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118 adhesion. Tspan18 is expressed in chick premigratory NCCs, but is downregulated prior to migration, suggesting that Tspan18 may negatively regulate neural crest EMT. Indeed, when NCCs express Tspan18 past the stage it is normally down regulated, migration is blocked. NCCs that fail to migrate maintain epithelial Cadherin6B (Cad6B) protein despite temporally normal down regulation of Cad6B mRNA, suggesting Tspan18 promotes cell adhesion by stabilizing Cad6B protein to antagonize EMT. In contrast, Tspan18 knockdown leads to premature loss of Cad6B protein, and as a result, some embryos exhibit enhanced migration. Curiously, Tspan18 knockdown also represses Cad6B transcription. This effect on Cad6B mRNA is independent of the Cad6B repressor Snail2, but correlates with increased nuclear b-catenin, perhaps as a consequence of its release from Cad6B-dependent adherens junctions. Finally, consistent with the fact that the NCC transcription factor FoxD3 regulates NCC adhesion and promotes migration, FoxD3 represses Tspan18 expression. Taken together, these data suggest that Tspan18, as a read-out ofFoxD3, plays an important role in antagonizing neural crest EMT by promoting adherens junctions. Supported by NIH F31 GM087951 and a U of MN Grant inAid. Program/Abstract # 357 Paladin is an antiphosphatase that modulates neural crest formation and migration Gammill, Laura S.; Roffers-Agarwal, Julaine; Hutt, Karla, University of Minnesota Genetics, Cell Biology & Development, Minneapolis, United States Cells in the vertebrate dorsal neural tube face a dilemma: delaminate and become neural crest, or remain epithelial and become roof plate. Because dorsal neural tube cells co-express neural crest and roof plate transcription factors, neural crest gene expression does not guarantee eventual migration as a neural crest cell. In order to investigate the importance of differential protein activity in determining when and where neural crest regulatory factors are active, we characterized the function of the putative phosphoregulatory protein Paladin (Pald). Pald contains two phosphatase active site motifs and is expressed in neural crest precursors and migratory neuralcrest cells throughout chick neural crest development. Pald knockdown delays expression of neural crest transcription factors Snail-2 and Sox10, but has no effect on FoxD3, indicating Pald differentially regulates genes previously thought to be co-regulated in the neural crest gene regulatory network. Moreover, Pald is not required for proper temporal expression of Cadherin6B or RhoB, although Pald is essential for timely migration away from the neural tube, suggesting Pald regulates specific features of migration downstream of delamination. Finally, because mutation of critical, catalytic cysteine residues within Pald’s predicted phosphatase active site motifs does not abolish Pald function in the neural crest, we conclude that Pald is an antiphosphatase. Altogether, our data indicate that Pald modulates the activity of specific regulatory factors by preventing their dephosphorylation in neuralcrest cells, providing an additional layer of regulation during early neuralcrest development. Funding: Minnesota Medical Foundation, F32DE019973, K22DE015309 Program/Abstract # 358 Loss-of-function analysis of RAC1 function in development of the zebrafish olfactory bulb Horne, Jack; Fisher, Kelly, Pace University Biology, Pleasantville, United States Development of the vertebrate brain requires that axons and dendrites grow and elaborate into specific projections, resulting in characteristic cell shapes that allow synaptic partners to appropriately connect. Through experiments in model organisms, much is known about the extracellular molecular cues, and their receptors expressed by neurons, that guide axons to their appropriate targets. What is less well understood is the molecular coupling between axon guidance receptors and the cytoskeletal regulatory apparati. Recent work has identified some prototypical regulatory molecules that feed directly into the cytoskeletal machinery for specific differentiation processes. For example, the Par3-Par6-aPKC complex is known to be important for establishing neuronal polarity 1, 2. The Rho family of GTPases, including Rho, Rac, and Cdc42, are known to regulate the elongation, growth, and guidance of neurites3. Also, the Ena/VASP family of actin regulators is known to be important for controlling the structure and motility of the growth cone – the motile end of a growing neurite4, 5. We have established a method that combines in vivo electroporation 6, 7 with Gal4-based transgenic zebrafish lines 8 that can be used to specifically target developing neurons of the olfactory bulb. Neurons targeted through this method show stereotypical axon projections of mitralcells 9, the major output cells of the olfactory bulb. In addition to spatial targeting of GFP expression, this technique also allows us to incorporate a loss-of-function reagent at a specific stage in neural development, providing excellent temporal control of the knockdown. Here we use this approach to determine the function of the Rac1 GTPase for the growth and guidance of the mitral cell axon projection. We target the function of Rac1 by co-electroporating an expression plasmid coding for a dominant negative form of Rac1 (T17N). We have characterized the phenotype of Rac loss-of-function using confocal microsopy and three-dimensional reconstruction of the morphology of the olfactory bulb projection. We show that embryos expressing DN Rac1 display shorter axon projections down the lateral olfactory tract, and havedrastically reduced crossing of axons to the contralateral side. Thus, Rac1 function appears to benecessary for proper formation of the axon projection of mitral cells from the olfactory bulb.
