Integration of Bmp and Wnt signaling by Hopx specifies commitment of cardiomyoblasts

Making cardiomyocytes In the heart, multiple cell types work together. Cardiac progenitor cells give rise to cardiomyocyte, endothelial, or smooth muscle lineages. However, the identity of a marker specific to cardiomyocyte formation has been elusive. Jain et al. now identify a specialized progenitor population that is committed exclusively to forming cardiomyocytes. They also identify the niche signals that promote lineage commitment and the mechanisms involved in making cardiomyocytes. The findings may help in the development of future cell-based regenerative therapeutics for heart disease. Science, this issue 10.1126/science.aaa6071 Identification of the committed cardiomyoblast that retains proliferative potential may inform cardiac regenerative therapeutics. INTRODUCTION Cardiac progenitor cells are multipotent, and lineage analyses of murine and chick cardiac development have demonstrated that these cells give rise to the cardiac endothelium, smooth muscle, and cardiomyocytes. However, the mechanisms governing commitment to the myocyte lineage in vivo remain largely unknown. Further understanding of these mechanisms, and of the identity of progenitors committed to the myocyte lineage, may advance cardiac regenerative therapies. RATIONALE Hopx is an atypical homeodomain expressed in cardiac mesoderm shortly after cardiac progenitor cells are first evident. Previous studies have demonstrated that Hopx functions as a nuclear transcription co-repressor and is expressed in adult, +4 intestinal stem cells and hair follicle bulge stem cells. We compare lineage tracing of multipotent cardiac progenitor cells marked by Islet1 and Nkx2-5 expression with lineage tracing of Hopx+ cells. We also perform functional studies of Hopx from endogenous tissue and differentiated embryoid bodies to identify mechanisms promoting commitment and myogenesis. RESULTS We define and characterize a Hopx-expressing cardiomyoblast intermediate that is committed to the cardiomyocyte fate. Hopx+ is initially expressed in a subset of cardiac progenitor cells residing in the precardiac mesoderm prior to the expression of troponin T, a component of the contractile sarcomere apparatus of myocytes. Lineage-tracing experiments demonstrate that Hopx+ cells give rise to cardiac myocytes exclusively. Early Hopx+ cardiomyoblasts expand during cardiogenesis. Overexpression of Hopx in cardiac progenitor cells leads to an increase in myocytes, whereas Hopx deficiency compromises myogenesis. Whole-genome analysis reveals that Hopx occupies regulatory regions of multiple Wnt-related genes, and Hopx–/– cardiac tissues are characterized by an expansion of Wnt signaling. Restoration of Wnt levels during differentiation of Hopx–/– embryoid bodies partially rescues myogenesis. Wnt signaling is a potent regulator of stemness of cardiac progenitor cells, and our data suggest that Hopx promotes myogenesis by repressing Wnt signaling. Cardiac progenitor cells down-regulate Wnt signaling as they enter the cardiac outflow tract, coincident with the expression of Hopx. The outflow tract is also enriched for bone morphogenetic protein (Bmp) signaling, known to influence differentiation of myocytes. Hopx physically interacts with activated Smad complexes in vitro and in vivo. Exogenous Bmp4 represses Wnt signaling in cardiac explants, and Bmp4-mediated Wnt repression requires Hopx. Thus, Hopx functions to couple Bmp signaling to repression of Wnt. CONCLUSION Our work defines an intermediate cardiac progenitor that expresses Hopx and is committed exclusively to the myocyte fate. Therefore, akin to an erythroblast in hematopoietic differentiation, we have termed these committed cardiac progenitor cells “cardiomyoblasts. ” The ability to identify committed, but undifferentiated, cardiomyocyte precursors may facilitate development of cardiac regenerative therapies, including those using embryonic stem cells and induced pluripotent stem cells. Hopx functions to promote myogenesis by physically interacting with Smad proteins to repress Wnt signaling. Our findings raise the possibility that Hopx-mediated integration of Bmp signaling to repress Wnt may be active in other progenitor populations and may potentially underlie the tumor suppressor function of Hopx. Lineage tracing of Hopx+ cells. Images depicting lineage tracing of early Hopx+ cardiomyoblasts that give rise to myocytes in the left ventricle and atria. Some images are duplicated and pseudocolored. Cardiac progenitor cells are multipotent and give rise to cardiac endothelium, smooth muscle, and cardiomyocytes. Here, we define and characterize the cardiomyoblast intermediate that is committed to the cardiomyocyte fate, and we characterize the niche signals that regulate commitment. Cardiomyoblasts express Hopx, which functions to coordinate local Bmp signals to inhibit the Wnt pathway, thus promoting cardiomyogenesis. Hopx integrates Bmp and Wnt signaling by physically interacting with activated Smads and repressing Wnt genes. The identification of the committed cardiomyoblast that retains proliferative potential will inform cardiac regenerative therapeutics. In addition, Bmp signals characterize adult stem cell niches in other tissues where Hopx-mediated inhibition of Wnt is likely to contribute to stem cell quiescence and to explain the role of Hopx as a tumor suppressor.