Endo-lysosomal dynamics during transcellular lumen formation in the vasculature. Analysis of the dynamics of late endosomal, lysosomal compartments during transcellular lumen formation by high spatial and temporal resolution microscopy

The formation of a functional vasculature is a crucial step during vertebrate development. In order to transport blood, the initial vascular sprouts interconnect and lumenize to form a patent vascular network. One of the main morphogenetic mechanisms to form interconnected lumens is transcellular lumen formation, where the lumen pushes through a single cell. During transcellular lumen formation, the apical membrane surface is extended in a highly dynamic fashion and it is not known, which trafficking pathways and membrane compartments contribute to the extension of the invaginating membrane and to the fusion of the apical membranes. Previous analyses in our laboratory of the transgenic line Tg(kdrl:mCherry-CAAX)S916 have shown that mCherry-CAAX dot-like structures elongate along the apical membrane suggesting a fusion of these dot-like structures. These dot-like structures are presumably vesicles or membrane compartments. Rab proteins are major regulators of membrane and protein trafficking. By expressing GFP-tagged Rab proteins in the vasculature of the zebrafish embryo and using high resolution in vivo imaging I showed that the mCherry-CAAX dot-like structures are labelled with late endosomal, lysosomal markers (Rab7a, Rab9a, Rab9b). I generated multiple new transgenic lines, which allowed me, in combination with a transgenic line expressing a fluorescently tagged lysosome-associated membrane protein TgBAC(lamp2- RFP)pd1117, to analyse the dynamics of late endosomal, lysosomal compartments during transcellular lumen formation. The results strongly suggest that late endosomal, lysosomal membrane compartments contribute to the apical membrane during transcellular lumen extensions. Further I have shown the first evidence that a Rab9b- positive, Lamp2-positive membrane compartment resides at the fusion site of the two apical membranes during transcellular lumen fusion in the zebrafish vasculature. These results suggest that a late endosomal, lysosomal or secretory lysosome might be important for the fusion of two apical membranes in transcellular lumen fusion in the zebrafish vasculature, indicating a similar mechanism as in transcellular lumen fusion in the trachea of Drosophila melanogaster previously described by Caviglia et al. (2016).