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126 The regulatory networks that control lineage specification in the developing central nervous system are not completely understood. We have recently identified a novel population of neurons that originate in the ventral neural tube at stages when spinal cord progenitors are committed to glial fates. They are a late-born subset of V2 cells and were identified as CerebroSpinal Fluid-contacting Neurons (CSF-cN). The aim of this work is to determine the mechanisms involved in the differentiation of CSF-cN. Genetic lineage tracing in vivo experiments and expression analysis in mice indicate that CSFcN progenitors express both the proneural transcription factor Ascl1 and the zinc finger containing transcription factor Gata3. We have found that CSF-cN are missing in Ascl1 mutant mice, while other neuronal cell types generated earlier from the same domain remain unaffected. In order to assess when Ascl1 is needed, we took advantage of the temporally restricted expression of Ascl1 conditional mice, revealing that Ascl1 exerts its critical actions around the time of CSF-cN differentiation. Moreover, our results indicate that Gata3 also plays a key role in CSF-cN development, as this population is severely affected in conditional CNS Gata3 knockouts. We determined that Ascl1 precedes Gata3 expression and that Gata3 is absent in ventral progenitors from Ascl1 mutants. On the contrary, we did not find a change in Ascl1 expression in Gata3 cKOs. In summary, we identified two key and specific regulators of CSF-cN specification. Our results show that Ascl1 and Gata3 act sequentially in controlling late neurogenic events in the ventral neural tube. Program/Abstract # 380 Transcription factor dynamics in single mouse ES cells during germ layer commitment Adele M. Doyle; Zou, Ling-Nan; Jang, Sumin; Ramanathan, Sharad (Harvard FAS Center for Systemzs Biology, Cambridge, United States Embryonic stem cells can differentiate into all the cell types in the body. It remains unclear though how intracellular signaling and gene expression dynamics control fate choices of individual cells. Our previous work showed that the dynamic expression ratio of two pluripotency factors governs the earliest fate choice an embryonic stem cell makes, between neuroectodermal and mesendodermal progenitors (Thomson, 2011). Here, our goal is to identify the key transcription factors and their dynamics that govern specification of single cells into progenitors of one of the three germ layers. We measured the expression dynamics of pluripotency factors (Oct4, Nanog) and germ layer-specific factors (Sox1, Brachyury, Gata6) in single mouse embryonic stem cells during early differentiation. We used these dynamics to identify and purify subpopulations of cells that appear en route to germ layer specification. Comparing gene profiles of these purified subpopulations, we identified other transcription factors whose expression dynamics correlated with differentiation. We found that expression of a subset of transcription factors (Dnmt3a, Eomes, Snail, Foxa2, Gata4) correlated with intermediate transitions between pluripotency and germ layer commitment. Future experiments to interfere with these transcription factors during differentiation will determine the importance of these dynamics on the fate choice single cells make between the three germ layer progenitors. Through these experiments, our aim is to develop a general approach to make quantitative predictions about the dynamics of key transcription factors that control developmental decisions. Program/Abstract # 381 The role of voltage-gated calcium channels in neuronal phenotype specification Herbst, Wendy; Saha, Margaret; Rabe, Brian; Welch, Zoe, The College of William and Mary, Williamsburg, United States In addition to their role in neurotransmission in the mature nervous system, voltage-gated calcium channels are implicated in a wide variety of developmental processes. Recent literature has demonstrated the role of calcium activity in neural induction, neurotransmitter phenotype specification, axon pathfinding, and dendrite outgrowth. Given that the voltagegated calcium channels are expressed at an appropriate time and place during development, we hypothesize that the alpha1 subunits mediate the spontaneous calcium activity that is important for neurotransmitter phenotype specification. To address this hypothesis, a morpholino “knockdown” approach is used to prevent the expression of the calcium channels. Thus far we have employed morpholinos to knockdown Cav2.1. This particular calcium channel subunit was chosen because it expressed widely inthe neural tube of Xenopus laevis during the early stages of development.The morpholinoinjected embryos are then raised to the swimming tadpole stage and observed for any morphological, behavioral, and gene expression differences. The morpholino-injected embryos have displayed less swimming movement and an underdeveloped nervous system. Additionally, we are using a bioinformatics approach to analyze the upstream regulatory elements of Cav2.1, by aligning this upstream sequence with the upstream regions of other calcium channels and among species. Using a transgenics approach, we are currently testing the hypothesis that the conserved sequences are important regulatory elements. Program/Abstract # 382 A low level of Hedgehog signaling in the notochord is sufficient for normal ventral pattering in the embryonic
