The LIM-homeodomain transcription factor Islet2a promotes angioblast migration.

Angioblasts of the developing vascular system require many signaling inputs to initiate their migration, proliferation and differentiation into endothelial cells. What is less studied is which intrinsic cell factors interpret these extrinsic signals. Here, we show the Lim homeodomain transcription factor islet2a (isl2a) is expressed in the lateral posterior mesoderm prior to angioblast migration. isl2a deficient angioblasts show disorganized migration to the midline to form axial vessels and fail to spread around the tailbud of the embryo. Isl2a morphants have fewer vein cells and decreased vein marker expression. We demonstrate that isl2a is required cell autonomously in angioblasts to promote their incorporation into the vein, and is permissive for vein identity. Knockout of isl2a results in decreased migration and proliferation of angioblasts during intersegmental artery growth. Since Notch signaling controls both artery-vein identity and tip-stalk cell formation, we explored the interaction of isl2a and Notch. We find that isl2a expression is negatively regulated by Notch activity, and that isl2a positively regulates flt4, a VEGF-C receptor repressed by Notch during angiogenesis. Thus Isl2a may act as an intermediate between Notch signaling and genetic programs controlling angioblast number and migration, placing it as a novel transcriptional regulator of early angiogenesis.

An α-smooth muscle actin (acta2/αsma) zebrafish transgenic line marking vascular mural cells and visceral smooth muscle cells.

Mural cells of the vascular system include vascular smooth muscle cells (SMCs) and pericytes whose role is to stabilize and/or provide contractility to blood vessels. One of the earliest markers of mural cell development in vertebrates is α smooth muscle actin (acta2; αsma), which is expressed by pericytes and SMCs. In vivo models of vascular mural cell development in zebrafish are currently lacking, therefore we developed two transgenic zebrafish lines driving expression of GFP or mCherry in acta2-expressing cells. These transgenic fish were used to trace the live development of mural cells in embryonic and larval transgenic zebrafish. acta2:EGFP transgenic animals show expression that largely mirrors native acta2 expression, with early pan-muscle expression starting at 24 hpf in the heart muscle, followed by skeletal and visceral muscle. At 3.5 dpf, expression in the bulbus arteriosus and ventral aorta marks the first expression in vascular smooth muscle. Over the next 10 days of development, the number of acta2:EGFP positive cells and the number of types of blood vessels associated with mural cells increases. Interestingly, the mural cells are not motile and remain in the same position once they express the acta2:EGFP transgene. Taken together, our data suggests that zebrafish mural cells develop relatively late, and have little mobility once they associate with vessels.

ßPix plays a dual role in cerebral vascular stability and angiogenesis, and interacts with integrin αvß8.

The growth of new blood vessels by angiogenesis and their stabilization by the recruitment of perivascular mural cells are thought to be two sequential, yet independent events. Here we identify molecular links between both processes through the ßPix and integrin α(v)ß(8) proteins. Bubblehead (bbh) mutants with a genetic mutation in ßPix show defective vascular stabilization. ßPix is a guanine nucleotide exchange factor and scaffold protein that binds many proteins including Git1, which bridges ßPix to integrins at focal adhesions. Here we show that the ability of ßPix to stabilize vessels requires Git1 binding residues. Knockdown of Git1 leads to a hemorrhage phenotype similar to loss of integrin α(v), integrin ß(8) or ßPix, suggesting that vascular stabilization through ßPix involves interactions with integrins. Furthermore, double loss of function of ßPix and integrin α(v) shows enhanced hemorrhage rates. Not only is vascular stability impaired in these embryos, but we also uncover a novel role of both ßPix and integrin α(v)ß(8) in cerebral angiogenesis. Downregulation of either ßPix or integrin α(v)ß(8) results in fewer and morphologically abnormal cerebral arteries penetrating the hindbrain. We show that this is coupled with a significant reduction in endothelial cell proliferation in bbh mutants or integrin α(v)ß(8) morphants. These data suggest that a complex involving ßPix, GIT1 and integrin α(v)ß(8) may regulate vascular stability, cerebral angiogenesis and endothelial cell proliferation in the developing embryo.