Hopx and the Cardiomyocyte Parentage
Authors: Michael D Schneider, Andrew H Baker, Paul Riley
Journal: Molecular Therapy (2015)

The dissection of steps that pattern primitive mesoderm toward the cardiovascular lineages is important to our current understanding of cardiovascular development and can instruct regenerative approaches to heart repair.1Mercola M Ruiz-Lozano P Schneider MD Cardiac muscle regeneration: lessons from development.Genes Dev. 2011; 25: 299-309Crossref PubMed Scopus (137) Google Scholar Primitive precursors that can adopt a cardiovascular fate, but also a subset of other mesoderm, are marked by the transcription factor Mesp1 (refs. 2Bondue A Lapouge G Paulissen C Semeraro C Iacovino M Kyba M et al.Mesp1 acts as a master regulator of multipotent cardiovascular progenitor specification.Cell Stem Cell. 2008; 3: 69-84Abstract Full Text Full Text PDF PubMed Scopus (291) Google Scholar, 3Lescroart F Chabab S Lin X Rulands S Paulissen C Rodolosse A et al.Early lineage restriction in temporally distinct populations of Mesp1 progenitors during mammalian heart development.Nat Cell Biol. 2014; 16: 829-840Crossref PubMed Scopus (171) Google Scholar). Later precursors, which selectively give rise to two or more of the cardiovascular cell types (bi- and multipotent cardiovascular progenitor cells), are marked by the transcription factors Nkx2.5 or Isl1, indicators of the first and second heart-forming regions of the embryo ("heart fields").4Wu SM Fujiwara Y Cibulsky SM Clapham DE Lien CL Schultheiss TM et al.Developmental origin of a bipotential myocardial and smooth muscle cell precursor in the mammalian heart.Cell. 2006; 127: 1137-1150Abstract Full Text Full Text PDF PubMed Scopus (443) Google Scholar,5Moretti A Caron L Nakano A Lam JT Bernshausen A Chen Y et al.Multipotent embryonic isl1+ progenitor cells lead to cardiac, smooth muscle, and endothelial cell diversification.Cell. 2006; 127: 1151-1165Abstract Full Text Full Text PDF PubMed Scopus (822) Google Scholar,6Kattman SJ Huber TL Keller GM Multipotent flk-1+ cardiovascular progenitor cells give rise to the cardiomyocyte, endothelial, and vascular smooth muscle lineages.Dev Cell. 2006; 11: 723-732Abstract Full Text Full Text PDF PubMed Scopus (598) Google Scholar What has been missing, however, is a molecular feature that uniquely marks cells destined to become cardiomyocytes within the heart. To illustrate, mapping the descendants of cells expressing either Nkx2.5 or Isl1, using the Cre/lox system to indelibly label their progeny in vivo, marks virtually all cell types of the mature heart, including smooth muscle, endothelium, epicardium, and fibroblasts.1Mercola M Ruiz-Lozano P Schneider MD Cardiac muscle regeneration: lessons from development.Genes Dev. 2011; 25: 299-309Crossref PubMed Scopus (137) Google Scholar This ubiquity results from expression of Nkx2.5 and Isl1 in the proepicardial organ,7Zhou B von Gise A Ma Q Rivera-Feliciano J Pu WT Nkx2–5- and Isl1-expressing cardiac progenitors contribute to proepicardium.Biochem Biophys Res Commun. 2008; 375: 450-453Crossref PubMed Scopus (107) Google Scholar a specialized structure on the surface of the early heart that gives rise to the external lining of the heart (epicardium) and its derivatives (coronary smooth muscle, coronary endothelial cells, cardiac fibroblasts). In short, what is missing is a marker of the immediate and specific precursor of cardiomyocytes, rather than earlier multipotent cells. A recent report by Jain et al. now shows that an atypical homeodomain transcription factor, Hopx, lacking a motif for direct DNA binding, is restricted to the direct precursors of cardiomyocytes, and not to those of the various other cardiac cells.8Jain R Li D Gupta M Manderfield LJ Ifkovits JL Wang Q et al.Integration of Bmp and Wnt signaling by Hopx specifies commitment of cardiomyoblasts.Science. 2015; 348: aaa6071Crossref PubMed Scopus (95) Google Scholar A fundamental mechanistic understanding of the heart's developmental origins in early life is instrumental to facilitate the creation of cutting-edge therapeutic opportunities for regeneration of the heart. Among them is the design of strategies that counteract the responses of the heart to damage that impede self repair. These advances include emerging ways to (i) block the transcriptional program that is induced in stressed cardiomyocytes, and that contributes to myocyte dysfunction; (ii) override the block to cell proliferation in postmitotic cardiomyocytes, which confounds restorative growth after injury; (iii) drive