Author response: Cellular localization of the cell cycle inhibitor Cdkn1c controls growth arrest of adult skeletal muscle stem cells (2018)

Article Figures and data Abstract Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Adult skeletal muscle maintenance and regeneration depend on efficient muscle stem cell (MuSC) functions. The mechanisms coordinating cell cycle with activation, renewal, and differentiation of MuSCs remain poorly understood. Here, we investigated how adult MuSCs are regulated by CDKN1c (p57kip2), a cyclin-dependent kinase inhibitor, using mouse molecular genetics. In the absence of CDKN1c, skeletal muscle repair is severely impaired after injury. We show that CDKN1c is not expressed in quiescent MuSCs, while being induced in activated and proliferating myoblasts and maintained in differentiating myogenic cells. In agreement, isolated Cdkn1c-deficient primary myoblasts display differentiation defects and increased proliferation. We further show that the subcellular localization of CDKN1c is dynamic; while CDKN1c is initially localized to the cytoplasm of activated/proliferating myoblasts, progressive nuclear translocation leads to growth arrest during differentiation. We propose that CDKN1c activity is restricted to differentiating myoblasts by regulated cyto-nuclear relocalization, coordinating the balance between proliferation and growth arrest. https://doi.org/10.7554/eLife.33337.001 Introduction Tissue regeneration is of vital importance for restoring tissue structure and function following damage. Skeletal muscle has a remarkable capacity to self-repair after severe injuries, a process dependent on muscle stem cells (MuSCs) (Relaix and Zammit, 2012). MuSCs originate from a PAX3/7+ progenitor cells that in late fetal life acquire their characteristic anatomical position between the basal lamina and the plasma membrane of muscle fibers (Mauro, 1961; Relaix et al., 2005). MuSCs are indispensable for postnatal muscle growth (White et al., 2010; Pawlikowski et al., 2015), maintenance (Keefe et al., 2015; Pawlikowski et al., 2015), and regeneration upon injury (Lepper et al., 2011; McCarthy et al., 2011; Murphy et al., 2011; Sambasivan et al., 2011). The majority of juvenile MuSCs acquire a non-proliferative, quiescent state around 3 weeks of post-natal life to ensure cellular/genomic integrity and long-term survival (Lepper et al., 2009; White et al., 2010; Cheung and Rando, 2013). However, once stimulated by homeostatic demand or damage, adult MuSCs exit quiescence, re-enter the cell cycle, and provide differentiated progeny for muscle repair, while a subpopulation self-renews to preserve the quiescent pool (Relaix and Zammit, 2012). The myogenic regulatory factors including MYOD and MYOGENIN orchestrate MuSCs commitment and progression through the myogenic lineage (Wang et al., 2014), while the signals that trigger cell cycle exit and (re-) entry into quiescence remain more elusive. Cell cycle is a tightly synchronized process responding to positive and negative signals, while inappropriate growth arrest can result in cancer, malformations during development, and defective stem cell renewal (Zhang et al., 1997; Matsumoto et al., 2011; Sherr, 2012). Cyclin-dependent kinase inhibitors (CDKIs) are negative cell cycle regulators. CDKIs are divided into two structurally and functionally defined families: the INK4 family (including Cdkn2a, Cdkn2b, Cdkn2c, and Cdkn2d) and the Cip/Kip family (including Cdkn1a, Cdkn1b, and Cdkn1c) (Borriello et al., 2011). Although members of the Cip/Kip family have been shown to inhibit proliferation and promote differentiation in embryonic muscle or in myoblasts in vitro, their involvement in postnatal MuSC cell cycle regulation is less well documented (Halevy et al., 1995; Reynaud et al., 1999; Zhang et al., 1999; Messina et al., 2005; Chakkalakal et al., 2014; Zalc