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Fig. 1 Genetic depletion of TNFRSF1B results in endothelial cell apoptosis and blood circulation disruption. (A) Scheme showing the main vessels of a 3-day-old zebrafish larvae. (B-G) Lateral view of 54(B) and 72 (C-F) hpf and tranverse and sagittal sections (G, bottom panels) of double transgenic fli1a:eGFP and gata1:dsRed larvae microinjected at the one-cell stage with standard control (STD-mo) and TNFRSF1B MOs. (B)TNFRSF1B depletion results in impaired differentiation of the CA and CV during the first sprouting, leading to blood pooling (bp) inside an enlarged unique dorsal vessel (asterisk). (C,D) At 72 hpf, blood pooling can still be observed in the caudal body part. In addition, hemorrhages appear throughout the body (arrowheads). (E,F) Zoomed views of trunk vasculature of 72 hpf larvae. TNFRSF1B deficiency results in the alteration of the second sprouting leading to the formation of a net of vessels that replace the CA and CV (E,F) and altered development of ISV (E). Arrows indicate ISA without blood circulation. (G,H) Confocal Z-stack sections of whole larvae (G) and sections (H) of the CHT of 60 hpf Tg(fli1a:eGFP) injected with standard and TNFRSF1B MOs showing TUNEL-positive cells (red) (arrowheads). Nuclei were counterstained with DAPI (blue). (I) Quantification of TUNEL-positive non-endothelial cells (eGFP) and endothelial cells (eGFP+) at 60 hpf from serial Z-stack sections. Each dot represents the number of TUNEL-positive cells per single larvae. The mean ± s.e.m. of the TUNEL-positive cells for each group of larvae is also shown; ***P<0.001. Scale bars: 100 μm unless otherwise indicated. CA, caudal artery; CV, caudal vein; CHT, caudal haematopoietic tissue; DA, dorsal aorta; DLAV, dorsal longitudinal anastomotic vessel; IPV, intersegmental primary vessels; ISV, intersegmental vessels; PCV, posterior cardinal vein; SIV, subintestinal vessels.