The cardiovascular system is one of the earliest organ systems to form in the developing embryo. Development of blood vessels is a strictly regulated process that is crucial for embryonic development as well as for organ functionality throughout the life of an organism. The circulatory system is necessary for the transportation of fluid, oxygen, metabolites, waste products, hormones, and immune cells and cytokines throughout the body. These functions cannot be carried out without proper formation of blood and lymphatic vessels. The focus of this dissertation is increasing the understanding of the mechanisms and pathways necessary for blood and lymphatic vessel development.
Previous studies have implicated mixed models of de novo vasculogenesis and angiogenesis in the development of blood vessels in organs. These studies, carried out in mouse and chick embryos, lack the ability to track vessel development in live embryos and therefore identify the source of cells forming these vessels. Here we identify a novel mechanism of organ vascularization in which endothelial cells in existing vessels migrate to form new vessels. We utilize zebrafish due to the rapid development, transparent embryos, and amenability to genetic manipulation to study vascularization of the embryonic gut tube. Vegfaa expression in the presumptive endoderm drives ventral migration of endothelial cells out of the posterior cardinal vein (PCV), which then coalesce to form the sub-intestinal vein (SIV) and supra-intestinal artery (SIA). These results represent a novel mechanism of organs vascularization that is likely to occur in other organs and in other vertebrate organisms.
We also investigate the role of ETS transcription factor, Etv2, in lymphatic development/lymphangiogenesis. Prior studies have demonstrated the requirement of Vegfc/Vegfr3 signaling in lymphatic development, however questions about the process remain, including how this signaling is regulated regarding lymphangiogenesis. Etv2 has previously been identified as a master regulator of vasculogenesis. In this study we demonstrate that Etv2 is required for lymphangiogenesis and directly regulates vegfr3 expression in lymphatic development. Knockdown of etv2 expression after vasculogenesis and primary angiogenesis results in a failure to develop the primary lymphatic vessel, the thoracic duct, as well as reduction in numbers of lymphatic progenitors. These results highlight a novel role for Etv2 in later development.
Finally, we utilized single cell RNA sequencing to investigate the heterogeneity of endothelial cells including how and when these cells acquire their specific identities. We identify several unexplored domains of etv2 expression. Prior studies have suggested that arterial and venous precursors acquire their vessel specific identity prior to the onset of circulation, but exactly when this occurs is still unknown. Here, we also identify co-expression of arterial and venous specific genes in individual cells. This study highlights the broad heterogeneity of endothelial cells and suggests that these cells are highly plastic during early development.
Together these studies highlight novel mechanisms and pathways in the development of the circulatory system. These discoveries represent identification of novel targets for the development of potential therapies for vascular defects and disease as well as allowing for possible regeneration of defective vascular tissues.