et al., 2014). Given the emerging importance of Cdkn1c in stem cell quiescence (Matsumoto et al., 2011; Zou et al., 2011; Furutachi et al., 2013), we explored its role in adult myogenesis and MuSC-supported regeneration. Using mouse mutants and ex vivo analysis, we provide evidence that Cdkn1c is involved in the early phase following MuSC activation events, with its genetic ablation leading to impaired muscle regeneration associated with decreased differentiation and increased proliferation. We further show that Cdkn1c is not detected in quiescent MuSCs but is induced upon activation and maintained in differentiating myogenic cells. Finally, we show that Cdkn1c subcellular localization is specifically regulated during adult myogenesis, with a progressive cytoplasmic to nuclear translocation as activated myoblasts proceed to differentiation. Our results suggest that muscle stem cells require Cdkn1c activity for the dynamic control of growth arrest during adult myogenesis. Results Cdkn1c is required for postnatal myogenesis Although Cdkn1c loss is generally associated with perinatal death (Yan et al., 1997; Zhang et al., 1997; Susaki et al., 2009; Mademtzoglou et al., 2017), a few Cdkn1c mutant mice survived in a mixed CD1;B6 background (4.2%; Figure 1—figure supplement 1A). Cdkn1c-deficient mice displayed reduced body weight compared to control littermates (Figure 1—figure supplement 1B–C). However, there was no significant difference in forelimb grip strength between Cdkn1c mutant and control mice at 1 or 2 months of age when the strength was calculated on a per weight basis. Strength was even slightly higher in 3- or 4-month-old Cdkn1c mutant mice compared to controls (Figure 1—figure supplement 1D). This difference could be explained by the smaller body weight of Cdkn1c mutants, possibly leading to increased relative grip strength (N/kg) in mutants. To evaluate the role of CDKN1c in muscle homeostasis, we examined sections of the hindlimb Tibialis anterior (TA) muscles in adult mice. Histological analysis showed that Cdkn1c knock-out muscles contained smaller fibers and displayed increased fibrosis (Figure 1A–D), implying hindered myogenic differentiation. The amount of centrally located nuclei, indicative of ongoing regeneration, was comparable in mutants and controls (Figure 1E). Myofiber culture conditions used allow MuSCs to become activated, start dividing (T24-48), and eventually, proceed to myogenic differentiation or self-renewal of the quiescent pool (T72) (Zammit et al., 2004). The number of PAX7+ MuSC on freshly isolated myofibers of Extensor digitorum longus (EDLs) was increased in Cdkn1c mutant mice compared to the controls (Figure 1F–G). Furthermore, PAX7+ MuSCs on Cdkn1c mutant myofibers were mostly MYOD-, at a similar percentage to controls (Figure 1H), indicating that Cdkn1c is not regulating MuSCs quiescence. When single myofibers were cultured for 72 hr (T72), Cdkn1c mutants displayed an increased ratio of PAX7+ MYOD- self-renewing cells and a decreased ratio of PAX7-MYOD+ differentiating myoblasts (Figure 1I–J). Taken together, our data suggest that in the absence of CDKN1c the MuSC compartment is correctly established, but a proportion of the MuSC population undergo increased self-renewal at the expense of differentiation. Figure 1 with 1 supplement see all Download asset Open asset Cdkn1c deficiency impairs normal muscle growth. (A) Hematoxylin and Eosin (HE) and Sirius red staining of control (Ctrl) and Cdkn1c mutant (Cdkn1c-Mut) mouse Tibialis anterior (TA) muscles were performed to examine muscle histology, centrally located nucleated myofibers, and fibrosis. Scale bars, 100 μm. (B) Histogram showing the average of myofiber diameters (μm). (C) Histogram of average fibrotic area per TA muscle. (D) Fiber size (μm) distribution in control and Cdkn1c mutant mice. (E) Histogram of number of