplants have been wellestablished. However, human tumor proliferation has not been studied in detail and,consequently, it has not been clear whether the zebrafish host could sustain thefundamental hallmark in cancer - proliferation.By using several proliferation markers, as ki67, PCNA and short pulse EdU labeling weshow that, indeed, zebrafish larvae are able to sustain proliferation of human colorectalcancer (CRC) cells. We also found that diverse human CRC cells are able to interactdifferently with the host vascular system (angiogenesis) and the primitive immune system.We also characterized metastatic capacities of human CRC cells and found that they exhibitdifferent intravasation potentials and are able to invade and metastasize to distant locations.Interestingly, CRC cells, once in circulation, have similar potential to extravasate andcolonize the CHT.Finally, we tested differential resistance to standard chemotherapy for advanced CRC.Indeed, not only we were able to detect different sensitivities between cell lines but also,within the same cell line, we were able to detect sensitivity to FOLFIRI but resistance toFOLFOX. Our results show that the zebrafish–larvae xenograft model provides a functionaland chemo-sensitive cancer profile that may be used in the future to assign the optimaltherapy for each cancer patient.908 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts

ZDM8 POSTERSZDM7Madison | USA 2015ZDM8Boston | USA 2015Modeling cytotoxic T cells recruitment to zebrafish melanomaAya Ludin 1 , Friedrich Kapp 1 , Yi Zhou 2 and Leonard I. Zon 1,2,31. Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, Massachusetts,US.2. Harvard Stem Cell Institute, Harvard University, Cambridge, MA 02138, USA3. Howard Hughes Medical Institute, Boston, MA 02115, USACytotoxic T lymphocytes (CTLs) eliminate neoplastic cells on a daily basis in healthy individuals.However, CD8α + CTLs, recruited to the established tumor, fail to attack tumor cells due tomechanisms that are not yet fully known. Melanoma is well modeled in zebrafish and T cells areconserved, but tools to report CTLs are not yet available. In order to gain insight into themechanism underlying CTLs infiltration into tumors we attempted to establish transgenic fishreporting CTLs in melanoma bearing fish. We adopted an approach evaluating the potency ofvarious enhancer regions in zebrafish, mouse and human genome that were suggested, or arelikely, to promote CD8α expression. We first identified possible CD8α enhancers in thezebrafish genome using ATAC-Seq of sorted zebrafish lymphocytes. We next cloned them as apromoter sequence together with a previously published short sequence shown to drive geneexpression, conjunct with GFP. In addition, we have utilized bioinformatic search and previousreports to identify mammalian sequences that are either conserved or reported to govern CD8αexpression in mature and differentiating CTLs. We cloned 4 possible enhancers of mouse andhuman origin each combined with the minimal promoter conjunct with GFP. To depict T cellinfiltration into tumors, we transplanted zebrafish melanoma cell lines into irradiated Rag2:GFPzebrafish. We found that as the tumor grew, the number of Rag2 + lymphocytes in the fish thymiand spleen were decreased, in correlation with tumor size, suggesting this model to be effectivein studying T cells recruitment to melanoma. Enhancing CTLs antitumor activity is currentlyshowing great promise in cancer treatment. Utilizing CTLs reported fish to gain farther insightinto the mechanisms underlying T cells regulation in the melanoma setting can lead to better,more efficient immunotherapies against cancer.Word count- 2908 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts91

ZDM8Boston | USA 2015ZDM8 POSTERSThe tumor suppressor adenomatous polyposis coli (apc)regulates mitochondrial pyruvate carrier 1 (mpc1): a novel rolefor apc in pyruvate metabolismImelda T. Sandoval 1 , Richard Glenn C. Delacruz 1 , Braden Miller 1 , ChristeenaSatterfield 1 , Kristofor A. Olson 3 , Shauna Hill 2 , Holly Van Remmen 2 , Jared Rutter 3and David A. Jones 11Immunobiology and Cancer Research Program, Oklahoma Medical Research Foundation, 825N. E 13 th St., Oklahoma City, OK 731042Free Radical Biology and Aging Research Program, Oklahoma Medical Research Foundation,825 N. E 13 th St., Oklahoma City, OK 731043Department of Biochemistry, University of Utah School of Medicine, 15 N. Medical Drive East,Salt Lake City, UT 84112Colon tumors undergo a series of genetic changes as they progress from normaltissue to carcinoma, with mutations in the tumor suppressor Adenomatous Polyposis Coli(APC) as the primary initiating mutation. Germline mutations in APC are found in a majorityof familial adenomatous polyposis (FAP), an inherited form of colon cancer, while somaticmutations in APC occur in 80% of sporadic colon tumors.Mitochondrial Pyruvate Carrier 1 (MPC1) is a member of the mitochondrial carrierfamily and has just been recently identified. It forms a heterocomplex with MPC2 and isresponsible for transporting pyruvate, a critical player in cellular metabolism, into the innermitochondrial matrix. Recent studies have shown that MPC1 is downregulated in varioustypes of cancers, particularly in colon adenocarcinoma, and exhibits a positive correlationwith APC. We, therefore, investigated whether APC regulates MPC1 and whether MPC1downregulation contributes to intestinal differentiation and development of colon cancer.Utilizing a zebrafish genetic mutant of apc (apc mcr ), we demonstrate that mpc1expression is reduced in apc mcr compared to apc +/mcr and wild type siblings. Antisensemorpholino knockdown of mpc1 in wild type embryos results in an array of developmentaldefects that most notably include failed intestinal differentiation. Knockdown of mpc1recapitulates other phenotypes of apc loss including pericardial edema, loss of pectoralfins and defective jaw formation. This is accompanied by mitochondrial dysfunction inmorphant embryos as evidenced by a decrease in metabolic respiration and triglyceridelevels. Interestingly, several other enzymes involved in pyruvate metabolism and transportare downregulated in apc mcr . Further studies into the regulation of MPC1 may lead to anew role for APC in pyruvate metabolism.928 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts

ZDM8 POSTERSZDM7Madison | USA 2015ZDM8Boston | USA 2015Myogenic Regulatory Factors And Their Role In EmbryonalRhabdomyosarcomaInês M. Tenente 1-3 , Myron Ignatius 1,2 , Eleanor Chen 1,2 , Madeline Hayes 1,2 , Qin Tang 1,2 ,David M. Langenau 1,21. Molecular Pathology Unit, Center for Cancer Research and Center for Regenerative Medicine,Massachusetts General Hospital, Boston, Massachusetts, USA2. Harvard Stem Cell Institute and Harvard Medical School, Boston, Massachusetts, USA3. Graduate Program in Areas of Basic and Applied Biology, Instituto de Ciências Biomédicas AbelSalazar, Universidade do Porto, Porto, PortugalRhabdomyosarcoma (RMS) is a common pediatric sarcoma of muscle withRAS pathway activation being the dominant oncogenic driver in EmbryonalRMS (ERMS). We have developed a kRASG12D-driven zebrafish ERMSmodel and identified that tumor-propagating potential is confined to amolecularly distinct cell type that expresses Myf5, m-Cadherin, c-Met andother satellite cell markers. MYF5, MYOD1 and MYOG are bHLH myogenicregulatory factors (MRFs) that orchestrate skeletal muscle differentiationduring development and regeneration and are sufficient to reprogramhuman mesenchymal cells into a myogenic fate. Importantly, these factorsare significantly up-regulated in mouse and human ERMS. Given that myf5expression defined the TPC compartment in our zebrafish model, wehypothesized that Myf5 and its transcriptional targets likely regulate growthand maintenance of TPCs. Using the zebrafish ERMS model, we have nowshown that induced expression of Myf5 was sufficient to confer tumorpropagatingability to differentiated populations of cancer cells. These“Induced TPCs” proliferate and generate aggressive tumors that retainmolecular markers of muscle differentiation but also re-express satellite cellmarkers. Next, we assessed if Myf5 is required for ERMS tumor initiation inmyf5 loss-of-function mutant zebrafish. Tumor onset, incidence, and regrowthfollowing transplantation were not impaired in myf5 -/- animals. Asthe identity of the cell-of-origin for RMS is currently controversial and giventhe redundancy of Myf5 and MyoD in muscle progenitors, our currenthypothesis is that tumors arising in myf5 -/- zebrafish recapitulate a distinctsubset of human ERMS. This idea is consistent with the fact that MYF5 isexpressed in only 50% of primary human ERMS and a small fraction ofhuman cell lines. Importantly, Knock down experiments in human ERMScell lines confirm independent roles of either MYF5 or MYOD1 in themaintenance of human ERMS cell viability. Collectively, our results supporta previously unappreciated role for bHLH MRFs in ERMS cell survival andintra-tumor functional heterogeneity.8 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts93

ZDM8Boston | USA 2015ZDM8 POSTERSNotch Signaling Increases the Number of Relapse-driving TumorPropagating Cells in Embryonal RhabdomyosarcomaMyron S. Ignatius 1 , Riadh Lobbardi 1 , Madeline Hayes 1 , Eleanor Chen 1 , Karin McCarthy 1 ,Aleksey Molodtsov 1 , Brian Beleya 2 , Deepak Bhere 3 , Kahlid Shah 3 , Petur Nielsen 1 ,Corinne Linardic 2 , Javed Khan 4 , Charles Keller 5 , David Langenau 11Department of Pathology, MGH-Cancer Center, Massachusetts General Hospital and Harvard MedicalSchool, Boston, MA2Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, NC3Department of Neurology, Massachusetts General Hospital, Harvard Medical School, Boston, MA4Oncogenomics Section, Pediatric Oncology Branch, Center for Cancer Research, National CancerInstitute, National Institutes of Health, Bethesda, MD5Pediatric Cancer Biology Program, Papé Family Pediatric Research Institute, Department of Pediatrics,Oregon Health & Science University, Portland, OREmbryonal rhabdomyosarcoma (ERMS) is a devastating pediatric muscle cancer withextremely poor prognosis at relapse. We have identified the tumor-propagating cell (TPC) in atransgenic zebrafish model of kRASG12D-induced ERMS that is responsible for drivingcontinued tumor growth and relapse. Building on these observations, we have identified theNotch pathway as a potent enhancer of ERMS self-renewal and TPC number. Specifically,limiting dilution cell transplantation experiments revealed that TPCs are increased 10-fold inERMS that co-express activated intracellular Notch1 (ICN1). This increase in TPC number ispartly the result of expansion of myf5-GFP+/mylz2-mCherry-negative ERMS-cell population.Unexpectedly, cell-transplantation experiments revealed that Notch pathway activation alsoconferred tumor-propagating ability to myf5-GFP+/mylz2-mCherry+ mid-differentiated ERMScells– a population of cells previously shown to lack self-renewal capacity. Single cellengraftment studies uncovered that NOTCH activation enabled ERMS cells to dedifferentiateinto myf5-GFP+/mylz2-mCherry-negative TPCs, suggesting that Notch has important roles inboth self-renewal and cell state transitions. We next wanted to validated our findings in humanERMS where NOTCH1 is highly expressed in >75% of patients. Specifically, human ERMScells were generated that expressed ICN1, these cells had elevated sphere-colony formation, asurrogate for self-renewal in vitro. By contrast, shRNA knockdown of NOTCH1 resulted indecreased sphere-colony formation, robust terminal differentiation of ERMS cells into latestagemyosin-expressing myoblasts, and suppressed growth in mouse xenograft experiments.Mechanistic studies uncovered that NOTCH1 binds directly to the SNAI1 promoter to activateexpression. SNAI1 is a zinc-finger transcriptional repressor with roles regulating survival,gastrulation, and EMT in cancer. Yet, a role for SNAI1 in ERMS has yet to be ascribed. SNAI1knockdown resulted in reduced sphere formation, suppressed differentiation, and inhibition ofgrowth in xenograft transplantation models. Our data indicate that Notch signaling regulatesboth TPC-self-renewal and differentiation in human ERMS and NOTCH/SNAI1 inhibition maybenefit patients with relapse disease.948 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts

ZDM8 POSTERSZDM7Madison | USA 2015ZDM8Boston | USA 2015Circadian Effect of MTX in Zebrafish Toxicity and ProliferationArianna Minuche 1 , Sergio Valencia 1 , Veronica Akle 11. Universidad de los Andes, School of Medicine. Bogota-ColombiaThe circadian system plays an important role in coordinating most cellular processes,including those implicated in the development of cancer. Previous studies have showncircadian variation in the pharmacokinetics, efficiency and side effects of multipleanticancer treatments, including methotrexate (MTX). However, the studies on the effect ofthe circadian system in the effectiveness and toxicity of this drug and its relation with theexpression of the enzymes in the folate pathway are limited, especially in diurnal animalmodels. The purpose of this investigation is to study the effect of MTX in embryo and adultzebrafish, and to assess its toxicity, doses response and expression of enzymes in thefolate pathway at different times of the day. The drug was administered during the earlyday and late in the afternoon and survival was determined during development. The ratesof proliferation were assessed using anti-BrdU staining in embryos and in the brain of adultzebrafish injected with MTX at different times of the day. The results from this study willestablish zebrafish as a model to study the circadian effect of anticancer drugs. It will alsohelp elucidate the mechanism of action and the pharmacokinetics of MTX to explain theempiric observation that this drug has a more beneficial effect when administered in theafternoon.8 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts95

ZDM8Boston | USA 2015ZDM8 POSTERSThe GAP-related Domain of NF1 is required for tumorsuppressionof MYCN-induced neuroblastoma in vivo.Shuning He 1 , Marc R. Mansour 1,2 , Dong Hyuk Ki 1 , Mark W. Zimmerman 1 ,Hillary M. Layden 1 , Eric D. de Groh 3 , Antonio R. Perez-Atayde 4 , ShizhenZhu 5 , Jonathan A. Epstein 3 , and A. Thomas Look 11 Department of Pediatric Oncology, Dana-Farber Cancer Institute, Harvard Medical School,Boston MA, 02115, USA.2 Department of Haematology, UCL Cancer Institute, University College London, WC1E 6BT, UK.3 Department of Cell and Developmental Biology, Penn Cardiovascular Institute, and the Institutefor Regenerative Medicine, Perelman School of Medicine at the University of Pennsylvania,Philadelphia, PA 19104, USA4 Department of Pathology, Children's Hospital Boston, Harvard Medical School, Boston MA,02115, USA.5 Department of Biochemistry and Molecular Biology/ Department of Molecular Pharmacology andExperimental Therapeutics, Mayo Clinic College of Medicine, Mayo Clinic Cancer Center andMayo Clinic Center for Individualized Medicine, Rochester MN, 55902, USA.Neuroblastoma accounts for 8-10% of all childhood malignancies and 15% of all cancerdeaths in children. Neuroblastoma is a malignant embryonic tumor of the peripheralsympathetic nervous system (PSNS) arising from dopaminergic cells of the neural crest,and is frequently associated with genomic amplification of the MYCN oncogene.Genomic aberrations leading to loss of the NF1 tumor suppressor gene have beenreported in primary and relapsed neuroblastomas, and low expression of NF1 iscorrelated with poor outcome, suggesting an important role for NF1 loss inneuroblastoma tumorigenesis. Here, we used genome engineering to develop zebrafishlines harboring loss-of-function mutations of the nf1a and nf1b genes (note nf1 isduplicated in the zebrafish). Loss of the nf1a orthologue greatly accelerated the onset ofneuroblastoma induced by MYCN overexpression in MYCN-transgenic zebrafish, with apenetrance of nearly 100% by 5 weeks of age, compared to 7% in MYCN-transgeniczebrafish with wildtype nf1a and nf1b (p

ZDM8 POSTERSZDM7Madison | USA 2015ZDM8Boston | USA 2015A role for Wnt Signaling in self-renewal and differentiation of EmbryonalRhabdomyosarcomaMadeline Hayes 1,2 , Eleanor Chen 3 , Myron Ignatius 1,2 , Ines Tenente 1,2 and DavidLangenau 1,21 Molecular Pathology, Cancer Centre, and Regenerative Medicine, Massachusetts GeneralHospital, Boston, MA 021292 Harvard Stem Cell Institute, Cambridge MA 021393 Department of Pathology, University of Washington, Seattle WA 98195Embryonal rhabdomyosarcoma (ERMS) is a common pediatric malignancy of musclewith relapse being the major clinical challenge. Sadly, 50% of relapse patients eventuallysuccumb to disease. A small population of undifferentiated, tumor propagating cells(TPCs) self-renew and ultimately drive continued tumor growth and relapse. If TPCscould be eliminated or induced to differentiate into non-proliferative, non-metastatic celltypes, tumors would regress and patients could remain relapse free. GSK3 antagonistshave recently been shown to differentiate TPCs and inhibit ERMS growth in bothzebrafish and human. These GSK3 inhibitors act by inducing canonical WNT/β-cateninsignaling. In skeletal muscle, Wnt/β-catenin signaling is required for stem celldifferentiation, whereas non-canonical Wnt/Planar cell polarity (PCP) signaling increasesmuscle stem cell self-renewal. To date, the relationship between Wnt/β-catenin andWnt/PCP signaling in ERMS is unknown. Here we utilize transgenic and live dynamicreal-time imaging approaches in live zebrafish to assess competing roles for canonicalWnt/β-catenin and Wnt/PCP pathways in regulating self-renewal and differentiation inERMS. We extend these findings to human ERMS and implicate a role for Wnt signalingin ERMS self-renewal and differentiation, suggesting a potential for Wnt pathwaymanipulation in the treatment of patients with ERMS.8 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts97

ZDM8Boston | USA 2015ZDM8 POSTERSLoss of function of ARID1A synergizes with MYCN in neuroblastomapathogenesisTing Tao 1 , Hui Shi 1,2 , Cigall Kadoch 1 , A. Thomas Look 1,*1 Department of Pediatric Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA02115, USA2 College of Animal Sciences, Zhejiang University, Hangzhou 310029, China.*Correspondence: Thomas_Look@dfci.harvard.eduNeuroblastoma is a solid tumor of the peripheral sympathetic nervous system (PSNS), whichaccounts for 15% of childhood cancer deaths. MYCN amplification is present in approximately20-25% of cases and is associated with poor prognosis. ARID1A is a member of the SWI/SNF(BAF) chromatin-remodeling complex that is known to modulate chromatin structure and genetranscription. It is frequently mono- or bi-allelically inactivated by mutation or deletion in high-riskchildhood neuroblastomas as well as in adult tumors, such as ovarian cancer and endometrialtumors. Upon bioinformatic analysis of microarray data sets, we demonstrated that low ARID1Aexpression is highly associated with MYCN amplification, which is due in part to loss of onecopy of ARID1A as part of a 1p deletion that often accompanies MYCN amplification. TheARID1A gene is duplicated in zebrafish, and the two genes are hence named ARID1Aa andARID1Ab. Whole-mount in situ hybridization (WISH) shows both ARID1Aa and ARID1Ab areubiquitously expressed in neuronal cells during early embryogenesis in zebrafish. We targetedeach allele of the two ARID1A genes using CRISPR/Cas genome editing and recovered stablelines with loss-of-function mutations in each gene. ARID1Ab but not ARID1Aa homozygousmutation showed embryonic lethality. Interestingly, heterozygous disruption of each orthologuedramatically accelerated the onset and increased the penetrance of MYCN-inducedneuroblastoma in our transgenic Tg (dβh:EGFP-MYCN) zebrafish neuroblastoma model,indicating that loss of only one of the four alleles was sufficient to interfere with the tumorsuppressor function of ARID1A in neuroblastoma. Our long-term goal is to fully utilize thezebrafish model to elucidate the mechanisms underlying the tumor suppressor role of thiscomponent of the BAF chromatin remodeling complex in neuroblastoma, as a first step towarddesigning targeted therapies that exploit vulnerabilities that arise from ARID1Ahaploinsufficiency in disease.988 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts

ZDM8 POSTERSZDM7Madison | USA 2015ZDM8Boston | USA 2015DEF plays a role in pre-rRNA processing that is upregulated tosynergize with MYCN in neuroblastoma pathogenesisHui Shi 1,2 , Ting Tao 1 , Samuel B. Sondalle 3 , Shizhen Zhu 4 , Susan J. Baserga 3 , JinrongPeng 2 , A. Thomas Look 1,*1Department of Pediatric Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA02115, USA2College of Animal Sciences, Zhejiang University, Hangzhou 310029, China3Departments of Genetics, Molecular Biophysics and Biochemistry, and Therapeutic Radiology, YaleUniversity School of Medicine, New Haven, CT 06520, USA4Department of Biochemistry and Molecular Biology, Department of Molecular Pharmacology andExperimental Therapeutics, Mayo Clinic College of Medicine, Mayo Clinic Cancer Center and Mayo ClinicCenter for Individualized Medicine, Rochester, MN 55902, USA*Correspondence: Thomas_Look@dfci.harvard.eduNeuroblastoma is a solid tumor of the peripheral sympathetic nervous system (PSNS), whichaccounts for 15% of childhood cancer deaths. The nucleolar factor DEF has been shown to playa key role in ribosome biogenesis through its role as a component of the small ribosomalsubunit (SSU) processome in pre-rRNA processing, and also to mediate p53 proteindegradation in collaboration with Calpain 3 in the nucleolus (Tao et al., 2013). DEF expression ishighly upregulated in most neuroblastomas and is highest in cases with MYCN amplification(http://r2.amc.nl). We report here that in two independent DEF transgenic lines, overexpressionof DEF dramatically accelerates the onset and increases penetrance of MYCN-inducedneuroblastoma in our transgenic Tg (dβh:EGFP-MYCN) zebrafish neuroblastoma model. Inaddition, DEF haploinsufficiency significantly decreases disease penetrance in a new MYCN_TTzebrafish neuroblastoma model established in this study. In human neuroblastoma, DEFknockdown induces cell growth inhibition and apoptosis in both Kelly and SH-SY5Yneuroblastoma cell lines. Overall, the elucidation of the molecular and pathogenic role of DEF inneuroblastoma will advance our understanding of the importance of DEF in ribosomebiogenesis, and the roles that increased levels of pre-rRNA processing play in allowingneuroblastoma cells to tolerate and be transformed by MYCN, which is amplified at the genelevel by one hundred-fold or more in these tumors. DEF and other components of the smallribosomal subunit (SSU) processome in pre-rRNA processing machinery would appear toconstitute a site of vulnerability in neuroblastoma cells, which could be exploited for the targetedtherapy of this disease.8 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts99

ZDM8Boston | USA 2015ZDM8 POSTERSModeling thyroid cancer in zebrafishAna Lourenço Almeida 1,2 , Miguel Godinho Ferreira 2 and Paula Soares 11. Cancer Biology Group, IPATIMUP, 4200-465 Porto, Portugal2. Instituto Gulbenkian Ciência, 2780-156 Oeiras, PortugalSomatic mutations in BRAF have been identified in an increasing number of tumorsnamely melanoma and papillary thyroid carcinoma. The most common BRAF mutationis the V600E and accounts for more than 90% of cases of cancer involving this gene.The oncogene induces constitutive ERK and NF-kB signaling due to hyperactivation ofthe RAS-MEK-ERK pathway. This pathway is critical in the transduction of signals bygrowth factors, hormones and cytokines and is involved in the regulation of cellproliferation, differentiation and apoptosis. Inhibitors that selectively target BRAFV600Emay provide clinical benefit for patients.We have modeled, for the first time, thyroid tumorigenesis in zebrafish by constitutivelyexpressing braf V600E oncogene in thyroid. The presence of the BRAF mutant form infollicular cells of tp53 +/+ zebrafish disrupts the thyroid tissue organization during larvalstages. However, it does not induce thyroid dysfunction during the adulthood and it isnot sufficient to promote cancer up to 12mpf.In contrast, larval tissue disorganization promoted by braf V600E is rescued in a p53-deficient background, suggestive of an overcome of the senescence induced by theoncogene in the follicular cells.In order to bypass the deleterious effects of early development, we have developed aheat shock-inducible CreER(T2) line to achieve inducible braf V600E expression in thethyroid.We hypothesize that, consistent with the braf V600E tp53 -/- melanoma zebrafish model,formation of thyroid lesions that develop into carcinomas requires both the BRAF gainof function mutation and the inactivation of the p53 pathway to promote cancer inzebrafish.1008 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts

ZDM8 POSTERSZDM7Madison | USA 2015ZDM8Boston | USA 2015Arsenic and DNA methylation: Using zebrafish to uncover themechanism of arsenic induced toxicity and methylomereprogrammingKathryn Bambino, Jaime Chu, Kirsten Sadler-Edepli1. Department of Preventive Medicine, Department of Developmental and Regenerative Biology, IcahnSchool of Medicine at Mount Sinai2. Department of Pediatrics, Icahn School of Medicine at Mount Sinai3. Department of Developmental and Regenerative Biology, Department of Medicine/Division of LiverDiseases, Icahn School of Medicine at Mount SinaiExposure to inorganic arsenic is a major public health concern with far reaching effects.Prenatal and childhood exposure elevates the risk of mortality and cancer in childhood, and therisk of adverse health outcomes after exposure persists throughout life. This suggests anepigenetic basis to arsenic induced disease. Despite ranking #1 on the Agency for ToxicSubstances and Disease Registry (ATSDR) watch list, the mechanism of arsenic toxicity isvirtually unknown. Some studies suggest that loss of DNA methylation is a contributing factor,yet a systematic examination of the epigenetic basis of arsenic induced disease in a wholeanimal is lacking. We established zebrafish as a novel in vivo system to test the hypothesis thatarsenic toxicity in embryos is caused by DNA hypomethylation. We found that the arsenicspecific methyltransferase (As3mt) is expressed in early zebrafish embryos and is then enrichedin the larval liver, as in mammals. Moreover, embryos exposed to inorganic arsenic demonstratereduced liver size and mortality beginning at 4 days post-fertilization. This was accompanied bya reduction of global DNA methylation by approximately 30% compared to untreated controls inembryos exposed from 4-24 hours post-fertilization. Further studies assessing changes inmethylation at specific loci by methylation-specific PCR and reduced representation bisulfitesequencing (RRBS) will enable us to identify functional links between arsenic metabolism,alterations to the methylome, and arsenic toxicity.8 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts101

ZDM8Boston | USA 2015ZDM8 POSTERSCentral Nervous System Infiltration by Zebrafish T LymphoblastMalignanciesChiara Borga 1,2 , Lance Batchelor 1 , Silvia Bresolin 2 , Ilaria Bronzini 2 , Giuseppe Basso 2 ,Nikolaus Trede 3 , Geertruy te Kronnie 2 , J. Kimble Frazer 11Section of Pediatric Hematology-Oncology, University of Oklahoma Health Sciences Center, OklahomaCity, USA2Department of Women’s and Children’s Health, Padua, Italy3Acetylon Pharmaceuticals, Boston, USACentral nervous system (CNS) invasion poses challenges in the treatment of Acute LymphoblasticLeukemia (ALL) and Lymphoblastic Lymphoma (LBL). Although patients with CNS + diseasereceive more intense CNS-directed treatment, they still have inferior outcomes. Little is knownabout molecular mechanisms mediating lymphoblast infiltration of the CNS, and such insights aredifficult to obtain from clinical samples, so studying this phenomenon requires animal models. Weinvestigated CNS involvement in two zebrafish models that both exhibit highly-penetrant T cellLBL/ALL (T-LBL/T-ALL): the ENU mutant, hlk, and a transgenic line expressing human MYC inlymphoblasts, hMYC. Histologic analyses of fish with localized thymic lymphomas (i.e., T-LBL)and disseminated T-ALL revealed that CNS invasion occurs at both disease stages in bothgenetic models at high frequency. Our results represent the first description of CNS + disease inD. rerio T cell cancers. Importantly, CNS involvement in zebrafish closely resembles that whichoccurs in human patients. Overall, qualitative and quantitative CNS infiltration was higher in hMYCfish compared to the hlk line. FACS-purified GFP + malignant cells from each CNS + fish wereanalyzed by RNA microarray (GeneChip Zebrafish Genome Arrays, Affymetrix), revealing upregulationof distinct gene networks governing cell motility in hlk and hMYC cancers, suggestingthat multiple mechanisms can drive lymphoblast CNS homing and invasion. Specifically, highexpression of cxcr4 correlated with the extensive CNS disease in hMYC fish, suggesting thispathway may promote the invasion, survival and growth of T-lymphoblasts in the CNSenvironment. Our findings in hlk and hMYC zebrafish position both lines as attractive models tostudy CNS + T-LBL/T-ALL and the mechanisms that control CNS dissemination, which has vitalclinical relevance.1028 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts

ZDM8 POSTERSZDM7Madison | USA 2015ZDM8Boston | USA 2015Using zebrafish to study the significance oftumour cell apoptosis and tumour-associated macrophagesin aggressive B-cell lymphomaZiyuan Chang, Thomas Ramezani, John Pound, Christopher Gregory, Yi FengMRC Centre for Inflammation Research, Queen’s Medical Research Institute, University ofEdinburgh, Edinburgh, UK.The combination of macrophage infiltration and abundant tumour cell apoptosis inaggressive B-cell lymphomas, eg. Burkitt’s lymphoma (BL), has led to the proposalthat apoptotic tumour cells drive a macrophage-promoted pro-oncogenicmicroenvironment in such tumours. Work described here was designed to testaspects of this hypothesis in: 1) a xenograft-based model of BL in zebrafish, and 2)an inducible zebrafish model of B-cell malignancy.A fluorescence-tagged BL cell line (BL2) has been generated and a xenograft modelconstructed by optimising parameters for transplantation of the cells into transgeniczebrafish embryos. 90% of BL2-transplanted larvae had a visible tumour mass 3days post injection (dpi) compared with only 55% of apoptosis-suppressed Bcl-2-transfected BL2 transplants. Furthermore, there was more metastasis of BL2 cellsthan Bcl2-transfectants at 3 dpi. These preliminary observations are consistent witha pro-oncogenic role for apoptotic BL cells. A role for macrophages in the decreasedmetastasis observed when tumour cell apoptosis is inhibited will be explored bymacrophage depletion and co-injection experiments.De-regulated expression of the c-MYC gene through its translocation toimmunoglobulin (Ig) loci is the hallmark of BL and mice bearing an Ig/mutated c-MYCtransgene develop BL-like tumours. We have placed a mouse c-MYC under thecontrol of an endogenous IgM1 promoter in an attempt to induce B cell malignancy inzebrafish. Transgenic founders, Tg (IgM1: Cre-ERT2) and Tg (IgM1: LoxP-dsRed-LoxP -cmyc-eGFP) were generated using a multisite gateway technique. By pairingfounders, Tg (IgM1: Cre-ERT2; IgM1: LoxP-dsRed-LoxP -cmyc-eGFP) will begenerated which should allow us to induce c-MYC expression at various stages of B-cell development by adding tamoxifen. Theoretically, by modulating c-MYCexpression, different B-cell malignancies will be induced. The myc-eGFP fusionprotein will allow us to observe tumour initiation, progression and metastasis in realtime and investigate the contributions of apoptosis and macrophages.Words: 2978 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts103

ZDM8Boston | USA 2015ZDM8 POSTERSIdentification of a zebrafish germline mutation that causes T cellcancer and apoptosis resistanceBarbara Squiban and J. Kimble FrazerSection of Pediatric Hematology/Oncology, Department of PediatricsUniversity of Oklahoma Health Sciences Center, Oklahoma City, USALeukemia and lymphoma are the 1 st and 3 rd most common pediatric cancers, and T cellcancers are common subtypes of both entities. Our group used a forward-genetic screen tocreate mutants prone to T cell cancer. The screen was performed using transgenic fishexpressing a T cell-specific GFP reporter, thus, GFP + thymic tumors detect fish with T cellcancer. One mutant line, oscar the grouch (otg), shows recessive cancer predisposition, andtheir cancers are resistant to treatments that normally induce T cell apoptosis. Larvae with biallelicotg mutation also have defects in radiation-induced apoptosis. My project seeks toidentify the mutation in otg fish using this larval phenotype and RNA-seq to find SNPs linkedto otg. RNA-seq will also be used to compare otg T cells and cancers, thus revealing otg’scandidate genomic locus and also yield information on downstream effectors in the otgpathway. Hence, if otg is a gene that impacts the apoptotic pathway as we hypothesize,identifying otg will inform our understanding of apoptosis and how this germline mutationcontributes to T cell oncogenesis.1048 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts

ZDM8 POSTERSZDM7Madison | USA 2015ZDM8Boston | USA 2015SCID zebrafish as a universal transplant platformJohn C. Moore 1-3 , Qin Tang 1-3 , Nora Torres 3,4 , Aleksey Molodtsov 1-3 , Nouran SAbdelfattah 1-3 , Tim Mulligan 5 , Caitlin Bourque 6 Jason Berman 7 , Robert S Liwski 7 , YarivHouvras 6 , A. Thomas Look 2,8 , Brant Weinstein 5 , John Rawls 9 , David M. Langenau 1-31. Division of Molecular Pathology, Department of Regenerative Medicine and Cancer Center,Massachusetts General Hospital, Boston, Massachusetts, USA.2. Harvard Medical School and Stem Cell Institute, Boston, Massachusetts, USA.3. Immune Compromised Zebrafish Resource Center, Massachusetts General Hospital, Boston,Massachusetts, USA.4. Harvard University, Cambridge, Massachusetts, USA.5. Laboratory of Molecular Genetics, NICHD, NIH, Bethesda, MD, USA.6. Departments of Surgery and Medicine, Weill Cornell Medical College, New York, New York, USA.7. Izaak Walton Killam Health Center, Dalhousie University, Halifax, Nova Scotia, Canada.8. Pediatric Oncology, Dana-Farber Cancer Institute, Boston, MA, USA.9. Department of Cell and Molecular Physiology, University of North Carolina at Chapel Hill, Chapel Hill,North Carolina, USA.Cellular transplantation into immune compromised mouse models is the standardexperimental technique used to understand cancer processes and to assess therapeuticresponse in vivo, but is low-throughput, expensive and difficult to directly visualize. To overcomethese challenges, we have developed immune compromised zebrafish (ICZ) in the casper strainusing genome editing techniques, by targeting genes required for immune cell function andknown to cause severe immune compromised deficient phenotypes (SCID) in human and mice.We are currently characterizing mutants in recombination-activating gene 2 (rag2), DNAdependentprotein kinase (prkdc), janus kinase 3 (jak3), interleukin 2 receptor gamma (Il2rg),zeta-chain (TCR) associated protein kinase 70 (zap70), and forkhead box N1 (foxn1/nude).Whole kidney marrow gene expression analysis, along with molecular and cellularcharacterization of ICZ lymphocytes demonstrates differential loss of T and B cell function,indicating a gene dependent phenotypic conservation with higher vertebrates. A subset ofmutants have reduced immune cell function, permitting engraftment of hematopoietic andmuscle satellite cells from allogeneic donors. Moreover, we demonstrate robust and persistentengraftment of primary or serial transplanted tumors including fluorescently labeled leukemias,rhabdomyosarcoma, and melanomas from a wide range of zebrafish strains. The ability totransplant non-immune matched cell types without the need for preconditioning or derivation ina clonal syngeneic line will revolutionize the types and scale of cell transplantation experimentsperformed in the zebrafish. The selective loss of immune cell subtypes will also facilitate ourunderstanding of immunological/tumor interactions. We are currently optimizing protocols toperform live cell imaging in optically clear casper strain lines and refining genetic models forengraftment of human cells. In coordination with the ICZ Resource Center run by the Langenaugroup, we are now distributing characterized lines to the zebrafish community. This work issupported by NIH grant R24OD016761.8 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts105

ZDM8Boston | USA 2015ZDM8 POSTERSOptical Control of Cancer Initiation from Single Cells inZebrafishZhiping Feng 1 , Tal Markus 2 , Sophie Vriz 3 , Michel Volovitch 3,4 , Ludovic Jullien 5 ,Shuo Lin 6 , Shimon Weiss 1,7 , David Bensimon 2,71. Department of Molecular, Cellular and Integrative Physiology, UCLA, USA2. Laboratoire de Physique Statistique, ENS, France3. College de France, Center for Interdisciplinary Research in Biology, France4. Department of Biology, ENS, France5. Department of Chemistry, ENS, France6. Department of Molecular, Cell and Developmental Biology, UCLA, USA7. Department of Chemistry and Biochemistry, UCLA, USAThe initiation of a cancer is believed to be a rare event taking place at the level ofindividual cells, yet current probes of cancer development are restricted to the collectiveproperties of many thousands of cells. Two key unresolved questions- the effectivenessof mutations of oncogenes for cancer initiation and the role of the localmicroenvironment on cancer initiation-require study of the fates of individual cells andtheir progenies. Recently, we developed a technology that allows for the control ofprotein activity and gene expression in single cells through light activation. In this work,we utilize this method to activate in individual cells of a zebrafish a typical oncogene, K-RasG12V and investigate effects of these changes on tumorigenesis under variedgenetic backgrounds. We have demonstrated the success of spatiotemporal control ofoncogene expression in live zebrafish. Furthermore, we investigated differenttumorigenic phenotypes by transiently or permanently activating K-Ras at varieddevelopmental stages. We believe our study could help open up a completely new basisfor understanding cancer growth and eventually testing anti-cancer drugs.1068 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts

ZDM8 POSTERSZDM7Madison | USA 2015ZDM8Boston | USA 2015Hemodynamic profiles tune the arrest and extravasation of circulatingtumor cellsSofia Azevedo 1 , Guillaume Allio 1 , Gautier Follain 1 , Nina Fekonja 1 , ShankarPattabhiraman 1 , Sébastien Harlepp 2 and Jacky G.Goetz 11 Inserm U1109, MN3T, Strasbourg, F-67200, France; Université de Strasbourg, Strasbourg, F-67000,France; LabEx Medalis, Université de Strasbourg, Strasbourg, F-67000, France; Fédération de MédecineTranslationnelle de Strasbourg (FMTS), Strasbourg, F-67000, France2 Institut de Physique et de Chimie des Matériaux de Strasbourg, UdS, UMR 7504, 23 rue du Loess,67034 Strasbourg, FranceMetastatic spread is a multistep process leading to the dissemination and growth of circulatingtumor cells (CTCs) at distant sites from the primary tumor. Interestingly, distant organcolonization does not occur randomly. In turn, two concepts have emerged: while the "seed andsoil" hypothesis states that metastasis will occur at sites where the local microenvironment isfavorable, the "mechanical" concept argues that adhesion and metastatic growth occur at sitesof optimal flow patterns. In both situations, arrest and extravasation of CTCs are the limitingfactor. It is thus very likely that arrest, survival and extravasation of CTCs are favored by optimalflow patterns.Here, we use the zebrafish embryos and exploit its multiple advantages for dissecting thecontribution of hemodynamic forces to adhesion and extravasation. The last steps of themetastatic spread are recapitulated by injecting a fluorescent metastatic mammary carcinomacell line (D2A1) in the circulation of zebrafish embryos. Subsequent adhesion and extravasationsteps, as well as analysis of local hemodynamic profiles, are assessed through a combination offast and long-term intravital imaging. Our results show that tumor cells disseminate all over theembryo, either as single or cluster of cells, but preferentially arrest in a specific region of thedeveloping vasculature, where the majority of extravasation events could also be observed. Fineflow mapping of this region revealed very specific blood flow profiles, favoring arrest of CTCs.We tuned blood flow patterns and observed that flow decrease could perturb the preferred sitesof arrest of CTCs. Interestingly, while extravasation of clusters of TCs involved active and localremodeling of the endothelium, this behavior and the subsequent extravasation of CTCs weredecreased in low-flow conditions. Altogether, our results suggest that local hemodynamicprofiles are very likely capable of tuning the last, and yet essential, steps of the metastasiscascade.8 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts107

ZDM8Boston | USA 2015ZDM8 POSTERSContinuous low frequency in vivo mutagenesis: a novel system fortumour inductionLara Sicouri 1 , Ganluca Deflorian 1 , Federica Pisati 1 , Svend K. Petersen-Mahrt 1 .1. IFOM - FIRC Institute of Molecular Oncology, Milan - ItalyIn somatic cells, DNA instability is associated with genetic diseases such as cancer. DNA damagecan be introduced by endogenous and exogenous agents, leading to mutations andrecombinations. Activation Induced Deaminase (AID) is a natural DNA mutator required forimmunoglobulin diversification in B cells. AID deaminates cytosine to uracil in single stranded DNA,leading to beneficial mutations and recombinations in the Ig locus, or to DNA instability and cancerin non-Ig loci. Because of the reduced success rate of human-mutations knock-ins into mouse/zebrafish genomes (GEM models), we generated a novel AID-dependent in vivo model-system forneoplasia. AID’s continuous mutagenesis activity was fused to gene targeting proteins, to createnew loci specific random mutations.In zebrafish, AID was targeted in vivo to the tumour suppressor p53 by fusing AID to TALEsrecognising p53. In established F1 transgenic lines females carrying TALE-AIDwt transgene haveimpaired fertility, with dead and non fertilised eggs. Histologic analysis (3 and 5 months postfertilisation), show that TALE-AIDwt females develop hyperplastic ovaries, with hyper proliferationof the stromal cells surrounding the oocytes, while TALE-AIDwt males are indistinguishable fromcontrols. These results suggest that the pre-neoplastic transformation of the ovary have a possibleimplication in fertility. Deep sequencing on normal and transformed tissue will reveal AID’s mutationfootprint.As proof of principle, TALE-AID fusion protein will be tested for in vitro cellular transformation, bytargeting the p53 locus of cells lines, deep sequencing the p53 locus for C-T transitions, andscoring for transformed cell clones.Targeting a natural DNA mutator, such as AID, to a specific tumour suppressor gene during thelifespan of the animal, is an innovative approach to provide novel insights into oncogenicdevelopment.1088 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts

ZDM8 POSTERSZDM7Madison | USA 2015ZDM8Boston | USA 2015The BRIP1/FANCJ DNA helicase as novel 17q driver oncogene inneuroblastomaSuzanne Vanhauwaert *1 , Annelies Fieuw *1 , Carina Leonelli 1 , Els Janssens 1 , Sara DeBrouwer 1 , Nadine Van Roy 1 , Shuning He 2 , A. Thomas Look 2 , Katleen de Preter 1 , FrankSpeleman 11: Center for Medical Genetics, Ghent University, Ghent, Belgium2: Department of Pediatric Oncology, Dana-Farber Cancer Institute and Harvard Medical School,Boston, Massachusetts, USA.Neuroblastoma (NB) is an aggressive neural crest derived childhood tumor with poor survivalrates, especially in older children. Thus far, three bona fide oncogenic driver genes havebeen identified in neuroblastoma: MYCN, amplified in roughly half of all aggressiveneuroblastoma cases, ALK mutations or amplifications in 10% of cases and rare LIN28Bamplifications. Chromosome 17q gain is by far the most common DNA copy numberalteration in NB but thus far the causal 17q drivers remain to be identified due to the largesize of the recurrently involved chromosome segments. In this study, we present the use ofan in silico integrative genomics approach to resolve this problem. First, we applied theCONEXIC algorithm (correlation analysis between copy number and expression in cancer) toan unique set of 211 primary NB tumor samples, consisting of combined mRNA/miRNAexpression and DNA copy number data to identify neuroblastoma candidate driver genes. Intotal, 144 unique candidate driver genes were identified and further prioritization usingENDEAVOUR identified BRIP1 (BRCA1 interacting protein, also known as FANCJ), locatedon 17q23.2, as top-ranked candidate driver gene in NB. Next, we tested the effects of stableshRNA BRIP1 knock down in NB cell lines and showed reduced cell viability and colonyforming capacity. Furthermore, in keeping with the role of BRIP1 in facilitating DNAreplication and preventing replication fork stalling, we observed increased DNA damagelevels as evidenced by increased γH2AX and RPA32 protein levels. Currently, DNA fiberanalysis is ongoing to visualize the effects of BRIP1 depletion on DNA replication velocity,replication fork stalling and restart. In addition, we are performing BRIP1 mosaic experimentsin a MYCN transgenic zebrafish NB model to monitor the effects of BRIP1 overexpression onacceleration of tumor development while also a DBH-BRIP1 stable overexpression zebrafishline is underway.8 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts109

ZDM8Boston | USA 2015ZDM8 POSTERSExploring the TLX1-PHF6 cooperative interrelationship in T-cellacute lymphoblastic leukemia through zebrafish modelingSiebe Loontiens * (1), Kaat Durinck * (1), Els Janssens (1), Suzanne Vanhauwaert (1),Pieter Rondou (1), Jan Cools (2), Charles De Bock (2), Tom Taghon (3), Inge Van deWalle (3), Finola Moore (4), David Langenau (4), Pieter van Vlierberghe (1), FrankSpeleman (1)(1) Center for Medical Genetics Ghent, Ghent University, Ghent, Belgium(2) Laboratory for the Molecular Biology of Leukemia, Center for Human Genetics, University ofLeuven and Center for the Biology of Disease, Vlaams Instituut voor Biotechnologie, Leuven,Belgium(3) Departement of Clinical Chemistry, Microbiology and Immunology, Ghent University, Ghent,Belgium(4) Harvard Stem Cell Institute, Boston, Massachusetts, United States of America* Shared first authorshipT-cell acute lymphoblastic leukemia (T-ALL) results from the malignant transformation ofthymocytes. In T-ALL, different oncogenic genetic lesions (e.g. TLX1/3, TAL1/2, LMO1/2,HOXA) are driving events while further additional mutations in oncogenes and tumorsuppressor genes collaboratively contribute to T-cell transformation. PHF6 is one of the mostcommonly affected tumor suppressor genes in T-ALL and predominantly occurring in TLX1driven cases. In order to explore this peculiar interrelationship, we first established the TLX1regulatory landscape. Unexpectedly, this revealed antagonistic interaction between TLX1and NOTCH1, explaining the observed thymic regression in TLX1+ T-ALL mouse modelsprior to leukemia formation and high frequency of NOTCH1 mutations in TLX1+ cases(Durinck et al., under review). Upon PHF6 knock down in T-ALL cells and immaturethymocytes we observed, amongst others, robust IL7R upregulation. Using CRISPR basedgenome editing, we generated PHF6 deficient MOHITO mouse T-ALL cells to assesssensitization for JAK inhibitors. To further test the role of PHF6 loss in counteracting TLX1mediated IL7R repression in vivo, we performed PHF6 gene inactivation by injection ofgRNAs and Cas9 protein into the one-cell stage zebrafish embryos yielding varying out-offramein/dels. In addition, we will use a TALEN based PHF6 knock out zebrafish line (Mooreet al., 2012). We will investigate the genetic interaction between TLX1 and PHF6 either bycrossing PHF6 deficient and Rag2-TLX1 overexpressing stable lines or injection of Rag2-TLX1 constructs into PHF6 deficient embryos. Finally, we also showed that upon PHF6knock down, marked perturbations occurred in maturation of human T- and B-cell andmyeloid lineages. Therefore, we are currently investigating altered expression of lineagespecific markers for primitive and definitive haematopoiesis in PHF6 deficient versus normalzebrafish embryos.1108 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts

ZDM8 POSTERSZDM7Madison | USA 2015ZDM8Boston | USA 2015Identifying novel combination therapies in BRAF V600E -drivenmelanoma in the zebrafish model.Michelle Dang and Leonard I. ZonStem Cell Program and Division of Hematology/Oncology, Children’s Hospital and Dana FarberCancer Institute, Howard Hughes Medical Institute, Harvard Stem Cell Institute, Harvard MedicalSchool, Boston, MA 02115, USA.Words: (282 out of 300)Targeted therapies in metastatic melanoma are ineffective long-term treatments due toacquired resistance. To address this challenge in melanoma therapy, we aim to testnovel anti-melanoma compounds in a clinical trial in the zebrafish. The oral gavagetechnique overcomes a major obstacle in studying drug efficacy in the adult zebrafish byoffering controlled, non-invasive and long-term drug delivery. As a proof of principle, weestablished drug efficacy of the FDA-approved BRAF V600E inhibitor, Vemurafenib, in theadult zebrafish. In the model, adult Casper zebrafish are irradiated, intraperitoneallytransplanted with a zebrafish melanoma cell line (ZMEL), and develop a large pigmentedtumor by 10 days post transplantation. Daily sub-lethal dosing at 100mg/kg ofVemurafenib, for 2 weeks via oral gavage resulted in a 90% decrease in tumor burdenand a 15% mortality rate. Extension of the study into 5 weeks of daily oral gavageresulted in a similar 90% decrease in tumor burden, but yielded a higher mortality rate of40%. In contrast, Vemurafenib-resistant ZMEL cell lines, generated in culture from lowdosedrug exposure for 4 months, did not respond to the oral gavage treatment regimenwhen transplanted into irradiated Casper zebrafish. Similarly, Caspers transplanted withprimary melanoma tumors resulted in an 80% decrease in tumor burden when dailygavaged for 2 weeks at 100mg/kg. Most importantly, this drug treatment regimen can beapplied to adult transgenic zebrafish harboring primary melanoma tumors and resulted inan 80% decrease in tumor burden. Taken together, we developed an effective drugtreatment system in both a transplantation model using adult Casper zebrafish and aprimary melanoma model using adult transgenic zebrafish. This technique will allowadult zebrafish to be used to identify more effective anti-melanoma combinationtherapies.8 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts111

ZDM8Boston | USA 2015ZDM8 POSTERSAn Orthotopic Model of Glioblastoma in Zebrafish Exhibits Key Hallmarksof Brain Tumors.1 Alessandra M. Welker, 1 Brian D. Jaros, 2 Jaime Imitola, 3 Balveen Kaur, and 1 Christine E.Beattie.The Ohio State University, Wexner Medical Center, Department of Neuroscience 1 , Neurology 2 , andNeurosurgery 3 . Columbus, OH 43210Glioblastoma (GBM) is a deadly brain cancer with few effective drug treatments available. There is aneed for better understanding of the highly aggressive biology of these tumors, including thepathways that regulate tumor characteristics such as migration and invasion of these cells in thebrain. Two of the many reasons supporting the development of novel animal models of GBM includethe inability to track individual tumor cells in-vivo during tumor development as well as a lack of arapid drug screening model. Therefore, we have generated a xenotransplant model of GBM inzebrafish by injecting human GBM neurospheres into the midbrain region of larvae. Analysis oflarvae over time showed progressive brain tumor growth through daily confocal imaging, behavioralabnormalities using touch-evoked swim assays, and premature death quantified by Kaplan-Meiercurves. Histological analysis including hematoxylin and eosin (H-E), Vimentin and Ki67 revealed thattumor cells were vimentin positive (~90%), proliferating (~40%) and that the tumors werehypercellular, composed of a variety of cell types including those with hyperchromatic nuclei,frequent mitoses, and necrosis. Two tumor phenotypes in larval zebrafish, diffuse (85%) andcompact (15%), were observed. In diffuse tumors, GBM9 cells migrated in the brain away from theinitial site of transplantation. In contrast, transplanted control mouse neural stem cells (mNSC) didnot migrate throughout the brain or engraft and form tumors. To determine whether GBM9 tumorswere responsive to currently used therapeutics, we performed a proof-of-principle study usingchemotherapeutic agents. Treating with either temozolomide or bortezomib caused a reduction intumor volume in-vivo and an increase in survival by 80% and 54% respectively. These data showthat we have successfully generated an orthotopic model of glioblastoma to study the biology ofglioma cells in-vivo as well as to perform drug screens.1128 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts

ZDM8 POSTERSZDM7Madison | USA 2015ZDM8Boston | USA 2015Using Zebrafish to Investigate the Synergistic Inhibition ofFocal Adhesion Kinase and Aurora Kinases on Ewing SarcomaProliferation and MetastasisNicole Melong 1 , Chansey Veinotte 1 , Brian Crompton 2 , Graham Dellaire 3 , KimberlyStegmaier 2,4 and Jason N. Berman 1,3 .1. Department of Pediatrics, IWK Health Centre, Halifax, Nova Scotia.2. Department of Pediatric Oncology, Dana-Farber Cancer Institute, Boston,Massachusetts.3. Department of Pathology, Dalhousie University, Halifax, Nova Scotia.4. Broad Institute of Massachusetts, Institute of Technology and Harvard University,Cambridge, Massachusetts.Ewing sarcoma (ES) is a tumor of bones and soft tissues. Outcomes have improved forlocalized disease with multi-agent chemotherapy but remain dismal for metastaticprogression. Focal adhesion kinase (FAK) increases cell adhesion, proliferation andsurvival, and is activated in ES (Crompton et al. 2013). We found that FAK inhibitorsimpair growth and colony formation in ES in vitro and in murine models. The aurorakinases (AK) are overexpressed in several cancers, suggesting a potential target formolecular therapies. In mouse xenografts, AK inhibitors hinder tumor growth andseveral AK inhibitors are currently in phase I trials. We optimized thexenotransplantation (XT) of human cancer cells into transparent zebrafish embryos asan innovative means for evaluating interactions between tumor cells, anti-cancer drugsand the microenvironment. We used this approach to evaluate established and noveltargeted therapeutics in leukemia (Bentley et al. 2015; Corkery et al. 2011; Smithen etal. 2012; Veinotte et al. 2014). ES A673 cells were microinjected into the yolk sac of 48hpf embryos and treated with FAK inhibitor alone, AK inhibitor alone, or in combination.Embryos were evaluated for proliferation by live-cell fluorescent microscopy andquantified in single cell suspensions ex vivo. Individually FAK inhibitor and AK inhibitortreatment showed no decrease in ES proliferation, but showed a significant folddecrease in combination. Cell migration was examined at 24 hr intervals in a predefinedlocation of the tail. The combination of FAK and AK inhibitors did not preventcell migration, suggesting that anti-tumor efficacy may be restricted to primary tumorexpansion with little impact on preventing metastases. Thus, the zebrafish XT modelhas the potential to serve as a rapid and cost-effective live animal screening platformthat can inform the prioritization of emerging combination therapies for furtherpreclinical testing and ultimate translation to Phase I studies.8 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts113

ZDM8Boston | USA 2015ZDM8 POSTERSGlypican4 modulates collective cell migration non-cell autonomouslyMarina Venero Galanternik 1,2 and Tatjana Piotrowski 1,21Department of Neurobiology and Anatomy, University of Utah, Salt Lake City, Utah 841322 Stowers Institute for Medical Research, Kansas City, Missouri 64110Proteoglycans are extracellular matrix glycoproteins known to be essential in thecontrol of several signaling pathways including Wnt/β-catenin, Fgf, BMP and Hh. Themodified sugar chains on proteoglycans are essential regulators of cell polarity, liganddistribution and signaling interactions between Wnt/β-catenin and Fgf signaling in theprimordium, a collectively migrating placode that gives rise to the zebrafish sensorylateral line. However, the specific individual function of primordium-expressedproteoglycans remains unknown. To address this problem, we investigated the functionof the Wnt/PCP regulator Glypican4 in the zebrafish primordium. We usedknypek fr6 /glypican4 null mutants to show that glypican4, a primordium expressedproteoglycan, does not regulate Wnt/β-catenin or Fgf signaling by itself but redundantlywith other proteoglycans in the primordium. Additionally, lateral line cell polarity inglypican4 fr6 mutants was lost but in a lesser degree compared to the defects observed inglobally sugar chain proteoglycan-depleted mutants. Yet, mutations in glypican4 lead tosevere non-cell autonomous primordium migration defects, due to the loss of cxcl12aexpressingmuscle precursors along the myoseptum. We confirmed this by transplantingwild type primordium clones into glypican4 fr6 mutants where these clones were not ableto rescue lateral line migration, but restoration of cxcl12a expression along themyoseptum in glypican4 fr6 mutants resulted in complete primordium migration,demonstrating that glypican4 non-cell autonomously affects lateral line development. 1148 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts

ZDM8 POSTERSZDM7Madison | USA 2015ZDM8Boston | USA 2015Investigating the Role of polκ in Melanoma Heterogeneity and DrugResistance Kelsey Temprine1, Erin Langdon2, and Richard M White11Department of Cancer Biology and Genetics, Memorial SloanvKettering Cancer Center, New York, NY100652Dartmouth College, Hanover, NH 03755Objectives: MAPK activation is central to melanoma, which is the 5 th most common cancer.However, despite the establishment of targeted inhibitors with profound activity in patients, theirultimate effectiveness is limited as resistance quickly develops, presenting a significant hurdle inlong-term patient survival. Drug resistance results from pre-existing or newly created mutations.In bacteria, the acquisition of new mutations under selection is referred to as stress-inducedmutagenesis (SIM), which is mediated by the error-prone DNA polymerase DinB. Maladaptedcells temporarily increase their mutation rates, and stochastically, a few mutations arebeneficial. We hypothesize that a similar mechanism exists in cancer via upregulation of DinB’svertebrate homologue DNA polymerase kappa (polκ) during the development of drug resistanceand are using human melanoma cells and transgenic zebrafish to study it.Methods: To demonstrate a link between polκ and drug resistance, we treated a melanoma cellline with a BRAF inhibitor until resistance developed. BRAF inhibition sensitivity was determinedusing proliferation assays, and expression levels were measured using Q-RT-PCR and WesternBlot. Finally, we generated transgenic zebrafish that overexpress polκ, which are beingobserved for effects on tumorigenesis. Results: Treatment of A375 cells with a BRAF inhibitor for 24 hours or 1 month (at which pointcells are still sensitive) led to a significant increase in the expression of polκ and moderateincreases in other Y-family polymerases. However, by 4 months of treatment when cellsbecome resistant, expression returned to basal levels. Conclusions: We demonstrated that polκ is upregulated in response to BRAF inhibition.Upregulation of error-prone DNA polymerases in melanoma would provide a mechanism linkingcellular stress to the acquisition of new mutations. We plan to define the mechanisms by whichBRAF inhibition causes polκ upregulation and determine the functional consequences of polκupregulation in terms of mutational spectrum and drug resistance. 8 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts115

ZDM8Boston | USA 2015ZDM8 POSTERSSATB2 accelerates melanoma progression and invasion byaltering cell fate and activating actin remodelingvan Rooijen, Ellen 1 , Thomas, Andrew 1 , van de Hoek, Glenn 1 , Laga Canales, Alvaro 2 ,Michelle Dang 1 , Elliott Hagendorn 1 , Song Yang 1 , Kaufman, Charles 1 , White, Richard 3 ,Trompouki, Eirini 4 , Zon, Leonard 11. Stem Cell Program, Division of Hematology/Oncology, Boston Children's Hospital and DanaFarber Cancer Institute, HHMI, Harvard Stem Cell Institute, Harvard Medical School, Boston, USA2. Department of Pathology, Brigham & Women’s Hospital, Boston, USA3. Cancer Biology & Genetics, Memorial Sloan Kettering Cancer Center, New York, USA4. Max Planck Institute of Immunobiology and Epigenetics, Freiburg, GermanyGenetic alterations and epigenetic changes in neural crest-derived melanocytes promotethe development of malignant melanoma, the most aggressive and deadliest form of skincancer. To interrogate the role of chromatin factors that stimulate melanoma in vivo, weperformed an overexpression screen in Tg(mitfa:BRAF V600E ); p53 -/- ; mitfa -/- zebrafish. Weidentified Special AT-rich Binding Protein 2 (SATB2) as a novel driver of melanoma.SATB2 is a chromatin organizer that controls gene expression by orchestrating higherorder chromatin structure, and regulating transcription. SATB2 plays a key role in callosalprojection neuron identity, and osteogenic differentiation. Loss of SATB2 induces defectsin neural crest lineages in fish and humans alike, while overexpression has been linked tocolorectal cancer, and head and neck squamous cell carcinoma. We examined SATB2protein levels in human melanoma, and found that compared to benign nevi, SATB2 washighly expressed in 61% (n=53) of tumors, which correlated to disease progression.Expression profiling by RNA-seq in combination with ChIP-seq to identify SATB2-boundgenes in zebrafish tumors, revealed that neural development/axon guidance, andRhoA/actin cytoskeletal programs are stimulated by SATB2 overexpression. SATB2 colocalizesto H3K27Ac-bound super enhancers that are enriched for EMT, cell migrationand cranial neural crest-associated loci. Interestingly, SATB2 melanomas display apredominant neuroid/spindle-like histopathology, compared to nevoid-type EGFP tumors.Additionally, we observed abundant cortactin/F-actin+ invadopodia in SATB2 primary celllines, which we have shown to locally degrade extracellular matrix. Invadopodia areassociated to cancer cell invasion and metastasis. Indeed, SATB2 melanomas are highlyaggressive and invasive, and rare metastases have been observed. We subcutaneouslytransplanted SATB2 and EGFP primary tumor cells into irradiated Caspers, and uniquelyobserved distant metastasis in 9/12 SATB2-recipients, 3.5 weeks post-transplant. Ourdata demonstrate that SATB2 misexpression induces a fate switch that contributes tomelanoma formation and invasion, which could lead the identification of therapeutictargets.1168 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts

ZDM8 POSTERSZDM7Madison | USA 2015ZDM8Boston | USA 2015Uncovering the function of Polycomb proteins in malignantmelanomaSara DiNapoli, Yariv Houvras1. Pharmacology Program, Weill Cornell Graduate School of Medical Sciences, New York, NY100652. Departments of Surgery and Medicine, Weill Cornell Medical College, New York, NY 10065Alterations in chromatin-modifying enzymes continue to be identified in multiple humancancers, suggesting that changes in chromatin structure make key contributions to tumorinitiation and progression. Methylation of histone H3 lysine 27 (H3K27) is catalyzed byPolycomb Repressive Complex 2 (PRC2), which is deregulated in numerous humancancers. In melanoma, the function of PRC2 remains incompletely understood due tothe identification of both gain and loss of function genetic alterations. In order to examinethe role of PRC2 in melanoma, we used a zebrafish melanoma model, in whichmelanocytes that express oncogenic BRAF-V600E and concurrently harbor a p53 loss offunction mutation develop melanoma. Surprisingly, overexpression of the histone H3.3-K27M mutation that inhibits PRC2 activity accelerates melanoma onset butoverexpression of wild-type EZH2 or tumor derived mutants EZH2-Y641F/N do not. Weperformed RNA-seq analysis of H3.3-K27M melanomas and identified an aberrantupregulation of Hox genes and genes specific to the neural lineage, suggesting analteration in the differentiation state of H3.3-K27M tumors. To test this, we are examininghow loss of PRC2 activity alters the differentiation of the neural crest duringembryogenesis. To uncover changes in chromatin structure in H3.3-K27M melanomas,we performed histone mass spectrometry analysis, which revealed local and genomewidechanges in specific histone modifications. These data suggest that loss of PRC2activity leads to global alterations in chromatin structure and perturbed differentiationthat promotes melanoma initiation. These studies will provide critical insight into the roleof PRC2 function in melanoma.!8 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts117

ZDM8Boston | USA 2015ZDM8 POSTERSHistone deacetylase inhibitors antagonize distinct pathways to suppresstumorigenesis of embryonal rhabdomyosarcomaTerra Vleeshouwer-Neumann, Michael Phelps, Theo K. Bammler, James W.MacDonald, Isaac Jenkins, and Eleanor Y Chen1. University of Washinton2. University of Washinton3. University of Washington4. University of Washington5. Fred Hutchinson Cancer Research Center6. University of WashintonEmbryonal rhabdomyosarcoma (ERMS) is the major subtype of Rhabdomyosarcoma(RMS), the most common soft tissue cancer in children, and is pathologicallycharacterized by myogenic differentiation arrest. The prognosis of patients with relapsedor metastatic disease remains poor due to the lack of effective treatment options. Froma previous large-scale chemical screen, histone deacetylase (HDAC) inhibitors havebeen identified as one class of lead compounds with the capacity to induce myogenicdifferentiation of ERMS cells. To gain insights into the role of HDACs in thepathogenesis of ERMS, we have characterized the anti-tumor effects of pan-HDACinhibitors, trichostatin A (TSA) and suberoylanilide hydroxamic acid (SAHA; also knownas vorinostat) in vitro using human cell lines and in vivo using a zebrafish model ofERMS. TSA and SAHA suppress ERMS tumor growth and progression by inducingmyogenic differentiation, reducing self-renewal as well as migratory capacity of tumorcells. Differential expression profiling studies demonstrate down-regulation of keyoncogenic pathways in HDAC inhibitor-treated cells. By integrating the results from gainof-function,loss-of-function and chromatin immunoprecipitation (ChIP) studies, we showthat Notch1- and EphrinB1-mediated pathways are epigenetically regulated by HDACsto inhibit differentiation and enhance migratory capacity of ERMS cells, respectively. Ourdissection of molecular networks modulated by HDAC inhibitors to bring about theirdistinct anti-tumor effects in ERMS promises translational impact particularly in the areasof novel therapeutics and prognostic biomarkers.1188 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts

ZDM8 POSTERSZDM7Madison | USA 2015ZDM8Boston | USA 2015A novel model of thyroid cancer in zebrafish reveals earlymorphologic and developmental abnormalities induced byoncogenic BRAF V600EViviana Anelli 1 , Jacques Villefranc 1 , Sagar Chhangawal 1 , Raul Martinez 1 , TheresaScognamiglio 2 and Yariv Houvras 11 Departments of Surgery and Medicine, Weill Cornell Medical College and New YorkPresbyterian, USA2 Departments of Pathology and Laboratory Medicine, New York Presbyterian Hospital/WeillCornell Medical Center, New York, NY, USAPapillary thyroid carcinoma (PTC) is the most common type of thyroid cancer, withBRAF V600E being the most frequent driver mutation. Here we describe the generation of anovel zebrafish model of thyroid carcinoma. We created a transgenic line expressinghuman BRAF V600E specifically in thyroid cells using a zebrafish thyroglobulin promoter.We utilize expression of fluorescent proteins (TdTomato or EGFP) in thyroid cells inorder to visualize cellular changes during the earliest stages of thyroid cancer initiationand progression, including migration, proliferation and invasion.During embryonic development, transgenic animals expressing oncogenic BRAF V600Eexhibited alteration in thyroid morphogenesis, with reduced or absent thyroid follicles,evident as early as 5 dpf. Treatment of transgenic embryos expressing BRAF V600E with acombination of BRAF and MEK inhibitors reversed the morphologic effects induced byoncogenic BRAF. To investigate the signaling pathways directly altered by BRAF V600Eexpression in thyrocytes, we sorted fluorescent cells from control and BRAF V600E -expressing thyrocytes at 5dpf and performed RNA-seq followed by gene set enrichmentanalysis (GSEA). This analysis revealed a BRAF V600E -induced upregulation of genesassociated with the epithelial to mesenchymal transition (EMT) signature. Among genesinvolved in EMT, we found that twist3, the zebrafish orthologue of human Twist2, a keyregulator of EMT, is upregulated in transformed thyrocytes. Interestingly, a BRAF V600E -induced transcriptional signature identified in zebrafish thyrocytes positively correlateswith Twist2 expression across a reference set of human PTC cancers, indicating aconserved EMT signature associated with Twist2 levels across human and zebrafishthyroid cancers. Adult transgenic zebrafish expressing BRAF V600E developed an invasivethyroid carcinoma with PCNA-positive cells, indicating the presence of activelyproliferating thyrocytes inside the tumor mass. Collectively, a new model of BRAF V600Einduced thyroid carcinoma in zebrafish offers novel avenues for studying tumor initiation,EMT, and identifying novel drug targets in human thyroid cancer.8 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts119

ZDM8Boston | USA 2015ZDM8 POSTERSInvestigation of MYC Collaborating Oncogenes in T-cell Acute LymphoblasticLeukemia Progression and RelapseElaine G. Garcia, Jessica S. Blackburn, Riadh Lobbardi, Aleksey Molodtsov, David M. Langenau1. Molecular Pathology, Cancer Centre, and Regenerative Medicine, Massachusetts General Hospital, Boston, MA 021292. Harvard Stem Cell Institute, Cambridge, MA 02139T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignancy of thymocytes, with less than30% of children and 8% of adults able to survive relapsed disease. In T-ALL, aberrant MYC activity is adominant oncogenic lesion. The MYC pathway can be activated or overexpressed by NOTCH1 activation ortrisomy 8 (+8) respectively. However to evolve T-ALL and accelerate its progression, additional mutations arelikely required. To identify MYC collaborating genes, we generated MYC-overexpressing T-ALL in zebrafishthat recapitulates the most common and treatment resistant subtype of human T-ALL. Through an unbiasedtransplantation screen, we have been able to assess functional differences between individual cells and usearray comparative genomic hybridization to identify focally amplified genes associated with high self-renewalcapacity, short latency, and/or increased aggression. Through this analysis, we identified 16 commonlyamplified genes, 10 of which are required for continued tumor growth in human T-ALL cell lines. Interestingly, 3of those 16 genes are co-amplified with MYC on +8 in human T-ALL – including phosphatase of regeneratingliver 3 (PRL3), TatD DNase Domain Containing Protein 1 (TATDN1), and cleavage and polyadenylationspecificity factor 1 (CPSF1). Moreover, PRL3, TATDN1, and CPSF1 were each independently amplified inzebrafish T-ALL, suggesting that they are not merely passenger genes in the context of human +8 T-ALL. Todate, I have confirmed that each gene collaborates with MYC to accelerate time to tumor onset in the zebrafishmodel and assessed their role in human T-ALL maintenance in vitro and in mouse xenograft studies. Ihypothesize that each gene has distinct roles in progression and relapse in T-ALL. My early studies suggestthat TATDN1 enhances LPC frequency and PRL3 suppresses apoptosis. The long-term goal of this work is toidentify novel molecular pathways that can be exploited for therapeutic innovation.1208 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts

ZDM8 POSTERSZDM7Madison | USA 2015ZDM8Boston | USA 2015Establishment of a zebrafish model for the study of metastaticprocess of human cancer cellsWen-Wei Chang 1,2 , Ju Chang-Chien 1,3 , Jiann-Jou Yang 1, 21. School of Biomedical Sciences, Chung Shan Medical University, No.110, Sec.1, Jianguo N.Rd., Taichung 40201, Taiwan.2. Department of Medical Research, Chung Shan Medical University Hospital, No.110, Sec.1,Jianguo N. Rd., Taichung 40201, Taiwan.3. Institute of Microbiology & Immunology, Chung Shan Medical University, No.110, Sec.1,Jianguo N. Rd., Taichung 40201, Taiwan.Cancer metastasis is the main reason for the high mortality in late stage of cancerpatients and there is no specific drug for treatment. Zebrafish could serve as an invivo model for study the metastatic process of human cancer cells since there is noadaptive immune system being developed until 14 days post-fertilization. Here we tryto use red fluorescence protein (RFP) expressing HeLa cells, a human cervicalcancer cell line, to establish a zebrafish model for the study of cancer metastasis. 150RFP-HeLa cells were suspended in 2.3 nl DMEM medium and injected into yolk sac of72 h zebrafish embryo followed by observation under inverted fluorescencemicroscopy to record the RFP signals at 6, 12, and 24 h post-injection. We found thatRFP-HeLa cells retained in yolk sac at 6 h, migrated to supraintestinal artery at 12 hand invaded into heart at 24 h. Unfortunately, all the injected zebrafish embryo diedafter 24 h. Our results show that zebrafish could be a model for study of metastaticprocess of human cancer cells but the further improvement for prolong the survivalperiod of injected embryo is required. This model could be further applied in fli1:EGFPzebrafish line and recording through time-lapse microscopy for study thedissemination process of metastatic HeLa cells.8 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts121

ZDM8Boston | USA 2015ZDM8 POSTERSMelanoma regression and recurrence in zebrafishSonia Wojciechowska 1 , Amy Capper 1 , Zhiqiang Zeng 1 , James Lister 2 and E.Elizabeth Patton 11MRC Institute of Genetics and Molecular Medicine, MRC Human Genetics Unit & EdinburghCancer Research Centre, University of Edinburgh, Western General Hospital, Crewe RoadSouth, EH4 2XR, UK2Department of Human and Molecular Genetics and Massey Cancer Center, VirginiaCommonwealth University, School of Medicine, Sanger Hall 11-014 1101 E. Marshall StreetRichmond, VA 23298-0033, USAEvidence from human melanoma indicates that the melanocyte lineage is critical formelanoma survival and contributes to therapeutic resistance. MITF (microphthalmiaassociatedtranscription factor) is a highly conserved “master melanocyte regulator”with a complex role in melanoma. Our lab has developed two temperature sensitivezebrafish melanoma models (BRAF V600E mitf and mitf vc7 p53), both carrying the MITFsplice site mutation that enables us to conditionally control its endogenous activity byadjusting the water temperature. We show that the MITF activity is crucial formelanocyte survival and that both mutated BRAF and p53 deficiency are oncogenicwith low levels of MITF, and result in fish nevi and melanoma which resembles thepathology of human disease. Complete inhibition of MITF activity leads to rapidtumor regression, but once its activity is restored the melanomas recur at the samesite as the original tumor. This suggests that a subpopulation of cancer initiating cellsremains following melanoma regression and is capable of repopulating the tumor.We have been able to show that some cells remaining at the sites of regressionexpress BRAF V600E and are currently searching for other markers. Our goal is toidentify the molecular signatures of these proposed melanoma stem cells and todevelop approaches to visualize and target these subpopulations. We are usinghistopathology studies, molecular and lineage tracing imaging methods andmelanocyte lineage transgenes in our genetic zebrafish models, as well as zebrafishmelanoma cell culture to study the nature of the cell of origin for the tumorrecurrence.1228 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts

ZDM8 POSTERSZDM7Madison | USA 2015ZDM8Boston | USA 2015A Novel Function for the PRL3 Phosphatase in the MelanocyteLineageNatasha Tracey, Judith Zich, Kerrie L. Taylor-Marie, Zhiqiang Zeng, WitoldRybski, Karthika Paranthaman, Hironori Ishizaki, Ian J. Jackson, and E.Elizabeth PattonMRC Human Genetics Unit, MRC IGMM, University of Edinburgh, Western General Hospital,Crewe Road, Edinburgh EH4 2XUThe melanocyte lineage is critical for melanoma development and survival. Toidentify new regulators of the melanocyte lineage, we performed a small moleculescreen in zebrafish embryos for regulators of melanocyte regeneration from themelanocyte stem cell (MSC). First, we targeted differentiated melanocytes withNFN1, a melanocytotoxic compound that we discovered (Zhou et al., 2012), and thenscreened for compounds that suppressed or enhanced melanocyte regeneration. Wediscovered that an inhibitor of the PRL3 phosphatase enhanced melanocyteregeneration from undifferentiated melanocytes following NFN1 melanocytotoxicity orfrom an mitfa genetic block. PRL3 (Phosphatase of Regenerating Liver 3) is aprenylated protein phosphatase that is highly expressed in metastatic cancers,transcriptionally regulated by p53, and is a validated anti-cancer target, but thefunction of PRL3 is unknown. We find that PRL3 inhibition can rescue loss of ErbBdependentmelanocytes in the embryo, suggesting that PRL3 substrates are presentdownstream of the ErbB receptor. To confirm these observations in vivo we mutatedprl3.1 and prl3.2 genes using TALE nucleases and CRISPR/Cas9, and find thatprl3.1 -/- mutant embryos show enhancement of melanocyte regeneration (albeit notas complete as PRL3 chemical inhibition). Critically, we find that overexpression ofprl3.1, but not a phosphatase dead mutant prl3.1 C104S or prl3.2, prevents melanocytedevelopment indicating PRL3 phosphatase activity can act as a negative regulator ofmelanocyte development. We are currently in the process of identifying the cellularand molecular substrates of PRL3 to understand its role in melanocyte regenerationfrom the MSC.8 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts123

ZDM8Boston | USA 2015ZDM8 POSTERSRole of oncogenic glutamate receptor signaling in melanocyteand melanoma developmentAna Neto and Craig CeolProgram in Molecular Medicine and Department of Molecular, Cellular and Cancer Biology,University of Massachusetts Medical School, Worcester, Massachusetts, USAGlutamate signaling has been shown to have a role in melanoma progression, in partthrough activating mutations in the metabotropic glutamate receptor 3 (GRM3) gene. Wehypothesize that altered glutamate signaling affects the development and function ofmelanocytes, endowing these cells with properties important for melanoma progression.By expressing mutant GRM3 variants in developing melanocytes, we determined howoncogenic GRM3 variants affected these cells. In embryonic melanocytes oncogenicGRM3 mutants disrupted trafficking of melanosomes, causing aggregation of thesepigment-producing organelles in the cell body. Wild-type GRM3 had no effect onmelanosome distribution. Melanosomes are trafficked in a cAMP-dependent manner,and drugs that directly or indirectly increase the cAMP levels were able to rescue themelanosome phenotype of oncogenic GRM3-expressing melanocytes. Our data indicatethat oncogenic GRM3 variants dysregulate cyclic AMP (cAMP) signaling, a heretoforeunknown role for these oncogenes. Recent data have implicated defective cAMPsignaling in the melanoma susceptibility of red-haired, fair-skinned individuals. DefectivecAMP signaling leads to an increase in the pheomelanin:eumelanin ratio, which resultsin overproduction of reactive oxygen species and poor UV absorbance ability. We arecurrently testing if oncogenic GRM3 variants are able to affect pheomelanin:eumelanincontent and, therefore, contribute to melanoma development.1248 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts

ZDM8 POSTERSZDM7Madison | USA 2015ZDM8Boston | USA 2015kit inhibits BRAFV600E-driven melanoma formation by facilitatingcompetition with wild-type RAFJames Neiswender 1 , Caitlin Bourque 2 , Leonard I. Zon 2 , Craig Ceol 11. UMass Medical School, Program in Molecular Medicine, Department of Molecular, Cell, and CancerBiology, Worcester, MA, USA2. Children’s Hospital Boston, Harvard Medical School, Boston, MA, USAThe receptor tyrosine kinase KIT promotes survival and migration of melanocytes duringdevelopment. Upon ligand binding, KIT stimulates the MAPK pathway by activating RAS,leading to the formation of active BRAF:CRAF dimers. Activating mutations in KIT drive theformation of a subset of melanomas arising on non-sun-exposed skin. However, the majority ofmelanomas arising on sun-exposed skin and do not express KIT. Our analyses of humangenomic data indicated that the loss of KIT expression co-occurred with the common melanomaoncogene BRAFV600E. To determine whether the loss of KIT facilitates BRAFV600E-drivenmelanoma formation, we introduced a kit(lf) mutation into melanoma-proneTg(mitfa:BRAFV600E); p53(lf) zebrafish. Melanoma onset was substantially accelerated inkit(lf); Tg(mitfa:BRAFV600E); p53(lf) fish, and tumors in these animals had higher MAPKactivation compared to Tg(mitfa:BRAFV600E); p53(lf) tumors. Furthermore, KIT knockdownalso increased MAPK activation in a BRAFV600E-mutant human melanoma cell line. Wehypothesized that KIT normally competes with BRAFV600E signaling by engaging wild-typeRAF proteins in downstream signaling. Because wild-type RAF proteins have 500-fold lesskinase activity than BRAFV600E, engaging wild-type RAF proteins could compete withBRAFV600E and diminish overall signaling activity. To test this hypothesis, we overexpressedwild-type BRAF during melanoma formation and found that it inhibited melanoma onset in a kitdependentmanner. We conclude that the loss of KIT observed in BRAFV600E mutantmelanoma contributes to disease progression by allowing BRAFV600E to stimulate MAPKactivity without interference from wild-type RAF.8 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts125

ZDM8Boston | USA 2015ZDM8 POSTERSUse of oral gavage to treat adult zebrafish with small molecule drugs.Raul Martinez-McFaline 1 , Jacques Villefranc 1 , Viviana Anelli 1 , Yariv Houvras 11. Departments of Surgery and Medicine, Weill Cornell Medical College, New York, NY, 10065, USAIn the last several years Zebrafish have proven to be a suitable animal model for cancer studies.Zebrafish embryos are amenable to small molecule screens, which can lead to novel drugdiscoveries and a better mechanistic understanding of drug effects on various cancer types.However, in adult Zebrafish drug delivery is limited to chemical treatment in water baths. Theamount of drug that is delivered can be indeterminate due to the individual differences involuntary consumption. As such, validated drug delivery methods in rodent models, such as oralgavage and intraperitoneal injection, are being explored in zebrafish. Oral gavage has proven tobe a reliable method for accurately delivering solutions to the adult zebrafish. However, itremains unclear as to whether a small-molecule compound can reach a target tissue in areliable and dependent manner. To address this, we treated adult transgenic zebrafishexpressing tdTomato under the control of a thyroid specific promoter Tg(tg:TdTomato-pA) withmethimazole (MTZ) for 8 days (q48hr). MTZ is an anti-thyroid drug that inhibits thyroidperoxidase and with prolonged exposure acts as a goitrogen. We hypothesize that exposure toMTZ would lead to thyroid specific effects in adult zebrafish given proper tissue targeting ofMTZ. Epifluorescent imaging revealed that upon exposure to MTZ via oral gavage there was anincrease in thyroid size. Using qPCR, thyroid markers, tg, tshr, tpo, slc5a5, maximally increasedafter 4 days of administration. These results suggest that MTZ reaches the target tissue,supporting oral gavage as an effective drug delivery method in adult zebrafish. In future studieswe hope to study the use of other small molecule compounds on adult zebrafish. With the rapidorganismal development and tumor onset in the fish it is important to be able to study the effectsof small molecules in the adult setting.1268 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts

ZDM8 POSTERSZDM7Madison | USA 2015ZDM8Boston | USA 2015A Genome-Editing Approach for the Stepwise Establishment ofZebrafish Models of Pediatric High-Grade Gliomas and MPNSTsFelix Oppel 1 , Shuning He 1 , Ting Tao 1 , Mark W. Zimmerman 1 , Adam D. Durbin 1 , NinaWeichert 1 , Dong H. Ki 1 , and A. Thomas Look 11Department of Pediatric Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA02115, USA.Resequencing of the genomes of primary human cancers has recently allowed the identificationof genomic alterations underlying pediatric high-grade glioma (HGG), a highly malignant braintumor in children. Recurrent mutations were most prominently found in the genes NF1, TP53,ATRX and H3F3A. Whereas NF1, TP53 and ATRX are known tumor suppressor genes, theimpact of precise H3F3A missense mutations on tumor biology is not well understood. In ourproject we used the genome-editing technology CRISPR-Cas9 to establish zebrafish models ofpediatric HGG, in order to examine the role of these mutations in tumor onset and progression.Our lab previously created zebrafish deficient for tp53 (tp53 -/- ) and for 3 of 4 alleles of nf1(nf1a +/- , nf1b -/- ). These fish develop HGGs with low penetrance and malignant peripheral nervesheath tumors (MPNSTs) with high penetrance. Using CRISPR-Cas9, we introduced earlyframeshift mutations of atrx into the background of tp53 -/- , nf1a +/- , nf1b -/- . All analyzed fish injectedwith Cas9-mRNA and CRISPR guide-RNAs (gRNAs) targeting atrx coding sequences displayedgermline transmission of mutant alleles with one third having frameshift mutations. PrimaryCRISPR-Cas9 atrx-gRNA injected tp53 -/- , nf1a +/- , nf1b -/- embryos developed as mosaics andexhibited higher tumor penetrance and accelerated onset of MPNSTs. ATRX mutations werefound in 7 of 7 early arising MPNSTs. This indicates cooperation of loss-of-function mutations inatrx with a deficiency in tp53 and nf1. Gliomas were not detected in mosaic atrx mutant fish, andthese will be reanalyzed in stable atrx deficient zebrafish lines. As a next step we will programmissense mutations of h3f3a into the zebrafish germline in a conditional fashion. Our zebrafishmodels of pediatric HGG and MPNST will help to discover how driver mutations cooperatemechanistically and serve as faithful models of childhood tumors for small molecule screens toreveal specific inhibitors of proliferation and survival in these malignancies.8 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts127

ZDM8Boston | USA 2015ZDM8 POSTERSUFD1: a novel contributor to MYC-driven T-cell acute lymphoblasticleukemogenesisLeah Huiting, Yasmina Samaha, Nicole M. Anderson, Fabrice J.F. Laroche, Boey Li, andHui FengDepartments of Pharmacology and Medicine, Cancer Research Center, Section of Hematology andOncology, Boston University School of Medicine, Boston, MA, USA.Eukaryotic cells activate the unfolded protein response (UPR) to promote cell survival or deathwhen facing endoplasmic reticulum (ER) stress. Tumor cells endure ER stress through the prosurvivalUPR, which restricts protein synthesis, limits de novo entry of proteins into the ER,and/or promotes protein folding and degradation through upregulation of chaperoneproteins. The ubiquitin fusion degradation protein-1 (UFD1) is one of such chaperone proteinsand interacts with p97 to retrotranslocate misfolded proteins from the ER to cytosol forproteasome-mediated degradation. However, its role in tumorigenesis remains unknown. T-cellacute lymphoblastic leukemia (T-ALL) and lymphoma (T-LBL) are malignancies of developingthymocytes and occur in both children and adults. To determine the role of UFD1 in T-ALLpathogenesis, we analyzed clinical patient samples and a zebrafish model of Myc-induced T-ALL, and found that UFD1 is upregulated in both zebrafish and clinical human T-ALL cells,compared to thymocytes and human T-LBL cells respectively. Heterozygous loss of ufd1significantly delays the progression of Myc-induced T-LBL to T-ALL, without affecting fishdevelopment. Interestingly, the heterozygous loss of ufd1 caused G 1 arrest in tumor cellsoverexpressing Myc, but not in thymocytes without Myc upregulation. Skp2 protein levels aredecreased in these G1-arrested Myc;ufd1+/- cells, compared to Myc;ufd1+/+ cells. Thedownregulation of Skp2 activity may stabilize its substrate p27, compromise MYC transcriptionalactivity, and cause cell cycle arrest. Consistent with our zebrafish data, shRNA knockdown ofUFD1 significantly decreased viability of human JURKAT T-ALL cells. Treatment of a panel ofhuman T-ALL cell lines with a small molecule inhibitor of p97, a critical functional partner ofUFD1, was lethal to these cells at micromolar concentrations. Collectively, our findings indicatea novel contributing role of UFD1 and UPR in MYC-driven leukemogenesis, and suggest thatUFD1 inhibition represents a promising approach for antileukemic treatment.1288 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts

ZDM8 POSTERSZDM7Madison | USA 2015ZDM8Boston | USA 2015Understanding the Role of DLST in MYCN-driven NeuroblastomaNicole Anderson 1* , Yun Zhou 1,2* , Ping Ren 3 , Ruijuan Xiu 3 , Ting Tao 4 , Xueli Pan 1 , NinaWiechert 4 , Shizhen Zhu 5 , A. Thomas Look 4 , Guoliang Qing 3 , Hui Fu 2 , Hui Feng 11 Departments of Pharmacology and Medicine, Cancer Research Center, Section of Hematologyand Medical Oncology, Boston University School of Medicine, Boston, MA, USA; 2 Departmentof Anatomy, Wuhan University School of Medicine,Wuhan, Hubei, China; 3 Department ofPharmacology, School of Basic Medicine, Huazhong University of Science & Technology,Wuhan, China; 4 Department of Pediatric Oncology, Dana-Farber Cancer Institute, Boston, MA,USA; 5 Department of Biochemistry/Molecular Biology, Mayo Clinic, Rochester, MN, USA;*equal contribution.Neuroblastoma is the most common extracranial tumor in children and is responsible for ~15% ofchildhood cancer-related deaths. Aberrant MYCN activity is found in ~30% of neuroblastomapatients and is associated with highly aggressive tumors and poor prognosis, underscoring theneed for effective therapeutics. Our previous studies implicated DLST as a critical regulator of c-Myc-driven leukemogenesis, where a 50% reduction of Dlst was sufficient to delay tumor onsetin our c-Myc-driven zebrafish model of T-cell acute lymphoblastic leukemia (T-ALL). DLST isthe E2 component of the α-ketoglutarate dehydrogenase complex (KGDHC), a key enzyme in theTCA cycle that converts α-ketoglutarate to succinyl-CoA. The TCA cycle plays a pivotal role incancer cell metabolism, where it fulfills the bioenergetic, biosynthetic, and redox balancerequirements of cells. Small molecule inhibition of DLST effectively reduces the viability ofhuman neuroblastoma cells in vitro, while minimally affecting normal cells. We have shown thathuman neuroblastoma cells with MYCN amplification exhibit increased sensitivity to DLSTinactivation by RNAi, compared to neuroblastoma cells without MYCN amplification.Interestingly, DLST protein levels are significantly elevated in human high-grade neuroblastomasamples, compared to low-grade tumors. To determine the effects of DLST inactivation in vivo,we are utilizing a zebrafish model of neuroblastoma, which was recently developed in Dr. AThomas Look’s laboratory and overexpresses the human MYCN gene under the control of thedopamine β-hydroxylase (dβh) promoter. The MYCN fish develop EGFP-expressingneuroblastoma as early as 5 weeks of age and by 13 weeks ~50% of the MYCN transgenic fishdevelop neuroblastoma. Our preliminary data suggest that the DLST is required for the growthand/or survival of neuroblastoma cells with aberrant expression levels of MYCN.8 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts129

ZDM8Boston | USA 2015ZDM8 POSTERSZebrafish Modeling Of PAX3-FOXO1 Driven Rhabdomyosarcoma Genevieve C. Kendall 1,2 , Koichi Kawakami 4 and James F. Amatruda 1-31. Department of Pediatrics, University of Texas Southwestern Medical Center, Dallas, TX USA. 2. Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, TX USA. 3. Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, TX USA.4. Division of Molecular and Developmental Biology, National Institute of Genetics, Mishima, Japan.Rhabdomyosarcoma is the most common soft tissue sarcoma in children, and is associated with amisregulation of skeletal muscle developmental pathways. Of the two histological subtypes, embryonal(ERMS) and alveolar (ARMS), ARMS is more aggressive and prone to metastases. The majority of ARMScases have a defining t(1;13) or t(2;13) chromosomal translocation, which fuses either PAX7 or PAX3 withFOXO1, creating a transcriptionally active chimeric protein. Clinically, the presence of PAX3-FOXO1 predictsreduced overall survival, yet the underlying mechanisms for this are not clear. Remaining questions includeunderstanding how PAX3-FOXO1 initiates and drives ARMS tumorigenesis in the context of a vertebratedevelopmental system, including susceptible cell types and targeted signaling pathways. To determine thedevelopmental consequences of PAX3-FOXO1 tissue or gene-specific expression, we performed a mediumthroughputhigh-content screen using the UAS/GAL4 and Tol2 system. We expressed UAS:PAX3-FOXO1 in26 GAL4 transgenic zebrafish lines, and found that PAX3-FOXO1 inhibits somitogenesis or induces cyclopiadepending on the targeted cell type(s). Of these 26 GAL4 lines, we selected 10 to develop ARMS tumormodels. The PAX3-FOXO1 induced cyclopia phenotype identified in the screen is recapitulated with mosaicembryonic expression of BetaActin:PAX3-FOXO1, but not BetaActin:PAX3. Developmental tolerance ofheterologous PAX3-FOXO1 and PAX3 also differs; PAX3-FOXO1 expression is significantly reduced by 14dpf,whereas PAX3 expression remains stable. To define the molecular pathways driving these distinct phenotypes,and identify PAX3-FOXO1 tolerant cell lineages, we FACS sorted cells expressing GFP tagged PAX3 orPAX3-FOXO1 from embryos, and performed microarray gene expression analysis. Contrasting PAX3 andPAX3-FOXO1 revealed distinct gene expression signatures and signaling pathways responsible for PAX3-FOXO1 driven cell transformation; including modifying expression of genes related to satellite cell specificationand maintenance. Overall, an ARMS zebrafish model with embryonic phenotypes that predict tumorigenesishas applications ranging from studying ARMS biology to translational in vivo drug discovery.1308 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts

ZDM8 POSTERSZDM7Madison | USA 2015ZDM8Boston | USA 2015Loss of chd5-mediated tumor suppression accelerates MYCN-drivenneuroblastoma tumorigenesis in zebrafishMark Zimmerman 1 , Shuning He 1 , Jimann Shin 1 , Shizhen Zhu 1 , Feng Guo 1 , Marc Mansour 1 ,Deepak Reyon 3 , Keith Joung 3 , Jinhua Quan 2 , Timur Yusufzai 2 and A. Thomas Look 11Department of Pediatric Oncology,2Department of Radiation Oncology, Dana-Farber Cancer Institute, Boston, MA 022153Department of Pathology, Massachusetts General Hospital, Charlestown, MA 02129Neuroblastoma is a childhood tumor of the peripheral sympathetic nervous system (PSNS) thatoriginates from cells of the primitive neural crest. For the 40% of patients with high-risk disease,current therapies are often ineffective and long-term survival remains obstinately low. A major riskfactor in neuroblastoma is hemizygous loss of the 1p36 chromosomal region, which has long beensuspected to harbor one or more powerful tumor suppressor genes. Our studies indicate that lossof Chromodomain helicase DNA-binding protein 5 (chd5), which is in this deleted region,cooperates with MYCN overexpression to accelerate in vivo neuroblastoma tumorigenesis. Thechd5 protein can serve as one of two enzymatic components of the nucleosome remodeling andhistone deacetylase (NuRD) complex, which is a repressor of gene expression and is reported tohave diverse roles in regulating chromatin organization, developmental signaling and genestability. Zebrafish chd5-null alleles were created utilizing both zinc-finger nuclease and TALENtechnology. The resulting chd5 mutant fish exhibit abnormal development of the PSNS in the formof expansion of the superior cervical ganglia and enlargement of the interrenal gland (adrenalmedulla). In order to examine the effect of chd5 haploinsuficiency on in vivo neuroblastomatumorigenesis, chd5 mutant fish were crossed with the dbh:MYCN transgenic model resulting inneuroblastoma tumors. Chd5 haploinsufficient fish exhibit an accelerated neuroblastomaphenotype with tumors present beginning as early as 6 weeks compared to 15 weeks observedin wild-type fish. Consistent with human neuroblastoma cases with 1p36 deletion, zebrafishtumors that are haploinsufficient for chd5 express high levels of the cell cycle regulator wee1,indicating that chd5 normally down-modulates the expression of this protein. Future studies willexamine the structural basis for the tumor suppressor function of chd5, so that the pathwaysdown-stream of chd5 can be elucidated and essential proteins in these pathways can be targetedin ways that specifically inhibit the growth of haploinsufficient neuroblastoma cells.8 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts131

ZDM8Boston | USA 2015ZDM8 POSTERSOverexpression of platelet-derived growth factor receptor alpha(PDGFRA) synergizes with loss of NF1 in the molecular pathogenesisof malignant peripheral nerve sheath tumor (MPNST)Dong Hyuk Ki 1 , Shuning He 1 , and A. Thomas Look 11Department of Pediatric Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston MA,02115, USA.MPNSTs are among the most frequently occurring sarcomas in children. They are locallyinvasive, metastatic, nerve sheath-associated sarcomas that are often found in children with type1 neurofibromatosis (NF1). Currently, complete surgical excision is the only curative therapy forMPNST, but these tumors are often inoperable. Chemotherapy regimens are for the most partineffective. Therefore, it is important to understand and dissect the aberrant molecular pathwaysleading to MPNST pathogenesis to identify novel therapeutic targets for the development of newtherapies for MPNSTs.To test whether PDGFRA overexpression accelerates the onset of MPNSTs in nf1 andp53 deficient zebrafish as an oncogene in MPNST pathogenesis, we cloned the cDNA encodingeither wild-type PDGFRA or constitutively activated PDGFRA under the control of the zebrafishsox10 neural crest-specific promoter. These constructs were co-injected with sox10:mCherry intofertilized eggs of nf1a +/- ;nf1b -/- ;p53 e7/e7 zebrafish and the mosaic progeny were monitored forMPNST onset and also outbred at 3 months of age to establish stable transgenic lines.Both wild type and constitutively activated PDGFRA injected zebrafish developed tumorsmuch more frequently than mCherry control nf1a +/- ;nf1b -/- ;p53 e7/e7 zebrafish. The tumors inMPNST prone zebrafish resembled human MPNSTs histologically and expressed high levels ofthe each of the respective human transgenic PDGFRA proteins by Western blotting. Interestingly,wild-type PDGFRA overexpressers developed MPNST much faster and at higher penetrance thanfish transgenic for mutationally activated PDGFRA or control fish. Our results indicate that thesehigh levels of PDGFRA expression are likely pathogenic in the tumor cell growth and survival.Thus, PDGFRA represents an ideal target for treatment with specific tyrosine kinase inhibitors intumors with either wild-type or mutant PDGFRA expression, and our transgenic zebrafish modelsare ideal to test the efficacy of PDGFRA inhibitors as well as other small molecule drugs in vivofor activity against MPNST.1328 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts

ZDM8 POSTERSZDM7Madison | USA 2015ZDM8Boston | USA 2015Modelling cancer Inflammation and treatments including surgeryand radiotherapyPaul MartinDepts of Biochemistry and Phys/Pharm, University of Bristol, UKWe have been investigating parallels between wound healing and cancer. Just as forwounds, we find H 2 O 2 to be a primary attractant enabling immune cells to sense earlyclones of transformed cells before they progress to cancers. We find that clones oftransformed cells deprived of immune cells proliferate at a slower rate suggesting thatgrowth signals, which we have now shown to include prostaglandins, are delivered to thetransformed cells by immune cells. This observation may, in part, explain clinical studiesshowing how low dose aspirin can stave off the onset of gut and other cancers. We knowthat inflammation at wound sites leads to deposition of collagenous scars and so wehave been examining matrix deposition and degradation driven by inflammation in thevicinity of growing clones of cancer cells. Because surgery and radiotherapy are two ofthe most effective means of treating cancer we have begun to use larval and adultzebrafish to model cancer surgery and radiotherapy, in particular investigating how theinduced inflammatory response impacts on immune cell recruitment to nearbytransformed cells and what might be the downstream consequences of this, andextrapolating this to human melanoma patients. We are also beginning to model theseinteractions in silico.8 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts133

ZDM8Boston | USA 2015ZDM8 POSTERSInvestigating the role of tetraploid intermediates in melanomaprogressionRevati Darp 1,2 , Sharvari Gujja 1 , Yvonne Edwards 1 , David Pellman 3 , Leonard Zon 4 , Neil Ganem 5 ,Craig Ceol 1,21 University of Massachusetts Medical School, Program in Molecular Medicine, Worcester, MA, USA2University of Massachusetts Medical School, Department of Molecular, Cellular and Cancer Biology,Worcester, MA, USA3Howard Hughes Medical Institute, Department of Pediatric Oncology, Dana-Farber Cancer Institute,Children’s Hospital and Department of Cell Biology, Harvard Medical School, Boston, MA, USA4 Howard Hughes Medical Institute, Stem Cell Program and Division of Hematology/Oncology of BostonChildren’s Hospital and Dana Farber Cancer Institute, Harvard Stem Cell Institute, Harvard MedicalSchool, Boston, MA, USA5 Departments of Pharmacology and Experimental Therapeutics and Medicine, Division of Hematologyand Oncology, Boston University School of Medicine, Boston, MA, USARecent bioinformatic analyses have indicated that at least one third of solid tumors haveundergone a genome-doubling event during their progression. Additional studies haveimplicated genome doubling as an important step early in tumor progression. In these studies ithas been proposed that nascent tumor cells that become tetraploid can more easily samplegenetic configurations that are advantageous for growth because they, as compared to diploidcells, are buffered against deleterious effects of mutations and changes in gene dosage.Studies using a melanoma-prone Tg(mitfa:BRAFV600E);p53(lf) strain suggest a role forgenome doubling in the genesis of tumors in these fish. We have found that, unlike wild-typemelanocytes, melanocytes in Tg(mitfa:BRAFV600E) animals are binucleate and tetraploid. Intissue culture, newly-formed tetraploid cells have been shown to undergo a p53-dependent G1cell cycle arrest. A similar arrest likely occurs in zebrafish melanocytes, as these cells inTg(mitfa:BRAFV600E); p53(lf) animals exhibit 8N and greater DNA content, suggesting bypassof an arrest that halts progression of Tg(mitfa:BRAFV600E) melanocytes. Recently, Hippopathway signaling has been shown to activate p53 specifically in response to tetraploidy. Toinvestigate the involvement of Hippo signaling, we placed an activated form of YAP, one that isinsensitive to Hippo pathway inhibition, under the mitfa promoter and monitored tumor formationin animals expressing BRAFV600E. Upon overexpression of activated YAP, we observedmelanoma formation in BRAFV600E-expressing zebrafish whereas no tumors were observed inanimals that expressed an EGFP control. Taken together, these data implicate tetraploid cellsand pathways that regulate progression of tetraploid cells in melanoma progression. Currentlywe are conducting retrospective bioinformatic analyses to determine whether melanomas inTg(mitfa:BRAFV600E); p53(lf) zebrafish progress through tetraploid intermediates, supportingthe notion that important activities of oncogenic BRAFV600E and p53 are to generate andsuppress the progression of nascent tetraploid melanoma cells, respectively.1348 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts

