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.

Regenerative adult myogenesis is crucial for recovery from injuries, but can be compromised by degenerative or disease states that affect the functional capacity of skeletal muscle stem cells (MuSC). The maintenance of MuSC function largely depends on the entry and maintenance of a non-cycling, reversible quiescent state. The molecular mechanisms that control MuSC cell cycle transitions and adult myogenesis have gained significant interest in recent years, as a way to understand post-trauma tissue restoration and, subsequently, to design efficient innovative therapies when it is defective.

Focusing on cell cycle exit signals and given our recent findings on the role of CDKN1c in cell fate decisions in embryonic/fetal myogenesis (Zalc et al., 2014), we hypothesized that CDKN1c may control cell cycle and differentiation of MuSCs in adult muscle. Indeed, Cdkn1c deficiency compromised muscle regeneration following in vivo injury (Figure 2A–D; Figure 4) and hindered primary myoblast differentiation and myotube formation (Figure 3; Figure 3—figure supplement 2). CDKN1c expression correlates with differentiation in many other cell types (Westbury et al., 2001), while in myogenic cultures CDKN1c has also been considered as a differentiation marker (Reynaud et al., 1999; Mounier et al., 2011). Nevertheless, the consequences of its ablation on adult muscle have not been examined previously, notably because of the perinatal lethality of Cdkn1c mutant mice. Maintaining the mice in a mixed CD1;B6 background allowed us to obtain some survivors, while analyzing floxed Cdkn1c mice allowed cell-specific studies. In vivo and in vitro analysis of both models demonstrated that lack of Cdkn1c compromised myogenic differentiation (Figures 1–4, Figure 3—figure supplement 2). In the case of Cdkn1c-null mice, there was increased MuSC self-renewal at the expense of differentiation (Figures 1–2), while conditional deletion of Cdkn1c in adult mice diminished the MuSC compartment after injury (Figure 4). These seemingly contradictory results might depend on compensatory mechanisms that are activated when Cdnk1c deletion is induced prenatally (mutant) versus postnatally (cKO). Moreover, in our cKO mice, compounding effects of inactivating both Pax7 (due to CreERT2 insertion in the PAX7 locus) and Cdkn1c cannot be excluded. However, comparing the Cdkn1c cKO mice to the Cre control, showed a much more severe regeneration impairment in the former (Figure 4). Together, our data on Cdkn1c mutant and cKO mice implicate an important function for CDKN1c in adult myogenesis, similarly to its emerging importance in the quiescence and renewal of several other stem cell types [Matsumoto et a., 2011; Zacharek et al., 2011; Furutachi et al., 2013).

Examining the myoblast populations expressing CDKN1c (i.e. quiescent vs. activated), we did not detect CDKN1c in quiescent MuSCs on sections or isolated myofibers of resting adult (8–12 weeks) muscle (Figure 5—figure supplement 1). On the contrary, CDKN1c was readily detected in interstitial cells (Figure 5—figure supplement 1). Our finding is consistent with previous reports of high CDKN1c levels in adult muscle (Matsuoka et al., 1995; Park and Chung, 2001) and lack of CDKN1c in FACS-isolated postnatal MuSC populations (Chakkalakal et al., 2014) or myofiber-associated MuSCs (Naito et al., 2016). In contrast, an early study detected CDKN1c in quiescent MuSCs (Fukada et al., 2007) which were isolated with a FACS protocol using an antibody previously described by the same group (Fukada et al., 2004). However, this antibody immuno-reacts with bone marrow cells (Fukada et al., 2004), while CDKN1c has a well-established role and presence in the hematopoietic lineage (Matsumoto et al., 2011; Zou et al., 2011). Furthermore, CDKN1c immunostaining in Fukada et al. (2007) was performed with an antibody against the CDKN1c carboxy-terminus, which might cross-react with the respective domain of CDKN1b (Matsuoka et al., 1995; Galea et al., 2008; Pateras et al., 2009). Combining our observation with previous reports (Fukada et al., 2004; Fukada et al., 2007; Chakkalakal et al., 2014; Naito et al., 2016); present study], we conclude that quiescent MuSCs do not express CDKN1c. Instead, it is established that they express CDKN1b, another member of the CDKI family including CDKN1c (Chakkalakal et al., 2014); our unpublished data].