fibers with centrally located nuclei. (F) PAX7+ (green) MuSCs (arrows) on the myofibers isolated from EDL muscles of Cdkn1c mutant and control mice. MYOD (red) is not normally expressed in PAX7+ MuSCs at T0 (quiescence). DAPI (blue) shows all nuclei. Scale bars, 50 μm. (G) Numbers of PAX7+ satellite cells on the myofibers isolated from EDL. (H) Ratio of MYOD+ activated cells per PAX7+ MuSC on the myofibers isolated from EDL muscles of Cdkn1c mutant and control mice. (I) Immunofluorescence for PAX7 (green) and MYOD (red) at T72 in single myofiber cultures. Arrows and arrowheads show PAX7+MYOD- quiescent satellite cells and PAX7-MYOD+ differentiating cells, respectively. Scale bars, 50 μm. (J) Quantification of ratios of PAX7+ and MYOD+ cells per fiber at T72. Nuclei were counter-stained with DAPI. *p≤0.05, **p≤0.01. https://doi.org/10.7554/eLife.33337.002 Next, we evaluated the impact of CDKN1c loss on skeletal muscle regeneration. We performed intramuscular cardiotoxin (CTX) injections into the Tibialis anterior (TA) and sacrificed the mice at 3, (d3), 4 (d4), 7 (d7), and thirty (d30) days post-injury, to evaluate early and late time points of the regeneration procedure. Once muscle degeneration is induced, MuSCs undergo: (1) activation, (2) proliferation to expand their population, (3) self-renewal of the quiescent pool for future needs, and (4) differentiation for newly generated fibers and muscle repair (Relaix and Zammit, 2012). At d3 post-injury, loss of Cdkn1c promoted myoblasts proliferation and counteracted differentiation, as shown by increased EdU+ incorporation and reduced MYOD+EdU+ fraction, respectively. (Figure 2—figure supplement 1A,B). At d4 post-injury, Cdkn1c-deficient muscles showed smaller embryonic myosin (eMyHC)+ myofibers, a marker for early myofiber formation, compared to controls (Figure 2A,C). At d7 post-injury, Cdkn1c-deficient muscles showed increased cell infiltration and smaller and heterogeneous myofiber formation (Figure 2A,B,D), suggesting a delay in the regeneration process. At d30, signs of impaired regeneration associated with smaller fibers were observed (Figure 2A,B,E), including deposition of fibrotic tissue (Figure 2A,F) in Cdkn1c-mutant mice. Next, we evaluated the MuSC population from isolated myofibers of EDLs at d30 and TA muscle sections. The number of PAX7+ MuSCs was increased in Cdkn1c mutants compared to the controls (Figure 2G–H; Figure 2—figure supplement 1C,D) while the proportion of MYOD+ MuSCs was not altered (Figure 2I). Therefore, our data suggest that Cdkn1c is required for postnatal muscle repair. In addition, Cdkn1c mutant myogenic cells demonstrated increased propensity for stem-cell self-renewal during both tissue establishment and regeneration. Figure 2 with 1 supplement see all Download asset Open asset CDKN1c deficiency delays muscle regeneration. (A) Embryonic myosin (eMyHC)/LAMININ/DAPI, Hematoxylin and Eosin (HE), and Sirius red staining of twelve- to fifteen-week-old control (Ctrl) and Cdkn1c mutant mouse TA muscles were performed for histological and fibrosis characterization 4, 7 or thirty days after cardiotoxin (CTX) injection. Scale bars, 100 μm. (B) Fiber size (μm) distribution in control (Ctrl) and Cdkn1c mutant (Cdkn1c-Mut) mice 7 (upper panel) or thirty (lower panel) days after CTX injection. (C) Histogram of average embryonic MyHC+ fiber diameters (μm) 4 days after CTX injection. (D) Histogram of average fiber diameters (μm) 7 and thirty days after CTX injection. (E) Fiber size (μm) distribution in control and Cdkn1c mutant mice thirty days after CTX injection. (F) Histogram of average fibrotic area per TA muscle. (G) PAX7+ (green) MuSCs (arrows) on the myofibers isolated from EDL muscles of Cdkn1c mutant and control mice thirty days after CTX injection. MYOD (red) is occasionally expressed in PAX7+ MuSCs (arrow heads). DAPI (blue) shows all