ZDM8 POSTERSZDM7Madison | USA 2015ZDM8Boston | USA 2015In vivo Modeling of a Single Nucleotide Polymorphism withinLMO1 Associated with High Risk Neuroblastoma Using TALENGenome Editing in ZebrafishNina Weichert 1 , Mark Zimmerman 1 , Ting Tao 1 , Andrew C.Wood 2 , Derek Oldridge 3 ,Shizhen Zhu 4 , Deepak Reyon 5 , Keith Joung 5 , John M. Maris 3 , A. Thomas Look 11. Department of Pediatric Oncology, Dana Farber Cancer Institute, Harvard Medical School,Boston, MA, USA2. Department of Molecular Medicine and Pathology, University of Auckland, Auckland, NewZealand3. Division of Oncology and Center for Childhood Cancer Research, Children’s Hospital ofPhiladelphia, Philadelphia, PA, USA.4. Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester MN, USA5. Department of Pathology, Massachusetts General Hospital, Charlestown, MA, USANeuroblastoma, an embryonal tumor of the peripheral sympathetic nervous system,accounts for 12% of all childhood cancer deaths. Using a genome-wide associationstudy approach, we discovered that a single nucleotide polymorphism (SNP) within thefirst intron of the LIM domain-only 1 (LMO1) oncogene locus is robustly associated withthe development of advanced neuroblastoma, including those with somatic MYCNamplification. We identified that the highly conserved regulatory SNP rs2168101 thatalters a GATA transcription-factor binding-site is essential for a super-enhancer activityat this site. We are now using our zebrafish model of MYCN-driven neuroblastoma tomodel allelic variation using TALEN genome editing to introduce the precisepolymorphism into the fish germline, which is predicted to be protective for thedevelopment of neuroblastoma.We injected mRNA encoding transcription activator-like endonucleases (TALENs) intosingle-cell zebrafish embryos to generate a double-strand break in the regioncorresponding to the conserved GATA motif within the first intron of the zebrafish lmo1gene. These TALENs were also injected together with a homologous single-strandedoligonucleotide containing the precise G-T SNP, which modifies the genomic sequenceto TATA.Here we show that TALEN-mediated genome editing is effective in disrupting the GATA3binding motif within the lmo1 locus by producing indels in the highly conserved sequencewith deletions up to 70 nucleotides, which were heritably transmitted through thezebrafish germline. This technology was also used to precisely introduce templateguidedalterations to mediate exact conversion of the GATA to TATA allele. We willbreed TATA/TATA; MYCN transgenic fish to recapitulate effects on the initiation andprogression of MYCN-induced neuroblastoma and compare it to the effect of lmo1-/-zebrafish.The zebrafish model system is ideal for functional genomics analyses to determinebiological relevance of both cancer-promoting human polymorphic variation andacquired somatic mutations that are currently under intense investigation in humancancers.8 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts135

ZDM8Boston | USA 2015ZDM8 POSTERSA Novel Missense Mutation in the Extracellular Domain ofPDGFRa Induces Functional Consequences in vivoAmanda K. Templeton, Imelda T. Sandoval, Richard Glenn C. Delacruz, ChristeenaSatterfield, Braden Miller, and David A. Jones1. Immunobiology and Cancer Research Program, Oklahoma Medical ResearchFoundation; Department of Pathology and Stephenson Cancer Center, University ofOklahoma Health Sciences Center, Oklahoma City, Oklahoma2. Immunobiology and Cancer Research Program, Oklahoma Medical ResearchFoundation, Oklahoma City, OK3. Immunobiology and Cancer Research Program, Oklahoma Medical ResearchFoundation, Oklahoma City, OK4. Immunobiology and Cancer Research Program, Oklahoma Medical ResearchFoundation, Oklahoma City, OK5. Immunobiology and Cancer Research Program, Oklahoma Medical ResearchFoundation, Oklahoma City, OK6. Immunobiology and Cancer Research Program, Oklahoma Medical ResearchFoundation; Department of Pathology Stephenson Cancer Center, University ofOklahoma Health Sciences Center, Oklahoma City, OklahomaPlatelet-derived growth factor receptor-α (PDGFRA) belongs to the class III receptortyrosine kinase family, which also includes c-KIT. They are characterized by theirstructural similarity in immunoglobulin-like extracellular domains, a transmembranedomain, and intracellular protein kinase domains. Additionally, mutations in PDGFRAand c-KIT have been described in skin malignancies. Exome sequencing of a patientwith basal cell carcinoma revealed PDGFRA E459K missense mutation. This mutationlies within the Ig5 domain of PDGFRA and may parallel mutations in c-KIT that result inconstitutive receptor activation or altered ligand affinity in skin malignancies. Additionally,bioinformatics analysis of skin malignancy tumors harboring mutations in PDGFRA,revealed that the most common mutation is the single amino acid change at codon 459.We were, therefore, interested in investigating whether mutations in PDGFRA are alsoimplicated in the pathogenesis of skin cancer. Assessment of functions of PDGFRA andthe novel variants were examined by performing loss-of-function and gain-of functionexperiments in zebrafish. Analysis of pdgfra expression in vivo revealed strong stainingin the developing lens, jaw, and pectoral fins. Knockdown of zpdgfra resulted in loss ofjaw cartilage development, which was rescued by injection of human wild-type PDGFRAmRNA. Injection of human PDGFRA carrying the E459K mutation alone resulted indevelopmental defects in structures derived from neural crest cells. These defectsincluded a decrease in epithelial pigmentation, altered migration of melanocytes, neuraltube defects, defective jaw formation, and ectopic development of eyes or secondarybody axis. Similar developmental defects were observed in embryos injected with humanPDGFRA carrying a characterized gain-of-function mutation, but not in those injectedwith only wild-type human mRNA. Collectively, these findings suggest that this novelPDGFRA variant carries functional consequences that may contribute to skin tumordevelopment. Therapeutic agents blocking PDGFRA may, therefore, represent novelapproaches to treating skin cancer.1368 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts

ZDM8 POSTERSZDM7Madison | USA 2015ZDM8Boston | USA 2015Tumor Suppression Mechanism of CCR4-NOT transcriptioncomplex subunit 3 (CNOT3) in T-cell Acute LymphoblasticLeukemiaZhaodong Li 1 , Marc R. Mansour 1,2 and A. Thomas Look 11 Department of Pediatric Oncology, Dana-Farber Cancer Institute, Harvard Medical School,Boston MA, 02115, USA.2 Department of Haematology, UCL Cancer Institute, University College London, WC1E 6BT, UK.T-cell acute lymphoblastic leukemia or T-ALL is an aggressive hematologic tumorarising from the malignant transformation of hematopoietic progenitors primedtoward T cell development, which characterizes a multistep oncogenic processinvolving constitutive activation of NOTCH signaling as well as the translocationand aberrant expression of other transcription factor oncogenes. CCR4-NOTtranscription complex subunit 3, or CNOT3, belongs to the evolutionarilyconserved multi-subunit CCR4-Not complex that regulates gene expression atmultiple levels. Very recently, scientists have identified a new class of recurrentsomatic mutations in patient T-ALL samples that inactivate CNOT3 in T-ALL(about 8% of high risk T-ALL cases). This finding indicates that loss of CNOT3contributes to the initiation and maintenance of T-ALL in humans. In this study,we capitalized on CRISPR-Cas technology and generated CNOT3 knockoutzebrafish lines. Cnot3 gene has two homologues in zebrafish, Cnot3a andCnot3b. Cnot3a-/- and Cnot3b-/- fish are viable and exhibit no obvious grossdevelopmental defects, indicating functional redundancy of the two homologues,considering that the Cnot3-/- mouse is embryonic lethal. To study the role of lossof the CNOT3 in the initiation and maintenance of T-ALL, Cnot3 mutant fish havebeen crossed with a conditional Myc-induced T-ALL zebrafish model wellestablishedin our laboratory to compare the tumor onset and progress under thewild-type and Cnot3a-/-; Cnot3b+/- or Cnot3a+/-; Cnot3b-/- backgrounds. Inaddition, we have also crossed Cnot3 mutant fish with Rag2-GFP transgenic fishand gene-profiling analysis will be performed on sorted GFP-positive cells toidentify genes that are regulated by CNOT3 in T cells. The study will allow us togain deep insight into the function of CNOT3 in T cell development and themechanisms of CNOT3 tumor suppression in T-ALL pathogenesis in vivo.8 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts137

ZDM8Boston | USA 2015ZDM8 POSTERSIdentifying GDF6 as a novel pro-survival melanoma oncogeneArvind M Venkatesan 1,2 , Rajesh Vyas 1,2 , Sanchita Bhatnagar 2 , Karen Dresser 3 ,Feng Qi 4 , Jian-Liang Li 4 , April Deng 3 , Michael Green 2 , Craig Ceol 1,21 Program in Molecular Medicine, UMass Medical School, Worcester, MA, USA2 Department of Molecular, Cell and Cancer Biology, Worcester, MA, USA3 Dept. of Dermatopathology UMass Medical School, Worcester, MA, USA4Sanford Burnham Medical Research Institute, Orlando, FL, USARegions of recurrent copy number variation (CNV) contain genes that affect tumorprogression. To identify these genes we defined regions of recurrent CNV in zebrafishmelanomas and compared these regions to ones recurrently altered in humanmelanomas. In the set of genes that were recurrently amplified in both species we foundthe BMP factor GDF6. Because we found BMP signaling to be upregulated inmelanomas, we investigated the role of GDF6 in melanoma progression. In analyses ofboth zebrafish and humans, GDF6 mRNA and protein were upregulated in melanomasas compared to normal melanocytes. In functional assessments, we found thatoverexpression of GDF6 accelerated melanoma onset in zebrafish. Furthermore,knockdown of GDF6 in melanoma cell lines led to apoptotic cell death in culture in vitroand in mice xenotransplants in vivo. Addition of recombinant GDF6 protein to the mediarescued the melanoma cells from undergoing GDF6 shRNA-induced apoptosis,suggesting that GDF6 acts as a secretory factor in aiding melanoma cell survival. GDF6,like other BMP factors, is predicted to signal through SMAD1/5/8 transcription factors,and similar growth defects were observed when SMAD1 was knocked down inmelanoma cells. To further define the relationship between GDF6 and SMAD1 inmelanoma, GDF6 knockdown was performed in cells expressing a constitutively activeSMAD1 variant. This variant rescued the death caused by GDF6 knockdown, suggestingthat, at least in part, GDF6 acts through SMAD1 to promote melanoma cell survival.These data establish a role for BMP signaling in melanoma and identify a novelsecretory factor, GDF6, which mediates this role. GDF6 is an excellent therapeutic targetdue to its secretory nature and its expression in a majority of human melanomas.1388 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts

ZDM8 POSTERSZDM7Madison | USA 2015ZDM8Boston | USA 2015Building personalized genetic models of human cancer inzebrafish to predict response to therapy and characterizedisease mechanismsJacques A. Villefranc 1,4 , Chantal Pauli 2,3,4 , Viviana Anelli 1,4 , TheresaScognamiglio 2,4 , Mark Rubin 2,3,4 and Yariv Houvras 1,4Departments of Surgery and Medicine 1 , Department of Pathology 2 Institute for PrecisionMedicine 3 , and Meyer Cancer Center 4 , Weill Cornell Medical College and New York PresbyterianHospital, New York, NY 10065.A key challenge posed by personalized medicine is the ability to functionally characterizespecific mutations identified in human tumors in order to guide treatment decisions.Transgenic approaches in zebrafish offer an opportunity to model individual patienttumors in order to develop personalized models. To test this concept, we performed agenetic characterization of a patient with thyroid carcinoma and created an in vivopersonalized thyroid cancer model in zebrafish. Our studies suggest that this approachcan be used to build preclinical models suitable for chemical-genetic studies.Chromosomal rearrangements in RET often lead to chimeric in-frame fusions of itskinase domain with one of several partners, resulting in ectopic, ligand-independentactivation of RET. RET/PTC1 encodes a CCDC6-RET fusion and results in ectopictranscriptional activation of RET in thyrocytes and is associated with papillary thyroidcancer (PTC). Cabozantinib, a selective RET inhibitor, restricts growth of RET/PTC1 celllines and shows marked activity in patients with RET-mutant medullary thyroid cancer.However, its efficacy is limited due unknown mechanisms of resistance. Geneticanalysis of the index patient's tumor revealed a RET/PTC1 rearrangement, geneticalterations in the TERT promoter and a focal loss of TP53. Transient, tissue-specificexpression of RET/PTC1 in zebrafish thyroid follicular cells resulted in tumor growth by14 days post fertilization, with histopathologic characteristics consistent with PTC. Exvivo treatment of zebrafish RET/PTC1 tumors with cabozantinib, blocks RET activationand downstream signaling. In parallel, we have generated 3D-cultures from establishedand primary cells, in order to model resistance to pharmacologic RET inhibition (RETi).Finally, we describe comparative approaches to model the effects of RETi in vivo usingtransgenic zebrafish and 3D culture model systems. These studies provide proof-ofconceptdata to develop a transgenic zebrafish platform for patient specific models ofthyroid carcinoma and other solid tumors.8 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts139

ZDM8Boston | USA 2015This is my Abstract Title for ZDM8Jill de Jong, Craig Ceol, Liz Patton, Jim Amatruda, Annemarie Meijer, Anna Huttenlocher.1. Affiliation for Jill2. Affiliation for Craig3. Affiliation for the restThis is my abstract that I would like to present for ZDM8 x max 300 wordsZDM8 POSTERSCharacterize the Role of Protein Kinase CK2 in MYCN-drivenNeuroblastomaHaiwei Lian 1,2,3,4 , Yun Zhou 1,2,3,4 , Dun Li 1,2 , Ting Tao 5 , Esther Landesman-Bollag 2 , AThomas Look 5 , David C. Seldin 2 , Hui Fu 4 ,Hui Feng 1,21Department of Pharmacology & Experimental Therapeutics, Center for Cancer Research, BostonUniversity School of Medicine, Boston, MA, USA 021182 Department of Medicine, Section of Hematology and Medical Oncology, Boston University School ofMedicine, Boston, MA, USA 021183 Wuhan University Renmin Hospital, Wuhan, P.R.China 4300604 Department of Anatomy, Wuhan University School of Medicine, Wuhan, P.R.China 4300715 Department of Pediatric Oncology Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA,USA 02215Amplification of the MYCN oncogene occurs in about 30-40% of advanced neuroblastomacases and is the most reliable predictor of poor prognosis. Protein kinase CK2 (casein kinase II)is a highly conserved serine/threonine kinase that can be effectively inhibited by smallmolecules. In fact, CX-4945, a potent, specific, ATP-competitive CK2 inhibitor, is currentlytested in clinical trials to treat multiple cancers. However, the role of CK2 and its targetability inMYCN-driven neuroblastoma remain unknown. To determine the relevance of CK2 to MYCNdrivenneuroblastoma pathogenesis, we analyzed published microarray data and compared theexpression of different CK2 subunits in primary neuroblastoma patient samples. We found thatboth CK2α and CK2α’ are upregulated in neuroblastoma patient samples with MYCNamplification, compared to the ones without MYCN amplification. To determine the effect ofCK2 inhibition on neuroblastoma cells, we treated a panel of human neuroblastoma cell lineswith CK2 inhibitor CX-4945. Importantly, CX-4945 treatment at low micromolar doses iseffective in decreasing viability of human neuroblastoma cells with MYCN amplification. Thisdecreased viability is partly due to reduced MYC protein levels and increased apoptosis of thesecells. To determine the in vivo effect of CK2 inhibition on neuroblastoma development, we arein the process of generating a ck2a knockout zebrafish by using the clustered, regularlyinterspaced short palindromic repeats (CRISPR)-CAS technology. We also obtained azebrafish model of MYCN-driven neuroblastoma through collaboration with Dr. A ThomasLook’s laboratory. The stable heterozygous ck2α fish (ck2α+/-) will be bred to the MYCN fish todetermine if ck2α heterozygous loss can significantly delay neuroblastoma initiation and/orprogression. Finally, we will use CK2 specific inhibitors (e.g.,CX-4945) to treat transgenic fishwith MYCN-driven neuroblastoma. These studies will allow us to determine the role of CK2 inneuroblastoma development, and provide valuable information about CK2’s targetability inMYCN-driven neuroblastoma.1408 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts

ZDM8 POSTERSZDM7Madison | USA 2015ZDM8Boston | USA 2015Tumor diversity and evolution revealed through restriction-siteassociated DNA sequencing.Elizabeth B. Perry 1 , Alvin Makohon-Moore 1 , Caihong Zheng 3 , Kajan Ratnakumar 1 ,Charles K. Kaufman 2 , Leonard Zon 2 , Jun Cai 3 , Christine Iacobuzio-Donahue 1 , &Richard M. White 11. Memorial Sloan Kettering Cancer Center, Cancer Biology & Genetics2. Dana Farber Cancer Institute, Department of Medical Oncology3. Laboratory of Disease Genomics and Personalized Medicine & Center of ComputationalBiology, Beijing Institute of GenomicsAll of the cells in the adult body of a multicellular organism are derived from asingle cell. Errors in replication and damage to DNA that occur during development canresult in an adult body that consists of a mosaic of genetically heterogeneous cellpopulations. Cancer has been described as a disease of abnormal somatic evolution inwhich DNA changes that occur in somatic cells can confer a fitness advantage allowingthose mutants to rapidly proliferate and ultimately cause disease. Although cancers aremost frequently diagnosed in older adults, the striking amount of genetic heterogeneitypresent in many human tumors is consistent with the hypothesis that initiating mutationalevents may occur many years prior to the appearance and diagnosis of cancer. Thepurpose of this project is to quantify the rates at which somatic mosaicism is generatedin normal tissues and tumors, and to understand factors that influence those rates. Tomeasure these rates, we are using a tool called RAD-seq, which is a reducedrepresentationsequencing approach initially developed for evolutionary genetic studies.We have generated transgenic zebrafish with an inducible fluorescent system (ubi:loxP-GFP-loxP-mCherry) to track lineages of melanocytes in a melanoma-prone (p53-/-; mitf-BRAFV600E +/+) and non-melanoma prone background (p53+/+ ;BRAF WT/WT). Usingflow sorting, we can specifically isolate mCherry+ melanocytes from both normal skinand melanomas these animals, and then use RAD-seq to assess mutation rates andpatterns. Our preliminary data shows that we can identify mutations and copy numberalterations that occur within normal tissue and tumors, both in zebrafish and humancancer patient samples. These preliminary data and tools are relevant to broaderquestions of cancer evolution and whether somatic mosaicism among normal cellpopulations influences cancer development later in life.8 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts141

ZDM8Boston | USA 2015ZDM8 POSTERSTitle: Bace2 in normal and malignant melanocytesAuthors: Yan Zhang 1,2 , Milena Zimmer 1 , Emily Kansler 1 , Nathaniel Campbell 1,2 , Richard White 1,2Affiliation: 1. Memorial Sloan Kettering Cancer Center, New York, NY; 2. Weill Cornell Medical College, NewYork, NY;BACKGROUND: Melanoma is a highly aggressive skin cancer that originates in the melanocytes.Pathways involved in melanocyte development are known to be subverted in the formation ofmelanoma. We have previously demonstrated that both human and zebrafish melanomas stronglyoverexpress several key genes involved in neural crest and melanocyte development, including a βsecretase, BACE2. The β-site APP cleaving enzymes 2 (BACE2) is a transmembrane aspartylprotease, and it cleaves substrates to release a shedding domain and a cytosolic domain. We aim toelucidate the roles of BACE2 in regulating melanocyte development and melanoma. RESULTS: We areinvestigating BACE2 in normal and malignant melanocytes using both zebrafish and human cell culturemodels. Zebrafish BACE2 -/- mutants show a defect in melanocyte number and patterning, withhyperdendritic melanocytes evident in the tail fin. Adult fish show a decrease in total melanocytenumbers. By in situ hybridization, BACE2 is highly enriched in zebrafish melanocytes at 24hpf.Because mammalian studies have suggested that PMEL is a substrate for BACE2, we determinedwhether loss of PMELA, or its close homolog GPNMB, was responsible for the abnormal melanocytepatterning. PMELA morpholinos did not recapitulate the BACE2 phenotype, while double knockdown ofPMELA and BACE2 resulted in a nearly complete loss of pigmented melanocytes, a phenotypedivergent from the BACE2 mutant. Similarly, CRISPR mediated F0 mosaic knockout of GPNMB did notrecapitulate the BACE2 mutant phenotype, although we can show that recombinant BACE2 efficientlycleaves GPNMB. This data suggests that either there is another substrate responsible for the BACE2phenotype, or alternatively that it is due to an accumulation of improperly processed proteins such asPMELA or GPNMB. To test this, we have created a PMELA-FLAG fusion protein to measure PMELAcleavage in the presence and absence of BACE2, which will facilitate overexpression of non-cleavablePMELA. Similar studies will be carried out for GPNMB. Finally, in order to understand the role ofBACE2 in melanoma, we have created triple transgenic mitf-BRAF V600E ;p53 -/- ;BACE2 -/- fish, along withhuman A375 and SKMel28 cells with a knockout of BACE2. The phenotype of these BACE2 deficientmelanomas is ongoing, but will allow us to identify a specific function for the BACE2 sheddase inhuman melanoma.1428 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts

ZDM8 POSTERSZDM7Madison | USA 2015ZDM8Boston | USA 2015Bace2 in zebrafish melanocyte developmentMilena A Zimmer, Yan Zhang, Richard White1. Cancer Biology and Genetics, Memorial Sloan Kettering Cancer Center2. Cancer Biology and Genetics, Memorial Sloan Kettering Cancer Center3. Cancer Biology and Genetics, Memorial Sloan Kettering Cancer CenterBACE1 and BACE2 are proteases classified as sheddases, which cleave the extracellular partof membrane-bound substrates generating a membrane-bound fragment. BACE2 is highlyoverexpressed in fish and human melanoma, prompting us to examine its function in normaland malignant melanocytes. The embryos of the zebrafish mutant for BACE2 have abnormalmelanocyte morphology, particularly in the head and tail regions and an increased number ofmelanocytes by 3-5 dpf. At 72hpf, tail melanocytes are hyperdendritic and suggestive ofaberrant migration. In adults, melanocytes are hyperdendritic and in contrast to the embryosreduced in numbers.Crossing the BACE2 mutant onto the tyrp1-mCherry background allowedfor detailed imaging of melanocytes by confocal microscopy. This confirmed highly abnormaldendrite and migration patterns of melanocytes. To examine melanosome structure, weperformed electron microscopy of the BACE2 mutant melanocytes at 72hpf, which showed asevere destruction of melanosomes and suggested spillage of free melanin into the cytosol. Todetermine which stage of development BACE2 is required, we performed in situ hybridization of24hpf and 72hpf embryos using probes for crestin, sox10, foxd3, mitfa, ednrba, c-kit, dct, tyr,tyrp1b, pmela and bace2 itself. At 24hpf, BACE2 is expressed in melanocyte precursors, similarto mitfa. By 72hpf, BACE2 mutants show increased staining for dct, tyrp1b and pmela,consistent with the increased melanocyte numbers in embryos. To determine what causes thediscordance between melanocyte numbers in the embryo and the adult we use time-lapseimaging and TUNEL staining. Finally, to gain insight into the molecular mechanisms by whichBACE2 regulates melanocyte morphology, we perform qRT-PCR and RNA-seq of isolatedtyrp1-mCherry melanocytes from WT and BACE2 mutant embryos and adults. These studieswill elucidate the mechanism which BACE2 regulates normal melanocyte development andmorphology, and point towards how this protease may be involved in melanoma pathogenesisduring transformation.8 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts143

ZDM8Boston | USA 2015ZDM8 POSTERSQuantifying Metastatic Spread In A Zebra-fish Melanoma ModelSilja Heilmann 2 , Kajan Ratnakumar 1 , Joao Xavier 2 , Richard White 1,31. Memorial Sloan Kettering Cancer Center, Cancer Biology & Genetics2. Memorial Sloan Kettering Cancer Center, Computational Biology3. Weill Cornell Medical CollegeUnderstanding the metastatic cascade remains a significant challenge. A broaderunderstanding of this complex, spatially and temporally dynamic process is needed. Theoften highly stochastic nature of metastasis formation means that a large number ofanimals are needed in order for patterns to emerge making zebrafish an ideal model forstudying metastases. Here we describe a quantitative system for measurement ofmetastasis using ZMEL1, a fluorescently labeled zebrafish melanoma cell line (mitfa-BRAF V600E ;p53 -/- ;mitf-GFP) transplanted into the casper strain. We transplanted 10 5 ,5x10 5 , or 10 6 cells into irradiated casper recipients, and then developed a customautomated image analysis algorithm to assess the pattern and number of metastases.The imaging suggested a tropism of the cancer cells for the kidney marrow area, afinding which was confirmed by histological sectioning and anti-GFP staining. Fish withkidney marrow metastases had a tendency towards overall more aggressive disease.We found that melanoma pigmentation was highly inversely correlated with metastaticcapacity, which is consistent with data from mammalian systems showing that metastaticcells tend to be initially less differentiated. Using quantification of several imagingparameters, we were able to develop a metastasis score, µ, that enables comparison ofmetastatic efficiency across groups. The µ score increased with increasing number oftransplanted cells, indicating that metastatic efficiency varies with the size of the primaryimplant. Using limiting dilution analysis, we find that the metastasis-initiating cellfrequency is approximately 1/120,000 cells in the ZMEL1 melanoma cell line, which isconsistent with mammalian studies showing that metastasis is overall an inefficientprocess. The development of these tools will now allow for large scale study of both cellintrinsicand microenvironmental modifiers of the metastatic process.1448 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts

ZDM8 POSTERSZDM7Madison | USA 2015ZDM8Boston | USA 2015Xiang Maomao Zhang AbstractThe successful establishment of metastases relies heavily on the suitability of the tumormicroenvironment (TME). Adipocytes constitute a major portion of the TME in several cancertypes but their contribution to tumor progression is poorly understood. However, epidemiologicalstudies demonstrate a positive correlation between obesity and cancer incidence, while clinicaldata show that tumors often preferentially metastasize to organs high in adipose tissue. Basedon these observations, we wish to investigate how tumor-associated adipocytes contribute tometastatic melanoma. We propose to use a zebrafish model of melanoma and molecularimaging of tumor metastases and tumor-associated adipocytes to understand how adipocytescontribute to the establishment of overt macrometastases.Our experimental strategy is to generate a transgenic adipocyte reporter line in zebrafish. Usingthis transgenic animal, in combination with a previously established zebrafish model ofmelanoma and a melanoma transplantation assay, we aim to characterize melanomaassociatedadipocytes. First, we will use molecular imaging to study the changes in adipocytemorphology, migration and density during tumor progression. Second, we will isolate adipocytesfrom cancer-bearing animals and perform gene expression analysis to characterize adipocytesubpopulations and their molecular activity. Importantly, we will determine whether our zebrafishdata are recapitulated in human melanoma by analyzing clinical sample sets. Finally, we willestablish a method for specific adipocyte ablation in our transgenic animal to functionally testthe role of adipocytes in tumor progression. Ultimately, we wish to identify clinically targetablepathways of cross-talk between adipocytes and melanoma cells, which represents a novel formof microenvironmental therapy for the treatment of melanoma.8 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts145

ZDM8Boston | USA 2015ZDM8 POSTERSCell of origin studies of malignant melanoma using bothtransgenic zebrafish and human pluripotent stem cell-derivedmelanocytesScott Callahan 1,2 , Yvonne Mica 2 , Lorenz Studer 2 , Richard White 11. Memorial Sloan Kettering Cancer Center, Cancer Biology & Genetics2. Memorial Sloan Kettering Cancer Center, Developmental BiologyAlbeit in an uncontrolled fashion, the ability of cancer to self-propagate is sharedwith adult stem cells. This shared trait led to the hypothesis that adult stem cells may bemore apt to transformation or may transform into distinct tumor phenotypes. To addressthe impact of the differentiation status of the cell of origin on melanoma this study willutilize two complementary systems: transgenic zebrafish and melanocytes differentiatedfrom hESCS.The established zebrafish melanoma model overexpresses mutant BRAF V600Eunder the mitfa promoter in a p53 -/- background. To test the importance of cell of origin,we have engineered transgenic fish that drive BRAF V600E expression in p53 -/- fish underthe control of neural crest promoters (crestin, sox10, ednrb1a), melanoblast promoters(mitfa and dct) and late stage melanocytes promoters (tyrp1 and pmela). Interestingly, inthe F0 mosaic animals, only the sox10, mitfa, and dct lines develop tumors. The mitfatumors grow aggressively and with 100% penetrance, the dct tumors are significantlysmaller with limited penetrance and the sox10 fish develop exclusively neuroblastomas.These findings suggest that there is a very fine developmental window for transformationof melanocytes using the BRAF V600E /p53 -/- system, but this will require confirmation instable F1 generations. Concurrently, we are engineering cre/lox inducible BRAF V600Ealleles, with stage specific CreER drivers.To complement the transgenic zebrafish, we have developed a protocol todifferentiate pigmented melanocytes via melanocyte progenitors from human pluripotentcells. The protocol mimics development by first proceeding through a neural crestprogenitor (sox10+) and melanoblast (mitf+) stage before becoming a mature pigmentproducing melanocyte (tyr+). We have demonstrated transformation via in vitro assaysupon applying viruses for BRAF V600E , sh_p53, and sh_p16. We hypothesize that thephenotype of each model will be strongly dependent upon the developmental stage.1468 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts

ZDM8 POSTERSZDM7Madison | USA 2015ZDM8Boston | USA 2015Nuclear Hormone receptors regulate transcriptional pausing inneural crestCristina Santoriello 1 , Bilguujin Dorjsuren 1 , Eugenia Custo Greig 1 , Riley Brian 1 , IsaacAdatto 1 and Leonard I. Zon 1-21.Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA 02138,USA2.Stem cell Program and Division of Hematology/Oncology, Children’s Hospital Boston and DanaFarber Cancer Institute, Boston, MA 02115; Harvard Medical School, Boston, MA 02115.Multipotent neural crest cells give rise to different cell types, including melanocytes,which are the pigment cells that are transformed in melanoma. A chemical screendesigned to look for repressors of neural crest gene expression revealed that the antiarthritisdrug leflunomide, an inhibitor of dihydrooratate dehydrogenase (DHODH)involved in de novo pyrimidine synthesis, modulates transcription elongation in neuralcrest and melanoma cells. Leflunomide is currently being tested in combination with aBRAF inhibitor in a clinical trial for metastatic melanoma. We performed a chemicalsuppressor screen in zebrafish embryos using whole mount in situ hybridization for theneural crest marker crestin, and looked for compounds that rescued crestin expressionafter leflunomide treatment. We identified 13 chemicals and, using metabolite profiling bymass spectrometry in human melanoma cells to evaluate nucleotide precursors andnucleotides levels, we classified chemicals into 3 distinct classes. The first chemicalclass specifically affects mitochondrial import or leflunomide activity, the secondincreases nucleotide pools. The third class acts on transcription and includes the nuclearhormone receptor progesterone (pgr). In support of this observation we found thatprogesterone rescued neural crest gene expression in several RNA polymerase IIassociated factor (PAF) mutants. To better understand the mechanism via which nuclearhormone receptor regulates transcription elongation in neural crest we performed RNAsequence analysis on isolated neural crest cells. RNA sequence analysis revealed thatleflunomide causes inhibition of pgr pathway and activation of other nuclear hormonesreceptor pathways. Morpholinos against nrli12, a nuclear hormone receptor involved inxenobiotic response rescues crestin expression in leflunomide treated embryos,suggesting that leflunomide itself acts via nuclear hormone receptors. We aim to betterdefine these nuclear hormone pathways that seem to be involved in transcriptionalelongation control in neural crest, melanocytes and melanoma and eventually find newtargets for melanoma therapies.8 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts147

ZDM8Boston | USA 2015ZDM8 POSTERSThe Apelin receptor and its ligand Elabela modulate Nodal/TGFβsignalling differently for proper cardiac developmentSerene C. Chng 1 , Ashish R. Deshwar 2,3 , Lena Ho 1 , Bruno Reversade 1,4,5* and Ian C.Scott 2,3,6*1 Institute of Medical Biology, A*STAR, Singapore2 Program in Developmental and Stem Cell Biology, The Hospital for Sick Children, Toronto,Ontario, M5G 1X8, Canada;3 Department of Molecular Genetics, University of Toronto4 Institute of Molecular and Cellular Biology, A*STAR, Singapore5 Department of Paediatrics, School of Medicine, NUS, Singapore6 Heart & Stroke/Richard Lewar Centre of Excellence in Cardiovascular Research, Universityof TorontoThe Apelin receptor (Aplnr) and its early ligand Elabela (Ela) are necessary for heartdevelopment by controlling the migration of cardiac progenitors to the anterior lateral platemesoderm. How Aplnr/Ela functions in this context is unclear. We found that Aplnr signallingmodulates Nodal/TGFβ signalling during gastrulation, a key pathway for mesendoderminduction and migration. Genetic loss-of-function studies of aplnra/b showed lowerexpression levels of a subset of Nodal target genes, whereas over-expression of Aplnra/bled to an increase expression of the same targets. Remarkably, increasing Nodal levels wassufficient to rescue the cardiac defects observed in aplnrb mutants. Unexpectedly, in thecontext of Nodal modulation, Ela appears to have opposing effects to that of Aplnr, since it isfound to dampen Nodal signalling. We will provide evidence that Ela may function as aninhibitory ligand to Aplnr. We propose a novel mechanism whereby Aplnr/Ela function as arheostat that fine-tunes Nodal signalling levels to allow proper mesendoderm specificationand heart formation.1488 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts

ZDM8 POSTERSZDM7Madison | USA 2015ZDM8Boston | USA 2015Unravelling the function of a novel unannotated gene responsiblefor Left-Right Asymmetry defects in HumansEmmanuelle Szenker 1 , Tania Attie-Bittach 2 , Patrice Bouvagnet 3 and Bruno Reversade 11. Human genetics and embryology laboratory, Institute of Medical Biology (IMB), A*STAR, Singapore2. Molecular genetics laboartory, Hôpital Necker Enfants Malades, Paris, France3. Congenital heart defects laboratory, CHU Lyon, FranceCiliopathies are genetic disorders disrupting ciliary function. As such, they can affect multipleciliary organs, causing a wide range of phenotypes such as left-right asymmetry defects,chronic respiratory infections, kidney and heart diseases, and/or infertility. Here, we studieda consanguineous family with situs inversus totalis and severe kidney / heart defects, withoutmutations in any of the reported heterotaxia genes (MIM, HTX1-6). Through homozygositymapping and whole exome sequencing, we uncovered the underlying mutation in this familyin a hitherto unannotated gene. This novel gene, which we named “TOUT DE TRAVERS(TDT)”, encodes a putative conserved metalloprotease. We show that tdt is maternallyexpressed and that only maternal-zygotic tdt mutant zebrafish embryos can reveal itsrequirement in L-R patterning and ciliary function.8 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts149

ZDM8Boston | USA 2015ZDM8 POSTERSAdult zebrafish have been recently exploited to study human cardiomyopathies, as indicated by thegeneration of two acquired models for cardiomyopathy, with one induced by anemia and the other bydoxorubicin. However, genetic models are yet to be reported, and it is unclear whether a zebrafishheart is useful for modeling different types of human cardiomyopathies. Here, we employed bothTALEN knock-out (KO) and transgenic technologies to model BCL2-associated athanogene 3 (BAG3) -based cardiomyopathies in adult zebrafish. We found that the heterozygous bag3 KO fish exhibitedprogressive heart dilation phenotype with hallmarks of dilated cardiomyopathy (DCM) such asactivation of atrial natriuretic factor (ANF), myofibrillar loss and cardiac dysfunction. In contrast,transgenic overexpression of a human mutation derivative BAG3 Pro209Leu (mBAG3 OE) in adult zebrafishresulted in a significantly reduced ventricular chamber size, suggesting a distinct pathophysiology.Through candidate pathway analysis, we detected an activated autophagy activity and increasedaccumulation of poly-ubiquitination proteins in the bag3 KO. In contrast, both of which were reduced inthe mBAG3 OE heart. A distinct set of genes that are differentiately regulated in these two bag3-basedmodels has also been identified via RNAseq analysis. Taken together, we conclude that genetic modelsof cardiomyopathy can be created in adult zebrafish via generating either loss-of-function mutants ortransgenic lines harboring a mutated human gene. Distinct phenotypes in these two bag3 -basedcardiomyopathy models suggest that a simple zebrafish heart is sophisticated enough to modeldifferent types of cardiomyopathies caused by different genetic changes in the same gene. Pathwayanalysis can be conducted in this vertebrate model with higher throughput, providing a new platform fordeveloping individualized therapies.1508 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts

ZDM8 POSTERSZDM7Madison | USA 2015ZDM8Boston | USA 2015Study of Developmental and Molecular Processes Regulated by Sorbs1using a Combination of in vitro and in vivo modelsAlexandra Veloso 1 , Anouk Bleuart 1 , Maud Martin 1 , Jonathan Bruyr 1 , and Franck Dequiedt 11 Laboratory of Protein Signaling and Interactions, GIGA-Research, University of Liège, B-4000 Liège,BelgiumSoHo proteins belong to a family that includes three members: Sorbs1 (Cbl associated proteinCAP/ponsin), Sorbs2 (Arg-Binding Protein 2, ArgBP2) and Sorbs3 (Vinexin). These proteinsshare a similar structure with a SoHo domain in N-terminal region and three SH3 domainsin carboxy terminal region. These characteristic domains bind to several signaling moleculesinvolved in a variety of cytoskeleton-related processes, and SoHo family members are thusthought to function as adaptor proteins. However, the precise role of these proteins in thecytoskeleton regulation and associated biological functions remains unknown.It is well established that cytoskeleton regulation is critical for various developmental eventsincluding angiogenesis, the process by which new blood vessels develop from pre-existingones, and myogenesis, the process responsible for muscle formation and regeneration.The goal of this project is to identify the developmental function of Sorbs1 and characterize theunderlying molecular events by exploiting a combination of in vivo (Zebrafish) and invitro models.Phenotype analysis revealed that Morpholino-mediated knock-down of Sorbs1 inducesabnormal development of cardiac, angiogenic and muscles structures. Knock-down zebrafishembryos were unable to form cardiac looping and present a cardiac edema. Also, it was noticedthat tail morphology was altered by Sorbs1 knock-down suggesting that Sorbs1 plays a role intrunk muscle formation. Finally, the development of venous angiogenic structures, such ascaudal vein plexus (CVP) and subintestinal veins (SIV), was specifically affectedby Sorbs1inactivation. Interestingly, Sorbs1 seems to have a specific role in venousangiogenesis (CVP and SIV), since arterial angiogenic structures, such as Intersegmentalvessels, were not affected in Sorbs1 morphants.In conclusion, these preliminary results of our work highlighted important developmental defectsby consequence of Sorbs1 inactivation in Zebrafish. Some of these defects appear to beregulated by angiogenesis and myogenesis, two developmental processes for which thetherapeutic implications are undeniable.8 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts151

ZDM8Boston | USA 2015ZDM8 POSTERSMissense Mutation in COL22A1 Is Associated With IntracranialAneurysmsQuynh V Ton PhD and Saulius Sumanas PhDCincinnati Children’s Hospital, Cincinnati, OHIntracranial saccular aneurysms (IAs) are small berry or balloon-like defects in the wall of themajor intracranial artery. The rate of mortality for the affected patients is high; survivors willrecover with major disability. Both environmental and genetic factors have been attributed to theaneurysm formation; however, the genetic factors are poorly understood. To identify additionalgenes responsible for IA development, whole exome sequencing of the patients from sevenselected families was performed. One of the identified mutant variants was a missense mutationin the highly conserved triple-helix region of the collagen COL22A1 isoform, function of whichhas not been previously known. Using zebrafish embryonic model we demonstrated thatcol22A1 is expressed in cranial tissues as well as in the cells between myotomes in the trunkand tail region. Early global overexpression of the human mutant but not wild-type COL22A1 inthe zebrafish embryos interfered with epiboly movements during gastrulation. Heat-shockinduced global overexpression of the mutant but not wild-type COL22A1 also resulted in highembryonic lethality. We used TALEN-engineered nucleases to generate a loss-of-functionmutant in zebrafish col22a1. The homozygous null mutants have now apparent morphologicaldefects and are viable as adults. However, 6-month-old adults exhibit increased abnormal bloodaccumulations in the eyes and cranial region, suggestive of hemorrhages. They also showcardiac dilation and a phenotype consistent with dilated cardiomyopathy. These results suggestthat the function of COL22A1 is important in maintaining vascular integrity and that mutations inCOL22A1 are one of the genetic causes for intracranial aneurysms in human patients.1528 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts

ZDM8 POSTERSZDM7Madison | USA 2015ZDM8Boston | USA 2015Congenital heart defects (CHDs) are the leading type of birth defect affecting 1% of livebirths and ~10% of stillbirth. We currently do not have a clear understanding of themolecular mechanisms underlying most CHDs. One of the signals that direct cardiacprogenitor (CP) specification is canonical Wnt signaling. While our previous studies havedemonstrated that canonical Wnt signaling promotes CP specification at the expense ofhematovascular progenitors, fate maps of pre-gastrula zebrafish embryos have shownthat the CP field is intermingled with pharyngeal muscle progenitors (PMPs).Interestingly, Wnt signaling has also been shown to inhibit pharyngeal muscledevelopment in chicken embryos. However, we do not yet understand if there is a directrelationship between CP and PMP specification within the vertebrate embryo. Todetermine if Wnt signaling promotes CP fate at the expense of PMP fate in the zebrafishembryo, we manipulated Wnt signaling using heat-shock inducible transgenic zebrafishlines and knockdown approaches at different stages of development. We found that priorto gastrulation Wnt signaling significantly inhibited dorso-anterior pharyngeal muscledevelopment, which is also the stage that Wnt signaling promotes CP specification.Moreover, we found that Wnt signaling prior to gastrulation is necessary and sufficient topromote an overlap of CP markers with PMP markers in a region of the anterior lateralplate mesoderm that normally only contains PMP markers. Together, our study providesa better understanding of the developmental mechanisms by which canonical Wntsignaling directs the fate decisions of CP and PMP fields within the early vertebrateembryo, which may provide insight into developmental syndromes with cardiac andcraniofacial abnormalities.8 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts153

ZDM8Boston | USA 2015ZDM8 POSTERSHeart development is severely affected by an impairment of the Hedgehogpathway in type II mucopolysaccharidosis (MPSII) animal models.Roberto Costa 1 , Stefania Bellesso 2 , Ilaria Zancan 1 , Marika Salavalaio 2 , Rosella Tomanin 2 ,Mirella Filocamo 3 and Enrico Moro 1 .1Department of Molecular Medicine, University of Padova, Italy.2.Department of Women’s and Child’s Health, University of Padova, Italy3Lab. Diagnosi Pre-Postnatale Malattie Metaboliche - IRCCS G. Gaslini, Genova, ItalyHunter syndrome (HS) is a rare disorder of the glycosaminoglycans (GAGs) metabolism, whichleads to the progressive accumulation of several GAGs. It is caused by the deficiency of iduronate2 sulfatase (IDS), an hydrolase able to remove the sulfate group from L-iduronate(ido) -2 sulfate. InMPSII patients the clinical manifestations include a neurologic involvement, severe airwaysobstruction, skeletal deformities and a severe cardiomyopathy. Accumulation of GAGsprogressively occurs in the ventricular chamber and in the aortic lumen of patients heart,contributing to the cardiac impairment. However, several evidences indicate that GAG depositionoccurs later than the first clinical symptoms. GAGs are structural polymers, which actively bindgrowth factors and surface receptors. Ido sulfatation is a reversible modification, which improvesthe binding potential of GAGs. Thus, the cellular matrix can be quickly modulated by GAGsmodifications affecting the mobility of several morphogens.In this work a preliminary characterization of heart development is provided in zebrafish andmouse loss of IDS function models. We demonstrate that IDS plays a key role in Shh and Wntsignaling pathways during early phases of heart development and loss of IDS function severelycompromise the early specification of heart cellular fates. In particular the formation of valvesrequire the cooperation of several signaling pathways, which define tissue specification, cellmigration and cell fate transition. The zebrafish loss of IDS function model display a progressiveAV malformation, which is reversed upon human IDS overexpression.Our results suggests that the onset of heart failure in Hunter patients may be due to adevelopmental impairment, unrelated to the belated GAGs accumulation. Moroever, this studydefine IDS as a key modifying enzyme for GAGs, given its ability to modulate the extracellularmatrix and morphogens.1548 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts

ZDM8 POSTERSZDM7Madison | USA 2015ZDM8Boston | USA 2015The role of inflammatory cell signalling during cardiac scarformation and regression in adult zebrafishR.J. Richardson 1 & P. Martin 11. Medical Sciences Building, Departments of Physiology and Pharmacology andBiochemistry, University of Bristol, University Walk, Bristol, BS8 1TD, UKOne of the first responses to tissue damage is the rapid and robust recruitment ofinflammatory cells to the injury site. These immune cells perform crucial protectiveand reparative functions but their influence at the repair site is not all positive.Studies of skin repair have demonstrated a direct link between the inflammatoryresponse and subsequent irreparable fibrosis/scar formation. By contrast to adultmammalian tissues, embryos and adult teleosts possess a remarkable capacity forcomplete, scar-free tissue regeneration in a variety of tissues including the skin andheart. Modeling cardiac damage in adult zebrafish reveals a robust inflammatoryresponse and initial collagen deposition within the myocardium; however, thisinterstitial collagen is gradually remodelled/removed leading to complete myocardialregeneration. Analysis of transgenic zebrafish lines and qPCR profiling of theinflammatory cells responding to cardiac injury reveals an early influx of neutrophils,which are later replaced by M1 and M2-subtype-marker-expressing macrophages.Ablation of these specific cellular sub-types will reveal their precise roles during scardeposition and resolution. Adult zebrafish models of cardiac damage offer uniqueopportunities for visualizing the inflammatory response in scarring and scar resolvingsituations. Live imaging of transgenic juvenile and adult zebrafish cardiac tissue vianewly established techniques including the creation of custom software (for imagingmoving tissue) will allow further cellular level characterization of immune cellphenotypes during these key injury response stages.8 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts155

ZDM8Boston | USA 2015ZDM8 POSTERSGenetic studies of histone demethylases kdm6ba and kdm6bbduring zebrafish cardiogenesis and regenerationAlexander A. Akerberg, Scott Stewart, Kryn StankunasInstitute of Molecular Biology, University of Oregon, Eugene, OREight of every one thousand infants are born with a congenital heart defect (CHD),affecting up to 1.3 million Americans. Improved diagnostics and therapeutics for thesecommon diseases rely on an improved understanding of the genetic and epigeneticbasis of heart development. Human mutations in cardiogenic transcription factors (TFs)implicate aberrant cardiac progenitor cell specification as a major contributor to CHD.However, it is unclear how these TFs are initially activated to promote cardiogenesis.Gene activation is often regulated by localized histone marks that confer either arepressive or active transcriptional state. For example, the Polycomb RepressiveComplex 2 (PRC2) deposits the repressive H3K27me3 mark on many cardiogenic genesat the earliest stages of embryogenesis. The current paradigm suggests that theserepressive marks must then be removed as mesoderm is specified into the cardiaclineage, possibly by the kdm6-family of H3K27me3-specific demethylases. To test thisidea, we undertook an expression and morpholino-based functional screen of zebrafishkdm6 family members. Our results suggest that kdm6bb has a specific anddemethylase-dependent role in gene activation during second heart field specification.We are pursuing this hypothesis using a series of CRISPR/Cas9-generated mutantsdesigned to either fully disrupt gene function or specifically disrupt the catalytic activity ofboth kdm6bb and its ohnolog kdm6ba. In parallel, we are using these lines to studyproposed roles of these demethylases in organ regeneration, when developmentalregulators must be re-activated as part of tissue damage-induced cellulardedifferentiation.1568 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts

ZDM8 POSTERSZDM7Madison | USA 2015ZDM8Boston | USA 2015High Throughput Forward and Reverse Screen forCardiotoxicology of Anticancer Drugs in ZebrafishToshio Tanaka, Noriko Umemoto, Miko Kawabata, Yasuhito Shimada, YuheiNishimuraDepartment of Pharmacogenomics and Systems Pharmacology, Mie University GraduateSchool of Medicine, 2-174Edobashi, Tsu, Mie 514-8507 JAPANMany anticancer drugs are associated with adverse cardiac effects, including leftventricular dysfunction. Moreover, the precise mechanism remains unclear in mostanticancer drugs. Here, we aimed to establish the methods to improve throughput inforward and reverse screen for anticancer drug-induced cardiotoxicity. In this context,the use of zebrafish in high throughput screening for drug discovery has beendocumented in several studies. However, in the protocols reported thus far cardiacfunction was analyzed manually and the methods were not applicable to a multiwellplate format. Therefore, a key challenge in the high throughput assessment of cardiacfunctional disturbances in zebrafish has been the development of a high contentmultiwell screening system. Here, cardiac dysfunction in zebrafish was assessed usinga novel automated imaging technology and ZF plate which is a specialized platform forzebrafish-based high throughput phenotype and functional screening in high contentimaging system. The method makes use of a transparent, transgenic line of zebrafishexpressing green fluorescent protein in the myocardium(MieKomachi 009, MK009).Fluorescent cardiac imaging using MK009 revealed that the ventricular dysfunction withseveral anticancer drug such as sorafenib. Transcriptome analysis of their heartsrevealed that stanniocalcin 1 (stc1) was downregulated by sorafenib. stc1 knockdown inzebrafish revealed that reduction of stc1 decreased the longitudinal dimensions ofzebrafish ventricles, similar to that which occurs during sorafenib treatment. STC1downregulation and cytotoxicity were also seen in human cardiomyocytes exposed tosorafenib. Reactive oxygen species (ROS) production significantly increased in bothspecies of human cardiomyocytes and zebrafish exposed to sorafenib and STC1knockdown compared with the controls. Finally, we found that forced expression of stc1normalized impairment, decreasing the longitudinal dimensions in zebrafish treated withsorafenib. The integrated high throughput forward and reverse screen system can beapplied for cardiotoxicology of anticancer drugs.8 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts157

