Supplementary Figure 7: muLEC sprouting is dependent upon Vegfc, Vegfd, Ccbe1 and Vegfr3 signaling.
From: Mural lymphatic endothelial cells regulate meningeal angiogenesis in the zebrafish
(a-d) Dorsal confocal images of 5dpf Tg(-5.2lyve1b:DsRed)nz101; Tg(fli1a:nEGFP)y7 embryos showing the lymphatic loop that forms in wildtype embryos (a, arrows, representative image based on analysis of n=7 embryos) is reduced in vegfchu5055 (b, arrows, n=6 embryos) and vegfduq7bh (c, arrows, n=6 embryos) single mutants, but is absent in vegfchu5055/vegfduq7bh double mutants (d, asterisk, n=9 embryos). Scale bars represent 100μm.
(e) Quantification of the number of muLECs present in a single putative lymphatic loop at 5dpf in wildtype (n=7 embryos), vegfchu5055 (n=6 embryos), vegfduq7bh (n=6 embryos) single mutants and vegfchu5055/vegfduq7bh double mutants (n=9 embryos). Error bars represent mean +/− sem; **** p<0.0001, from one way ANOVA (F (3, 25) = 166.6).
(f-i) At 5 dpf, the lyve1/prox1a positive cells in Tg(-5.2lyve1b:DsRed)nz101; Tg(prox1a:KalTA4, 4xUAS:uncTagRFP)nim5; Tg(10xUAS:Venus) have formed a lymphatic loop in wildtype embryos (f, n=6 embryos), however in vegfchu5055 (g, n=6 embryos) and vegfd (h, n=6 embryos) mutants, the development of the lymphatic loop is reduced (arrows). In vegfchu5055 / vegfduq7bh double mutant embryos (i, n=6 embryos) the lymphatic loop is completely absent (asterisks). Scale bars represent 100μm.
(j-l) The lyve1 lymphatic loop structure (control, j) is absent in vegfr3 morphant (n=15 embryos) (k) and (l) ccbe1 morphant (n=15 embryos) embryos at 5dpf (asterisks).
(m) Quantification of the number of muLECs present in a single putative lymphatic loop at 5dpf in uninjected controls (n=15 embryos), vegfr3 morphant (n=15 embryos) and ccbe1 morphant (n=15 embryos). **** p<0.0001, from two-tailed student t-test (t=33.62 df=28). Scale bars represent 100μm.
(n,o) Dorsal confocal images from 24 mm Tg(-5.2lyve1b:DsRed)nz101 vegfchu5055 (n) and vegfduq7bh (o) mutants showing the presence of either ligand is sufficient for development of the muLECs. Representative image based on analysis of n=4 adult brains. Scale bars represent 200μm.
