Congress Abstracts - Society for Developmental Biology

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Program/Abstract # 124 Analysis of the space/time sonic hedgehog expression using luciferase as a dynamic reporter Bastidas-Ponce, Aimée; Covarrubias, Luis; Wood, Christopher (Instituto de Biotecnología-UNAM, Mexico) In the embryonic developing midbrain, the dopaminergic neurons (mDAN) form, at the ventral midline, from floor plate cells that express the morphogen Sonic Hedgehog (Shh). The niche for differentiation is formed and maintained by Shh in concert with other morphogens, whose expression levels vary dynamically over time. In this study, we designed a strategy to characterize the dynamics of shh transcription through monitoring the activity of bioluminescent reporter genes coupled to transcriptional regulatory elements (enhancer/promoter) of shh gene. We used the SBE1 and SFPE1 enhancers to direct expression of reporter genes to specific regions of the neural tube (midbrain and caudal regions, respectively). We analyzed the expression of these vectors in HEK293T, ESCs during differentiation into mDAN and cells isolated from the embryonic mesencepahlon (E10.5), using population bioluminescence assays and monitoring single cells on a microscope. For all the cell types, our results indicate that luciferase activity was generally extremely low, although in primary cells, regulatory elements did direct and limit expression of luciferase to the ventral mesencephalic-derived cells in a dynamic manner. During the in vitro differentiation of ESCs we observed changes in the transcriptional activity of shh. These lines could be used to generate transgenic mice to analyze in vivo the dynamic transcription of shh during embryonic development. Supported by CONACyT 132478. Program/Abstract # 125 The transcriptional regulatory logic of Sonic Hedgehog target genes in the mouse limb Lewandowski, Jordan; Powell, Marian (University of Texas at Austin, USA); Ji, Hongkai (Johns Hopkins Bloomberg School of Public Health, USA); Vokes, Steven (University of Texas at Austin, USA) The Sonic Hedgehog (Shh) pathway is required to properly form the vertebrate limb by directing a complex transcriptional response that is mediated by GLI transcription factors. Although Shh signaling has been extensively studied, little is known about how the transcriptional output is regulated. Previous work using microarray gene expression data and GLI3 chromatin immunoprecipitation identified 205 putative Shh-target genes and their associated GLI binding regions (GBRs). The goal of this study is to identify additional factors enriched in GBRs, and determine if the factors contribute to regulate Shh-target genes. In order to identify genes likely to be regulated by Shh signaling we performed whole-mount in situ hybridization on the 205 genes, and identified 42 expressed in the posterior limb. To determine if these genes are regulated by a common set of transcription factors we searched for enriched DNA motifs in GBRs associated with the 42-gene group. One DNA motif of particular interest is Sp1, which is enriched in GBRs associated with 23/42 posterior genes. Inhibiting Sp1-mediated transcription with mithramycin A in mouse limb bud cultures inhibits 15/23 genes associated with GBRs containing Sp1 and Gli motifs. We propose a model a model where GLI and SP1 function to regulate the transcription of a subset of Shh-target genes in the developing limb bud. Program/Abstract # 126 Characterization of Spry2 Cis-Acting Elements Responsive to FGF Signals Zhang, Ying; Lewandoski, Mark (National Cancer Institute, USA) A challenge in developmental biology is to understand how signaling pathways regulate downstream genes. The Fibroblast Growth Factor (FGF) family is one of the first signaling pathways discovered to act during development, but ironically, compared to other pathways, we know little about the downstream mediators and FGF-responsive elements (FREs) in target genes. Therefore, we are investigating potential FREs in Spry2, which encodes an important modulator of FGF signaling and is expressed in FGF signaling centers during development. We have constructed a Spry2-luciferase construct that contains 8 kb of Spry2 sequences upstream of the initiation codon, which is fused to luciferase. This construct is responsive to exogenous FGF in NIH3T3 cells. We then generated a series of transgenic mouse lines with a similar transgene but with Spry2 sequences driving lacZ activity. This construct recapitulates endogenous Spry2 expression in most regions from embryonic day E7.0 through E11.5. These include the primitive streak (PS), presomitic mesoderm, somites, limb buds, branchial arches, anterior neural ridge and the mid-hindbrain organizer. We are performing the appropriate genetics to determine FGF-responsiveness. Thus far we have found that transgenic expression is reduced in the PS when Fgf4 and Fgf8 are inactivated in that tissue with TCre-activity and in the midhindbrain junction when Fgfr1 is inactive with En1Cre. Also, limb bud Spry2-lacZ activity is absent in mutants lacking the apical ectodermal ridge, a source of FGF activity. By using different Fgf4 and 8 loss-of-function alleles, we found that Spry-lacZ activity is more sensitive to FGF loss than the endogenous Spry2. Thus we have generated useful FGF-reporter mouse lines and are refining our analysis to determine the minimal sequences that act as FREs. We will use these sequences in DNA-centered techniques to determine the trans-acting proteins that target gene activation via the FGF signaling cascade. Program/Abstract # 127 Mechanisms of Tbx4 action on hindlimb development Luxey, Maëva (IRCM, Canada); Nemec, Stephen; Drouin, Jacques (Montreal, Canada) Limb development is governed by a genetic program common to all appendages. Morphological and functional differences between forelimbs (FL) and hindlimbs (HL) appear to result from modulation of this developmental program. Mechanisms for specification of limb identity have focused on three transcription factors with limb-restricted expression patterns: Pitx1 and Tbx4 that are expressed in HL, and Tbx5 expressed in FL. Characterization of the transcriptional properties of these factors revealed that Pitx1 acts upstream of 36
