Fig. 6

Heat-shock inducible Bmp2b expression partially rescues nfatc1 expression in hand2 mutants and MTZ-treated embryos.

(A-C) Endocardial nfatc1 expression is restored in heat-shocked hand2^-/-; hsp70:bmp2b transgenic embryos (C) when compared with hand2^-/- embryos (B) at 30hpf. Ubiquitous bmp2b induction does not rescue the shape of the endocardial cone. The embryos were obtained by crossing hsp70:bmp2b^+/?; hand2^+/-×hand2^+/- carriers in myl7:GFP background, where a mixture of heterozygous and homozygous hsp70:bmp2b adults were used; therefore, 50-100% of the embryos have hsp70:bmp2b transgene. hand2^-/- embryos were identified based on the bilateral myl7:GFP expression. (D-I) hsp70:bmp2b induces nfatc1 expression in hand2 mutants in the endothelial/endocardial cells. Confocal maximum intensity projections of flat-mounted embryos (ventral views) analyzed by fluorescent in situ hybridization/immunohistochemistry for nfatc1 expression (in situ hybridization, blue, E,F,H,I) and Cdh5/VE-Cadherin (immunofluorescence, red, D,F,G,I) at 24hpf. (F,I) Merged channels. (D-F) Wild-type embryo; (G-I) hand2^-/-; hsp70:bmp2b embryo. nfatc1 is expressed in the endocardial tube in E,F (arrows), whereas Cdh5 staining outlines the surfaces of endocardial cells, as well as endothelial cells of blood vessels. Patches of mislocalized nfatc1 expression are present in hand2^-/-; hsp70:bmp2b embryos (arrows, H,I), which overlap with Cdh5 staining, confirming their endocardial identity. (J-L) Partial rescue of nfatc1 expression at 46hpf stage in heat-shocked myl7:mCherry-NTR; hsp70:bmp2b transgenic embryos treated with MTZ to deplete the myocardium. nfatc1 expression is absent in the MTZ-treated myl7:mCherry-NTR embryo (arrow, K), and partially restored in a heat-shocked myl7:mCherry-NTR; hsp70:bmp2b embryo (arrows, L). Heat-shock or MTZ treatment alone did not affect nfatc1 expression (not shown).