Congress Abstracts - Society for Developmental Biology

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Program/Abstract # 561 Rab23 is a novel regulator of epithelial morphogenesis, polarity and lumen formation Gual Soler, Maria Margarita; Luo, Lin; Taguchi, Tomohiko; Venturato, Juliana (Inst for Molecular Bioscience, Australia); Bryant, David M.; Mostov, Keith E. (Univ of California, San Francisco, USA); Martin Belmonte, Fernando (Centro de Biología Molecular Severo Ochoa, Consejo Superior de Investigaciones Científicas, Spain); Wicking, Carol; Stow, Jennifer L. (Inst for Molecular Bioscience, Australia) The small GTPase Rab23 has been linked to developmental defects, vertebrate ciliogenesis and cancer, but its intracellular functions remain largely unknown. Here we show that GFP-Rab23 localizes on recycling endosome (RE) membranes and on the plasma membrane of epithelial Madin-Darby Canine Kidney (MDCK) cells. We explored the role of Rab23 in epithelial polarity and morphogenesis using loss and gain-of-function approaches in MDCK monolayers and cysts. In monolayer cultures, overexpression of GFP-Rab23 altered monolayer morphology by increasing cell height and packing, whereas siRNA-mediated Rab23 depletion resulted in mislocalization of adherens junction markers E-cadherin and ß-catenin, irregular monolayer morphology and altered tight junction integrity. However, the most profound defects were found in three-dimensional cyst cultures, in which GFP-Rab23 overexpression produced aberrant cysts with loss of cell polarity, absence of lumen and increased cell proliferation. Furthermore, de novo lumen formation was disrupted by overexpression of wild-type and a dominant-negative version of Rab23 (S23N), as well as by shRNAmediated Rab23 knockdown. Both overexpression and depletion of Rab23 impaired the delivery of podocalyxin to the cyst interior and the generation of an apical surface to initiate the lumen. Together, our findings reveal a key role for Rab23 in polarity and lumen formation, suggesting that precise control of Rab23 dosage is critical for epithelial morphogenesis. Program/Abstract # 562 Characterization of Combover/CG10732, a Novel Drosophila Rho Kinase Substrate and its Potential Role in Planar Cell Polarity Signaling Fagan, Jeremy K. (Albert Einstein Coll Med, USA); Lu, Qiuheng; Adler, Paul (U Virginia, USA); Jenny, Andreas (Albert Einstein Coll Med, USA) Cellular polarization is essential for nutrient transport, cell-cell communication and other cellular processes including spindle orientation during cell division, cell migration and cell differentiation. In addition to apical-basal polarity, polarity across the plane of an epithelium is a fundamental phenomenon required for the formation of complex tissues. This phenomenon is known as Planar Cell Polarity (PCP) and is controlled by the non-canonical Wnt/Fz-PCP signaling pathway. While some are known, most downstream effectors of the PCP pathway remain elusive. Particularly, it is not understood how Rho kinase, a known PCP effector also implicated in tumor cell migration, exerts its function. In a genome-wide Rho kinase substrate screen we identified Combover/CG10732 as a novel Rho Kinase substrate in vitro. RNAi knockdown of Combover in the Drosophila wing yielded a multiple wing hair phenotype, indicative of a defect in planar cell polarity signaling. Using mass spectrometry, we have identified several sites of Rho Kinase phosphorylation and site-directed mutagenesis has confirmed that these are bona-fide sites of Drok phosphorylation. Using a Yeast-2- Hyrbid approach, we have identified the planar cell polarity effector Multiple wing hair (Mwh) as a Combover interacting protein. We are currently performing GST pull-down and co-immunoprecipation experiments to support the hypothesis of a physical interaction between Combover and Mwh. A combover mutant allele generated by homologous recombination was homozygous viable that failed to phenocopy the initial multiple wing hair RNAi phenotype. Nevertheless, overexpression of both isoforms of Combover (RA and RB) causes a strong multiple wing hair phenotype observed when combined to wing specific Gal4 drivers. Importantly, this mwh phenotype can be enhanced by removal of certain members of the mwh group of PCP effector genes. Future work will further investigate the role of Combover as a novel Rho Kinase substrate and potential wing-specific planar cell polarity effector gene. Program/Abstract # 563 Lens placode apical actin network: the role of PAR3 and ROCK Melo, Maraysa de Oliveir; Borges, Ricardo; Yan, Chao (Univ de São Paulo, Brazil) Vertebrate lens originates from pre-lens ectoderm, a simple cuboidal epithelium that overlies the optic vesicle. Thereafter, the pre-lens ectoderm cells elongate become columnar and form the lens placode.We showed previously that actomyosin filaments are distributed along the apico-basal cell sides of the chick pre-lens ectoderm and become enriched apically in the lens placode. Here, we investigate the role of the PAR complex in the apical localization of actin filaments. PAR3 is a scaffold protein that forms a complex with PAR6/aPKC and establishes and maintains epithelial cell polarity in a diverse set of models. PAR3, PAR6 and aPKC are already at the apical domain in the pre-lens ectoderm and remain apical during lens placode elongation. We overexpressed aPKCΔN and different deletion mutants of PAR6 in pre-lens ectoderm and these did not alter the polarization of the actin filaments. In contrast, PAR3(T833A) caused a slight increase in apical actin filaments in lens placode. PAR3(T833A) is not phosphorylated by ROCK and increases the stability of the PAR complex. Surprisingly, this same mutant significantly increased apical actin in the adjacent ectoderm as well. This phenotype is unexpected because the placode´s surrounding ectoderm does not polarize actin. Inhibition of ROCK by Y27632, disrupted the polarization of actin and myosin II but not of PAR3. Taken together, these data suggest that: 1) Phosphorylation of Thr833 in PAR3 inhibits actin polarization in non-placodal ectoderm; 2) ROCK activity is required for maintenance of actin polarization in the lens placode. 162
