Extended Data Fig. 4: Additional data related to cell death, senescence and Fig. 4.
(a) Confocal projections of Tg(-5.2lyve1b:DsRed2) (green) stained with TUNEL (magenta) in siblings and a ddx21uq20bh mutant at 48 hpf. Arrows indicate TUNEL-positive parachordal lymphatic endothelial cells (PLs). Scale bars= 50μm. (b) Confocal projections of IF for endothelial nuclei (Tg(fli1a:nEGFP), green) and cleaved Caspase-3 (magenta) in siblings and ddx21uq20bh mutants at 48 hpf. Arrows indicate cleaved Caspase-3-positive PLs or gut cells. Scale bars= 50μm. (c) Quantification of TUNEL-positive endothelial cells in siblings (n = 10) and ddx21uq20bh mutants (n = 10). p = 0.4737 for PCV. p = 0.5124 for LECs. p = 0.4737 for vISV. (d) Quantification of the number of cleaved Caspase-3 and GFP double positive cells in siblings (n = 11) and mutants (n = 25). p = 0.6239 for PCV. p = 0.2539 for LECs. p = 0.8707 for vISV. (e) Quantification of number of cell death in LECs between 36-56 hpf in siblings (n = 13) and ddx21uq20bh mutants (n = 13). p>0.9999. (f) Quantification of number of cell deaths in venous intersegmental vessel ECs between 36-56 hpf in siblings (n = 13) and ddx21uq20bh mutants (n = 13). p>0.9999. n=biologically independent samples (embryos) analysed. (g) Representative bright field images of senescence-associated ß-galactosidase (SAß-gal) stained 5 dpf sibling (18) and ddx21uq20bh mutant (14) larvae. Scale bars= 100μm. (h) Representative images of p21 in situ hybridisation on transverse sections of a sibling (7) and a mutant (7) at 2 dpf. 6 out of 7 mutants displayed darker stanning. Scale bars= 20μm. The number of animals (embryos) analyzed in g and h is indicated in parentheses. (i) Quantification of DDX21 (foci, left) intensity on control, siDDX21-1 and siDDX21-2 transfected HUVECs (n = 6 independent replicates for both siDDX21s). The variance in DDX21 foci intensity is larger in siDDX21-1 when compared to siDDX21-2. p<0.0001. (j) Quantifications of p53-positive aISV and PCV cells in 48 hpf siblings (n = 17) and ddx21uq20bh mutants (n = 17) from Fig. 4j. p = 0.2157 for aISV. p = 0.2265 for PCV. (k) Quantifications of total ECs and PCV cell numbers in siblings and ddx21uq20bh mutants across 3 somites. p = 0.016 for EC (siblings n = 40, ddx21uq20bhmutants n = 28). p = 0.8012 for PCV (siblings and ddx21uq20bhmutants n = 10). aISV= arterial intersegmental vessel, DA = dorsal aorta, LEC = lymphatic endothelial cell, PCV = posterior cardinal vein, vISV= venous intersegmental vessel. Mean values + /-SEM shown except for (i) which shows median, quartiles, and maximum/minimum values. Two-tailed student’s t-test for (j, PCV) and (k). Two-tailed Mann Whitney test for (c-f) and (j, aISV). Kruskal-Wallis test with Dunn’s multiple comparisons test for (i).
