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expression was observed in the limb buds close to but not within the cartilage elements, we focused on the effect of EVI-1 on chondrogenesis in stage 24 limb micromass cultures. Cells were transfected with an siRNA to target chicken EVI-1 at the time of plating. After six days of cultivation, the level of chondrogenesis was evaluated in cultures by Alcian blue staining. Perinodular inhibition of chondrogenesis in cultures was observed. Downregulation of EVI-1 after siRNA treatment was confirmed by qPCR. Since EVI-1 was expressed adjacent to the apical ectodermal ridge and this structure is a source of FGFs, we wanted to analyse the influence of FGFR inhibitors on EVI-1 gene expression. Local injection of FGF inhibitor PD161570 into the limb bud at stages 20–22, downregulated EVI-1 expression was determined by qPCR. Next, we performed gain-of-function experiment, where FGF2 beads were implanted into the right wings at stage 20. We observed upregulation of EVI-1 expression 16h after treatment. In summary, it was found that EVI-1 levels need to be maintained in order for chondrogenesis to occur. In addition EVI-1 may be a novel gene mediating the effects of FGF on chondrogenesis. This work was supported by GACR (grant 304/09/0725 ) to MB and CIHR grant to JMR. Program/Abstract # 244 Effects of homocysteine on mesenchymal cells during limb development on chick embryos Bourckhardt, Gilian; Kobus, Karoline; Cecchini, Manuela; Müller, Yara; Ammar, Dib; Nazari, Evelise (Universidade Federal de Santa Catarina (UFSC), Brazil) Hyperhomocysteinemia is a metabolic condition resultant of folic acid dietary deficiency. This condition is related with the occurrence of congenital anomalies that include limb defects. High levels of homocysteine (Hcy) can induce DNA damage and cell cycle arrest due to non-remethylation of Hcy to methionine. The aim of this study was to investigate whether high levels of Hcy can affect the mesenchymal cell dynamics d uring limb development. Chick embryos were treated with 20 µmol D-L Hcy/50 µL saline at E2 and analyzed at E6. Control embryos were treated with 50 µL saline. To identify cells in proliferation and proteins involved in cell cycle we performed immunolocalization and flow cytometry analyses using antibodies anti-phosphohistone H3 (mitosis marker), anti-p53, anti-p21 and anti-PCNA. No significant differences on cell proliferation rate were observed between Hcy-treated and control embryos. Thus, we observed a downregulation of proliferating cell nuclear antigen (PCNA) and the p21 protein, both involved in the G1 phase of cell cycle progression. On the other hand, the Hcy induces in mesenchymal cells of the limbs, an upregulation in expression of p53 protein, which can be activated by DNA damage. Additionally, we observed an increase of apoptosis rates. Our results indicate that the Hcy-treatment changes the mesenchymal cell dynamics during limb development of the G. domesticus. Program/Abstract # 245 Inhibition of Hedgehog Signaling is Necessary for ß-Catenin-Regulated Interzone Differentiation and Joing Morphogenesis Rockel, Jason; Yu, Chunying; Whetstone, Heather (The Hospital for Sick Children, Canada); Craft, April (University Health Network, Canada); Reilly, Katherine; Alman, Benjamin (The Hospital for Sick Children, Canada) The mechanisms responsible for articular chondrocyte (AC) development are incompletely elucidated. ACs derive from Gdf5- expressing interzone cells and differentiate through a distinct pathway compared growth plate chondrocytes (GPCs), which do not derive from interzone cells . Hedgehog (HH) signalling is active in chondrocytes, primarily in GPCs. In osteoarthritis, a degenerative disease of articular cartilage, the HH-regulated GPC developmental program is r ecapitulated in ACs. Thus inhibition of HH signalling may be necessary for normal interzone cell differentiation, joint morphogenesis, and the maintenance of ACs. Using transgenic mice and ex vivo cultured embryos, we found that inhibition of HH signalling maintained interzone populations cell autonomously but had no effect on joint or skeletal morphogenesis. In contrast, activation of HH signalling inhibited interzone cell differentiation and maintenance in a cell non-autonomous manner. Interestingly, transgenic mice that had activated HH signalling in interzone cells developed osteochondrodysplasias and morphological abnormalities including ectopic joint cartilage, reduced AC differentiation and undifferentiated cells within the joint space. HH signalling also reduced Wnt/β -catenin activity in interzone progeny. Constitutive activation of β-catenin rescued HH-induced knee joint abnormalities and partially rescued the osteochondrodysplasias. Treatment of hindlimb organ cultures with FGF18, a β-catenin target gene, also rescued HH-induced joint abnormalities. These data indicate that HH signalling needs to be downregulated in interzone cells for β-catenin-regulated AC differentiation and joint morphogenesis. Program/Abstract # 246 Characterizing gene expression dynamics between Shox2 and Hox genes during limb development Neufeld, Stanley John (University of Calgary, Canada), Scott, Alexandra; Wang, Fan (Durham, USA); Cobb, John (University of Calgary, Canada) The proper development of the vertebrate limb relies on homeobox genes of both the Hox and Shox families of genes. In mice and humans, mutation of certain members of either gene family results in similar phenotypes, such as malformed or shortened limb segments. We have previously established that Shox2 and Hox genes genetically interact in the mouse limb, supporting the view that these genes function together. To gain further insight, we are analyzing their relative expression dynamics through double mRNA FISH in whole embryos. This analysis reveals an intriguing pattern where Shox2 expression prominently overlaps with proximalacting Hox genes, and is complementary to the expression of distal-acting Hox genes. This dichotomy is established as early as E10.5, suggesting that these relative dynamics could be important for the proper development of the discrete segments of the limb. We are currently assessing the possibility of cross-regulatory control between Hox genes and Shox2, and also the possibility that these genes 70
coordinately regulate the expression of a downstream target gene. Overall, this analysis will provide insight into transcriptional control that operates during normal limb development, and aspects that fail during genetic disease. Program/Abstract # 247 Interdigital mesoderm acts as a signaling center instructing digit joint formation Huang, Bau-Lin (National Cancer Institute-Frederick, USA); Koyama, Eiki; Pacifici, Maurizio (Children's Hospital of Philadelphia, USA); Mackem, Susan (National Cancer Institute-Frederick, USA) The number and position of joints is one of the morphologic hallmarks of digit identity. Interdigital mesoderm (IDM) has been implicated in regulating digit identity, but its exact role and whether it regulates digit joint formation are unclear. Digital joints are lost in the 5'HoxdDel (Hoxd11,12,13 -/- ) mouse. Using Gdf5Cre lineage tracing analysis to mark presumptive joint progenitors (interzone, IZ) in 5'HoxdDel, Gdf5+ cells are absent from, but surround the IZ region, which consists of Sox9+ chondrogenic cells. Wnt/βcatenin signaling has been shown to play a key role in joint formation by repressing the chondrogenic program in the IZ. Our genetic analysis of a conditional 5' Hoxd allele reveals that " late " 5'Hoxd function is required for digit joint formation (to E12.5) and we tested whether 5 ' Hoxd genes act in the βcatenin pathway. Introducing a conditional, stabilized βcatenin allele (exlox3 βcatenin; βCatGOF) restores normal joint formation and joint markers in 5'HoxdDel digits. To determine tissue requirements for rescue by βCatGOF, either cartilage- (Sox9CreER) or interdigit-specific (OsrCre) Cre lines were used. Surprisingly, joint formation was restored in 5'HoxdDel digits only by OsrCre/βCatGOF but not Sox9CreER/βCatGOF. Genetic lineage tracing demonstrates that OsrCre/βCatGOF cells are found only in IDM and do not contribute to IZ. These results indicate a non-cell autonomous role for βcatenin acting in IDM to regulate digit joint formation, and reveal that IDM can act as a signaling center to instruct digital joint formation. BMP signaling has been proposed to regulate digit identity from the IDM and we are currently testing the role of Bmp and other potential targets regulated by 5'Hoxd and βcatenin. Program/Abstract # 248 Interdigit BMP signaling is essential for programmed cell death and is implicated in digit formation Kaltcheva, Maria M.; Pajni-Underwood, Sangeeta (National Cancer Institute-Frederick, USA); Harfe, Brian (University of Florida College of Medicine, USA); Lewandoski, Mark (National Cancer Institute-Frederick, USA) Shaping of the embryonic limb involves many processes including growth, differentiation, and programmed cell death (PCD). These processes integrate complex information from multiple signaling cascades such as the BMP and FGF pathways. Our previous work has