Genetic analysis of genes linked to lumen formation in the zebrafish vasculature

Formation of a functional vascular network is a crucial step in vertebrate early development. After formation of the first two major vessels, the dorsal aorta and the posterior cardinal vein, the vasculature expands through sprouting angiogenesis. Vascular network formation requires the interconnection of individual sprouts by the process of anastomosis. This process can be subdivided into different steps: initial filopodial contact, junction formation and lumen formation. With respect to lumen formation, two distinct mechanisms have been described: Transcellular lumen formation – driven by apical membrane invagination - and cord hollowing, which requires by cell rearrangements. It was proposed that during transcellular lumen formation, the growth of the invaginating apical membrane is dependent on vesicular trafficking, since punctae of the apical membrane marker mCherry-CAAX were seen to disperse in the growing apical membrane. It was shown that the observed vesicles colocalize with late endosomal/lysosomal markers Lamp2, Rab9 and Rab7, therefore, labeling them as part of the late endocytic/ lysosomal pathway. An essential step during transcellular lumen formation is the fusion between the apical membrane within the endothelial cell. Previous work in tracheal lumen formation in D. melanogaster identified the tethering factor staccato as a critical component of this process. In this study, we want to further investigate the role of late endocytic trafficking for lumen formation in the zebrafish vasculature. We therefore generated mutant lines for all three rab7 paralogues and the zebrafish homologue of stac, unc13d. Targeted mass spectrometry validated the isolated rab7 mutant alleles and showed that all wild-type protein and residual fragments thereof were absent in the respective mutant. rab7 single and double homozygous mutants are viable and fertile but triple homozygous rab7 mutants are lethal. Taken together, the phenotypic analysis of single and compound mutants showed, that rab7a and the newly described copy rab7bb are functionally redundant and that the third copy rab7ba seems to have a distinct function, potentially similar to what was proposed for mammalian rab7b. Furthermore, of the three rab7 genes in zebrafish, at least two provide maternal contribution and embryos maternal homozygous for these two alleles show phenotypes in the yolk of laid eggs. This Phenotype resembles what was previously reported in C. elegans. However, none of the mutants displayed any strong phenotypes linked to lumen formation or lumen fusion, but a conclusion on the role of this pathway cannot be drawn due to the early death of triple homozygous embryos, or the presence of additional unc13 genes. In order to be able to follow rab7 depleted endothelial cells and its role on lumen formation, inducible systems are required.