Fig. 4

Hhex is required cell-autonomously in endothelial cells to promote venous and lymphatic sprouting in zebrafish. a, b Transplantation of Tg(fli1ep:DsRedEx) donor cells into TgBAC(etv2:EGFP) hosts derived from hhex^+/− incrosses. Wild-type endothelial cells contribute to arteries, veins, and lymphatics in wild-type sibling (a) and mutant (b) hosts at 5 dpf (arrowheads point to vISVs; asterisks indicate TD). c, d Quantification of vISVs (c) and TD extensions (d) across four somites in hhex^−/− larvae with transplanted wild-type cells (n = 13) vs. hhex^−/− larvae without transplanted wild-type cells (n = 6) at 5 dpf. Wild-type endothelial cells can partially rescue both vISV and TD formation in hhex^−/−. e hhex endothelial overexpression strategy using the fli1a promoter partially rescues the hhex^−/− vascular phenotype (arrowheads point to vISVs; asterisks indicate TD). f, g Quantification of vISVs (f) and TD (g) extensions across four somites in hhex^−/− (n = 6) and Tg(fli1a:tdTomato-2A-hhex); hhex^−/− (n = 6). h RNA sequencing of 48 hpf FACS-sorted hhex^−/− endothelial cells and hhex-overexpressing endothelial cells. Heat map comparisons between these datasets identify Hhex as a regulator of genes implicated in lymphatic specification (prox1a, prox1b, mafba, sox18, nr2f2) and Flt4 signaling (nrp2a, nrp2b, flt4, vegfc) while endothelial cell markers are modulated only by endothelial-specific hhex overexpression. Values represent means ± s.e.m. ****P ≤ 0.0001 and ***P ≤ 0.001 by t-test. Scale bars: 50 μm