Single cell analysis of endothelial morphogenesis in vivo

Abstract

Vessel formation has been extensively studied at the tissue level, but the difficulty in imaging the endothelium with cellular resolution has hampered study of the morphogenesis and behavior of endothelial cells (ECs) in vivo. We are using endothelial-specific transgenes and high-resolution imaging to examine single ECs in zebrafish. By generating mosaics with transgenes that simultaneously mark endothelial nuclei and membranes we are able to definitively identify and study the morphology and behavior of individual ECs during vessel sprouting and lumen formation. Using these methods, we show that developing trunk vessels are composed of ECs of varying morphology, and that single-cell analysis can be used to quantitate alterations in morphology and dynamics in ECs that are defective in proper guidance and patterning. Finally, we use singlecell analysis of intersegmental vessels undergoing lumen formation to demonstrate the coexistence of seamless transcellular lumens and single or multicellular enclosed lumens with autocellular or intercellular junctions, suggesting that heterogeneous mechanisms contribute to vascular lumen formation in vivo. The tools that we have developed for single EC analysis should facilitate further rigorous qualitative and quantitative analysis of EC morphology and behavior in vivo.

Auto-fusion and the shaping of neurons and tubes

“…These vacuoles fuse, enlarge and eventually connect to similar vacuoles in adjacent cells through exocytosis or anastomosis. Some imaging studies in zebrafish have supported this mechanism [ 31 , 32 ].…”

Cell behaviors and dynamics during angiogenesis

“…Subsequent studies showed that such sprouts often exhibit differing cellular architectures, making conclusions about individual cells and how they coordinate lumen formation rather difficult (Blum et al, 2008). Using morespecific cell markers, a recent study reinvestigated this and concluded that heterogeneous mechanisms contribute to lumen formation in ISVs in vivo (Yu et al, 2015). This study provides further evidence for vesicle (vacuole) formation and the generation of an intracellular lumen, but also observed the formation of a lumen between paired ECs, that is most likely generated by a mechanism of cord hollowing, similar to that observed during anastomosis of ISVs (Herwig et al, 2011;Lenard et al, 2013;see below).…”

Time to make the doughnuts: Building and shaping seamless tubes

“…1B) [11, 12]. In zebrafish, where live imaging has been possible, some seamless tubes are transient, developmental precursors to larger multicellular tubes or to pruned vessels [13–16]. However, in mammals, TEM studies suggested that as many as 30% – 50% of the tubes in capillary beds of adults can be seamless tubes [10].…”