Morphogenetic mechanisms of blood vessel fusion in the Zebrafish embryo

The formation of a vascular network requires the connection and formation of a lumen between individual endothelial sprouts, a process called vessel fusion or anastomosis. In the vertebrate trunk of the zebrafish (Danio rerio), the intersegmental vessels (ISVs) develop by angiogenesis, i.e. the formation of new vessels by pre-existing vessels, in a conserved metameric manner, which allows the analysis of morphogenetic mechanisms of blood vessel development. From the dorsal aorta (DA) individual endothelial sprouts, led by tip cells and trailing stalk cells, grow between the somite boundaries dorsally towards the roof of the neural tube where they extend anterior and posterior. Neighboring extensions of sprouts fuse to each other and eventually form a continuous lumen, which results in the formation of a new vessel, the dorsal longitudinal anastomotic vessel (DLAV). In the past, many molecular key players of blood vessel formation have been identified and a number of studies described endothelial lumen formation in vivo. However, how individual cellular behaviors contribute to the process of vessel fusion have not yet been described. Thus, I engineered a transgenic zebrafish reporter system labeling endothelial cell-cell junctions by an EGFP-hZO1 (human Zonula Occludens-1 protein fused to enhanced green fluorescent protein) fusion protein. This reporter system allowed analysis of the dynamics of endothelial tight junctions during intersegmental vessel fusion in vivo and thus, the relative behavior of individual endothelial cells (ECs). We previously proposed that neighboring ISVs make contact to each other by filopodial extensions. This behavior is reflected by the de novo formation of point-like junctional complexes (i.e. Vascular endothelial cadherin (VE-cadherin) and ZO1) which elaborate into ring-like structures as the contacting sprout extensions enlarge their mutual surface, suggesting the formation of new apical membrane compartments. Following individual cell-cell junctions in the fusing ISVs and analyzing a novel apical marker, podocalyxin2 (Pdxl2), we confirmed the formation of apical membrane compartments during the initial phase of vessel fusion and deciphered two distinct cellular mechanisms of blood vessel fusion, a cord hollowing and membrane invagination mechanism, that result in the formation of different types of biological tubes. In cord hollowing, cell rearrangements of ECs in fusing neighboring sprouts result in the formation of a multicellular tube. Here, a tip cell forms two apical membrane compartments, one with a stalk cell and another one with a neighboring tip cell. These are then brought together by cell rearrangements and coalesce into one larger membrane compartment that eventually integrates into a continuous lumen within the ISV/DLAV. During membrane invagination, the membrane of a tip cell, adjacent to a preexisting lumen, undergoes apicobasal polarization and invaginates through its own cell body and coalesces with one of the previously formed apical membrane compartments at the sites of contact between neighboring tip cells, which results in the formation of a seamless tube.