Program/Abstract # 564 Withdrawn Program/Abstract # 565 ADAM10 and ADAM19 proteolytically process Cadherin6B during epithelial-to-mesenchymal transitions of the cranial neural crest Schiffmacher, Andrew T.; Taneyhill, Lisa (U Maryland, USA) Epithelial-to-mesenchymal transitions (EMTs) occurring in chick cranial neural crest cells permit these cells to delaminate from the neuroepithelium en masse, acquire motility, and emigrate from the dorsal neural tube. Epithelial adherens junction disassembly is an essential component of this EMT process. Within the premigratory neural crest domain, N-cadherin and Cadherin6B (Cad6B) are downregulated prior to completion of EMT, and this loss of protein is necessary for proper cranial neural crest emigration. While Cad6B downregulation is controlled in part by Snail2-mediated transcriptional repression, we hypothesize that post-translational mechanisms are also utilized to promote the observed rapid turnover of Cad6B protein due to its usual long half-life. For the first time in vivo, we demonstrate the presence of a putative N-terminal fragment (NTF) generated by Cad6B proteolysis that correlates with a reduction in full-length Cad6B and the onset of EMT. Importantly, we note the concomitant appearance of two C-terminal cleavage fragments (CTFs) that are absent in embryos treated with a broad-spectrum metalloproteinase inhibitor (GM6001). By co-expressing relevant proteases with Cad6B in vitro, we identified that A Disintegrin and Metalloproteinases (ADAM) ADAM10 and ADAM19, along with γ-secretase, cleave Cad6B to give rise to the NTF and CTFs previously observed in vivo. As both ADAMs are expressed in the appropriate spatio-temporal pattern to process Cad6B in vivo, they were assessed for Cad6B proteolytic activity in vivo. Overexpression of either ADAM within premigratory cranial neural crest cells prior to EMT results in premature loss of Cad6B protein. Together, these results suggest a two-part mechanism for Cad6B proteolysis involving ADAM10, ADAM19, and γ-secretase during cranial neural crest cell EMT. Program/Abstract # 566 Ephrin signaling maintains apical adhesion of neural progenitors Davy, Alice; Arvanitis, Dina (CBD, France); Behar, Annie (IPBS, France); Tryoen-Toth, Petra (INCI, France); Bush, Jeff (UCSF, USA); Jungas, Thomas (CBD, France); Vitale, Nicolas (INCI, France) Apical neural progenitors are polarized cells whose apical membrane is the site of cell-cell and cell-extracellular matrix adhesion events that are essential to maintain the integrity of the developing neuroepithelium. Apical adhesion is important for several aspects of the nervous system development including morphogenesis and neurogenesis, yet the mechanisms underlying its regulation remain poorly understood. Herein, we show that ephrin-B1, a cell surface protein that engages in cell signaling upon binding cognate Eph receptors, controls normal morphogenesis of the developing cortex. Efnb1 deficient embryos exhibit morphological alterations of the neuroepithelium which correlate with neural tube closure defects. Using loss-of-function experiments by ex vivo electroporation we demonstrate that ephrin-B1 is required in APs to maintain their apical adhesion. Mechanistically, we show that ephrin-B1 controls cell/ECM adhesion by promoting apical localization of integrin-beta1 and we identify ADP-ribosylation factor 6 (ARF6) as an important effector of ephrin-B1 reverse signaling in apical adhesion of APs. Our results provide evidence for an important role for ephrin-B1 in maintaining the structural integrity of the developing cortex and highlight the importance of tightly controlling apical cell/ECM adhesion for neuroepithelial development. Program/Abstract # 567 The regulation of epithelial cell adhesive forces by the MID1/Alpha4/PP2Ac complex and its implications for cleft lip susceptibility Cox, Timothy C. (U Washington, USA), Huang, Yongzhao; Koto, Cathy (Seattle Children's Res Inst, USA) The embryonic orofacial epithelium facilitates the fusion of converging facial prominences that is essential for proper oronasal form and function. Failure to properly fuse these prominences results in cleft lip/palate (CLP), one of the most common birth defects, yet little is known about the role of causative genes. MID1 is a microtubule-associated E3 ubiquitin ligase, the loss of which results in a syndromic form of CLP. MID1 interacts strongly with Alpha4, the mammalian homolog of yeast Tap42. As in yeast, Alpha4 binds the catalytic subunit of protein phosphatase 2A (PP2Ac) to uniquely regulate its activity and that of the TOR pathway. We report here that perturbation of MID1 function in polarized MDCK cells results in reduced cell-cell adhesive strength and altered cell-ECM interactions, with concomitant changes in mTOR phosphorylation, Rac1 activation, and phospho-FAK levels. In non-polarized cells, disruption of MID1 reduces adhesion to laminin but not collagen, and perturbs collective migration. Taken together, our data suggest that mTOR/Rac1 signaling is the downstream pathway affected by the MID1/Alpha4 axis in the regulation of epithelial cell adhesion. In support of the importance of this epithelial function of MID1 during formation of the upper lip, in ovo electroporation of the same dominant-negative form of MID1 into chick pre-fusion orofacial epithelia was found to result in cleft lip in this species. Ongoing studies using fabricated micropost technology are aimed at determining the relative contributions of different downstream effectors of MID1 and quantifying their impact on intercellular adhesive forces. These studies provide the first clues as to the cellular mechanisms responsible for CLP. 163
