An in vivo repertoire of zebrafish cardiomyocyte-specific cis-regulatory elements [RNA-Seq]
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ABSTRACT: cis-Regulatory elements (cREs) are essential for the spatio-temporal control of gene expression during development and disease. However, cRE activity is highly dependent on cell and tissue type. The developing heart is composed of several cell-types, predominantly cardiomyocytes. Therefore, cardiomyocytes-specific modelling is required to understand the cis-regulation of the developing heart. Zebrafish are an ideal model to study heart development, as they share a number of physiological features with the human heart during development. Therefore, we present a comprehensive cardiomyocyte-specific repertoire of cREs isolated from zebrafish larvae. This data combines live transcriptomics and epigenetic profiling, providing insights into cREs and their associated genes involved in heart development. We further perform a transgenic reporter assay for the identified cREs of bmp10 and popdc2 genes, validating these genomic regions as cardiac regulatory elements. We share this comprehensive, reproducible cardiomyocyte-specific cRE resource as an interrogable web tool for understanding the epigenetic and transcriptomic mechanisms underlying heart development and emergence of congenital heart defects.
Project description:cis-Regulatory elements (cREs) are essential for the spatio-temporal control of gene expression during development and disease. However, cRE activity is highly dependent on cell and tissue type. The developing heart is composed of several cell-types, predominantly cardiomyocytes. Therefore, cardiomyocytes-specific modelling is required to understand the cis-regulation of the developing heart. Zebrafish are an ideal model to study heart development, as they share a number of physiological features with the human heart during development. Therefore, we present a comprehensive cardiomyocyte-specific repertoire of cREs isolated from zebrafish larvae. This data combines live transcriptomics and epigenetic profiling, providing insights into cREs and their associated genes involved in heart development. We further perform a transgenic reporter assay for the identified cREs of bmp10 and popdc2 genes, validating these genomic regions as cardiac regulatory elements. We share this comprehensive, reproducible cardiomyocyte-specific cRE resource as an interrogable web tool for understanding the epigenetic and transcriptomic mechanisms underlying heart development and emergence of congenital heart defects.
Project description:For a short period of time in mammalian neonates, the mammalian heart can regenerate via cardiomyocyte proliferation. This regenerative capacity is largely absent in adults. In other organisms, including zebrafish, damaged hearts can regenerate throughout their lifespans. Many studies have been performed to understand the mechanisms of cardiomyocyte de-differentiation and proliferation during heart regeneration however, the underlying reason why adult zebrafish and young mammalian cardiomyocytes are primed to enter cell cycle have not been identified. Here we show the primed state of a pro-regenerative cardiomyocyte is dictated by its amino acid profile and metabolic state. Adult zebrafish cardiomyocyte regeneration is a result of amino acid-primed mTOR activation. Zebrafish and neonatal mouse cardiomyocytes display elevated glutamine levels, predisposing them to amino acid-driven activation of mTORC1. Injury initiates Wnt/β-catenin signalling that instigates primed mTORC1 activation, Lin28 expression and metabolic remodeling necessary for zebrafish cardiomyocyte regeneration. These studies reveal a unique mTORC1 primed state in zebrafish and mammalian regeneration competent cardiomyocytes.
Project description:Congenital heart disease is among the most frequent major birth defects. Epigenetic marks are crucial for organogenesis, but their role in heart development is poorly understood. Polycomb Repressive Complex 2 (PRC2) trimethylates histone H3 at lysine 27, establishing H3K27me3 repressive epigenetic marks that promote tissue-specific differentiation by silencing ectopic gene programs. We studied the function of the catalytic subunit of PRC2, EZH2, in murine heart development. Early EZH2 inactivation by Nkx2-5Cre caused lethal congenital heart malformations, but slightly later EZH2 inactivation by cTNT-Cre did not. To study how the cardiomyocytes gene expression program is properly established in the early heart development, we combined the technologies of RNA sequencing and chromatin immunoprecipitation sequencing to identify the functional target genes directly repressed by EZH2. Intriguingly, these were enriched for transcriptional regulators of non-cardiac expression programs, such as transcription factors that regulate neuronal (Pax6) and cardiac progenitor genes (Isl1 and Six1). EZH2 was also required to maintain spatiotemporal regulation of cardiac gene expression, as Hcn4, Mlc2a, and Bmp10 were inappropriately upregulated in ventricular RNA. Furthermore, EZH2 was required for normal cardiomyocyte proliferation, establishing H3K27me3 epigenetic marks at cell cycle inhibitors Ink4a/b and repressing their expression. Our study reveals a previously undescribed role of EZH2 in regulating heart formation and shows that perturbation of the epigenetic landscape early cardiogenesis has sustained disruptive effects at later developmental stages. 8 E12.5 heart apex were used for RNA preparation each group.
Project description:During heart regeneration in the zebrafish, fibrotic tissue is replaced by newly formed cardiomyocytes derived from pre-existing ones. It is unclear whether the heart is comprised of several cardiomyocyte populations bearing different capacity to replace lost myocardium. Here, using sox10 genetic fate mapping, we identified a subset of pre-existent cardiomyocytes in the adult zebrafish heart with a distinct gene expression profile that expanded massively after cryoinjury. Genetic ablation of sox10+ cardiomyocytes severely impaired cardiac regeneration revealing that they play a crucial role for heart regeneration.