127 spinal cord. Iulianella, Angelo, Dalhousie University Anatomy and Neurobiology, Halifax, Canada; Trainor, Paul (Stowers Institute, Kansas City, United States) Current models of neural patterning emphasize a role for the length of time progenitor cells are exposed to the morphogen Sonic Hedgehog (Shh) as the key determinant in generating pattern in the ventral vertebrate spinal cord. Although a Shh concentration gradient is thought to pattern the neural tube, a temporal mechanism implies that small amounts of morphogen are required to initiate the diversification of ventral spinal cord cell types. We utilized a genetic approach to test this idea during mouse spinal cord development. Because Patched1 is both the receptor and negative regulator of Shh signaling, its mutation sensitizes the embryo to Hedgehog signalling. We employed a novel Patched1 mutation (called Wiggable) that removes its carboxyl-terminus, which is required to inhibit Hedgehog signal transduction. This mutant is less severe than the Patched1-null and shows expanded ventral character in the developing spinal cord, consistent with increased Shh signalling. We then tested whether Wiggable mutants can rescue ventral patterning in situations where Shh levels were either entirely absent or severely reduced. We accomplished this by using the Shh-null and Hedgehog acteyl transferase (Hhat)-null mouse mutants, respectively. Hhat encodes a palmitoylase that is required to produce secreted forms of Hedgehog proteins, and is thus crucial for the establishment of the Shh signaling gradient. In its absence, the spinal cord develops without the ventral-most cell types such as floorplate cells and V3 interneurons. Surprisingly, Hhat/Wiggable double mutants show completely normal spinal cord patterning, while Shh/Wiggable mutants lacked only floorplate cells. The principal difference between Shh and Hhat mutants being that Hhat mutants still have low amounts of Hedgehog signaling in the notochord. Thus, our findings show that remarkably little Hedgehog signalling from the notochord is all that is required to initiate the full range of neuronal identities in the vertebrate spinal cord. Program/Abstract # 383 CCAR1 is required for Ngn3-mediated endocrine differentiation Lu, Chung-Kuang, National Chung Cheng University, Chiayi County,Taiwan Neurogenin3 (Ngn3) is a basic helix-loop-helix transcription factor that specifies pancreatic endocrine cell fates during pancreas development. It can also initiate a transdifferentiation program when expressed in pancreatic exocrine and ductal cells. However, how Ngn3 initiates a transcriptional cascade to achieve endocrine differentiation is still poorly understood. Here, we show that cell cycle and apoptosis regulator 1 (CCAR1), which is a transcriptional coactivator for nuclear receptors, also interacts with Ngn3. The association between Ngn3 and CCAR1 was verified by pull-down assays and coimmunoprecipitation analyses. Using gene reporter assays, we found that CCAR1 is essential for Ngn3 to activate the expression of the reporter genes containing the NeuroD promoter. Moreover, down-regulation of endogenous CCAR1 in the PANC-1 pancreatic ductal cell line inhibits the transdifferentiation program initiated by Ngn3. CCAR1 is, therefore, a novel partner of Ngn3 in mediating endocrine differentiation. Program/Abstract # 384 Cell cycle exit is required for cell invasive behavior Matus, David Q.; Kelley, Laura; Schindler, Adam; Chi, Qiuyi; Sherwood, David, Duke University, Durham, United States Cell invasion through basement membranes is an essential cell biological process that occurs during development, immune surveillance, and is mis-regulated during cancer metastasis. While cell cycle exit is a hallmark of differentiated cell types (i.e., muscle and neurons), it is unknown whether cells must exit the cell cycle to acquire the ability to invade. To investigate cell cycle control of the invasive phenotype, we are using a simple in vivo model of cell invasion that occurs during C. elegans larval development, where a specialized gonadal cell, the anchor cell (AC), invades through the uterine and ventral epidermal basement membrane to contact the developing vulval epithelium. We performed a uterine-specific RNAi screen targeting 598 C. elegans transcription factors, identifying the tailless/TLX ortholog, NHR-67, as a novel regulator of the AC cell