shown that BMP signaling regulates interdigit (ID) PCD indirectly by modulating the secretion of FGFs from the apical ectodermal ridge, which act as cell survival factors to the ID mesenchyme. However, this does not exclude a direct role for BMPs in PCD. We therefore genetically examined the direct role of BMPs as triggers of ID PCD. To reduce BMP signaling to the ID mesenchyme we inactivated the gene encoding receptor BMPR1A within the ID. This results in retention of ID tissue, syndactyly, in adult mice due to a decrease of ID PCD during embryogenesis. To test redundancy between BMPR1A and BMPR1B in PCD we inactivated ID Bmpr1A in a Bmpr1B null background. This compound mutant has a further decrease in PCD and a significant upregulation of Gdf5 expression in the ID mesenchyme. GDF5 is a TGFβ family ligand that can bind BMP receptors but has not been previously implicated in regulating ID PCD. During our analysis we also discovered a potential role of the ID tissue in digit formation. Bmpr1B null digits are short with abnormal development of their phalanges. This defect is completely rescued in digit one when we inactivate Bmpr1A in the ID. To fully understand the role of ID BMP signaling on normal limb development we are inactivating ID Bmp2, 4, and 7. Preliminary analysis of these mutants reveals a syndactylous phenotype, supporting a role of BMP signaling in ID PCD. This work will elucidate the role of ID BMP signaling in directly regulating PCD and digit formation. Program/Abstract # 249 Tramtrack69 regulates epithelial tube expansion in the Drosophila ovary through Paxillin and the homeobox protein Mirror Peters, Nathaniel C.; Berg, Celeste (University of Washington, USA) Epithelial tubes are the infrastructure for multicellular organs and tissues. Faithful tube morphogenesis requires the precise orchestration of cell signaling, shape, polarity, migration, and adhesion. The Drosophila ovary provides a robust, accessible model for epithelial tube morphogenesis. The somatic epithelium that encases each developing egg chamber forms and then expands a pair of tubes during late oogenesis, and the lumens of these tubes mold the eggshell’s two dorsal respiratory appendages (DAs). Thus, the DAs of the laid egg provide an external readout for the efficacy of DA-tube morphogenesis. The Tramtrack69 (TTK69) transcription factor is required for DA-tube expansion; the twin peaks (ttk twk ) mutation reduces TTK69 levels during late oogenesis and disrupts DA-tube expansion, resulting in stunted DAs. Microarray and in situ hybridization analyses comparing wild-type and ttk twk ovaries implicate the focal-adhesion scaffold Paxillin and the homeobox-protein Mirror as TTK69 effectors of DA-tube expansion. Tissuespecific RNAi against Paxillin and mirror produces DA defects that are enhanced in ttk twk heterozygotes, demonstrating function in DA-tube formation and genetic interactions with ttk twk . Although Mirror patterns the DA tubes prior to morphogenesis, we show that Mirror regulates DA-tube expansion independently of patterning. Mirror positively influences Paxillin expression, and overexpression of Paxillin can partially suppress the mirror RNAi defect. Additionally, shibire (Drosophila Dynamin) and lamina ancestor regulate DA-tube expansion downstream of TTK69. Thus, we have identified TTK69 effectors required for tube expansion, several of which are novel regulators of epithelial tube morphogenesis. 71
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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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Page 23 and 24: Program/Abstract # 78 In vivo recep
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Page 29 and 30: Program/Abstract # 98 A gradient of
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Page 33 and 34: NSCs, but not in neurons, bind not
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Page 45 and 46: Program/Abstract # 156 Systematic I
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Page 51 and 52: Penaeid shrimp represent an importa
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Page 61 and 62: condition. Kanyon embryos have a mi
Page 63 and 64: Program/Abstract # 218 Lfng regulat
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Page 67 and 68: medaka genome. These miRs are encod
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Page 89 and 90: Program/Abstract # 308 Mechanistic
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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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