nuclei. Scale bars, 50 μm. (H) Numbers of PAX7+ MuSCs on the EDL isolated myofibers . (I) Ratio of MYOD+ activated cells per PAX7+ MuSC on the myofibers isolated from EDL muscles of Cdkn1c mutant and control mice. Nuclei were counter-stained with DAPI. Scale bars, 100 μm. *p≤0.05, **p≤0.01. https://doi.org/10.7554/eLife.33337.004 CDKN1c regulates the balance between proliferation and differentiation in activated myoblasts We next abrogated CDKN1c specifically in adult MuSC to avoid cell non-autonomous effects and bypass the impact of CDKN1c loss during development. To conditionally ablate Cdkn1c, we have generated a floxed Cdkn1c allele (Cdkn1cFlox) to enable conditional knock-out of Cdkn1c using the Cre/loxP system (Mademtzoglou et al., 2017). Given that Cdkn1c is an imprinted gene expressed only by the maternal allele (Matsuoka et al., 1995), we used heterozygotes with maternal inheritance of Cdkn1cFlox, hereafter indicated as Cdkn1cFlox(m)/+. To specifically target the MuSCs and their progeny, we intercrossed Cdkn1cFlox mice with the Pax7CreERT2 line (Lepper et al., 2009). In the compound mice, tamoxifen (TMX) administration results in CreERT2 translocation to the nucleus and Cdkn1c excision in the MuSC lineage. One week of TMX administration led to 84.5% recombination efficiency (Figure 3—figure supplement 1A–B). We then inserted Cdkn1cFlox and Pax7CreERT2 in the ROSAmTmG background (Muzumdar et al., 2007). These double-fluorescent mice report Cre activity by expressing membrane-Tomato (mT) prior to Cre-mediated excision and membrane-GFP (mG) after excision. This allowed us to selectively isolate recombined (GFP+) MuSC cells. We isolated by flow cytometry GFP+ MuSCs of control (Pax7CreERT2; Cdkn1c+/+; ROSAmTmG/+) and Cdkn1c-deficient (Pax7CreERT2; Cdkn1c Flox(m)/+; ROSAmTmG/+) mice. Collected cells grew in expansion conditions (20% fetal bovine serum, 10% horse serum) up to 70% confluence and then were serum-deprived to differentiate for 1 or 3 days (Figure 3A). Cdkn1c transcript and protein were induced by differentiation in control cells (Figure 3B–C). In contrast, they were not detected in cells from mutant animals (Figure 3B,D), demonstrating efficient Cre-mediated recombination of the Cdkn1c allele in FACS-isolated cells. We then performed primary myoblast cultures from control and Cdkn1c-deficient mice. We monitored proliferating cells under 'growth' (high serum) or 'differentiation' (low serum) conditions, and at d1 and d3 post-differentiation we stained with early (e.g. MYOGENIN) or late (i.e. Myosin heavy chain, MyHC) differentiation markers, respectively. Consistent with the function of CDKN1cas a cell cycle inhibitor, we observed increased proliferation in primary myoblasts derived from MuSC-specific Cdkn1c mutant mice. When cells were maintained in growth conditions for 5 days, we observed 50% and 38% more EdU+ and KI67+ cells, respectively, in the absence of CDKN1c (Figure 3E–H), suggesting that CDKN1c is involved in restraining cell cycle progression. Slightly more PAX7+ EdU+ cells were observed in the Cdkn1c-deficient myoblast cultures, while differences in MYOD+ EdU+ or MYOGENIN+ EdU+ populations did now show significant differences (Figure 3—figure supplement 1C–F). Furthermore, in cultures of FACS-isolated MuSCs from MuSC-specific Cdkn1c-deficient mice, myogenic differentiation was impaired. One-day post-differentiation, MYOGENIN was significantly decreased (Figure 3I,K,L). In addition, myotube formation 3 days post-differentiation was severely compromised (Figure 3J,M,N). We also detected a similar increase in cell proliferation and reduction in myogenic differentiation in primary myoblasts isolated from global Cdkn1c mutant mice (Figure 3—figure supplement 2). In conclusion, both MuSC-specific and global ablation of CDKN1c led to increased primary myoblasts proliferation at the expense of