Tbx4 and contributes to Tbx4 repression. Tbx4 and Tbx5 share a conserved transcriptional activation domain that may control limb bud growth as Tbx5-/- mice fail to develop FLs. In addition, Tbx4 has a unique C-terminal repressor domain, the activity of which is correlated with the role of Tbx4 in HL identity. Interestingly, the Tbx4 repressor activity appears to be context-dependent, suggesting a role for critical corepressor(s). We use biochemical approaches to investigate the putative Tbx4 coregulators, in particular through the purification of protein complexes and GS/MS analyses. Further, we have implemented genomic approaches (RNA-Seq on FL and HL from Wt and Pitx1 -/- buds and ChIP-Seq for Pitx1, Tbx4 and chromatin marks) t o identify targets of Tbx4 and Pitx1 action. Collectively, these analyses will define the gene regulatory networks and mechanisms reponsible for Tbx4 function(s) in HL development. Program/Abstract # 128 Hoxa5 Function in Organogenesis is Controlled by a Complex Regulatory Network Involving YY1 Transcription Factor Berube-Simard, Felix-Antoine; Olivier, Boucherat; Jeannotte, Lucie (CRC-HDQ, Canada) The development of the fertilized egg into a multicellular organism depends on a hierarchy of molecular events requiring the specific spatio-temporal expression of multiple genes. Among the key players are Hox genes encoding transcription factors essential in specifying the regional identity along the embryonic axes and orchestrating morphogenesis. Hox mutant mice present homeotic transformations affecting the skeleton and anomalies in organogenesis that can impair viability. We are interested in deciphering the function and the regulation of Hox genes using as a model the Hoxa5 gene. The characterization of the Hoxa5 mutant mouse line generated in the laboratory has led to a better understanding of the role of Hoxa5 in several pathologies including COPD and cancer. Moreover, our work on Hoxa5 developmental regulation allowed us to position Hoxa5 in the molecular cascade controlling lung development. Hoxa5 mutant mice present a panoply of phenotypes indicative of the broad range of Hoxa5 actions throughout life. Most of Hoxa5 -/- mice die at birth from respiratory distress due to tracheal and lung dysmorphogenesis and surviving mutants display lung airspace enlargement and goblet cell metaplasia. Hoxa5 expression is confined to the mesenchyme of the entire respiratory tract suggesting that it provides regional cues to the contiguous epithelia and participates to cell fate determination. Indeed, lung goblet cell metaplasia results from Clara to goblet cell transdifferentiation, a process accompanied by an increased activity of Notch signaling. Hoxa5 expression in the developing lung is under the control of cis-acting sequences located in the Hoxa4-Hoxa5 intergenic region that specifically bind the Zn finger transcription factor YY1. Our findings unveil Hoxa5 as a micromanager controlling not only organ formation and axial patterning but also details of cell morphogenesis and function. Hoxa5 function is intimately linked to an accurate regulation of its expression. (Financial support from CIHR and NSERC) Program/Abstract # 129 Protein-protein interaction of Antp/Scr Homeodomains in the genetic control of development in D. melanogaster Elizondo-Rodríguez, F. Salomé; Reséndez-Pérez, Diana (FCB-UANL, Mexico) Hox genes encode for a group of transcription factors involved in the regulation of diferentiation networks that lead to the proper development of Drosophila. Homeodomains in addition to its DNA binding ability, also have functions of RNA binding, secretion, penetration and protein-protein interactions. These broad functions of HD-containing genes are essential for development suggesting that HD-containing proteins cross react with other HD-containing partners. This cross-regulation of HDs interactions prompted us to investigate further protein-protein interactions of Hox HDs. Since previous work showed that Sex comb reduced (Scr) repression is mediated by Antennapedia in vivo , we focused on HD protein interaction of Antp and Scr Drosophila Hox genes. Here we describe protein-protein interaction of Antp/Scr using Bimolecular fluorescence complementation and transcriptional activation assay. Our results indicated that Antp HD has a crucial role in protein-protein interaction of Antp/Scr in which YPWM was not absolutely required. We also found Extradenticle (Exd) is a requisite cofactor for the HD interactions of Antp and Scr. Currently, we are also analyzing residues in the HDs involved in this interaction and transcriptional activity using cofactos as Exd. In addition, we are verifying the presence of trimeric interactions and participation of Exd to the Scr repression by Antp. These results indicate that HDcontaining genes f orm protein complex that regulates downstream genes through protein-protein interaction modifying DNA binding affinity of transcription factors that may explain epistatic relations of Hox genes in D. melanogaster. Program/Abstract # 130 Protein-protein interaction of Antennapedia with TFIIE-ß in Drosophila melanogaster Altamirano Torres, Claudia Dalila; Reséndez-Pérez, Diana; Cárdenas-Chávez, Diana L.; Elizondo-Rodrígiuez, F. Salomé (FCB- UANL, San Nicolás de los Garza, Mexico) Segmental