Downloaded from genesdev.cshlp.org on October 3, 2021 - Published by Cold Spring Harbor Laboratory Press Oscillations of MyoD and Hes1 proteins regulate the maintenance of activated muscle stem cells Ines Lahmann,1,11 Dominique Bröhl,1,11 Tatiana Zyrianova,1 Akihiro Isomura,2 Maciej T. Czajkowski,1 Varun Kapoor,3 Joscha Griger,1 Pierre-Louis Ruffault,1 Despoina Mademtzoglou,4 Peter S. Zammit,5 Thomas Wunderlich,6 Simone Spuler,7 Ralf Kühn,8,9 Stephan Preibisch,3 Jana Wolf,10 Ryoichiro Kageyama,2 and Carmen Birchmeier1 1Developmental Biology/Signal Transduction, Max-Delbrück-Center for Molecular Medicine, 13125 Berlin, Germany; 2Institute for Frontier Life and Medical Sciences, Kyoto University, Kyoto 606-8507, Japan; 3Microscopy/Image Analysis, Max-Delbrück- Center for Molecular Medicine, 13125 Berlin, Germany; 4IMRB U955-E10, Institut National de la Santé et de la Recherche Médicale (INSERM), Faculté de Medicine, Université Paris Est, 94000 Creteil, France; 5Randall Centre for Cell and Molecular Biophysics, King’s College London, London SE1 1UL, United Kingdom; 6Max Planck Institute for Metabolism Research, 50931 Cologne, Germany; 7Muscle Research Unit, Experimental and Clinical Research Center, Max-Delbrück-Center, Charité Medical Faculty, 13125 Berlin, Germany; 8Transgenic Core Facility, Max-Delbrück-Center for Molecular Medicine, 13125 Berlin, Germany; 9Berlin Institute of Health, 10178 Berlin, Germany; 10Mathematical Modelling, Max-Delbrück-Center for Molecular Medicine, 13125 Berlin, Germany The balance between proliferation and differentiation of muscle stem cells is tightly controlled, ensuring the maintenance of a cellular pool needed for muscle growth and repair. We demonstrate here that the transcriptional regulator Hes1 controls the balance between proliferation and differentiation of activated muscle stem cells in both developing and regenerating muscle. We observed that Hes1 is expressed in an oscillatory manner in activated stem cells where it drives the oscillatory expression of MyoD. MyoD expression oscillates in activated muscle stem cells from postnatal and adult muscle under various conditions: when the stem cells are dispersed in culture, when they remain associated with single muscle fibers, or when they reside in muscle biopsies. Unstable MyoD oscillations and long periods of sustained MyoD expression are observed in differentiating cells. Ablation of the Hes1 oscillator in stem cells interfered with stable MyoD oscillations and led to prolonged periods of sustained MyoD expression, resulting in increased differentiation propensity. This interfered with the maintenance of activated muscle stem cells, and impaired muscle growth and repair. We conclude that oscillatory MyoD expression allows the cells to remain in an undifferentiated and proliferative state and is required for amplification of the activated stem cell pool. [Keywords: ultradian oscillation; MyoD; muscle stem cell; Hes1] Supplemental material is available for this article. Received November 20, 2018; revised version accepted February 19, 2019. Skeletal muscle has a remarkable regenerative capacity, They proliferate during the postnatal period and generate which is attributed to tissue resident stem cells (Lepper differentiating cells for muscle growth. In the adult, mus- et al. 2011; Sambasivan et al. 2011). Stem cells of the skel- cle stem cells are quiescent but become activated and pro- etal muscle represent a small cell population in the post- liferate upon muscle injury to generate new muscle tissue. natal muscle that were originally defined as satellite Understanding the mechanisms controlling the balance cells by their anatomical location between the basal lam- between proliferation and differentiation of muscle stem ina and plasma membrane of the myofiber (Mauro 1961). cells holds promise for regenerative medicine and is the These stem cells derive from myogenic progenitor cells subject of intense research. and are marked by Pax7 (Seale et al. 2004; Gros et al. Stem cell maintenance depends on exogenous sig- 2005; Kassar-Duchossoy et al. 2005; Relaix et al. 2005). nals. Notch signaling plays an important role in their maintenance in both development and the adult. Genetic ablation of Notch signaling by mutation of the genes 11These authors contributed equally to this work. Corresponding authors: [email protected], [email protected] Article published online ahead of print. Article and publication date are © 2019 Lahmann et al. This article, published in Genes & Development, online at http://www.genesdev.org/cgi/doi/10.1101/gad.322818.118. Free- is available under a Creative Commons License (Attribution-NonCom- ly available online through the Genes & Development Open Access mercial 4.0 International), as described at http://creativecommons.org/li- option. censes/by-nc/4.0/. 