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12 murine heart growth than previously thought. Further, as congenital heart disease is often associated with defects in second heart field development, the embryological and genetic advantages of the zebrafish model can be applied to study the vertebrate second heart field. Program/Abstract # 36 Live imaging of hematopoietic niche colonization reveals distinct endothelial and stem cell interactions Tamplin, Owen J.; Durand, Ellen; Lawson, Katy; Li, Pulin; Zon, Leonard, Children's Hospital/ Harvad, Boston, United States Hematopoietic stem and progenitor cells (HSPCs) self-renew and give rise to all blood cell types throughout adulthood. Definitive HSPCs arise from the hemogenic endothelium of the dorsal aorta, are released into circulation, and then seed an intermediate hematopoietic tissue before colonizing the adult marrow. In mammals this intermediate tissue is the fetal liver and in the zebrafish it is the caudal hematopoietic tissue (CHT), a vascular plexus in the ventral tail of the embryo. We created an HSPC-specific transgenic reporter line using the previously described mouse Runx1 +23 kb intronic enhancer. Together with an endothelial reporter (flk1:dsRed), we could use time-lapse live imaging to follow HSPCs as they migrate to the CHT. Upon arrival, HSPCs underwent a number of distinct steps to engraftment, including adherence to the vessel wall, extravasation, and triggering of niche formation—endothelial cells actually remodel around the HSPC to create a niche. To determine if this endothelial niche formation is conserved in mammals, we performed live imaging of mouse fetal liver explants at embryonic day 11.5, the earliest stage of seeding by HSPCs. Strikingly, we observed CD31+ endothelial cells adhere to and form a rosette around single c-kit+ HSPCs, similar to the cellular behaviors observed in zebrafish. To find clues to the molecular mechanisms that regulate these distinct cellular behaviors during hematopoietic niche colonization we performed a chemical genetic screen. We found compounds that both increased and decreased CHT hematopoiesis. We are now applying these chemical hits during live imaging of CHT colonization and are gaining important insights into stem cell engraftment and hematopoietic niche formation. Program/Abstract # 37 Neuronal guidance cues direct early blood vessel formation Meadows, Stryder M.; Fletcher, Peter, UT Southwestern Med Ctr, Dallas, United States; Moran, Carlos (U Arizona, Tucson, United States); Ratliff, Lyndsay; Xu, Ke (UT Southwestern Med Ctr, Dallas, United States); Neufeld, Gera (Haifa, Israel); Chauvet, Sophie; Mann, Fanny (Marseille, France); Krieg, Paul (U Arizona, Tucson, United States); Cleaver, Ondine (UT Southwestern Med Ctr, Dallas, United States) Neuronal guidance molecules are known to influence endothelial cell (EC) behavior. These molecules shape the vasculature by acting as attractive or repulsive signals; however, it is unclear if these molecules affect patterning of the initial blood vessel network. Our analysis of neural guidance cues during embryonic mouse development indicates that multiple repulsive cues are present in the notochord. We hypothesize that overlapping sets of repulsive guidance cues expressed in the notochord direct EC migration and patterning of the first blood vessels that form in the mammalian embryo, the paired dorsal aortae (DA). We have analyzed mutant mice embryos lacking a notochord and similar to previous studies in avian embryos, we observed dramatic vascular abnormalities characterized by scattered aortic ECs that fail to form vessels. In these embryos, all repulsive guidance cues are lost at the midline and ECs crossed the normally avascular midline. Furthermore, we identify a single repulsive guidance cue, Semaphorin 3E (Sema3E), expressed from the lateral plate mesoderm that creates avascular zones which define the lateral edges of the DA. Rather than a single smooth vessel, Sema3E null embryos display a tree-like plexus of aortic vessels with ‘branches’extending into the lateral avascular spaces. Interestingly, despite such a severe phenotype, Sema3E-/-mice survive throughout adulthood. We find that these defective vessels resolve back into normal DA during subsequent development, likely due to additional, undetermined repulsive guidance cues. Overall, these studies demonstrate how multiple, functionally redundant and non-redundant repulsive cues work coordinately to shape the early embryonic blood vessels. Program/Abstract # 38 Novel role for an Aquaporin gene in neural tube closure Van Antwerp, Daniel; Weber, Mackenzie; Merzdorf, Christa, Montana State University, Bozeman, United States As a result of a micro array screen for genes regulated by the Zic family of transcription factors in Xenopus laevis, we identified a membrane water channel, or aquaporin protein, which we named aquaporin 3b (aqp3b). Characterizing aqp3b expression through embryogenesis showed that it localized very specifically to a narrow column of cells at the edges of the closing neural plate. As zic genes play a role in neural tube closure, we tested whether aqp3b is involved as a downstream effector of this process. Microinjection of aqp3b morpholino oligos suggested that this gene is important for proper neural tube closure. Further analysis of the cellular identity of aqp3b expression has been aided by detailed, yet decades old, light
