Study on the regulation of morphogen secretion during Xenopus embryonic development

Abstract

Morphogen is defined as a paracrine factor which drives multiple cell fates in accordance with its concentration gradient. During embryonic development, several morphogens are used for cell-cell communication to accomplish differential cell fate decisions during body axis formation, stem cell differentiation and organ development. It is relatively well understood how these morphogens initiates signaling cascades at the membrane, within the cytosol or in the nucleus of receiving cells. Also, the consequences of the signalings driven by these morphogens have been well characterized. For example, activation of Wnt signaling cascades by Wnt morphogens confers a number of developmental processes such as dorsoventral body axis formation, mesoderm induction, anteroposterior neural patterning and heart development. Despite of well characterized mechanisms how morphogens affect intracellular signaling cascades and thus how they drive developmental processes, relatively little is known about the mechanisms how morphogens are secreted. Therefore, I carried out this study to understand how morphogens are secreted and how these processes are regulated. Here, I describe two proteins, Wntless and Rab3d, in the regulation of Wnt and Noggin secretions, respectively. Wnt signaling is implicated in a variety of developmental and pathological processes. The molecular mechanisms governing the secretion of Wnt ligands remain to be elucidated. Wntless, an evolutionarily conserved multi-pass transmembrane protein, is a dedicated secretion factor of Wnt proteins that participates in Drosophila embryogenesis. In the first part of this study, I showed that Xenopus Wntless (XWntless) regulates the secretion of a specific Wnt ligand, XWnt4, and that this regulation is specifically required for eye development in Xenopus laevis. Moreover, the Retromer complex is required for XWntless recycling to regulate XWnt4-mediated eye development. Inhibition of Retromer function by Vps35 morpholino (MO) resulted in various Wnt deficiency phenotypes affecting mesoderm induction, gastrulation cell movements, neural induction, neural tube closure, and eye development. Overexpression of XWntless led to the rescue of Vps35 MO-mediated eye defects, but not other deficiencies. These results collectively suggest that XWntless and the Retromer complex are required for the efficient secretion of XWnt4, facilitating its role in Xenopus eye development.Rab3d is a member of Ras-related small GTPase family of secretory Rab, Rab3. In the second part of this study, I showed that Xenopus Rab3d is expressed specifically in the anterior border of neural plate at neurula stage when neural plate converges and folds to initiate neural tube formation. Morpholino-mediated knockdown of Rab3d resulted in neurulation defects both in neural plate convergence and folding. Interestingly, perturbation of BMP signaling rescued neurulation defects of Rab3d morphants, suggesting that Rab3d inhibits BMP signaling during neurulation. By secretion assay in Xenopus animal cap, I found that Rab3d specifically regulates secretion of a BMP antagonist, Noggin but not Chordin and Wnts. I also showed that Rab3d is co-localized and co-immunoprecipitated with Noggin and this interaction is dependent on the GTP/GDP cycle of Rab3d. Together, these findings suggest that Rab3d-mediated secretion regulation of a BMP antagonist, Noggin, is one of the mechanisms of BMP antagonism during Xenopus anterior neurulation.