myogenic differentiation. Together, our data demonstrate that CDKN1c is required in MuSCs for correct cell cycle regulation and differentiation during postnatal myogenesis. Figure 3 with 2 supplements see all Download asset Open asset Cdkn1c deficiency impairs myogenic differentiation. (A) Time-course of tamoxifen (TMX) administration, muscle satellite cell harvest (FACS arrow) and culture (light gray bar for growth culture conditions, dark gray bar for differentiation culture conditions). Analyzed animals were Pax7CreERT2/+; Cdkn1cFlox(m)/+;RosamTmG (Cdkn1c cKO) and Pax7CreERT2/+; Cdkn1c+/+;RosamTmG (control; Ctrl); maternal inheritance of the imprinted Cdkn1c is indicated by superscript (m). (B) Cdkn1c transcript levels of control and Cdkn1c cKO myoblast cultures 3 days post-differentiation. ND; not detected. (C–D) Control (C) and Cdkn1c cKO (D) myoblast cultures were examined for CDKN1c protein (red) following three days under differentiation conditions. (E–N) Control and Cdkn1c cKO myoblast cultures were examined for EdU+ (light blue) cells (E, G, H), KI67+ cells (F), MYOGENIN+ cells (green; I, K, L), and myotube formation (J, M, N). Nascent myotubes were marked with myosin heavy chain (MyHC; green; M, N). Nuclei were counter-stained with DAPI (blue). Graphs show quantification of EdU and KI67 expression under growth conditions (E, F), MYOGENIN expression following 24 hr under differentiation conditions (I), and MyHC+ cells following 72 hr under differentiation conditions (J). Data show mean +SD, n = 3 animals. Asterisks indicate significance; *p≤0.05, ***p≤0.001. Scale bars, 40 μm (C, K), 1000 μm (G, M). https://doi.org/10.7554/eLife.33337.006 Satellite-cell-specific CDKN1c loss compromises muscle regeneration We next evaluated the impact of MuSC-specific Cdkn1c ablation, driven by Pax7CreERT2 (Lepper et al., 2009), on skeletal muscle regeneration after injury. Following 4 weeks of tamoxifen administration to induce recombination in adult (8- to 12-week-old) mice, we injected control and Pax7CreERT2/+; Cdkn1cFlox (Cdkn1c cKO) TA muscles with CTX and evaluated muscle repair at 7 days post-injury (Figure 4A), having achieved 94.5% recombination efficiency in MuSCs (Figure 4—figure supplement 1A–B). Of note, Pax7CreERT2/+ (hereafter, Cre control) animals only express one allele of Pax7, as the Cre recombinase is inserted into the Pax7 gene. Hence, in all experiments we also included Cre control mice, as we found that Pax7CreERT2 (Lepper et al., 2009) heterozygous mice display a mild regeneration phenotype (Figure 4B–G'), hence potentially acting as sensitizing background. Histological analysis at d7 post-injury showed normal tissue repair in wild-type (Wt) muscles (Figure 4B,C). In contrast, Cre control muscles showed increased cell infiltration and reduced regenerated myofibers of smaller sizes compared to Wt (Figure 4B'; Figure 4—figure supplement 1C–D), while muscle regeneration was severely compromised in Cdkn1c cKO muscles, in which newly formed myofibers were rare and small and most of the tissue consisted of cell infiltration and fat deposits (Figure 4B'', C''; Figure 4—figure supplement 1C–D). Furthermore, induction of early differentiation was delayed, as evidenced by the presence of embryonic eMyHC+ fibers in Cdkn1c cKO and Cre control, but not in Wt muscles at this time point (Figure 4D–E''). Consistent with the lack of tissue regeneration, we observed a massive diminution of the MuSC compartment in Cdkn1c cKO muscles (Figure 4F–H). Together, our data reveal that CDKN1c expression in the myogenic lineage is required for muscle repair. Figure 4 with 1 supplement see all Download asset Open asset MuSC-specific Cdkn1c ablation hinders muscle regeneration. (A) Time-course of tamoxifen administration, intramuscular injury of TA muscle (CTX arrow), and muscle harvest (D7 arrow). (B–G) Cryosections of TA muscle