plxnd1 acts cell-autonomously to guide lymphatic vessel growth. (A-B″) Confocal images of the lyve1b:EGFP expression (anti-EGFP, green) (A,B), plxnd1 expression (anti-plxnd1, magenta) (A′,B′) or both (A″,B″) in the head (A,A″) or trunk (B,B″) of 48 hpf lyve1b:EGFP larvae. Yellow arrowheads indicate either the facial lymphatic sprout (A) or the parachordal LECs (B). Blue arrowheads indicate blood vessels. There is non-specific staining in the yolk extension (YE). (C) Schematic of the transplantation protocol. Cells were transplanted from 3 hpf lyve1b:DsRed; kdrl:EGFP donor embryos to 3 hpf fli1a:EGFP host embryos, resulting in chimeric larva with host-derived blood and lymphatic vessels indicated in light green and donor-derived lymphatic vessels indicated in white. (D,E) Confocal images of the facial vasculature in chimeric animals at 6 dpf showing donor-derived LECs (white) from either wild-type (D) or plxnd1nz75 (E) donors. For clarity, EGFP-positive vessels are not displayed. Aberrant plxnd1nz75 facial lymphatic vessels are highlighted by yellow arrowheads in E. (F,G) Confocal images of the trunk vasculature in chimeric animals at 6 dpf showing donor-derived LECs (white) from either wild-type (F) or plxnd1nz75 (G) donors. Note the misaligned plxnd1nz75 vessel highlighted by the yellow arrowhead in G. (H,I) Quantitation of aberrant facial lymphatics (H) or the number of misaligned ISLVs (I) at 6 dpf. **P<0.01 (Mann–Whitney test); data are mean±s.d. Scale bars: 50 µm. DA, dorsal aorta; FLS, facial lymphatic sprout; ISV, intersegmental vessel; ISLV, intersegmental lymphatic vessel; LDA, lateral dorsal aorta; PCV, posterior cardinal vein; PCeV, posterior cerebral vein; PL, parachordal LEC; PHS, primary head sinus; YE, yolk extension. Numbers in graphs represent numbers of larvae.
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