Figure 3. IFT cardiomyocyte number is unaffected by increased Hh signaling.

(A-D) Control uninjected embryos (A,B) and embryos injected with shh mRNA (C,D) are shown after immunofluorescence (A,C) or in situ hybridization (B,D) at 48 hpf. Frontal views; arrowheads indicate the IFT. (A,C) In Tg(myl7:H2A-mCherry) embryos, mCherry fluorescence (green) marks cardiomyocyte nuclei, and immunofluorescence reveals Amhc (magenta) and Isl1 (white) localization; red dashed lines outline the ventricle. (A’,C’) Isl1 localization is shown in white; white dashed lines outline the ventricle. Although injection with shh mRNA alters cardiac morphology, the population of Isl1+ cardiomyocytes is comparable in injected embryos and controls. (B,D) Embryos injected with shh mRNA retain bmp4 expression in the IFT (n=12) in a narrow ring similar to that in uninjected controls (n=9); black dashed lines outline the heart. Scale bars: 50 μm. (E,F) Graphs indicate the number of Isl1+ cardiomyocytes in the IFT (E) and the number of Isl1− atrial cardiomyocytes (F) at 48 hpf. See Materials and Methods for cell counting technique. Injection of shh mRNA increases the number of Isl1− atrial cardiomyocytes but does not change in the number of Isl1+ IFT cardiomyocytes relative to uninjected controls. **p<0.01.

(G-I) Fluorescent in situ hybridization indicating expression of ltbp3 (green) is combined with MF20 immunofluorescence (magenta); lateral views at 26 hpf, arrowheads indicate the arterial pole. In wt (G), ltbp3 is expressed in OFT progenitor cells located at the arterial pole (n=13). In embryos injected with shh mRNA (H), ltbp3 expression is expanded (n=9). In smo mutants (I), ltbp3 expression is typically absent (n=7/12) or reduced (n=4/12).