ZDM8Boston | USA 2015ZDM8 POSTERSInteractions between a pair of cell adhesion molecules controlthe production of outflow tract progenitor cells from the secondheart fieldXin-Xin Zeng and Deborah YelonDivision of Biological SciencesUniversity of California, San DiegoLa Jolla, CA, 92093, USAHeart formation requires the contributions of progenitor cells from both the first andsecond heart fields. Notably, the cardiac outflow tract (OFT) is built through recruitmentof second heart field (SHF)-derived cells to the arterial pole of the heart. Abnormal OFTgrowth and morphogenesis are prevalent in patients with congenital heart defects, yetour understanding of OFT formation remains incomplete. Our research utilizes thezebrafish to elucidate the mechanisms responsible for the precise selection of anappropriate number of OFT cells from the SHF. Our recent studies demonstrated thatcell adhesion molecule 4 (cadm4) plays an essential role in restricting the size of theOFT. Zebrafish cadm4 morphants and mutants exhibit a dramatically expanded OFT,and, conversely, overexpression of cadm4 results in a greatly diminished OFT.Moreover, cadm4 activity limits the production of OFT progenitor cells and the durationof their accumulation at the arterial pole. In order to determine how this cell adhesionmolecule regulates OFT progenitor production, we are investigating which proteins actas binding partners for Cadm4 and which domains of Cadm4 are crucial for its functions.Our latest data indicate that cell adhesion molecule 3 (cadm3) is an additional memberof the Cadm family that is relevant to the regulation of OFT formation. We havegenerated several mutations in cadm3 through CRISPR/Cas9 genome editing, and wefind that cadm3 mutants display an elongated OFT and an enlarged pool of OFTprogenitors, similar to the phenotype observed in embryos lacking cadm4 function.Furthermore, our structure-function analysis demonstrates that the extracellular Ig-likedomains of Cadm4 are important for its control of OFT progenitor recruitment. Together,our data suggest a model in which heterophilic interactions between the ectodomains ofCadm4 and Cadm3 control OFT size by restraining the deployment of SHF progenitorsto the OFT.1588 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts

ZDM8 POSTERSZDM7Madison | USA 2015ZDM8Boston | USA 2015Transgenic and Patient Derived Xenograft Zebrafish Model forPersonalized Medicine of Hepatocellular CarcinomaHan-Syuan Lin 1,2 , Bo-Han Tseng 1,2 , Yi-Lien Huang 1,2 , Hsing-Pang Hsieh 3 ,Tsu-An Hsu 3 , Horng-Dar Wang 2 1, 4, 5, and Chiou-Hwa Yuh1 Institute of Molecular and Genomic Medicine, National Health Research Institutes, 2 College ofLife Science and Institute of Biotechnology, National Tsing-Hua University, 3 Institute ofBiotechnology and Pharmaceutical Research, National Health Research Institutes, 4 Institute ofBioinformatics and Structural Biology, National Tsing-Hua University, 5 Department of BiologicalScience & Technology, National Chiao Tung UniversityHepatocellular Carcinoma (HCC) is the third most common cancer worldwide andranked as a leading cause of mortality in Taiwan. Currently, there is only oneFDA-approved target therapy (Sorafenib) which can inhibit liver cancer growth andprolong patients’ life. The development of new target therapy is therefore urgentlyneeded. In addition, due to the heterogeneity of HCC, predicting the drug efficacy foreach patient is the difficult challenge for cancer clinicians. Therefore, we propose touse patient-derived tumor xenograft zebrafish model to determine which drug iseffective for each specific cancer patient for physician’s precise prescription. We haveused the HBx-induced HCC mouse model and identified four common regulators (Src,Edn1, Bmp4 and Bmp7) which can be used as molecular targets for HCC treatment.We also have established the transgenic fish overexpressing HBx or src in the p53mutant, and edn1 transgenic zebrafish developed into HCC. The HCC from thosetransgenic zebrafish is much more similar to human HCC in histopathology and thepattern of gene expression, which allow them more suitable as drug screeningplatform to identify the drugs to effectively treat human liver cancer. In this report, I willdemonstrate the advantage of zebrafish HCC model as well as zebrafish embryos.We have developed a high-throughput drug screening platform to identify novel andsafe anti-HCC therapeutic means to save more life. Due to the heterogeneity of HCC,there are no effective clinical tests that can predict the effectiveness of the anti-canceragents for patients. Using patient-derived xenograft zebrafish model, we are able toidentify safe and personalized medicine for the future use in clinical therapy.8 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts159

ZDM8Boston | USA 2015ZDM8 POSTERSOverfeeding accelerates thioacetamide-induced liver damage inzebrafish.Elena Turola 1,4 , Fabiola Milosa 1,4 , Rosina Critelli 1,4 , Marisa di Giovanni 2 , Franco Cotelli 3 , EricaVilla 1,4 .1.Gastroenterology Unit, Department of Internal Medicine, University of Modena and Reggio Emilia, Modena,Italy2. Department of Pathology, University of Modena and Reggio Emilia, Modena, Italy3. Department of Biosciences, University of Milan, Milan, Italy4. WomenInHepatology NetworkNon-alcoholic fatty liver disease (NAFLD) is becoming a worldwide epidemics. A relevantproportion of NAFLD patients progress to non-alcoholic steatohepatitis (NASH), fibrosis/cirrhosisand hepatocellular carcinoma (HCC). The relationship between NAFLD, inflammation, obesity,gender, reproductive status and age are known although the reciprocal roles are not completelyunderstood. We previously demonstrated that simply hyper-feeding zebrafish for 24 weeks leads tosteatosis and substantial fibrosis in young and old male fish and old female fish but not in youngfemales fish. However, relevant chronic liver disease takes >54 weeks to develop. In the overfeedingmodel inflammation is lacking. We have therefore investigated the effect of thioacetamide(TAA), a hepatotoxin causing centrilobular necrosis and periportal inflammation, as additionalhepatotoxic agent.Adult zebrafish (young/old, males/females) were fed with Artemia in either high-dose or standardquantity and intraperitoneally injected with 300mg/kg of TAA thrice a week for 9 weeks.Histopathological examination and qRT/PCR on genes involved in lipid metabolism (PPARgamma,PPARalpha), fibrosis (TGFbeta), inflammation (IL-6, TNFalpha) and HCC (c-MYC, PSMD10, IGF-2) were performed on liver samples collected at 1,6,24,36 weeks. In over-fed but not in standardfedfish the administration of TAA induced, already at 12 weeks, severe liver damage compatiblewith histologic liver cirrhosis. In over-fed/TAA fish only relevant steatosis was present until week 4(as in the previously described simple overfeeding model) which substantially decreased, leavingplace to marked fibrosis and nodules, after week 8. Moderate inflammation was present in bothgroups.Our data suggest that TAA plus overfeeding is able to quickly induce a NASH model very close tohuman condition, which very rapidly leads to severe liver damage. This underlines the importanceof the inflammatory hit in the transition from NAFLD to NASH. Further observation will showwhether HCC will also develop, allowing to evaluate the differential mechanisms activated in thesub-models.1608 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts

ZDM8 POSTERSZDM7Madison | USA 2015ZDM8Boston | USA 2015Adenylate Kinase 2 is essential for zebrafish gastrointestinaldevelopmentRissone Alberto 1 , Bishop Kevin 2 , Jones Marypat 3 , Wincovitch Stephen 4 , SoodRaman 2 , Burgess Shawn 1 and Candotti Fabio 51Translational and Functional Genomics Branch, NHGRI - National Institutes of Health,Bethesda, MD2Zebrafish Core, National Human Genome Research Institute, NIH3Genomics Core, National Human Genome Research Institute, NIH4Cytogenetics & Microscopy Core, National Human Genome Research Institute, NIH5 Division of Immunology and Allergy, University Hospital of Lausanne, Lausanne, Switzerland Adenylate kinase 2 (AK2) is a mitochondrial enzyme critical for cellular energyhomeostasis. Mutations of the AK2 gene are responsible for reticular dysgenesis (RD),one of the most rare and severe forms of severe combined immunodeficiency (SCID).RD is characterized by the lack of granulocytes and lymphocytes, leaving RD patientsvulnerable to recurrent serious infections and death early in life. Using zinc-fingernucleases technology, we introduced frameshift mutations in the ak2 gene (ak2 del2 ). Inaddition, we isolated an ENU-induced ak2 mutant carrying a missense mutation withinexon 4 (ak2 L124P ). Contrary to ak2 del2/del2 mutants that resulted in a loss of ak2 mRNA, theL124P substitution did not affect the mRNA transcript. Notably both the deletion and themissense ak2 mutants presented identical hematopoietic defects. Surprisingly, despitethe severity of the hematopoietic phenotype, only ak2 del2/del2 mutants were lethal,suggesting the presence of a more severe condition than the ak2 L124P/L124P mutants.WISH analysis showed that ak2 expression was not restricted to the hematopoieticsystem, but was also observed in the liver, gut and exocrine pancreas, suggesting thatthe gastrointestinal development could be affected. Using specific probes forgastrointestinal system and histological and in-vivo time-lapse confocal analyses onak2 del2/del2 transgenic mutants we observed a strong reduction in liver size, severeabnormalities in the gut and lack of exocrine pancreas without affecting pancreatic β-celldevelopment. Moreover, such organs were characterized by increased oxidative stressresulting in a block of cell proliferation, as indicated by the lack of pcna expression andBrdU staining, followed by apoptosis as shown by the ectopic expression of p53 andcdkn1a/p21 in liver and gut. Overall, our data describe a previously unknown function ofak2 gene in gastrointestinal development and indicated that ak2 loss-of-function doesnot affect only hematopoietic development but it can induce a broader spectrum ofphenotypes. 8 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts161

ZDM8Boston | USA 2015ZDM8 POSTERSUsing zebrafish to characterize the role of dachb and theeffects of diet on the development of the pancreatic islet andglycemia.Lingling Yang 1,3 , Sarah E. Webb 4 , Heung Man Lee 1,3 , Gang Xu 1,2,5 , Juliana C. N.Chan 1,2,3 , Andrew L. Miller 4,6 , Ronald C.W. Ma 1,2,31 Department of Medicine and Therapeutics, 2 Hong Kong Institute of Diabetes and Obesity, and3 Li Ka Shing Institute of Health Sciences, CUHK, Prince of Wales Hospital, Shatin, Hong KongSAR, China4 Division of Life Science and State Key Laboratory of Molecular Neuroscience, Hong KongUniversity of Science and Technology, Clear Water Bay, Hong Kong SAR, China.5 Teaching and Research Division, Hong Kong Baptist University, Hong Kong SAR, China6 Marine Biological Laboratory, Woods Hole, MA 02543, USA.All metazoans utilize insulin to control their energy metabolism but they do so bydifferent mechanisms. In vertebrates, insulin is secreted from endocrine cells in thegut or pancreas. Insulin expression is controlled by several transcriptional regulators,including Dachshund. In a genome-wide association study, we have reported theassociation of familial young onset type 2 diabetes in the Chinese population with asingle nucleotide polymorphism within intron 1 in DACH1, the latter gene beingimplicated in developmental biology. In zebrafish, the dachshund homologues,dacha, dachb and dachc are expressed in the pancreas, pronephros, central nervoussystem and sensory organs. Here, we used zebrafish as a model to study thepossible role of dachb in islet development and type 2 diabetes. Embryos wereco-injected with dachb morpholino (MO) and p53 MO, and then fixed at 24 hpf and 30hpf, followed by immunolabelling of the pancreatic β-cells and endocrine cells.Embryos co-injected with dachb MO and p53 MO displayed a smaller β-cell andendocrine cell area compared with the control group co-injected with a dachb4-mismatch and p53 MO. To establish a type 2 diabetes zebrafish model, weover-fed the adult fish with egg yolk or normal diet with dried artemia (control). After8 weeks of over-feeding with egg yolk, the body weight increased significantlycompared to no change in the control fish. After a significant increase in bloodglucose levels in the first four weeks, this returned to a level comparable to the controlgroup in the subsequent four weeks. These data supported the notion that geneticpolymorphism of DACH1 may cause abnormal β-cell development with increased riskof diabetes in an obesogenic environment.1628 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts

ZDM8 POSTERSZDM7Madison | USA 2015ZDM8Boston | USA 2015Epigenetic regulation of liver development in zebrafishShuang Wang, Vinitha Jacob, Yelena Chernyavskaya, and Kirsten Sadler Edepli.1. Department of Medicine, Division of Liver Diseases, Icahn School of Medicine atMount Sinai2. Department of Developmental and Regenerative Biology, Division of Liver Diseases, IcahnSchool of Medicine at Mount SinaiCellular proliferation is a tightly regulated biological process that controls liverdevelopment and regeneration under physiological conditions, and may lead to livercancer when dysregulated. A forward genetic screen in zebrafish identified Uhrf1(Ubiquitin-like and PHD and RING finger domains 1) as a key regulator of both liverdevelopment and liver regeneration following resection. Further work from our laboratorydemonstrated that the overexpression of human UHRF1 in zebrafish hepatocytes leadsto liver cancer as early as 15 days post-fertilization. UHRF1 is a key mediator inmaintaining DNA methylation patterns and we hypothesize that this epigenetic functionof UHRF1 is critical to regulating hepatocyte cell proliferation under physiological andpathological conditions. Here, we examined how the loss of DNA methylation impactsthe hepatocyte cell cycle during zebrafish liver development. We found that global DNAhypomethylation precedes the appearance of a morphological phenotype includingdefective liver growth in uhrf1 mutants. Surprisingly, this is accompanied by anincreased number of hepatocytes undergoing S-phase, marked by BrdU incorporation.Furthermore, the BrdU signal is retained in mutant hepatocytes, suggesting an inabilityto proceed through the cell cycle. These results suggest that UHRF1 regulates livergrowth by stimulating DNA synthesis and simultaneously inhibiting cell division, either orboth of which may be a result of DNA hypomethylation. In future studies we wish tofurther characterize the cell cycle defects observed in the liver of uhrf1 mutant fish and,ultimately, to elucidate the mechanistic link between global DNA hypomethylation and S-phase regulation during liver development.8 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts163

ZDM8Boston | USA 2015ZDM8 POSTERSA Balancing Act: Dysregulated differentiation and proliferationas a Novel Mechanism of HepatocarcinogenesisChad Walesky, Wolfram GoesslingDepartment of Medicine – Genetics Division, Brigham and Women’s Hospital, Harvard MedicalSchool, Boston, MAHepatocellular carcinoma (HCC) is among the deadliest cancers worldwide,with only ~16% of patients surviving more than five years. The incidence of HCChas increased, and there are no effective strategies for prevention, diagnosis, ortreatment, indicating a significant unmet medical need. Typically, cancersdevelop from an imbalance between cellular growth and differentiation signals.While most advances in cancer therapy have been made by inhibition of cellproliferation, the re-establishment of cell differentiation has resulted in the cure ofcertain forms of leukemia. In this study, we have focused on the interplaybetween hepatocyte nuclear factor 4α (HNF4α), the master regulator of hepaticdifferentiation, and the WNT/β-catenin signaling pathway, which enhances cellproliferation.We demonstrate crosstalk between the two pathways in the developingzebrafish liver: Knockdown of HNF4α results in significantly decreased β-cateninexpression at 72 hours post fertilization (hpf), concomitant with impaired liverdifferentiation as evidenced by diminished liver fatty acid binding protein (lfabp)expression. Reciprocally, knockdown of β-catenin results in a significantdecrease in HNF4α expression at 72 hpf along with dysregulation of HNF4αtarget genes and impaired liver differentiation. Further, zebrafish with overactivationof β-catenin not only reveal up-regulated β-catenin target genes, butalso dysregulation of HNF4α targets. To demonstrate conservation of ourfindings in mammals, we examined mouse livers with loss of the central β-catenin regulator Apc (Apc Fl/Fl; AlbCreERT2), resulting in up-regulation of Axin2and dysregulation of HNF4α targets of hepatic differentiation and tumorigenesis.Taken together, these findings indicate a delicately balanced crosstalk betweenthe HNF4α and β-catenin signaling pathways, as well as a block in hepaticdifferentiation. Further investigation into the mechanisms by which thesepathways interact can provide valuable knowledge in understanding liverdevelopment and how alterations in these pathways may contribute to livercancer pathogenesis.1648 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts

ZDM8 POSTERSZDM7Madison | USA 2015ZDM8Boston | USA 2015The novel G-protein coupled estrogen receptor GPER enhances livergrowth via PI3K/Akt and mTOR pathway activationEstrogen plays essential roles in reproductive organ development and function;however, the impact on non-reproductive organs such as the liver is poorlyunderstood. A chemical genetic screen in zebrafish identified estrogen as a novelregulator of liver development: estrogen exposure enhances hepatocyte numberand liver size, as determined by in situ hybridization for the hepatocyte markerliver fatty acid binding protein (lfabp) at 72 hours post fertilization (hpf). Thedevelopmental effect of estrogen on liver growth is not mediated by classicalnuclear hormone estrogen receptors, but by a novel membrane G-coupledestrogen receptor (GPER), which is expressed in the developing liver from 48hpf: Exposure to GPER modulators during liver outgrowth (>96hpf) dramaticallyaffected liver size, cell number and proliferation, while modulators of estrogennuclear receptor activity had no impact. Estrogen or the GPER agonist G1enhanced cellular proliferation, while the GPER inhibitor G15 blocked the positivegrowth effects of estrogen on the maturing liver, indicating that estrogen signalsthrough GPER to increase liver size. Similarly, morpholino knockdown of GPERdecreased liver size and blocked the positive effects of estrogen, which could berescued by GPER mRNA co-injection, further confirming the results of thechemical exposures. GPER exerts its effects on hepatocytes via the PI3K/mTORpathway: co-exposure of estrogen with the PI3K inhibitor LY924002 inhibited liverenlargement, while treatment of GPER morphants with the PI3K activator 740 Y-P partially rescued liver size and cell number. Estrogen and G1 exposureincreased phosphorylation of PI3K, AKT and S6, as measured by Western blot.The mTOR inhibitor Rapamycin or loss of mTOR1 limited the estrogenic effect onliver differentiation. Our data identify GPER as a novel regulator of liver formationand growth during zebrafish development, acting through PI3K/mTOR signalingto regulate the differentiation and expansion of mature hepatic tissue in vivo.8 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts165

ZDM8Boston | USA 2015ZDM8 POSTERSA Post-Developmental Genetic Screen for Zebrafish Models of Inherited LiverDiseaseSeok-Hyung Kim 1,*, Simon Wu 2, Gary T. Hardiman 1, Kevin C. Ess 3, Joshua H. Lipschutz 1, 4 andDon C. Rockey11 Dept. of Medicine, Medical University of South Carolina, Charleston, SC2 Dept. of Biological Sciences, Vanderbilt University, Nashville, TN3 Dept. of Pediatrics, Vanderbilt University School of Medicine, Nashville, TN4 Dept. of Medicine, Ralph H. Johnson Veterans Affairs Medical Center, Charleston, SC* CorrespondenceNonalcoholic fatty liver disease (NAFLD) is one of the most common causes of chronic liver disease such assimple steatosis, nonalcoholic steatohepatitis (NASH), cirrhosis and fibrosis. However, the molecularpathogenesis and genetic variations causing NAFLD are poorly understood. The high prevalence andincidence of NAFLD suggests that genetic variations on a large number of genes might be involved in NAFLD.To identify genetic variants causing inherited liver disease, we used zebrafish as a model system for a largescalemutant screen, and adopted a whole genome sequencing approach for rapid identification of mutatedgenes found in our screen. Here, we report on a forward genetic screen of ENU mutagenized zebrafish. From250 F2 lines of ENU mutagenized zebrafish during post-developmental stages (5 to 8 days post fertilization),we identified 19 unique mutant zebrafish lines displaying visual evidence of hepatomegaly and/or steatosiswithout obvious developmental defects. Histological analysis of mutants revealed several specific phenotypes,including common steatosis, micro/macrovesicular steatosis, hepatomegaly, ballooning, and acutehepatocellular necrosis. Using whole genome sequencing we have identified and confirmed the geneticdefects in 7 of mutants. Molecular characterization of mutants suggests that impaired mitochondrial, lysosomalfunction, altered mitophagy, lipophagy or lipid metabolism could be underlying mechanism of liverpathogenesis in identified mutants. As results, we identified at least four novel models of human geneticdiseases, including Tuberous Sclerosis Complex (TSC), Multiple Acyl-CoA Dehydrogenase Deficiency (MADD),Chediak-Higashi syndrome (CHS) and Alpers syndrome (AS), and we already published TSC and MADDpreviously. Thus, this work has identified multiple post-developmental mutants and establishes zebrafish as anovel animal model for post-developmental inherited liver disease.1668 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts

ZDM8 POSTERSZDM7Madison | USA 2015ZDM8Boston | USA 2015Functional Analysis of Hirschsprung Patient Exome Sequence Datausing ZebrafishIain Shepherd 1 , Colin Harrison 1 , Sijia Wang 1 , Tara Wabbersen 1 , Ludmila Petrova 1 , William Cheng 2 , BintaJalloh 1 , Yunia Sribudiani 2 , Rajendra.K.Chauhan 2 and Robert Hofstra 21 Department of Biology, Emory University, Atlanta, USA and 2 Department of Clinical Genetics, ErasmusMedical Center, Rotterdam, The NetherlandsWe have utilized the zebrafish to rapidly determine the functional significance of newlyidentified mutated genes in Hirschsprung disease (HSCR) patients. HSCR is a pediatriccondition where the enteric nervous system (ENS) fails to form properly. The diseaseoccurs once in every 5,000 live births and occurs both familial and sporadically. HSCRis a classic multifactorial genetic disorder for which 14 genes have been identified.. Ofthese genes, RET accounts for >80% of all identified mutations. However the geneticbasis of the disease in the vast majority of HSCR patients is unknown. Studies by ourlab and others have shown there is a strong evolutionary conservation in the genesrequired for ENS development and all of the known HSCR genes have been shown tobe required for zebrafish ENS development. We exome sequenced a large Dutchpedigree which we has linkage of HSCR to a locus on chromosome 4. We identified amutation in the LRBA gene in all patients. Significantly, the MAB21L2 is a gene locatedwithin intron 41of LRBA. In addition we also identified a RET mutation in one branch ofthe family and a mutation in Indian Hedgehog (IHH) in another branch of the family. Itappears that two of these genes combine (LRBA/MAB21L2 and RET orLRBA/MAB21L2 and IHH) in different patients to cause the disease phenotype.Perturbation of function of ret, ihh, or mab21l2 in zebrafish results in intestinalaganglionosis. We have also exome sequenced 10 long-segment Hirschsprung patients.Bioinfomatic analysis followed by sanger sequencing showed that there were 2 provende novo mutations per patient. We selected 10 of these de novo mutations for furtherfunctional analysis in zebrafish. Preliminary results show that several of these de novogenes are critical for normal zebrafish ENS development suggesting that these arepotentially new HSCR gene loci.8 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts167

ZDM8Boston | USA 2015ZDM8 POSTERSZebrafish Blastomere Culture Screening Establishes Adenosine as aRegulator of Hematopoietic Stem and Progenitor CellsJoseph Mandelbaum 1 , Ilya Shestopalov 1 , Lili Jing 1 , and Leonard I. Zon 1 .1. Stem Cell Program and Division of Hematology/Oncology, Children’s Hospital and Dana Farber CancerInstitute, Howard Hughes Medical Institute, Harvard Stem Cell Institute, Harvard Medical School, Boston,MA 02115, USA.Hematopoietic stem cells (HSCs) give rise to all differentiated blood cells and are used clinicallyto reconstitute the bone marrow. Patients who do not have a matched donor marrow wouldbenefit from iPSC-derived HSCs, and finding factors that promote HSC generation frompluripotent sources would help develop these personalized therapies. We reported a chemicalscreen identifying inducers of myogenesis using a zebrafish blastomere culture system (Xu etal., Cell, 2013). Here, we have adapted this culture screening system to find inducers of c-myb,a marker of hematopoietic stem and progenitor cells (HSPCs), to identify factors that expandHSPCs. In this study, c-myb:GFP zebrafish are grown to 50% epiboly and dissociated intosingle cells. The cultured cells are treated with 3,840 compounds in duplicate for two days andthen GFP fluorescence is read out. We identified 14 chemicals (z score >10; hit rate 0.36%) thatinduce c-myb expressing cell expansion, including the adenosine analog 3-deazaadenosine,suggesting that adenosine modulates HSPC formation. We treated whole embryos with theadenosine receptor antagonist CGS15943 and agonist 5’-N-ethylcarboxamidoadenosine(NECA) to observe effects on HSPC development. Confocal imaging of the aorta-gonadmesonephros(AGM) region, the site where HSPCs are specified, showed a significantlydecreased number of HSPCs in cmyb:GFP embryos (36.0±2.4) vs DMSO control (43.2±2.4)due to CGS15943 treatment, and significantly increased (52.8±4.8) due to NECA treatment.We next examined the effect on endothelial cells since HSPCs originate from hemogenicendothelium by undergoing endothelial-to-hematopoietic transition (EHT). Knockdown of theadenosine A 2b receptor by morpholino injections caused more endothelial cells, marked byflk1:GFP, to abort EHT by time-lapse imaging. Taken together, we conclude that adenosinesignaling is required for proper HSPC emergence, and further validates our zebrafish culturesystem to identify factors that promote HSPC expansion from pluripotent sources.1688 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts

ZDM8 POSTERSZDM7Madison | USA 2015ZDM8Boston | USA 2015THYROID REGULATION OF GLOBIN SWITCHINGDavid M. Wiley 1-2 , Jared J. Ganis 1-2 , Daniel E. Bauer 1-2 , Matthew C. Canver 1-2 , Jian Xu 1-2 ,Sonja Boatman 2 , Rebecca Maher 2 , Elizabeth B. Riley 2 , Grant Rheingold 2 , James Palis 3 ,Stuart H. Orkin 1-2, 4 , Leonard I. Zon 1-2, 4 –[1] Harvard Stem Cell Institute, Harvard Medical School, 1Blackfan Cir., Karp 7, Boston, MA 02115, [2] Stem Cell Program and Division of Hematology/Oncology,Children's Hospital and Dana Farber Cancer Institute, 1 Blackfan Cir., Karp 7, Boston, MA 02115, [3]Department of Pediatrics, Center for Pediatric Biomedical Research, University of Rochester MedicalCenter, Rochester, NY, USA, [4] Howard Hughes Medical InstituteThe maturation of cells into an adult state directly affects aging, cancers, and β-globin disorders,such as sickle cell anemia and β-thalassemia. In erythroid cells, embryonic, fetal and adultcellular states are clearly demarcated by the expression of ε, γ, and β-globin respectively andectopic induction of fetal globin ameliorates the symptoms of the aforementioned β-globindisorders. To identify critical and novel regulators of cell maturation, we preformed two screens:a candidate based morpholino screen and a large scale chemical screen in zebrafish embryoslooking for factors that induced the expression of an adult globin gene, α a1, in zebrafishembryos. The top hits from both screens converged on the thyroid hormone (TH) signalingpathway. The TH ligands robustly induce adult globin in zebrafish embryos and antisensemorpholinos against TH pathway components block the ectopic induction of adult globin.Interestingly, treating embryonic thalassemia mutant zebrafish, zinfandel, with TH partiallyrescues this embryonic anemia. In addition, treating adult zebrafish with a TH antagonist,Dronedarone, induces ectopic fetal globin expression. These effects are conserved in humanK562 cells and primary fetal proerythroblasts, as treatment with TH robustly increases adultglobin. Chip-Seq analysis of THR shows binding at the β-globin locus and treating cells with THinduces H3K27ac at the β-globin promoter, indicating that the TH pathway is directly regulatingdownstream epigenetic effects. At the receptor level, the THRA is known to regulate erythroidcell differentiation and THRB is preferentially expressed in adult proerythroblasts.Consequently, we hypothesized that THRB is a critical regulator of cellular maturation, whileTHRA was primarily responsible for differentiation. Knockdown of THRB in proerythroblastsrobustly induces fetal globin with negligible effects on differentiation. Our studies provideimportant insight into how endocrine signaling pathways coordinate pleiotropic effects andidentified a new therapeutic target for activating fetal globin.8 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts169

ZDM8Boston | USA 2015ZDM8 POSTERSUsing zebrafish to study human ALA-dehydratase-deficientporphyria (ADP) and hereditary coproporphyria (HCP)Shuqing Zhang 1,2 , Zhijie Niu 1,2 , Jingjing Wang 1,2 , Han Wang 1,2*1 Center for Circadian Clocks, Soochow University, Suzhou 215123, Jiangsu, China2 School of Biology & Basic Medical Sciences, Medical College, Soochow University, Suzhou215123, Jiangsu, ChinaDefects in the enzymes involved in heme biosynthesis result in a group of humanmetabolic genetic disorders known as porphyrias. Using a zebrafish model for humanhepatoerythropoietic porphyria (HEP), caused by defective uroporphyrinogendecarboxylase (UROD), the fifth enzyme in the heme biosynthesis pathway, we recentlyhave found a novel aspect of porphyria pathogenesis. However, no hereditable zebrafishmodels with genetic mutations of alad and cpox, encoding the second enzyme deltaaminolevulinatedehydratase (ALAD) and the sixth enzyme coproporphyrinogen oxidase(CPOX), have been established to date. Here we employed site-specific genome-editingtools transcription activator-like effector nuclease (TALEN) and clustered regularlyinterspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) togenerate zebrafish mutants for alad and cpox. These two zebrafish mutants displayphenotypes of heme deficiency, hypochromia, abnormal erythrocytic maturation andaccumulation of heme precursor intermediates, reminiscent of human ALA-dehydratasedeficientporphyria (ADP) and hereditary coproporphyrian (HCP). Further, we observedaltered expression of genes involved in heme biosynthesis and degradation andparticularly down-regulation of exocrine pancreatic zymogens in ADP (alad-/-) and HCP(cpox-/-) fishes. These two zebrafish porphyria models can survive at least 7 days andthus provide invaluable resources for elucidating novel pathological aspects ofporphyrias, discovering new therapeutic targets and developing effective drugs for thesecomplex genetic diseases. Our studies also highlight generation of zebrafish models forhuman diseases with two new versatile genome-editing tools.1708 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts

ZDM8 POSTERSZDM7Madison | USA 2015ZDM8Boston | USA 2015Tet2 and Tet3 are essential for hematopoietic stem celldevelopmentCheng Li 1,2 , Yahui Lan 3 , Todd Evans 3 , Mary Goll 11 Developmental Biology Program, Memorial Sloan Kettering Cancer Center, New York, NY,10065, USA.2 Program in Biochemistry and Structural Biology, Cell and Developmental Biology, and MolecularBiology, Weill Cornell Graduate School of Medical Sciences, Cornell University, New York, NY,10065, USA3 Department of Surgery, Weill Cornell Medical College, New York, NY, 10065, USAHematopoietic stem cells (HSCs) are self-renewing precursors that are capable ofproducing all adult blood cells. A number of genes required for HSC production havebeen identified, including runx1 and scl. However, it is not clear how these genes arecoordinately regulated during development. The ten eleven translocation proteins Tet1,Tet2 and Tet3 convert the epigenetically modified base 5-methylcytosine to 5-hydroxymethylcytosine, providing a mechanism for activation of genes repressed by 5-methylcytosine. TET2 mutations are frequent in myeloid leukemias, but roles for Tetenzymes during hematopoietic development are less well defined. Here, wedemonstrate that tet2 and tet3 are essential for HSC development. Zebrafish carryingTALEN induced mutations in tet1, tet2, or tet3 were viable with modestly reduced 5hmCand normal hematopoiesis. In contrast, tet2/3 double mutants had a 30-fold reduction in5hmC and lacked definitive blood. Embryos produced few hematopoietic stem cells(HSCs) correlating with fewer endothelial to hematopoietic transition events and reducedexpression of early markers of the hemogenic endothelium including gata2b. Gata2bexpression is known to be dependent on Notch signaling, and we observe specific lossof expression of a Notch reporter in the hemogenic endothelium of tet2/3 doublemutants. Our results identify Tet2 and Tet3 as early regulators of the homogenicendothelium and uncover the Notch signaling pathway as a target of 5-hydroxymethylcytosine mediated gene regulation.8 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts171

ZDM8Boston | USA 2015ORAL or POSTERZDM8 POSTERSCharacterization of the caudal hematopoietic tissue revealshotspots of niche interactions with hematopoietic stem cellsFriedrich G. Kapp, MD 1,2 , Owen J. Tamplin, PhD 2 , Ivie Tokunboh 1 , Doug S.Richardson, PhD 3 , Yawei Kong, PhD 4 , Eric Liao, MD, PhD 4 , Leonard I. Zon, MD1,21. Department of Stem Cell and Regenerative Biology and Harvard Stem Cell Institute,Harvard University, MA 02138, USA2. Stem Cell Program and Division of Hematology/Oncology, Boston Children's Hospital andDana Farber Cancer Institute, Howard Hughes Medical Institute, Harvard Stem Cell Institute,Harvard Medical School, Boston, MA 02115, USA3. Harvard Center for Biological Imaging, Harvard University, MA 02138, USA4. Center for Regenerative Medicine, Massachusetts General Hospital, Harvard MedicalSchool, Boston, MA 02114, USA; Division of Plastic and Reconstructive Surgery,Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114, USA; ShrinersHospitals for Children, Boston, MA 02114, USAThe importance of the interactions between hematopoietic stem cells (HSC) and theirniche has been highlighted in recent years. The caudal hematopoietic tissue (CHT) isthe equivalent of the fetal liver in mammals. Here, we characterize this HSC niche byperforming time-lapse confocal imaging. Inpp5d has been implicated as beingimportant for niche function of mesenchymal stromal cells. We generated aninpp5d:zsgreen transgenic line that preferentially labels CHT-specific sinusoidialvessels at 3 dpf suggesting specialized endothelial cells in the HSC niche. We alsoestablished a cxcl12b:zsgreen transgenic line that labels neural crest cells andperivascular cells, the latter surrounding sinusoidal vessels in the CHT. Neural crestcells invade the CHT starting at 32-36 hpf and give rise to perivascular melanocytesand to Schwann cells on nerves that reach the space between the aorta and thecaudal vein plexus. Using the recently published runx:mCherry line we identifiedhotspots of HSC activity between the dorsal aorta and the upper branch of the caudalvein plexus, where most HSCs lodging and cell division events take place. HSCactivity was associated with sox10 positive melanocytes and cxcl12b positiveperivascular cells. We treated embryos with the erbb3 inhibitor AG1478 that inhibitsthe migration of Schwann cells along nerves but does not affect melanocytemigration. While Schwann cells were depleted in the CHT after treatment, cxcl12bpositive perivascular cells and melanocytes remained unchanged and we did not seean effect on HSC numbers or on HSC/melanocyte interactions. In summary, weidentified three populations in the CHT - endothelial, stromal and neural crest derived- that co-localize with HSC activity and showed that inhibition of Schwann cellmigration does not affect HSC numbers in the CHT, highlighting the importanceperforming functional studies in addition to analysis of spatial associations.1728 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts

ZDM8 POSTERSZDM7Madison | USA 2015ZDM8Boston | USA 201511,12-epoxyeicosatrienoic acid regulates hematopoiesis via a distinctG-protein coupled receptorJamie L. Lahvic 1 , Pulin Li 1 , Michelle Ammerman 2 , Leonard I. Zon. 11. Harvard Medical School/Boston Children’s Hospital, Boston, MA2. Boston Children’s Hospital, Boston, MAIn a screen for enhancers of competitive hematopoietic stem cell transplantation in thezebrafish, we identified the endogenous lipid signaling molecule, 11,12-epoxyeicosatrienoic acid(EET). This eicosanoid has anti-inflammatory and vasodilatory effects, and we found that itspro-hematopoietic phenotype is conserved in mice and embryonic zebrafish. EET signalingactivates a G-protein coupled receptor (GPCR) whose identity remains unknown, hinderinggenetic dissection of EET signaling mechanisms. We have used a novel bioinformatic GPCRexpression method to develop a list of candidate EET receptors. We performed RNAseq geneexpression profiling in duplicate on three human cell lines with known EET-binding activity toanalyze basal levels of GPCR expression. We found only 37 GPCRs expressed above athreshold FPKM of 0.3 in all three cell lines. We further profiled HEK293T cells, which lackEET-binding ability, and found that 10 of these receptors have absent or low expression in thiscell line. These 10 candidate EET receptors include well-studied GPCRs, eicosanoid receptors,and orphan receptors. To assay signaling in this diverse group of GPCRs, we are employingthe PathHunter assay, which measures β-arrestin recruitment to the membrane by a specificGPCR. We have determined that 11,12-EET does not signal via either of two prostaglandinreceptor candidates, PTGER2 or PTGER4. Identification of the 11,12-EET receptor is animportant prerequisite for clinical modulation of EET signaling, and will open new avenues togenetic manipulation of EET signaling for mechanistic studies. Additionally, this method ofcomparative basal gene expression analysis across multiple cell lines may be useful for deorphaningother GPCR ligands.8 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts173

ZDM8Boston | USA 2015ZDM8 POSTERSZDM8Elucidation of novel genetic regulators of hematopoietic stem cell development using azebrafish forward genetic screenJulie Perlin 1 , Samantha Collins 1 , Logan Carr 1 , Song Yang 1 , Yi Zhou 1 , Weijun Pan 2 , and Leonard Zon 11. Stem Cell Program and Division of Hematology/Oncology, Boston Children's Hospital, Harvard Medical School, Boston,MA.2. Institute of Health Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences & ShanghaiJiao Tong University School of Medicine, Shanghai, ChinaMarrow transplantation of hematopoietic stem cells (HSCs) is an important therapy for patients with a variety ofanemias, immunodeficiencies, and leukemias. The transplant relies on the homing and engraftment of donorHSCs to the recipient marrow, yet the genetics of such cell mobility are not understood. We have undertakenthe first large-scale genetic screen to uncover pathways essential for HSC engraftment into the stem cell nichein the zebrafish. 35 mutants were found that were morphologically normal, but that had specific alterations inthe expression of cmyb, a critical hematopoietic transcription factor, in the fetal liver equivalent of the zebrafish,or caudal hematopoietic tissue (CHT). In development, HSCs arise from the aorta, enter circulation, and thenundergo dynamic interactions with associated endothelial cells to form the blood stem cell niche. Six mutantlines have been cloned and the mutated genes responsible for the cmyb phenotype participate in cell cycleregulation, cell proliferation, and cell-cell interactions. In an additional mutant line, LDD764, HSCs are born inthe aorta, yet fail to engraft into the CHT. In this mutant a conserved serine is mutated in the RNA bindingprotein Nol9. Nol9 is highly expressed in Burkitt’s Lymphoma, CD34+ hematopoietic cells, and T cells, and isrequired for ribosome biogenesis. Defects in ribosome biogenesis lead to very specific hematological diseasesand malignancies such as 5q- syndrome, a subtype of Myelodysplastic syndrome, Diamond Blackfan anemia,and Schwachman-Diamond syndrome. RNA sequencing has revealed that expression of niche factors arereduced in LDD764 mutants. It is therefore possible that a non-autonomous defect in the niche causes areduction of HSCs and a subsequent reduction of T cells. Further studies will provide insight into HSCdevelopment and hematopoietic niche formation, and the genes and pathways uncovered may be able to bemanipulated to augment therapeutic marrow transplantation.1748 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts

ZDM8 POSTERSZDM7Madison | USA 2015ZDM8Boston | USA 2015SXR and PPARα activation bypass the requirement of tif1γ in erythroiddifferentiation.Marlies P. Rossmann 1 , Xiaoying Bai 2,3 , Eugenia Custo Greig 1 , Bilguujin Dorjsuren 1 , Chang-Ying Chiang 1 , Lauren Krug 1 , Wahaj Chaudhry 1 , Shakkaura Kemet 1 , Isaac Adatto 1 , LeonardI. Zon 1,2 .1 Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA;2 Stem Cell Program and Hematology/Oncology, Children's Hospital Boston, Howard Hughes MedicalInstitute, Boston, MA;3 Cecil H. and Ida Green Center for Reproductive Biology Sciences, Department of Obstetrics/Gynecology,The University of Texas Southwestern Medical Center, Dallas, TXAberrant erythroid differentiation can give rise to anemias and leukemias. Regulated transcriptionelongation at erythroid loci is vital for specific differentiation steps during blood development.Transcriptional intermediary factor 1 gamma (TIF1γ), the gene mutated in the blood deficientzebrafish moonshine mutant, recruits positive elongation factors to erythroid genes to relievepaused Pol II. To elucidate the TIF1γ-mediated mechanisms in erythroid differentiation, weperformed a chemical suppressor screen in moonshine mutant embryos with 3,400 chemicalcompounds. Employing this strategy we have identified two clinically used compounds, Clofibrateand Leflunomide, to rescue formation of βe3 globin-expressing cells at 22 hpf in 70 - 90% ofmoonshine embryos as assessed by in situ hybridization. Leflunomide, an inhibitor ofdihydroorotate dehydrogenase, blocks de novo pyrimidine synthesis and reduces wild-typeβe3 globin expression. However, Leflunomide rescues the moonshine bloodless phenotype, atleast partially through the activation of the steroid xenobiotic receptor (SXR). Clofibrate is anagonist of both, SXR and the peroxisome proliferator-activated receptor alpha (PPARα). Toidentify the PPARα-interacting proteome in erythroid progenitors, we established a human K562erythroleukemia cell line with dox-inducible expression of PPARα and PPARα target genes.Large-scale Flag-immunoprecipitation followed by mass spectrometric analysis identifiedPPARα’s heterodimerization partner RXR, co-activators and -repressors, 24 subunits of themediator complex, six of the cohesin complex, seven RNA Pol II subunits as well as DSIF andp-TEFb, two positive regulators of transcription elongation. Morpholino-mediated knockdown ofthe mediator complex subunit med1 prevents Clofibrate-mediated rescue of βe3 globinexpression in moonshine embryos. Given the association with specific elongation and chromatinfactors, we hypothesize that PPARα integrates these factors to facilitate elongation of erythroidtranscripts. Our studies provide a basic understanding of the processes regulating transcriptionelongation in the differentiation of hematopoietic cells, and could lead to novel therapeuticstrategies.8 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts175

ZDM8Boston | USA 2015ZDM8 POSTERSRole of autophagy in vertebrate hematopoiesis and human myelodysplastic syndromeKH Chan, ACH Ma & AYH Leung.Department of Medicine, Li Ka Shing Faculty of Medicine, University of Hong Kong, Hong KongAutophagy is a conserved cellular mechanism critical for degrading expired cytoplasmic constituents.Its molecular machinery consists of induction, autophagosome formation and degradation. Autophagyhas been demonstrated to play a pivotal role in hematopoiesis, with reference to pathogenesis ofmyelodysplastic syndrome (MDS). MDS is a clonal bone marrow disease characterised by insufficienthematopoiesis leading to cytopenias. Cellular consequences of defective autophagy, e.g. damagedmitochondria and increased apoptosis, have been observed in MDS specimens, suggesting the role ofautophagic dysregulation in MDS pathogenesis.It was reported that key players of autophagy machinery, FIP200 and Atg7, are required for properHSC maintenance and differentiation, and ablating them induces anemia and abnormalmyeloproliferation in murine models. However, functional studies of autophagy-related genes arelacking and the precise role of autophagy in vertebrate hematopoiesis remains obscure. The presentstudy focuses on zebrafish atg13 (adaptor protein essential for autophagy initiation complex) and atg7(ubiquitin-like modifier-activating enzyme for autophagosome formation) and examines the role ofautophagy and its dysregulation in hematopoiesis.Spatial and temporal expressions of zebrafish atg13 and atg7 were examined by whole-mount in situhybridisation (WISH) and semi-quantitative RT-PCR. To knock-down atg13 and atg7, translationblockingmorpholinos (MOs) were injected into embryos and their effects were evaluated by WISHand whole-mount autophagy assays (LysoTracker-Red and Autophagy Detection Kit). Moleculartargeting of atg13 and atg7 MOs was confirmed by its ability to quench atg13-EGFP and atg7-EGFPchimeric reporters, respectively. Knock-down of atg13 or atg7 induces no significant defects inzebrafish hematopoiesis. Yet, LysoTracker-Red assay of atg7-knocked-down embryos displayedreduced staining pattern.This serves as a pilot study which warrants the use of GFP-LC3 transgenic lines to monitorhematopoietic-specific autophagy activity in knocked-down embryos. TALEN- and CRIPSR/Cas9-mediated mutagenesis will also be employed to generate autophagy-related mutant lines for furtherinvestigation of their functions in embryonic and definitive hematopoiesis.1768 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts

ZDM8 POSTERSZDM7Madison | USA 2015ZDM8Boston | USA 2015A novel role of Aldh16a1 in vertebrate erythropoiesisAlvin CH Ma, CH Man, Heron HC Han, Anskar YH LeungDepartment of Medicine, LKS Faculty of Medicine, The University of Hong Kong, Hong KongBackground: Aldehyde dehydrogenase (aldh) are a group of NAD(P)+-dependent enzymesinvolved in the biosynthesis of retinoic acid and a wide-range of metabolism. We previouslydemonstrated that inhibition of aldh activity with diethylaminobenzaldehyde (DEAB)expanded primitive hematopoeisis in zebrafish embryos and conducted a morpholino(MO)-mediated reverse genetic screen on the hematopoietic function of 15 individual aldhisoforms. Although none of the individual knock-down recapitulated DEAB treatment, weidentified that aldh16a1, an uncharacterized isoform, may play a novel role in regulatingerythropoiesis.Methods: Expression pattern of zebrafish aldh16a1 was examined by RT-PCR andwhole-mount in situ hybridization (WISH). aldh16a1 knock-out model was generated withTALEN and effect on embryonic hematopoiesis was examined. ALDH16A1 was alsoknock-down using shRNA in human cord blood (CB) CD34+ population and the effect onhematopoietic lineage differentiation was examined by colony-forming assay.Results: aldh16a1 expressed along the mesoderm at early stages and predominantly indeveloping head and anterior pronephric duct at 24 hpf. Later at 48 hpf, it also expressed incausal hematopoietic tissue. TALEN-mediated aldh16a1 mutagenesis was confirmed byRFLP assay and DNA sequencing. In aldh16a1 mutant embryos, although early initiation oferythropoiesis as shown by gata1 expression at 12 hpf was not affected, erythropoiesis at 18hpf was significantly reduced. Other hematopoietic lineages were not affected. TUNEL assayshowed that apoptosis was induced in erythroid cells and erythroid maturation was alsoaffected as shown by cell morphology. In human CB CD34+ population, knock-down ofALDH16A1 significantly reduced the number of CFU-E without affecting CFU-GEMM/GM.Conclusion: aldh16a1 knock-out significantly suppressed erythropoiesis by inducingapoptosis and also perturbing erythroid maturation. Known-down of ALDH16A1 in human CBCD34+ population also perturbed erythroid lineage differentiation, suggesting that Aldh16a1may have a conserved role in regulating vertebrate erythropoiesis. Further studies arewarrant to investigate the unknown underlying mechanism of Aldh16a18 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts177

ZDM8Boston | USA 2015ZDM8 POSTERSPrimitive Myeloid Cells Facilitate Definitive Blood Stem CellMigrationElliott J. Hagedorn, Julie R. Perlin, Christopher R. D’Amato, Owen J. Tamplin andLeonard I. Zon.Boston Children’s Hospital, 300 Longwood Avenue, Boston, MA 02115As in mammals, zebrafish hematopoietic stem and progenitor cells (HSPCs) are born inthe aorta-gonad-mesonephros (AGM) region, then enter circulation and transientlycolonize the caudal hematopoietic tissue (CHT, hematopoietic equivalent to themammalian fetal liver), before seeding the adult organs of the thymus and kidney(mammalian bone marrow equivalent). Using HSPC-specific transgenes (cd41:eGFP orrunx1+23:NLS-mCherry) and spinning disk confocal microscopy, we have been able tovisualize with high spatial and temporal resolution, the distinct steps of HSPC migration.To investigate the different cell types that regulate these migration events, we undertooka targeted cell ablation approach and have identified a role for primitive myeloid cells.Whole-embryo ablation of macrophages, using a macrophage-specific NTR transgene orby injection of clodronate liposomes, results in a phenotype in which HSPCs fail tomigrate out of the AGM and instead remain embedded in the fibronectin-richextracellular matrix between the aorta and posterior cardinal vein (PCV). Live cellimaging of the AGM in wild-type embryos double positive for cd41:eGFP andmpeg1:mCherry (macrophages) revealed direct physical interactions between HSPCsand macrophages that lasted 30-45 minutes and often resolved with the HSPC migratinginto the PCV (7/10 HSPCs examined interacted with a macrophage before entering thePCV). Chemical inhibition or morpholino knockdown of matrix metalloproteinases(Mmps) resulted in an accumulation of HSPCs in the AGM, similar to that observed withmacrophage depletion. Colocalization analysis using immunohistochemistry and in situhybridization, however, revealed the two myeloid-specific Mmps (mmp-9 and -13a) areexpressed primarily by neutrophils, suggesting a mechanism whereby mmp-expressingneutrophils coordinate with macrophages to release HSPCs from the AGM. Thesestudies advance the basic understanding of the cell-cell interactions that regulate HSPCmigration and may have important clinical implications for transplantation-basedtherapies for blood diseases.1788 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts

