24 research outputs found
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
Expression of <i>abl1</i> and <i>abl2</i> in different cell types during zebrafish embryogenesis based on single-cell RNA seq expression atlas.
Data were generated by Daniocell atlas https://daniocell.nichd.nih.gov/ [31]. (TIF)</p
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
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><sup><i>ci26</i></sup> and <sup><i>ci30</i></sup> 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
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
CRISPR/ Cas9 knockdown of <i>abl1</i> and <i>abl2</i> function results in reduced DA size.
(A) A diagram illustrating the targeting sites of sgRNAs against abl1 and abl2 genes. Each gRNA was more than 90% effective based on DNA sequencing analysis. (B-D) Analysis of DA size in kdrl:GFP embryos at 28 hpf. Note the reduced DA diameter in embryos injected with abl1 and 2 gRNA mixture. Data from two replicate experiments are shown in different colors. Mean±SD is shown. **p (TIF)</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
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