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Endoglin knockdown affected the development of the vasculature (A) 72 hpf Tg(fli1a:EGFP)y1 transgene zebrafish embryos were selected to demonstrate the phenotypic change in the Con-MO, eng-MO, eng mRNA and eng-MO + eng mRNA group. (All Tg(fli1a:EGFP)y1 zebrafish embryos were injected with 2 ng morpholinos and 500 pg mRNA. For example, Con-MO group: 2 ng 5-mispair control MO and 500 pg mCherry mRNA). The red arrows point to the disrupted structure of ISVs. Scale bars are 100 μm. (B) Quantification of phenotypic defects in the Con-MO, eng-MO and eng-MO + eng mRNA group (Normal: most vascular and intersegmental blood vessels do not have obvious defects; Mild: most vascular vessels do not have obvious defects, but some intersegmental blood vessels are twisted and defective; Severe: most vascular and intersegmental blood vessels are disrupted, and some intersegmental blood vessels have disappeared). (C) FACS analysis of fli-GFP+ cells in the 24 hpf Negative Con, Con-MO and eng-MO groups (Negative Con: AB zebrafish injected with 5-mispair control MOs; Con-MO: Tg(fli1a:EGFP)y1 zebrafish injected with 5-mispair control MOs; eng-MO: Tg(fli1a:EGFP)y1 zebrafish injected with endoglin MOs). (D) Quantification of fli-GFP+ cell number from FACS analysis (mean ± SD; the experiments were repeated three times). (E) qPCR analysis of endoglin downstream gene expression in 24 hpf zebrafish embryos, including id1, id3 and jdp2. Gapdh was used as an internal control (mean ± SD; the experiments were repeated three times). A value of P was considered statistically significant (*P<0.05) for D and E.
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