Fig. 4

Pan-endothelial expression of a zebrafish Bmpr1aa^p.R438H mutant protein does not affect embryonic valvulogenesis. (A–E) Shown are reconstructions of confocal z-stack images of the zebrafish embryonic atrioventricular canal (AVC) region. (A) Embryonic cardiac ventricle in a Tg(fli1a:Gal4FF)^ubs3; Tg(UAS:bmpr1aa^WT_IRES_EGFP)^md65; Tg(7xTCF-Xia.Sia:NLS-mCherry)^ia5 zebrafish at 120hpf. The AVC region is outlined. Scale bar = 20 µm. (B) AVC region in a Tg(fli1a:Gal4FF)^ubs3; Tg(UAS:bmpr1aa^WT_IRES_EGFP)^md65; Tg(7xTCF-Xia.Sia:NLS-mCherry)^ia5 or (C) Tg(fli1a:Gal4FF)^ubs3; Tg(UAS:bmpr1aa^p.R438H_IRES_EGFP)^md60; Tg(7xTCF-Xia.Sia:NLS-mCherry)^ia5 zebrafish at 120hpf. Indicated is the diameter of the AVC (d). Scale bar = 10 µm. (D,D′) Embryonic valve formation in the WT zebrafish embryo at 72hpf. Box indicates the region shown in (D′). Valve leaflets in WT embryos are characterized by double-layering with an abluminal population of AVC cells that has active Wnt signalling marked by Tg(7xTCF-Xia.Sia:NLS-mCherry)^ia5 (arrow). Cell membranes of luminal cells are marked by immuno-labelling against ALCAM (cells marked by asterisks). (E,E′) Similarly, valvulogenesis is not affected in Tg(fli1a:Gal4FF)^ubs3; Tg(UAS:bmpr1aa^p.R438H_IRES_EGFP)^md60; Tg(7xTCF-Xia.Sia:NLS-mCherry)^ia5 embryos that show a normal double-layered leaflet morphology with Wnt signalling in abluminal cells (arrow) and ALCAM-positive luminal cells (asterisks). Scale bars = 20 µm. (F) The diameter of the AVC in zebrafish embryos with pan-endothelial overexpression of bmpr1aa^p.R438H does not significantly differ from that upon bmpr1aa^WT overexpression [total number of embryos analysed: bmpr1aa^WT, n = 6; bmpr1aa^p.R438H, n = 8; two-sided student’s t-test, p = 0,066].