Upon MuSC activation and differentiation, we show that CDKN1c is strongly upregulated (Figures 5–6). Accordingly, activation of MuSCs during muscle regeneration has been shown to induce CDKN1c, with its levels peaking at d3-4 (Yan et al., 2003); our unpublished observations]. The early induction of CDKN1c upon activation might be associated with MYOD expression, a transcription factor with which CDKN1c has been implicated in a positive feedback loop (Reynaud et al., 2000; Osborn et al., 2011). Specifically, MYOD has been shown to induce Cdkn1c both by disrupting a chromatin loop to release the Cdkn1c promoter and by upregulating intermediate factors (Vaccarello et al., 2006; Figliola et al., 2008; Busanello et al., 2012; Battistelli et al., 2014). Furthermore, we previously identified a muscle-specific Cdkn1c regulatory element that MYOD binds and transactivates (Zalc et al., 2014), while co-immunoprecipitation assays revealed direct CDKN1c-MYOD binding (Reynaud et al., 2000). Moreover, MyoD mutant mice present a similar muscle phenotype as our Cdkn1c-deficient mice (Megeney et al., 1996), also consistent with Cdkn1c being a direct downstream gene of MYOD, and possible association of MYOD and CDKN1c. MYOD is expressed within hours after MuSC activation (Zammit et al., 2004; Zhang et al., 2010) and sustains the transition from quiescence to cell cycle via the replication-related factor CDC6 (Zhang et al., 2010). MYOD induces growth arrest in non-myogenic cell lines (Crescenzi et al., 1990; Sorrentino et al., 1990) and has a well-established role for entry into the myogenic lineage. While the possibility of CDKN1c associating with MYOD to regulate CDKN1c translocation was an attractive hypothesis, CDKN1c was not found associated with MYOD binding in muscle-specific gene regulatory regions (Cao et al., 2010) evaluated by ChIP (Figure 6—figure supplement 1). Remarkably, despite robust expression of MYOD, myoblasts continue to proliferate and do not proceed to differentiation for several days (Tajbakhsh, 2009). In fact, in dividing myoblasts MYOD activity is inhibited by Id proteins and CDK/Cyclin complexes (Wei and Paterson, 2001). Furthermore, additional factors, including MYOGENIN, were suggested to initiate or enhance transcription of some MYOD targets (Blais et al., 2005; Cao et al., 2006).

We demonstrate robust CDKN1c presence in activated MuSCs, including expression in proliferating myoblasts in isolated myofiber cultures (e.g. KI67+ cells at T72, cycling cells at T24-T48; Figures 5–6). These observations might contradict its traditional function as cell cycle exit factor (Lee et al., 1995; Matsuoka et al., 1995) and its role in growth arrest during embryonic myogenesis (Zhang et al., 1999; Zalc et al., 2014). Our data, however, suggest that the uncoupling of CDKN1c growth arrest activity in proliferating myoblasts of isolated myofiber cultures is related to its subcellular localization. CDKN1c is cytoplasmically restricted in activated myoblasts but is progressively detected in the nucleus as differentiation takes place (Figure 6). Indeed, forced CDKN1c expression in myoblasts of isolated myofiber cultures led to nuclear CDKN1c and growth arrest, while overexpression of Nuclear localization signal-deficient CDKN1c was compatible with myoblast proliferation (Figure 6; Figure 6—figure supplement 2). On the contrary, CDKN1c cyto-nuclear shuttling was not related to myogenic differentiation, suggesting an uncoupling of cell cycle exit and myogenic differentiation (Figure 6—figure supplement 2), in line with our previous observations in embryonic muscles (Zalc et al., 2014).

