Morphogenetic Domains and Signaling Centers in Zebrafish Organizer Endoderm

University Of Washington, Seattle WA

Investigators

Abstract

The goal of this study is to determine the morphological transformations and inductive signaling functions of Zebrafish Organizer Endoderm during embryonic axis formation. The zebrafish Nieuwkoop Center (inducer of dorsal cell fate in the Organizer Region), as well as a putative signaling center involved in head induction, are thought to reside in the dorsal Yolk Syncytial Layer (YSL) (i.e. the dorsal cortex) of the zebrafish embryo's giant endodermal yolk cell. During blastulation and gastrulation, several hundred YSL nuclei within the zebrafish syncytial yolk cell undergo highly-organized morphogenetic movements. These movements may serve to spatially transform and translocate early-acting inductive signaling centers located within the dorsal YSL. Laser photoablation will be used to selectively inactivate specific domains of fluorescently-labeled dorsal YSL nuclei, to determine whether putative signaling centers in the dorsal YSL actually induce and/or maintain gene expression in the overlying zebrafish blastoderm Actomyosin-based contractility within the YSL may serve to drive directional flows of (i) YSL nuclei, (ii) the YSL plasma membrane, as well as (iii) mesendodermal cells that are directly attached to the YSL plasma membrane. Cytoskeletal inhibitors will be applied to the YSL to test for these putative mechanical interactions. In separate experiments, the blastoderm will be mechanically removed from the yolk cell to determine whether the yolk cell is capable of expressing its characteristic morphogenetic domains without inductive or mechanical influences from the blastoderm. These combined experiments will determine the extent to which the morphogenesis of the endodermal yolk cell is coupled to the overlying blastoderm. The morphogenesis of the zebrafish yolk cell will also be examined in double mutant embryos that are deficient in cellular endoderm and dorsal mesoderm. By studying YSL morphogenesis in mutants with these specific genetic deficiencies, altered morphogenetic movements of YSL nuclei may be identified and linked to failed gene expression. NEM/forerunner cells, a small endocytic cellular domain derived from the putative zebrafish Nieuwkoop Center, have recently been implicated in the establishment of right-left asymmetry in the zebrafish embryo. Experiments will be performed to determine whether secreted signals from the dorsal YSL and/or Eph/ephrin interactions are involved in the endocytic and morphogenetic behaviors of the endodermal NEM/forerunner cell cluster. The results of the above project should reveal (1) the sequence of endodermal cell behaviors that are involved in the morphological transformation the zebrafish dorsal axis, and (2) whether the signaling activities of the dorsal YSL and associated NEM/forerunner cells play roles in organizing zebrafish morphogenesis. Resolving these issues will contribute to a fundamental understanding of the sequence of endodermal cell behaviors involved in the construction of a vertebrate Organizer Region, as well as the restructuring of germ layers that lie near the Organizer Region during the course of gastrulation and segmentation.