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International Society of Developmen
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neural crest cells (hNCC)) human em
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activity may determine the orientat
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Developmental cell signaling pathwa
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opposed roles during early gastrula
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functions by the recruitment of add
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function validated with explant stu
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Program/Abstract # 48 Modeling regu
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surface of the embryo. In zebrafish
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morphologies. Our analyses not only
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junctional proteins (RPE patterning
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Program/Abstract # 78 In vivo recep
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Program/Abstract # 85 Guts and gast
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Program/Abstract # 92 The C. elegan
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Program/Abstract # 98 A gradient of
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Program/Abstract # 106 Developing a
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NSCs, but not in neurons, bind not
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abnormalities in the development of
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Tbx4 and contributes to Tbx4 repres
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downregulated by polycomb-group pro
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Zac1 expression in the developing c
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understanding the mechanisms that u
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Program/Abstract # 156 Systematic I
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Program/Abstract # 163 Size regulat
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TACC3 was localized around the DNA
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Penaeid shrimp represent an importa
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tubule. Using live imaging of cultu
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show that cell polarity is disrupte
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process. However, a clear mechanist
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Zhang, Haoran; Wong, Elaine Yee-man
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condition. Kanyon embryos have a mi
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Program/Abstract # 218 Lfng regulat
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works in concert with basal cues fr
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medaka genome. These miRs are encod
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facilitan cambio en patrón morfoge
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coordinately regulate the expressio
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downstream asymmetric signals. The
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viability and morphology of over 20
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owser. The genomic differences obse
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evolutionary analysis to study the
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teleostei. Our data evidenced that
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ontogeny. The typical condition for
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examples whose Shh expression requi
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insights into the ancestral role of
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Program/Abstract # 308 Mechanistic
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Patients with Joubert Syndrome and
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Program/Abstract # 323 Drosophila g
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signalling during gut and enteric n
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normally form, hence producing prox
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survival in Shh mutant embryos foll
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conserved in amniotes other than ch
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maturation and function. We strive
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Urness, Lisa D; Bleyl, Steven B (Un
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development is already unknown. Emb
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in A-P and P-D patterning by establ
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Page 133 and 134: Program/Abstract # 462 Retinaldehyd
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Congress Abstracts - Society for Developmental Biology

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