Fig. 5: Quantitative analysis of LEC expansion.
From: Direct specification of lymphatic endothelium from mesenchymal progenitors
Representative whole-mount immunofluorescence for ERG and PROX1 in E9.5 (a,a′), E10 (b,b′), E10.5 (c,c′) and E11.0 (d,d′) embryos. 3D projections of regions of interest were segmented (a′–d′) for temporal quantification of ERG+PROX1− ECs within the venous endothelium, ERG+PROX1+ ECs within the venous endothelium and ERG+PROX1+ ECs outside of the venous endothelium. e, Quantification of total ECs and PROX1+ ECs inside and outside of the venous endothelium between E9.5 and E11.0 (E9.5, n = 3; E10, n = 6; E10.5, n = 6; E11, n = 3). f, Schematic representation of the dual-pulse labeling strategy for analysis of cell cycle dynamics. g–g″, Representative immunofluorescence for EMCN, PROX1, EdU and BrdU on a transverse vibratome section from an E10.5 embryo. h, Quantification of labeling of PROX1+ ECs inside and outside of the venous endothelium with EdU and/or BrdU (n = 4). i, Quantification of cell cycle duration inside and outside of the venous endothelium at E10.5 (n = 4, *** P < 0.001). j,j′, Representative immunofluorescence for EMCN, PROX1 and KI67 on a transverse vibratome section from an E10.5 embryo. k, Quantification of growth fraction for PROX1+ ECs present inside or outside of the venous endothelium (n = 3). l–l″, Representative immunofluorescence for EMCN, PROX1, mCherry (G1/early S) and mVenus (S/G2/M) on a transverse vibratome section from an E10.5 Rosa26Fucci2 embryo. m, Quantification of PROX1+ ECs in S/G2/M phases of the cell cycle inside or outside of the venous endothelium (n = 3, **** P < 0.001). DA, dorsal aorta. Scale bars, 500 μm (a–d), 50 μm (g–g″), 100 μm (j,j′ and l–l″). Statistical analyses were performed using unpaired Student’s t-test. Data are presented as mean ± s.d.
