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The cNRE drives expression in atrial cells. Mutational analysis of the SMyHC III promoter in zebrafish reveals a dual role in atrial activation and ventricular repression. A Schematic representation of the SMyHC III promoter sequence highlighting the position of the cNRE sequence and mutated sites. B Confocal images in frontal views, anterior to the top, of a representative zebrafish embryo. Ventricular expression is demonstrated by overlapping eGFP expression driven by SMyHC IIIΔcNRE and stable mCherry fluorescence driven by the ventricular stable line. C Representative panel of eGFP expression patterns in cardiac chambers of zebrafish embryos in lateral views, anterior to the left, injected with SMyHC III::eGFP. (at) atrium. (vt) ventricle. D Graphic representation of eGFP chamber expression patterns of cohorts of embryos injected with SMyHC III::eGFP, SMyHC IIIΔcNRE::eGFP, and constructs containing point mutations in the cNRE Hexads A, B, and C (Mut A, B, and C, respectively) as well as a non-Hexad control mutation (Mut S). Embryos were analyzed at 48 h post-fertilization (hpf) and classified into three categories of cardiac expression patterns: exclusive atrium (at), exclusive ventricular (vt), and atrium plus ventricular (at+vt). chi-square test, ΔcNRE: **p = 0.0026; ****p < 0.0001, Mut A: ****p < 0.0001, Mut B: ***p = 0.0002; ****p < 0.0001, Mut C: ***p = 0.0005, Mut S: *p = 0.0388 (vt); *p = 0.0116 (at+vt), comparing SMyHC III to each mutation and condition. E Frontal views, anterior is to the top, mouse embryos. SMyHC III::HAP (line 5, n = 18) isolated heart at 10.5 days post coitum (dpc), showing intense, dark blue, atrial coloring indicative of conspicuous HAP expression. F HAP assays in homogenates of atrial and non-atrial cardiac tissues in SMyHC III::HAP (n = 18), unpaired t test, p < 0.0001. GSMyHC IIIΔcNRE::HAP (line 110, n = 16) isolated heart at 10.5 dpc, showing absence of HAP expression. H HAP assays in homogenates of atrial and non-atrial cardiac tissues in the SMyHC IIIΔcNRE::HAP mutant (n = 16), unpaired t test, p = 0.0013. I Confocal image in lateral view, anterior is to the left, of a representative zebrafish embryo. Exclusive ventricular eGFP expression is observed at 48 hpf when injected with the vmhc promoter. J Confocal image in lateral view, anterior is to the left, of a representative zebrafish embryo. Expression of eGFP is detectable in both heart chambers at 48 hpf when injected with the 5xcNRE-vmhc construct. K Graphical analysis of chamber expression patterns of the cohort of embryos injected with vmhc or 5xcNRE-vmhc promoter constructs. (at) atrium. (vt) ventricle. chi-square test, p < 0.05, comparing vmhc::eGFP and 5xcNRE-vmhc::eGFP embryos for each condition. Scale bars are 30 µm.
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