ZDM8 POSTERSZDM7Madison | USA 2015ZDM8Boston | USA 2015The Role of Calreticulin (Calr) in Vertebrate HematopoiesisKF Man, Anskar YH Leung, Alvin CH Ma.Department of Medicine, LKS Faculty of Medicine, University of Hong Kong, Hong KongIntroduction Calreticulin (CALR) is a multi-functional protein mainly controllingprotein folding and calcium homeostasis in endoplasmic reticulum. Recently, recurrentmutations in CALR have been found in essential thrombocythemia (ET) and primarymyelofibrosis (PMF) patients without Janus kinase 2 (JAK2) or thrombopoietin (MPL)mutations. Previous studies have shown that CALR mutation might act through theJAK2-STAT5 signaling pathway in myeloproliferative neoplasm. However, thefunctional role of CALR in hematopoiesis remains unknown. In this study, we madeuse of zebrafish model to examine the unknown roles of calr in vertebratehematopoiesis. Methods Gene expression of calr and other hematopoietic geneswere evaluated by whole-mount in situ hybridization (WISH) and quantitative RT-PCR(Q-PCR). Morpholino (MO) and transcription activator like effector nuclease (TALEN)were used to knock down and knock out calr. Results Zebrafish calr is expressedpredominantly in head region during embryonic stage. It is also expressed in thehatching glands and axial vasculature at 18-24 hours post-fertilization (hpf), in caudalhematopoietic tissue (CHT) at 48hpf and in the lateral line in later stage. In adultzebrafish, calr is highly expressed in kidney, liver, intestine, muscles and skin tissues.Knock-down of calr resulted in a decrease in expression of genes associated withmyeloid lineages at 24hpf, including l-plastin, mpo and mpeg1. It also led to anincrease in expression of c-myb (associated with HSCs) at 48, 72 and 96hpf. Toconfirm these MO-mediated phenotypes and further examine the role of calr in adulthematopoiesis, TALENs targeting exon-1, -5 and -9 were used to generate calrknock-out models and their mutagenic activities were shown by restriction fragmentlength polymorphism (RFLP) assay. Conclusion Knock-down of calr altered myeloidand HSCs lineages during zebrafish embryonic development. It provides importantground for further understanding of the unknown role of Calr in vertebratehematopoiesis8 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts179

ZDM8Boston | USA 2015ZDM8 POSTERSA screen for epigenetic regulators of hematopoiesis reveals a requirement for Ing4 inHSC specificationKatie L. Kathrein 1,2 , Ashley McNeil 1 , Ellen Durand 1,2 , Rebecca Maher 1 , and Leonard I. Zon 1,2,31 Stem Cell Program, Division of Hematology/Oncology, Boston Children's Hospital and Dana Farber Cancer Institute,2 Harvard Stem Cell Institute, Harvard Medical School, 3 HHMI, Boston, MADuring development, hematopoietic stem cells (HSCs) are specified from hemogenic endothelium. TheseHSCs self-renew, proliferate and differentiate to form mature blood cells. Regulation of this process is achievedin part by factors that orchestrate chromatin structure and establish an epigenetic code for hematopoiesis. Togenerate a complete compendium of factors that regulate the epigenetic code in HSCs, we have undertakenthe first large-scale in vivo reverse genetic screen targeting 488 chromatin remodeling factors to determinetheir role in HSC specification. We identified 29 genes that alter HSC marker expression upon knockdown.Some hits are members of complexes already known to regulate hematopoiesis, such as the polycomb, NuRD,and trithorax complexes. Other complexes, like the Hbo1 complex, have no known role in hematopoiesis. TheHbo1 complex functions to promote gene expression through the binding of trimethylated histone H3K4 byIng4, which results in localized chromatin acetylation by Hbo1. Four members of the complex, Ing4, Phf16(Jade3), Hbo1, and Brd1, were identified in the screen. To test the function of the complex in HSCspecification, we minimally inhibited expression of Ing4, Brd1, and Phf16 and found that they geneticallyinteract to regulate HSC specification. Ing4 has been shown to regulate the transcription factor NF-kB throughsequestration of the RelA component of NF-kB at target loci. Using ChIP-seq for ING4 in human CD34+ cells,we show that ING4 is bound to many regulators of blood development including c-MYB, LMO2, RUNX1 andIKAROS. ING4 is also bound to several NF-kB target genes. Ing4 negatively regulates NF-kB, suggesting thatloss of Ing4 results in an overabundance of NF-kB signaling. We show that loss of both Ing4 and NF-kB targetgenes can rescue HSC specification. These results suggest that Ing4 and the Hbo1 complex are criticalregulators of HSC specification.1808 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts

ZDM8 POSTERSZDM7Madison | USA 2015ZDM8Boston | USA 2015Investigating the Role of Wnt16 Signaling during HSC Niche SpecificationNikki Glenn 1 , Chaithu Parupalli 1 , Wilson Clements 11 Department of Experimental Hematology, St. Jude Children’s Research Hospital, Memphis, TNHematopoietic Stem Cells (HSCs) are born from endothelial cells in the floor of the primitive descending aorta,known as the dorsal aorta in zebrafish. A better understanding of the cellular specification niche that regulatesthe birth of these cells might inform attempts to instruct their specification in vitro. Recently, Wnt16, a noncanonicalWnt, was demonstrated to be required for HSC development in zebrafish. Wnt16 signals through aseries of downstream relay signals, but the final signal(s) most proximal to HSC specification remain unknown.In Wnt16 loss of function animals, there is also an earlier defect in a compartment of the somite, thesclerotome, which may house vascular smooth muscle cells. These results suggest that sclerotome-derivedcells might contribute to the regulatory environment, or “niche”, that directs HSC specification, explaining thefailure of HSC specification when this compartment develops incorrectly. We have found that loss of thesclerotomal genes results in loss of HSCs and are further investigating the role of this somite compartment inHSC niche formation through generation of a transgenic sclerotomal zebrafish line. We have identifiedputative receptors for the Wnt16 signaling cascade, including Frizzled 2 (Fzd2). Elucidating additionalcomponents of the Wnt16 pathway will reveal how the HSC specification niche is formed during development,and test the contribution of the somite sclerotome compartment to hematopoietic ontogeny. Our studies willprovide a foundation to define the in vivo cellular specification environment and elucidate the complete set ofsignals involved in HSC specification, with the ultimate goal of informing clinical efforts at directeddifferentiation of pluripotent stem cells to an HSC fate.8 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts181

ZDM8Boston | USA 2015ZDM8 POSTERSDecreased leukemia initiating cells in hdac1 haploinsufficient T-ALLMargaret Pruitt, Sean McConnell, Anthony Restaino, Wilfredo Marin, and Jill deJongDepartment of Pediatrics, University of Chicago, Chicago, ILT-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematologic malignancythat accounts for 10-15% of pediatric and 25% of adult ALL cases. While treatment of T-ALL has improved, relapse is still common and is associated with a poor prognosis. Ashistone deacetylases (hdacs) are aberrantly expressed in hematopoietic malignancies,and hdac inhibitors are used to treat some cancers, we hypothesized that hdac1 mightplay a role in leukemogenesis. Zebrafish T-ALL can be generated efficiently by coinjectingplasmids driving expression of the murine c-myc oncogene and the fluorophoremCherry in immature lymphocytes using the rag2 promoter. When T-ALL is generatedin hdac1+/- fish, we find no significant difference in the tumor latency or in the survivaltime between hdac1+/- and wild type (WT) fish. However, when primary tumor cells aretransplanted into syngeneic recipient fish, the hdac1 haploinsufficient tumors grow at aslower rate when compared to WT tumors. We determined that the slower rate ofgrowth is due to a lower frequency of leukemia initiating cells (LICs) in the tumorpopulation of hdac1+/- fish (1 in 125 cells for hdac1+/-; compared with 1 in 39 cells forWT; p

ZDM8 POSTERSZDM7Madison | USA 2015ZDM8Boston | USA 2015Using oncogene combinations to model myeloid leukaemia inzebrafishElisa Alghisi 1 , Martina Konantz 1 , Marina Mione 3 , Claudia Lengerke 1,21 Department of Biomedicine, University Hospital Basel, Basel – Switzerland2Clinic for Hematology, University Hospital Basel, Basel, Switzerland3Institute of Toxicology and Genetics, Karlsruhe Institute of Technology (KIT), Eggenstein-Leopoldshafen, GermanyThe zebrafish is versatile experimental model for hematopoietic studies. The conservedfunctions of genes involved in leukaemogenesis indicate that it may also be used to studymalignant haematopoiesis. Several oncogenes involved in human leukaemia have beentransiently overexpressed in zebrafish embryos. Despite first encouraging results theseexperimental models often failed to fully recapitulate human myeloid malignancy perhaps dueto difficulties in targeting the right cellular compartment.We took advantage of the Gal4/UAS binary system and existing transgenic lines tooverexpress oncogenes in specific zebrafish blood cells. We first used the Friend leukaemiavirus integration 1 (fli1) promoter to drive the expression of oncogenic HRAS and observedthe myelo-erythroid proliferation characterized by the expansion of the caudal hematopoietictissue, enhanced expression of myelo-erythroid genes and delayed erythrocyte maturation(Alghisi et al. 2013). Currently, we are using this model to investigate the effect of HRASoverexpression at other maturation stages by using runx1, pu.1 and mpeg reporter lines.Preliminary data surprisingly suggest that HRAS overexpression in runx1-positive cells doesnot induce a similarly consistent CHT expansion. More detailed analyses are underway andwill be presented at the meeting.To investigate the role of potential cooperating genetic events, we have generated a UAStransgenic line for the zebrafish homologue ecotropic viral insertion site 1 (evi1). EVI1 is apotent myeloid oncogene associated with aggressive leukaemia and recent mutationalanalyses of EVI1 positive human acute myeloid leukaemias revealed concomitant RASmutations in a majority of cases (Lavallée et al. 2014). Currently we generate zebrafish linesto test the effect of HRAS and evi1 co-expression under the control of different hematopoieticpromoters. Our aim is to investigate the interaction between HRAS and EVI1 duringleukaemogenesis and to establish zebrafish leukaemia models that can be used for furtheranalyses and eventually also for small molecule screens.8 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts183

ZDM8Boston | USA 2015ZDM8 POSTERSDevelopment of Multi-lineage Hematopoiesis in Two Novel Zebrafish runx1MutantsErica Bresciani 1 , Blake Carrington 2 , Erika M. Kwon 1 , MaryPat Jones 3 , Stephen Wincovitch 4 , RamanSood 1,2 , Paul Liu 11. Oncogenesis and Development Section, National Human Genome Research Institute, NIH2. Zebrafish Core, National Human Genome Research Institute, NIH3. Genomics Core, National Human Genome Research Institute, NIH4. Cytogenetics and Microscopy Core, National Human Genome Research Institute, NIHLong term hematopoietic stem cells are essential for the life-long maintenance of the hematopoietic systemof an organism. The transcription factor RUNX1 is required for the emergence of definitive hematopoieticstem cells (HSCs) from the hemogenic endothelium during the embryo development. Runx1 knockoutmouse embryos lack all definitive blood lineages and cannot survive past embryonic day 13. However, wepreviously showed that zebrafish homozygous for an ENU-induced truncation mutation in runx1(runx1 W84X/W84X ) were able to recover from a larval “bloodless” phase and develop adult definitivehematopoiesis, suggesting the formation of runx1-independent adult HSCs. Therefore, in order to furtherinvestigate if RUNX1-independent pathway(s) exists for the formation of adult HSCs, we generated two newrunx1 mutants that present a deletion of 8 bp (runx1 del8/del8 ) and a deletion of 25 bp (runx1 del25/del25 ) withinexon 4 of runx1 using the TALEN technology. These mutations cause frameshifts and prematureterminations, resulting in loss of function of runx1 (runx1 -/- ). Both runx1 del8/del8 and runx1 del25/del25 mutantembryos lack expression of the HSC marker c-myb and fail to develop definitive blood lineages.Interestingly, about 40% of runx1 del8/del8 and runx1 del25/del25 mutants recover from a bloodless phase anddevelop to adulthood. runx1 del8/del8 larvae gradually lose circulating blood cells and become bloodlessbetween 8 and 14 dpf. However they gradually regain circulating blood cells between 15 and 20 dpf. Takentogether, our data is consistent with the previously described runx1 W84X/W84X phenotype and supports thepossibility of a runx1-independent mechanism for HSC formation.1848 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts

ZDM8 POSTERSZDM7Madison | USA 2015ZDM8Boston | USA 2015Distinct roles for matrix metalloproteinases 2 and 9 in zebrafishhematopoietic stem cell emergenceLindsay N. Theodore 1 , Mauricio Cortes 1 , Kelsey Natsuhara 2 , Sarah Y. Liu 1 , WolframGoessling 3 , Trista E. North 11. Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA.2. Harvard University, Cambridge, MA.3. Brigham and Women’s Hospital, Harvard Medical School, Boston, MA.Matrix-metalloproteinases (MMPs) are a group of extracellular matrix (ECM) proteinsinvolved in regulating cell interactions, migration and signaling. Despite the importanceof ECM remodeling in tissue homeostasis, the role of MMPs in hematopoietic stem cell(HSC) formation is poorly understood. A screen of MMP-inhibitors in the zebrafishidentified MMP2 and MMP9 as modulators of hematopoiesis. HSCs are born in theaorta-gonad mesonephros (AGM) region and migrate to/expand in the caudalhematopoietic tissue (CHT) before colonizing the thymus and kidney. Chemical andgenetic inhibition of MMP2 during HSC emergence retained runx1+ cells in the AGM,delaying their migration to the CHT. These findings are consistent with the broadexpression of MMP2 during the onset of hematopoiesis and its enrichment in thevascular niche. In contrast, MMP9 expression is restricted to a subpopulation of myeloidcells, and its loss did not affect HSC migration, instead resulting in aberrant HSCexpansion within the CHT. Additionally, MMP2/9 inhibition led to abnormal colonizationof the thymus. Repression of MMP2/9 activity prior to thymus colonization by AGMderivedHSCs reduced the number of rag+ cells in the thymus; however, inhibition afterthe initial seeding of the thymus led to an increase in rag+ cells, indicating a temporaldependencefor MMP activity on lymphoid migration and maturation. We previouslyidentified MMP9 as a downstream target of prostaglandin-E2 (PGE2). Co-treatment withPGE2 and an MMP9 inhibitor increased HSC numbers within the CHT compared toPGE2-only treatment. Conversely, co-treatment with MMP2 attenuated the PGE2-mediated enhancement of AGM HSC production, supporting the idea that MMP2 andMMP9 have distinct functions in hematopoiesis. In sum, our findings indicate that MMP2is required for HSC budding and migration between the embryonic sites ofhematopoiesis, whereas MMP9 is involved in HSC expansion in the CHT, and bothMMP2/9 are required for colonization of the thymus.8 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts185

ZDM8Boston | USA 2015ZDM8 POSTERSIdentification of approved drugs that are selectively active againsthematopoietic stem and progenitor cells with inactivating mutationsof TET2Chang-Bin Jing 1 , Evisa Gjini 1 , and A. Thomas Look 1,*1 Dana-Farber Cancer Institute, 450 Brookline Ave, Boston, MA 02215*Corresponding author: A. Thomas Look Thomas_Look@dfci.harvard.eduThe Ten-ElevenTranslocation-2 (TET2) gene encodes a member of the TET family of DNAmethylcytosine oxidases that converts 5-methylcytosine (5mC) to 5-hydroxymethylcytosine(5hmC) to initiate the demethylation of DNA within genomic CpG islands. Somatic loss-offunctionmutations of TET2 are frequently observed in human myelodysplastic syndrome (MDS),which is a clonal malignancy characterized by dysplastic changes of developing blood cellprogenitors leading to ineffective hematopoiesis. These mutations represent a transformationalevent in hematopoietic stem and progenitor cells (HSPCs), inducing a premalignant state ofclonal dominance that predisposes to the acquisition of additional mutations, which then lead toovert malignancies. The “preleukemic state” of patients in remission provides an opportune timeto wipe out the mutant clone. We have developed tet2-/- zebrafish and have shown thatheterozygous and homozygous tet2 mutant zebrafish are viable and fertile and develop clonalmyelodysplasia of hematopoietic cells in the kidney marrow at 11 months of age. By 24 monthsof age, the tet2 mutant zebrafish have progressed to full-blown MDS with anemia. Thesefindings indicate that the clonal dominance by tet2-mutated HSCs represents a critical precursorevent in the development of additional mutations leading to myeloid cell malignancies. We areusing the zebrafish tet2 model to screen the FDA-approved drugs that are synthetic lethal withtet2 loss in HSPC. With whole in situ hybridization, we are analyzing approved drugs to identifycompounds that selectively cause diminished numbers of definitive HSPCs in the caudalhematopoietic tissue of tet2+/- and tet2-/-, but not wild-type, zebrafish embryos treated from 24to 120 hours post-fertilization. We anticipate that this analysis of HSPCs in zebrafish embryoswill yield candidate small molecule drugs that are synthetic lethal with tet2 loss in hematopoiesis,and that these drugs will be ideal for testing in mammalian systems to selectively eliminate Tet2mutant HSPCs.1868 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts

ZDM8 POSTERSZDM7Madison | USA 2015ZDM8Boston | USA 2015The role of transcription pausing in hematopoietic stem celldevelopmentQiwen Yang, Xiuli Liu, Ting Zhou, Jennifer Cook, Kim Nguyen, Xiaoying BaiGreen Center for Reproductive Biology Sciences, Dept. of Ob/Gyn, UT Southwestern Medical Center,Dallas, TX 75390The emergence of hematopoietic stem cells (HSCs) in vertebrate embryos is tightly regulated byRNA polymerase II (Pol II)-mediated gene transcription. Increasing evidences in recent yearshave demonstrated that transcriptionally engaged Pol II often pauses shortly after initiation bypausing factors DSIF and NELF, and requires positive elongation factors P-TEFb for pauserelease. How this pausing-to-elongation transition contributes to cell fate determination is largelyunknown. In a zebrafish spt5 hypomorph mutant, we detected a dramatic loss of definitive HSCsin 36hpf embryos. Spt5 is a key subunit of DSIF and this mutant Spt5 protein has been reportedto specifically disrupt its pausing function. Similar reduction of HSCs was also detected inembryos lacking the other pausing factor NELF. We further found that the HSC defect in spt5mutant could be rescued by inhibiting Pol II pausing-to-elongation transition with either P-TEFbinhibitors or knockdown of P-TEFb subunit cdk9. Intriguingly, paused Pol II has been suggestedto play a positive role in maintaining transcription by keeping an accessible chromatin structureat some gene promoters in Drosophila. In line with this positive role of pausing, in spt5 mutantwe detected a consistent downregulaion of multiple genes involved in inflammatory signalingpathways, especially the genes regulating interferon signaling. Recent studies havedemonstrated that inflammatory signaling, including IFNγ and its downstream effector JAK-STAT, plays an essential role in HSC specification in both zebrafish and mouse embryos.Consistent with these studies, we detected a reduction of JAK2 activity in mutant embryos, anda partial rescue of HSCs by activating JAK-STAT. Furthermore, in a chemical suppressorscreen we found that GSK3 inhibitors could rescue HSCs in spt5 mutants through upregulatingstat3 transcription. Our data therefore suggest that transcription regulators and developmentalsignals converge at the paused Pol II to regulate HSC emergence in vertebrate embryos.8 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts187

ZDM8Boston | USA 2015ZDM8 POSTERSPDGFRB signaling acts downstream of metabolic alteration to affecthematopoietic stem cell induction in the zebrafish embryoSung-Eun Lim 1 , Virginie Esain 1 , Mauricio Cortes 1 and Trista E North 11BIDMC and Harvard Medical School, Boston MA 02115Hematopoietic stem cells (HSCs) have the ability to both self-renew and differentiate into all themature blood cell lineages and thereby reconstitute the entire blood system. In addition to theirendogenous role in maintaining blood homeostasis, HSCs are also therapeutically valuable forthe treatment of hematological malignances and bone marrow failure. Thus, a betterunderstanding the factors and pathways involved in the induction and maintenance of HSCs isof great therapeutic potential. While the function of PDGFRB signaling is well established invascular development, its role in hematopoiesis is less well understood. We recently showedthat transient glucose elevation elicited dose-dependent effects on HSCs through elevatedmetabolic activity and subsequent ROS-mediated induction of hif1a. PDGFB, a hif1a-targetgene, was identified as the most upregulated target gene in response to metabolic stimulationby gene expression analysis. Morpholino knockdown of PDGFRB led to decreased HSPCformation by runx1 WISH and flk1+myb+ HSPC cell counts; chemical inhibition of PDGFRBsignaling using AG1295 during the period of hematopoietic development also led to decreasedrunx1+ expression at 36hpf by WISH. Similar results were seen in the context of hif1alphastimulation, indicating that PDGFRB signaling is a key downstream mediator in metabolicinduction of HSCs. No gross vascular abnormalities were observed in PDGFRB morphants asdetermined by ve-cadherin and fli/flk expression, suggesting the effects on HSCs are occurringindependently of PDGFB function in vascular development. Significantly, overexpression ofPDGFB led to enhanced runx1+ HSPC formation at 36hpf as well as cmyb+ HSPCs in the CHTat 48hpf. A survey of known PDGFB regulatory targets by qPCR showed a significant increasein inflammatory cytokines including IL-6. Epistatic investigations are ongoing to confirm the roleof IL-6 as the relevant downstream mediator of PDGFRB signaling in HSPC formation.1888 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts

ZDM8 POSTERSZDM7Madison | USA 2015ZDM8Boston | USA 2015Phenotypic Assays using Zebrafish Hematopoesis Models forElucidation of Hematopoietic ToxicityAnna Lenard 1 , Claudia McGinnis 2 and Claudia Lengerke 11. University Hospital Basel, Hebelstrasse 20, 4031 Basel, Switzerland.2. Pharma Research and Early Development, Mechanistic Safety, Roche Innovation Center,Grenzacherstrasse 124, 4070 Basel, Switzerland.Hematologic findings are one of the most common side effects encountered in preclinicalsafety testing of new drug candidates. The consequences of direct or indirect damage toblood cells and their precursors can be potentially life-threatening, and hence, hematotoxicitycan lead to the termination of a promising drug candidate.Current hematotoxicity testing employs in vitro models with a cell viability read-out. However,this approach only allows a limited read-out and, for example, does not capture effects onlater maturation stages of blood cell progenitors. Here, we propose zebrafish as analternative animal model that captures the full range of hematopoietic lineages andmaturation stages in an in vivo setting. Using flow cytometry and high-content in vivo imagingof fluorescent reporter expression we analyze defined hematopoietic lineages in regards tocell numbers, distribution and activity.Currently, the first set of reference compounds is being evaluated, in order to validate theutility and sensitivity of this model system.A double transgenic reporter system was established for flow cytometry analyses of erythroidlineage (Tg(gata1:DsRed);(globin:GFP)) allowing distinguishment of mature globin/gata1double positive erythrocytes from gata1-only positive erythroid progenitors. The line wasvalidated in a proof-of-principle phenylhydrazine-induced hemolytic anemia assay wheredepletion of mature erythrocytes was documented. Furthermore, treatments with antimitoticcancer drugs showed time-dependent depletion of progenitors and/or mature erythroid cells.Additionally, an automated image analysis tool was established for assessment of neutrophiland macrophage numbers using 3D-imaged, live embryos from transgenic linesTg(mpo:GFP), Tg(mpeg1:Gal4;UAS:Kaede). Further transgenic lines, e.g. for lymphoid andstem cell lineages, are currently generated and assessed for imaging and flow cytometryassays.It is expected that upon successful validation, this new multiplexed in vivo model will findimmediate application in predictive and mechanistic preclinical safety testing, complementingexisting in vitro hematopoietic approaches and more complex in vivo studies in mammals.8 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts189

ZDM8Boston | USA 2015ZDM8 POSTERSTransgenic zebrafish expressing mCherry under the control of thehemogen promoter recapitulate the erythropoietic program andknockdown of hemogen causes pronounced embryonic anemiaMichael Peters, Sandra Parker, and H. William Detrich, IIINortheastern UniversityAn increasing number of erythroid genes have been identified in zebrafish that functionsimilarly to their human orthologs. Hemogen is a transcription factor expressed in hematopoieticprogenitors that plays a role in erythroid cell survival and differentiation in mammals. Theoverexpression of hemogen induces tumor formation in mice, and this putative oncogene isupregulated in many leukemias and other cancers. To date, the expression profile of hemogenand the function of the Hemogen protein have not been examined in teleosts. Using zebrafish,we show that the spatiotemporal expression pattern of hemogen occurs at sites of primitive anddefinitive hematopoiesis, as is true for mammals. We created transgenic Tg(hemgn:mcherry)zebrafish that recapitulate hemogen expression in the intermediate cell mass, the caudalhematopoietic tissue, and the pronephros. In double transgenic Tg(hemgn:mcherry, LCR:GFP)zebrafish, the reporters colocalize in both primitive and definitive erythroid lineages. Gata1 ispredicted to bind evolutionarily conserved motifs in the hemogen promoter to regulate itsexpression. We show that hemogen transcripts are absent in vlad tepes, a gata1 mutant linethat may model congenital dyserythropoietic anemias in humans. We find that morpholinoknockdown of hemogen leads to a significant loss of mature erythrocytes as quantified by invivo flow analysis. Morphants produce half as many erythrocytes as controls (p

ZDM8 POSTERSZDM7Madison | USA 2015ZDM8Boston | USA 2015Role of Sclerotome in Hematopoietic Stem Cell DevelopmentClair Kelley 1 , Nicole Glenn 1 , Robert Wilkinson and Wilson Clements 11. Department of Hematology, St. Jude Children’s Research Hospital2. Department of Cardiovascular Science, University of Sheffield, Medical SchoolHematopoietic stem cells (HSCs) that develop within the AGM (aorta, gonad & mesonephric)region of the early embryo self renew and ultimately provide a life-long supply of multi-lineageblood cells. In vertebrates, HSCs derive from hemogenic endothelium in the ventral wall of thedorsal aorta. A specialized microenvironment, the hematopoietic niche, supports specificationand maintenance of HSCs, but the origin of these niche cells is not known. HSC-promotingsignals that arise from the niche cells are not completely defined and a better understanding ofthese signals would benefit stem cell therapies. Our previous studies showed that Wnt16 isrequired for the development of HSCs and for normal patterning of sclerotome. Sclerotomearises from the ventromedial portion of the somite and here, we investigate a role for sclerotomein the development of HSCs. We have developed sclerotome transgenic fish using Pax1regulatory elements and use fluorescent imaging to show that sclerotome derivatives surroundthe dorsal aorta prior to and during HSC specification. In addition, we have used loss offunction analyses to show a requirement for several sclerotome genes, including Pax1a, for thedevelopment of HSCs and definitive hematopoietic lineages. Our data suggests that sclerotomederivatives contribute to the HSC niche of the early embryo and forms a basis for defining theniche architecture and signals that drive HSC specification.8 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts191

ZDM8Boston | USA 2015ZDM8 POSTERSLightsheet live imaging reveals the earliest stages of zebrafish kidneymarrow colonization by hematopoietic stem cellsOwen J. Tamplin, PhD 1 , Friedrich G. Kapp, MD 1 , Hunter L. Elliott, PhD 2 , Douglas S.Richardson, PhD 3 , and Leonard I. Zon, MD 11 Hematology/Oncology and Stem Cell Program, Boston Children’s Hospital; Harvard Stem Cell Institute;Harvard Medical School; Howard Hughes Medical Institute2 Image and Data Analysis Core, Department of Cell Biology, Harvard Medical School3 Harvard Center for Biological Imaging, Harvard UniversityHematopoietic stem cells (HSCs) support the entire blood system and reside in a specializedmicroenvironment called the niche. In mammals the adult niche is in the bone marrow and inzebrafish it is in the kidney marrow. The kidney marrow niche is colonized by HSCs between 4and 5 days post fertilization (dpf). We previously described a Runx:mCherry transgenic line thatmarks HSCs in all sites of definitive hematopoiesis throughout development and into adulthood.We have used this line to observe the earliest immigration events of HSCs as they arrive in thekidney marrow. First, we paralyzed embryos by injecting alpha-bungarotoxin protein directly intothe circulation. Next we performed live imaging of the early kidney marrow niche. At 4-5 dpf thekidney marrow is at a depth of ~100 microns from the surface of the embryo and cannot beclearly imaged live using standard confocal microscopy. To overcome this challenge, we usedlightsheet microscopy because its perpendicular objective lenses allow illumination from oneangle and observation from another angle. We performed long term imaging (~16 hours) of theentire kidney marrow and quantified the total number of stem cells. The first wave of HSCcolonization between 4-5 dpf reached a steady state of ~50-60 cells. Shortly before 5 dpf thestem cell pool began to expand at a rate of ~5 cells per hour. This increase could be fromproliferation of HSCs present in the niche and/or migration of more HSCs into the niche—thetemporal resolution of the experiment does not distinguish between these two possibilities. Wehave visualized colonization of what will become the adult HSC niche and estimated the size ofthe stem cell pool that will persist throughout life.1928 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts

ZDM8 POSTERSZDM7Madison | USA 2015ZDM8Boston | USA 2015A Novel Phytosterol increases Hematopoietic Stem Cell HomingYuliana Astuti, Ashley Kramer, Troy C. LundPediatric Blood and Marrow Transplant, University of Minnesota, Minneapolis, MN 55455Inefficient hematopoietic cell homing can result in graft failure after hematopoietic celltransplant (HCT) which has > 90% mortality. Novel compounds that increase homingcould prevent graft failure and reduce mortality. Using a novel bioluminescent-basedimaging (BLI) platform to perform a functional HCT screen, we were able to quantifyhematopoietic cell homing and engraftment activity in the zebrafish as early as 24 hoursafter HCT. We have optimized the BLI acquisition conditions, HCT injection, as well asradiation dosage and show that < 2000 donor hematopoietic cells can routinely bedetected and quantified 1 day post HCT in recipient marrow. We have screened over900 compounds for new small molecules that lead to more robust homing/engraftmentafter HCT. Our first screen generated 78 “hits” of which 30 compounds showed goodreproducibility. Specifically, we discovered a novel phytosterol, ergosterol, was able toincrease the number of hematopoietic cells homing to the zebrafish marrow by 30% afterHCT. Ergosterol is a member of the vitamin D family and a precursor to vitamin D 2 .Experiments in rodents showed that ergosterol treated hematopoietic cells hadincreased homing of lin-Sca+kit+ (LSK) stem cells by 50% versus control 16 hours afterHCT (n = 6 animals/group, p= 0.02). Ergosterol treated LSK cells to expanded 2.5 timesgreater in cell number versus control (p=0.02) in in vitro cell expansion assays and hadincreased viability suggesting that ergosterol has novel pleitropic effects on cellularhoming, cell division, and cell survival. Whether these findings are working through aclassic vitamin D receptor mediated pathway is under current investigation. Inconclusion, we have employed a luciferase-based functional screen to find novel agentsthat increase hematopoietic cell homing and determined that a novel phytosterol canimprove homing, cell survival, and promote cell expansion after HCT that may havefuture clinical implications.8 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts193

ZDM8Boston | USA 2015ZDM8 POSTERSZebrafish: A novel tool to study 3-O sulfated heparan sulfate andHSV-1 infectionVaibhav Tiwari 1, 2 , Deepak Shukla 21 Department of Microbiology & Immunology, Chicago College of Osteopathic Medicine, MidwesternUniversity, Downers Grove, IL 606152 Department of Ophthalmology & Visual Sciences, University of Illinois at Chicago, Chicago, IL 60612For many years, zebrafish have been the prototypical model for studies in developmentalbiology. In recent years, zebrafish has emerged as a powerful model system to study infectiousdiseases including viral infections. In case of herpes simplex virus-1 (HSV-1) a key interactionbetween viral envelope glycoprotein D (gD) and 3-O-sulfated heparan sulfate (3-OS HS)mediates virus-cell fusion and promotes viral spread in primary cultures of corneal stromaderived from the eyes. The 3-O-Sulfation of HS is catalyzed by 3-O-sulfotransferase (3-OST)enzyme. Multiple isoforms of 3-OST are differentially expressed in tissues of zebrafish embryos.Here, we provide evidence that the eyes of zebrafish embryos can be successfully infected withHSV-1 and that the model can be used for the study of 3-OST enzymes in HSV-1 infection. Ourcomprehensive analysis of the role of zebrafish 3-OST isoforms (3-OST-1, 3-OST-5, 3-OST-6,and 3-OST-7) in HSV-1 entry shows some very interesting results. We found that a group of 3-OST gene family isoforms (3-OST-2, -3, -4, and -6) with conserved catalytic and substratebindingresidues of the enzyme mediates HSV-1 entry and spread, while the other group (3-OST-1, -5, and -7) lacks these properties. These results highlight the differences in the finalsproducts generated by various 3-OSTs and help establish zebrafish as a novel and highlyrelevant tool to study HSV-1 infection and pathogenesis.1948 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts

ZDM8 POSTERSZDM7Madison | USA 2015ZDM8Boston | USA 2015T lymphocytes control microbial composition by activelysuppressing overgrowth of pathogenic bacteria in the zebrafish gutS. Brugman 1,91. Department of Paediatric Gastroenterology, Wilhelmina Children’s Hospital, University MedicalCentre Utrecht, Utrecht, the Netherlands9. Animal Sciences Group, Cell Biology and Immunology, Wageningen University, Wageningen,the NetherlandsDysbiosis of the intestinal microbial community is considered a risk factor forthedevelopment of chronic intestinal inflammation as well as other diseases such as diabetes,obesity and even cancer. Study of the innate and adaptive immune pathways controllingbacterial colonization has however proven difficult in rodents, considering the extensivecross-talk between bacteria and innate and adaptive immunity. Here, we used the zebrafishto study dysbiosis. Zebrafish lack a functional adaptive immune system in the first weeks oflife, enabling study of the innate immune system in the absence of adaptive immunity. Weshow that in larval zebrafish, lacking adaptive immunity, Vibrionales species (knownpathobionts) are able to grow out. These species are effectively suppressed upon adaptiveimmune development, whereas in Rag1-deficient zebrafish (lacking adaptive immunitythroughout life) Vibrionales abundance stays high. Using cell transfer experiments, weconfirmed that adoptive transfer of T lymphocytes, but not B lymphocytes into Rag1-deficientrecipients suppresses outgrowth of Vibrionales. Further studies showed that T lymphocytesare necessary for epithelial Cxcl8 expression in the zebrafish intestines and ex vivo exposureof intestinal T lymphocytes to Rag1-deficient microbiota results in increased interferongammaexpression by these T lymphocytes, which does not occur upon exposure to wildtypemicrobiota. In conclusion, we show that T lymphocytes control microbial composition byeffectively repressing the outgrowth of pathobionts in the zebrafish intestine via induction ofcytokines and activation of intestinal epithelial cells.8 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts195

ZDM8Boston | USA 2015ZDM8 POSTERSA zebrafish embryonic model for comparative studies of Aspergillusinfection.Bjørn Koch 1 , Ellen Lagendijk 2 , Natalia Hajdamowicz 1 , Arthur Ram 2* , Annemarie Meijer 1*1 Molecular Cell Biology, Leiden University2 Molecular Microbiology & Biotechnology, Leiden University*These authors contributed equally to the workAmong the species of filamentous fungi of the Aspergillus genus, A. fumigatus stands out asdrastically more pathogenic than the rest. Utilizing a recently published zebrafish embryonic hindbraininfection scheme we aim to explore this trait, by characterizing the infectious development of twoAspergillus species, A. fumigatus and A. niger.Surprisingly, A. niger appears to cause the same overall mortality in infected embryos as A. fumigatus,but at an accelerated pace. The temporal difference in infectious development is manifested as afaster transition to filamentous growth of A. niger as compared to A. fumigatus. Applying confocaltime-lapse microscopy to investigate the interactions between host immune cells and fungal conidia,we noticed a difference in the behavior of incoming macrophages to the hindbrain in response to thetwo species of fungi, within a narrow temporal window at the onset of infection. Whereas A. fumigatusconidia are efficiently phagocytized by macrophages within 6-8 hours after infection, A. niger conidiaare to a significant extent left untouched by macrophages in this timeframe after infection. In the caseof A. niger, unphagocytized conidia will germinate and rapid hyphal growth precedes embryonic death.By contrast, in the case of A. fumigatus, germination and hyphal growth occurs from insidemacrophages heavily laden with engulfed spores. From our observations we have devised a model ofA. niger versus A. fumigatus infectious progression in zebrafish embryos. Further investigationsencompassing mutant genotypes in the host as well as the pathogen will improve our model andunderstanding of the differences in disease progression and virulence factors between A. niger and A.fumigatus.1968 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts

ZDM8 POSTERSZDM7Madison | USA 2015ZDM8Boston | USA 2015System-wide analysis of the T-cell responseMerja Jaronen 1* , Ruxandra Covacu 1* , Hagit Philip 2* , Jorge Almeida 3 , Samuel Darko 3 , JessicaKenison 1 , Chun-Cheih Chao 1 , Gur Yaari 2 , Yoram Louzoun 2 , Liran Carmel 2 , Daniel C. Douek 3 ,Sol Efroni 2 and Francisco J. Quintana 11 Ann Romney Center for Neurologic Diseases, Brigham and Women’s Hospital, Harvard Medical School, 77Avenue Louis Pasteur, Boston, MA 02115, USA.2 The Mina and Everard Goodman Faculty of Life Science, Bar Ilan University, Ramat Gan 52900, Israel.3 Human Immunology Section, Vaccine Research Center, National Institute of Allergy and Infectious Diseases,US National Institutes of Health, Bethesda, Maryland, USA.The T-cell repertoire (TCR), constituted by the pool of TCR specificities, governs the ability of theimmune system to respond to both foreign and self-derived immune challenges. However, due to thelimited power of the research tools used for its investigation, the complexity and the dynamics of theTCR repertoire have remained enigmatic. Zebrafish (Danio rerio) is an ideal model system to study theadaptive immune system because it has the most ancient recognizable adaptive immune systemwhose features match those in its mammalian counterparts. In addition, the number of T-cells in thezebrafish has been approximated to about 2×10 5 cells, a 10 6 fold lower number compared to the T-cellnumbers found in mice. Therefore, in contrast to TCR sequencing studies performed in mammals,where isolated T-cell populations have been used, the zebrafish offers the possibility to perform farmore complete immune repertoire studies.We have combined high-throughput and computational approaches with the experimental advantagesoffered by the zebrafish as an immunological model to study the complete TCR repertoire underhomeostatic conditions and in response to stimulation with self and non-self antigens. We found thatthe T-cell response in zebrafish is consistently dominated by a limited number of cross-reactive TCRsshared by different individuals. Interestingly, immunization with self and non-self antigens expandedspecific TCR sequences but did not increase the total repertoire, leading to a net decrease in TCRdiversity. These features, classically attributed to the innate immune system, might also characterizeadaptive systems operating in lower vertebrates and represents an early step in the evolution of theadaptive immune response. Moreover, the results demonstrate that zebrafish might provide a platformto design vaccines that trigger broad pathogen- or tumor-specific T-cell responses.8 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts197

ZDM8Boston | USA 2015ZDM8 POSTERSAnalysis and manipulation of phagocyte calcium dynamics ininfection and inflammation.Rebecca W. Beerman 1 , Molly A. Matty 1 , Gina Au 1 , Loren L. Looger 3 , Kingshuk RoyChoudury 2 , Philipp J. Keller 3 , and David M. Tobin 11. Molecular Genetics and Microbiology, Duke University, Durham, NC, 27710, USA2. Duke Radiology, Duke University, Durham, NC, 27710, USA3. Howard Hughes Medical Institute, Janelia Research Campus, Ashburn, VA 20147, USAGenetically encoded calcium indicators enable in vivo imaging of calcium dynamics, buthave generally been deployed largely in the context of neuronal activity. Here weexamine macrophage and neutrophil calcium dynamics in vivo during sterileinflammation and infection. Using light sheet microscopy, we find leading edgeenrichment of calcium influx in migrating neutrophils, and orchestrated calciumtransients as macrophages and neutrophils reach a wound site. Using cell-specificexpression of the mammalian capsaicin receptor TRPV1, we have generated ageneralizable tool for the temporal and spatial control of calcium influx in specificimmune cells. These experiments suggest that calcium dynamics are tightly coupled tospecific migratory behaviors as well as macrophage function during infection withintracellular pathogens.1988 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts

ZDM8 POSTERSZDM7Madison | USA 2015ZDM8Boston | USA 2015XPR1 is specifically required for the differentiation of tissueresidentmacrophagesAna M. Meireles 1 , Celia E. Shiau 1 , Catherine Guenther 1 , David Kingsley 1 , William S.Talbot 11. Department of Developmental Biology, Stanford University School of Medicine, Stanford, USAMacrophages are phagocytic cells which populate all tissues of a complex organism. Indifferent tissues, specialized resident macrophages adopt different morphologies andperform different functions. For example, whereas microglia cells (brain macrophages) areimportant for the brain’s immune defense and neuronal synapse elimination, osteoclasts(bone resident macrophages) are important for correct bone development, bone remodellingin adults and mineral homeostasis at the organismal level. Despite the importance of tissuespecific macrophages for immune function and whole-organism homeostasis, the precisegenetic control of resident macrophage differentiation remains elusive.In a zebrafish genetic screen for microglia mutants, we identified xpr1b st87 as a mutation thatseverely reduces the number of microglia cells. Further analysis showed that the numberand morphology of Langerhans cells (epidermal macrophages) is also compromised.Surprisingly, some xpr1b st87 mutants survive to adulthood, but exhibit profound defects inbone architecture that are characteristic of osteoclast disruption. Rescue experiments haveshown that Xpr1b acts autonomously in the macrophage lineage. XPR1 was recentlyidentified in vertebrates as a dedicated phosphate exporter and has been proposed as themain effector of phosphate efflux. We constructed a targeted mutation of xpr1a, a duplicateof xpr1b in the zebrafish genome, to determine if Xpr1a and Xpr1b have redundantfunctions. Interestingly, single mutants for xpr1a were viable and displayed no defects in themacrophage lineage, while double mutants for xpr1b;xpr1a were similar to xpr1b singlemutants. Our genetic analysis reveals a specific role for the phosphate exporter Xpr1 in thedifferentiation of tissue macrophages. We are currently investigating what makes themacrophage lineage so sensitive to alterations in intracellular phosphate levels and thepossible link between Xpr1 mediated phosphate export and osteopetrosis.8 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts199

ZDM8Boston | USA 2015ZDM8 POSTERSThe protozoan parasite Trypanosoma carassii is an extracellular blood flagellate that caninfect several fish species and cause a chronic infection. It is phylogenetically closely relatedto the mammalian parasite Trypanosoma brucei that is causing the fatal sleeping sicknessdisease. Infection of fish with T. carassii presents several pathological features ofmammalian infections with T. brucei parasites such as polyclonal B cell activation, anaemia,and splenomegaly. In the current study we present the establishment and characterization ofa trypanosome infection model in zebrafish. To characterize the progression of the infectionat various stages of (immune) development 5 dpf larvae, 1-month-old juvenile and 3-montholdadult zebrafish were injected with low, medium and high doses of T. carassii andanalysed at various time points after infection. The optimal dose for each stage wasdetermined by monitoring the survival. As expected, we found that a fully developed adaptiveimmune system is required to survive the infection. Parasite levels were quantified byrealtime quantitative-PCR analysis using parasite-specific primers. Immune gene expressionprofiling was performed and correlated with the kinetics of parasite replication. Next, 3-5 dpfmpeg:mCherry and mpx:gfp transgenic larvae were used to investigate the involvement ofmacrophages and neutrophils in the response to the infection, to monitor cell migration, cellproliferation and host cell-parasite interaction. Altogether, in this study we present theestablishment, kinetics of development and severity of a trypanosome infection in zebrafish.In addition we describe the real-time dynamics of host-parasite interactions, particularlyfocusing on the interaction with innate immune cells. To our knowledge this is the first studydescribing a trypanosome infection model in zebrafish and will provide additional tools toinvestigate the in vivo dynamics of host-pathogen interaction in a developing immunesystem, in a complicated model such the one of trypanosome infections.2008 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts

ZDM8 POSTERSZDM7Madison | USA 2015ZDM8Boston | USA 2015Shuttling spores between leukocytes in zebrafish fungalinfection modelsVahid Pazhakh 1 , Felix Ellett 1 , Stefan Greulich 1 , Alex Andrianopoulos 2 , GrahamLieschke 11. Australian Regenerative Medicine Institute, Monash University, Clayton, Victoria 3800,Australia.2. Department of Genetics, University of Melbourne, Parkville, Victoria 3010, Australia.Although many aspects of leukocyte biology have been studied in detail, theirindividual behaviours during infection is yet to be fully examined. One way tounderstand host/pathogen interactions is to observe leukocytes live and in vivofollowing an infective challenge. We have therefore exploited the opticaltransparency of zebrafish embryo, combined with an infection model, to studyhost/pathogen interactions in a zebrafish model of the fungal opportunistic pathogenPenicillium marneffei, the cause of penicilliosis.We demonstrated that P. marneffei is actively phagocytosed by macrophages in thismodel, as in mammalian hosts. Neutrophils display a myeloperoxidase-dependantfungicidal activity, while macrophages provide a protective niche for the pathogen.During high-resolution intravital imaging of infections, we observed a previouslyundescribed phenomenon: neutrophil-to-macrophage shuttling of P. marneffeispores. By collecting multiple examples, we have defined the morphological featuresof spore shuttling. Shuttling is a unidirectional transfer between alive and motileleukocytes, occurring during infection establishment, and is directed from neutrophilstowards both naive and spore-laden macrophages.Furthermore, we have now demonstrated shuttles in Aspergillus fumigatus infection,again where spores phagocytosed by neutrophils are transferred to macrophages.Therefore, shuttling is not pathogen-specific, and appears to be a generalmechanism of pathogen transfer between leukocytes.Whether shuttling is a mechanism evolved by pathogens to facilitate their transfer toa more hospitable leukocyte or a mechanism evolved by the immune system totransfer the pathogen to an antigen-presenting cell is not yet established. Thelongitudinal survival of the shuttled spores inside recipient macrophages is anothercritical question that is experimentally challenging to demonstrate.These data demonstrate the utility of zebrafish infection model for studying leukocytebehaviour. Neutrophil to macrophage shuttling of pathogens now needs to beconsidered to as an integral component of the host-pathogen interaction that mayhave a bearing on fungal infection outcome.8 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts201

ZDM8Boston | USA 2015ZDM8 POSTERSThe role of vascular dysfunction during mycobacterial infectionStefan Oehlers, David TobinDepartment of Molecular Genetics and Microbiology, Center for Microbial Pathogenesis, DukeUniversity Medical Center, Durham, NC 27710, USAPathogenic mycobacteria induce the formation of complex cellular aggregates calledgranulomas that are the hallmark of tuberculosis. Here we examine the developmentand consequences of vascularisation of the tuberculous granuloma in the zebrafish-Mycobacterium marinum infection model characterised by organised granulomas withnecrotic cores that bear striking resemblance to those of human tuberculosis. Usinglarval and adult zebrafish infected with M. marinum, we have illustrated a web ofinteraction between granuloma formation, hypoxia, and the induction of angiogenesis.We have shown that VEGFR-targeting drugs are a potential host directed therapy thatreduce infection burden, limit mycobacterial dissemination and synergise with antibioticsto protect the host from mycobacterial infection. Here we present novel data fromexperiments targeting other aspects of vascular biology during infection. Specifically, wehave investigated the outcome of augmenting the host vascularisation response and ofmanipulating vascular permeability on mycobacterial infection. We will discuss theimplications of these findings in the context of novel host directed therapies formycobacterial infection.2028 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts

ZDM8 POSTERSZDM7Madison | USA 2015ZDM8Boston | USA 2015Ancient collection of antigen processing and presentation genesuncovered by genomic sequencing of clonal zebrafishSean C. McConnell and Jill L.O. de JongDepartment of Pediatrics, Section of Hematology/Oncology, University of Chicago, Chicago, IL 60637Antigen presentation genes are exceptionally polymorphic, with human populationscarrying hundreds of alleles for major histocompatibility complex (MHC) Class I genes.Co-inherited antigen processing genes such as proteasomal subunits and transporterassociated with antigen processing (TAP) genes are much less polymorphic.Nevertheless, allelic differences in human proteasomal subunit and TAP genes haverecently been shown to be associated with a number of complex disorders, includingsusceptibility to infection, cancer, and autoimmune diseases. We have identifieddifferences in the antigen processing and presentation genes of zebrafish by wholegenome sequencing of the clonal zebrafish line CG2. Examination of the divergent coreMHC haplotype on chromosome 19 from this clonal line provided sequences for sixunique genes that are not found in the zebrafish reference genome: mhc1uga, abcb3l2,psmb9b, psmb8f, and novel genes psmb10b and abcb3l3. Immunoproteasomalsubunits psmb8f and psmb10b each have substitutions predicted to alter peptidecleavage specificity. In addition we identified two TAP2 genes, abcb3l2 and abcb3l3,that are likely to transport distinct peptide subsets due to unique substitutions in theirspecificity loops. Psmb9a was replaced by psmb9b in the CG2 haplotype, andadditional genomic changes were observed including the deletion of psmb9l along withmhc1uga replacing two reference MHC genes. One explanation for dichotomoushaplotypic associations of these genes is their functional specialization; an attractivemodel includes balancing selection maintaining alternative pathways, each with uniquepeptide repertoires. Our data provide evidence that these antigen processing andpresentation genes represent ancient lineages derived during formation of the adaptiveimmune system, leading to deeply divergent sequences preserved in zebrafish. Work isnow proceeding to determine how these different haplotypes influence immuneresponse. These data demonstrate important immunogenetic differences betweenindividual zebrafish that are likely to impact disease modeling of infection, cancer, andautoimmunity.8 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts203

