Investigation of Apelin signaling as a regulator of vascular morphogenesis

This work addresses the role of the Apelin signaling pathway in blood vessel development. In order to form the complex vascular network required to supply all organs and tissues with oxygen and nutrients, endothelial cells must leave existing vessels. These endothelial cells migrate along chemotactic and haptotactic signals and subsequently form new vessels. This process is called sprouting angiogenesis. During angiogenesis, specialized cells (tip cells) at the tip of the new vessel lead the way, while so-called "stalk cells" follow the tip cells and form the new lumen of the vessel. The Apelin signaling pathway has been shown to be involved in embryonic, regenerative and pathogenic angiogenesis. In zebrafish, the Apelin signaling pathway consists of two ligands (Apln and Apela) that bind to the G-protein coupled receptors Aplnra and Aplnrb. In particular, Apln and Aplnrb play a role in angiogenesis. Despite these findings, however, little is known about the intracellular signals by which the Apelin signaling pathway controls dynamic cell behavior during angiogenesis. Both the ligand Apln and the receptor are expressed by newly forming vessels, and Apln is particularly enriched in tip cells. Therefore, it was hypothesized that vascular Apln as an autocrine signal is a regulator of angiogenesis. In this work, we investigated the molecular and cellular mechanisms by which the Apelin signaling pathway controls cell behavior during vessel formation. For this purpose, we used embryonic zebrafish as a model for angiogenesis. By generating novel transgenic reporter lines, we were able to show that the ligand Apln is not only expressed in blood vessels, but also in neural stem cells in the dorsal neural tube. The combination of the new reporter lines with high-resolution time-lapse imaging of living embryos revealed that the tip cells elongate towards the apln-expressing neural stem cells and then migrate along them. These behaviors are lost in both apln and aplnrb mutants, in which the tip cells have a truncated morphology. To disprove the dogma that autocrine Apelin from the vasculature controls angiogenesis, we performed rescue experiments using newly generated transgenic lines. We were able to show for the first time that paracrine Apln from neural stem cells controls vascularization. During angiogenesis, tip cells form many dynamic membrane extensions (filopodia). These act like antennae to perceive signals from their environment and thereby control vascular growth. A detailed comparison of the cellular behavior of tip cells during vessel formation in wild-type and in aplnrb mutants was performed by high-resolution time-lapse imaging of the endothelial membrane and the cytoskeleton of transgenic embryos. Here we could show that tip cells form specialized protrusions that arise from filopodia and are crucial for cell migration when stimulated by apln from neural stem cells. We were thus not only able to further investigate the effect of the Apelin signaling pathway on vessel formation but were also able to describe the formation of a novel membrane protrusion induced by Apelin signaling that makes tip cell migration more efficient. Furthermore, by analyzing novel biosensors, we were able to investigate signal transduction at the single cell level in real time in living organisms. This enabled us to describe a positive regulation of ERK and PI3K enzyme activity in tip cells by the Apelin signaling pathway. By studying these biosensors, we also observed that tip cells exhibit higher ERK activity than stalk cells. This cell behavior is preceded by asymmetric cell division of tip cells. In aplnrb mutants, we were able to show that instead of asymmetric cell division, symmetric cell division occurs, whereby the higher ERK activity in tip cells is also lost compared to stalk cells. This "equalization" of tip cells and stalk cells means that the newly forming vessel always has alternating tip cells at the tip and no tip cell can assert itself. This results in very inefficient vessel growth. In summary, we were not only able to disprove the dogma of the autocrine function of apelin, but also demonstrated a new way of neurovascular communication through apelin signals that controls the vascularization of the neural tube. These results provide a deeper understanding of angiogenesis and tip cell biology, which may contribute to the development of new therapies for cardiovascular diseases.