Fig. 4

The cardiac and blood/endothelial programmes are cross-antagonistic. (A) Nkx2.5 RNA over-expression (50 pg) from the 1- to 2-cell stage reduced etsrp, scl and pu.1 and expanded hand2 expression in the anterior lateral plate mesoderm (ALPM; arrowheads) at the 7-somite stage. The anterior mesodermal marker gata6 was unaffected in the same cells. Nkx2.5-GFP fusion expression was visualised at the shield stage to confirm injection and expression. Dorsal views of 7-somite whole zebrafish embryos are shown, anterior to the top; for fluorescence, lateral views of shield stage whole embryos are shown, dorsal to the right. (B) An increase in endothelium and myelopoiesis was evident throughout development upon depletion of Fgf. Embryos treated with SU5402 at 3 hpf were assessed for endocardium by expression of kdrl and cdh5 at the 16-somite stage, whereas pu.1 was assessed for myeloid development. Increased endocardial (arrows) and myeloid gene expression was observed. (C) Similar treatment increased kdrl::GFP⁺ endothelial cell numbers, with concomitant loss of myocardium at 36 hpf (white arrow; stained with MF20 antibody). (D) Increased differentiated myeloid cells (l-plastin⁺) seen at 30 hpf. (E) Nkx2.5-induced downregulation of the haemangioblast programme is not sustained throughout development. Myeloid gene expression (pu.1) was downregulated at the 16-somite stage upon nkx2.5 over-expression, and at 26 hpf l-plastin remained depleted. However, expression of cmlc2, kdrl and cdh5 appeared similar to wild type at 26 hpf. Dorsal views are shown, anterior to the top, except in panel D and 26 hpf embryos in panel E in which lateral views are shown, anterior to the left. Scale bars: 100 μm.