Fig. 4

The amount of cardiac jelly is increased in double mutant embryos and the AVC expansion is downstream of Bmp4 signalling. (A) Representative confocal stacks of the cardiac jelly in siblings versus double mutant embryos (ventral view, head towards the top), demonstrating thicker cardiac jelly (outlined) in double mutant embryos at 72 hpf. (B) Dot plot depicting the measurement of cardiac jelly area, showing no significant difference (ns) in jelly thickness of ventricles of siblings versus mutants, whereas significantly more jelly in the atrium of double mutants compared with siblings is observed (n=10; *P<0.05; two-tailed t-test). Data are mean±s.d. (C) Representative images of embryos following in situ hybridisation for has2 expression in sibling and double mutant embryos. Images show categories of has2 expression in embryos at 72 hpf. The heart is outlined. (D) The proportion of embryos with different categories of has2 expression in wild-type (n=13), double heterozygotes (n=34) and double mutant (n=10) embryos in uninjected controls or in wild-type (n=5), double heterozygotes (n=9) and double mutant (n=8) embryos following bmp4 knockdown. The proportion of phenotypes in double mutant embryos injected with bmp4 morpholino most closely resembles uninjected double heterozygotes, demonstrating restoration of has2 expression upon bmp4 knockdown. (E) Representative confocal stacks of Tg(ubi:ssNcan-EGFP) to visualise the cardiac jelly in siblings versus double mutant embryos at 72 hpf, either uninjected or injected with bmp4 morpholino. (F) Dot plots depicting the measurement of cardiac jelly area in atria of sibling versus double mutant embryos. A significant increase in the jelly area is observed between uninjected siblings and double mutants (n=11; ***P<0.01; two-way ANOVA for multiple comparisons with Tukey's post hoc correction). No significant difference (ns) is observed in the jelly area between siblings and double mutants following bmp4 morpholino injection (n=16 siblings; n=11 double mutants), demonstrating that the increase in the cardiac jelly thickness is restored following bmp4 morpholino injection. Two-tailed t-test; data are mean±s.d. Scale bar: 30 μm.