Figure 3: Ephrin-B3 promotes angiogenesis through inhibition of EphA4-induced cell death during zebrafish development. (A) Phylogenetic tree showing the inferred evolutionary relationships between ephrins members among various biological species. ML on protein sequences, under JTT model, with 100 bootstrap replicates. EFNB3 orthologs in zebrafish, ephrinB3-b and ephrinB3-Like are highlighted. (B, C) EphrinB3-Like silencing triggers defects in intersegmental vessels (ISV) formation, which are significantly rescued by caspases inhibition through BAF treatment or by co-silencing of EphA4. (b) Representative images of trunk vasculature are shown. (c) Quantification of the percentage of embryos with ISV defects in each condition is presented. ***p < 0.001; χ² test. (D) BAF treatment or EphA4 silencing are both sufficient to rescue apoptosis in MO ephrinB3-Like-injected embryos, as shown by TUNEL staining [36]. Representative images of trunk vasculature are shown. Red dots correspond to apoptotic cells. (E) EphrinB3-Like silencing also triggers caspase-3 activation in zebrafish embryos, which is significantly rescued by BAF treatment or EphA4 co-silencing. Caspase-3 activity was assessed on whole embryos extracts and is expressed as mean relative index to control. Error bars correspond to standard deviation. *p < 0.05; **p < 0.01; U-test.