Nr2f1a maintains atrial nkx2.5 expression to repress pacemaker identity within venous atrial cardiomyocytes of zebrafish

  1. Kendall E Martin
  2. Padmapriyadarshini Ravisankar
  3. Manu Beerens
  4. Calum A MacRae
  5. Joshua S Waxman

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    Evaluation Summary:

    This manuscript will be of interest to developmental biologists and pediatric cardiologists. Addressing the role of NR2F transcription factors in the fish heart, it provides novel insight into atrial chamber patterning and the formation of pacemaker cells. High-quality data are presented supporting the novel finding of a requirement of nr2f1a for restricting the production of pacemaker cells. Yet, data are currently not conclusive in claiming transdifferentiation of atrial cells in the mutants.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #3 agreed to share their name with the authors.)

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Abstract

Maintenance of cardiomyocyte identity is vital for normal heart development and function. However, our understanding of cardiomyocyte plasticity remains incomplete. Here, we show that sustained expression of the zebrafish transcription factor Nr2f1a prevents the progressive acquisition of ventricular cardiomyocyte (VC) and pacemaker cardiomyocyte (PC) identities within distinct regions of the atrium. Transcriptomic analysis of flow-sorted atrial cardiomyocytes (ACs) from nr2f1a mutant zebrafish embryos showed increased VC marker gene expression and altered expression of core PC regulatory genes, including decreased expression of nkx2.5 , a critical repressor of PC differentiation. At the arterial (outflow) pole of the atrium in nr2f1a mutants, cardiomyocytes resolve to VC identity within the expanded atrioventricular canal. However, at the venous (inflow) pole of the atrium, there is a progressive wave of AC transdifferentiation into PCs across the atrium toward the arterial pole. Restoring Nkx2.5 is sufficient to repress PC marker identity in nr2f1a mutant atria and analysis of chromatin accessibility identified an Nr2f1a-dependent nkx2.5 enhancer expressed in the atrial myocardium directly adjacent to PCs. CRISPR/Cas9-mediated deletion of the putative nkx2.5 enhancer leads to a loss of Nkx2.5-expressing ACs and expansion of a PC reporter, supporting that Nr2f1a limits PC differentiation within venous ACs via maintaining nkx2.5 expression. The Nr2f-dependent maintenance of AC identity within discrete atrial compartments may provide insights into the molecular etiology of concurrent structural congenital heart defects and associated arrhythmias.

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  1. Version published to 10.7554/elife.77408 on eLife
  2. eLife

    Author Response

    Reviewer #1 (Public Review):

    Following previous publications showing that NR2F2 controls atrial identity in the mouse and human iPS cells, the authors address in the fish the role of the transcription factor Nr2f1a, which is specific to the atrial chamber. This had been initiated in a previous publication (Duong et al, 2018) and is extended in this manuscript. In mutant fish, the atrial chamber is smaller and mispatterned. Markers of the atrioventricular canal and of the pacemaker are expanded. Transcriptomic analyses and electrophysiological measures further support this observation. A putative enhancer of nkx2.5 is identified by ATAC-seq and shown to be repressed in nr2f1a mutants, suggesting that Nkx2.5, a known repressor of pacemaker identity, may be a mediator of Nr2f1a. Overexpression of nkx2.5 delays the appearance of pacemaker cells, and is proposed to partially rescue the absence of nr2f1a.

    Overall, this work provides novel insight into the mechanism of atrial chamber patterning in the fish and discusses the conservation of the role of nr2f1a. However, the claim that atrial cells switch their identity into ventricular and pacemaker cells is currently not demonstrated. Alternative hypotheses of mispatterning, cell number changes by proliferation, survival, or ingression are not ruled out by the data presented. The claim that "Nr2f1a maintains atrial nkx2.5 expression" or of a "progressive loss of Nkx2.5 within the ACs" needs to be further supported. The definition of "atrial cells (AC)" varies between figures.

