Nr2f1a maintains atrial nkx2.5 expression to repress pacemaker identity within venous atrial cardiomyocytes of zebrafish
- Molecular Genetics, Biochemistry, and Microbiology Graduate Program, University of Cincinnati College of Medicine, United States
- Molecular Cardiovascular Biology Division and Heart Institute, Cincinnati Children’s Hospital Medical Center, United States
- Divisions of Cardiovascular Medicine, Genetics and Network Medicine, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, United States
- Department of Pediatrics, University of Cincinnati College of Medicine, United States
Figures
Figure 1 with 4 supplements
The atrioventricular canal (AVC) resolves to Vmhc-expressing cardiomyocytes in nr2f1a mutant hearts.
(A) Schematic for isolation of atrial cardiomyocytes (ACs) using Tg(amhc:EGFP) transgene for RNA-seq and assay for transpose-accessible chromatin sequencing (ATAC-seq) at 48 hr post-fertilization …
Figure 1—figure supplement 1
Individual channels showing the resolution of atrioventricular canal (AVC) cardiomyocytes to ventricular cardiomyocyte (VC) identity in nr2f1a mutants.
(A–H’’) IHC for Amhc (blue) and Vmhc (red) at 30, 48, 72, and 96 hr post-fertilization (hpf) in wild-type (WT) and nr2f1a mutant embryos. Forty-eight to 96 hpf images (B–D) and (F–H) are the same as …
Figure 1—figure supplement 2
Quantification of cardiomyocytes in nr2f1a mutants.
(A) Quantification of only Amhc+/Vmhc- cardiomyocytes (CMs) within the wild-type (WT) and nr2f1a mutant hearts. (B) Quantification of only Amhc+/Vmhc+ (AVC) cardiomyocytes within the WT and nr2f1a …
Figure 1—figure supplement 3
Nr2f1a mutant hearts do not have increased cardiomyocyte death.
(A–D) z-Slices of IHC for cleaved Caspase 3 (green) and Mhc (red) in wild-type (WT) and nr2f1a mutant embryos. The cleaved Caspase 3+ cells shown in the heart were not cardiomyocytes. Number of …
Figure 1—figure supplement 4
Cardiomyocytes within the nr2f1a mutant atria do not have increased proliferation.
(A–F) z-Slices of IHC for phosphohistone H3 (pHH3) (green), Mhc (pan-cardiac; purple), and myl7:DsRed2-NLS (pan-cardiac; red) in wild-type (WT) and nr2f1a mutant hearts from 48 to 96 hr …
Figure 2 with 3 supplements
Pacemaker cardiomyocyte (PC) identity expands from the venous pole across the atrium in nr2f1a mutant hearts.
(A–F) IHC for Amhc (blue), Vmhc (red), and fgf13a:EGFP (green). White arrows indicate boundaries of Et(fgf13a:EGFP) expression within the atrium. Number of embryos examined - 48 hr …
Figure 2—figure supplement 1
Individual channels showing that pacemaker cardiomyocyte (PC) identity expands from the venous pole across the atrium in nr2f1a mutant hearts.
(A–F’’’) IHC for Vmhc (red), Amhc (blue), and fgf13a:EGFP (green) at 48, 72, and 96 hr post-fertilization (hpf). White arrows indicate boundaries of fgf13a:EGFP expression within the atrium. Images …
Figure 2—figure supplement 2
Isl1 expression expands throughout the atrium in nr2f1a mutant embryos.
(A–F) Isl1 (green), Amhc (purple), and Mhc (red) expression in wild-type (WT) and nr2f1a mutant embryos at 48, 72, and 96 hr post-fertilization (hpf). White arrows mark boundaries of Isl1 expression …
Figure 2—figure supplement 3
Newly differentiating cardiomyocytes are not added to the atrium in nr2f1a mutant hearts.
(A) Schematic depicting strategy for assessing newly differentiating cardiomyocytes using the Tg(myl7:NLS-KikGR) and Tg(fgf13a:EGFP) transgenes. (B–E”) Wild-type (WT) and nr2f1a mutant embryos …
Figure 3 with 7 supplements
Atrial cardiomyocytes (ACs) in nr2f1a mutants function as pacemaker cardiomyocytes (PCs).
(A) Schematic of point placements to measure heart rate (cyan) and action potential duration at 20% repolarization (APD20) (magenta) in a 48 hr post-fertilization (hpf) heart. (B) Representative …
Figure 3—figure supplement 1
Prolonged repolarization and decreased atrial conduction velocity in nr2f1a mutants compared to wild-type (WT) and heterozygous nr2f1a atria.
(A) Action potential duration at 20% repolarization (APD20) of WT, nr2f1a+/-, and nr2f1a-/- hearts. (B) Atrial velocity of WT, nr2f1a+/-, and nr2f1a-/- hearts. (C) Vmax of WT, nr2f1a+/-, and nr2f1a-/…
Figure 3—video 1
Heart from 48 hr post-fertilization (hpf) wild-type (WT) embryo.
Figure 3—video 2
Heart from 48 hr post-fertilization (hpf) nr2f1a mutant embryo.
Figure 3—video 3
Heart from 72 hr post-fertilization (hpf) wild-type (WT) embryo.
Figure 3—video 4
Heart from 72 hr post-fertilization (hpf) nr2f1a mutant embryo.
Figure 3—video 5
Heart from 96 hr post-fertilization (hpf) wild-type (WT) embryo.
