Signaling through Y1226/7 is essential for initial lympho-venous sprouting and lymphatic differentiation. (A-C) Confocal microscopy of Tie2 immunostaining at 36 hpf. Arrows denote lympho-venous sprouts which give rise to trunk lymphatic and intersomitic vein (ISV) in wild-type embryos; arrowheads indicate intersomitic arteries (ISAs), which display Tie2 faint immunostaining in all embryos. Bracket indicates PCV. (D) Percentage of ISA or ISV connections at 72 hpf in embryos of the indicated genotype as determined by angiography. Total numbers of vessels counted for analysis are indicated. (E-G) Confocal microscopy of live Tg(fli1a:egfp)y1 embryos of the indicated genotype at 48 hpf. White arrows denote parachordal cells (PACs) in wild-type embryos. Blue and red arrowheads indicate ISVs and ISAs, respectively. Asterisks denote normal position of PACs; occasional fluorescence is observed in non-endothelial cells at the horizontal myoseptum (e.g. panel F). (H,I) Confocal images of Prox1 (red) and GFP (green) immunostaining in Tg(fli1a:egfp)y1 embryos of the indicated genotype at 36 hpf. Arrows denote Prox1-expressing endothelial cells in lymphatic sprouts; Corpuscle of Stannius (cs) is indicated and asterisk denotes Prox1-positive muscle pioneer. (J,K) Average number of Prox1-positive venous endothelial cells and lymphatic sprouts per embryo of the indicated genotype and at 36 hpf (J) or the indicated stage (K). Total number of embryos scored in each category is indicated on the x-axis. (D,J,K) **P<0.01, ***P<0.001; ns, not significant. Error bars are ±s.d. (A-C,E-I) Lateral views, anterior to the left, dorsal is up. Genotype is indicated for each panel.
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