Abstract
Notch signaling is a conserved cell fate regulator during development and postnatal tissue regeneration. Using skeletal muscle satellite cells as a model and through myogenic cell lineage-specific NICDOE (overexpression of constitutively activated Notch 1 intracellular domain), here we investigate how Notch signaling regulates the cell fate choice of muscle stem cells. We show that in addition to inhibiting MyoD and myogenic differentiation, NICDOE upregulates Pax7 and promotes the self-renewal of satellite cell-derived primary myoblasts in culture. Using MyoD−/− myoblasts, we further show that NICDOE upregulates Pax7 independently of MyoD inhibition. In striking contrast to previous observations, NICDOE also inhibits S-phase entry and Ki67 expression and thus reduces the proliferation of primary myoblasts. Overexpression of canonical Notch target genes mimics the inhibitory effects of NICDOE on MyoD and Ki67 but not the stimulatory effect on Pax7. Instead, NICD regulates Pax7 through interaction with RBP-Jκ, which binds to two consensus sites upstream of the Pax7 gene. Importantly, satellite cell-specific NICDOE results in impaired regeneration of skeletal muscles along with increased Pax7+ mononuclear cells. Our results establish a role of Notch signaling in actively promoting the self-renewal of muscle stem cells through direct regulation of Pax7.
SUPPLEMENTAL MATERIAL
Supplemental material for this article may be found at http://dx.doi.org/10.1128/MCB.06753-11.
ACKNOWLEDGMENTS
Contributions of the authors were the following: Yefei Wen, conception and design of experiments, collection and assembly of data, data analysis and interpretation, manuscript writing, and final approval of the manuscript; Pengpeng Bi, collection and assembly of data, paper revision, and final approval of the manuscript; Weiyi Liu, collection and assembly of data and final approval of the manuscript; Atsushi Asakura, provision of MyoD−/− primary myoblasts and final approval of the manuscript; Charles Keller, provision of Pax7-CreER mouse and final approval of the manuscript; Shihuan Kuang, conception and design of experiments, financial support, administrative support, provision of study materials, data analysis and interpretation, manuscript writing, and final approval of the manuscript.
We thank Yong-Xu Wang (University of Massachusetts School of Medicine) for the adeno-Cre virus, Philip Soriano (MSSM) for Myf5-Cre mice, and Kazuki Kuroda for the NICDOE plasmid. We appreciate the advice of Michael Rudnicki and the technical assistance of Jian Luo.
The project was supported by funding from the MDA, NIH, and USDA to S.K.
We declare no conflicts of interest.