Figure 3—video 6
Heart from 96 hr post-fertilization (hpf) nr2f1a mutant embryo.
Figure 4 with 2 supplements
Nkx2.5 expression recedes from venous pole in nr2f1a mutant atria.
(A–F’’’) IHC for Nkx2.5 (purple), Amhc (blue), and fgf13a:EGFP (SAN - green) in wild-type (WT) and nr2f1a mutant embryos from 48 to 96 hr post-fertilization (hpf). White arrows indicate border of …
Figure 4—figure supplement 1
Nkx2.5 is predominantly excluded from pacemaker cardiomyocytes (PCs) at the venous pole of the atrium.
(A–A”) IHC for Nkx2.5 (green), Isl1 (red), and Amhc (blue) in wild-type (WT) hearts at 72 hr post-fertilization (hpf) (n=4). White arrows indicate cardiomyocytes expressing both Nkx2.5 and Isl1. …
Figure 4—figure supplement 2
Nkx2.5:ZsYellow expression recedes toward the arterial pole of the atrium in nr2f1a mutants.
(A–D’’’) IHC for nkx2.5:ZsYellow (nkx:ZY - purple), Amhc (blue), and fgf13a:EGFP (green) in the atria of wild-type (WT) and nr2f1a mutant embryos. White arrows indicate border of nkx2.5:ZsYellow+ …
Figure 5 with 2 supplements
Nkx2.5 induction at 20 hr post-fertilization (hpf) represses pacemaker cardiomyocyte (PC) marker expansion in nr2f1a mutant atria.
(A) Schematic of timeline for heat-shock experiments. (B–I) IHC of representative hearts for Amhc (blue), fgf13a:EGFP (green), and myl7:DsRed2-NLS (red) used to quantify Amhc+/fgf13a:EGFP+ …
Figure 5—figure supplement 1
Induction of Nkx2.5 at 20 hr post-fertilization (hpf) does not rescue heart rate.
Quantification of heart rate in wild-type (WT) and nr2f1a mutant embryos at 96 hpf following induction of Nkx2.5 at the 20 somite stage; 96 hpf WT: Nkx2.5-EGFP- (n=7), Nkx2.5-EGFP+ (n=8); 96 hpf nr2f…
Figure 5—figure supplement 2
Nkx2.5-EGFP induction at 40 hr post-fertilization (hpf) partially represses the expansion of pacemaker cardiomyocyte (PC) identity.
(A) Schematic of heat-shock experiments. (B–I) IHC for Amhc (blue), fgf13a:EGFP (green), and myl7:DsRed2-NLS (red) of representative hearts from wild-type (WT) and nr2f1a mutant embryos with and …
Figure 6 with 3 supplements
A putative nkx2.5 enhancer is expressed in atrial cardiomyocytes (ACs) adjacent to the sinoatrial node (SAN).
(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 …
Figure 6—figure supplement 1
Peaks showing open chromatin surrounding representative loci from the assay for transpose-accessible chromatin sequencing (ATAC-seq) data.
(A–C) Regions of open chromatin using images from the UCSC genome browser were found at the promoters and adjacent to nr2f1a, tbx3a, and isl1 in the sorted amhc:EGFP+ cardiomyocytes from wild-type …
Figure 6—figure supplement 2
Deletion of the putative nkx2.5 enhancer in crispants.
(A,B) Transgenic Tg(–55nkx2.5:EGFP) control and crispant embryos at 96 hr post-fertilization (hpf). Images are lateral views with anterior to the left and dorsal up. White arrows in (A) indicate …
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Figure 6—figure supplement 2—source data 1
Uncropped gel pictures of PCR analysis for efficacy of the dgRNA pairs in generating deletions of the transgenic and endogenous –55nkx2.5 loci.
Numbers in the labeled gels indicate individual control uninjected and CRISPR/Cas9-injected embryos. Control uninjected embryos 6–8 and CRISPR/Cas9-injected embryos 1–3 are shown in Figure 6—figure supplement 2.
- https://cdn.elifesciences.org/articles/77408/elife-77408-fig6-figsupp2-data1-v1.zip
Figure 6—figure supplement 3
Nr2f1a is expressed throughout cardiomyocytes in the atrium.
(A–A”) IHC for Nr2f1a (purple), fgf13a:EGFP (green), Amhc (blue), and Mhc (red) in wild-type (WT) hearts at 72 hr post-fertilization (hpf) (n=7). White arrows indicate Nr2f1a+ and fgf13a:EGFP+ …
Figure 7
Model summarizing the consequences of Nr2f1a loss on atrial development.
Following its initial requirement promoting atrial cardiomyocyte (AC) differentiation (Duong et al., 2018), Nr2f1a is required to maintain AC identity within the arterial (outflow) and venous …
Additional files
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Supplementary file 1
(xlsx) Sheets with integrated and primary RNA-seq and assay for transpose-accessible chromatin sequencing (ATAC-seq) data with differential gene expression, changes in called peaks, and putative Nr2f binding sites.
- https://cdn.elifesciences.org/articles/77408/elife-77408-supp1-v1.xlsx
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Supplementary file 2
Antibody information.
- https://cdn.elifesciences.org/articles/77408/elife-77408-supp2-v1.xlsx
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Supplementary file 3
Primer information.
- https://cdn.elifesciences.org/articles/77408/elife-77408-supp3-v1.xlsx
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MDAR checklist
- https://cdn.elifesciences.org/articles/77408/elife-77408-mdarchecklist1-v1.pdf