were stained for histological and satellite cell population characterization 7 days after CTX injection. Analyzed animals at (B-G) were wild-type littermates (Wt; Pax7+; Cdkn1c+; B–G), Cre control (Pax7CreERT2; B'–G'), and Cdkn1c cKO (Pax7CreERT2; Cdkn1cFlox; B''–G''). (B) HE staining for histologic characterization of the muscles. (C) Oil Red O staining for evaluation of fat infiltration of the muscles. (D–E) embryonic myosin (eMYHC, red)/LAMININ (LAM, green) immunofluorescence to mark newly formed myofibers post-regeneration. (F–G) PAX7 (red)/LAMININ (LAM, green) immunofluorescence to mark PAX7+ satellite cells. Nuclei in (D-G) were counter-stained with DAPI (blue). Scale bars, 50 μm. (H) Quantification of (F-G). Data show mean +SD, n ≥ 5 animals. Asterisks indicate significance; **p≤0.01. https://doi.org/10.7554/eLife.33337.009 Dynamic CDKN1c expression in adult myogenesis To analyze the expression of CDKN1c during MuSC-mediated myogenesis, we isolated single myofibers with their associated PAX7+ MuSCs from EDL muscles of adult wild-type mice. Immunostaining experiments, using a CDKN1c-specific antibody, showed that quiescent MuSCs were not labeled (Figure 5—figure supplement 1A). Similarly, MuSCs of resting adult TA muscles were CDKN1c-negative following labeling of cryosections (Figure 5—figure supplement 1B). To evaluate the kinetics of CDKN1c expression during activation, self-renewal, and differentiation, we analyzed myofibers that were cultured from 24 to 72 hr. Zammit et al., 2004During the initial proliferation state (T24-T48) MuSCs co-express PAX7 and MYOD (Zammit et al., 2004). Activated PAX7+ and MYOD+ myoblasts at T24-T48 presented with increasing amounts of CDKN1c. Unexpectedly, this negative cell cycle regulator was not expressed in the quiescent population, but in activated MuSCs entering the cell cycle. However, during this early proliferation phase, CDKN1c was restricted to the cytoplasm (Figures 5A–B and 6A–B). Consistent with these findings, CDKN1c was mainly restricted to the cytoplasm at d3 following CTX injection in vivo, a time point when most cells are in a proliferative state (Figure 5C). Figure 5 with 1 supplement see all Download asset Open asset Cdkn1c cytoplasmic expression after satellite cell activation. (A) Satellite-cell-derived myoblasts (T24–T48) of single EDL myofibers stained with PAX7 (green) and CDKN1c (red). Arrowheads indicate PAX7+ cells. (B) Satellite cell-derived myoblasts of single EDL myofibers stained with MYOD (green) and CDKN1c (red). Arrowheads indicate MYOD+ cells. Nuclei were counter-stained with DAPI. n ≥ 3. Scale bars, 40 μm. (C) CDKN1c (red) staining of TA muscle at 3 (D3) or t (D13) days after CTX injection. Asterisks indicate regions with cytoplasmic CDKN1c. # indicates central nuclei of newly formed fibers during muscle regeneration. Nuclei were counter-stained with DAPI. Scale bars, 20 μm. https://doi.org/10.7554/eLife.33337.011 Figure 6 with 2 supplements see all Download asset Open asset Cdkn1c expression and subcellular localization during satellite cell activation and differentiation. (A–D) Immunofluorescence for PAX7 (A; green), MYOD (B; green), MYOGENIN (C; green) or KI67 (D; green) and Cdkn1c (red) at T72 in single myofiber cultures of EDL muscles and quantification of PAX7+ (A), MYOD+ (B), MYOGENIN+ (C) or KI67+ (D) cells that co-expressed CDKN1c over the time-course of the culture. Cytoplasmic (light blue) or nuclear (dark blue) localization of CDKN1c is indicated in the graphs. Scale bars, 40 μm. (E) Immunofluorescence for Cyant fluorescent protein (CFP, green), KI67 (purple), and CDKN1c (red) in transduced (i.e. CFP+) myoblasts at T72 in single myofiber cultures. Fibers were transduced with empty (control; expressing Cdkn1c (Cdkn1c panel) or localization Cdkn1c (F) Quantification of transduced myoblasts that were proliferating Nuclei were