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Epigenetic control of coordinated hematopoietic and cardiovascular development by Rnf2 in zebrafish

XX Peng, G Feng, YH Sun

Preprint posted on December 16, 2020 https://www.biorxiv.org/content/10.1101/2020.12.14.422669v2

Polycomb orchestrates blood and heart development in zebrafish

Selected by Tim Petzold

Background

Haematopoiesis and cardiovascular development each depend upon highly regulated spatiotemporal gene expression programmes during embryogenesis. Furthermore, associations between these two processes have been demonstrated, such as the developing endocardium contributing to to haematopoiesis (Nakano et al, 2013). However, whether the factors which control haematopoiesis and cardiovascular development overlap, or are interconnected, is understudied. Zebrafish are a powerful system in which to study this, since they produce a large number of externally fertilized, rapidly developing embryos which can be easily manipulated with a large number of available genetic tools. In this preprint, Peng et al use zebrafish to investigate whether Polycomb group proteins play a role in cardiovascular and/or haematopoietic development. The authors report that the Polycomb protein Rnf2 is important for the maintenance of the delicately balanced genetic programmes required for cardiogenesis and haematopoiesis.

Key findings

Rnf2 represses haemato-endothelial gene expression and increases cardiac specification gene expression

The authors generated rnf2 mutant (rnf2-/-) zebrafish using CRISPR/Cas9 technology. Subsequently, they performed whole-mount in situ hybridisation to analyse the expression of haemato-endothelial and cardiac specification genes. Expression of the haemato-endothelial genes lmo2, tal1 and etv2 was found to be upregulated in rnf2-/- embryos in compared to controls during early embryogenesis, whilst expression of hand2, a gene involved in cardiomyocyte production, was reduced. The authors then examined the expression of these and other genes later during development (22-30 hpf). At these time points, the haematopoietic progenitor specification genes tal1 and etv2 were more highly expressed in rnf2-/- embryos whilst the expression of numerous genes required for cardiac chamber development, including nkx2.5 and tbx20, was reduced. Together, these data, along with a reduction in heart rate observed in the rnf2-/- embryos, suggest that loss of rnf2 results in upregulation of genes required for haemato-endothelial specification, whilst reducing both the expression of cardiogenesis-related genes as well as cardiac function.

rnf2 expression balances cardiac and HSC developmental gene expression programmes

Whole-mount in situ hybridisation was again utilised to further investigate cardiac development as well as primitive and definitive haematopoiesis in rnf2-/- embryos. Markers of primitive haematopoiesis showed that erythropoiesis is increased in the absence of rnf2. Furthermore, rnf2 mutants displayed an increase in expression of the HSC marker genes runx1 and cmyb. However, differentiation of HSCs into granulocytes, macrophages and erythrocytes was found to be defective. Next, the authors demonstrate that loss of Rnf2 not only results in edema, defective cardiac looping and bradycardia, but also in a disrupted antrial-ventricular canal structure, which may be due to the reduced expression of bmp4, alcama and vcana. Primitive heart valve development was also found to be affected in the rnf2 mutant, as determined by H&E staining.

Rnf2 directly regulates haemato-endothelial genes via its catalytic activity

By using both ChIP-seq and RNA-seq, Peng et al further explored how Rnf2 regulates the expression of cardiac and haemato-endothelial genes. Numerous cardiac specification and haemato-endothelial genes were shown to display Rnf2 ChIP-seq signals at their promoters. Notably however, the authors’ RNA-seq analyses demonstrated that in the rnf2 mutant embryos, expression of haemato-endothelial genes was elevated, something not found to be the case for cardiac specification genes. Subsequently, an elegant experiment was performed in which mRNA encoding a mutant Rnf2, lacking its H2A ubiquitination activity, was injected into one cell-stage rnf-/- embryos. Partial rescue of cardiac defects and the expression of a subset of cardiac chamber genes was observed in the injected embryos, suggesting that the catalytic activity of Rnf2 is important for the repression of key haemato-endothelial genes.

Significance

This preprint contributes to existing evidence that loss of Rnf2 results in cardiac development defects (Chrispijn et al, 2019), whilst providing the first strong evidence of haematopoietic defects in the absence of Rnf2 in zebrafish. There is increasing evidence for epigenetic regulation of cardiac development and haematopoiesis and this study not only enhances this, but also contributes to our knowledge of the molecular mechanisms governing cardiogenesis and haematopoiesis and how some of these may be interlinked. Together, the data in this work may contribute to the development of treatments for congenital heart defects and blood disorders such as leukaemia.

