Extended Data Fig. 1: The Prox1 −11kb enhancer drives reporter gene expression in lymphatic endothelial cells and at high levels in valves.

a, ChIP in hLECs demonstrates binding of GATA2, FOXC2, NFATC1 and PROX1 at the Prox1 −11 kb enhancer and promoter regions. Data are independent experiments and shown as mean ± SEM when n > 2. b, Schematic of construct used to generate stable transgenic reporter mice. c, Strategy for CRISPR-Cas9 mediated deletion of the Prox1 −11 kb element. Guide RNA sequence targeting GATA2 binding site (underlined) and resulting 5 bp deletion are indicated. d, CRISPR-Cas9 mediated deletion series. e, Immunofluorescent analysis of mouse embryos carrying the Prox1 −11 kb enhancer driven lacZ reporter transgene. Transverse sections at E11.5 show β-galactosidase activity is detected in PROX1⁺ endothelial cells lining the cardinal vein (arrowheads). Coronal sections in the jugular region at E12.5 and E14.5, reveal high levels of reporter activity in lymphovenous valves (arrows) while there is no detectable reporter gene expression in PROX1⁺ hepatocytes in E14.5 liver. f, Wholemount X-gal staining of tissues from transgenic mouse pups at post-natal day 4. β-galactosidase activity is present in the lung, dermis, thoracic duct, and mesenteric lymphatic vasculature. In lymphatic vasculature at early post-natal stages, reporter activity is restricted to larger collecting vessels and is not observed in lymphatic vessels in tissues analysed by wholemount X-gal staining at adult stages >P28. Black arrows indicate valves; left side (LS); right side (RS). g, The Prox1 −11 kb enhancer drives reporter gene expression in venous and cardiac valves. Transverse sections of transgenic mouse embryos at E18.5 show reporter activity in venous valves (arrows), semilunar and atrioventricular cardiac valves. Scale bars, 100 µm (e, g), 200 µm (f).