We have not identified the molecular events underlying the CDKN1c nucleo-cytoplasmic shuttling, although some observations might explain this pattern. Firstly, CDKN1c could be implicated in cell cycle progression through CDK/Cyclin assembly, similarly to its Cip/Kip siblings (Michieli et al., 1994; Harper et al., 1995; LaBaer et al., 1997; Peschiaroli et al., 2002). In the absence of CDKN1a and CDKN1b, CDKN1c is solely responsible for CDK/Cyclin complex stabilization in mouse embryonic fibroblasts (Cerqueira et al., 2014). However, this might be less likely in myoblasts, where Cdkn1c-MYOD binding engages the CDKN1c helix domain (Reynaud et al., 2000) that was found indispensable for CDK/Cyclin binding and inhibition (Hashimoto et al., 1998; Reynaud et al., 2000). Moreover, in agreement with CDKN1c function as a cell cycle inhibitor, we observed reduced differentiation and increased proliferation in its absence (Figures 1I–J, 2A–E and 3E–N). Secondly, CDKN1c could be involved in nucleo-cytoplasmic distribution of cyclins or CDKs, as previously observed in other cell types. CDKN1c has been shown to interfere with the nuclear translocation of cyclin D1 (Zou et al., 2011) and to relocalize a fraction of CDK2 into the cytoplasm (Figliola and Maione, 2004). Thirdly, while in the cytoplasm, CDKN1c might participate in MuSC mobilization, one of the earliest manifestations of their activation (Siegel et al., 2009). Cytoplasmic CDKN1c was described to regulate cell motility together with LIM-kinase1 (Vlachos and Joseph, 2009; Chow et al., 2011; Guo et al., 2015). Although we did not detect LIM-kinase1 in myogenic cells, we cannot exclude association with other, yet uncharacterized, partners. Future studies are expected to elucidate the roles of CDKN1c in different sub-cellular compartments of myoblasts.

In conclusion, our data indicate that CDKN1c plays essential roles at the initial phase following MuSC activation. We suggest that CDKN1c acts at that early phase, as a) in vivo, Cdkn1c mutants display a strong phenotype during the first days after injury and b) Cdkn1c mutant myoblasts have compromised functions in cultures that last a few days, thus resembling the first events after MuSC activation. CDKN1c presence is compatible with activation/proliferation and possibly represents an early activation event. Its loss profoundly affects ex vivo myogenic differentiation and delays in vivo post-injury recovery, which may lead to increased MuSC self-renewal. Therefore, the reduced regeneration capacity of Cdkn1c-mutant muscle is not caused by decreased numbers of MuSCs, but results from the reduction of myogenic differentiation, which increases propensity for stem-cell self-renewal. Defective stem cell cycle dynamics and continuous activation/proliferation can lead to DNA damage accumulation, apoptosis, pool exhaustion, and inability to support homeostatic or regenerative demands. A better understanding of cell cycle regulation in MuSCs is imperative to define the molecular events underlying their long-term preservation.

All data generated or analysed during this study are included in the manuscript and supporting files.

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Thank you for submitting your article "Cellular localization of the cell cycle inhibitor p57kip2 controls proliferation and growth arrest of adult skeletal muscle stem cells" for consideration by eLife. Your article has been reviewed by three peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen by Didier Stainier as the Senior Editor. The reviewers have opted to remain anonymous.

The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this letter to draw your attention to significant concerns that you would need to address before this work could be considered for publication.

Summary:

This manuscript describes a role for the cell-cycle inhibitor p57 in adult skeletal muscle stem cells (MuSCs). Previous work by the same authors indicated that p57 deletion during embryonic development leads to defects in muscle progenitors' proliferation and differentiation. Similarly, a role for p57 in the proliferation and differentiation of the myogenic C2C12 cell line has been reported.

Here, the authors report that a very small percentage (4.2%) of germline p57 mice survived to adulthood in a mixed background. The surviving animals displayed reduced body weight, decreased myofiber cross-sectional area and increased fibrosis. A marginal increase (1%) of Pax7+ MuSCs was observed on freshly isolated fibers of p57 knock-out mice. Such small increase in Pax7+ MuSCs persisted 30 days after muscle injury. At this time point, no statistically significant difference was noted in Pax7+/MyoD+ committed muscle progenitors of p57 knock-out versus control. To circumvent the lethality observed in germline p57 knock-out mice, the authors selectively deleted p57 in MuSCs by crossing maternal p57fl(m)/+ with tamoxifen-inducible Pax7-CreERT2 mice. Unfortunately, p57 was not efficiently ablated at high frequency. FACS-isolated p57-deleted MuSCs had increased proliferation and decreased differentiation potentials. Quiescent MuSCS did not express p57 which was detected in activated in MuSCs.

Subcellular localization of p57 progressively shifted from the cytoplasm during activation to the nucleus upon differentiation. Overexpression of full-length p57, but not of a mutant incapable of nuclear localization, negatively regulates cell proliferation.

Essential revisions:

1) Beside causing perinatal lethality, germline p57 ablation induces gross developmental defects owing to its role in controlling quiescence of hematopoietic and neural stem cells. Thus, the combination of very few (~4%) p57 knock-out surviving animals and the extensive non-cell specific function of p57 make the results of the presented experiments difficult to interpret.

