Fig. 2
From: Temporospatial inhibition of Erk signaling is required for lymphatic valve formation
Repressed Erk activity in FCLVs during valve formation. a Development of the lymphatic valve (arrowheads) and FCLV-PHS LVV (arrows) labeled with gata2a:EGFP in the anterior part of facial lymphatic vessels (dashed box) at different developmental stages. Middle panel shows gata2a:EGFP expression presented in Fire LUT (Fiji). The arrowheads indicate the putative valve-forming sites. Scale bars, 50 μm. b Detection of valve-forming LECs labeled by high expression of Prox1a at different developmental stages. Middle panel shows Prox1a expression presented in Fire LUT (Fiji). The numbers of embryos with the Prox1a expression pattern (cyan) are indicated. Arrowheads point out valve-forming regions. Scale bars, 50 μm. c Lineage tracing of the valve cells from the surrounding lymphatic vessels with photoconverted Kaede. The LECs at FCLV, LFL near the FCLV, or LFL away from the FCLV were photoconverted at 3 dpf and the valve formation was observed at 5 dpf. The middle two panels are the enlarged and section views of the boxed regions on the left, and valve structures are labeled by dotted lines. The numbers of embryos with the indicated phenotype are shown. Photoconversion results are summarized as schematic drawings on the right. Scale bars, 50 μm. d Upper panel depicts the formation of anterior facial lymphatic vessels. Two populations of LEC precursors from PHS, PHS-LPA and PHS-LPP, contribute to the FCLV, and LEC precursors from ventral aorta, VA-L, contribute to the anterior part of LFL (aLFL) along with PHS-LPP. Lower panels, Erk activity detected by ERKKTR biosensor in Tg(gata2aECE:ERKKTR;gata2aECE:nls-mCherry) fish during lymphatic development, from 52 hpf to 77 hpf. The FCLV region (white solid lines) and anterior part of the LFL (aLFL, yellow solid line) are shown. White arrows indicate cells with high Erk activity, and yellow arrows indicate cells with relative low Erk activity. The individual cells used for ERKKTR-EGFP analysis are listed in Supplementary Fig. 2c, d. Scale bars, 50 μm. e Quantification of the cytoplasmic/nuclear (C/N) intensity ratios used as readout for Erk activity in individual cells. Unpaired two-tailed t test, n = 2 independent experiments, one of them is shown here. All images are anterior to the left, dorsal upward
