Abstract
Vascular patterning is a highly organized process in angiogenesis. It involves precise control of endothelial cell (EC) migration, branching, and connection. Based on previous studies, directed EC migration and branching depends on correct protrusions of ECs. However, the underlying mechanism of directional EC protrusions is still unclear. Here we propose a novel secreted protein, THSD7A, which is expressed in the zebrafish developing neuronal system, contributes to EC protrusion regulation. From our data, knockdown of THSD7A expression by injecting morpholino disrupted intersegmental vessel (ISV) sprouting of zebrafish at 30 hour-post-fertilization (hpf) stage. To acquire more information, we further examined the behavior and morphology of endothelial tip cells on the ISVs of Thsd7a-knockdown zebrafish by confocal microscopy. The data showed that morpholino knockdown of THSD7A slowed down ISV growth by misleading endothelia cells to stay longer in early stage of dynamic protrusion process, in which ECs extended numerous protrusions like a fan-shape. This phenotype implied malfunction of retracting excessive EC protrusions. Besides, ECs of ISVs formed ectopic EC branching and false connection, resulting in complex ISV pattern in 34hpf zebrafish. On the other hand, an in vitro system, 3D collagen angiogenesis assay, was constructed for investigating the specific role of THSD7A. The data from in vitro 3D collagen angiogenesis assay proved that THSD7A was related to angiogenic patterning, including EC protrusions and connections. Statistic analysis also implied that THSD7A played a dual role in angiogenesis depending on its concentration. Taken the in vivo and in vitro data together, this work suggests that THSD7A may mediate EC protrusion during angiogenic sprouting and plays an essential role in angiogenic patterning as a regular of EC branching and connection.