"In vivo" analysis of junctional dynamics underlying angiogenic cell behaviors

The blood circulatory system delivers nutrients and oxygen to tissues and organs. Moreover, the circulatory system retains high plasticity throughout an organism’s life, which is important for physiological processes, such as wound healing, but also during pathological processes, such as tumor growth.
Blood vessels can sprout from existing ones, a process called angiogenesis, to create a more branched network that reaches avascular and the most distal parts of an animal body. The morphogenetic processes that underlie angiogenesis can be studied at cellular resolution in the zebrafish embryo. Moreover, the fast development of the embryo allows to follow these processes in real-time. In the trunk, where metameric vessels sprout from the dorsal aorta, cells migrate collectively in a hierarchy defined by a leading endothelial cell at the tip of the sprout and several following cells, called stalk cells. Once the tip cells reach the level of the dorsal neural tube, they extend laterally and initiate the anastomosis process with tip cells from the neighboring segments. Eventually, cells rearrange and form a patent lumen, which allows blood circulation.
Angiogenic sprout outgrowth and anastomosis involve complex cell behaviors and cell-cell interactions, which need to be precisely orchestrated. The molecular mechanisms underlying these cellular activities are not known. However, proteins that mediate endothelial specific cell adhesion are good candidates to promote concerted cell behaviors. The goal of my thesis was (i) to characterize the cell shape changes that occur during angiogenic sprouting, (ii) to analyze the function of VE-cadherin in this process and (iii) to analyze the role of VE-cadherin and Esama, as candidate proteins, in the initiation of endothelial cell-cell interaction at the onset of vascular anastomosis.
I found that two major cell behaviors contribute to angiogenic sprouting. While cell migration is predominantly used by the tip cell, elongation of the stalk is mainly achieved by extensive stalk cell elongation, rather than by pulling forces exerted by the tip cell.
VE-cadherin, which is the major component of adherens junction of endothelial cells, is required for concerted angiogenic cell junctional elongation. The absence of VE-cadherin in stalk cells leads to a disorganized cortical F-actin network, which reflects the elongation defects. Furthermore, the loss of VE-cadherin function can be phenocopied by inhibiting actin polymerization.
Anastomosis is initiated by filopodial contacts between endothelial cells. The formation of these contacts is thought to be mediated by endothelial specific adhesion molecules, which provide adhesion as well as cell type specificity. We have previously shown that VE-cadherin plays an important role in anastomotic contact formation. However, our observation that tip cells can still generate contacts in the absence of VE-cadherin, prompted us to investigate the role of a second endothelial-specific adhesion molecule, Esama, during anastomosis.
In my thesis I generated a targeted mutation in the zebrafish esama gene using TALEN technology and started to analyze the loss of function. Embryos mutant for esama are viable and do not show major vascular defects, except for small, transient gaps in junctional rings. However, zebrafish embryos lacking both, VE-cadherin and Esama, show frequent detachments of stalk from tip cells, ineffective cell-type specific recognition and strongly protrusive cell morphologies. All together, the phenotypes of the ve-cadherin mutants are aggravated by the simultaneous absence of Esama. Moreover, the double mutant shows junctional discontinuities, seen as big gaps within the junctional rings between stalk cells.
Our results support a model for angiogenic sprout elongation by cell shape changes orchestrated by VE-cadherin. VE-cadherin connects the actin cytoskeletons of neighboring stalk cells and drives the cell elongation by localized actin polymerization at the edges of the elongating junctions. Esama and VE-cadherin have partly overlapping functions during angiogenic sprouting and anastomosis. Both proteins are required for endothelial contact formation during anastomosis, but also for the maintenance of structural integrity during angiogenic sprouting.