Fig. 4

rasa1 regulates lymphatic valve specification through inhibiting Erk signaling. a p-Erk1/2 immunostaining reveals increased Erk signal in FCLV LECs in rasa1a^−/−;rasa1b^−/− and rasa1a(-3)^−/−;rasa1b^−/− mutant embryos at 77 hpf. Solid lines, FCLV; yellow dashed lines, other tissues with p-Erk1/2 signals. Arrows indicate the obvious p-Erk1/2 signals in FCLV. Right, enlargement of the boxed regions in the left panels. Scale bars, 20 μm (left) or 10 μm (right). b Relative p-Erk1/2 intensity compared to lyve1b:DsRed2 expression in FCLV in wild-type and mutant embryos. Unpaired two-tailed t test (at least 3 independent experiments; Wild-type n = 7; rasa1a^−/−;rasa1b^−/− n = 5; rasa1a(-3)^−/−;rasa1b^−/− n = 8). c Selumetinib treatment from 2–4 dpf restored the valve structure in rasa1a(-3)^−/−;rasa1b^−/− labeled with gata2a:EGFP and also induced ectopic gata2a:EGFP expression in the FLV. Arrowheads, LVs; arrows, FCLV-PHS LVVs; yellow arrows, RFLS-CCV LVVs; yellow arrowheads, ectopic gata2a:EGFP positive cells; asterisks, OLVs. The numbers of embryos with exhibited valve structures are shown. Scale bars, 50 μm. d Selumetinib treatment restores the LV and LVV formation in rasa1a(-3)^−/−;rasa1b^−/− mutants at 4 dpf. Prox1a immunostaining was used to label the valve-forming LECs. Prox1a expressions were also presented in Fire LUT (Fiji). Arrowheads, LVs; arrows, FCLV-PHS LVVs. N = 3 independent experiments. Scale bars, 20 μm. e Statistical analysis of the valve-forming LECs in siblings with DMSO (n = 11), rasa1a(-3)^−/−;rasa1b^−/− mutant (Mt) with DMSO (n = 10), and Mt with 100 μM selumetinib (n = 20) in (d). Unpaired two-tailed t test. All images are anterior to the left, dorsal upward