Project description:Heart failure is a leading cause of mortality and morbidity in the developed world, partly because mammals lack the ability to regenerate heart tissue. Whether this is due to evolutionary loss of regenerative mechanisms present in other organisms or to an inability to activate such mechanisms is currently unclear. Here, we decipher mechanisms underlying heart regeneration in adult zebrafish and show that the molecular regulators of this response are conserved in mammals. We identified miR-99/100 and Let-7a/c, and their protein targets smarca5 and fntb, as critical regulators of cardiomyocyte dedifferentiation and heart regeneration in zebrafish. Although human and murine adult cardiomyocytes fail to elicit an endogenous regenerative response following myocardial infarction, we show that in vivo manipulation of this molecular machinery in mice results in cardiomyocyte dedifferentiation and improved heart functionality after injury. These data provide a proof-of-concept for identifying and activating conserved molecular programs to regenerate the damaged heart. RNA-Seq expression profiles of rat cardiomyocytes after knockdown of miR-99/100 and Let-7 miRNAs
Project description:The heart suffers from a severe loss of cardiomyocyte after myocardial infarction (MI). However, it is not known which type of cell death is the major contributor of the loss of cardiomyocytes. By studying a mouse heart MI model at a regenerative and a non-regenerative stage, we demonstrate that ferroptosis, not apoptosis or necroptosis, is the major contributor to cardiomyocyte death starting at 1 day-post-MI. Intrinsic Pitx2 signaling in cardiomyocyte prevents ferroptosis. Meanwhile, cardiac fibroblasts expressing high level of Fth1 interact with cardiomyocytes to share the iron burden, therefore inhibit cardiomyocyte ferroptosis. Cardiomyocyte Pitx2 also inhibits Tsp1 expression, therefore negatively regulate fibroblast-to-myofibroblast activation to limit fibrosis.
Project description:Heart failure is a leading cause of mortality and morbidity in the developed world, partly because mammals lack the ability to regenerate heart tissue. Whether this is due to evolutionary loss of regenerative mechanisms present in other organisms or to an inability to activate such mechanisms is currently unclear. Here, we decipher mechanisms underlying heart regeneration in adult zebrafish and show that the molecular regulators of this response are conserved in mammals. We identified miR-99/100 and Let-7a/c, and their protein targets smarca5 and fntb, as critical regulators of cardiomyocyte dedifferentiation and heart regeneration in zebrafish. Although human and murine adult cardiomyocytes fail to elicit an endogenous regenerative response following myocardial infarction, we show that in vivo manipulation of this molecular machinery in mice results in cardiomyocyte dedifferentiation and improved heart functionality after injury. These data provide a proof-of-concept for identifying and activating conserved molecular programs to regenerate the damaged heart. Analysis of miRNA levels in regenerating zebrafish hearts
Project description:Unlike human hearts, zebrafish hearts efficiently regenerate after injury. Regeneration is driven by the strong proliferation response of its cardiomyocytes to injury. In this study, we show that active telomerase is required for cardiomyocyte proliferation and full organ recovery, supporting the potential of telomerase therapy as a means of stimulating cell proliferation upon myocardial infarction. Heart transcriptomes of WT and telomerase defective adult zebrafish animals were profiled by RNASeq, in control conditions and 3 days after heart cryoinjury.
Project description:While the heart regenerates poorly in mammals, efficient heart regeneration occurs in certain amphibian and fish species. Zebrafish has been used extensively to study heart regeneration, resulting in a model in which preexisting cardiomyocytes dedifferentiate and reinitiate proliferation to replace the lost myocardium. However, there is limited knowledge about the cellular processes that occur in this rare population of proliferating cardiomyocytes during heart regeneration. To identify such processes, we generated a transgenic line based on nppa expression that marks proliferating cardiomyocytes located at the wound border zone. Next we have used a single-cell RNA-sequencing approach in the regenerating adult zebrafish heart and we uncovered that proliferating border zone cardiomyocytes have very distinct transcriptomes compared to the non-proliferating remote cardiomyocytes and that they resemble embryonic cardiomyocytes. Moreover, these cells have reduced expression of mitochondrial genes and reduced mitochondrial oxidative phosphorylation activity, while glycolysis gene expression and glucose uptake are increased, indicative for metabolic reprogramming. Mechanistically, this metabolic reprogramming is induced by Nrg1/Erbb2 signaling and this mechanism is conserved in murine hearts. Furthermore, pharmacological inhibition of glycolysis impairs cardiomyocyte proliferation of both zebrafish and murine cardiomyocytes. Together these results reveal a conserved mechanism in which cardiomyocytes undergo metabolic reprogramming, which is essential for cardiomyocyte proliferation and heart regeneration and could ultimately help to develop novel methods to promote mammalian heart repair.
Project description:The Gata4 transcription factor is essential for normal heart development, but the molecular basis for its function remain poorly understood. We profiled at the whole genome level transcript changes in cardiomyocytes when Gata4 is depleted from zebrafish embryos. Our objective was to elucidate the cardiomyocyte-specific molecular program functioning downstream of Gata4 in order to better understand the role of Gata4 in cardiac morphogenesis. Six samples in total are deposited. Three replicate control samples and three replicate Gata4 morphant samples were analyzed.