    Major comments:

    1. The definition of "AC" varies from figure to figure: amhc+ in Fig 1A, amhc+vmhc- in Fig.1S1A, amhc+fgf13a- in Fig. 2 and 5, morphological area in Fig. 3. Please clarify how the atrial chamber is delineated in mutants in Fig. 3 since the avc constriction is not obvious.

    a. As stated in the response to Essential Revisions comment 1.B, we have tried to clarify the definitions of the cardiomyocytes populations in the revised text by indicating the specific markers used in the text and the figures. We then provide our interpretation for what this means regarding the different cardiomyocyte populations.

    b. Since the analysis of the electrophysiology cannot be performed with markers or the transgenic zebrafish embryos using GFP, we chose areas for analysis closer to the middle of the morphological atrium in the nr2f1a mutant and WT sibling control embryo hearts that would be consistent with having Amhc+ expression and fgf13a:EGFP+ transgenic and Isl1 markers that were found from the analysis with immunohistochemistry. This strategy was schematized in Figure 3A and is now explicitly stated on lines 266 and 267 of the revised manuscript.

    1. The claim of a switch in cell identity or transdifferentiation is not demonstrated. This would require cell tracking or single-cell transcriptomics. I don't see how "AVC (..) [is] resolving to ventricular identity", since amhc seems to be maintained throughout the atrial chamber at all stages. The claim that "the number of vmhc+ only cardiomyocytes progressively increased" is not supported by Fig1S1. The expansion of pacemaker cells may result from cell ingression at the arterial pole. This hypothesis is in keeping with the expression of nr2f1a outside the heart tube in putative atrial progenitors (Duong, 2018). The phenotype upon nkx2.5 overexpression may also be interpreted along this line: ingression of pacemaker cells is delayed. The claim that "PC identity progressively expands throughout nr2f1a mutant atria" is not supported by the quantifications of a mean of 12 fgf13a+amhc+ cells at 96hpf (Fig. 2H), which is as many as fgf13a-amhc+ cells (Fig. 2G) and a quarter of the total amhc+ cells in Fig. 1J. The schema in Fig 6 does not reflect quantifications at 96hpf, which indicate the persistence of amhc+vmhc+ cells, amhc+ only, or amhc+fgf13a- in Fig 1S1 and 2G.

    "We did not observe effects on cell death or proliferation in the hearts of nr2f1a mutants": please provide the data, since proliferation was shown to be affected in mouse mutants (Wu, 2013).

    a. As indicated above in our response to the Essential Revisions comment 1.D, our quantification of cardiomyocytes indicates there are progressively fewer Amhc+/Vmhc+ cardiomyocytes in the nr2f1a mutant hearts (Figure 1J-L). The total number of Vmhc+ cardiomyocytes (Amhc+/Vmhc+ and Amhc-/Vmhc+) cardiomyocytes is increased in the nr2f1a mutant hearts relative to the WT sibling hearts. However, the number of Vmhc+-only (Amhc-/Vmhc+) cardiomyocytes, which reflect the ventricles, does not increase significantly in the n2f1a mutants and are not statistically different than their WT siblings at each of the stages, despite their trending that way (Figure 1 – figure supplement 2C). The total number of cardiomyocytes in the nr2f1a mutant hearts also is not increasing during these stages (Figure 1L). Along with the lack of cardiomyocyte death or proliferation (Figure 1 – figure supplements 3 and 4), this suggests that these hearts have more total Vmhc+ cardiomyocytes and the addition of Vmhc+-only cardiomyocytes is primarily coming from the cardiomyocytes in the Vmhc+/Amhc+ atrioventricular canal progressively losing Amhc expression. As indicated in the response to Essential Revisions comment 1.D, we have provided the individual image channels in a revised Figure 1 – figure supplement 1 and proportions of Vmhc+ cardiomyocytes in Figure 1 – figure supplement 2D to help clarify this issue.

    b. Regarding the transdifferentiation vs ingression of newly-differentiating cardiomyocyte hypotheses for the expansion of pacemaker markers, was addressed in the response to Essential Revision comment 2. Please see that comment for how we addressed this concern.

    1. The claim that "Nr2f1a maintains atrial nkx2.5 expression" or of a "progressive loss of Nkx2.5 within the ACs" needs to be further supported by quantification of the number of nkx2.5 positive cells in nr2f1a mutants. It seems that some cells in Fig. 4 co-express nkx2.5 and pacemaker markers in the mutant, which questions the repressive role of Nkx2.5. Following the observation of an nkx2.5 enhancer active next to pacemaker cells in control heart but absent in nr2f1a mutants, shouldn't we expect a gap of nkx2.5 expression next to pacemaker cells in mutants? It is unclear why pacemaker cells express nr2f1a (Fig. 6S1) but not nkx2.5. This needs clarification.

