Cell fate decisions of pluripotent embryonic stem (ES) cells are dictated by activation and repression of lineage-specific genes. Numerous signaling and transcriptional networks progressively narrow and specify the potential of ES cells. Whether specific microRNAs help refine and limit gene expression and, thereby, could be used to manipulate ES cell differentiation has largely been unexplored. Here, we show that two serum response factor (SRF)-dependent muscle-specific microRNAs, miR-1 and miR-133, promote mesoderm formation from ES cells but have opposing functions during further differentiation into cardiac muscle progenitors. Furthermore, miR-1 and miR-133 were potent repressors of nonmuscle gene expression and cell fate during mouse and human ES cell differentiation. miR-1's effects were in part mediated by translational repression of the Notch ligand Delta-like 1 (Dll-1). Our findings indicate that muscle-specific miRNAs reinforce the silencing of nonmuscle genes during cell lineage commitment and suggest that miRNAs may have general utility in regulating cell-fate decisions from pluripotent ES cells.
Guiding multipotent cells into distinct lineages and controlling their expansion remain fundamental challenges in developmental and stem cell biology. Members of the Wnt pathway control many pivotal embryonic events, often promoting self-renewal or expansion of progenitor cells. In contrast, canonical Wnt ligands are thought to negatively regulate cardiomyogenesis in several species. However, the cell-autonomous role of canonical Wnt signaling within precardiac mesoderm, through its obligatory transcriptional mediator, -catenin, is unknown. Using tissue-specific in vivo genetic manipulation, we found that -catenin is required for development of cardiac progenitors and is a positive regulator of proliferative expansion of such progenitor cells. At discrete windows of development in embryonic stem cells, activation of canonical Wnt signaling promoted expansion of cardiac progenitors after initial commitment and was required for cardiac differentiation. Together, these data provide in vivo and in vitro evidence that canonical Wnt signaling promotes the expansion of cardiac progenitors and differentiation of cardiomyocytes.cardiac development ͉ embryonic stem cells ͉ -catenin ͉ second heart field
The regulation of multipotent cardiac progenitor cell (CPC) expansion and subsequent differentiation into cardiomyocytes, smooth muscle, or endothelial cells is a fundamental aspect of basic cardiovascular biology and cardiac regenerative medicine. However, the mechanisms governing these decisions remain unclear. Here, we show that Wnt/β-Catenin signaling, which promotes expansion of CPCs1–3, is negatively regulated by Notch1-mediated control of phosphorylated β-Catenin accumulation within CPCs, and that Notch1 activity in CPCs is required for their differentiation. Notch1 positively, and β-Catenin negatively, regulated expression of the cardiac transcription factors, Isl1, Myocd and Smyd1. Surprisingly, disruption of Isl1, normally expressed transiently in CPCs prior to their differentiation4, resulted in expansion of CPCs in vivo and in an embryonic stem (ES) cell system. Furthermore, Isl1 was required for CPC differentiation into cardiomyocyte and smooth muscle cells, but not endothelial cells. These findings reveal a regulatory network controlling CPC expansion and cell fate that involve unanticipated functions of β-Catenin, Notch1 and Isl1 that may be leveraged for regenerative approaches involving CPCs.
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