Nodal-related signaling during zebrafish embryogenesis

I describe the Nodal-related family of signaling molecules that have been implicated in mesodermal and neural patterning, and left-right asymmetry, in mouse, frog, and chicken embryos. My studies focus on the isolation and characterization of one zebrafish nodal orthologue, squint (sqt). The early expression pattern of sqt begins dorsally in both the embryonic and extraembryonic tissue. Overexpression of sqt in whole embryos expands or duplicates axial cell fates. Furthermore, Sqt overexpression, exclusively in the extraembryonic yolk syncytial layer (YSL), causes broadened or duplicated dorsal mesodermal marker gene expression in the overlying blastoderm. Additionally, the inductive properties of Sqt are compared to another fish Nodal-related ligand, Cyc. Sqt is an efficient inducer of organizer-type mesendodermal markers, while Cyc induces both mesodermal and neural fates. Additionally, using cyc;sqt double mutants, I describe the process of neural posteriorization/transformation, which is essential for anterior-posterior (A-P) regionalization of the vertebrate central nervous system (CNS). I explore the molecular identity of the influence present in lateral mesodermal precursors that are maintained in Nodal-deficient embryos, which exerts a posteriorizing effect on anterior neural tissue. Employing a variety of genetic situations that vary the extent of mesendodermal precursor formation, I find that expression of wnt8, a previously implicated neural transformer, is necessary for posteriorization. Moreover, specific inhibition of wnt8 abrogates posteriorization in vivo. My findings provide strong evidence that Wnt8 acts in lateral mesendodermal precursors, independent of Sqt and Cyc, as an essential component of the neural transformation process. Lastly, I define cis-acting regulatory regions within the sqt upstream region that drive GFP reporter gene expression in the endogenous sqt expression domains in transient transgenic assays. A ?9 kb segment upstream of the start ATG drives GFP throughout the extraembryonic yolk syncytial layer (YSL), a region of the embryo shown to induce overlying blastomeres to adopt a mesendodermal fate, and in deep and superficial cells of the marginal zone. Using standard deletion analyses, we narrowed the region mostly responsible for YSL and deep marginal blastodermal expression to a 1 kb fragment (?1.3 to ?0.3 kb) upstream of the translational start site.