Date of Award
Master of Science
Genetics, Development and Cell Biology
Jeffrey J. Essner
Proper development of complex organ systems depends on the formation of vascular networks to transport fluids and exchange gases and metabolites. Understanding the processes that guide vascular network and endothelial tube formation is imperative because these mechanisms are also likely used to maintain homeostasis in the vessel wall. Most of what we understand about the molecular mechanisms that guide vascular tube formation is from knock down studies performed in vitro. These studies have shown that vesicles fuse to form a primary vessel lumen. Here, we focus on two small GTPases, Rab5c, which was identified in screen as being required for endothelial tubulogenesis in zebrafish, and Rac1, which is required for endothelial lumen formation in vitro. We have created zebrafish transgenic lines that label these proteins with the enhanced green fluorescent protein (EGFP) to understand their role in vascular formation in early embryonic development in vivo. EGFP fusion with Rab5c and Rac1 along with co-injection with Tol2 transposase has allowed us to establish transgenic zebrafish to follow and observe protein the dynamics of Rab5c and Rac1 localization during the forming lumen. By crossing these EGFP lines to an flk1a:mcherry lines, which expresses the red fluorescent protein, mcherry, in vascular endothelial cells, we determined where the proteins localize during the forming vasculature. Confocal microscopy was used to create time-lapse movies when the embryos were at 30 hours post fertilization (hpf). These images show that the Egfp-rab5c protein likely localizes to forming lumen and putative cellular junctions in the intersegmental vessels. The UAS:egfp-rac1 transgenic line shows consistent GFP localization to the putative luminal or apical membranes. Overall, this project defines critical cellular components that regulate vesicle transport and adherens junction remodeling during tube formation. This knowledge can then lead to discovery of new targets for novel angiogenic therapies to ultimately alleviate cancer and vascular diseases.
Ellen Elizabeth Tisdale
Tisdale, Ellen Elizabeth, "Analysis of blood vessel tube formation using Rab and Rac fusion proteins in zebrafish" (2015). Graduate Theses and Dissertations. 14434.