524 GENES & DEVELOPMENT 33:524–535 Published by Cold Spring Harbor Laboratory Press; ISSN 0890-9369/19; www.genesdev.org Downloaded from genesdev.cshlp.org on October 3, 2021 - Published by Cold Spring Harbor Laboratory Press Oscillations of MyoD and Hes1 in muscle cells encoding the ligand Dll1 or the transcriptional mediator ing of muscle stem cells expressing luciferase-reporters. of Notch signals, RBPj, results in up-regulated MyoD ex- Activated muscle stem cells on isolated muscle fibers pression, premature myogenic differentiation, and the and in muscle biopsies also displayed oscillatory MyoD depletion of the muscle stem cell pool (Schuster-Gossler expression. The oscillatory period was short, i.e., ∼3h, et al. 2007; Vasyutina et al. 2007; Bjornson et al. 2012; and thus much shorter than the cell cycle or circadian Bröhl et al. 2012; Mourikis et al. 2012b; Czajkowski et al. rhythm. In contrast, MyoD expression was sustained in 2014). Conversely, forced Notch activation suppresses differentiating cells. We also demonstrate that Hes1 myogenic differentiation (Kopan et al. 1994; Shawber et drives oscillatory MyoD expression: MyoD oscillations al. 1996; Kuroda et al. 1999; Delfini et al. 2000; Hirsinger become unstable and MyoD is sustained when Hes1 is ab- et al. 2001; Conboy and Rando 2002; Mourikis et al. lated. This was accompanied by a higher propensity of 2012a). Available evidence indicates that suppression of Hes1 mutant cells to differentiate. Our analysis indicates MyoD expression is an important aspect of Notch signal- that the oscillatory expression of MyoD allows activated ing, and that uncontrolled MyoD expression is responsible myogenic stem cells to remain in a proliferative state. for premature myogenic differentiation and the depletion However, when MyoD oscillations become unstable and of the muscle stem cell pool (Bröhl et al. 2012). are replaced by sustained MyoD expression, cells are driv- The bHLH transcription factor MyoD is a master regula- en out of the proliferating state and differentiate. Thus, os- tor of myogenic differentiation. Ectopic expression of cillatory MyoD expression allows for the amplification of MyoD in fibroblasts suffices to induce myogenic differen- the activated stem cell pool to ensure correct muscle tiation, demonstrating that sustained expression of MyoD growth and regeneration. activates the myogenic program (Weintraub et al. 1991). In vivo, MyoD cooperates with Myf5 and Mrf4 to control myogenesis, and MyoD is required for efficient muscle re- Results generation (Megeney et al. 1996; Kassar-Duchossoy et al. 2004). Many molecular inputs that modulate the balance Notch signals suppress myogenic differentiation and are between proliferation and differentiation in muscle stem required for the maintenance of the muscle stem cell cells, among them Notch signals, regulate MyoD and, pool (Vasyutina et al. 2007; Bröhl et al. 2012). The Hes/ thus, cellular behavior. MyoD is up-regulated when Notch Hey family of transcriptional repressors are important tar- signaling is ablated during development, leading to the get genes of the Notch pathway (Weber et al. 2014). Vari- loss and premature differentiation of myogenic progenitor ous members of the Hes/Hey family are activated by cells. This is demonstrated by the fact that muscle stem Notch signaling in muscle stem cells (Supplemental Fig. cells are maintained in double mutants where both S1A; see also Mourikis et al. 2012b). We systematically an- MyoD and Notch signals are ablated (Bröhl et al. 2012; alyzed mice with mutations in genes of the Hes/Hey fam- Czajkowski et al. 2014). However, the molecular mecha- ily in order to identify the functionally dominant members nism by which the Notch pathway suppresses MyoD func- of this family in skeletal muscle. Ablation of Hes1 (coHes1 tion and/or expression has remained open (Buas and mutant mice; see also Supplemental Fig. S1B; Supplemen- Kadesch 2010). tal Material) affected the number of Pax7+ muscle stem Recent studies in neuronal precursor cells demonstrate cells in late fetal development. In other tested mutants that Notch signaling components are expressed in an oscil- (i.e., Hey1, Hes5, and Hes7), no pronounced changes in latory manner, in particular the Notch ligand Dll1 and the the muscle or muscle stem cell numbers were apparent Notch target Hes1 (Shimojo et al. 2008). Furthermore, the (Supplemental Fig. S1B–H). Our results indicate that protein product of the pro-neural gene Ascl1 oscillates in Hes1 is an important and dominant member of the Hes/ neuronal precursor cells (Imayoshi et al. 2013). These mol- Hey family in the skeletal muscle although functional re- ecules oscillate with short periods