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Figure 6
(A) Comparison of assay for transpose-accessible chromatin sequencing (ATAC-seq) peaks from wild-type (WT) and nr2f1a mutant amhc:EGFP+ cardiomyocytes at 48 hr post-fertilization (hpf), ~55 kb upstream of nkx2.5. Asterisk denotes canonical Nr2f binding site. (B) Schematic of –55 kb nkx2.5 enhancer reporter construct. (C) IHC for Tg(–55nkx2.5:EGFP) enhancer (green), Amhc (blue), and Vmhc (red) in hearts at 72 hpf. White arrow indicates –55nkx2.5:EGFP expression. (D–D’) IHC for –55nkx2.5:EGFP (green), Isl1 (purple), Amhc (blue), and sarcomeric myosin heavy chain (Mhc: pan-cardiac - red). The transgenic enhancer reporter is expressed in the ACs directly adjacent to the Isl1+ pacemaker cardiomyocytes (PCs). (E) IHC for Tg(–55nkx2.5:EGFP) enhancer (green), Amhc (blue), and Vmhc (red) in an nr2f1a mutant heart at 72 hpf. Expression of transgenic enhancer reporter is lost in nr2f1a mutants. Yellow arrow indicates venous pole devoid of –55nkx2.5:EGFP expression. (F) Comparison of percentage of WT and nr2f1a mutant embryos with –55nkx2.5:EGFP expression in the heart at 72 hpf WT (n=16); nr2f1a-/- (n=16), using Fisher’s exact test. (G,H) IHC for Nkx2.5 (green), Amhc (purple), and Mhc (red) in uninjected control and –55nkx2.5 crispant embryo hearts at 96 hpf. White arrow in (G) indicates the border of Nkx2.5+ cardiomyocytes extends close to the venous pole. Yellow arrow in (H) indicates that the Nkx2.5+ cardiomyocyte border is located farther from the venous pole of the atrium in crispant embryos. (I) Quantification of Nkx2.5+/Amhc+ cardiomyocytes in uninjected control (n=11) and –55nkx2.5 crispant (n=11) embryos at 96 hpf. (J,K) IHC for fgf13a:EGFP (green), Amhc (blue), and myl7:DsRed2-NLS (red) in uninjected control and –55nkx2.5 crispant Tg(fgf13a:EGFP)+ embryo hearts at 96 hpf. White arrows in (J) indicate the region of fgf13a:EGFP+ cardiomyocytes at the venous pole. Yellow arrows in (K) indicates that the region of fgf13a:EGFP+ cardiomyocytes is expanded from the venous pole in the atrium of crispant embryos. (L) Quantification of fgf13a:EGFP+/Amhc+ cardiomyocytes in uninjected control (n=10) and –55nkx2.5 crispant (n=11) embryo hearts at 96 hpf. Differences between uninjected control and –55nkx2.5 crispants were analyzed using Student’s t-test. Error bars indicate s.e.m. *p=0.05–0.001, **p<0.001. Scale bars in all images indicate 50 μm.
A putative nkx2.5 enhancer is expressed in atrial cardiomyocytes (ACs) adjacent to the sinoatrial node (SAN).