identity along the anteroposterior axis of bilateral animals is specified by Hox genes. Antennapedia homeoproteín (Antp) is responsible for thoracic segments and head formation in Drosophila. Antp acts by DNA binding through the conserved homeodomain (HD) and by protein-protein interactions of YPWM motif with the transcription factor BIP2 linking this homeoprotein with the basal transcription machinery. Therefore, we analyzed if other general transcription factors (GTFs) are involved in Antp functional specificity. Here we describe protein-protein interactions of Antp HD with the GTF TFIIE-β by Bimolecular Fluorescence Complementation (BiFC) and transactivation assays. Our results indicated that helix II of the Antp HD has a crucial role in this protein-protein interaction with TFIIE-β and is not mediated by YPWM motif . We also found that aminoacids Iso-32 and His-36 of helix II of Antp HD are required for this interaction. Transcriptional activity of Antp was analyzed by the presence of TFIIE-β and we 37
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Page 3 and 4: neural crest cells (hNCC)) human em
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Page 15 and 16: Program/Abstract # 48 Modeling regu
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Page 19 and 20: morphologies. Our analyses not only
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Page 23 and 24: Program/Abstract # 78 In vivo recep
Page 25 and 26: Program/Abstract # 85 Guts and gast
Page 27 and 28: Program/Abstract # 92 The C. elegan
Page 29 and 30: Program/Abstract # 98 A gradient of
Page 31 and 32: Program/Abstract # 106 Developing a
Page 33 and 34: NSCs, but not in neurons, bind not
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Page 45 and 46: Program/Abstract # 156 Systematic I
Page 47 and 48: Program/Abstract # 163 Size regulat
Page 49 and 50: TACC3 was localized around the DNA
Page 51 and 52: Penaeid shrimp represent an importa
Page 53 and 54: tubule. Using live imaging of cultu
Page 55 and 56: show that cell polarity is disrupte
Page 57 and 58: process. However, a clear mechanist
Page 59 and 60: Zhang, Haoran; Wong, Elaine Yee-man
Page 61 and 62: condition. Kanyon embryos have a mi
Page 63 and 64: Program/Abstract # 218 Lfng regulat
Page 65 and 66: works in concert with basal cues fr
Page 67 and 68: medaka genome. These miRs are encod
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Page 71 and 72: coordinately regulate the expressio
Page 73 and 74: downstream asymmetric signals. The
Page 75 and 76: viability and morphology of over 20
Page 77 and 78: owser. The genomic differences obse
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Page 81 and 82: teleostei. Our data evidenced that
Page 83 and 84: ontogeny. The typical condition for
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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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perturbed in arco piccolo mutants w
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Hoxb7-Cre line, leads to multiple u
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differentiation from common progeni
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investigate this issue, we used tar
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stem cell‐like characteristics. H
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diminished. Interestingly, we found
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y invading osteoblasts. Quite diffe
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coimmunoprecipitation experiments d
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cells and sub-cellular endosomal co
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accumulation is decreased. This wor
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Sex determination in vertebrates de
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Program/Abstract # 462 Retinaldehyd
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Program/Abstract # 469 Search for n
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growth factors used to keep them al
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it is still required to promote mig
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on the outside of small clear vesic
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Program/Abstract # 497 mef2ca contr
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were colocalized along the epidermi
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neural tube morphogenesis, is conse
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Hypoxia-inducible factors (HIFs) ar
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To address this issue, we have take
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Program/Abstract # 530 The MADS pro
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Better understanding of the underly
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NPCs blunted proliferation in the S
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Program/Abstract # 551 Evolutionari
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group is interested in inferring an
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Program/Abstract # 564 Withdrawn Pr
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Program/Abstract # 573 Fluoride ind
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oligodendrocytes of the central ner
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cycle, no changes were observed in
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have begun to document the targets
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Congress Abstracts - Society for Developmental Biology

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