119 Program/Abstract # 359 The methyltransferase NSD3 is required for neural crest migration Jacques-Fricke, Bridget; Gammill, Laura, University of Minnesota Genetics, Cell Biology and Development, Minneapolis, United States Neural crest cells are avertebrate stem cell population that arise from the dorsal neural tube, migrate extensively to reach their final targets, and form a variety of structures. While methylation is known to impact neural crest specification, we have identified aneural crest-essential methyl transferase that shows methylation independently regulates neural crest migration as well. The lysinemethyl transferase nuclear receptor SET domain-containing 3 (NSD3) is expressedin premigratory and migratory neural crest cells. Disrupting NSD3 by preventing its production or blocking its function results in reduced premigratory Sox10 expression and decreased migration distance, but not alterations in cell death or proliferation. Temporally restricting NSD3 loss of function to migratory stages reveals that NSD3 directly regulates neural crest migration. NSD3 overexpression also impairs neural crest migration, suggesting that a precise level ofNSD3- mediated methylation is required for proper neural crest development. As NSD3 methylates histone H3K36 in vitro, we are currently evaluating NSD3-dependent changes in histone methylation status in chick cells. Surprisingly, we found NSD3 is expressed in both the nucleus and cytoplasm of migratory neural crest cells, suggesting that NSD3 maymethylate nonhistone substrates as well. Altogether, our work reveals an indispensable role for NSD3 in neural crest cell migration. Funded by NSF IOS-1052102 and NIH F32 DE021651. Program/Abstract # 360 The interplay of actomyosin contraction and post-translationally modified microtubules regulates adhesion maturation and cell migration Joo, E. Emily; Yamada, Kenneth, NIH/NIDCR/CBS, Bethesda, United States Although much is known about how individual cytoskeletal systems contribute to cellular locomotion, how these different systems coordinate their functions to achieve physiological migration is still poorly understood. Here we show that human fibroblasts and organ explants reciprocally coordinate levels of acetylated microtubules and activity of actomyosin contraction to modulate the surface density of integrin and the progression of adhesion maturation, which dictate the migration rates of fibroblasts. Experimentally reducing contraction increased the level of acetylated microtubules. Conversely, increasing microtubule acetylation decreased cellular contraction. This inverse, reciprocal interaction between acetylated microtubules and contraction was achieved by competitive myosin phosphatase interactions with either MLC or HDAC6, which affected the activation state of either protein. This balance of contractility and acetylated microtubules controlled the surface density of the alpha5beta1 integrin, which affected adhesion maturation into fibrillar adhesions. Hyper acetylation of microtubules decreased endocytosis of the alpha5beta1 integrin, and the decreased rate of migration due to hyperacetylation of microtubules was partially rescued by inhibiting the alpha5beta1 integrin. Thus, a homeostatic balance between contractility and acetylated microtubules is achieved through controlled activation and deactivation of myosin II and HDAC6, which regulates the surface density of alpha5beta1 integrin and maturation of adhesions, thereby governing the rate of cell migration. Program/Abstract # 361 Golgi orientation directs early cerebellar Purkinje cells migration through axon specification Kwan, Kin Ming; Au, June Sin Man, School of Life Sciences, The Chinese University of Hong Kong, Shatin, Hong Kong Neuronal migration is a fundamental process for central nervous system development. Neuronal migration is accomplished together with cytoskeletal and secretory pathway polarization. Golgi apparatus is an early compartment in protein secretory pathway. Golgi reorientation has been found to occur during neocortical and cerebellar neuronal migration. During early cerebellar Purkinje cell (PC) migration, Golgi reorientates from the base of the leading process to the opposite pole. However, it is not clear whether Golgi polartiy leads to a directional neuronal migration. Moreover, how Golgi orientation governs neuronal migration remains unknown. In this current work, we show that Golgi relocates to the base of the leading process during early PC migration, which may specify the leading process into axon. Disruption of Golgi orientation suppresses axon specification in cultured PCs. In conditional inactivation of Smad1/5 in mouse cerebellum, a subpopulation of PCs failed to migrate and displayed random and dispersed Golgi positioning, which suggests a close relationship between Golgi orientation and PC migration. We further demonstrated that, in later developmental stages of Smad1/5 mutant mice, those PCs which failed to migrate were not only lack of polarized Golgi but also axon protrusions. Our results suggest a correlation between Golgi orientation and early PC migration. It implicates that Golgi orientation may specify axon during PC early polarization, which maybe important for PC further migration.
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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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9 Program/Abstract # 26 On the comb
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15 paracellular space, and interact
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19 Dominguez-Castellano, Maria; Gar
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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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Page 69 and 70: 69 Program/Abstract # 209 Drosophil
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Page 73 and 74: 73 experimental analysis. The jerbo
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Page 83 and 84: 83 understood. Here, we have establ
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Page 91 and 92: 91 Program/Abstract # 278 Heterogen
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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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