    a. The repressive role of Nkx2.5 with respect to pacemaker identity has been well documented in zebrafish and mice (Colombo et al., 2018). Nkx2.5 and Isl1 expression at the venous pole of zebrafish hearts are predominantly mutually exclusive, although there are a few cardiomyocytes at their borders that the express both Nkx2.5 and pacemaker markers. We recgonize that there are still some Nkx2.5-expressing cardiomyocytes that overlap with the pacemaker maker cardiomyocytes in the nr2f1a mutant hearts, as shown in Figure 4F. However, the majority of these cardiomyocytes have lower expression than the adjacent cardiomyocytes that form a border and do not have overlapping expression. Furthermore, as shown in Figure 4D-F and Figure 4 – figure supplement 2, the overall effect appears to be a regression of Nkx2.5+ expression in cardiomyocytes and corresponding expansion of pacemaker markers from the venous pole from 48 though 96 hpf in the nr2f1a mutant hearts, consistent with the established role of Nkx2.5 in repressing pacemaker identity. In the revised manuscript, we have provided each of the individual channels for the images in Figure 4 to better allow visualization of the different cardiomyocyte markers and a new supplemental figure showing the predominantly mutually exclusive expression of Nkx2.5 and Isl1 at the venous pole of zebrafish embryo hearts (Figure 4 – figure supplement 1).

    b. The expression of Nkx2.5 within the heart, like any gene, is likely controlled by multiple different regulatory elements. It is not clear to us why Reviewer #1 feels one would expect to see a gap in expression between Nkx2.5+ and pacemaker cardiomyocytes in the nr2f1a mutant hearts, unless Nkx2.5 was not required to repress pacemaker identity or there was a significant delay between loss of Nkx2.5 and gain of pacemaker markers. As indicated in the response to Essential Revisions comment 3.C, in the revised manuscript, we show experiments in which we have deleted the putative nkx2.5 enhancer element and found there is a loss of Nkx2.5+ and gain of fgf13a:EGFP+ cardiomyocytes in the atrium, as one might expect if the enhancer promotes or maintains Nkx2.5 expression in atrial cardiomyocytes that border the pacemaker cardiomyocytes. In the revised manuscript, this experiment is described in the Results (lines 348-364 and included in a revised Figure 6 and new Figure 6 – figure supplement 2.

    c. Please see our response to Essential Revision comment 3.A regarding the issue of Nr2f1a expression in pacemaker cardiomyocytes.

  3. eLife

    Evaluation Summary:

    This manuscript will be of interest to developmental biologists and pediatric cardiologists. Addressing the role of NR2F transcription factors in the fish heart, it provides novel insight into atrial chamber patterning and the formation of pacemaker cells. High-quality data are presented supporting the novel finding of a requirement of nr2f1a for restricting the production of pacemaker cells. Yet, data are currently not conclusive in claiming transdifferentiation of atrial cells in the mutants.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #3 agreed to share their name with the authors.)

  4. eLife

    Reviewer #1 (Public Review):

    Following previous publications showing that NR2F2 controls atrial identity in the mouse and human iPS cells, the authors address in the fish the role of the transcription factor Nr2f1a, which is specific to the atrial chamber. This had been initiated in a previous publication (Duong et al, 2018) and is extended in this manuscript. In mutant fish, the atrial chamber is smaller and mispatterned. Markers of the atrioventricular canal and of the pacemaker are expanded. Transcriptomic analyses and electrophysiological measures further support this observation. A putative enhancer of nkx2.5 is identified by ATAC-seq and shown to be repressed in nr2f1a mutants, suggesting that Nkx2.5, a known repressor of pacemaker identity, may be a mediator of Nr2f1a. Overexpression of nkx2.5 delays the appearance of pacemaker cells, and is proposed to partially rescue the absence of nr2f1a.
    Overall, this work provides novel insight into the mechanism of atrial chamber patterning in the fish and discusses the conservation of the role of nr2f1a. However, the claim that atrial cells switch their identity into ventricular and pacemaker cells is currently not demonstrated. Alternative hypotheses of mispatterning, cell number changes by proliferation, survival, or ingression are not ruled out by the data presented. The claim that "Nr2f1a maintains atrial nkx2.5 expression" or of a "progressive loss of Nkx2.5 within the ACs" needs to be further supported. The definition of "atrial cells (AC)" varies between figures.

