Sorbs1 functions independently of Vegfc signaling during in vivo lymphangiogenesis. A RT-qPCR analysis of prox1a relative expression at 48 hpf in endothelial cells (ECs) sorted from WT and sorbs1−/− Tg(fli1a:eGFP)y1 embryos using FACS as described in Fig. 1D (ns = non-significant, unpaired t-test). Results are means from 5 experiments. B Confocal imaging (Z-maximum intensity projections) of WT or sorbs1−/− Tg(fli1a:eGFP)y1 embryos after immuno-staining against Prox1 was used to quantify the number of Prox1-positive endothelial cells per 2 body segments in the dorsal (light purple arrows) and ventral side (dark purple arrows) of the posterior cardinal vein at 32 hpf. Scale bar represents 50 μm (n = number of embryos; ns = non-significant; two-tailed Mann–Whitney U-test). C Quantification of PLs in 10 somites of Tg(kdrl:GFP) embryos injected with sorbs1 and/or flt4 morpholino, at different concentrations (indicated in the figure) (n = number of embryos; ns = non-significant, ***P < 0.001; Mann–Whitney U-test). Representative confocal microscopy images (Z-maximum intensity projections) of the trunk vasculature are shown on the right. Scale bar represents 75 μm. D Confocal images (Z-maximum intensity projections) were used to quantify PL extent within the trunk region of 54 hpf Tg(fli1a:eGFP)y1 WT or sorbs1−/− embryos expressing transgenic constructs coding for human VEGFC fused with RFP, or the RFP alone, under the shh promoter (expression in the roof plate, white asterisks) (n = number of embryos; **P < 0.01; Mann–Whitney U-test). PLs are indicated with green arrows and magnified insets on the right provide detailed views of PL morphology. Scale bar represents 100 μm
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