Fig. S3

Overexpression of Xenopus dominant-negative Brg1 (dn-xBrg1) inhibits brg1 function in zebrafish. (a) Schematic of the Tol2-based construct of conditional expression of dn-xBrg1 driven by the heat-shock promoter 70 (hsp70). (b) Western blots showing that heat-induced dn-xBrg1 protein increased in transgenic adult hearts (tg) compared with those in non-transgenic wild-type siblings (wt) with (+HS) or without heat shock (-HS), or transgenic adult hearts without heat shock (-HS). Alpha-actin served as a loading control. (-) HS, without heat shock; (+) HS, with heat shock. (c-f) Heart tube became stenotic and abnormal looping in tg embryos compared with wt sibling embryos at 48 hpf after heat shock (30 min each time at 5 hpf, 17 hpf, 29 hpf, and 41 hpf) or wt and tg embryos without heat shock. The heart tube was labeled by in situ hybridization with cmlc2, vmhc, amhc, or nppa probes. In situ hybridization showing that cardiac genes bmp4, tbx2b, and notch1b were abnormally expressed in tg embryos compared with wt sibling embryos after heat shock (30 min each time at 9 hpf, 33 hpf and 57 hpf) (g, h, i). Note the expanded bmp4 (g) and tbx2b (h) domains and decreased expression of notch1b in the atrioventricular canal of tg embryos (i). (j) Heart tube, labeled by cmlc2, became stenotic but the atrium and ventricle were specified in tg embryos compared with wt sibling embryos at 60 hpf after heat shock. Red brackets indicate expression domains of atrioventricular canal markers (bmp4 and tbx2b); black arrowheads point to decreased expression of notch1b in tg embryos; number of the right-upper corners showing the number of phenotypic embryos out of the total embryos analyzed. Scale bars, 100 μm.