nonmyocytes to a cardiac fate, using a cocktail of heart-forming transcription factors; (iv) graft cardiomyocytes created from pluripotent stem cells by sequential exposure to specific growth factors and their inhibitors so as to recapitulate native developmental signals; and (v) exploit dormant stem cells in adult hearts, for use either as a cell therapy product that is possibly superior to other grafted cell types, or, uniquely, as a target for activation in situ.1Mercola M Ruiz-Lozano P Schneider MD Cardiac muscle regeneration: lessons from development.Genes Dev. 2011; 25: 299-309Crossref PubMed Scopus (137) Google Scholar,9Xin M Olson EN Bassel-Duby R Mending broken hearts: cardiac development as a basis for adult heart regeneration and repair.Nat Rev Mol Cell Biol. 2013; 14: 529-541Crossref PubMed Scopus (333) Google Scholar,10Burridge PW Keller G Gold JD Wu JC Production of de novo cardiomyocytes: human pluripotent stem cell differentiation and direct reprogramming.Cell Stem Cell. 2012; 10: 16-28Abstract Full Text Full Text PDF PubMed Scopus (479) Google Scholar,11Smart N Bollini S Dube KN Vieira JM Zhou B Davidson S et al.De novo cardiomyocytes from within the activated adult heart after injury.Nature. 2011; 474: 640-644Crossref PubMed Scopus (522) Google Scholar,12Noseda M Harada M McSweeney S Leja T Belian E Stuckey DJ et al.PDGFRα demarcates the cardiogenic clonogenic Sca1+ stem/progenitor cell in adult murine myocardium.Nat Commun. 2015; 6: 6930Crossref PubMed Scopus (109) Google Scholar Hopx is a homeodomain protein that lacks residues for binding DNA, and was shown through loss-of-function mutations to be required for normal myocardial development in mice and zebrafish, its absence resulting in hyperplasia.13Chen F Kook H Milewski R Gitler AD Lu MM Li J et al.Hop is an unusual homeobox gene that modulates cardiac development.Cell. 2002; 110: 713-723Abstract Full Text Full Text PDF PubMed Scopus (231) Google Scholar Although not contacting DNA directly, Hopx is tethered indirectly by serum response factor and other DNA-binding proteins, and acts as a corepressor of genes to which it is bound13Chen F Kook H Milewski R Gitler AD Lu MM Li J et al.Hop is an unusual homeobox gene that modulates cardiac development.Cell. 2002; 110: 713-723Abstract Full Text Full Text PDF PubMed Scopus (231) Google Scholar through the recruitment of histone deacetylases (Hdacs) responsible for repressed chromatin.14Kook H Lepore JJ Gitler AD Lu MM Wing-Man Yung W Mackay J et al.Cardiac hypertrophy and histone deacetylase–dependent transcriptional repression mediated by the atypical homeodomain protein Hop.J Clin Invest. 2003; 112: 863-871Crossref PubMed Scopus (271) Google Scholar Hdacs also act as transcription factor deacetylases: e.g., corepression of Gata4 target genes by Hopx involves a ternary complex with Hdac2, the latter deacetylating Gata4 protein.15Trivedi CM Zhu W Wang Q Jia C Kee HJ Li L et al.Hopx and Hdac2 interact to modulate Gata4 acetylation and embryonic cardiac myocyte proliferation.Dev Cell. 2010; 19: 450-459Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar Jain et al. used a formidable series of refined genetic and genomic tools to investigate the involvement of Hopx in early cardiac development. These include the use of (i) epitope-tagged Hopx fused to green fluorescent protein, to track early expression; (ii) Hopx-driven Cre recombinase and tamoxifen-dependent Cre, for fate mapping; (iii) Hopx-deficient embryonic stem cell lines, for in vitro differentiation; (iv) transcriptome-wide expression profiling; (v) the identification of gene sequences bound indirectly by Hopx, a corepressor, in embryonic hearts by chromatin immunoprecipitation followed by massively parallel sequencing (ChIP-seq); and (vi) mass spectrometry of Hopx protein–protein complexes immunoprecipitated from epitope-tagged midgestation hearts. Hopx was expressed at the onset of heart formation following Nkx2.5 and Isl1, contributed to all four chambers of the heart (Figure 1), and preceded cardiac troponin T (Tnnt2), a gene encoding an early muscle-specific contractile (sarcomeric) protein. Fate mapping determined that most cardiomyocytes were derived from the Hopx+ lineage. Interestingly, those described as specialized at the inflow region, which is associated with the pulmonary veins, and the interatrial septum escaped this parentage, at least within the limits of the Cre/lox method used. This subset of cardiomyocytes might therefore be developmentally distinct. Ablation of Hopx reduced cardiomyocyte formation