Abstracts - Society for Developmental Biology

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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.