SH2 domain protein E and ABL signaling regulate blood vessel size.

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

Numerical original data used for graphs and summary statistics.

Tab numbers in the Excel table match to the figure numbers of corresponding graphs. (XLSX)</p

Consensus ABL phosphorylation sites YXXP are required for SHE function.

(A) Consensus ABL phosphorylation sites are conserved between zebrafish, mice and humans. (B) Vascular endothelial expression of a mutant construct fli1:sheFXXP-2A-mCherry, where all four consensus tyrosines have been substituted into phenylalanine, fails to rescue the pericardial edema in she mutants at 4 dpf. The first bar showing she-/- embryos (no Tg) is copied from Fig 3I. ****pfli1:sheFXXP-2A-mCherry embryos and sibling mCherry-negative embryos at 28 hpf. 5 measurements were performed in each embryo, which were then averaged for statistical calculations. 2 replicate experiments were performed, shown in different colors. n corresponds to the number of embryos. Mean ± SD is shown. *p<0.05, Student’s t-test.</p

A proposed model for SHE and ABL signaling during vascular tubulogenesis.

Activated ABL promotes enlarged vascular lumen through a downstream effector P-CRKL which increases endothelial cell proliferation and increases Cldn5 expression, thus affecting tight junctions and cell adhesion. Activated ABL phosphorylates SHE, which then interacts with ABL to dampen its activity resulting in the lumen of appropriate size.</p

Diagrams and sequence chromatograms of <i>she</i>^<i>ci26</i> and ^<i>ci30</i> mutations.

(A) sheci26 mutants have a 7 bp deletion. Alignment of wild-type and sheci26 mutant sequences is shown starting with the first coding ATG. (B) Genomic DNA sequence chromatogram of sheci30 mutants. (C) sheci30 mutants carry a 575 bp deletion and 24 bp insertion present between exons 3 and 4 within she gene. (TIF)</p

<i>she</i> mutants show enlarged diameter of the dorsal aorta.

(A,B) Overall vascular patterning of she-/- mutants is normal when compared to their sibling wild-type embryos at 28 hpf. Embryos are in kdrl:GFP background. (C-F) A wider DA is observed in she mutant embryos compared to their wild-type (she+/+) siblings at 1 and 2 dpf (28 and 48 hpf respectively). Red line indicates DA diameter. (G,H) DA is narrower in she mutants at 4 dpf compared to their siblings. (I,J) Qtracker dots were injected into the circulatory system at 2 dpf (48 hpf) stage. Wider DA is apparent in she mutants (red lines), indicating enlarged vascular lumen size. (K) Diameter of the DA and PCV at 1–4 dpf in she mutants and their wild-type siblings. * phe mutant and sibling embryos were obtained by in-crossing sheci26+/-; kdrl:GFP carriers. Embryos at 1 and 2 dpf were genotyped after imaging. Embryos at 4 dpf were separated based on the phenotype, and wild-type siblings include she+/+ and she+/- embryos at this stage. Numbers at the bottom of the bars indicate the total number of embryos analyzed.</p

Blood flow does not affect <i>she</i> expression or <i>she</i> mutant phenotype.

(A,B) In situ hybridization analysis for she expression at 28 hpf in tnnt2 MO-injected embryos and uninjected controls. Trunk region is shown, anterior is to the left. Numbers in the lower right indicate embryos that showed normal expression pattern out of the total number of embryos in 3 replicate experiments. (C-H) DA size analysis at 28 hpf in wt (she+/+) and she-/- sibling embryos, injected with tnnt2 MO, compared to uninjected controls. Embryos were obtained from cross of she+/- parents in kdrl:GFP background and subsequently genotyped. Note that DA diameter is greatly reduced in tnnt2 MO-injected embryos compared to wild-type uninjected embryos, and increased in she mutants, injected with tnnt2 MO compared to wild-type embryos injected with tnnt2 MO. Data are combined from 3 replicate experiments, shown in different color. Mean±SD is shown. The number of embryos analyzed is shown at the bottom of each bar. The same wt+tnnt2 MO embryos were used for comparisons in (G) and (H). *p (TIF)</p

Hybridization chain reaction (HCR) analysis of <i>cldn5b</i> mRNA expression at 24 hpf.

(A,B) cldn5b (purple) and kdrl:GFP fluorescence in the trunk region of she mutant and wild-type sibling embryos. DA, dorsal aorta; PCV, posterior cardinal vein. cldn5b fluorescence is shown in A’,B’. (C) Quantification of cldn5b fluorescence in the DA. p = 0.17, Student’s t-test. Error bars show SEM. Data show combined results from two independent experiments. (TIF)</p

Blood flow in the tail region of wild-type zebrafish embryos.

Wild-type siblings obtained from an incross of sheci26 heterozygous adults in kdrl:GFP; gata1:dsRed background were imaged at 4 dpf using confocal microscopy. (MP4)</p

Inhibition of SHE in HUVECs results in enlarged tubulogenesis.

(A-F) HUVEC cells were transfected with either control or SHE siRNA and analyzed in 3D collagen matrix assay at 48 (A-C) or 72 h (D-F) after transfection. The values (± standard deviation) are derived from 6–7 representative fields from 3 replicate wells where total EC tube area was measured. *** pS11 Fig. (H) Relative intensity ratio in siSHE / siControl samples. Note increased intensity in pPAK4 and pCRKL samples compared to siControl (which equals to 1); *p<0.05, **p<0.01, Student’s t-test.</p