Morphogenesis of the zebrafish organizer region

The organizer region of vertebrate embryos has important patterning functions during gastrulation that help define the embryonic axis. The goal of this thesis was to determine the cell movements and behaviors that transform the zebrafish (Danio rerio ) organizer region during axis formation. Using timelapse confocal microscopy to examine vitally stained zebrafish embryos, it was determined that a superficial endodermal cell domain, the Non-involuting, Endocytic Marginal (NEM) cells, predicts the location of the zebrafish organizer. During gastrulation, NEM cells segregate from other deep cells and move in front of the blastoderm margin, where they remain throughout epiboly (and are called forerunner cells). Because of the dorsal location of NEM cells, their nuclear localization of beta-catenin, and their early segregation, it was hypothesized that NEM cells are specified by the Wnt signaling pathway. This was confirmed by finding that ectopic and expanded NEM cell clusters form in embryos treated with LiCl, a hyperdorsalizing agent that affects Wnt signaling by inhibiting GSK3 (inhibits beta-catenin). In order to understand cell movements occurring in other domains of the organizer region, cell populations in individual embryos were analyzed for their collective 3-dimensional movements. The results of this analysis support the view that involution is the predominant mechanism of hypoblast formation during zebrafish gastrulation. Regional patterns of convergence and extension movements of cells are also expressed during dorsal axis formation. Moreover, cells move in collective cellular flows that are specific to their regional positions in the embryo. The yolk syncytial layer (YSL) is involved in DV patterning and mesoderm induction in teleost embryos, but little was previously known about YSL morphogenesis. During late blastula and early gastrula stages, a directed movement of YSL nuclei away from the margin and toward the animal pole occurs. Starting at midgastrula stages, patterned movements of YSL nuclei begin, including extensive convergent-extension movements, which result in widespread and patterned nuclei flow patterns. Together, these studies show the zebrafish organizer region is sub-divided into multiple cellular domains that can be distinguished by their unique morphogenetic behaviors. These domains of cell and nuclei behavior are responsible for the formation of the zebrafish dorsal embryonic axis.