in differentiating embryonic stem cells, as measured in multiple ways. The presence of Tnnt2+ cells and beating clusters, and microarray expression profiling of myocyte-related genes were assessed. Analogously, in Hopx–/– embryos, Tnnt2 expression was delayed in the second heart field. Delayed cardiomyocyte formation and delayed Tnnt2 expression, rather than complete loss in both cases, combine to suggest functional redundancy in early myogenesis, although the compensatory players remain elusive. Notably, however, Hopx was more often associated physically with genes encoding Wnt ligands and the Wnt signaling pathway as opposed to sarcomeric genes as the most common targets of Hopx; Wnt2, Wnt5b, and Wnt6 expression was increased in embryoid bodies lacking the corepressor Hopx, whereas Hopx normally occupies the proximal promoter regions of these genes. Consistent with these findings, expression of the Wnt target gene Axin2 increased more than twofold in Hopx-null cells. XAV939, an inhibitor of the β-catenin-dependent Wnt pathway, restored normal levels of both Axin2 and Nkx2.5, and partially corrected the defects in sarcomeric gene expression (Myh6, Myh7, Myl7, Myl3, Tnnc1, Actn2, Actc1). From this it can be inferred that normal cardiomyogenesis requires inhibition of Wnt signals by Hopx. Hopx-interacting proteins included Hdac2, as expected, plus other members of the nucleosome-remodeling deacetylase complex. Less foreseeably, Hopx also bound Smad4, a transcription factor that is crucial to signaling by the transforming growth factor-β superfamily, and that was recovered together with Smad4 and activated (phosphorylated) Smad1/5/8 from midgestation embryo lysates. These ligand-activated partners of Smad4 are selective for bone morphogenetic proteins (BMPs), superfamily members that have particular importance to driving cardiac myogenesis. Confirming their physical association, Smad4 and Hopx were coenriched at the Wnt loci discussed above, suggesting further that inhibition of Wnt signals by Hopx is driven by developmentally and spatially regulated BMPs. As further proof of this circuit, the Wnt-responsive gene Axin2 was repressed by BMP4 in wild-type embryoid bodies but not in those lacking Hopx. A possibly similar function coupling BMPs to Wnt suppression and differentiation has been hypothesized for other progenitor cell types that express Hopx, including stem cells of the intestine and hair follicle.8Jain R Li D Gupta M Manderfield LJ Ifkovits JL Wang Q et al.Integration of Bmp and Wnt signaling by Hopx specifies commitment of cardiomyoblasts.Science. 2015; 348: aaa6071Crossref PubMed Scopus (95) Google Scholar The need to silence Wnt signaling for cardiac myogenesis to proceed is evident from diverse studies both in model organisms and in pluripotent stem cells, including those derived from humans.16Noseda M Peterkin T Simoes FC Patient R Schneider MD Cardiopoietic factors: extracellular signals for cardiac lineage commitment.Circ Res. 2011; 108: 129-152Crossref PubMed Scopus (93) Google Scholar The specificity of Hopx as a prospective marker of future cardiomyocytes, together with its biological role coupling BMPs to Wnt suppression, adds importantly to the developmental insights that can guide future strategies in cardiac regenerative medicine.1Mercola M Ruiz-Lozano P Schneider MD Cardiac muscle regeneration: lessons from development.Genes Dev. 2011; 25: 299-309Crossref PubMed Scopus (137) Google Scholar,8Jain R Li D Gupta M Manderfield LJ Ifkovits JL Wang Q et al.Integration of Bmp and Wnt signaling by Hopx specifies commitment of cardiomyoblasts.Science. 2015; 348: aaa6071Crossref PubMed Scopus (95) Google Scholar,9Xin M Olson EN Bassel-Duby R Mending broken hearts: cardiac development as a basis for adult heart regeneration and repair.Nat Rev Mol Cell Biol. 2013; 14: 529-541Crossref PubMed Scopus (333) Google Scholar,10Burridge PW Keller G Gold JD Wu JC Production of de novo cardiomyocytes: human pluripotent stem cell differentiation and direct reprogramming.Cell Stem Cell. 2012; 10: 16-28Abstract Full Text Full Text PDF PubMed Scopus (479) Google Scholar,11Smart N Bollini S Dube KN Vieira JM Zhou B Davidson S et al.De novo cardiomyocytes from within the activated adult heart after injury.Nature. 2011; 474: 640-644Crossref PubMed Scopus (522) Google Scholar,12Noseda M Harada M McSweeney S Leja T Belian E Stuckey DJ et al.PDGFRα demarcates the cardiogenic clonogenic Sca1+ stem/progenitor cell in adult murine myocardium.Nat Commun. 2015; 6: 6930Crossref PubMed Scopus (109) Google Scholar