    Major comments:

    - The definition of "AC" varies from figure to figure: amhc+ in Fig 1A, amhc+vmhc- in Fig.1S1A, amhc+fgf13a- in Fig. 2 and 5, morphological area in Fig. 3. Please clarify how the atrial chamber is delineated in mutants in Fig. 3 since the avc constriction is not obvious.

    - The claim of a switch in cell identity or transdifferentiation is not demonstrated. This would require cell tracking or single-cell transcriptomics. I don't see how "AVC (..) [is] resolving to ventricular identity", since amhc seems to be maintained throughout the atrial chamber at all stages. The claim that "the number of vmhc+ only cardiomyocytes progressively increased" is not supported by Fig1S1. The expansion of pacemaker cells may result from cell ingression at the arterial pole. This hypothesis is in keeping with the expression of nr2f1a outside the heart tube in putative atrial progenitors (Duong, 2018). The phenotype upon nkx2.5 overexpression may also be interpreted along this line: ingression of pacemaker cells is delayed. The claim that "PC identity progressively expands throughout nr2f1a mutant atria" is not supported by the quantifications of a mean of 12 fgf13a+amhc+ cells at 96hpf (Fig. 2H), which is as many as fgf13a-amhc+ cells (Fig. 2G) and a quarter of the total amhc+ cells in Fig. 1J. The schema in Fig 6 does not reflect quantifications at 96hpf, which indicate the persistence of amhc+vmhc+ cells, amhc+ only, or amhc+fgf13a- in Fig 1S1 and 2G.
    "We did not observe effects on cell death or proliferation in the hearts of nr2f1a mutants": please provide the data, since proliferation was shown to be affected in mouse mutants (Wu, 2013).

    - The claim that "Nr2f1a maintains atrial nkx2.5 expression" or of a "progressive loss of Nkx2.5 within the ACs" needs to be further supported by quantification of the number of nkx2.5 positive cells in nr2f1a mutants. It seems that some cells in Fig. 4 co-express nkx2.5 and pacemaker markers in the mutant, which questions the repressive role of Nkx2.5. Following the observation of an nkx2.5 enhancer active next to pacemaker cells in control heart but absent in nr2f1a mutants, shouldn't we expect a gap of nkx2.5 expression next to pacemaker cells in mutants? It is unclear why pacemaker cells express nr2f1a (Fig. 6S1) but not nkx2.5. This needs clarification.

  5. eLife

    Reviewer #2 (Public Review):

    In this manuscript, Martin and colleagues use the zebrafish embryo model to evaluate how the transcription factor Nr2f1a regulates early heart development. Previous work by the authors' lab and others has shown that NR2F homologues in mice and zebrafish play important roles in maintaining an atrial cardiomyocyte identity and repressing ventricular fate. Mutation of NR2F2 in humans is associated with congenital heart disease, further underscoring the importance of this family of genes in heart development. Here the authors show via further analysis of a zebrafish nr2f1a mutant that two events are occurring in the atria: 1) and expansion of ventricular cardiomyocyte gene expression in the region of the atrioventricular canal; and 2) and expansion in cells with a pacemaker cardiomyocyte character at the venous pole of the heart. This second point is in particular a novel observation and the focus of this manuscript.

    Following transcriptome analysis that shows changes in gene expression associated with atrial, ventricular, and pacemaker cardiomyocytes in mutant nrf21a atria, detailed counts of cardiomyocytes coupled with analysis of markers of cardiomyocyte fates are used to show that changes in atrial cardiomyocyte cell state occur over 48 to 96 hours post-fertilization in nr2f1a mutants. Electrophysiological analysis of mutant hearts supports a model where pacemaker cardiomyocyte fate is expanded at the venous pole of mutant hearts, whereas cells at the atrioventricular canal behave more like ventricular cardiomyocytes. Mechanistically, it is shown that Nr2f1a may act to maintain the expression of nkx2.5 in atrial cardiomyocytes bordering pacemaker cells at the venous pole, consistent with the known role of Nkx2.5 in repressing pacemaker identity. This is supported by experiments showing that transgenic overexpression of nkx2.5 can rescue some aspects of atrial/pacemaker cell identity defects in nr2f1a mutants.