Open questions

1. The definitive haematopoiesis phenotypes in the rnf2 mutants are very intriguing, with HSC specification seemingly enhanced, whilst differentiation of these HSCs is blocked. I’m wondering:

a) Do the RNA- and ChIP-seq data indicate which downstream genes may be resulting in this HSC differentiation defect?

b) Which phenotypes (if any) are observed when injecting rnf2 mRNA into wild-type embryos to overexpress the gene?

2. Which do you propose results in the cardiac defects in the rnf2 mutants, the reduced expression in genes such as bmp4, alcama and vcana, or the ectopic expression of the haemato-endothelial genes nfatc1 and gata2 and the lymphoid commitment gene rag1 in the heart? Alternatively, do you think both contribute?

References

Nakano, H., Liu, X., Arshi, A., Nakashima, Y., van Handel, B., Sasidharan, R., Harmon, A.W., Shin, J.H., Schwartz, R.J., Conway, S.J., Harvey, R.P., Pashmforoush, M., Mikkola, H.K.A., and Nakano, A., (2013). Haemogenic endocardium contributes to transient definitive haematopoiesis. Nat Commun 4, 1564.

Chrispijn, N.D., Elurbe, D.M., Mickoleit, M., Aben, M., de Bakker, D.E.M., Andralojc, K.M., Huisken, J., Bakkers, J., and Kamminga, L.M. (2019). Loss of the Polycomb group protein Rnf2 results in derepression of tbx-transcription factors and defects in embryonic and cardiac development. Sci Rep 9, 4327.

 

 

Posted on: 18th January 2021 , updated on: 13th April 2021

doi: https://doi.org/10.1242/prelights.27011

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Author's responses

Professor Yuhau Sun shared

1. The definitive haematopoiesis phenotypes in the rnf2 mutants are very intriguing, with HSC specification seemingly enhanced, whilst differentiation of these HSCs is blocked. I’m wondering:

a) Do the RNA- and ChIP-seq data indicate which downstream genes may be resulting in this HSC differentiation defect?

The combined ChIP-seq and RNA-seq analysis may allow us to identify the DEGs that are directly regulated by Rnf2. As Rnf2 primarily functions as transcriptional repressor, we focused on the up-regulated DEGs. We found that 32 genes fits into the criteria (figure 7E). These genes can be grouped into 4 subgroups, which include hox family of transcription factors such as hoxa3a, hoxb4a, hoxb5a, hoxc5a, hoxd9a, hoxa9b; Neural genes such as nkx3.2, otx1b, otx2, pax1a, six2a, six3a, sox3; cardiac genes such as tbx2/3/18, and hematoendothelial progenitor genes such as etv2, tal1, fev and gata2a. The up-regulation of the hox, cardiac and neural genes in alternative lineages may disrupt the normal hematopoietic gene program, therefore blocking HSC differentiation. Also, the up-regulation of hematoendothelial progenitor genes may also inhibit the differentiation of HSCs. In wild type embryos, these progenitor genes need to be shut down to allow differentiation to occur. However, in the absence of Rnf2, these genes are persistently expressed, which may block or delay HSC differentiation.

To comprehensively understand the mechanism, one could perform single cell RNA sequencing for wild type and Rnf2-/- using hematopoietic-reporter fish lines.

b) Which phenotypes (if any) are observed when injecting rnf2 mRNA into wild-type embryos to overexpress the gene?

We have not done this experiment yet. And we would like to do this to see if there are heart and blood phenotypes.

2. Which do you propose results in the cardiac defects in the rnf2 mutants, the reduced expression in genes such as bmp4alcama and vcana, or the ectopic expression of the haemato-endothelial genes nfatc1 and gata2 and the lymphoid commitment gene rag1 in the heart? Alternatively, do you think both contribute?

This is a good question. We think that the cardiac defects at later time points (say 48 and 72 hpf) are the consequences of both direct and indirect effect by loss of Rnf2. In this manuscript, we are actually trying to propose the earliest possible direct effect: the up-regulation of Rnf2-bound hematoendothelial progenitor genes.

One important observation was that the expression of Rnf2-bound cardiac specifying genes (except tbx2/3) remained largely unchanged in mutant hearts at 36 and 48 hpf when cardiac phenotypes are already manifest, suggesting that normal cardiac program must be disturbed earlier in the absence of Rnf2. Based on this work, we tentatively propose that the up-regulation of hematoendothelial progenitor genes might be responsible for that, which can begin as early as early somitogenesis in the ALPM where hematoendothelial progenitors are known to antagonize cardiac progenitors. In addition, the hematoendothelial progenitor genes are persistently expressed in the embryonic heart (after 24 hpf) to repress cardiac genes. Thus, we have addressed the earliest gene expression change that may affect cardiogenesis in zebrafish, which is epigenetically controlled by PRC1/Rnf2.

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