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Analysis of flow regulated angiogenesis during zebrafish development

Final Report Summary - ZEBRAFISHANGIO (Analysis of flow regulated angiogenesis during zebrafish development)

My ERC proposal aimed at an understanding of the processes that regulate angiogenesis in response to changes in blood flow and therefore hemodynamic cues. We analyzed the function of the blood flow regulated chemokine receptor cxcr4a and its ligand cxcl12. We generated mutants in cxcl12b and compared their phenotypes with those of the previously generated cxcr4a mutants. Our studies showed that both genes were critically involved during the formation of the brain vasculature. Importantly, our work identified an important hemodynamic feedback loop regulating cxcr4a expression, thereby ensuring proper arterial-venous patterning in the brain. We furthermore showed in collaborative work that cxcr4a is required during the formation of lymphatic blood vessels and in coronary arteries. We then set out to understand how blood vessels form during tissue regeneration. In order to do so, we analyzed blood vessel sprouting during zebrafish fin regeneration. We first developed an intubation-based anesthesia protocol that allowed us to continuously image adult zebrafish for up to two days. This work provided evidence that newly forming arteries are derived from venous endothelial cells and that these cells undergo specific migratory behaviors in order to ensure proper arterial morphogenesis. Importantly, we went on to show that these migratory behaviors critically depended on cxcr4a signaling. Comparing blood vessel formation in the embryo with regenerative settings revealed that arteries usually sprout from pre-existing veins and that the proper pathfinding of the new blood vessel sprouts depends on cxcr4a signaling. Thus, our work greatly advances our understanding of blood vessel development in different settings, both in the embryo and during tissue regeneration.

Our analysis on the influence of blood flow on vascular patterning revealed that the removal of unwanted blood vessel connections during development, also referred to as pruning of blood vessels or regression, was greatly influenced by changes in hemodynamics. We investigated the pruning of the ocular vasculature and for the first time showed the cellular dynamics during this process. Our imaging approach revealed that initially multicellular blood vessels rearrange to form a unicellular tube before regression. We hypothesize that this rearrangement reduces the likelihood of vascular leakage. We furthermore show that differences in blood flow between neighboring vessels can induce blood vessel pruning, while an absence of blood flow per se does not induce this behavior in blood vessels. Together, our studies elucidate several key morphogenetic principles during blood vessel development and regression that are shared between embryonic and regenerative processes and identified the chemokine receptor cxcr4a as an important player in regulating endothelial cell migratory behaviors. Additionally, we established technological advances concerning long-term anesthesia that will facilitate the imaging of regenerative processes in adult zebrafish.