Supplementary Figure 1: lyve1-expressing cells in the adult zebrafish meninx co-express the lymphatic marker genes prox1a and fli1a
From: Mural lymphatic endothelial cells regulate meningeal angiogenesis in the zebrafish
(a) Confocal images of adult zebrafish brain in Tg(-5.2lyve1b:DsRed)nz101; TgBAC(prox1a:KalTA4 uq3bh;10xUAS:Venus) animals showing cells that co-express the lymphatic markers prox1a and lyve1. Expression of prox1a is mosaic due to the Gal4 system. Full quantification of marker co-expression can be found in Supplementary Figure 1e. Scale bar represents 100μm.
(b) Confocal images of adult zebrafish brain in Tg(-5.2lyve1b:DsRed)nz101; Tg(fli1a:EGFP)y1 showing co-expression (arrows) of the lymphatic markers fli1a and lyve1. Full quantification of marker co-expression can be found in Supplementary Figure 1f. Scale bar represents 100μm.
(c) Quantification of muLEC density on the surface of the brain (meningeal surfaces n=6 adult brains. Error bars represent mean +/− sem, Statistical testing N/A.)
(d) Quantification of the number of lyve1-positive meningeal cells (over the surface of the tectum, or adjacent to the central arteries (cTA)) compared with non-meningeal lyve1-positive cells from cross sections of the adult zebrafish brain. n=4 adult brain cross sections, p<0.0001, from two-tailed student t-test (t=8.926 df=6).
(e) Quantification of co-expression of cells from Tg(prox1a:KalTA4uq3bh;10xUAS:Venus) and lyve1 (Tg(-5.2lyve1b:DsRed)nz101) and the macrophage marker mpeg1 (Tg(mpeg1:mcherry)). 215 cells from n=4 larvae (lyve1) and 255 cells from n=5 larvae (mpeg1) were scored from confocal Z-stack images. Error bars represent mean +/− sem, Statistical testing N/A.
(f) Quantification of co-expression of cells in Tg(-5.2lyve1b:DsRed)nz101 and Tg(fli1a:EGFP)y1, TgBAC(pdgfrβ:EGFP)uq15bh, TgBAC(acta2:EGFP)uq17 and Tg(nkx2.2a:EGFP)vu16Tg. 100 cells from n=5 larvae were scored from confocal Z-stack images. Error bars represent mean +/− sem, Statistical testing N/A.
(g) Quantification of the distance from the nucleus of muLECs to the center of the parenchyma (defined as the centre of the unlabeled space surrounded by a vascular loop) shows that muLECs are more closely associated with blood endothelial cells than the space between vessels. 77 muLEC nuclei from n=3 larvae, p<0.0001, from two-tailed student t-test (t=15.64 df=130). Error bars represent mean +/− sem.
(h) Quantification of the distance from the nucleus of a given muLEC to the nearest vessel branch point, compared with the distance from the nucleus of a given muLEC to the midpoint between branches of the closest vessel. muLECs are quantitatively closer to vessel branch points than vessel midpoints. 77 branch and 77 midpoints points from n=3 larvae, p<0.0001, from two-tailed student t-test (t=4.74 df=151). Error bars represent mean +/− sem.
