Fig. 5
From: Temporospatial inhibition of Erk signaling is required for lymphatic valve formation
Inhibition of MAPK/Erk signaling leads to valve hyperplasia. a erk1−/−;erk2−/− double mutants develop obvious edema at 5 dpf. Arrowheads indicate the edema in the heart and gut regions. Scale bar, 200 μm. b Statistical data for pericardial edema in the offspring of erk1-/+;erk2−/− mutants intercrossing at 5 dpf. Two-sided Fisher’s exact test. c erk1−/−;erk2−/− double mutants show defects in both lymphatic vessel and valve formation. In erk1−/−;erk2−/− double mutants, two types of valve phenotypes are observed, which are summarized in the schematic drawings on the right. Asterisks, anterior LFLs; arrowheads, lymphatic valves; arrows, FCLV-PHS LVVs. Scale bars, 50 μm. d At 5 dpf, Prox1a immunostaining reveals an increase of valve cells in the FCLV-PHS LVV and type II FCLV-LV in erk1−/−;erk2−/− double mutants. The number of mutants with type II valve is indicated. Solid lines indicate FCLV and dashed lines indicate LFL. Scale bars, 50 μm. e Statistical analysis of the valve-forming LECs with high Prox1a expression in erk1+/+;erk2−/− (n = 7) siblings or erk1−/−;erk2−/− (n = 16) mutants at 5 dpf in (d). Unpaired two-tailed t test (LV for erk1+/+;erk2−/− n = 7; LV for erk1−/−;erk2−/− n = 16; FCLV-LVV for erk1+/+;erk2−/− n = 5; FCLV-LVV for erk1−/−;erk2−/− n = 11). f Detection of the valve-forming LECs by Prox1a and gata2a:EGFP immunostaining in embryos treated with different doses of selumetinib from 2 to 4 dpf. Brackets indicate the LV structures. Two different types of LVs are observed. The numbers of embryos with the Prox1a expression pattern (magenta) in LVs are indicated. Scale bars, 20 μm. g Statistical analysis of LV cells after selumetinib treatment in (f). Unpaired two-tailed t test, (DMSO n = 7; 10 n = 7; 50 n = 6; 100 μM n = 3 and 5). h Statistical analysis of FCLV cells, excluding the valve-forming LECs with high Prox1a expression in (f). Unpaired two-tailed t test, (DMSO n = 7; 10 n = 6; 50 n = 7; 100 μM n = 8). i Detection of FCLV-PHS LVV formation in embryos treated with different doses of selumetinib from 2 to 4 dpf. The numbers of embryos with the Prox1a expression pattern (magenta) are indicated. Scale bars, 20 μm. j Statistical analysis of FCLV-PHS LVV cells in (i). Unpaired two-tailed t test (DMSO n = 7; 10 n = 6; 50 n = 7; 100 μM n = 8). k Live imaging of the ectopic valve-forming LECs labeled with gata2a:EGFP in embryos treated with selumetinib from 3 to 4 dpf. gata2a:EGFP fluorescence intensities at positions 1–6 are shown. Ectopic gata2a:EGFP expression was found at position 5 in 50 μM treated embryos and positions 5 and 3 in 100 μM treated embryos. All images are anterior to the left, dorsal upward