    Quite a bit is known with respect to NR2F1/2 in mammalian heart development, including the promotion and maintenance of atrial at the expense of ventricular cell identity. Dr. Waxman's group has also previously shown in zebrafish that Nr2f1a similarly promotes atrial identity. What is new here is a role for Nr2f1a in preventing an atrial to pacemaker fate switch at the venous pole of the heart via maintaining nkx2.5 expression. The question of how different regions (AVC vs venous pole) of the heart may respond differently to the same transcription factor (Nr2f1a in this case) is a truly interesting one, and the regionalized regulation of nkx2.5 by Nr2f1a shown in this work certainly provides another piece for this puzzle.

    Strengths:
    This work includes a comprehensive quantification of cell numbers along with multiple markers to describe the dynamics in changes of cell state in the developing atrium of nr2f1a mutants. This is coupled with detailed electrophysiological analyses that support the overall conclusion that an atrial to pacemaker CM cell state change is occurring in nr2f1a mutants. The analysis of a putative Nr2f1a-regulated nkx2.5 enhancer and the demonstration of the rescue of some aspects of pacemaker expansion in nr2f1a mutants by restoration of nkx2.5 expression present a compelling potential mechanism for Nr2f1a activity.

    Weaknesses:
    In some cases, there is a reliance on a few markers (the fgf13a:GFP transgene, in particular) to ascribe cell lineage/state. An examination of if nkx2.5 expression can rescue cardiac function and electrophysiology is not shown but would have been a great addition to this work.

  6. eLife

    Reviewer #3 (Public Review):

    Martin and coworkers have investigated the consequences of nr2f1a gene disruption for heart development, focusing on the period between 48 and 96 hours after fertilization. Nr2f1a is the functional homologue of mouse Nr2f2 (Coup-TFII) which was previously found to be required for atrial development and maintenance and suppression of ventricular gene expression and phenotype in the atrial compartments in mice. Using a marker (amhc-gfp), the transcriptomes of 48 hpf atrial cardiomyocytes wt and nr2f1a null mutants were defined. Among the differentially expressed genes, several markers of atrial identity were downregulated, of ventricular identity were upregulated (as expected) and some genes associated with pacemaker cardiomyocytes (sinoatrial node cardiomyocytes in mouse) were up (or de-)regulated.

    Using markers vmhc for ventricular cardiomyocytes, amhc for atrial cardiomyocytes, and fgf13-gfp (enhancer trap) for pacemaker cardiomyocytes, the number of cardiomyocytes of each type was counted at subsequent stages of development in wt and nr2f1a mutants. The authors found a strong reduction of atrial cells in mutants at 48, 72, and 96 hpf (all stages investigated). Notably, the amhc+ population did not change during these developmental stages in wt and mutant hearts (suppl fig 1A). Vmhc+ cell populations were not different between genotypes. In mutants, but not in wt, the number of fgf13a-gfp+ cells increased during development. The number of amhc+/fgf13a-gfp- cells declined only in mutants during development.

    The authors measured several functions of wt and mutant hearts and found the heart rate of mutants did not increase during development, whereas it does in wt, prolonged repolarization, prolonged AP20 (slower depolarization), atrial conduct howed differential accessibility (loss of accessibility in mutants) that also harboured a motif for Nr2f factors. When testing the function of the enhancer in an embryo reporter assay, they found that the enhancer was active in atrial cells adjacent to pacemaker cells and that the activity domain of the enhancer decreased in nr2f1a mutants. The authors conclude that nr2f1a maintains atrial identity and limits the differentiation of venous atrial cardiomyocytes into pacemaker cells by maintaining nkx2-5 expression.

    The study is well done, and the results interesting. While several observations in this study validate previous results (Nr2f1a/Nr2f2 function in determining atrial phenotype, suppressing ventricular gene expression, Nkx2-5 function to repress pacemaker program), the main novelties are the expansion of the pacemaker marker expression in the atrial domain in nr2f1a mutants, the identification of nkx2-5 as a target of Nr2f1a involved in the pacemaker phenotype expansion, and the identification of a putative Nr2f1a target enhancer. The transdifferentiation of atrial cells into pacemaker cells is less convincingly demonstrated, as is the phenotype of the pacemaker cells in mutants. An in-depth analysis of the ATAC-seq (and RNA-seq) analysis of wt and mutant atrial cells and its implications and comparison with epigenetic state data from other models is lacking, which is a missed opportunity, as this would provide novel insights complementing this study.

  7. Version published to 10.1101/2022.02.24.481762v1 on bioRxiv