Establishment of cell polarity in zebrafish: Role of Staufen and GRB2 in cell migration

Abstract

I have tried to investigate mechanisms by which cells establish polarity in zebrafish for my thesis study. Cell polarity is partly dependent on asymmetric localization of molecules. In zebrafish, the role of localized maternal factors influencing embryo development is poorly understood. Staufen is an RNA binding protein, required for correct localization of transcripts required for specification of the anterior-posterior axis and the germ line in D. melanogatser. I investigated the role of Staufen proteins during zygotic development of zebrafish. I isolated and characterized stau1 and stau2 cDNAs from zebrafish. I tested the function of stau1 and stau2, using antisense morpholino knockdown and dominant negative peptide interference studies. Staufen proteins are not required for patterning of the germ layers during zygotic development of zebrafish. Interference of Staufen by morpholinos or disruption of function of Staufen proteins by dominant negative peptides abolishes maintenance of germ cell specific transcripts vasa or nanos1 during later development. In the absence of Staufen proteins, PGCs do not migrate properly and undergo cell death. These results suggest that zebrafish Staufen1 and Staufen2 are required for maintenance and survival of primordial germ cells (PGCs). Staufen proteins are also required for the survival of CNS neurons in zebrafish. We have thus identified a new function for Staufen proteins in PGC maintenance and survival, which is distinct from its known role in germ line specification in D. melanogatser. To identify additional factors that may be involved in localization in zebrafish egg, we developed a screen to over-express zebrafish ovary specific cDNAs in the yeast (S. Pombe) and analyzed for cell polarity (b tb shape, bent shape) defects. Grb2, an SH3-SH2-SH3 domain containing protein, was one of the candidates identified from the screen. Knockdown of grb2 by morpholinos, showed no phenotype. Over expression of dominant negative Grb2 peptides in zebrafish embryo causes defects in convergent extension cell movements during gastrulation. The convergent extension defects caused by the dominant negative Grb2 peptides can be rescued by co-injection with wild type grb2 mRNA. Disruption of Grb2 proteins by dominant negative peptides causes rounded cell shape of enveloping layer (EVL) and the prechordal plate (PCP) cells. Actin accumulation is abolished in the yolk and the PCP cells, affecting morphogenesis of the EVL or the PCP. The PCP cells do not undergo normal convergence and extension cell movements in Grb2 disrupted embryos. The EVL cell morphology influences delay in epiboly in Grb2 disrupted embryos. As a result of improper cell movements during gastrulation, the organization of body plan is abnormal in Grb2 dominant negative peptide injected embryos in zebrafish. Thus grb2 may regulate cell movements by modulating cell shape.