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Figure 4 Insets are magnification of dashed boxes. (a) Tg(fli1:EGFP);Tg(prox1a:KalTA4-UAS:uncTagRFP) 16 wpf (fish size 23–28 mm) heart showing isolated lymphatic clusters (n = 8). (b,c) Double labeled prox1a+;flt4+ isolated LECs are first detected at ~13 wpf (20–22 mm) (b, arrows) and coalesce to generate isolated capillaries by 16wpf (fish size 25–28 mm) (c, arrows). (d) Quantification of double-labeled prox1a+;flt4+ isolated LECs in the ventricles of 14–28 mm fish (n14-19mm=4, n20-22mm=5, n23-28mm=5). (e) prox1a+ isolated LECs are also labeled by Prox1 antibody (inset, arrow). (f) 20 wpf (fish size 28 mm) double-transgenic prox1a;flt4 hearts demonstrate that isolated LECs are not labeled following intravascular injection of Qdot705 (inset, arrow) (image in f) is an additional view of Figure 2—figure supplement 1c). (g) Isolated LEC clusters develop normally in 22 wpf (fish size 25–30 mm) Tg(flt1_9a_cFos:GFP);Tg(lyve1b:dsRed2); cxcr4a-/- hearts, quantified in (h) (nwt20-22mm=8, ncxcr4a-/- 20-22mm = 6, nwt23-28mm=7, ncxcr4a-/-23-28mm = 8). (i,j) lyve1b+ isolated LEC clusters are not precociously detected in Vegfaa-OE hearts (12.5 wpf, fish size 19–22 mm), in (i) (nveh = 7, nTam = 8) or PHZ treatment (j) (ncontrol = 9, nPHZ = 9) (k). No isolated LECs are detected in 19-23wpf (fish size 23–28 mm) Tg(fli1:EGFP);Tg(lyve1b:dsRed2);flt4 -/- hearts, quantified in (l) (nwt = 5, nflt4-/-=6). (m) Significantly reduced numbers of isolated LECs are detected in Tg(fli1:EGFP);Tg(lyve1b:dsRed2);vegfc+/- animals at 26 wpf (fish size 25–28 mm), quantified in (n) (nwt = 3, nvegfc+/-=5). Scale bars are 200 µm. Error bars, mean ± s.e.m. Anterior view in a-c, e,f,k,m. Posterior view in g.