Title: Hematopoiesis and angiogenesis in the zebrafish
Description: Blood and blood vessels function in concert to provide oxygen, defense, and wound healing to the body. The blood lineages are generated by hematopoiesis, by which hematopoietic stem cells divide and differentiate to form the mature blood cells. Angiogenesis, remodeling of the vascular network, ensures that tissues are sufficiently vascularized and prevents aberrant blood vessel formation. The mechanisms of hematopoiesis and angiogenesis are highly conserved across vertebrate species and the zebrafish has been successfully used to study the genetic regulation and molecular signaling pathways of these complex processes. Erythropoiesis is the division and differentiation of erythroid precursors to form mature red blood cells. This process is modulated by the binding of erythropoietin ( epo ) to its cognate epo receptor ( epor ) on the surface of erythroid progenitors, which initiates a signaling cascade to direct their division and differentiation into erythrocytes. This thesis describes the cloning and functional characterization of the zebrafish epo and epor genes. Analysis of their expression revealed marked parallels between zebrafish and mammalian gene expression patterns. The results demonstrated that zebrafish epo expression was induced by anemia and hypoxia, overexpression of epo mRNA caused polycythemia, disruption of epor blocked erythropoiesis, and that there was a requirement for STAT5 in epo signaling. Together, these findings reveal the conservation of an ancient program that ensures proper red blood cell numbers under all conditions.

Angiogenesis requires the coordination of signaling pathways that regulate the shape and motility of endothelial cells. Small GTPases, (Rho Rae, Cdc42) and Arf translate extracellular stimuli into intracellular regulation of the actin cytoskeleton, and thus control polarity, shape, movement, and adhesion. The activities of Rho and Arf GTPases are regulated by GTPase activating proteins (GAPs). We identified a zebrafish mutant, grenache ( gre ), in which small vessels formed by angiogenesis are compromised, resulting in hemorrhage. Molecular cloning revealed a mutation in arap3 , which is a GAP for Arf and Rho GTPases, thus providing a means to coordinate multiple signaling pathways. We postulate that arap3 is important for mediating endothelial morphology, adhesion, or motility, and that abrogation of this coordination leads to leaky blood vessels and subsequent blood loss.