Extended Data Fig. 5: Additional data related to DDX21 loss, cell cycle arrest and Fig. 4.
(a) Gating strategy for the cell cycle analysis on sorted LECs and VECs from sibling and ddx21uq20bh mutants. Top to bottom panels represent the cell population selection based on size and granularity (X-axis reflects FSC = Forward Scatter and Y-axis SSC = Side Scatter, A = Area), identification of double positive cells for fli1a:GFP and lyve1b:DsRed to select LECs and VECs (Y-axis reflects DsRed and X-axis GFP intensities), selection of single cells obtained by gating based on Hoechst staining (Y-axis reflects A = area and X-axis W = width), histogram of cell numbers with Hoechst staining (Y-axis reflects cell counts and X-axis W = width). Black line reflects Hoechst intensity distribution in cells, coloured lines reflect fitted model for cell cycle profiles based on analysis with Watson method (G1-phase=blue, S-phase=yellow and green=G-M-phase). (b) Gating strategy for the cell cycle analysis on control and siDDX21-treated HUVECs. Top to bottom panels represent the cell population selection based on size and granularity (Y-axis reflects FSC = Forward Scatter and X-axis SSC = Side Scatter, A = Area), selection of single cells obtained by gating based on Hoechst staining (Y-axis reflects A = area and X-axis W = width), histogram of cell numbers with Hoechst staining (Y-axis reflects cells counts and X-axis W = width. Black line reflects Hoechst intensity distribution in cells, coloured lines reflect fitted model for cell cycle profiles based on analysis with Dean-Jett-Fox method (G1-phase=blue, S-phase=yellow and green=G-M-phase). (c) The cell cycle profile obtained by FACS of LECs and VECs in siblings and ddx21uq20bh mutants (n = 4). G1-phase p = 0.0332, S-phase p = 0.737, G2-M-phase p = 0.0290. (d) The cell cycle profile obtained by FACS in control and siDDX21 transfected HUVECs (n = 9). G1-phase p = 0.004 (siDDX21-1) and p < 0.0001 (siDDX21-2), S-phase p = 0.0277 (siDDX21-1) and p = 0.0082 (siDDX21-2), G2-M-phase p = 0.3013 (siDDX21-1) and p = 0.0216 (siDDX21-2). (e) Left: Confocal projections showing endothelial nuclei (Tg(fli1a:nEGFP), green) and 5-Ethynyl-2′-deoxyuridine (EdU) staining (magenta) in a 36 hpf sibling and a ddx21uq20bh mutant. Arrows indicate EdU-positive secondary sprouts. Scale bars= 50μm. Right: Quantification of EdU-positive PCV or secondary sprout endothelial cells represented as a ratio of EdU-positive PCV or secondary sprout endothelial cells and total number of PCV or secondary sprout endothelial cells in siblings (n = 7) and ddx21uq20bh mutants (n = 7). p = 0.1200 for PCV. p = 0.5224 for secondary sprouts. (f) Confocal projections showing endothelial nuclei (green) and 5-Ethynyl-2′-deoxyuridine (EdU) staining (magenta) in a 48 hpf sibling and a ddx21uq20bh mutant. Arrows indicate EdU-positive parachordal lymphatic endothelial cells (PLs). Scale bars= 50μm. (g) Left: Confocal projections of IF for DAPI (blue), p53 (magenta) and phospho-histone H3 (PHH3, green) on control or siDDX21 transfected HUVECs. Scale bars= 25μm Right: Quantification of ratio of PHH3-positive cells in p53-low or p53-high cells on control or siDDX21 transfected HUVECs (n = 6 independent replicates). p < 0.0001 for control. p = 0.0143 for siDDX21. n = 6 biologically independent samples from 2 independent experiments. DA = dorsal aorta, PCV = posterior cardinal vein. Mean values + /-SEM shown. Paired one-tailed student’s t-test for (c). Two-tailed student’s t-test for (e, secondary sprout). Two-tailed Mann Whitney test for (e, PCV). Ordinary one-way ANOVA test with Dunnett’s or Tukey’s multiple comparisons test for (d) and (g), respectively.
