Fig. 4

Epicardium-specific inactivation of shha expression during heart development.

(A) Semi-qRT-PCR analysis of purified cardiomyocytes (CM) and epicardial cells (Epi) from 96 hpf hearts (see also Flow Cytometry, Materials and methods). Cardiomyocyte (vmhc) and epicardial (tcf21) markers were used to confirm the specificity of cell sorting. (B) Semi-qRT-PCR analysis of shha and ptch1 expression in hearts dissected from tcf21:CreER; shha^ct/ct embryos treated with the vehicle (−) or 4-HT (+). (C) Phenotype of tcf21:CreER; shha^ct/ct embryos treated with the vehicle or 4-HT. Severe cardiac edema was observed in 4-HT–treated embryos at 96 hpf (six abnormal in eight analyzed; right, arrowheads) but not in vehicle-treated embryos (zero abnormal in eight analyzed; left, arrowheads; n = 8 each; p<0.01, Fisher’s exact test). (D) Immunofluorescence of heart sections obtained from vehicle- or 4-HT–treated tcf21:CreER; shha^ct/ct embryos. Insets, single-channel images of Mef2 immunofluorescence. Dotted yellow lines in insets depict the outline of the ventricle. (E) Quantification of Mef2⁺ nuclei from the sections obtained from the vehicle- or 4-HT–treated tcf21:CreER; shha^ct/ct embryos in D (n = 13 and 12). The data are presented as the mean ± SEM (***p<0.001, Mann–Whitney U test). (F) Semi-qRT-PCR analysis of epicardial marker gene expression in hearts dissected from tcf21:CreER; shha^ct/ct embryos treated with the vehicle (−) or 4-HT (+). (G) Immunofluorescence staining of heart sections obtained from vehicle- or 4-HT–treated tcf21:CreER; shha^ct/ct embryos. Raldh2 immunofluorescence was detected in tcf21:DsRed2⁺ epicardial cells in vehicle-treated embryos (left, arrowheads) but not in 4-HT-treated embryos (right, arrowheads). Bottom panels, single-channel images of Raldh2 immunofluorescence. (H) Semi-qRT-PCR analysis of the expression of myocardial growth factor genes in hearts dissected from tcf21:CreER; shha^ct/ct embryos treated with the vehicle (−) or 4-HT (+). (I) Semi-qRT-PCR analysis of shha-dependent myocardial growth factor genes in purified cardiomyocytes (CM) and epicardial cells (Epi) obtained from 96 hpf hearts. Single confocal sections are shown in D and G. ve, ventricle. Scale bar, 10 μm.