13 and electron microscopy studies of Xenopus neural tube closure. In these studies, Thomas Schroeder identified a cell positioned at the interface between the converging epidermal layer and invaginating neural epithelium. Termed the “IS cell”, for intermediate superficial cell, it has characteristics of both epidermal and neural epithelial cells, allowing a transitional zone between these diverse cell types. We hypothesize that aqp3b is specifically expressed in IS cells and suggest that IS cells and their expression of aqp3b are required for proper closure of the neural tube. IS cells do not themselves undergo apical constriction, but, upon aqp3b inhibition, we observe a failure of apical constriction in cells that normally form the dorsal-lateral hingepoints. The possibility of intercellular action of IS cells on the neighboring hinge point cells has not been previously predicted. We are attempting to broaden these results to other species and to define the mechanism by which IS cells and aqp3b participate in neural tube morphogenesis. Program/Abstract # 39 Maintenance of axial patterning in the sea urchin embryo: A role of Wnt1 signaling Angerer. Lynne M., National Institutes of Health, Bethesda, United States The initial patterning along the anterior-posterior (AP) and dorsal-ventral (DV) axes of sea urchin embryos depends on Wnt and Nodal signaling, respectively, and occurs before gastrulation. The secondary DV axial patterning relies on a Wntdependent process that removes a suppressor of nodal expression from non-anterior ectoderm. Here we report that unexpectedly, later, when gastrulation begins, Wnt signaling continues to affect Nodal signaling, not by supporting it, but rather by preventing nodal expression in the ventral-posterior region of the embryo. This region normally gives rise to the posterior-transverse ciliary band, the supra-anal ectoderm and ventral endoderm. When Wnt1 is knocked down, expression of nodal and its target genes, gsc and bra, extends ectopically on the ventral side toward the blastopore. As a consequence, initial fates of cells in this region are changed to oral ectoderm, as shown by lineage tracing. Strikingly, the ciliary band, which forms adjacent to the nodal expression domain, is shifted significantly in the dorsal direction, toward and sometimes beyond the position of the blastopore. This results in the blastopore and stomodeal regions being positioned in the same ventral plane instead of approximately at a 90° angle. Before gastrulation, wnt1 expression is radial in posterior blastomeres. But when gastrulation begins, it is lost from the dorsal side by a Nodal-dependent process and maintained only on the ventral side, where it suppresses nodal. Thus, Wnt- and Nodal-dependent processes mutually antagonize each other to maintain the body plan established at earlier stages by these same pathways. Program/Abstract # 40 Membrane morphogenesis during tracheal tube development in Drosophila Jayan Nandanan, N; Mathew, Renjith (EMBL, Heidelberg, Germany); Leptin, Maria, EMBO, Germany The terminal cells of the Drosophila respiratory (tracheal) network contain seamless, membrane bounded intracellular tubes through which air travels. The tube-containing extensions of these cells are elaborated during chemotactic growth directed by developmental cues in embryos and subsequently ramify in response to signals from hypoxic tissues in larvae. Extensive membrane traffic occurs during growth, as the cell rapidly and concomitantly elaborates two membrane domains of opposite characteristics. The outer (basal) membrane migrates and grows with highly dynamic filopodial extensions, while at the same time the inner tube of apical characteristics is constructed from as yet unknown membrane sources. We find that the membranes forming the intracellular tubules contain lipids and proteins typical of apical plasma membranes in polarized epithelial cells. The Drosophila synaptotagmin-like protein Bitesize (Btsz) and the activated form of its interaction partner Moesin are also located at the growing luminal membrane. Our functional studies indicate that the actin cytoskeleton, through its interaction with Btsz via Moesin directs apical membrane morphogenesis to create and maintain distinct intracellular tubules. Using real-time in vivo imaging we have analysed the assembly of the intracellular tube. We find that ER and Golgi rapidly distribute into the developing cellular extensions prior to the assembly of the membrane bounded tube within. Redistribution of secretory machinery ahead of the growing tube seems to be a prerequisite for proper tube extension. Program/Abstract # 41 Asymmetric division of luminal cells produces low-polarity high-motility cells that collectively migrate to form mammary ducts Huebner, Robert J., Johns Hopkins Sch of Med, Baltimore, United States; Lechler, Terry (Duke, Durham, United States); Ewald, Andrew (Johns Hopkins Sch of Med, Baltimore, United States) We seek to understand the cellular and molecular mechanisms that underlie branching morphogenesis. To overcome the limitations of optical imaging in the intact mouse, we rely on organotypic cultures of primary mammary epithelium. Using 3D time-lapse imaging and transgenic fluorescent reporters, we have resolved cell migration, proliferation, and tight junction dynamics throughout branching morphogenesis. The initial stage of morphogenesis involves a growth factor
