Figure 4.
LPHN2 controls EC YAP/TAZ mechanosensing and vascular morphogenesis in zebrafish embryo.(A) Confocal fluorescence microscopy analysis of trunk cross-sections at 72 hpf of Tg(Kdrl:EGFP)s843 WT (lphn2a+/+) and CRISPR/Cas9-mediated lphn2a knock-out (lphn2a−/−) zebrafish embryos carrying EC-specific EGFP expression (green) and stained for Lphn2 (purple) and phalloidin (red). Nuclei are stained with DAPI (blue). Lphn2 is enriched in ECs of DA and posterior cardinal vein (PCV). The first panel on the left (scale bar, 30 µm) displays a whole cross-section whose boxed area is magnified and depicted in the five panels located on its right (scale bar, 10 µm). (B) Lateral view in brightfield (top) and fluorescence confocal (bottom) microscopy of WT (lphn2a+/+) and CRISPR/Cas9-mediated lphn2a−/− zebrafish embryos in the Tg(kdrl:EGFP)s843 background. Scale bar, 100 µm. (C) Yap1/Taz reporter activity is prominent in the endothelium trunk vasculature of zebrafish embryos. Left: Representative confocal images of Tg(Hsa.CTGF:nlsmCherry)ia49 /Tg(kdrl:EGFP)s843 double-transgenic lphn2a+/+ and lphn2a−/− siblings at 60 hpf. Yap1/Taz activation signal was automatically segmented on fluorescent confocal mCherry image z-stacks, inside a EGFP fluorescent mask identifying ECs. Scale bar, 50 µm. The EC-restricted Yap1/Taz signal intensity only was represented in 3D analysis and quantified. Right: Relative quantification of integrated density of Yap1/Taz Hsa.CTGF:nlsmCherry reporter activity signal colocalized with kdrl:GFP in lphn2a+/+ (n = 9) and lphn2a−/− (n = 7) zebrafish embryos (56–72 hpf). Results are the mean ± SD. Statistical analysis: Mann–Whitney test; **, P ≤ 0.01. (D) Real-time quantitative PCR analysis of ctgfa, ctgfb, and cyr61 mRNAs of FACS-sorted ECs isolated from lphn2a+/+ or lphn2a−/− zebrafish embryos, at 48 hpf, relative to the housekeeping gene actb1 and normalized on the mRNA levels measured in lphn2a+/+ animals. Results are the mean ± SD of three or more independent assays (n > 80 embryos for condition). Statistical analysis: one-way ANOVA followed by Tukey’s multiple comparison test; NS, P > 0.05; *, P ≤ 0.05; **, P ≤ 0.01. (E) Representative confocal images of kdrl:GFP positive cells (left) and represented in the 3D analysis (right) used to quantify EC size (bottom) in lphn2a+/+ (n = 5) and lphn2a−/− (n = 7). Scale bar, 20 µm. Results are the mean ± SD. Statistical analysis: Mann–Whitney test; *, P ≤ 0.05. (F) TEM analysis of endothelium area (left), used to determine the EC area normalized on the diameter of the vessel (right) in lphn2a+/+ (n = 3) and lphn2a−/− (n = 5). Color code identifies the ECs facing the vascular lumen (L). Scale bar, 10 µm. Results are the mean ± SD. Statistical analysis: Mann–Whitney test; *, P ≤ 0.05. (G) Representative confocal images of ZO-1 staining in intersomitic blood vessels (ISVs) of lphn2a+/+ and lphn2a−/− at 48 hpf. Arrowheads point at continuous ZO-1–stained intercellular contacts between ISV ECs of lphn2a+/+ zebrafish embryos. The ZO-1 intercellular staining between ISV ECs is instead reduced, discontinuous, and fragmented in lphn2a−/− zebrafish embryos. Scale bars, 20 µm (left) and 30 µm (right).

LPHN2 controls EC YAP/TAZ mechanosensing and vascular morphogenesis in zebrafish embryo. (A) Confocal fluorescence microscopy analysis of trunk cross-sections at 72 hpf of Tg(Kdrl:EGFP)s843 WT (lphn2a+/+) and CRISPR/Cas9-mediated lphn2a knock-out (lphn2a−/−) zebrafish embryos carrying EC-specific EGFP expression (green) and stained for Lphn2 (purple) and phalloidin (red). Nuclei are stained with DAPI (blue). Lphn2 is enriched in ECs of DA and posterior cardinal vein (PCV). The first panel on the left (scale bar, 30 µm) displays a whole cross-section whose boxed area is magnified and depicted in the five panels located on its right (scale bar, 10 µm). (B) Lateral view in brightfield (top) and fluorescence confocal (bottom) microscopy of WT (lphn2a+/+) and CRISPR/Cas9-mediated lphn2a−/− zebrafish embryos in the Tg(kdrl:EGFP)s843 background. Scale bar, 100 µm. (C) Yap1/Taz reporter activity is prominent in the endothelium trunk vasculature of zebrafish embryos. Left: Representative confocal images of Tg(Hsa.CTGF:nlsmCherry)ia49 /Tg(kdrl:EGFP)s843 double-transgenic lphn2a+/+ and lphn2a−/− siblings at 60 hpf. Yap1/Taz activation signal was automatically segmented on fluorescent confocal mCherry image z-stacks, inside a EGFP fluorescent mask identifying ECs. Scale bar, 50 µm. The EC-restricted Yap1/Taz signal intensity only was represented in 3D analysis and quantified. Right: Relative quantification of integrated density of Yap1/Taz Hsa.CTGF:nlsmCherry reporter activity signal colocalized with kdrl:GFP in lphn2a+/+ (n = 9) and lphn2a−/− (n = 7) zebrafish embryos (56–72 hpf). Results are the mean ± SD. Statistical analysis: Mann–Whitney test; **, P ≤ 0.01. (D) Real-time quantitative PCR analysis of ctgfa, ctgfb, and cyr61 mRNAs of FACS-sorted ECs isolated from lphn2a+/+ or lphn2a−/− zebrafish embryos, at 48 hpf, relative to the housekeeping gene actb1 and normalized on the mRNA levels measured in lphn2a+/+ animals. Results are the mean ± SD of three or more independent assays (n > 80 embryos for condition). Statistical analysis: one-way ANOVA followed by Tukey’s multiple comparison test; NS, P > 0.05; *, P ≤ 0.05; **, P ≤ 0.01. (E) Representative confocal images of kdrl:GFP positive cells (left) and represented in the 3D analysis (right) used to quantify EC size (bottom) in lphn2a+/+ (n = 5) and lphn2a−/− (n = 7). Scale bar, 20 µm. Results are the mean ± SD. Statistical analysis: Mann–Whitney test; *, P ≤ 0.05. (F) TEM analysis of endothelium area (left), used to determine the EC area normalized on the diameter of the vessel (right) in lphn2a+/+ (n = 3) and lphn2a−/− (n = 5). Color code identifies the ECs facing the vascular lumen (L). Scale bar, 10 µm. Results are the mean ± SD. Statistical analysis: Mann–Whitney test; *, P ≤ 0.05. (G) Representative confocal images of ZO-1 staining in intersomitic blood vessels (ISVs) of lphn2a+/+ and lphn2a−/− at 48 hpf. Arrowheads point at continuous ZO-1–stained intercellular contacts between ISV ECs of lphn2a+/+ zebrafish embryos. The ZO-1 intercellular staining between ISV ECs is instead reduced, discontinuous, and fragmented in lphn2a−/− zebrafish embryos. Scale bars, 20 µm (left) and 30 µm (right).

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