FIGURE 9.

Transcription profiles and apoptosis of tsu3994 mutant ECs. A–C, transcription profiling of tsu3994 ECs. kdrl:GFP⁺ ECs were sorted from tsu3994;Tg(kdrl:GFP) mutant or sibling embryos at 44 hpf for RNA-Seq analysis. A, gene numbers mapped to the zebrafish genome assembly version Zv10 in different samples. B, number of differentially expressed genes (DEGs) between the two samples (>2-fold with a false discovery rate of <0.001). C, Gene Ontology term categories with enriched expression for up-regulated genes in mutant ECs. D–F, expression levels of several vesicle coat genes and ER stress/apoptosis-related genes were analyzed at indicated stages by real time quantitative PCR. *, p < 0.05; **, p < 0.01; ns, statistically nonsignificant. G, tsu3994 embryos have more apoptotic endothelial cells. tsu3994;Tg(kdrl:GFP) Mt and wild-type sibling (Sib) embryos were immunostained for active caspase 3 (red) and GFP (green) at 36 and 48 hpf. A trunk region was shown. The apoptotic endothelial cells in mutants were indicated by arrowheads. Note that mutant embryos also had more apoptotic cells outside the vasculature that are likely to include blood cells leaking from broken vessels and other types of cells. The average number of Caspase 3⁺ endothelial cells within four intersegmental vessels at comparable regions in Mt and siblings was shown on the right. H, effect of GSK2656157 and TUDCA treatments and knockdown of several apoptotic genes on hemorrhage in tsu3994 mutants. The treated or MO-injected embryos were subjected to O-dianisidine staining at 48 hpf. Following photograph, embryos were individually genotyped, and the mutants were classified into three categories as exemplified on the top. n, number of observed embryos; ***, p < 0.001.