ZDM8Boston | USA 2015ZDM8 POSTERSIdentification of benzopyrone as a common structural feature incompounds with anti-inflammatory activity in a zebrafish phenotypicscreen.Anne Robertson 1 , Nikolay Ogryzko 1 , Katherine Henry 1 , Catherine Loynes 1 , Philip Ingham 2 ,Heather Wilson 1 , Roderick Flower 3 , Simon Jones 4 , Moira Whyte 5 , Stephen Renshaw 11The Bateson Centre, University of Sheffield, Sheffield, UK2Institute of Molecular and Cell Biology, 61 Biopolis Drive, Proteos, Singapore3William Harvey Research Institute, Barts and The London, Queen Mary's School of Medicine and Dentistry,John Vane Science Centre, London, UK4Department of Chemistry, University of Sheffield, Sheffield, UK5MRC/UoE Centre for Inflammation Research, University of Edinburgh, The Queen's Medical ResearchInstitute, Edinburgh, UK!Neutrophils are essential for host defence and are recruited to sites of inflammation in response totissue injury or infection. For inflammation to resolve, these cells must be cleared efficiently and ina controlled manner, either by apoptosis or reverse migration. If the inflammatory response is notwell regulated, persistent neutrophils may cause damage to host tissues and contribute to thepathogenesis of chronic inflammatory diseases, which respond poorly to current treatments. It istherefore important to develop drug discovery strategies that can identify new therapeuticsspecifically targeting neutrophils, either by promoting their clearance or by preventing theirrecruitment. Our recent in vivo chemical genetic screen for accelerators of inflammation resolutionidentified a subset of compounds sharing a common chemical signature, the bicyclic benzopyronerings. Here, we further investigate the mechanisms of action of the most active of this chemicalseries, isopimpinellin, in our zebrafish model of neutrophilic inflammation. We found that thiscompound targets both the recruitment and resolution phases of the inflammatory response.Neutrophil migration towards a site of injury is reduced by isopimpinellin and this occurs as a resultof PI3K inhibition. We also show that isopimpinellin induces neutrophil apoptosis to driveinflammation resolution in vivo, using a new zebrafish reporter line detecting in vivo neutrophilcaspase-3 activity and allowing quantification of flux through the apoptotic pathway in real-time.Finally, our studies reveal that clinically available ‘cromones' are structurally related toisopimpinellin and have previously undescribed pro-resolution activity in vivo. These findings mayhave implications for the therapeutic use of benzopyrones in inflammatory disease.!2048 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts

ZDM8 POSTERSORAL or POSTERZDM7Madison | USA 2015ZDM8Boston | USA 2015Tracking PGE2 effect on HSPC progenitor populations via thetranscription factor CREBEva M. Fast 1,2 , Ellen M. Durand 2 , Audrey Sporrij 1 , Leslie Ojeaburu 1 , Leonard I.Zon 1,21. Department of Stem Cell and Regenerative Biology and Harvard Stem Cell Institute, HarvardUniversity, MA 02138, USA2. Stem Cell Program and Division of Hematology/Oncology, Boston Children's Hospital andDana Farber Cancer Institute, Howard Hughes Medical Institute, Harvard Stem Cell Institute,Harvard Medical School, Boston, MA 02115, USAProstaglandin E2 was the first compound identified in a zebrafish chemical screenthat entered clinical trials. The beneficial effect on hematopoietic stem and progenitorcells (HSPC) is conserved in zebrafish, mouse and humans but the exactmechanistic foundation is still unknown. We have acquired several RNA-seq, ChIPseqand ATAC-seq datasets detailing molecular changes induced by PGE2. To testpotential PGE2 target genes we aim to take advantage of the relative ease andspeed of genetic perturbation in zebrafish embryos compared to a transplantationmodel. In mouse transplantation studies we determined that PGE2 treatment inducesa fate switch towards long-term engraftment potential. Time lapse imaging of HSPCbirth in the zebrafish embryo also suggested that PGE2 increases HSC fate byenhancing HSPC specification by 2-fold. Treatment of PGE2 at an early time pointbefore HSPCs arise was sufficient to lead to increased HSPC numbers,corroborating that PGE2 is affecting a progenitor population. Our molecular dataimply the transcription factor CREB as the major downstream effector of PGE2. Thefast and immediate effect of PGE2 is conserved in zebrafish since a 10 minutetreatment was sufficient to dramatically upregulate Creb1 phosphorylation byimmunohistochemistry. To track and identify cells receiving the Creb1 signal we havebuilt a reporter line using the promotor region of CREM, whose expression was 120-fold upregulated by RT-qPCR in hCD34+ cells after PGE2 treatment. Zebrafishreporter lines using both the human and zebrafish CREM/crema promotors showedsignificantly increased florescence upon PGE2 treatment. Together these resultsestablish parallels of PGE2 target cell populations and timeframes between thezebrafish embryo and mammalian transplants. These results lay the groundwork forfuture genetic studies of PGE2 target genes in the zebrafish embryo. We expect torelate these findings further to human hematopoietic stem cell transplantation.8 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts205

ZDM8Boston | USA 2015ZDM8 POSTERSThe role of mir-223 in neutrophil reverse migrationWenqing Zhou, Qing DengDepartment of Biological Sciences, Purdue University, West Lafayette, IN 47907The presence of neutrophils is a hallmark of inflammation and the first line of defenseagainst invading pathogens. To reduce the non-specific damage to the host,inflammation is actively restricted by neutrophil apoptosis at sites of inflammation.Recently live imaging in zebrafish embryos revealed that neutrophil reverse migrate.After their initial recruitment, neutrophils migrate away from the inflammation site,return to the vasculature and persist in the body. To date, how neutrophils reversemigrate and the signals triggering the process are unknown.MicroRNAs are evolutionarily conserved, small non-coding RNAs that posttranscriptionallyregulate protein synthesis by binding to partially complementaryregions in target messenger RNAs. Mir-223 is a hematopoietic-specific microRNA andthe role of mir-223 is increasingly appreciated in infection, inflammation and cancer.We performed microRNA quantitative RT-PCR using zebrafish as a model and foundan enrichment of mir-223 in neutrophils compared with the entire fish. In woundingassay, when inhibited the function of mir-223, we detected an accumulation ofneutrophils around wound sites during the resolution phase (6 hours post wounding).By inhibiting the function of mir-223 specifically in neutrophils, we found that neutrophilintrinsic expression of mir-223 was required for neutrophil reverse migration.The precise control of neutrophil-mediated inflammation is critical for both host defenseand the prevention of immunopathology. Understanding the mechanism that regulatesneutrophil reverse migration by mir-223 may bring insight into new and innovativeapproaches that promote or inhibit neutrophil reverse migration to prevent or alleviateinflammatory diseases.2068 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts

ZDM8 POSTERSZDM7Madison | USA 2015ZDM8Boston | USA 2015The role of lipid mediators in zebrafish inflammation resolutionelucidated by a novel ptges reporter line.Loynes, CA, Griffiths, JD, Levy, BD, Renshaw, SA.1. Department of Infection and Immunity and The Bateson Centre, University of Sheffield2. Department of Infection and Immunity and The Bateson Centre, University of Sheffield3. Department of Internal Medicine Pulmonary and Critical Care Medicine Division, Brigham andWomen's Hospital, Boston4. Department of Infection and Immunity and The Bateson Centre, University of SheffieldResolution of inflammation is critical to restore tissue homeostasis and is a pre-requisite fornormal wound healing. Failure of inflammation resolution contributes to tissue injury in chronicinflammatory diseases. Resolution of inflammation is an active process where clearance ofapoptotic cells by macrophages dictates leukocyte phenotype, lipid mediator production andthe course of wound healing. Prostaglandin E2 (PGE 2) has an important role in the course ofthe inflammatory response in a time and dose dependant manner and in the production ofspecialised pro-resolving mediators (SPMs), determining the balance of host-defence andinflammation in wound healing.Tail transection was performed on zebrafish larvae inducing an inflammatory response.Blocking apoptosis using a pan-caspase inhibitor, zVAD-fmk leads to failure of inflammationresolution. Similarly, macrophage ablation using metronidazole treatment of nitroreductaseexpressingtransgenics led to persisting tissue neutrophilia. Exogenous PGE 2 is able to correctthis phenotype in both cases, suggesting PGE 2 is sufficient for inflammation resolution in thissystem. Using an assay for reverse migration, we found that PGE 2 accelerates reversemigration. To study the cells expressing PGE 2. We generated a Prostaglandin E synthase(ptges) GFP reporter line and expression of ptges was captured by time-lapse microscopyduring the inflammatory response. Increased ptges expression can be seen upon injury andwithin proliferating cells.To further assess the role of specific pro-resolving mediators in this process, larvae wereinjected or incubated with maresin-(R)-1 and resolvin D2. Maresin-(R)-1 and resolvin D2 alsosignificantly reduce neutrophil numbers at the site of injury. Macrophage recruitment issignificantly enhanced by maresin-(R)-1 only.Finally, we assessed the role of lipid mediators on regeneration of the caudal tail fin. PGE 2and Maresin-(R)-1 significantly enhance fin regeneration over 24 hours.These data indicate that lipid mediators play a vital role in inflammation resolution andregeneration in the zebrafish, and may prove to have therapeutic potential.8 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts207

ZDM8Boston | USA 2015ZDM8 POSTERSIntegrating human and zebrafish studies to find new drugtargets in the neutrophil kinomeKatherine M Henry 1 , Julien Rougeot 2 , Anne L Robertson 1 , Zakia Kanwal 2 ,Joseph Burgon 1 , Caroline Tabor 1 , Annemarie H Meijer 2 , William J Zuercher 3and Stephen A Renshaw 11 Infection and Immunity and The Bateson Centre, University of Sheffield, UK2 Institute of Biology, Leiden University, The Netherlands3 Department of Chemical Biology, GlaxoSmithKline, Research Triangle Park, NC, USANeutrophils are the first cells recruited to sites of inflammation where they defendagainst invading pathogens. However, when neutrophils do not leave theinflammatory site appropriately, chronic inflammation contributes to diseases such asrheumatoid arthritis. Enhancing neutrophil clearance may be beneficial in thesecircumstances.We have previously published that inhibition of serum and glucocorticoid regulatedkinase1 (SGK1) reversed the effect of the potent neutrophil survival factor, GM-CSF,in primary human neutrophils. Pharmacological and genetic inhibition of SGK1 alsoenhanced resolution of inflammation in our zebrafish model.Including SGK1, there are 518 protein kinases in humans. Many kinase inhibitors arein clinical use, but the neutrophil kinome is largely unexplored. The aim of this studywas therefore to define the neutrophil kinome of both human and zebrafishneutrophils and to explore the functional role of kinases in inflammation byperforming a compound screen in our zebrafish model.Probing of a published RNAseq dataset identified 284 (of 518) annotated proteinkinases in human neutrophils. A subset, including SGK1, was regulated by GM-CSF.Zebrafish neutrophil kinases were determined by RNAseq performed on GFPpositive neutrophils from transgenic zebrafish larvae. Zebrafish neutrophilsexpressed 296 kinases, 200 of which were also expressed in human neutrophils.To assess the functional role of these kinases in neutrophilic inflammation, thePublicly-available Kinase Inhibitor Set 1 was screened. Zebrafish underwent tail fintransection and were arrayed in a 96-well plate containing kinase inhibitors.Zebrafish were imaged at 12 hours post injury. Scoring of these images revealed 20‘hit’ compounds.Kinase profiling of ‘hit’ compounds revealed potential new drug targets in theneutrophil kinome. Further investigation of the relevant kinases is now beingundertaken by the generation of zebrafish mutants using CRISPR technology.Compounds targeting kinases identified here may have potential as pro-resolutiontherapies for the treatment of chronic inflammatory conditions.2088 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts

ZDM8 POSTERSZDM7Madison | USA 2015ZDM8Boston | USA 2015Interleukin 1 induction in sterile tissue injuryNikolay V. Ogryzko, Stephen A. Renshaw, Heather L. Wilson1. The Bateson Centre and Department of Cardiovascular Science, University of Sheffield,Sheffield, United Kingdom2. The Bateson Centre and Department of Infection and Immunity, University of Sheffield,Sheffield, United Kingdom3. The Bateson Centre and Department of Cardiovascular Science, University of Sheffield,Sheffield, United KingdomIL-1 is a critical regulator of the inflammatory response and understanding its role ininflammation is therefore of great therapeutic interest. The mammalian IL-1 familyconsists of a multiple expansion of a single locus, retained as a unique gene on thezebrafish genome. Zebrafish IL-1 is closest to mammalian IL-1 α and β and appearsto correspond to a common ancestral precursor. To investigate IL-1 in zebrafish wehave generated an Interleukin-1 reporter fish based on BAC recombineering and theGRCz10 assembly; allowing us to express EGFP under the control of 60kb of thenative IL-1 promoter. Our reporter recapitulates our IL-1 WISH data, with expressiondependent on inflammatory insult. During S. aureus infection, IL-1 is expressedduring the initial stages of infection with subsequent loss of signal in a successfullycleared infection. Persistent infection results in continued IL-1 expression. Duringinjury, IL-1 expression is leukocyte specific with pu.1 morpholino based leukocytedepletion resulting in loss of measureable IL-1 fluorescence. Macrophages andneutrophils appear to have differential timecourse of IL-1 expression. Both leukocytesubtypes show IL-1 High and IL-1 Low populations, but the distinct roles of thesesubtypes in inflammation and inflammation resolution, and the role of IL-1 in thesespecific pathways remains unclear.8 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts209

ZDM8Boston | USA 2015ZDM8 POSTERSCigarette smoke-induced inflammation and tissue remodellingof zebrafish gillsFränze Progatzky 1 , Terence H. Cook 2 , Jonathan R. Lamb 1 , Trevor T. Hansel 3 ,Laurence Bugeon 1 , Margaret J. Dallman 11. Department of Life Sciences, Faculty of Natural Sciences, Imperial College London, UK2. Department of Medicine, Faculty of Medicine, Imperial College London, UK3. National Heart and Lung Institute, Faculty of Medicine, Imperial College London, UKInflammatory diseases of the respiratory system such as asthma and chronicobstructive pulmonary disease are on the rise globally and remain poorly understoodconditions. While attention has long focused on the activation of type 1 and type 2helper T cells of the adaptive immune system in these disease, there is also a needto understand the interactions between the innate immune cells and the epitheliallining of the respiratory system. Cigarette smoke predisposes the respiratory tissueto a higher incidence of inflammatory disease and here we have used zebrafish gillsas a model to study the effect of cigarette smoke on a respiratory epithelium.Zebrafish gills fulfil the same gas-exchange function and have a similar structure tothe mammalian airways, with a mucus-covered respiratory epithelium scattered withimmune cells and smooth muscle cells at the base of the lamella. A major advantageis that gill tissue is directly exposed to ambient water, which makes it easy to targetwith waterborne substances. Exposure to cigarette smoke extract resulted in anincrease in transcripts of the pro-inflammatory cytokines IL-1β and TNFα in the gilltissue, which was at least in part mediated via NF-κB activation within epithelial cellsof the gill. Longer-term exposure of fish for 6 weeks to cigarette smoke extractresulted in gill tissue remodelling with lamellar fusion and mucus cell formation. Thisshows, for the first time, that zebrafish gills are a relevant model for studying theeffect of inflammatory stimuli on a respiratory epithelium, since they mimic theimmunopathology involved in respiratory inflammatory diseases of humans.2108 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts

ZDM8 POSTERSZDM7Madison | USA 2015ZDM8Boston | USA 2015Presenting speaker: Jessica KenisonTNBS-induced enterocolitis as an inflammatory model for largescalescreening of environmental factors.Jessica Kenison 1 , Merja Jaronen 1 , Chun-Cheih Chao 1 and Francisco J. Quintana 11 Ann Romney Center for Neurologic Diseases, Brigham and Women’s Hospital, Harvard MedicalSchool, 77 Avenue Louis Pasteur, Boston, MA 02115, USA.Inflammatory Bowel Disease (IBD) is a group of inflammatory disorders, primarilyincluding ulcerative colitis and Crohn’s disease, that are thought to arise mainly as aresult of genetic susceptibility and environmental factors. Murine models of IBD,including IL-10 deficient mice and dextran sodium sulfate (DSS) treated mice, havecontributed greatly to helping us understand the pathology of IBD. However, there aresome limitations, including the associated time, cost and ethics of using mouse models.For these reasons and more, an additional model of IBD has been introduced utilizingzebrafish (Danio rerio) larvae immersed in 2, 4, 6-trinitrobenzenesulfonic acid (TNBS).The zebrafish is an ideal model for the study of IBD as it has a digestive tract verysimilar to mammals, and an immune system very strongly related to both humans andmice. Additionally, zebrafish embryos can conveniently be produced in large numbers,and zebrafish larvae are transparent, allowing for easy observation of changes in gutmorphology and movement. We have utilized the zebrafish model of IBD to completelarge-scale screenings of close to 100 compounds that have been found to be potentiallyimportant environmental factors. Our screenings suggested a number of compoundswhich influenced the disease phenotype and could be further validated and tested inmurine models of inflammation.8 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts211

ZDM8Boston | USA 2015ZDM8 POSTERSInflammatory regulation of hematopoietic stem cell formation andfunction in the normal and hyperglycemic niche.V Esain 1 , S-E Lim 1 , and TE North 11 Beth Israel Deaconess Medical Center, Harvard Medical School Boston, MAMany intrinsic factors required for Hematopoietic Stem Cell (HSC) formation and function havebeen elucidated, but less is known about extrinsic regulation in the HSC niche. Both infectionsand altered metabolism are known to cause inflammatory responses in vivo; while therelationship between infectious challenge and pediatric leukemia appears dependent on age atexposure, offspring of mothers with type 1 diabetes are known to exhibit a higher risk ofchildhood leukemia. We have previously reported that heightened glucose metabolism increasedthe onset and magnitude of HSC formation in the zebrafish embryo. Additionally, we, and others,recently showed that a network of locally produced pro-inflammatory cytokines are necessary tostimulate embryonic HSC production across vertebrates. However, the impact of heightenedpro-inflammatory cytokine stimulation occuring during HSC maturation and differentiation in ametabolically-altered embryonic niche environment are poorly characterized. Embryos exposedto increased physiological doses of glucose during the entire onset of hematopoiesis (24-120hpf)or select windows of HSC development: specification (12-36hpf), or maturation and seeding ofsecondary organs (72-120hpf), exhibit a higher number of runx1/cmyb+ HSCs in allhematopoietic sites. Interestingly, embryos with chronic, but not acute, hyperglycemia alsoexhibit a higher number of mpo+ myeloid cells and a decreased rag1 expression in the thymus,suggesting impaired HSC function. RT-qPCR analysis of embryos exposed to heightenedglucose for a short (12-36hpf), intermediate (72-120hpf) or long (24-120hpf) developmentalwindow displayed increased expression of IFNg, IFN-phi, IL-1b, IL-6 TNFa and/or their receptorsconfirming dysregulated glucose metabolism increased inflammation in the embryo. Preliminaryexperiments using morpholino-mediated knockdown suggest that IFNg, TNFa and IL-1b actdownstream of glucose stimulation to regulate different aspects of HSC function. Together thesestudies will further characterize the effect of the maternal environment on HSC function and willhelp elucidate the factors contributing to the development of childhood leukemia.2128 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts

ZDM8 POSTERSZDM7Madison | USA 2015ZDM8Boston | USA 2015The molecular mechanism underlying glucocorticoid resistance ofinflammatory leukocyte migrationMarcel SchaafInstitute of Biology, Dept. Animal Sciences and Health, Leiden University, The NetherlandsGlucocorticoids are widely used as anti-inflammatory drugs. In general they are very effectivedrugs, but unfortunately resistance to glucocorticoid therapy occurs in many patients. Theeffects of glucocorticoids are mediated by the Glucocorticoid Receptor (GR), which acts as aligand-activated transcription factor. In the present study, we have used the zebrafish toinvestigate the anti-inflammatory effects of glucocorticoids, in particular their effects ofleukocyte migration. Neutrophil and macrophage migration was studied upon amputation ofthe tail fin of zebrafish larvae, and the results showed that neutrophil migration wassuppressed by glucocorticoid treatment, but macrophage migration was not. Using a GRmutant fish line we demonstrated that the effect was mediated by the GR. Furthermore, weshowed that both neutrophil and macrophage migration were dependent on de novo proteinsynthesis. In order to study which proteins are required for leukocyte migration we performeda microarray study. The results of this study revealed that amputation upregulates many proinflammatorygenes, but that most of this upregulation is inhibited by glucocorticoidtreatment, which generally dampens the transcriptional response to amputation. Theexpression of two genes involved in the biosynthesis of leukotrienes, arachidonate 5-lipoxygenase (alox5) and arachidonate 5-lipoxygenase activating protein (alox5ap), wasupregulated upon amputation and this was not affected by glucocorticoid treatment. Using anAlox5 inhibitor it was shown that leukocyte migration is dependent on the activation of thispathway, and by mass spectrometry we analyzed the effects of amputation andglucocorticoid treatment on the concentrations of a large panel of leukotrienes andprostaglandins. In conclusion, we have demonstrated that macrophage migration is resistantto glucocorticoid treatment because its induction involves a leukotriene biosynthesis pathwaywhich is not affected by glucocorticoids. We therefore suggest that inhibition of leukotrienefunction and/or synthesis may represent an interesting anti-inflammatory therapycomplementary to glucocorticoids, especially in glucocorticoid-resistant patients.8 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts213

ZDM8Boston | USA 2015ZDM8 POSTERSGenome editing of factor X reveals unexpected early survival and lateonset hemorrhageZhilian Hu 1 , Michael C. Huarng 1 , Marzia Menegatti 2 , Deepak Reyon 3 , Yang Liu 1 , J. KeithJoung 3 , Flora Peyvandi 4 , and Jordan A. Shavit 1 .1. Department of Pediatrics, University of Michigan, Ann Arbor, MI2. Pathophysiology and Transplantation, Università degli Studi di Milano, Milan, Italy3. Molecular Pathology Unit, Massachusetts General Hospital, Charlestown, MA4. Angelo Bianchi Bonomi Hemophilia and Thrombosis Center, Fondazione IRCCS Ca` Granda, Milan,ItalyPathologic hemorrhage is a major contributor to mortality in the general population as well as inindividuals with bleeding disorders. Study of bleeding in the mouse through targetedmutagenesis of common pathway coagulation factors, including factor V, prothrombin, andfactor X (F10), is complicated by embryonic/neonatal lethality due to spontaneous hemorrhageand suspected vascular defects. We produced a zebrafish knockout of f10 through genomeediting with TALENs. Although indistinguishable morphologically from f10 +/+ and f10 +/- siblings atearly stages, f10 -/- mutants demonstrated a spontaneous bleeding phenotype with progressivelethality between 1-6 months of age and extensive hemorrhage in multiple tissues, particularlybrain. f10 -/- larvae did not develop cardinal vein clots in response to laser-mediated venousendothelial injury, and this was rescued by transient expression of zebrafish f10. Known humanF10 disease causing substitutions G222D and C350F were engineered into the orthologouspositions in zebrafish f10 cDNA, and each failed to rescue the bleeding phenotype. Weproceeded to screen sequence variants from 5 newly identified patients with F10 deficiency, andwere able to localize the causative mutation in each individual. Whole mount in situ hybridizationof markers for arterial/venous endothelial specification revealed no differences between f10 +/+and f10 -/- larvae. In summary, we have produced a model of human F10 deficiency that exhibitsa spontaneous lethal adult bleeding phenotype, yet embryos and larvae only display inducedbleeding, in contrast to observations in mammals. Although data from mouse knockouts suggesta vascular defect due to loss of thrombin signaling, our studies to date reveal no such defect inf10 -/- mutants. Further study of this mutant may identify species specific factors enablingprotection from early hemorrhage. This platform may also be useful to screen variants frompatients with F10 deficiency and other coagulation disorders, as well perform small moleculescreens to identify novel therapeutics.2148 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts

ZDM8 POSTERSZDM7Madison | USA 2015ZDM8Boston | USA 2015Idiopathic Scoliosis development in zebrafish and humans.Simone Laupheimer 1 and Bruno Reversade 11. Institute of Medical Biology, Human Genetics and Embryology Laboratory, A*STAR, 138648,SingaporeIdiopathic scoliosis is defined as an abnormal sidewise curvature of the spine, developingafter birth without any muscular or spinal injuries. Its exact cause is still unknown but geneticcomponents have been suggested.We generated a knockout zebrafish line for a cell surface receptor of the Tgf superfamily.Heterozygous and homozygous mutant zebrafish show spine deformations ranging from akinked tail to a severe combination of kyphosis, lordosis and scoliosis. The first visibledeformations can be documented around 4 weeks of development and show a progressionsimilar to that seen in human patients.To determine if Tgf also plays a role in spine curvature in humans, we screened a cohort ofpatients with idiopathic scoliosis. We discovered a couple of new causative genes whoseinterplay with Tgf will be discussed.8 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts215

ZDM8Boston | USA 2015ZDM8 POSTERSAlterations in actin capping protein Capzb causes craniofacialmalformation and hypotonia in zebrafishKusumika Mukherjee 1,4 , Kana Ishii 1,4 , Michael Talkowski 2,4 , Cynthia C. Morton 3,4 , RichardMaas 3,4 , and, Eric C. Liao 1,41 Center for Regenerative Medicine, Massachusetts General Hospital, 2 Center for Human GeneticResearch, Massachusetts General Hospital, 3 Brigham and Women’s Hospital, 4 Harvard Medical School,Boston, USA.Craniofacial malformations are among the most common congenital anomalies with a stronggenetic basis. We have identified a new candidate gene, CAPZB that encodes an actin-cappingprotein and is also important in maintaining the structural integrity of muscles. The isolateddisruption of CAPZB was first identified in a patient who presented with cleft palate (CP),micrognathia and hypotonia. We have exploited the zebrafish to model the phenotypesobserved in the human case and to carry out detailed analysis of molecular and developmentalprocesses to determine the function of capzb in craniofacial morphogenesis. The spatiotemporalgene expression of capzb shows that capzb is maternally inherited and ubiquitously expressed.Analysis of the musculo-skeletal structures of capzb mutants generated from an insertionalmutagenesis screen, show that the lower jaw elements are smaller and retrusive (micrognathia),the palate is only partially fused with a cleft in the anterior palate (CP) and the myofibrils arehighly disorganized (hypotonia). Lineage tracing experiments suggest that defects in migrationof the neural crest cells (NCCs) cause the craniofacial anomalies. There is no observed defectin cell proliferation, survival or intercalation in the mutants. Importantly, over-expression ofcapzb by mRNA injection also perturbs normal craniofacial development, suggesting that acorrect stoichiometry of Capzb binding to polymerizing actin is critical to tissue morphogenesis.We have successfully modeled the CP, hypoplastic mandible and hypotonia exhibited by theproband in a capzb animal model. We identified CAPZB to be important during embryogenesis,with consequent neural crest phenotypes affecting craniofacial and muscle development.Ultimately, the capzb mutant has provided us with a molecular example of how form followsfunction by revealing how disruption of a basic cellular process such as regulation of actincytoskeletal dynamics can cause morphological change in NCCs, impacting NCC migrationthereby resulting in craniofacial dysmorphism.2168 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts

ZDM8 POSTERSZDM7Madison | USA 2015ZDM8Boston | USA 2015Neuromuscular regulation in zebrafish by a large AAA+ATPase/ubiquitin ligase, mysterin/RNF213Yuri Kotani 1 , Daisuke Morito 1 , Satoru Yamazaki 2 , Kazutoyo Ogino 3 , KoichiKawakami 4 , Seiji Takashima 5 , Hiromi Hirata 3 , Kazuhiro Nagata 11. Faculty of Life Sciences, Kyoto Sangyo University, Kyoto, Japan.2. Department of Cell Biology, National Cerebral and Cardiovascular Center, Suita, Japan.3. Department of Chemistry and Biological Science, School of Science and Engineering,Aoyama Gakuin University, Sagamihara.4. National Institute of Genetics and Department of Genetics, Sokendai, Mishima, Japan.5. Department of Medical Biochemistry, Graduate School of Medicine, Osaka University, Suita,Japan.Mysterin (also known as RNF213) is a huge intracellular protein with two AAA+ ATPasemodules and a RING finger ubiquitin ligase domain. Mysterin was originally isolated asa significant risk factor for the cryptogenic cerebrovascular disorder moyamoya disease,and was found to be involved in physiological angiogenesis in zebrafish. However, thefunction of mysterin and the physiological significance of mysterin expression other thanin blood vessels remain largely unknown. In this study, we performedantisense-mediated suppression of a mysterin orthologue in zebrafish larvae andrevealed that mysterin-deficient larvae showed significantly fragile fast muscle fibersand immature projection of primary motoneuron, leading to severe motor deficits.Muscle-specific overexpression of mysterin caused the restoration of those phenotypesand interestingly the AAA+ ATPase and ubiquitin ligase activities of mysterin wereshown to be both essential for proper fast muscle formation, demonstrating an essentialinvolvement of mysterin in the neuromuscula development of zebrafish.8 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts217

ZDM8Boston | USA 2015ZDM8 POSTERSSuccessful treatment with inhibitors of the mitochondrial permeabilitytransition pore in a zebrafish model of collagen VI muscular dystrophyMarco Schiavone 1 , Alessandra Zulian 1 , Justina Šileikytė 1 , Erika Rizzo 1 , FrancescaTagliavini 2 , Luciano Merlini 2 , Patrizia Sabatelli 2 , Sudeshna Roy 3 , Frank Schoenen 3 , Nikolai V.Naoumov 4 , Michael Forte 5 , Francesco Argenton 6 , Paolo Bernardi 11 Department of Biomedical Sciences, University of Padova, Padova, Italy2 Laboratory of Musculoskeletal Cell Biology, Istituto Ortopedico Rizzoli, Bologna, Italy3 University of Kansas Specialized Chemistry Center, Lawrence KS, USA4 Novartis Pharma AG, Basel, Switzerland5 Vollum Institute, Oregon Health and Science University, Portland OR, USA6 Department of Biology, University of Padova, Padova, ItalyUllrich Congenital Muscular Dystrophy (UCMD) and Bethlem Myopathy (BM) are inherited musclediseases due to mutations in the genes encoding the extracellular matrix protein collagen VI.Opening of mitochondrial permeability transition pore(s) (mPTP) is the causative event in diseasepathogenesis, and a potential pharmacological target for therapy. For this purpose, we investigatedtwo groups of compounds: (i) cyclophilin inhibitors able to desensityze the mPTP in vitro, i.e.cyclosporin (Cs) A and two non immunosuppressive cyclosporine derivatives, D-MeAla 3 -EtVal 4 -undecapeptide (Alisporivir) and N-methyl-4-isoleucine-undecapeptide (NIM811); and (ii) compound147, a novel mPTP inhibitor selected after high-throughput screening of a library of 363,827compounds from the NIH Molecular Libraries Small Molecule Repository. We used a zebrafishmodel of collagen VI myopathy obtained by deletion of the N-terminal region of the col6a1 triplehelical domain, a common dominant mutation of UCMD. Treatment with antisense morpholinosequences targeting col6a1 exon 9 at the 1-4 cell stage (within 1 hour post fertilization, hpf) causedsevere ultrastructural and motor abnormalities as assessed by electron and fluorescencemicroscopy, birefringence, spontaneous coiling events and touch-evoked responses measured at24-48 hpf. These structural and functional abnormalities were largely corrected via cyclophilin Dinhibition with NIM811 or Alisporivir (which both proved significantly more effective than CsA)administered at 21 hpf. In contrast, FK506 (tacrolimus), a calcineurin inhibitor that does not bind tocyclophilins, was ineffective. Motor abnormalities and birefringence were significantly rescued bycompound 147, which acts independently of cyclophilin inhibition. Since the mPTP of zebrafishshares the key regulatory features of the mammalian mPTP, these results suggest that earlytreatment with mPTP inhibitors could be beneficial as a potential therapy for UCMD and BM. Theexisting large clinical database of Alisporivir makes it a drug candidate ready for clinicalinvestigation in these patients.2188 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts

ZDM8 POSTERSZDM7Madison | USA 2015ZDM8Boston | USA 2015A zebrafish model for human FOPMelissa LaBonty 1 , Pamela C. Yelick 11. Program in Cellular, Molecular, and Developmental Biology, Sackler School of GraduateBiomedical Sciences, Tufts University, Boston, MAThe zebrafish Type I BMP/TGFβ family member receptor, Alk8, modulates BMP/TGFβsignaling to promote cartilage and bone formation. Activating mutations in the humanortholog of Alk8, ACVR1, are associated with the disease Fibrodysplasia OssificansProgressiva (FOP). This disease is characterized by the gradual ossification of fibroustissues, including skeletal muscle, tendons, and ligaments. The objective of this work isto develop a zebrafish model for FOP to elucidate the poorly understood molecularmechanisms driving heterotopic bone formation in FOP and other human diseases.Gateway cloning was used to generate a vector containing the Hsp70 heat shockpromoter driving the expression of mCherry-tagged constitutively active (CA) Alk8.Constructs were injected into single cell stage BMP response element reporter(BRE:GFP) zebrafish to create stable transgenic CA-Alk8 lines. Developmentally stagedtransgenic animals were subjected to short term (3 weeks) or long term (3 months) dailyheat shock intervals to induce CA-Alk8 expression. We are currently performing µCT,and Alcian blue/Alizarin red staining to characterize overall changes in mineralizationpatterns in these animals, and to identify sites of heterotopic ossification (HO).Preliminary results showed that CA-Alk8-expressing zebrafish exhibited enhancedmineralization and ectopic bone formation as compared to heat-shocked wildtype andnon-heat shocked CA-Alk8 transgenic zebrafish controls. IHC and IF analyses ofactivated BMP/TGFβ signaling (pSmad1/5, pSmad2/3), and chondrocyte (Collagen II,Sox9), endothelial cell (Tie2, vWF) and osteoblast differentiation markers (Runx2,Osteocalcin) are being used to examine heterotopic cartilage and bone formation on acellular level. We anticipate that heat shock inducible CA-Alk8 transgenic zebrafish willprovide an informative model for HO in human FOP. These studies were supported byNIH/NIDCR R01DE018043 (PCY), NIH/NIDCR R21 AR065761 (PCY), and NSFGRFP NS9344 (ML).8 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts219

ZDM8Boston | USA 2015ZDM8 POSTERSIntegration of Hedgehog signaling into networks controllingzebrafish regenerative osteogenesisBenjamin E. Armstrong 1,2 , Scott Stewart 1 , Kryn Stankunas 1, 31 Institute of Molecular Biology2 Department of ChemistryUniversity of OregonEugene, OR 97403, USA3 Department of BiologyUniversity of OregonEugene, OR 97403, USAWhile humans have the innate ability to repair minor bone fractures, this processbecomes less effective with age and severely damaged or lost bone is unable toregenerate. In contrast, zebrafish can fully regenerate complex tissues and organs, suchas their caudal fin, which contains bone, epidermis, nerve, and blood vessels. Zebrafishfin bone regeneration is achieved through a unique process by which mature osteoblasts(Obs) dedifferentiate to form a pool of lineage-restricted progenitors (pObs) uponamputation. The pObs are maintained in a balance between proliferation in response todistal Wnt signals and differentiation promoted by cell intrinsic BMP signaling. Hedgehog(Hh) signaling has also been implicated in regenerative osteogenesis, however it isunclear how this pathway is integrated within the Wnt-BMP circuit. Using the photoconvertibleptch2:kaede line as a Hh reporter, we resolved Hh activity as first initiating inde-differentiated osteoblasts within the regenerating blastema around 24 hours postamputation (hpa). Later, at 72 hpa, two distinct domains of active Hh-responsive cellsare found in the basal epidermis and adjacent re-differentiating pObs. Neither Wnt norBMPR inhibitors perturb ptch2:kaede expression, suggesting Hh signaling functions inparallel to the Wnt-BMP network. Expression analysis confirmed that sonic hedgehog a(shha, epidermis, initiating at 36 hpa) and indian hedgehog a (ihha, osteoblasts) are thepredominant Hh ligands expressed in the regenerating fin. We investigated the role ofihha in fin regeneration using ihha null (ihha hu2131/ hu2131 ) adult fish. Ihha is not required forWnt-driven proliferation of pObs or BMP-promoted initiation of Ob differentiation.However, the ihha-null fish exhibit defects in calcification after amputation, albeit withincomplete penetrance. Since ptch2:kaede reporter activity is largely maintained in ihhamutants, we suspect Ob Hh signaling can be promoted by epidermal-origin shha or thatlow levels of ihhb partially compensate for the loss of ihha.2208 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts

ZDM8 POSTERSZDM7Madison | USA 2015ZDM8Boston | USA 2015Intra-ray mesenchymal cells generate the Wnt-producing progenitorniche that orchestrates caudal fin regenerationGabriel A. Yette, 1 Scott Stewart, 1 Kryn Stankunas 1,21. Institute of Molecular Biology, University of Oregon, Eugene, OR 97403, USA2. Department of Biology, University of Oregon, Eugene, OR 97403, USAZebrafish have the remarkable capability to completely regenerate lost or damaged bone. Athorough understanding of the underlying mechanisms could aid the development of innovativetherapies for human bone injuries. Bone regeneration in zebrafish fins is mediated by a pool ofdedifferentiated progenitor osteoblasts (pObs) that subsequently redifferentiate to producereplacement bone. We showed that a spatial segregation of two opposing pathways, Wnt/cateninand BMP establishes a balance of growth and differentiation of pObs, respectively, untilregeneration is complete. Wnt-producing cells in the distal regenerating fin, the RegeneratingFin Progenitor Niche (RPN), promote self-renewal of adjacent distal pObs until the fin isreformed. However, the source of cells that form the RPN and the mechanisms that confer itsWnt-producing ability are poorly understood. We used an immunostaining screen fortranscription factors (TFs) to identify Snail and Dach as having expression patterns suggestiveof roles in RPN establishment or function. Snail expression is confined to intra-raymesenchymal (IRM) cells residing between non-regenerating hemi-rays in the medial finmesenchyme at 72 hours post amputation. In contrast, Dach expression is restricted to distalRPN cells that are organizationally distinct from the adjacent Snail + population and undetectablein non-regenerating tissue, medial mesenchyme, or pObs. Snail + /Dach + cells at the interface ofthese two populations are transient intermediates between dedifferentation/redifferentiation. Weused genetic lineage tracing studies to determine that Dach + RPN cells are exclusively derivedfrom IRM cells. Our current efforts are to further characterize the niche population and pursuethe hypothesis that the extent and “lifespan” of the Dach + niche serves as a “timer” to ensurecorrect fin pattern and size is restored at the end of the regenerative process.8 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts221

ZDM8Boston | USA 2015ZDM8 POSTERSInvestigating potential therapeutics for merosin-deficientcongenital muscular dystrophySarah J. Smith 1 , Eric J. Horstick 2 , and James J. Dowling 11 Genetics and Genome Biology, The Hospital for Sick Children, Toronto, Canada2 Eunice Kennedy Shriver National Institute of Child Health and Human Development, Bethesda,USACongenital muscular dystrophies (CMDs) are a heterogeneous group of inheritedmuscular disorders with devastating consequences. Merosin-deficient congenitalmuscular dystrophy (MDC1A) is one of the most common CMDs in Western countries,and is caused by recessive loss-of-function mutations in the laminin-α2 gene resultingmuscle fiber detachment from the extracellular matrix. This pathology leads todevastating physiological consequences including scoliosis, feeding difficulties, jointcontractures, wheelchair dependence, and respiratory dependence. There are nocurative therapies for MDC1A.Recently, we observed abnormal lysosome localization in MDC1A patient musclebiopsies. Lysosomes have many roles in the cell, including plasma membrane repair.Accumulating evidence also suggests improper lysosome function is associated withdystrophic damage. Mice lacking the lysosome-specific Ca 2+ sensor Syt-VII, as well asthose with disrupted lysosome calcium channel ML1, exhibit a dystrophic phenotypesimilar to muscular dystrophy patients. We predict that in the case of MDC1A,lysosomes are acting to prevent disease progression, localizing to the membrane torepair sarcolemma damage. As such, we hypothesize that lysosomes are acting in acompensatory manner in response to muscle degeneration in MDC1A. We haveconfirmed the redistribution of lysosomes in the candyfloss zebrafish model of MDC1A,and are currently determining whether we can improve disease pathology by chemicallyor genetically targeting lysosome pathways. We have also identified a new phenotypethat allows us to identify mutants prior to fiber detachment, allowing us to target our drugtreatment efforts more specifically.2228 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts

ZDM8 POSTERSZDM7Madison | USA 2015ZDM8Boston | USA 2015Connexin 34.4 may play important role in the myogenesis ofzebrafishChing-Hsuan Hsu, Jiann-Jou YangDepartment of Biomedical Sciences, Chung Shan Medical University, Taichung, Taiwan, ROCConnexins (Cxs) are homologous four transmembrane domain proteins that arethe major components of gap junctions. Gap junctional intercellular communication(GJIC) has numerous functions, each of which meets the particular needs of organs,tissues or groups of cells. These gap junctions facilitate direct intercellularcommunication between adjoining cells, allowing for the transmission of bothelectrical and chemical signals. In this study, we presented expression pattern ofzebrafish cx34.4 (zfcx34.4). This gene shared 62% similarity with CX30.3 gene ofHomo sapiens. The cDNA were isolated from zebrafish embryos by semi-quantitativeRT-PCR. During embryogenesis, zebrafish cx34.4 gene expressed from 1.5 hourspost-fertilization (hpf) to 5 days post-fertilization (dpf), especially in 12hpf to 48hpf. Inaddition, whole-mount in situ hybridization and parafilm section revealed that zfcx34.4was expressed in slow muscle of 36 to 48 hpf. Moreover, we injected zebrafish cx34.4anti-sense Morpholino (MO) knockdown zebrafish cx34.4 gene at zebrafish one-cellstage. Observed at 72hpf, 22% of the knockdown embryos showed severe alterationsin their phenotype. In addition, we increase the concentration of MO, the mortality rateshowed dose dependent. Our results demonstrate the importance of cx34.4 gene inzebrafish embryonic early stage. From our results, we suggested that zfcx34.4probability affect the myogenesis in zebrafish development early stage.8 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts223

ZDM8Boston | USA 2015ZDM8 POSTERSLinking motile ciliary dysfunction to adolescent idiopathic scoliosisCurtis Boswell 1 , Daniel Grimes 2 , Nicholas Morante 2 , Cedric Julien 3 , Alain Moreau 3 , Carol Wise 4 ,Rebecca Burdine 2 and Brian Ciruna 11Developmental and Stem Cell Biology, SickKids Research Institute, Toronto, ON2Department of Molecular Biology, Princeton University, Princeton, NJ3Université de Montréal, CHU Sainte-Justine Research Centre, Montreal, QC4Texas Scottish Rite Hospital for Children, Dallas, TXAdolescent idiopathic scoliosis (IS) is a debilitating disease characterized by a threedimensionalcurvature of the spinal column that arises in the absence of observablephysiological or anatomical defects. Although there has been investigation into geneticcomponents of IS, the underlying cell biological and molecular mechanism for diseaseprogression remains unknown. Recently, our lab described the first genetically defineddevelopmental model of IS in ptk7 mutant zebrafish. These fish are defective in a keymodulator of planar cell polarity (PCP) signalling, implicating this pathway in the onsetof disease. PCP signalling controls the polarized orientation and localization of motilecilia, which is essential for proper cerebrospinal fluid flow. We performed scanningelectron microscopy on adult ptk7 mutant brains to examine CSF-propelling motileciliated populations and observed a striking decrease in the cilia density andorganization. To determine whether loss of Ptk7 in motile ciliated tissue was the causeof these curvatures in our zebrafish model, we generated a transgene that expressesptk7 specifically in this tissue. Preliminary investigation into these tissue-specific rescuetransgenics shows a rescue of the IS phenotype. To further demonstrate therequirement for motile cilia function in spine homeostasis, we analyzed multiplezebrafish mutants that specifically lack motile cilia function. These mutants alldeveloped late onset spinal curvatures similar to our ptk7 model, strengthening the linkbetween motile cilia dysfunction and disease. In ongoing work, we have beenscreening whole exome sequencing cohorts of IS patients for mutations that disruptPCP and motile cilia function and are currently evaluating novel variants forpathogenicity and dominant activity. Together, our data suggest that PCP-directedmotile cilia function is required for proper adolescent spine development.2248 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts

ZDM8 POSTERSZDM7Madison | USA 2015ZDM8Boston | USA 2015Zebrafish Protein-Trap Gene-Break Transposon System to ExploreVertebrate Proteome of Skeletal Muscle RemodelingNoriko Umemoto, Mark D. Urban, Rhianna M. Harm, MaKayla R. Berg, Camden L. Daby, Karl J.Clark and Stephen C. EkkerDepartment of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MNIn response to physiological and pathological signals myofibers transform and remodel to adaptto environmental demands. Exercise, electrical stimulation, disease states and microgravity allimpact muscle remodeling, but the molecular mechanism of this process remains unclear.Protein-trapping, gene-break transposon (GBTs) mutants generate mRFP tagged insertionalalleles with more than 97% knockdown of endogenous transcript levels in zebrafish. The mutantalleles are reversible via Cre recombinase or splice-site-blocking morpholinos. We have over 60annotated GBT zebrafish lines with mRFP expression in skeletal muscle including 11 lineswhere we have already identified the disrupted gene. One such muscle-specific mRFP line hasa gene break insertion in ryr1b, which is responsible for calcium homeostasis during contractionand relaxation of skeletal muscle. Heterozygous ryr1b larvae swam significantly slower than WTand homozygous ryr1b mutations are lethal. These GBT muscle lines have the potential todiscover new genes responsible for muscle remodeling during physiological and pathologicalconditions. To identify muscle remodeling phenotypes of the GBT lines, we are performing afunctional phenotype screening based on quantification of tcap expression, which is known as astretch sensor in Z-disk of sarcomeres. We report details about our muscle collections and thenew findings with the unique functional phenotype assay.8 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts225

ZDM8Boston | USA 2015ZDM8 POSTERSMODELING HUMAN MITOCHONDRIAL DISEASES CAUSED BYMUTATIONS IN NUCLEAR GENOMEAuthors: Jarryd M. Campbell 1 , Tanya L. Poshusta 1 , Mark D. Urban 1 , Danielle E.Hunter 1 , Hirotaka Ata 1 , Melissa S. McNulty 1 , Roberto Lopez Cervera 1 , Camden L.Daby 1 , Karl J. Clark 1 , Stephen C. Ekker 11. Mayo Clinic, Rochester, MNMitochondria play a role in diverse biological processes including ATP production,calcium homeostasis, regulation of metabolism, apoptosis, and vascular endothelial cellmigration. They maintain their own genome that encodes 37 genes but also require theimport of over 1500 genes from the nuclear genome. Mutations in either genome canlead to diseases that are currently difficult to diagnose with no curative treatment. Theirfunction appears to be tissue-specific, leading to an incredibly heterogeneous group ofhealth issues that include heart, liver, muscle, eye, and nervous system breakdowns.The heterogeneous dimension of mitochondrial diseases makes it difficult to reach broadconclusions about mitochondrial disease biology in vivo. To address this problem, weare making 27 mutant zebrafish with indel mutations in nuclearly encoded mitochondrialgenes and four Gene-Break Transposon (GBT) fish lines. We chose the 27 genes usinga combination of their link to human disease and function, with the end result of primarilyknocking out electron transport chain genes (17 mutants) but complement with a non-ETC genes made up primarily of the calcium uniporter (10 genes). Our goal is togenerate two frameshift alleles per gene from the collection of 27 and phenotype eachline using observational analysis, respiration measurements, metabolic profiling,behavioral studies, and, in some cases, subcellular and tissue imaging. Afterphenotyping we will treat mutant fish with drugs prescreened in cell culture withpromising results and ask if the drugs are able to ameliorate disease in differentsubclassifcations of mitochondrial disease. Currently, we are characterizing thesemutants for this project, and we will outline the specific genes, their function, and themutations that are complete. We encourage collaborations for those interested inmitochondrial biology.2268 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts

ZDM8 POSTERSZDM7Madison | USA 2015ZDM8Boston | USA 2015Mutation of zebrafish kif5B genes causes craniofacial malformationand myopathyPhilip D. Campbell 1 , Adrian Santos-Ledo 1 , Marta Gronska 2 , Florence L. Marlow 1,21 Albert Einstein College of Medicine. Department of Developmental and Molecular Biology, 1300 MorrisPark Avenue, Bronx, NY 104612 Albert Einstein College of Medicine. Dominick P. Purpura Department of Neuroscience, Bronx, NY 10461Microtubule motors transport diverse cargoes that are crucial for cellular morphology andfunction. Kinesin, Dynein, and Myosin motors actively transport or tether components to specificsubcellular destinations. Mutations within the motor proteins have been linked to peripheralneuropathies associated with Charcot-Marie-Tooth disease and rare forms of Hereditary SpasticParaplegia, to the motor neuron disease, amyotrophic laterals sclerosis, to Multiple sclerosisand to muscular atrophy. The Kinesin superfamily is composed of 45 Kinesin motor proteins(called Kifs) in mammals. Mammals have three kif5 genes, kif5A, kif5B, and kif5C, which mayallow for diversification of Kinesin-I-mediated biological processes as evidenced by the distinctdiseases associated with mutations in kif5A and kif5C in humans. To date mutations in humankif5B have not been linked to disease; however, the mouse global knockout is embryonic lethaland skeletal muscle specific elimination of Kif5B causes myotendinous junction deficits andmuscular dystrophy. Here we used CRISPR mediated mutagenesis to disrupt zebrafish kif5Baand kif5Bb. Whereas single mutants show mild (kif5Ba) or no kif5Bb) overt phenotypes and areviable to adulthood, compound mutant genotypes lead to myopathy reminiscent of the skeletalmuscle specific mouse knock-out. In addition, compound mutants showed craniofacialabnormalities. Immunohistochemical and analysis of fluorescent reporters indicate craniofacialdeficits are associated with abnormal chondrocyte morphology, including impaired elongationand stacking. Our data shows that the duplicated zebrafish kif5B genes cooperate to promotenormal myogenesis and reveal a novel role for Kif5B in chondrogenesis and craniofacialdevelopment; thereby, providing insight into in vivo kif5B functions that when compromised maycontribute to human disease.8 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts227