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Page 31 and 32: 31 and migration (ADAM13). They act
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63 Incubation of regenerating cells
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65 learn which dynamic behaviors oc
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67 cell proliferation and for norma
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69 Program/Abstract # 209 Drosophil
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71 cranial neural crest cells, as t
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73 experimental analysis. The jerbo
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75 The through-gut, consisting of s
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77 thereby increasing the chance of
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79 into the role snx5 playsin the N
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81 Lee, Hu-Hui, National Chiayi ers
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83 understood. Here, we have establ
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85 assays, chromatin immunoprecipit
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87 approach. A similar strategy was
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89 verify the functional specificit
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91 Program/Abstract # 278 Heterogen
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93 Program/Abstract # 284 Investiga
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95 Program/Abstract # 290 A molecul
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97 dizygotic twin studies have show
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99 Program/Abstract # 301 Dual role
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101 trafficking of cargo proteins a
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103 Sonic Hedgehog (Shh) is a secre
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105 tube was aberrant. Our data ind
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107 degradation of Smad proteins. W
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109 strong affects on organogenesis
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111 that maternally-supplied Lkb1 i
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113 Program/Abstract # 341 Coordina
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115 partners. Our transgenic gain o
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117 delayed and stunted as monitore
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119 Program/Abstract # 359 The meth
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121 Misregulation of molecular path
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123 back to the midbody in cbp-1 RN
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125 versus asymmetric cell division
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127 spinal cord. Iulianella, Angelo
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129 determine cell cycle activity i
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131 in expanded Lmx1a/b+ progenitor
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133 ROS levels. Collectively, our r
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135 limbs. Apoptosis was also pertu
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137 Henkels, Julia; Zamir, Evan, Ge
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139 however, and its role in CSF ma
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141 embryonic precursors to form an
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143 with fork retarding Replication
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145 homeodomain (HD) transcription
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147 fate, potentially acting downst
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149 involved in Drosophila ventral
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151 Program/Abstract # 456 Thoracic
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153 ureteric insertion into the bla
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155 of p16, a known cyclin kinase i
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157 rax mutant was recently found i
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Abstracts - Society for Developmental Biology

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