Fig. 6

efnb2-ephb4b mutants have defects in lymphatic system. a efnb2a^−/− and efnb2a^−/−;efnb2b^−/− double mutants exhibit pericardial edema (arrowheads) and blood filling of facial lymphatic vessels (arrows) at 4 dpf, whereas efnb2b^−/− develops normally. Casper background was introduced to facilitate observation. Scale bar, 200 μm. b Statistical summary of pericardial edema in the offspring of efnb2a^-/+;efnb2b^−/− mutants intercrossing. Two-sided Fisher’s exact test. c Pericardial and gut edema (arrowhead) with blood filling of lymphatic vessels (arrows) phenotype in ephb4b^−/− mutants. Casper background was introduced to facilitate observation. Scale bar, 200 μm. d Percentage of ephb4b^−/− mutants with pericardial edema. Each dot represents the percentage of edema embryos (total embryos >40) from one pair of fish with the indicated genotype. Unpaired two-tailed t test (WT n = 8; ephb4b^−/− n = 15). e gata1:DsRed labeled red blood cells (arrows) enter the lyve1b:TopazYFP labeled facial lymphatic vessel (green) at 77 hpf in ephb4b^−/− mutants. The numbers of embryos with indicated phenotype are shown. Scale bars, 50 μm. Uncropped images are presented in Supplementary Fig. 9k. f Lack of the lymphatic valve (arrowhead), FCLV-PHS LVV (arrow), and RFLS-CCV LVV (yellow arrow) at 77 hpf in efnb2a^−/−;efnb2b^−/− double mutants. The numbers of embryos with the indicated valve morphology are shown. Scale bars, 50 μm. g Top, ephb4b^−/− mutants have defective LVs (arrowheads) and FCLV-PHS LVVs (arrows) at 77 hpf. Bottom, ephb4b^−/− mutants have defective RFLS-CCV LVVs (yellow arrows) and red blood cells (RBCs) entry of the RFLS at 4 dpf. Scale bars, 20 μm. h, Immunofluorescence of Prox1a in siblings and efnb2a^−/−;efnb2b^−/− mutants at 77 hpf. The blood and lymphatic fluid have spontaneous red fluorescence. Scale bars, 20 μm. i Statistical analyses of the valve-forming LECs in siblings (n = 4) and efnb2a^−/−;efnb2b^−/− mutants (n = 8) in (h). Unpaired two-tailed t test. j Immunofluorescence of Prox1a in siblings and ephb4b^−/− mutants on the Tg(gata2a:EGFP) transgenic background at 77 hpf. Arrows, FCLV-PHS LVVs; arrowheads, LVs. Valve-forming LECs are labeled by high Prox1a expression. Scale bars, 20 μm. k Statistical analyses of the valve-forming LECs in siblings (n = 15) and ephb4b^−/− mutants (n = 22) in (j). Unpaired two-tailed t test. l Transmission electron microscopy reveals the absence of LVs in efnb2a^−/−;efnb2b^−/− mutants at 77 hpf. Scale bars, 10 μm. m Confocal imaging of RBC flow around the FCLV-PHS LVV in siblings and ephb4b^−/− mutants on the Tg(gata2a:EGFP;gata1:DsRed) background. Primary head sinus (PHS) is marked by white dotted lines. RBCs (arrows) are labeled by gata1:DsRed, and FCLV-PHS LVVs (arrowheads) are gata2a:EGFP-positive. RBCs enter the FCLV through the defective LVV in ephb4b^−/− mutants, but are blocked by the well-formed LVV in siblings at 77 hpf. Right, diagrams of RBC flow in the siblings and ephb4b^−/− mutants. Scale bars, 20 μm. n LV and LVV defects in the caudal heart in ephb4b^−/− mutants. Right, enlarged caudal hearts in the boxed regions. Arrow, CH-CV LVV; arrowhead, LV in the caudal heart. The numbers of embryos with indicated valve morphology are shown. SL, standard length. Scale bars, 50 μm. All images are anterior to the left, dorsal upward