ZDM8Boston | USA 2015ZDM8 POSTERSNervous System Abnormalities in a Zebrafish Model of DuchenneMuscular DystrophySarah Kucenas, Rebecca Nash, Jenna Eckardt, Lindsay Marjoram, Vani Hariharan1. University of Virginia, Department of Biology, BIG Center, Charlottesville, VA2. University of Virginia, Department of Biology, Charlottesville, VA3. University of Virginia, Department of Biology, Charlottesville, VA4. Duke University, Department of Cell Biology, Durham, NC5. University of Virginia, Department of Biology, Charlottesville, VADuchenne Muscular Dystrophy (DMD) is an X-linked recessive disorder caused by mutations inthe dystrophin gene. Currently, there is no cure for DMD, although several novel genomemodification treatments and translation read-through drugs are currently in clinical trials.Interestingly, in addition to the muscle weakness and atrophy observed in DMD patients,approximately one third of this population also has significant cognitive defects. Althoughcurrently thought to be unaffected in children with DMD, several previous studies demonstrate aprofound and significant defect in motor conduction velocity in at least a subset of children withthis disease. Therefore, although the mechanism underlying muscle fragility and atrophy areextensively studied and well understood, the nervous system phenotypes observed in thispopulation are not. For this reason, elucidation of the role of dystrophin in the nervous system isof profound clinical importance. Using an N-ethyl-N-nitrosourea (ENU) forward mutagenesisscreening approach in zebrafish, we identified mutants for an allele we designated as runaway(ray) vu267 . These fish were morphologically indistinguishable from WT embryos at 48 hours postfertilization (hpf), but by 72 hpf, had smaller jaws/heads, disrupted pigmentation and evidence ofmuscle fiber degeneration. Closer analysis revealed that spinal motor nerve development wasalso perturbed, with perineurial glia exiting the spinal cord ectopically and disorganized neuralcrest migration. Intriguingly, these nervous system defects always preceded any obviousmuscle atrophy. Using whole genome sequencing and complementation testing with anotherallele of dystrophin, sapje-like, we confirmed that ray vu267 mutants harbor a mutation indystrophin. Because of the early neural patterning defects we observe in these mutants, wehypothesize that there is a neurogenic component to DMD that precedes any overt musclephenotype and that these early defects may exacerbate disease etiology.2288 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts

ZDM8 POSTERSZDM7Madison | USA 2015ZDM8Boston | USA 2015Zebrafish Models for Human Craniofacial and Skeletal Diseases.1Viktoria Andreeva, Ph.D., 1 Melissa LaBonty, B.A., 1 John Lyons, B.A., 2 Len Zon, M.D.,3Wolfram Goessling, M.D., Ph.D. and 1 Pamela C. Yelick, Ph.D.1Tufts University, Boston MA, 2Department of Pediatrics, Boston Children’s Hospital, Boston MA3Department of Medicine, Harvard Medical School, Boston MAResearch in my laboratory focuses on mineralized tissue development, homeostasis, diseaseand regeneration. Craniofacial and skeletal defects are prevalent birth defects, occurring in1/700 live births. Craniofacial and skeletal defects can also result from cancer resections,sports injuries, accidents and battlefield trauma, resulting in significantly reduced quality oflife. We perform gene discovery and functional characterizations in zebrafish to identifymolecular signaling pathways regulating normal and syndromic mineralized tissuedevelopment relevant to human diseases. We apply knowledge generated from studies inzebrafish to inform tissue engineering strategies for effective repair and regeneration ofhuman mineralized craniofacial and skeletal tissues. To this end, we performed a large-scale,chemical mutagenesis screen in zebrafish to identify early lethal and adult viable mineralizedtissue mutants. We identified a variety of zebrafish mutants as models for human mineralizedtissue diseases including: Treacher Collins Syndrome; vertebral disc disease; scoliosis;hemifacial microsomia; midface hypoplasia; osteoporosis; degenerative disc disease;Osteogenesis Imperfecta (OI) and extremity malformations. We are currently performingNext Generation Sequencing to identify the causative mutation for each mineralized tissuezebrafish mutant. These studies are anticipated to significantly improve our currentknowledge and understanding of molecular signaling pathways regulating normal mineralizedtissue development and disease, and to provide inroads into new molecular targets andstrategies to treat and prevent human mineralized tissue diseases. These studies weresupported by NIH/NIDCR R01DE018043 (PCY) and NIH/NIDCR R21 AR065761 (PCY).8 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts229

ZDM8Boston | USA 2015ZDM8 POSTERSExtraocular Muscle Regeneration in Adult Zebrafish. A ParadigmChange?Alfonso Saera-Vila, Phillip E. Kish, Alon Kahana.Department of Ophthalmology and Visual Sciences, Kellogg Eye Center, University of Michigan,Ann Arbor, MI, USABinocular vision requires control of eye movement by 6 pairs of extraocularmuscles (EOMs). EOM dysfunctions cause misaligned binocular inputs, whichcan lead to amblyopia (neurologic vision loss) or diplopia, affecting 5-10% of theU.S. population. More broadly, skeletal muscle injury and degenerativeconditions are common, debilitating, and represent important causes of morbidityand mortality. Zebrafish is an established model for the study of tissueregeneration and human disease. We developed a zebrafish model for EOMregeneration following myectomy, focusing on the lateral rectus muscle (LR) dueto its anatomical isolation from the other EOMs and its surgical accessibility. Aftera myectomy of approximately 50% of the LR, adult zebrafish regenerate ananatomically correct and functional muscle. Interestingly, this regenerativeprocess appears to be independent of Pax7+ satellite cells. Instead, our datareveal that residual EOM myocytes undergo dedifferentiation, losing the Z-bandarchitecture and becoming mesenchymal cells expressing markers such asvimentin, fibronectin and smooth-muscle actin. Autophagy is strongly activatedduring this process to degrade the sarcomeric machinery and precedes a robustproliferation burst that involves 72% of the regenerating muscle nuclei with a cellcycle length of 19 hours. Different approaches were followed to characterize thesource of the proliferating cells. These approaches included single- and doublelabelingexperiments of proliferating myocyte nuclei undergoing multiple roundsof regeneration, and dextran fate-mapping. Surprisingly, MEF2-expressingmyonuclei were labeled with the proliferation marker EdU immediately after theonset of proliferation, indicating that the “post-mitotic” nuclei of the EOM haveregained the ability to proliferate after severe injury. I conclusion, zebrafish EOMsregenerate after myectomy by reprograming the extant myocytes to undergo amuscle-to-mesenchymal transition, proliferate, migrate and redifferentiate to forma new and functional muscle.2308 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts

ZDM8 POSTERSZDM7Madison | USA 2015ZDM8Boston | USA 2015Study of the koliber mutant points to the novel role of cholesterolsynthesis gene in skeletogenesisRebecca A. Anderson 1 , Kevin Schwalbach 1 , Stephanie R. Mui 1 , Elizabeth E. LeClair 3 ,Jolanta M. Topczewska 2 , Jacek Topczewski 11 Department of Pediatrics, 2 Department of Surgery, Northwestern University Feinberg School of Medicine,Stanley Manne Children’s Research Institute, Chicago, IL3 Department of Biological Sciences, DePaul University, Chicago, ILWe have identified a spontaneous mutant, referred to as koliber(kol nu7 ), which displays a lateonsetphenotype characterized by a misshapen body, reduced body length and small head, firstdetectable by gross morphologic examination at six weeks. We observed hyperossification ofboth endochondral bones, which develop through mineralization of cartilaginous molds, andintramembranous bones, which develop directly from condensed mesenchymal cells. Thedefects appear to be a result of enhanced, but not premature ossification. Analysis of the growthplate reviled aberrant stratification, particularly in the hypertrophic zone, and ectopic ossificationat later stages of development. In adult kol, normally persistent, growth plates are frequentlylost. Retention of negatively charged proteoglycans was observed in the growth plates ofjuvenile fish accompanied with an abnormal extracellular matrix environment. Expressionanalyses of kol growth plates indicated defect of the hypertrophic chondrocytes and elevatedexpression ossification markers.Using the positional cloning approach we identified critical region for a kol mutation.None of the genes located in this region were mutated, suggesting a lesion to a potentialregulatory element. qRT-PCR analysis, of the gene surrounding kol locus, revealed dramaticdown regulation of msmo1 in the mutant. Using the CRISPR technique, we have mutagenizedmsmo1 gene. Genetic complementation test indicate that the kol nu7 :msmo1 transheterozygotesdisplay a severe koliber phenotype by five weeks of development, supporting the role of msmo1in the skeletogenesis. Elevated expression of msmo1 was detected in the pre-hypertrophicchondrocytes of the growth plate indicating its specific role in the cartilage maturation. Themsmo1 gene encodes methylsterol monooxygenase 1, enzyme responsible for critical steps inthe “postsqualene” cholesterol biosynthesis. Interestingly, in several mutations of human genesinvolved in this pathway are associated with skeletal disorders. Our study indicates critical roleof sterols in the bone development and provide a new model of human skeletal dypslasias.8 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts231

ZDM8Boston | USA 2015ZDM8 POSTERSPurpose: Progressive protein destabilization and unfolding, followed by self-assembly offibrils or aggregates are hallmarks of cellular degeneration during aging.Characterization of the mechanisms of cellular degeneration in vivo is limited by thedifficulties of studying the dynamics of molecular aging and degeneration in animalmodels. The purpose of the current study is to express human amyloid proteins fused toyellow fluorescent protein (YFP) in the transparent cells of the lens of the eye to evaluatethe cellular distribution and dynamics of progressive aggregation in vivo.Methods: Human cDNAs for alphaB crystallin (αB), betaB crystallin (βB) and amyloidbeta (Aβ) proteins were cloned into Invitrogen Gateway Tol2 vectors in frame with theYFP (Aβ was generously provided by J. Sullivan). The fusion proteins were controlled bya zebrafish αA crystallin promoter, which limits expression to eye lens cells (Kurita et al.(2003) Dev Biol 255:113). Control and experimental vectors were injected into fertilizedwild-type ABWIK zebrafish embryos at the one- or two-cell stage. At 4-6 days postfertilization,the lenses of F1 transgenic developing zebrafish were imaged usingfluorescence or multiphoton microscopy.Results: YFP, αB-YFP and βB-YFP were expressed in alternating bright and dark layersthroughout the lens. In contrast, amyloid Aβ-YFP expressed primarily in bands of corticalfiber cells as bright condensed fluorescent aggregates. The cellular distribution wasasymmetric and Aβ-YFP was not observed in every cell. Aβ-YFP was found in peripheralcells only. Core cells were either protected against aggregate formation or did notexpress Aβ. The patterns of Aβ fluorescence changed with age.Conclusions: αA crystallin promoter driven expression allowed visualization of Humanamyloid YFP-fusion proteins in vivo in zebrafish lens. The patterns of gene expressionvaried with the selected protein and with age. Supported by EY04542 from the NEI.2328 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts

ZDM8 POSTERSZDM7Madison | USA 2015ZDM8Boston | USA 2015Feeling the sleep pressure in health and diseaseSabine Reichert and Jason RihelDepartment of Cell and Developmental Biology, UCL, London, UKThe control of sleep and wakefulness is a fundamental biological process thatensures health and cognitive performance across species. The need to sleep (sleeppressure) is determined by two processes: the sleep homeostat and the circadianclock. Compared to the circadian drive for wakefulness, the mechanism regulatingsleep homeostasis is not well understood. It has been previously demonstrated thatsleep-modulatory circuits locate to the hypothalamus and include wake-promotingorexinergic neurons in the lateral hypothalamus as well as GABA/galaninergic sleeppromotingneurons in the ventrolateral preoptic area (VLPO). However, how thesesleep/wake promoting centres sense and process increased homeostatic pressure tomodulate behaviour remains largely unknown. Because sleep disruption is acommon feature of numerous disorders, including epilepsy, autism spectrumdisorder, Prader Willi Syndrome and also has negative consequences on health, howthese processes go awry during disease is of great interest.In order to dissect the neuronal underpinnings of sleep homeostasis, we combineddrug-induced sleep deprivation and molecular anatomical analysis with functionalimaging in zebrafish larvae. Following sleep deprivation, zebrafish enter a prolongedphase of sleep coinciding with an increase in sleep-inducing hypothalamicneuropeptides such as galanin. Using cfos staining and dynamic two photonimaging,we are investigating the identity of the neuronal populations that arespecifically active in sleep deprived animals and are testing their functionalimportance during sleep rebound. Interestingly, preliminary data suggests thatphysical activity is not sufficient to accumulate sleep homeostatic pressure but thatspecific increases in neuronal activity are required.Taken together, we are employing drug-induced sleep deprivation to identify theneurochemical mechanisms and neural circuits involved in sleep homeostasis andtheir disruption in genetic models of neurodevelopmental diseases.8 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts233

ZDM8Boston | USA 2015ZDM8 POSTERSDomoic acid targets developing oligodendrocytes and causesdevelopmental neurotoxicityJennifer Martinez Panlilio 1,2,3 , Neelakanteswar Aluru 2,3 , Mark E. Hahn 2,31 MIT/WHOI Joint Graduate Program in Oceanography and Oceanographic Engineering2 Biology Department, Woods Hole Oceanographic Institution3 Woods Hole Center for Oceans and Human HealthAlterations in glutamate homeostasis during development can have adverse consequences forlong-term nervous system function. The goal of the study is to determine how exposure to anexcitatory glutamate analog, domoic acid (DA), during development affects glial celldevelopment and behavior. Zebrafish embryos were microinjected with different concentrations(1.42, 2.6 and 7.1ng DA) of DA at the 2-8 cell stage. At 4 dpf, oligodendrocyte precursor cells(OPCs) were counted, at 5 dpf the degree of myelination was quantified, and at 7 dpf the larvalstartle response was tested. Exposure to 7.1 ng DA caused a 3.5-fold reduction in the numberof OPCs along the dorsal spinal cord (p=.0002). However, DA exposure did not reduce themRNA expression of myelin basic protein, suggesting that DA can perturb oligodendrocytedevelopment but does not decrease myelination. Nonetheless, DA exposure was shown to alterbehavior. DA-exposed animals showed a muted startle response to vibrational taps (14V, 18Vand 22V intensity) compared to water-injected controls. Furthermore, the long-latency startleresponses (12ms after stimuli) of larvae exposed to the highest concentration of DA had asmaller bend angle during their first c-bend (p=.005) and were slower (p=.03) in executing theircounter turn. We conclude that developmental exposure to DA alters startle response in a dosedependentmanner, while oligodendrocyte development is only perturbed at the highest dose(7.1ng DA). Further analyses will determine which processes in oligodendrocyte developmentare perturbed, and whether altering OPCs is related to the aberrant startle responses.(P01ES021923, OCE-1314642, WHOI APO)2348 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts

ZDM8 POSTERSZDM7Madison | USA 2015ZDM8Boston | USA 2015Behavioural, Neuroanatomical and Pharmacological Profiling inZebrafish Models of AutismMarcus Ghosh 1 , Ellen Hoffman 2,3 , Antonio Giraldez 4 , Matthew State 5 , and Jason Rihel 11. Department of Cell and Developmental Biology, University College London, Gower Street,London WC1E6BT, UK2. Child Study Centre, Yale School of Medicine, New Haven, CT 06510, USA3. Program on Neurogenetics, Yale School of Medicine, New Haven, CT 06510, USA4. Department of Genetics, Yale University School of Medicine, New Haven, CT 06510, USA5. Department of Psychiatry, University of California San Francisco, San Francisco, CA 94143, USARecent multi-genome comparisons, such as genome-wide association studies and wholeexome sequencing, suggest a large number of genes that may play causative roles in autismspectrum disorder. The impact of these mutations on brain development, function andsubsequently behaviour remains however largely unknown, precluding a mechanisticunderstanding of autism and consequently targeted pharmacological therapeutics.To address this issue we have developed a zebrafish phenotyping pipeline to systematicallystudy a library of zebrafish mutants in high confidence autism risk genes. Our approach hasthree main lines of inquiry; behaviour, neuroanatomy and pharmacological profiling. Bysubjecting mutants to a battery of quantitative behavioural assays we are working to identifydeficits in clinically relevant behavioural phenotypes, including seizure liability and sleepdisruption; common and important comorbidities in autistic individuals. To investigate theaetiology of these phenotypes we are employing both confocal and two photon imaging toidentify neuroanatomical and functional deficits in our models, providing insight into how thesedeficiencies translate to behavioural abnormalities. Finally by applying drug clusteringmethodologies we are not only pharmacologically probing both behavioural andneuroanatomical phenotypes, but also identifying novel drug-gene interactions and phenotypicsuppressors.While the specific profile of each of our mutants is highly informative and perhaps highlightsthe need for personalised treatments for these individuals, we hope to identify commonthemes amongst our models that may provide exciting evidence for convergent mechanismsand broad spectrum therapeutics.Ultimately through the application of this methodology we hope to gain insight into howmedically relevant defects in brain architecture and function give rise to clinically observablebehavioural phenotypes as well as to identify potentially translatable phenotypic suppressors.8 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts235

ZDM8Boston | USA 2015ZDM8 POSTERSA Novel Zebrafish Model of Neurodegeneration-Induced AstrocyteActivationMerja Jaronen, Ruxandra Covacu, Jessica Kenison and Francisco J. QuintanaAnn Romney Center for Neurologic Diseases, Brigham and Women’s Hospital, Harvard Medical School, 77Avenue Louis Pasteur, Boston, MA 02115, USA.Astrocytes play an important role in the pathology of numerous neurodegenerative diseases, suchas multiple sclerosis (MS), Alzheimer’s disease (AD), Parkinson’s disease (PD), amyotrophiclateral sclerosis (ALS) and schizophrenia. However, the molecular pathways controlling astrocytefunction and activation are still relatively unknown. This has resulted in a lack of astrocyte-targetedtherapies for the management of central nervous system (CNS)-related disorders. Current studiescharacterizing the molecular mechanisms of astrocyte involvement in CNS disorders will results ina large list of candidate genes and proteins, which need to be further validated in simplerexperimental organisms. Both primary astrocytes and cell lines have been used for validationpurposes. However, they lack influence over neighboring cells. Therefore there is a clear need fornew experimental models to study astrocytes in their physiological context. Zebrafish offers asuperior platform for such validation studies, while it enables the use of both genetic and chemicalscreens to identify candidates of interest that can be further investigated in other model systems.We have developed a reporter zebrafish line to follow astrocyte activation, and established azebrafish model of inducible neurodegeneration. This reporter zebrafish line is useful to studyastrocyte signaling pathways controlling astrocyte physiology. This zebrafish reporter line can becombined with existing zebrafish models of CNS-related disorders. Thus, this reporter zebrafishline is a unique tool that allows us to investigate in vivo targets and genes identified in genomicand transcriptional studies performed in murine and human models, and also to identify newpathways of interest.2368 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts

ZDM8 POSTERSZDM7Madison | USA 2015ZDM8Boston | USA 2015Novel genes critical for hypoxic preconditioning in zebrafishare regulators of insulin and glucose metabolismT. Manchenkov * , M. P. Pasillas * , D. Zhang * , G. G. Haddad §,† , and F. B. Imam *,†Department of Pediatrics (Divisions of * Neonatology and § Respiratory Medicine), University ofCalifornia San Diego School of Medicine, La Jolla, California, and † Rady Children’s Hospital, SanDiego, United States of AmericaFine metabolic regulation to adjust for changes in oxygen and energy availability is aconserved, ubiquitous survival strategy of cells and tissues to unpredictableenvironments. Severe oxygen deprivation can overwhelm these protective strategiesand is a common cause of major brain, heart, and kidney injury in adults and newbornsalike. Intriguingly, mild hypoxia can be preventative against a later, more severe hypoxiaexposure via “hypoxic preconditioning”—a protective phenomenon that is not yet fullyunderstood. We have therefore established and optimized an embryonic zebrafish modelto study hypoxic preconditioning in detail using a functional genomic approach. Usingthis developmental zebrafish model, we validated five novel hypoxia-protective genesfrom hundreds of hypoxia-regulated genes we identified via differential expressionmicroarray: irs2, crtc3, and camk2g2 have been previously implicated in insulin andglucose metabolism, while btr01 and ncam2 are previously uncharacterized. Theseresults extend our understanding of the mechanisms of hypoxic preconditioning andaffirm the discovery potential of this novel vertebrate hypoxic stress model.8 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts237

ZDM8Boston | USA 2015ZDM8 POSTERSScreening natural products for neuroprotective potential in thezebrafish larvae PD modelSuzita Noor, David Wong, Vickneswaran Kanthasamy, Anwar NorazitDepartment of Biomedical Science, Faculty of Medicine, University of Malaya, Kuala Lumpur, MalaysiaNeurodegenerative diseases are debilitating conditions that have thus far remainedincurable. Parkinson ’s Disease (PD) is the second most common neurodegenerativedisease and is characterized by progressive loss of dopaminergic (DA) neurons in thesubstantia nigra pars compacta. Current PD treatments are able to alleviate symptomsbut do not halt disease progression. The focus is now on discovering natural productsand antioxidants with potential neuroprotective properties that might delay diseaseprogression, or be applicable for use as adjunctive therapy.In zebrafish, DA neurodegeneration is induced by exposure to MPTP, a neurotoxin thatcauses profound reduction of tyrosine hydroxylase(th)-positive neurons and impairslocomotor behaviour. By exposing embryos to MPTP, a zebrafish PD model isavailable to assess the neuroprotective potential of antioxidant compounds. Therehave been many anecdotal reports of curcumin being used as a neuroprotectant. Weapplied the zebrafish model to assess curcumin extracts in protecting zebrafish larvaefrom neuronal insult. Larvae at 3 dpf were immersed in system water with dissolvedcurcumin for 24 hours before commencing with MPTP exposure at 4 dpf. Wildtypelarvae were exposed to MPTP and co-incubated with curcumin up to 7 dpf, with thesolutions refreshed daily. Controls were larvae that were untreated, as well as embryosthat were exposed to curcumin alone, and MPTP alone. All larvae were assessed at 5dpf for signs of impaired locomotor behavior, and at 7 dpf, all larvae were euthanisedand homogenized in Trizol for RNA extraction and gene expression analyses.Larvae were also observed for curcumin biocompatibility. For larvae that were pretreatedwith curcumin prior to MPTP exposure, locomotor impairment brought about byMPTP neurotoxicity was ameliorated, correlating with gene expression data. Thisscreening method could be applied to other natural product extracts to quickly identifyneuroprotective potential.2388 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts

ZDM8 POSTERSZDM7Madison | USA 2015ZDM8Boston | USA 2015Modeling the Chemical Pathways of Fast-acting Antidepressants inZebrafishAndrew J Rennekamp 1,2,3 , Seth Brownmiller 1 , Xi-Ping Huang 4,5 , Bryan L Roth 4,5 andRandall T Peterson 1,2,31. Cardiovascular Research Center and Division of Cardiology, Department of Medicine, MassachusettsGeneral Hospital, Charlestown, MA, USA.2. Department of Systems Biology, Harvard Medical School, Boston, MA, USA.3. Broad Institute, Cambridge, MA USA.4. National Institute of Mental Health Psychoactive Drug Screening Program. University of NorthCarolina, Chapel Hill, Chapel Hill, NC, USA.5. Department of Pharmacology, University of North Carolina, Chapel Hill, Chapel Hill, NC, USA.Target-based drug discovery approaches have the potential to miss systems-modulatingcompounds, which may be useful for the treatment of complex polygenic diseases likedepression. Zebrafish larvae can be used for rapid high-throughput discovery of behaviormodifying small molecule drugs with complex circuit-modifying pharmacology. The dissociativedrug Ketamine has fast-acting antidepressant effects in treatment-resistant major depressivedisorder (MDD), however its use is limited due to its hallucinogenic side effect and abusepotential. We found that in zebrafish larvae Ketamine causes a dose-dependent switch fromfreezing to escape behavior in the strobe light assay. We then used this assay to find novelclasses of small molecules that either phenocopy or block the effects of Ketamine. We foundthat the effects of Ketamine are mimicked by neurotrophin agonists and GABA-A blockers butsuppressed by AMPA receptor antagonists and L-type Ca 2+ channel blockers. This suggeststhat the chemical pathways thought to be responsible for the antidepressant effects of Ketaminein mammals are likely also intact in fish. Therefore, zebrafish present a novel approach for thediscovery of new fast-acting antidepressants that is both holistic and target-agnostic.8 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts239

ZDM8Boston | USA 2015ZDM8 POSTERSCharacterizing vertebrate stress response with mutant zebrafishstrains in the hypothalamic-pituitary-adrenal axisHan B. Lee 1 , Tanya L. Poshusta 2 , Randall G. Krug II 1 , and Karl J. Clark 1,21. Neurobiology of Disease program, Mayo Graduate School2. Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN USAStress-related neuropsychiatric disorders (ND), such as major depressive disorder, are leadingcauses of disability worldwide. A hallmark physiological change in patients with NDs isalterations in the hypothalamic-pituitary-adrenal (HPA) axis activity that manifest as hyper- orhypo-cortisolemia. Vertebrate-specific systemic stress response (SR) signals through theneuroendocrine HPA axis and sympathetic nervous system (SNS). However, we only havelimited knowledge on how molecular components in the HPA axis and SNS interact to producean integrated behavioral response. Hyperosmotic stress (100 mM NaCl) invokes an increasedlocomotor response in larval zebrafish within minutes. We hypothesized that zebrafishlocomotor response to hyperosmotic stimulation is a behavioral SR that is composed of multiplemolecular pathways. We have begun our genetic analysis by examining key receptors in theHPA axis. By injecting highly efficient TALENs (transcription activator-like effector nucleases)that target genes in the HPA axis and generate DNA lesions in both chromosomes (bi-allelic),we have found that locomotor response to hyperosmotic stress is significantly influenced bycrhr1 (corticotropin releasing hormone receptor 1), mc2r (melanocortin receptor 2; receptor foradrenocorticotropic hormone (ACTH)), and nr3c1 (glucocorticoid receptor), but not bymineralocorticoid receptor (MR; nr3c2). In addition, mc2r germline mutants showed a significantdecrease in locomotor response. To our surprise, an nr3c1 germline mutant that results in aframeshift-induced truncation of the glucocorticoid receptor did not show any deficits inlocomotor response. We are currently investigating potential compensatory mechanisms bycharacterizing locomotor response in nr3c1 mutants with pharmacological MR down-regulationand alternative alleles of nr3c1 mutants. Understanding how multiple signaling systemscontribute to this seemingly simple behavioral response will help us better model vertebratespecificSR.2408 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts

ZDM8 POSTERSZDM7Madison | USA 2015ZDM8Boston | USA 2015Expression of the transcription factor gs homeobox 1 definesneurons that are required for prepulse inhibitionSadie A. Bergeron, Nicole Carrier, Grace H. Li, Sohyun Ahn, Harold A. BurgessNIH, NICHD, Bethesda, MD, USA 20892Prepulse inhibition (PPI) of the acoustic startle response is a behavioral test used as ameasure of effective sensory information processing by the central nervous system. DisruptedPPI is reported in schizophrenia and is an endophenotype with which genetic andneuroanatomical changes can be linked to neurodevelopmental disturbances. To identify neuralcircuitry for PPI, we performed a novel genetic circuit-breaking screen. For this, we firstdeveloped a new technique to enrich for brain-specific expression of Gal4 in stable transgeniclines. These lines were then used to conditionally express a cytotoxin in neurons before testinglarvae for PPI. Line y252, an enhancer trap for gs homeobox 1 (gsx1), has disrupted PPI whichwas recapitulated by direct optogenetic inhibition of gsx1 neurons expressing archaerhodopsin.Brainstem gsx1 neurons synapse in close proximity to the command neuron for startleresponses, the Mauthner cell, and they express glutamate (vglut2). Consistent with thissurprising implication of glutamate signaling in a functionally inhibitory pathway,pharmacologically blocking NMDA receptors reproduces the gsx1 neuron ablation phenotype.Intriguingly, the pattern of Gsx1 expression is highly conserved during neural development inmice and zebrafish, therefore we analyzed Gsx1 knockout mice for PPI to determine if ourfindings were relevant to mammals. We found reduced PPI in Gsx1 knockout mice compared towild type siblings. Since Gsx1 knockout mice die by three weeks of age, we generated gsx1zebrafish mutants which recapitulate phenotypes of the mutant mice including reduced size,neuroendocrine changes, and altered PPI. Our data support a model in which Gsx1 derivedneurons form an integral part of the PPI circuit across vertebrates, and future work in zebrafishwill reveal important roles for gsx1 in the development of neural circuits across brain regionsthat are involved in sensory motor gating disruptions in neurological disorders.8 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts241

ZDM8Boston | USA 2015ZDM8 POSTERSAs a participant in the NIH Undiagnosed Diseases Network (UDN), a 21-year-old malepatient was seen and demonstrated signs of upper motor neuron dysfunction, deafness,and seizures, and has since declined into a state of paralysis. After SNP and exomeanalysis of the patient and his family, Gem associated protein 5 (GEMIN5) was identifiedas a strong candidate disease allele. GEMIN5 is the snRNA-binding component of thesurvival of motor neuron (SMN) complex. The SMN complex plays an important role inmRNA splicing by cytoplasmic assembly of spliceosomal small nuclearribonucleoproteins that subsequently catalyze the splicing reaction. While GEMIN5 is notcurrently associated with a human disease, the gene SMN1 (and the related SMN2), thecore protein of the SMN complex, is associated with spinal muscular atrophy (SMA)types 1-4. Reduction of SMN1 is also associated with severe motor neurondegeneration. Therefore, due to the known interaction of the SMN complex andGEMIN5, we hypothesize the missense mutation found in one copy of GEMIN5 iscontributing to this patient’s disease. We tested the function of GEMIN5 in zebrafish bytargeted inactivation using CRISPR-Cas9. We observed fatality between 7-21 dpf in fishhomozygous for frame shifting deletions in GEMIN5. We tested the mutant fishes’ abilityto repair neural damage by performing a hair cell regeneration assay after copperexposure. The mutant animals had no regenerative response despite theirdevelopmentally normal appearance. We have created a transgenic line ubiquitouslyexpressing wild-type human GEMIN5 or the variant found in the patient to rescue thezebrafish mutant phenotypes. We examined the mutants’ motor neuron development bysensory motor neuron staining using Znp-1 antibody. These results confirm a role forGEMIN5 in survival and reinforce our interest in testing the association between theidentified variants in the patient and the manifestation of the disease.2428 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts

ZDM8 POSTERSZDM7Madison | USA 2015ZDM8Boston | USA 2015A new model for Multiple Sclerosis: Using zebrafish to directlyinvestigate demyelination and remyelination in vivoAngela D. Morris, Sarah Kucenas1. University of Virginia, Departments of Neuroscience & Biology, BIG Center, Charlottesville, VA2. University of Virginia, Department of Biology, BIG Center, Charlottesville, VAMultiple Sclerosis (MS) is a chronic, neurodegenerative disease in which the immune systeminappropriately attacks the insulating myelin sheath surrounding axons of the central nervoussystem (CNS), leading to myelin destruction, axonal degeneration and a life often dependentupon walking aids and assistance. Oligodendrocytes, an essential myelinating glial population inthe CNS, are targeted during MS attacks, leaving axons exposed and vulnerable to theirenvironment. Unfortunately, how demyelination occurs and the cellular mechanics involved inremyelinating naked axons within demyelinated lesions are unknown. Mammalian modelscommonly used to study MS, including experimental autoimmune encephalomyelitis (EAE) anddrug-induced demyelination, as well as patient data or clinical samples, do not allowresearchers to investigate the processes underlying myelin destruction and repair in vivo.Although a generalized demyelination model exists in zebrafish that utilizes the nitroreductasemediatedablation system, a major limitation of this model is the inability to create focal lesions,which are a hallmark of MS pathology. Therefore, we have a created a novel, drug-inducedfocal demyelination model in zebrafish. Using microinjection, we deliver a demyelinatinglysolecithin cocktail solution focally and precisely into the spinal cord of zebrafish larvae.Preliminary characterization of this model demonstrates that the lysolecithin cocktail has anaffect on oligodendrocytes as we observe a significant reduction in the number of Sox10 + cellsat 8 hours post injection and a decrease in sox10:mrfp fluorescence only within the drugdispersal region of the lysolecithin cocktail injection group. In the future, we will use this modelto visualize the temporal dynamics and cellular mechanics underlying myelin destruction, debrisclearance and subsequent myelin repair as it occurs in real time, in a living vertebrate organism.8 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts243

ZDM8Boston | USA 2015ZDM8 POSTERSEffects of cocaine on visual processing in zebrafishElizabeth Riley, Konstantin Kopotiyenko, Irina Zhdanova.1. Riley: Graduate Program in Neuroscience and Department of Anatomy andNeurobiology, Boston University School of Medicine2. Kopotiyenko: Department of Anatomy and Neurobiology, Boston UniversitySchool of Medicine3. Zhdanova: Department of Anatomy and Neurobiology and Graduate Program inNeuroscience, Boston University School of MedicinePsychostimulants are known to alter visual function and improve visual attention inpatients with attention deficit. To determine the impact of prenatal and acute cocaineexposure on visual processing, we employed two-photon microscopy and a transgeniclarval zebrafish expressing the calcium indicator GCaMP-HS to document neuronalresponses to contrasting visual stimuli, red light (LF) and dark (DF) flashes. We foundthat both LF and DF elicited similar responses in the optic tectum neuropil (TOn), whilethe dorsal telencephalon (dTe) responded only to LF. Acute cocaine (0.5 µM) reducedneuronal responses to LF in both brain regions but not to DF. Repeated stimuluspresentation led to habituation of dTe neurons to LF and acute cocaine prevented this.TOn habituated to DF, but not LF, and DF habituation was not modified by cocaine.Remarkably, prenatal cocaine exposure prevented the effects of acute cocaine on LFresponse amplitude and habituation later in development in both regions, but did notaffect DF responses. The characteristic effects of acute cocaine on LF responses inboth TOn and dTe were absent in zebrafish lacking the dopamine transporter, thecanonical target of cocaine. We discovered that, in spite of similar neural responses toLF and DF in the TO (superior colliculus in mammals), responses to LF are morecomplex, involving dTe (homologous to the cerebral cortex), and are more vulnerable tococaine. We demonstrated that acute cocaine exposure affects visual processingdifferentially by brain region, likely via the dopamine system, and that prenatal cocaineexposure can modify visual processing in multiple brain structures and in a stimulusdependentmanner. Our result offers new methodological approaches for studying theadverse and therapeutic effects of psychostimulants on visual attention, and for thedevelopment of new pharmacological interventions.2448 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts

ZDM8 POSTERSZDM7Madison | USA 2015ZDM8Boston | USA 2015Tools for zebrafish research, from activity analysis to detailed observation inembryos and larvaeBrooke AprilNoldus Information Technology Inc., Leesburg, VirginiaZebrafish are an emerging model organism, for both importance in the market and their relative lowcost.Behavioral and physiological analysis of zebrafish disease models, genetics, pharmacology,toxicology, and neuroscience require tools that combine accuracy, reliability, and validity with highthroughput. Automated recording, data collection, and analysis using the video tracking systems presentedhere increase efficiency, standardization, and throughput in zebrafish research across the life span.For studies of embryos and larvae, we developed a comprehensive system by combining the latestin video tracking, image processing, and pattern recognition technology with high-end optics and camerasin a completely controlled environment. The transparency of zebrafish embryos and larvae also lendsperfectly to studying activity and tail coiling of embryos, as well as gut, blood flow, and cardiovascularfunctioning in larvae. New imaging technologies have recently been developed to measure these detailedphysiological responses as well.Beyond individual behavior and physiology, adult zebrafish are an important model of complexsocial phenotypes as they engage in shoaling behavior that is highly sensitive to experimentalmanipulations. Using our software to track an entire shoal, several studies with psychotropic drugs havedemonstrated significant effects on group size and shoaling activity. The availability of such tools forzebrafish research will further enhance experimental modeling of genetic, pharmacological, anddevelopmental mechanisms underlying human social deficits.Three-dimensional analysis of adult zebrafish swimming paths offers another promisingmethodology. Compared to traditional 2D traces, 3D swim path reconstructions enable both macro(general) and micro-level (specific/repeated) analyses of potentially meaningful behavioral patterns,providing a complete picture of fish behavior. 3D track analysis delivers movement parameters (e.g.distance traveled, velocity, angular velocity, path tortuosity) within 3D space, which has shown to be highlysensitive to psychoactive substances.The various techniques presented here offer scientists valuable quantitative descriptions ofphysiological and behavioral profiles of disease, illness, toxicological substances, genetic alterations, andpharmaceutical compounds.8 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts245

ZDM8Boston | USA 2015ZDM8 POSTERSImaging the dynamics of tumor cell heterogeneity followingtransplantation in optically clear, immune compromisedzebrafishQin Tang 1,2 , John C. Moore 1,2 , Jessica S. Blackburn 1,2 , Elaine G. Garcia 1,2 , Myron S.Ignatius 1,2 , Madeline N. Hayes 1,2 , Ines M. Tenente 1,2 , Caitlin Bourque 4 , ShuningHe 2,3 , A. Thomas Look 2,3 , Yariv Houvras 4 , David M. Langenau 1,2*1. Molecular Pathology, Cancer Center, and Regenerative Medicine, Massachusetts GeneralHospital, Boston, MA 02129;2. Harvard Stem Cell Institute, Cambridge MA 02139;3. Pediatric Oncology, Dana-Farber Cancer Institute, Boston, MA 02114;4. Departments of Surgery and Medicine, Weill Cornell Medical College, New York, New York10065Cancers contain a wide diversity of tumor cell types that are defined by differentiationstate, unique genetic mutations, and altered epigenetic programs that ultimately impartfunctional diversity to individual cancer cells. Elevated tumor heterogeneity is linked withincreased risk of progression, therapy resistance, and relapse. Yet, imaging of cancerprocesses, including the dynamic behavior of heterogeneous cancer cell populations inlive animals, has been both a technical and biological challenge in the field.Investigators have developed imaging modalities and complex surgical approaches inmice to gain optical access to developing tumors – requiring implantation of imagingwindows for repeated high-resolution intravital microscopy (IVM). Using theseapproaches, several hallmarks of cancer, including migration, intravasation, invasion andmetastasis, as well as responses to chemotherapies in vivo, have been successfullyimaged. Building on the previously reported rag2 E450fs mutant zebrafish that are deficientin T and B cells and engraft fluorescently labeled zebrafish cancers, we have createdrag2 E450fs (casper) fish and achieved direct visualization of heterogeneous cancer cellpopulations at single cell resolution by non-invasive, repeated, live animal confocalmicroscopy, without the need for surgical construction of imaging windows. Tumorengraftment into this model permitted the dynamic visualization of neovascular growthover time, niche partitioning of tumor-propagating cells in embryonal rhabdomyosarcoma(ERMS), the emergence of clonal dominance in T-cell acute lymphoblastic leukemia (T-ALL), and tumor evolution resulting in increased proliferation and metastatic potentialmelanoma. In total, cell transplantation approaches using the rag2 E450fs (casper)zebrafish provide many unique opportunities for uncovering novel biology underlyingcancer and will likely provide a means to dynamically image cancer processes in largescalestudies.2468 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts

ZDM8 POSTERSZDM7Madison | USA 2015ZDM8Boston | USA 2015CRISPR MultiTargeter: A Web Tool to Find Common and UniqueCRISPR Single Guide RNA Targets in the ZebrafishSergey V. Prykhozhij 1 , Vinothkumar Rajan 2 , Daniel Gaston 3 , Jason N. Berman 1,2,31. Department of Pediatrics, Dalhousie University, Halifax, Nova Scotia, Canada2. Department of Microbiology and Immunology, Dalhousie University, Halifax, Nova Scotia, Canada3. Department of Pathology, Dalhousie University, Halifax, Nova Scotia, CanadaGenome engineering in the zebrafish has been revolutionized by the discovery of clusteredregularly interspaced palindromic repeats (CRISPR) and CRISPR-associated system genes(Cas) in bacteria. The type IIB Streptococcus pyogenes CRISPR/Cas9 system functions inmany species and additional types of CRISPR/Cas systems are under development. In the typeII system, expression of CRISPR single guide RNA (sgRNA) targeting defined sequencestogether with Cas9 generates a sequence-specific nuclease inducing small deletions orinsertions. Moreover, large DNA molecules can be knocked-in at the targeted sites. Severaltools available for designing sgRNAs identify unique locations in the genome. However, theability to find sgRNA targets common to several similar sequences or, by contrast, unique toeach of these sequences, would also be advantageous. To provide such a tool, we developedthe CRISPR MultiTargeter website (www.multicrispr.net). Similar DNA sequences in questioninclude duplicated genes and sets of exons of different transcripts of a gene. Thus, weimplemented a basic sgRNA target search of input sequences for single-sgRNA and twosgRNA/Cas9nickase targets, as well as common and unique sgRNA target searches in 1) a setof input sequences; 2) a set of similar genes or transcripts; or 3) transcripts a single gene. Wedemonstrate potential uses of the program by identifying unique isoform-specific sgRNA sites in71% of zebrafish alternative transcripts and common sgRNA target sites in approximately 40%of zebrafish duplicated gene pairs. The design of unique targets in alternative exons is helpfulbecause it will facilitate functional genomic studies of transcript isoforms. Similarly, theapplication of CRISPR Multitargeter to duplicated genes may simplify multi-gene mutationaltargeting experiments, for example, to generate disease models in zebrafish. Given the growingpopularity of the CRISPR/Cas system for disease modeling in zebrafish, CRISPR Multitargeterprovides a unique interface to enhance the versatility of CRISPR/Cas targeting.8 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts247

ZDM8Boston | USA 2015ZDM8 POSTERSZebrabow clonal analysis with fate-mapping defines the number ofstem cells arising during hematopoietic developmentJonathan E. Henninger 1,2 , Ellen M. Durand 1 , Jessica L. Moore 1 , Leonard I. Zon 1,21Division of Hematology/Oncology, Boston Children’s Hospital, Boston, MA 021552Harvard Medical School, Boston, MA 02115Lifelong hematopoiesis involves the proliferation and differentiation of successive clones intomature blood lineages. These clones arise in the hemogenic endothelium of the developingaorta. To date, evidence for the number of stem cells that arise is based on genetic reporters ortransplantation. These studies define the origins of hematopoiesis, but the heterogeneity of thearising pools has never been assessed in a native setting. We have optimized the Zebrabowsystem to induce endogenous and heritable barcoding during stem cell development inzebrafish. Zebrafish that express the Zebrabow construct, containing floxed dTomato, CFP, andYFP, were crossed with fish that express CreER T2 driven by the draculin promoter, which isexpressed in hematopoietic stem cells and tissues derived from lateral mesoderm. Zebrabowembryos were treated with tamoxifen at distinct stages of embryogenesis. Activation of the Creprotein leads to stochastic recombination of the fluorophores to generate unique color barcodes.Our system can achieve up to 95% labeling efficiency, and it allows for clonal analysis in allblood lineages including erythrocytes, which are nucleated in zebrafish. Activation ofdraculin:CreER T2 by tamoxifen at 24-36 hpf induced labeling of stem cells with multiple colors forover one year post-label induction. At 3 months after Cre recombination, peripheral blood andmarrow smears displayed multicolored blood. FACS and confocal microscopy were used tomeasure intensity levels of the three fluorescent proteins. We applied supervised, density-basedclustering of the intensity values to identify color barcodes. Measuring these barcodes ingranulocytes as a read-out of stem cell activity, we find that 19 HSC clones (16.6-21.6, 95% CI)are responsible for sustaining hematopoiesis throughout development and in adulthood. Ourstudies provide insight into early clonal events that regulate the development of hematopoiesis,and provide a novel system to examine the genetics of clonality.2488 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts

ZDM8 POSTERSZDM7Madison | USA 2015ZDM8Boston | USA 2015Zebrafish Transgenic Embryos Specifically Respond toEndoplasmic Reticulum StressHung-Chieh Lee 1 , Chih-Wei Tseng 1 , Hui-Lan Huang 1 , Kai-Yen Lin 1 , Huai-JenTsai 21. Institute of Molecular and Cellular Biology, National Taiwan University, Taipei, Taiwan2. Institute of Biomedical Sciences, MacKay Medical College, New Taipei City, TaiwanUpstream open reading frame (uORF)-mediated translational inhibition isimportant in controlling the expression of key regulatory genes. However,understanding the underlying molecular mechanism of such uORF-mediated controlsystem in vivo is challenging by the absence of an animal model. We generated azebrafish transgenic line, termed huORFZ, harboring a construct which the uORFsequence from human CCAAT/enhancer-binding protein homologous protein gene(huORF chop ) is fused with GFP and driven by a cytomegalovirus promoter. Thetranslation of transgenic huORF chop -gfp mRNA is absolutely inhibited by the huORF chopcassette in huORFZ embryos during normal conditions. However, when the huORFZembryos were treated with ER stresses, such as ER-stress inducing drugs, alcohol,heat shock, hypoxia, heavy metals and endocrine disrupting chemicals, thedownstream gfp mRNA was thereof translated and GFP was apparent in embryos.Interestingly, we found that the GFP expression patterns in huORFZ embryos weretissue-specific responses to various stresses and the intensity of GFP signal inhuORFZ embryos was correlated to the strength of stresses. Specifically, whenhypoxia stress was given to the huORFZ embryos for 2 hr, followed by 10 hr of oxygenrecovery, we observed that GFP signal was apparent only in the central nervoussystem. The GFP patterns were consistent with the patterns of endogenous chopmRNA and protein. By immunofluorescence and flow cytometry analysis, we foundthat GFP-(+) cells were mostly neuronal stem/progenitor cells, along with some wereastrocytes, and a small portion of oligodendrocytes. We also demonstrated thathypoxia-responding GFP-(+) cells after recovery could proliferate, migrate anddifferentiate into neurons.8 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts249

ZDM8Boston | USA 2015ZDM8 POSTERSDissection of normal, aberrant, and malignant hematopoiesis atsingle cell resolution in zebrafishFinola E. Moore, Elaine G. Garcia, Jessica S. Blackburn, Qin Tang, John C. Moore,Aleksey Molodtsov, David M. Langenau1. Molecular Pathology, Cancer Center, and Regenerative Medicine, Massachusetts GeneralHospital, Boston, MA 021292. Harvard Stem Cell Institute, Cambridge, MA 02139Hematopoeisis culminates in the production of functionally diverse blood celltypes. This heterogeneity is best understood at the single cell level; common single cellapproaches utilize antibodies that recognize cell surface markers for fluorescentactivated sorting (FACS). In zebrafish, the lack of antibodies has compelled researchersto use transgenic lines that drive fluorescent protein expression within specific blood celltypes. However, these approaches are limited by the availability of transgenic lines andfluorescent proteins combinations that can be distinguished by FACS. Here, we appliedthe Fluidigm BioMark technology to transcriptionally analyze single blood cells, allowingfor the study of zebrafish hematopoietic heterogeneity at high resolution. We developeda panel of 96 primers encompassing general blood lineage markers and transcriptionallyprofiled cells from the whole kidney marrow and thymus (n=456 cells). In total, 97% ofcells could be categorized into the major blood lineages; the marrow comprised 16% Tcells, 19% B cells, 39% erythroid cells, and 27% myeloid cells. We could also definespecific stages of T and B cell differentiation (n=252 cells) and NK cells (n=3). We nextused this platform to characterize animals deficient in rag2, jak3, and prkdc, establishingthat these lines have reduced T cells in both thymus and marrow. Moreover, the marrowof prkdc D3613fs fish contained only immature B cell subsets. Finally, we examinedmalignant cells from Myc-driven T-cell acute lymphoblastic leukemia and found theywere arrested at the CD4+/CD8+ cortical thymocyte stage (n=347 cells). Interestingly,leukemic cells did not cluster with normal T cells based by gene expression, whichreflected inappropriate expression of both differentiation genes and other hematopoeticspecific genes. In total, we have used single cell transcriptional analysis in zebrafish toparse different lineages, distinguish differentiation states, and reveal new cell typeswithin heterogeneous cell populations.2508 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts

ZDM8 POSTERSZDM7Madison | USA 2015ZDM8Boston | USA 2015High-Throughput Gene Targeting and Phenotyping using CRISPR/Cas9 formodeling Human diseasesGaurav K. Varshney 1 , Wuhong Pei 1 , Matthew LaFave 1 , Blake Carrington 1 , Kevin Bishop 1 ,Viviana Gallardo 1 , Wenbiao Chen 2 , Raman Sood 1 , Johan Ledin 3 , and Shawn M. Burgess 11. National Human Genome Research Institute, National Institutes of Health, Bethesda, MD, USA2. Vanderbilt University School of Medicine, Nashville, TN, USA3. Science for Life Laboratory, Uppsala University, Uppsala, SwedenHuman candidate disease genes are being identified rapidly from different exome sequencing andGWAS projects, and these candidates need to be functionally validated in a model organism. The useof CRISPR/Cas9 as a genome-editing tool in various model organisms has radically changed targetedmutagenesis. We developed a high-throughput targeted mutagenesis staregy using CRISPR/Cas9technology in zebrafish that will make possible both saturation mutagenesis of the genome, and largescalephenotyping efforts. We describe a cloning-free sgRNA synthesis, coupled with streamlinedmutant identification methods utilizing fluorescent PCR and multiplexed, high-throughput sequencing.We report germline transmission data from 162 loci targeting 83 genes in the zebrafish genome, inwhich we obtained a 99% success rate for generating mutations and an average germlinetransmission rate of 28%. We further verified 678 unique alleles from 58 genes by high-throughputsequencing. We demonstrate that our method can be used for efficient multiplexed gene targeting. Wealso demonstrated phenotyping can be done in the F 1 generation by inbreeding two injected founderfish, significantly reducing animal husbandry and time. We used this strategy in generating geneknockouts of orthologs of 50 non-syndromic human deafness genes in zebrafish. Preliminary resultsshows three out of eight gene knockouts recapitulate human deafness phenotypes based on startlingresponse assay.8 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts251

ZDM8Boston | USA 2015ZDM8 POSTERSWnt and BMP signaling pathways alterations are a hallmark of bonedefects in a fish model for Gaucher diseaseIlaria Zancan 1 , Stefania Bellesso 2 , Roberto Costa 1 , Marika Salvalaio 2 , Chrissy Hammond 3 ,Francesco Argenton 4 , Mirella Filocamo 5 and Enrico Moro 1 .1 Department of Molecular Medicine, University of Padova, Italy.2 Department of Women’s and Children’s Health, University of Padova, Italy3 Department of Biochemistry, Physiology & Pharmacology, University of Bristol, BS8 1TD, UK4 Department of Biology, University of Padova, Italy5Centro di Diagnostica Genetica e Biochimica delle Malattie Metaboliche Istituto Giannina Gaslini,Genova, Italy.Type I Gaucher disease (GD) is caused by mutations in the gene encoding the lysosomal enzymeglucocerebrosidase (GBA 1). Impaired enzymatic activity leads to considerable lysosomalamassment of undegraded glucocerebroside (GC). Patients affected by type 1 GD manifestdebilitating bone defects like osteonecrosis, osteopenia and bone fractures. Despite thedevelopment of novel therapeutic approaches, bone response to current enzymatic replacement isslow and bone manifestations may worsen or persist. The pathogenetic mechanisms responsiblefor bone alterations are currently unknown. Using a morpholino-based GBA knockdown approachand an available genetic mutant we have analyzed the effects of GBA loss of function on differentcell signaling pathways. Our results suggest that the impaired GBA function leads to canonical Wntsignaling activity decrease, which in turn severely affect fish osteogenesis. Furthermore, increasedlevels of the negative regulators Gsk3β, Axin1 and Dkk1 were observed thus providing a potentialmechanistic explanation of the diminished Wnt pathway activity (Zancan et. al, 2014).To further elucidate the pathogenetic cascade underlying bone defects in the GD zebrafish model,we have extended our analysis to the BMP signaling, which is crucial for skeletogenesis and boneformation during development. Using a recently developed fish reporter to detect BMP signalingactivity we have observed up-regulation of this pathway in GBA morphants. RQ-PCR analysis oncDNA from pooled morphant and controls cDNAs showed increased expression levels of BMPtransduders, Smad1, 5 and 8. This preliminary data, shed new light in the complexity of Gaucherdisease bone pathogenesis.2528 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts

ZDM8 POSTERSZDM7Madison | USA 2015ZDM8Boston | USA 2015FGF signaling impairment substantially contribute to bone pathologicalmanifestations in animal models for Mucopolysaccharidosis Type II(MPSII)Stefania Bellesso 1 , Marika Salvalaio 1 , Elisa Tognon 2 , Roberto Costa 3 , Ilaria Zancan 3 , RosellaTomanin 1 and Enrico Moro 3 .1Department of Women’s and Children’s Health, University of Padova, Italy.2 Department of Biology, University of Padova, Italy.3 Department of Molecular Medicine, University of Padova, Italy.Mucopolysaccharidosis are rare lysosomal storage disorders caused by the deficiency in one ormore lysosomal enzymes involved in glycosaminoglycans (GAGs) catabolism. Traditionally, thepathogenesis of these diseases has been attributed to progressive intracellular storage ofundegraded glycosaminoglycans. However it’s now evident that more complex pathogenicmechanisms may contribute to the pathological manifestations.Lysosomes are key organelles involved in essential regulatory functions. Therefore, we havehypothesized that mutations altering lysosomal function may induce early cellular anddevelopmental defects that concur to patients’clinical symptoms.In order to analyze the pathogenic mechanism underlying Mucopolysaccharidosis type II (MPSII orHunter Syndrome; OMIM +309900), we have created a zebrafish model using a morpholino-basedknock down technology.MPSII is caused by a deficient activity of the iduronate 2-sulfatase (IDS) enzyme and, sinceskeletal abnormalities are one of the major disabling aspects in patients, we have decided to studyhow IDS functional alterations lead to the pathological skeletal phenotype.Morphant fish display defects in chondrogenesis and osteogenesis and show an early alteredexpression of bone-related markers and bone ossification impairment.Using different approaches we have demonstrated that in zebrafish IDS knockdown affects theexpression of key FGF signaling markers at early life stages, before a clear lysosomal impairmentis detectable. Moreover, we have extended our analysis to IDS knockout mice and Hunter patientssamples to confirm this signaling pathway involvement in the pathologic defects. Our results showthat in all the analyzed samples FGF markers expression is altered thus supporting the role of FGFsignaling in MPSII-related bone defects.FGF signaling is strictly involved in many stages of ossification and its regulation is finely controlledby extracellular glycosaminoglycans composition. Therefore, the impairment of this signaling, dueto IDS deficiency, may cause an early dysregulated expression of genes involved in bonedevelopment.8 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts253

ZDM8Boston | USA 2015ZDM8 POSTERSA Pak1/Erk signaling module as a therapeutic target inRasopathiesDaniela Araiza-Olivera, Jonathan Chernoff.1. Fox Chase Cancer Center, 333 Cottman Avenue, Philadelphia, PA, USARasopathies are developmental abnormalities in newborns caused by germlinegain-of-function mutations in the Ras pathway. The Rasopathy syndromes have incommon sustained activation of Erk during embryonic development, and often aremanifest as craniofacial aberrancies, skin lesions, cardiac defects, and cognitive defects.In zebrafish, gain-of-function mutants of N-Ras, H-Ras, Sos, Shp2, B-Raf, and Mek1induce aberrant jaw cartilage development, as well as heart defects. These abnormalilitesare associated with defective migration of neural crest cells.p21-activated kinase1 (Pak1) is a serine/threonine kinase that acts downstream ofthe small GTPases Cdc42 and Rac1 to regulate a multitude of cellular processes. Pak1phosphorylates c-Raf and Mek1, and we and others have shown that loss of Pak1 activityleads to loss of c-Raf, Mek1, and subsequent Erk activation in many cell types.Importantly, we have recently shown that zPak1 is essential for Erk activation and neuralcrest migration in zebrafish.Because Pak1 is involved in activation of Erk, we speculated that this kinase mightplay a role in Rasopathy pathogenesis. To test this we chose to evaluate if the phenotypicconsequences of Ras pathway overactivation during development can be blocked by Pakinhibitors. We therefore introduced gain-of-function Rasopathy-associated genes into onecellzebrafish embryos, and asked if Pak specific small molecule inhibitors could suppressthe phenotypes. We observed that embryonic abnormalities associated with expression ofRasopathy-associated genes could be suppressed by Pak specific small molecule. Thesedata suggest that Pak might serve as a useful therapeutic target in Ras-driven pathologies.2548 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts

ZDM8 POSTERSZDM7Madison | USA 2015ZDM8Boston | USA 2015Autophagy confers the therapeutic effects of mTOR inhibitionin PKDPing Zhu, Xiaolei Xu, and Xueying LinDepartment of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine,Rochester, MN 55905, USAAutophagy is an important cellular mechanism that clears misfolded proteinaggregates and recycles long-lived proteins and organelles, ensuring cells to survivestresses. Autophagy dysfunction contributes to the pathogenesis of a growing numberof diseases such as neurodegenerative disorders and cardiac diseases, andautophagy activation has been pursued as a therapeutic strategy. Polycystic kidneydisease (PKD), including Autosomal Dominant PKD (ADPKD) and AutosomalRecessive PKD (ARPKD), is one of the most common heritable human diseases.Alteration in autophagy has been noted in CY/CY rat and cpk mice, two PKD models;however, the role of autophagy in the pathogenesis of PKD remains unclear. Here, weinvestigated the relationship between autophagy and cystogenesis using bothzebrafish and rodent models. We generated stable TALEN mutants for a zebrafishhomologue of PKD1, mutations in which account for 85% of ADPKD. These zebrafishpkd1a mutants exhibited cystic kidney, mTOR activation, and therapeutic benefits ofmTOR inhibition, indicating a conserved PKD pathogenesis. Moreover, pkd1amutants displayed dysfunctional autophagic flux and accumulation of ubiquitin-taggedproteins in the insoluble fraction of cell lysates, a marker of protein aggregates. Theseautophagic defects were validated in mammalian kidney cells derived from Pkd1nullmice and human patients. Importantly, autophagy activators, includingmTOR-dependent and mTOR-independent reagents, are able to attenuate cysticphenotype and restore kidney function. At the cellular level, we found that theseautophagy activators facilitate clearance of protein aggregates. Taken together, ourdata demonstrated an important role of autophagy in cystic kidney and suggestedautophagy activation as a potential therapy for PKD.8 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts255

ZDM8Boston | USA 2015ZDM8 POSTERSBioEYES: A Partnership to Advance K-12 ScienceValerie ButlerBioEYES Program Manager, Carnegie Institution for ScienceBioEYES is a standards-aligned, K-12 science outreach program that improves teacherquality and science education through the use of live zebrafish. Founded in 2002 by Drs.Jamie Shuda and Steven Farber, its mission is to foster enthusiasm for science byoffering students opportunities to explore life science through real world applicationsusing a hands-on approach to learning. BioEYES provides live zebrafish, microscopes,and program materials and students mate adult zebrafish and use the scientific method toobserve behavior, genetics, and animal development. Each day, just like researchscientists in the laboratory, students ask questions, hypothesize and test ideas, record andanalyze data, and think critically about the impact scientific research has on society. Sincethe program’s inception, BioEYES has reached over 86,000 students in the U.S. andAustralia, and has centers at the University of Pennsylvania, the Carnegie Institution forScience, the University of Notre Dame, the University of Utah, and Monash University.BioEYES directly addresses the nation's call for improvement in science education withmeasureable results in: better teacher preparedness and increases in students’ scientificliteracy, practice, and engagement. In addition, the program seeks to close theachievement gap for minority populations. Student learning and attitude data as well asfeedback from teachers and students who have participated in the program will bepresented.2568 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts

ZDM8 POSTERSZDM7Madison | USA 2015ZDM8Boston | USA 2015Generation of a MLIV zebrafish disease model using both ZFN andCRISPR/Cas9 genome editingHuiqing Li, Silvia Vergarajauregui and Rosa PuertollanoLaboratory of Cell Biology, National Heart, Lung and Blood Institute, National Institutes of Health,Bethesda, MD 20892, USAAbstract: Mucolipins are members of a family of endo/lysosomal cation channels thatregulate trafficking along the endocytic pathway and whose cellular function is linked tohuman disease. In mammals, the mucolipin family includes three members, mucolipin-1, -2,and -3 (MCOLN1-3). Loss-of-function mutations in MCOLN1 are associated with a humandisease known as mucolipidosis type IV (MLIV). MLIV is an autosomal recessive diseasecharacterized mental retardation, poor muscle function, impaired vision and possibleblindness. To better understand the pathology of this disease, we aimed to generate aMLIV disease model in zebrafish. Two putative zebrafish MCOLN1 co-orthologs have beenidentified, mcoln1.1 and mcoln1.2. By using specific Zinc Finger Nucleases (ZFN), wesuccessfully created two independent mcoln1.1 knockout lines. Initial characterization ofmcoln1.1 homozygous null embryos revealed noticeable cell death in the eye. Cell deathwas confirmed as cell apoptosis by TUNEL staining in both mcoln1.1 knockout lines. Whenmcoln1.1 -/- fish embryos were injected with mcoln1.2 morpholino, the observed phenotypebecome even more apparent and increased apoptosis was detected in the whole body ofthe mcoln1 lost embryos, thus suggesting some level of redundancy between mcoln1.1 andmcoln1.2. To further confirm these observations, we used the CRISPR-Cas9 system togenerate mcoln1.2 mutant animals. Ten founders was screened, 8 of them carriedmutations, and two of the mutations created premature stop. Two independent mcoln1.2knockout lines have been made and crossed to the mcoln1.1 knockout line to make adouble knockout line for a MLIV zebrafish disease model.8 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts257

ZDM8Boston | USA 2015ZDM8 POSTERSHealthier communities through Prescription Education and InSciEd OutStephen C. Ekker 1 , David Argue 1 , Kirk P. Balderes 2 , and Chris Pierret 11. Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN, USA2. Balderes LLC, Programing Consultant, Rochester, MN, USAIntegrated Science Education Outreach (InSciEd Out) is a partnership between researchers, educators andtheir local communities. We describe the use of education as an intervention into the health of our communityas “Prescription Education” (PE). Due to the accessibility of the Zebrafish Model System and specific mutantand transgenic modeling of our core clinical areas of study, PE has been focused on identifying health andscience partners near zebrafish research facilities.The use of the PE model with partners around the world has necessitated the development of an infrastructurefor assessment of community level health needs, the alignment of disparate partners to a common philosophy,and the construction of a complex communication and technological network for the support of educationpartners and the analysis of countless data sources. We have assembled a new technological portal for realtimeaccess to these materials.Our digital hub has been constructed to include onboarding tools at a resolution from community level all theway to single students such that education and health outcomes can be tracked longitudinally. Data securedthrough the context of the classroom partnerships are drawn into a cloud-based work center where InSciEdOut personnel are directed through a semi-automated workflow to analyze complex data sources includingzebrafish assays and imaging, surveys, free response reflections, and talking drawings. Volunteer and techresource lending centers are managed through proprietary library software. Finally, the students, teachers,scientists, and clinicians of the broader InSciEd Out global community are drawn together through an openaccess, peer-reviewed, interactive scientific publication system for all experiential levels.InSciEd Out and PE provide a scaffold for meaningful work between the scientific and clinical zebrafishresearchers and the communities they serve, including the added benefit of manageable “big data” and thepotential for rapid scaling of this approach around the globe.2588 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts

ZDM8 POSTERSZDM7Madison | USA 2015ZDM8Boston | USA 2015Resource Construction at the China Zebrafish ResourceCenterLuyuan Pan, Kuoyu Li, Feng Xiong, Xunwei Xie, Liyue Liu, Yonghua SunChina Zebrafish Resource Center, State Key Laboratory of Freshwater Ecology andBiotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072,ChinaIn China, zebrafish was firstly adopted in lab research in early 1990s, and thezebrafish research community has been rapidly growing since 2000. To meet thegrowing needs of zebrafish strains and technical support, in October, 2012, the ChinaZebrafish Resource Center (CZRC, website: http://zfish.cn) was established at theInstitute of Hydrobiology (IHB), Chinese Academy of Sciences (CAS) in Wuhan. Aftertwo years’ development, CZRC has maintained more than 500 mutant and transgeniczebrafish lines, and a number of DNA plasmids and cell lines till late 2014. All the fishlines provided by CZRC are tightly linked to the web information of ZebrafishInformation Network (ZFIN). In addition to providing the regular fish resources, CZRCalso provides technical training and technical services, i.e., gene knock-out andtransgenic services. In 2013, 40 labs in China started the Zebrafish All GeneKnock-out Project (ZKO Project) for Chromosome 1. The final goal of ZKO project isto knock-out all the 1333 genes on zebrafish chromosome 1 using CRISPR/CAS9system. Up to now, more than 900 genes have been successfully knocked out. CZRCis the project coordinator, and all mutant lines built in the project will be verified andpreserved by CZRC and published as open resources to the whole research field.With the progress of ZKO project, CZRC will expand its fish stocks to 2000 lines bythe end of 2015, and it is likely to become the largest zebrafish resource center in Asia.Resource-based research, including systemic construction of certain human diseasemodels and chemical screening, is also in the works.8 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts259

ZDM8Boston | USA 2015ZDM8 POSTERSThe T Protein: Vertebrae Fit to a TAuthors: Karen Tiffany 1 , N Anderson 1 , A Arnholt 1 , I Baumgartner 1 , A Butt 1 , O DeBuhr 1 , SDyke 1 , M Griffin 1 , J Hu 1 , I Kalmer 1 , L Ketelhohn 1 , J Lawniczak 1 , N Minerva 1 , A Naas 1 , MRoddy 1 , A Satchie 1 , E Squires 1 , M Wandsnider 1 , J Wankowski 1 , A Wilde 1 , EZietlow 1 , and Michael Pickart 2Author Affiliations1Science Cedarburg High School, Cedarburg, WI United States2Concordia University Wisconsin, School of Pharmacy, Mequon, WI United StatesPresenters (indicated in bold)AbstractCongenital vertebral malformations (CVMs) comprise a group of spinal abnormalities thatinclude alterations in vertebral shape or number. Evidence suggests CVMs have a genetic link,possibly resulting from mutations in multiple genes. One gene implicated is T. T protein, atranscription factor found in a variety of animals including humans, is essential for correctembryonic development and guides the development of bone and cartilage from embryonicmesodermal tissue. T protein accumulates in the nuclei of notochord cells, interacts with DNA atspecific genes, and acts as a genetic switch to activate the genes. T protein binds to the majorand minor grooves of DNA as a dimer. Mutations in T (turning “off” the T protein switch) arehypothesized to result in defects in spinal development. The Cedarburg SMART (StudentsModeling A Research Topic) Team has designed a partial model of T protein using 3D printingtechnology to investigate its structure-function relationship, focusing primarily on the residuesimportant for dimerization of T (Pro125, Asp126, and Pro128) and for binding DNA (Arg67)using PDB File 1XBR.pdb. A 3D model facilitates the understanding of how the location of themutations may impact the function of T. Furthermore, it has facilitated the continued generalinvestigation of proteins impacting vertebral development in zebrafish by the Cedarburg SMARTTeam. Program supported by a grant from NIH-CTSA.2608 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts

ZDM8 POSTERSZDM7Madison | USA 2015ZDM8Boston | USA 2015The Regulation of Endoderm Development by Nuclear Receptor Nr5a2Olivia Weeks 1 , Sahar Nissim 1 , Jared Talbot 2 , John Hedgepeth 1 , Elisabeth Busch-Nentwich 3 , Derek Stemple 3 , Sharon Amacher 2 , Wolfram Goessling 1 .1. Department of Genetics, Brigham and Women’s Hospital, Harvard Medical School, Boston,Massachusetts, USA.2. Department of Molecular Genetics, The Ohio State University, Columbus, Ohio, USA.3. Wellcome Trust Sanger Institute, Wellcome Trust Genome, Campus Hinxton, Cambridge, UK.The endoderm emerges as one of three essential germ layers during development, giving riseto the gastrointestinal and respiratory tracts. Abnormal endodermal development can lead tofetal death or debilitating congenital malformations, affecting the patient’s health for life.Developmental studies are important for understanding the basis of endoderm organdysfunction and can provide an opportunity to identify the many context-dependent functions offactors that are dysregulated in disease. NR5A2 is an orphan nuclear receptor with describedfunctions in endoderm cancers, cellular reprogramming, and lipid metabolism. Recent studieshave established that NR5A2 can regulate the development and maturation of pancreasprogenitors; however, its role in endoderm specification and liver development is largelyunknown due to the embryonic lethality of Nr5a2 -/- mice. Here, we elucidate novel roles fornr5a2 at multiple points during endoderm development, including extraembryonic yolk syncytiallayer (YSL) formation, endoderm induction, and liver differentiation. We demonstrate thatmorpholino-mediated knockdown and chemical inhibition of nr5a2 causes defects in YSLformation, epiboly, and endoderm induction by shield stage. nr5a2 morphants exhibit prematureYSL development with upregulation and misexpression of endoderm-inducing YSL genes, suchas nanog, mxtx-1/2, gata5, and sox32. Moreover, loss of nr5a2 blocks the formation andmigration of sox17+ endoderm cells, suggesting that downstream hepatopancreas defects may,in part, result from a reduction in endoderm progenitor cells. Furthermore, we demonstrate thatliver development is disrupted in nr5a2 mutant fish, as demonstrated by an absence of thehepatocyte-specific marker liver fatty acid binding protein at 72 hours post fertilization. Based onthese findings, we hypothesize that nr5a2 functions at multiple steps to control endodermdevelopment: it provides spatiotemporal regulation over yolk syncytial layer formation andendoderm induction and regulates hepatic development.8 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts261

ZDM8Boston | USA 2015ZDM8 POSTERSAdapting zebrafish disease assays to the K-12 education spaceMadeleine E.M. Hammerlund, Mark Urban, Melissa Folstad1. Mayo Clinic for Madeleine and Mark2. University of MN – Rochester for MelissaZebrafish have become a prevalent and dynamic tool in disease research. As a modelthey can be manipulated using assays to better understand diseases such as addictionand stress related disease. Assays for these diseases can be adapted for accessibility tostudents in K-12 education. This transition requires adjustment to the lab setting,scientists’ research capabilities, and available technology.Integrated Science Education Outreach (InSciEd Out), a program focused uponimproving health and science literacy, introduces stress, addiction, and behavior assaysto K-12 students through classroom curriculum with related disease content to build acontextual understanding of the function of the assay for students. Using disease assaysprovides students with tools and techniques to answer their own questions aboutdisease. To perform the stress locomotion assay in a traditional lab setting software andhigh-end equipment is used, but it can be adjusted to the classroom lab space throughthe use of common tools such as petri dishes, a flip camera, and transparency film. Thesame adjustments can be made to the nicotine locomotion assay to fit the classroomtechnology and materials. Behavior assays have been adjusted through the use ofmating tanks and adult zebrafish to answer student scientists’ questions to run studentexperiments. Transitioning assays from leading disease research labs to the classroomprovides the techniques and tools for scientists’ of multiple capacities to build newquestions, add to current scientific research, and improve understanding of disease forthe scientific community.2628 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts

ZDM8 POSTERSZDM7Madison | USA 2015ZDM8Boston | USA 2015In vivo imaging of mammalian HSCs engraftment and colonizationinto the CHT of zebrafish embryosMargarita Parada K. 1,2 , Anne Clatworthy 2 , Humberto Jijon 3 , Eduardo J. Villablanca 4 &Miguel Allende 1 .1 Centro FONDAP de Regulación del Genoma. Universidad de Chile.2 Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts,USA.3 Center for Computational and Integrative Biology, Massachusetts General Hospital, HarvardMedical School, Boston, Massachusetts, USA.4 Translational Immunology Unit, Department of Medicine, Solna, Karolinska Institutet and UniversityHospital, Stockholm, Sweden.Hematopoietic stem cells (HSCs) generate the full repertoire of blood and immune celltypes throughout an organism’s life. They are produced during ontogeny, and activelyrespond to niche-specific environmental cues to mobilize, expand or differentiate. Thesenourishing niches dynamically change locations throughout development, beginning in theaorta–gonad–mesonephros (AGM) region and the yolk sac, followed by the placenta, fetalliver, spleen and bone marrow. Similar to mammals, HSCs in zebrafish originate early inthe aorta, move transiently to the caudal hematopoietic tissue (CHT) and ultimatelymobilize to the thymus and kidney (equivalent to the mammalian bone marrow).Capitalizing on these similarities, we are developing a xenograft model to study HSCshoming and niche-interactions in vivo and in real time using mammalian bone marrowderived HSCs transplanted into fish larvae. To this aim, CD117+ cells where isolated frommice bone marrows by means of negative selection, fluorescently stained, andtransplanted into the blastoderm of 5 hpf zebrafish embryos. Live imaging of transplantedembryos throughout development revealed that CD117+ cells colocalized with theintermediate cell mass (ICM) at 24 hpf, with the AGM region at 48 hpf, and CHT at 72 hpf.CD117+ cells would evidence in situ proliferation, and in some cases, moved intocirculation. The engraftments do not suggest mayor alteration of endogenoushematopoiesis as zebrafish neutrophil development and function is not impaired. Usingthis approach, murine HSCs could be followed for at least 7 dpf. Engraftment with humanpromyelocytic HL-60 cells also shows that they are able to colonize the CHT. Collectively,these data reveal conserved mechanisms and cues among vertebrates duringhematopoiesis development, positioning the zebrafish as a good model to furtherinvestigate in an intact living organism the mechanisms that promote hematopoiesisdevelopment and related diseases. Funding: FONDECYT 1140702/FONDAP 15090007.8 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts263

ZDM8Boston | USA 2015ZDM8 POSTERSLow cost technology for science education partnershipsMichael P. Ekker 1,2 , Chok Koth 1,3 , Austin S. McCoy 1,4 , Biswadeep Das 1 , Chris Pierret 1,51. InSciEd Out, Mayo Clinic Center for Clinical and Translational Science, Rochester, MN,USA2. University of Minnesota, Minneapolis, MN, USA3. Alternative Learning Center, Rochester, MN, USA4. Century High School, Rochester, MN, USA5. Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN, USAExisting technologies for science education can be very expensive. Dissectingmicroscopes cost between $1500 and $4000, and have a large number of shortcomingsfor the classroom environment. Shared use requires the addition of an expensivewireless camera that may further necessitate a tablet or laptop, increasing the alreadyhighbarrier of entry. For most schools, these technologies are simply inaccessible.Science partnerships with educators require lower cost devices, low maintenancesoftware and hardware, and versatile equipment. This equipment must often be storedand transported. Here we address the high cost of classroom microscopy with a ‘focus’on zebrafish. We purchased a $60 microscope and WiFi camera from internationaldistributors that can be used with tablets and laptops. We then designed a new 3Dprintable base with a mobile stage that includes a mirror and light for proper imaging ofspecimens like zebrafish. A second capillary-integrated base allows full rotation ofzebrafish embryos for imaging. We are currently performing an environmental toxicitystudy to validate the new microscope against our laboratory models. A second projectincludes the transport of 40-150 computers to classrooms for microscopy image captureand other technological needs. Charging, storage, transport, and WiFi access are allconsidered in our designs. We are conducting end user interviews to make finalrevisions to both of these products. Our intent is to share these products through thepublication of their CAD drawings for use by the zebrafish education community.2648 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts

ZDM8 POSTERSZDM7Madison | USA 2015ZDM8Boston | USA 2015HIF-Independent Hypoxia-Induced Genes are Requiredfor Hypoxic Preconditioning in ZebrafishDanhua Zhang 1 , Tania Manchenkov 1 , Martina Pasillas 1 , Farhad Imam 11. Department of Pediatrics, University of California, San DiegoHypoxia induces cellular damage and contributes to many ischemia-related diseases,including stroke and coronary heart disease. Hypoxic preconditioning (hPC), a brief noninjurioushypoxic exposure, has been shown to improve hypoxia tolerance in differenttissues and model organisms. Accordingly, a detailed mechanistic study underlying hPCcould provide insights into novel therapeutic targets for hypoxic injury. It has been shownthat changes in both cellular metabolism and systemic blood supply contribute to thehPC-mediated hypoxia tolerance. While the well-studied hypoxia-inducible factor (HIF)signaling plays a central role in regulating the adaptations to hypoxia, other non-HIFpathways also contribute to protection and recovery from hypoxic injury. We havedeveloped an effective hPC protocol for in depth in vivo analysis of hPC in zebrafishembryos. With this hPC protocol, we found HIF signaling to be necessary, but notsufficient, to generate the full extent protective effect of hPC. In addition, constitutivelyactive HIF signaling compromised hPC outcome, suggesting excessive HIF activationcan be detrimental. Further, via differential expression microarray and morpholinoknockdown we have recently discovered several novel factors critical for hPC, includingbtr01, irs2 and crtc3. These genes are hypoxia-inducible in multiple zebrafish tissues butare not transcriptionally upregulated in a vhl mutant with constitutive HIF activation,suggesting their functions are HIF-independent. Next, we will investigate the detailedfunction of these factors in hPC using the mutants generated via the CRISPR strategy. 8 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts265

ZDM8Boston | USA 2015ZDM8 POSTERSLow cost, low power, mobile genotypingAustin S. McCoy 1,2 , Michael P. Ekker 1,3 , Chok Koth 1,4 , Biswadeep Das 1 , Bart McCoy 5 , StephenC. Ekker 1,6 , Chris Pierret 1,61. InSciEd Out, Mayo Clinic Center for Clinical and Translational Science, Rochester, MN, USA2. Century High School, Rochester, MN, USA3. University of Minnesota, Minneapolis, MN, USA4. Alternative Learning Center, Rochester, MN, USA5. Special Support Processor Development Group, Mayo Clinic, Rochester, MN, USA6. Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN, USAPolymerase Chain Reaction is the backbone of genetic-based testing, research anddisease detection. However, the technology required to perform PCR is immobile, expensive,and power intensive. To combat these limitations, and with a focus on diagnostics in thedeveloping world, we created a thermocycler that is low cost (~$100), low power (10-15 Watts),portable (< 2 kilograms), and easily maintainable (95% 3D printable components). Our ThermalCycler uses a robotic arm to transport test tubes between heated aluminum blocks kept atstable temperatures. Engineering experiments using calibrated in-well thermistor measurementscharacterize temperature accuracy, uniformity, and transition times inside standard 0.2 ml testtubes. The prototype was then validated against a laboratory-grade thermocycler for PCRamplification. Temperature transition speed is also 5X faster than conventional units. Utilizingthe Launchpad business planning program, we identified multiple possible applications for thistechnology including genotyping, community hospital diagnostics, disease outbreak teamdiagnostics, GMO testing, environmental testing, water testing, and wildlife research. However,the thermocycler function alone does not yield the full diagnostic analysis that would be requiredby these markets. We considered additional functionality to include gel electrophoresis, probebasedstrip tests, and fluorescent detection. It was decided that fluorescent detection would bepursued for its mobility and on-site responsivity. When combined with our thermocycler, qPCRfunctionality can be achieved for an assembly cost below $200. Current project work includesthe validation of our low cost, low power fluorescent detection system and deeper Launchpadcharacterization of our end user market.2668 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts

ZDM8 POSTERSZDM7Madison | USA 2015ZDM8Boston | USA 2015Supporting Zebrafish and Human Disease Research at ZFIN.Yvonne M. Bradford, Sridhar Ramachandran, Sabrina Toro, Douglas G. Howe, AnneEagle, Patrick Kalita, Prita Mani, Ryan Martin, Sierra Taylor Moxon, Christian Pich, KevinSchaper, Xiang Shao, Monte WesterfieldZFIN, 5291 University of Oregon, Eugene, OR 97403-5291, USAThe Zebrafish Model Organism Database (ZFIN; http://zfin.org) is the central resource forgenetic, genomic and phenotypic data for zebrafish (Danio rerio) research. ZFIN continuouslyassesses trends in zebrafish research, adding new data types and providing data repositoriesand tools that members of the research community can use to navigate data available in theirfields of interest. Zebrafish are increasingly being used to model and study human disease. Tofacilitate disease related research, ZFIN is developing support to annotate and report zebrafishmodels of human disease.Disease term pages at ZFIN will display the human genes involved in the disease, thecorresponding zebrafish orthologs, and any reported zebrafish models of the disease. Througha collaboration with the Monarch Initiative (http://monarchinitiative.org), a phenotype comparisongrid will relate zebrafish and disease phenotypes, facilitating the visualization of zebrafish genesassociated with phenotypes similar to those of the human disease. Our site wide search allowsfor the exploration and retrieval of disease terms as well as publications, zebrafish models andgenes associated with a specific disease.Implementation of human disease support at ZFIN is ongoing, with completion expected in theFall of 2015. ZFIN is seeking input from the zebrafish community and is welcoming suggestionson how we can better support human disease related research.8 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts267

ZDM8Boston | USA 2015ZDM8 POSTERSCrush injury in zebrafish bmp1 mutant as a model to study humanOsteogenesis Imperfecta fracture repair.Monika Jagoda Tomecka 1,2 , Luis Medina Sanchez 2 , Henry Roehl 2 , Tom Carney 1,31Institute of Molecular and Cell Biology, Proteos, Singapore2Department of Biomedical Science, The University of Sheffield, UK3Lee Kong Chian School of Medicine, Nanyang Technological University, SingaporeOsteogenesis imperfecta (OI) is a heterogeneous genetic disease characterised by brittlebones, due to defect in generation of mature collagen I. Patients suffer multiplespontaneous bone fractures which severely reduce quality of life and there is a pressingneed for a tractable and accessible model of these OI fractures. 90% of OI cases are due tomutations in Collagen I genes, however several other collagen processing and transportgenes have been described to contribute. Amongst these, BMP1 is required to process theC-terminal region of the Collagen I triplex. Zebrafish Collagen I or Bmp1 mutants bothdisplay many aspects of OI. We have begun to investigate the bone repair process in theseOI models. Through examination of introduced bone crushes, we are trying to understandtemporal, physical as well as biochemical differences in the fracture repair process incomparison to WTs. We show that not only do these models display spontaneous breakformations, but also that the lack of bone strength leads to injury enlargement, instead ofhealing. The repair process is inefficient and delayed. Such analyses will provide bettercharacterization of the molecular and cellular defects of recurrent and irreparable OI fracture.2688 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts

ZDM8 POSTERSZDM7Madison | USA 2015ZDM8Boston | USA 2015Ldb2 Regulates Vegf-A-dependent Arterial Specification viaExpression of Delta-like 4 in ZebrafishSeung-Sik Rho 1, 2 , Shigetomo Fukuhara 1 , Naoki Mochizuki 1 , Young-Guen Kwon 21Department of Cell Biology, National Cerebral and Cardiovascular Center Research Institute, 5-7-1 Fujishirodai, Suita, Osaka 565-8565, Japan2Department of Biochemistry, College of Life Science and Biotechnology, Yonsei University, Seoul120-749, KoreaDuring early vascular development, vascular endothelial growth factor-A (Vefg-A)regulates arterial specification by inducing expression of Delta-like 4 (DLL4), a ligand forNotch receptor. However, the molecular mechanisms underlying Vegf-A-induced Dll4expression remain unknown. We previously identified the transcription cofactor LIMdomain-binding protein 2 (LDB2) as one of the genes that are induced during ECdifferentiation. Here, we have identified LDB2 as a key regulator of VEGF-A-dependentDLL4 expression and subsequent arterial specification. The zebrafish homolog of LDB2,ldb2a, was expressed in the dorsal aorta (DA), but not in the posterior cardinal vein (PCV).Arterial expression of ldb2a was suppressed by inhibiting Vegf-A signaling. ldb2amorphants exhibited impaired separation of the DA and PCV, and downreguation ofarterial marker efnb2a and upregulation of venous marker flt4 in the DA, suggesting thatLdb2a regulates Vegf-A-induced arterial specification. In addition, dll4 expression in thearterial vessels was suppressed not only by inhibition of Vegf-A signaling but also byknockdown of Ldb2a. In vitro luciferase reporter assays revealed that LDB2 augmentedNotch signal-dependent activation of DLL4 intron 3 enhancer by cooperating with SOXFtranscription factor. Consistently, ldb2a morphants co-injected with either soxf or rbpjmorpholino oligonucleotide exhibited single axial vessels, implying the loss of DA.Furthermore, defects in arterial specification caused by Ldb2a deficiency were partiallynormalized by injecting dll4 mRNA. Collectively, these findings indicate that Vegf-Ainduces expression of Ldb2a, which in turn potentiates the Notch signal-mediatedstimulation of dll4 intron 3 enhancer by cooperating with SoxF to induce expression ofDll4 leading to arterial EC specification.8 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts269

ZDM8Boston | USA 2015ZDM8 POSTERSA signal through the noise: quantitative evaluation of the scienceeducation intervention InSciEd OutJoanna Yang 1 , Thomas J. LaBounty 2 , Linnea R. Archer 3 , Kyle M. Casper 3 , James D. Sonju 3 ,Stephen C. Ekker 1, 4 , Chris Pierret 1,41. InSciEd Out, Mayo Clinic Center for Clinical and Translational Science, Rochester, MN, USA2. LaBounty Consulting, LLC, Woodbury, MN3. Lincoln K-8 School, Rochester, MN, USA4. Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN, USAThe K-12 years are formative for development of scientific interest and skills, presenting acritical target demographic for science education intervention. Here we assess the impact of onescience intervention, Integrated Science Education Outreach (InSciEd Out), upon scienceengagement and learning at Lincoln K-8 School in Rochester, Minnesota. Using five years ofpost-implementation InSciEd Out data including three distinct cohorts, quantitative measuresshow that InSciEd Out is an efficacious program for sustained student election into the sciencepipeline and improved Minnesota state standardized testing performance. High-resolution valueaddedanalysis for specific strand topics reveals focused gains in Life Sciences and History andNature of Science correlated with student enrollment post-implementation of InSciEd Out.Quantification of any singular intervention’s signal is difficult in the noise of competing initiativesbut is necessary for well-informed policy-making and betterment of student education.2708 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts

ZDM8 POSTERSZDM7Madison | USA 2015ZDM8Boston | USA 2015Live intracellular calcium dynamics during podocytedevelopment and injury in zebrafishRitu Tomar 1 , Erin Merkel 1 , Aleksandr Vasilyev 1 and Iain A. Drummond 11 Nephrology Division, Massachusetts General Hospital/Harvard Medical School, Boston,Massachusetts 02129, USANephrotic syndrome genes, including TrpC6 and PLCe1, affect podocyte calciumsignaling. However, the role of calcium signaling during podocyte developmentremains unknown. We aim to understand podocyte calcium signaling duringdevelopment using live imaging of zebrafish embryos. We generated multipletransgenic lines to express calcium biosensor, GCaMP3, and YFP/GFP specificallyin podocytes. Live imaging showed that immature podocytes (48hpf) are dynamicand interact with the dorsal aorta to form glomerular capillaries. By 4 dpf podocytesstabilize and the filtration barrier is functionally mature. We observed spontaneouscalcium transients in podocytes at 2-3 dpf, which were silenced by 4 dpf suggesting arole for calcium signaling in podocyte maturation. Elimination of blood pressure byblocking heartbeat with the drug or Tnnt2 knockdown resulted in glomerular collapsebut did not block calcium transients. Calcium transients were blocked bycyclopiazonic acid, thapsigargin and 2APB but not by cilnidipine or nifedipine,indicating calcium release was from intracellular stores via PLC pathway. plce1knockdown resulted in podocyte defects, disorganized capillaries, and loss ofpodocin expression, suggest a requirement for calcium signaling in podocytedifferentiation. Significantly, calcium transients were reinitiated in mature podocytes(6dpf) by puromycin injury emphasizing the role of impaired calcium signaling inkidney diseases. Further we found that plce1 is required downstream of wt1 and osr1for podocyte maturation. Further studies on upstream regulators and downstreameffectors of calcium signaling in podocyte are ongoing. These studies will help toidentify new targets for glomerular diseases and establish zebrafish as a model forglomerular diseases caused by impaired calcium signaling.8 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts271

ZDM8Boston | USA 2015ZDM8 POSTERSAnnotation of the Expression and Function of Mitochondrial GenesThrough Zebrafish Insertional MutantsWeibin Liu 1,2 , Jingchun Yang 1,2 , Yonghe Ding 1,2 , Yun Deng 1,2,3 , Roberto Lopez Cervera 1 ,Jarryd M. Campbell 1 , Stephen C. Ekker 1 , Xiaolei Xu 1,21 Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, Rochester, MN,United States of America2 Division of Cardiovascular Diseases, Department of Medicine, Mayo Clinic College of Medicine,Rochester, MN, United States of America3 Present address: Department of Genetics and Development Biology, College of Life Sciences, HunanNormal University, P.R. ChinaMitochondrial diseases are a diverse, complex, and understudied set of disorders, with geneticcontributions coming from both nuclear and mitochondrial genomes. Mitochondrial diseasesmainly affect the nervous system, muscle, and liver, leading to defects in sensory organs,muscular coordination, and liver metabolism. Though many mitochondrial genes encoded by thenucleus are known, the function and expression of most are unknown. In this study, wedemonstrate the power of zebrafish forward genetics using a gene-breaking transposon (GBT)to trap three nuclearly encoded mitochondrial genes (lrpprc, idh2, and tefm), which allowedspatiotemporal and functional annotation of the loci. Spatiotemporal localization was madepossible by a red fluorescence protein (RFP) reporter in the GBT vector that becomes fused tothe endogenous gene upon insertion in the genome. Gene disruption occurs due to a prematurestop codon that follows the exogenous RFP, which results in a truncated form of theendogenous gene product. We observed larval lethal phenotypes in homozygousGBT0235/lrpprc and GBT0360/tefm, but not in GBT0360/idh2. Particularly interesting is thelrpprc mutant due to its overlap with the pathogenesis of human Leigh Syndrome, FrenchCanadian type (LSFC). As in humans, mitochondrial morphology in hepatocytes was aberrant inhomozygous fish and contained decreased levels of respiratory chain proteins when comparedto wild type siblings. By 7 dpf, homozygotes develop hepatic steatosis and die shortly thereafter.Interestingly, the survival rate of these fish is improved upon fasting. This presents anopportunity to explore the mechanism by which health is improved in the fish for use in humantherapeutics. Together, our data show that our high-throughput zebrafish GBT mutants arevaluable tools for annotating the expression and function of mitochondrial genes. These mutantscan then serve as in vivo vertebrate models of mitochondrial disease to study diseasepathogenesis, as well as to find effective drug therapeutics.2728 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts

ZDM8 POSTERSZDM7Madison | USA 2015ZDM8Boston | USA 2015Prostaglandin signalling regulates ciliogenesisby modulating intraflagellar transportDaqing Jin, Terri T. Ni, Jianjian Sun, HaiyanWan, Jeffrey D. Amack, Guangju Yu,Jonathan Fleming,Chin Chiang 4 ,Wenyan Li, Anna Papierniak, Satish Cheepala,Gwenaëlle Conseil, Susan P. C. Cole, Bin Zhou,Iain A. Drummond, John D.Schuetz, Jarema Malicki and Tao P. Zhong1 State Key Laboratory of Genetic Engineering, Department of Genetics, Fudan UniversitySchool of Life Sciences, Shanghai 200433, China. 2 Department of Medicine,VanderbiltUniversity School of Medicine, Tennessee 37232, USA. 3 Department of Cell & DevelopmentalBiology, State University of New York Upstate Medical University, New York 13210, USA.4 Department of Cell & Developmental Biology, Vanderbilt University School of Medicine,Tennessee 37232, USA. 5 MRC Centre for Developmental and Biomedical Genetics, TheUniversity of Sheffield, Sheffield S10 2TN, UK. 6 Department of Pharmaceutical Science, StJude Children’s Research Hospital, Tennessee 38163, USA. 7 Division of Cancer Biology andGenetics, Queen’s University, Kingston, Ontario K7L 3N6, Canada. 8 Institute for NutritionalSciences, Chinese Academy of Sciences, Shanghai 200031, China. 9 Department of Medicine,Massachusetts General Hospital, Harvard Medical School,Massachusetts 02148, USA.1. Affiliation for Daqing, Jianjian, Guangju, Wenyan, Tao P2. Affiliation for Terri T, Haiyan, Tao P3. Affiliation for Jeffrey D4. Affiliation for Jonathan, Chin5. Affiliation for Anna, Jarema6. Affiliation for Satish, John D7. Affiliation for Gwenaëlle, Susan P. C8. Affiliation for Bin9. Affiliation for Iain ACilia are microtubule-based organelles that mediate signal transduction in a variety oftissues. Despite their importance, the signalling cascades that regulate ciliumformation remain incompletely understood. Here we report that prostaglandinsignaling affects ciliogenesis by regulating anterograde intraflagellar transport (IFT).Zebrafish leakytail (lkt) mutants show ciliogenesis defects, and the lkt locus encodesan ATP-binding cassette transporter (ABCC4). We show that Lkt/ABCC4 localizes tothe cell membrane and exports prostaglandin E2 (PGE2), a function that is abrogatedby the Lkt/ABCC4T804M mutant. PGE2 synthesis enzyme cyclooxygenase-1 and itsreceptor, EP4, which localizes to the cilium and activates the cyclic-AMP-mediatedsignaling cascade, are required for cilium formation and elongation. Importantly,PGE2 signalling increases anterograde but not retrograde velocity of IFT andpromotes ciliogenesis in mammalian cells. These findings lead us to propose thatLkt/ABCC4-mediated PGE2 signalling acts through a ciliary G-protein-coupledreceptor, EP4, to upregulate cAMP synthesis and increase anterograde IFT, therebypromoting ciliogenesis.8 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts273

ZDM8Boston | USA 2015ZDM8 POSTERSBBS1 localization and functional changes due to a disease-associatedmutationC. Anthony Scott 1 , Lisa M. Baye 1 , Qihong Zhang 2 , Val C. Sheffield 2 , Diane C. Slusarski 11. Department of Biology, University of Iowa2. Department of Pediatrics and Howard Hughes Medical Institute, University of Iowa Carver College ofMedicineBardet-Beidl syndrome (BBS) is a genetically heterogeneous disorder caused by defects in thefunction of the cilia, thereby classifying it as a ciliopathy. To date, mutations in 19 differentgenes have been associated with BBS. BBS1, accounts for ~25% of all BBS cases. Of those,the most common mutation is a missense mutation, BBS1M390R yet the functional properties ofthe mutation have not yet been fully characterized. The M390R mutation is predicted to behighly disrupting due to the introduction of a positive charge into the hydrophobic core of thefolded protein. We used cell lines and zebrafish to understand the disease mechanism ofBBS1M390R. Zebrafish bbs1 localizes to the centrosome, while bbs1M390R does not. We alsofind that bbs1M390R interacts with components of the BBS complex but not with bbs4 (anothercentrosome localized BBSome component). To evaluate functional properties, suppression ofknockdown defects was tested. Knockdown of bbs1 shows characteristic phenotypes of BBSdisruption in zebrafish: reduced Kupffer’s vesicle and intracellular transport delays. Additionally,cardiovascular and lateral line defects also occur. Sequential injection of BBS1 mRNA intomorpholino (MO) injected embryos suppressed all defects. In contrast, sequential injection ofBBS1M390R mRNA does not suppress the characteristic BBS phenotypes but does rescuecardiovascular and lateral line defects, suggesting some function of BBS1 not associated withthe BBSome, as it exists in cilia function. The extent to which the interaction of BBS1 with BBS4influences stability and localization as well as recently generated BBS1 genetic lines will bediscussed. We conclude that defective function of BBS1M390R arises from its inability to bindthe final BBSome subunit and localize to the centrosome, and the ability of BBS1M390R to beable to suppress some, but not all, of the bbs1 knockdown phenotypes strongly suggestsdifferential activity in a tissue-specific manner.2748 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts

ZDM8 POSTERSZDM7Madison | USA 2015ZDM8Boston | USA 2015Regeneration of zebrafish melanocytes involves directdifferentiation and concurrent replenishment of progenitor cellsSharanya Iyengar, Melissa Kasheta, Craig CeolProgram in Molecular Medicine and Department of Molecular, Cell and Cancer Biology, Universityof Massachusetts Medical School, Worcester, MA, USADuring regeneration, cells must coordinate proliferation and differentiation to rebuildtissues that are lost. Understanding how source cells execute the regeneration processhas been a longstanding goal in regenerative biology with implications in wound healingand cell replacement therapies. Melanocytes are pigment-producing cells in the skin ofvertebrates that can be lost during hair graying, injury and disease-relateddepigmentation. To gain insight into melanocyte regeneration we set out to identify thecellular source of regeneration melanocytes in adult zebrafish and the path throughwhich source cells reconstitute the pigment pattern. Using a targeted cell ablationapproach we identified that the transcription factor mitfa is expressed not only indifferentiated melanocytes but also in the cells that mediate melanocyte regeneration.We then used mitfa-positivity to perform single cell lineage-tracing analyses and foundthat a majority of regeneration melanocytes arise through direct differentiation of mitfaexpressingprogenitor cells. During regeneration, other mitfa-expressing cells dividesymmetrically to generate additional mitfa-positive progenitors. We performed EdUincorporation experiments to show that cells that divided in the first round ofregeneration could differentiate into melanocytes in a subsequent round of regeneration.We also used reporter assays and drug studies to identify pathways important inmelanocyte regeneration. We found that Wnt signaling gets turned on duringregeneration and that Wnt inhibition after ablation of differentiated melanocytes blocksregeneration, suggesting that mechanisms used in ontogenetic melanocyte developmentare also important during regeneration. Direct differentiation coupled with symmetricdivision allows for regeneration to occur while also maintaining a progenitor pool. Thiswork defines a heretofore unappreciated role for direct differentiation in melanocyteregeneration and suggests a broader role for this process in the maintenance ofepithelial sheets.8 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts275

ZDM8Boston | USA 2015ZDM8 POSTERSDevelopmental Control of DNA ReplicationJoseph C. Siefert 1,2 , Amnon Koren 3 , Christopher L. Sansam 1,21. Oklahoma Medical Research Foundation, Cell Cycle and Cancer Biology Research Program,Oklahoma City, OK2. University of Oklahoma Health Sciences Center, Department of CellBiology, Oklahoma City, OK 3. Department of Genetics, Harvard Medical School, Boston, MAOur DNA replicates in a spatiotemporal order called the replication-timingprogram. Recent evidence has indicated that the replication-timing program caninfluence transcription and genome stability. For any particular cell type, thereplication-timing program is a very stable epigenetic trait, but duringtumorigenesis and cellular differentiation, segments of the genome undergoearly-to-late or late-to-early replication-timing switches that correspond tochanges in transcription and chromatin and nuclear organization. Although theexistence of the replication-timing program has been known for over fifty years,we know little about how it is controlled and what it does. We are using thezebrafish as a model to study the function of replication-timing during vertebrateembryonic development and tumorigenesis. We have profiled replication-timingacross the zebrafish genome in fibroblast cell lines and in embryos at pre-MBT,dome, shield, bud, and 28 hpf stages. The overall replication-timing programchanges during development, and particularly striking changes occur betweenshield and bud stages. Similar replication-timing changes have been reported formammalian embryonic stem cells as they undergo differentiation. We have alsostarted to analyze the developmental functions of replication-timing control genesin zebrafish. Our data provides a foundation to use the zebrafish to study themechanisms of functions of developmental changes in DNA replication andnuclear organization. 2768 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts

ZDM8 POSTERSZDM7Madison | USA 2015ZDM8Boston | USA 2015CRISPR mediated targeted mutagenesis at the NHGRI ZebrafishCore: Efficient protocols to generate mutant fishBlake Carrington 1 , Gaurav Varshney 1 , Kevin Bishop 1 , Anupam Prakash 1 , MaryPatJones 1 , Gabrielle Robbins 1 , Shawn Burgess 1 , and Raman Sood 11. National Human Genome Research Institute, National Institutes of Health, Bethesda, MD, USARecent advances in genome editing by zinc finger nucleases (ZFNs), TAL effectornucleases (TALENs) and clustered regularly interspersed palindromic repeat/Cas9nucleases (CRISPRs) have revolutionized the use of zebrafish model in functionalgenomics. These nucleases have been used to generate gene knockout mutationscaused by indels at the target site by NHEJ repair pathway and knockin of specificmutations mediated by homology directed repair pathway. Previously, we generatedknockout mutants for 26 genes using ZFNs and TALENs. Since last year, we have beenusing CRISPRs due to their flexible design options and cloning-free single guide RNAsynthesis at the cost of a nucleotide primer. Here, we will present the pipeline we haveimplemented to quickly generate knockout mutants. To save time and costs associatedwith fish husbandry and founder screening, we adopted a fluorescent PCR basedmethod, termed CRISPR-STAT (Somatic Tissue Analysis Test) to evaluate somaticactivity in injected embryos. Our data revealed a strong correlation between thedistribution profile of peaks observed by fluorescent PCR in the injected embryos andgermline transmission activity of the corresponding CRISPRs. Using this method wehave been able to generate mutations in over 100 genes in a few months. CRISPR-STAT can also be used to prescreen CRISPS for knockin of desired mutations since ahighly efficient CRISPR is a pre-requisite for homology directed repair pathway. We willalso present data on our efforts to generate knockin mutants with specific point mutationat the target site and an insertion of a transgene.8 th ANNUAL ZEBRAFISH DISEASE MODELS MEETING | 24 August - 27 August, 2015, Boston, Massachusetts277