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Blood vessels in different vascular beds vary in size, which is essential for their function and fluid flow along the vascular network. Molecular mechanisms involved in the formation of a vascular lumen of appropriate size, or tubulogenesis, are still only partially understood. Src homology 2 domain containing E (She) protein was previously identified in a screen for proteins that interact with Abelson (Abl)-kinase. However, its biological role has remained unknown. Here we demonstrate that She and Abl signaling regulate vessel size in zebrafish embryos and human endothelial cell culture. Zebrafish she mutants displayed increased endothelial cell number and enlarged lumen size of the dorsal aorta (DA) and defects in blood flow, eventually leading to the DA collapse. Vascular endothelial specific overexpression of she resulted in a reduced diameter of the DA, which correlated with the reduced arterial cell number and lower endothelial cell proliferation. Chemical inhibition of Abl signaling in zebrafish embryos caused a similar reduction in the DA diameter and alleviated the she mutant phenotype, suggesting that She acts as a negative regulator of Abl signaling. Enlargement of the DA size in she mutants correlated with an increased endothelial expression of claudin 5a (cldn5a ), which encodes a protein enriched in tight junctions. Inhibition of cldn5a expression partially rescued the enlarged DA in she mutants, suggesting that She regulates DA size, in part, by promoting cldn5a expression. SHE knockdown in human endothelial umbilical vein cells resulted in a similar increase in the diameter of vascular tubes, and also increased phosphorylation of a known ABL downstream effector CRKL. These results argue that SHE functions as an evolutionarily conserved inhibitor of ABL signaling and regulates vessel and lumen size during vascular tubulogenesis.

Intracranial aneurysms (IAs) represent a major clinical concern due to their risk of rupture and the resulting morbidity and mortality. Both environmental and genetic factors contribute to IA susceptibility, yet the genetic causes of IA remain poorly understood. We previously identified several single nucleotide variants (SNVs) in collagen XXII ( ) in affected individuals with IA. However, the functional impact of these variants has not been determined, and it remains unclear whether and how they increase IA susceptibility. Here, we tested the functional effect of these variants in a zebrafish embryo model. Inducible overexpression of six human SNVs increased the incidence of cranial hemorrhage in zebrafish embryos, while overexpression of wild-type had no significant effect. Overexpression of DNA construct encoding COL22A1 P989L variant disrupted intracranial vascular architecture, leading to reduced vessel length, altered vascular surface parameters, and abnormal arterial patterning. Overexpression of the P989L SNV also caused pronounced vascular leakage, reduced pericyte number, and decreased expression of the tight junction proteins Claudin-5 and ZO-1. P989L SNV overexpression was also associated with increased expression of the endoplasmic reticulum stress marker . In silico modeling suggested that the P989L variant likely perturbs triple-helix formation in COL22A1, thereby causing protein misfolding and compromising its function. Together, these findings demonstrate the deleterious effects of IA-associated variants on vascular function and stability and suggest that these variants may increase the incidence of IA in humans.

The mechanisms regulating the formation of organ-specific vasculature are still poorly understood. We have previously identified a population of late-forming endothelial progenitor cells in zebrafish embryos (termed secondary vascular field, SVF), which emerge from the lateral plate mesoderm after 24 hpf stage, after blood circulation has been initiated. Here we investigate the functional role of SVF cells and the molecular mechanisms that govern the emergence of these SVF cells and their contribution to vasculature. We identified that the bHLH transcription factor Hand2 and Junctional Adhesion Molecule Jam2b are expressed in the SVF-forming region and are required for the emergence of SVF cells in zebrafish embryos. Time-lapse imaging and based lineage tracing showed that SVF cells serve as the major source of the intestinal vasculature, including the supraintestinal artery (SIA) and subintestinal vein (SIV), which are subsequently remodeled to provide the blood flow to many internal organs. To analyze the functional role of and a related gene in vascular development, we generated double maternal-zygotic mutants, which display a greatly reduced number of SVF cells and show defects in the intestinal vasculature development. Further analysis showed that functions in the SVF-forming region upstream of and is required to induce expression of transcription factor a known master regulator of vasculogenesis. In summary, our results identify new roles for Jam2 signaling and Hand2 function in the emergence of organ-specific vascular progenitors. The presence of similar progenitors in mammalian embryos suggests that this mechanism is evolutionarily conserved.

Blood vessels in different vascular beds vary in lumen diameter, which is essential for their function and fluid flow along the vascular network. Molecular mechanisms involved in the formation of a vascular lumen of appropriate size, or tubulogenesis, are still only partially understood. protein was previously identified in a screen for proteins that interact with Abelson (Abl)-kinase. However, its biological role has remained unknown. Here we demonstrate that She and Abl signaling regulate vascular lumen size in zebrafish embryos and human endothelial cell culture. Zebrafish mutants displayed increased endothelial cell number and enlarged lumen size of the dorsal aorta (DA) and defects in blood flow. Vascular endothelial specific overexpression of resulted in a reduced diameter of the DA lumen, which correlated with the reduced arterial cell number and lower endothelial cell proliferation. Chemical inhibition of Abl signaling in zebrafish embryos caused a similar reduction in the DA diameter and alleviated the mutant phenotype, suggesting that She acts as a negative regulator of Abl signaling. Enlargement of the DA lumen in mutants correlated with an increased endothelial expression of and ), which encode proteins enriched in tight junctions. Inhibition of expression partially rescued the enlarged DA in mutants, suggesting that She regulates DA lumen size, in part, by promoting expression. SHE knockdown in human endothelial umbilical vein cells resulted in a similar increase in the diameter of vascular tubes, and also increased phosphorylation of a known ABL downstream effector CRKL. These results argue that SHE functions as an evolutionarily conserved inhibitor of ABL signaling and regulates lumen size during vascular tubulogenesis.