cycle. Loss of function of NHR-67 results in the presence of multiple ACs that fail to breach the basement membrane. Through the use of cell cycle reporters we show that depletion of NHR-67 results in the normally post-mitotic AC failing to exit the cell cycle and proliferating. To determine whether cell cycle defects were functionally correlated with defects in invasion we manipulated the AC cell cycle machinery. Loss of genes required for cell cycle exit phenocopies depletion of NHR-67, resulting in multiple ACs that fail to invade. Conversely, prevention of cell cycle entrance in NHR-67-depleted animals rescues the invasion defect. To better understand the link between cell cycle exit and invasion, we are quantifying the dynamics ofthe actin cytoskeleton during proliferative and invasive states. Additionally, we are examining AC-specific gene expression during these disparate states and have identified that proliferative ACs fail to express MMPs that are transcriptional targets of the AP-1 transcription factor FOS-1A. Together, our data demonstrates a functional link between cell cycle exit and invasion and raises the possibility that invasive cells should be classified as a
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1 Program/Abstract # 1 Role of the
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3 Program/Abstract # 7 Forward gene
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7 Program/Abstract # 19 Lethal gian
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9 Program/Abstract # 26 On the comb
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11 Program/Abstract # 32 Imaging in
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13 and electron microscopy studies
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15 paracellular space, and interact
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17 the proximal to distal axis of t
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19 Dominguez-Castellano, Maria; Gar
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21 We are interested in the role of
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23 more likely than students who di
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25 was a significant loss of cells
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27 Fgfr3 expression in the limb cho
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29 division, but rapidly loses its
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31 and migration (ADAM13). They act
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33 Program/Abstract # 101 The influ
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35 Program/Abstract # 107 Shroom3-d
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37 Program/Abstract # 113 Requireme
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39 (Queens College, Flushing, Unite
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41 these data imply that Dynamin is
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43 Congenital renal malformations a
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45 Elevated nuclear translocation o
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47 progenitor cells that cover the
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49 abnormally dilated capillaries i
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51 malformations in murine limb bud
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53 Yuqing (Mouse Imaging Centre, To
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55 in the villi at E14. At E11, the
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57 Program/Abstract # 173 The role
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59 Boldt, Clayton, UT Southwestern,
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61 examine stem cell-based CNS rege
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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
Page 75 and 76: 75 The through-gut, consisting of s
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Page 81 and 82: 81 Lee, Hu-Hui, National Chiayi ers
Page 83 and 84: 83 understood. Here, we have establ
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Page 91 and 92: 91 Program/Abstract # 278 Heterogen
Page 93 and 94: 93 Program/Abstract # 284 Investiga
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Page 97 and 98: 97 dizygotic twin studies have show
Page 99 and 100: 99 Program/Abstract # 301 Dual role
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Page 119 and 120: 119 Program/Abstract # 359 The meth
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Page 133 and 134: 133 ROS levels. Collectively, our r
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Page 151 and 152: 151 Program/Abstract # 456 Thoracic
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Abstracts - Society for Developmental Biology

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