counter-stained with DAPI (blue). Scale bars, 20 μm. Data show mean +SD, n ≥ 3 compared to control At of cells at are including differentiation of PAX7 expression associated with expression of MYOD and MYOGENIN) and self-renewal of loss of and lack of MYOGENIN) (Zammit et al., 2004). there is a mixed population of self-renewing expressing PAX7 activated/proliferating PAX7 and and differentiating expressing MYOGENIN myogenic cells. At this we observed of CDKN1c expression in of these populations (Figure as differentiation by MYOGENIN Cdkn1c expression nuclear (Figure Although Cdkn1c was mostly cytoplasmic in T72 myoblasts, Cdkn1c nuclear presence in around of and of T72 myoblasts (Figure In line with Cdkn1c protein was mainly restricted to cell nuclei at late of regeneration following in vivo muscle injury Figure 5C). Finally, to the T24-T48 populations of myoblasts in single myofibers ex vivo, Cdkn1c to be in KI67+ proliferating cells at to their cytoplasm (Figure Next, to evaluate Cdkn1c nuclear translocation was with its with MYOD et al., et al., et al., et al., 2011; et al., et al., 2014; Zalc et al., 2014), we used data generated by a MYOD et al., to at muscle regulatory We performed experiments with an to Cdkn1c these with and we found no significant at of the (Figure supplement Finally, to the subcellular localization of CDKN1c the of myoblasts, we generated Cdkn1c (Cdkn1c or Cdkn1c the localization (Cdkn1c using protein as a to transduced cells (Figure was used as control (Figure of Cdkn1c in single myofiber cultures was associated with nuclear CDKN1c protein (Figure and a marked in myoblasts (Figure In contrast, expression of the restricted CDKN1c to the cytoplasm (Figure and did not induce cell cycle exit as Cdkn1c (Figure contrast, of Cdkn1c or Cdkn1c did not the of cells entering myogenic differentiation, as evidenced by MYOD MYOGENIN expression (Figure supplement in to with in these experiments, we in Cdkn1c-deficient myoblasts following (Figure supplement to the ex vivo system of single myofibers (Figure Figure supplement we observed cell cycle from myogenic differentiation, in Cdkn1c but not Cdkn1c (Figure supplement Together, these results show that nuclear CDKN1c growth while cytoplasmic localization of the protein observed at early of MuSC activation is with proliferation. Furthermore, our data suggest of cell cycle exit and myogenic differentiation. Discussion adult myogenesis is for from injuries, but can be compromised by or that the capacity of skeletal muscle stem cells The maintenance of MuSC function on the entry and maintenance of a quiescent The molecular mechanisms that control MuSC cell cycle and adult myogenesis have significant in as a to tissue to efficient when is on cell cycle exit signals and our on the role of CDKN1c in cell in myogenesis et al., 2014), we that CDKN1c control cell cycle and differentiation of MuSCs in adult muscle. Cdkn1c deficiency compromised muscle regeneration following in vivo injury (Figure Figure and hindered primary myoblast differentiation and myotube formation (Figure Figure 3—figure supplement 2). CDKN1c expression with differentiation in cell et al., while in myogenic cultures CDKN1c has also been as a differentiation marker et al., 1999; et al., 2011). the of its ablation on adult muscle have not been examined of the perinatal of Cdkn1c mutant mice. the mice in a mixed CD1;B6 background allowed us to while floxed Cdkn1c mice allowed In vivo and in analysis of both demonstrated that lack of Cdkn1c compromised myogenic differentiation (Figures Figure 3—figure supplement 2). In the of mice, there was increased MuSC self-renewal at the expense of differentiation (Figures while conditional of Cdkn1c in adult mice the MuSC compartment after injury (Figure These results depend on mechanisms that are activated when is induced in our cKO mice, effects of both Pax7 to