Extended Data Figure 6. The Vav1+ lineage does not contribute to LECs emerging from the common cardinal or jugular veins but contributes to VEGF-C induced LECs emerging from yolk sac explants.

Vav1-Cre;R26R-tdTomato lineage tracing revealed no recombination nor labeling of the nascent LECs budding from common cardinal vein endothelium in E10.0 embryos (a) as confirmed by co-staining for Emcn, Prox1 and tdTomato fluorescence (a). White inset box in (a) highlighted by labeling for Emcn, Tomato and Prox1 (b-d). Vav1-Cre;R26R-EYFP lineage tracing revealed no recombination nor labeling of LECs forming the jugular lymph sacs in E12.5 embryos (e) as confirmed by co-staining for GFP, Emcn and Prox1 (e). White inset box in (e) highlighted by individual α-GFP (f), Emcn (g) and Prox1 (h) staining (n=3 embryos analyzed per time-point) ccv, common cardinal vein; jls, jugular lymph sac; jv, jugular vein; sv, sinus venosus. Representative staining for Prox1 and native Tomato fluorescence of ex-vivo cultures of explanted Vav1-Cre;R26R-tdTomato concepti at E8.0, including the intact yolk sac (i) and outgrowth of Tomato+ cells (j). Explants were cultured with 100ng/mL of recombinant VEGF-C-Cys(156)Ser²⁸ (R&D Systems), a potent selective lymphangiogenic cue that only signals via VEGFR-3 (i). High resolution images of the specification of Tomato+/Prox1+ LECs (indicated by white inset boxes) in the yolk sac explants (k-n) and in the surrounding cellular outgrowth (o-z) was observed (Tomato+ in red; Prox1+ in blue; single and merged channels shown). Co-staining was confirmed by z-stack reconstructions for each four high resolution panel set (n, r, v, z); n=6 explants analyzed. Scale bars: a, e 50μm; b-d and f-h 12.5μm; i 100μm; j 50μm; m, q, u, y 15μm