FIGURE 5

FIGURE 5

Delayed development is observed in vessels but not blood cells or the heart. (A) The expression levels of artery (cldn5b), vein (mrc1a), heart (myh7, myh6), lymphatic (prox1a, vegfc, flt4), and endothelial integrity (klf2a, egfl7, amotl2a) markers at 21, 24 and 27 hpf embyros were analyzed by quantitative RT-PCR (qRT-PCR). The gray bars represent the −ΔΔCt values comparing dKO to WT. ns, no significance; *P < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. (B) Whole-mount in situ hybridization (WISH) analysis at 30 hpf embryo showing the developmental patterns of arteries (cldn5b), veins (mrc1a), red blood cells (gata1a), macrophages (lyz), neutrophils (mfap4.1), atrium (myh7), and ventricle (myh6). The lower panel of cldn5b and mrca1 show dorsal views of embryos, while all other images show lateral views. The arrow head indicated the CCV. Scale bar, 200 μm. (C) The CCV in WT and dKO embryos at 24 hpf. The length of the extended CCV is outlined by a white dotted line. Scale bar: 100 μm. (D) Quantification of CCV length shows significant shortening in Tg(fli1a:EGFP); swell1a^{twu0421/twu0421}; swell1b^+/twu0422 (Tg(fli1a:EGFP)^−/−,±) embryos. The lower normal boundary (72.43 μm) was defined as the mean value minus two standard deviations in WT embryos (indicated by a red dotted line). Shortened CCV was observed in 41.67% (5/12) of Tg(fli1a:EGFP)^−/−,± embryos. Sample sizes (n) are indicated above the scatter plot. The data were collected from two biological replicates. *p < 0.05. (E) Delayed development of the dorsal longitudinal anastomotic vessel (DLAV) and ISVs in Tg(fli1a:EGFP)^−/−,± embryos at 30, 48, and 72 hpf. White arrowheads indicate impaired DLAV and ISV development, while red arrowheads highlight impaired CV development. Scale bar: 100 μm.