2) To circumvent the limitations related to germline deletion, the authors have attempted to specifically delete p57 in MuSCs using the Pax7-CreERT2 driver. This approach has been proven unsuccessful in ablating p57 with high frequency. This is a technical issue that should be rectified. Without deleting p57 in the majority of MuSCs, in vivo experiments aimed at evaluating the role of p57 in homeostatic as well as regenerative conditions are simply not possible and the major tenet of this study, i.e., the function of p57 in adult MuSCs, cannot be conclusively determined.

3) Because of the low recombination of the p57 allele, in vitro experiments were presumably conducted with rare recombined MuSCs. Were there a selection process, the results may be difficult to interpret.

4) Based on the results presented in Figure 2, the authors conclude that p57 counteracts self-renewal. It remains unclear whether Pax7+ p57-null MuSCs proliferate or return to quiescence 30 days after injury. For this, it is necessary to evaluate cell proliferation by EdU labeling in vivo at later stages after tissue repair (30 days), as well as to determine cell anatomical position and expression of quiescent genes.

5) The cytoplasmic-nuclear shuttling of p57 should be documented upon MuScs in vivo activation. P57 subcellular localization could be determined in activated MuSCs on cultured myofibers with analysis of p57 protein localization upon in vivo activation. Analysis could be done on muscle stem cells at different early time points after tissue injury, either in tissues or on fixed cells after FACS isolation.

6) The fate of cells in which p57 has been overexpressed should be investigated to determine whether they are differentiating (Myogenin staining). Moreover, p57 overexpression experiments should be conducted in p57-null MuSCs.

7) No mechanistic insight of how p57 regulate different function in the cytoplasm and the nucleus is offered. P57 has been reported to associate with MyoD. Does nuclear p57 colocalize with MyoD at muscle regulatory regions? Even data that helps to exclude certain mechanisms would be useful in determining the subcellular functional role of p57.

We are also including the full reviews below in case the additional comments are helpful.

Reviewer #1:

The authors of this manuscript report that the cyclin-dependent kinase inhibitor p57 regulates skeletal muscle cell differentiation. Constitutive as well as conditional p57 knock-outs display increased proliferating muscle stem cells (MuSCs) and decreased differentiating muscle cells following muscle injury and regeneration. P57 was not expressed in quiescent MuSCs and activated in MuSCs entering the cell cycle. During cell differentiation p57 relocated from the cytoplasmic compartment to the nucleus where it is required to induce growth arrest.

Overall, the experiments are well conducted and the conclusions supported by the results.

While p57 has not been previously ablated in adult muscle cells, there have been several independent reports indicating that p57 either along with p21 or on its own regulates skeletal muscle cell proliferation and differentiation (Zhang et al., 1999; Naito et al., 2016).

Suggestions:

1) Pax7^CreERT2; p57+/-; ROSA^mTmG/+ mice should be challenged with muscle injury and MuSC-mediated repair investigated.

2) The nuclear role of p57 in promoting growth arrest should be experimentally addressed.

P57 has been reported to associate with MyoD (Reynaud et al., 1999). The authors could evaluate whether p57 is recruited on selected MyoD targets in differentiating myocytes.

Reviewer #2:

In the manuscript entitled 'Cellular localization of the cell cycle inhibitor p57kip2 controls proliferation and growth arrest of adult skeletal muscle stem cells' the authors are investigating the role of the cell cycle inhibitor p57kip2 during adult skeletal myogenesis for the first time. p57kip2 has been previously studied as a regulator of stem cell quiescence in other stem cell system such as HSC and neural stem cells (Matsumoto et al., 2011; Zou et al., 2011;Furutachi et al., 2013). Previous work in myogenesis has shown that p57kip2 inhibits proliferation and controls muscle differentiation in embryonic muscle or in myoblasts cell line (C2C12) in vitro.

The manuscript by Mademtzoglou et al., attempts to demonstrate for the first time a role for p57kip2 during proliferation and differentiation of adult skeletal muscle satellite cells (MuSC). In particular, the authors showed that p57kip2 protein is not expressed in quiescent muscle SCs, but its expression is increased upon MuSC activation and retained during differentiation. By using genetic mouse model and ex vivo experiments they showed that p57kip2 is required for proper myogenic lineage progression. Genetic ablation of p57kip2 lead to increased proliferation and self-renew, but muscle differentiation is impaired. Interestingly, they showed that p57kip2 subcellular localization is progressively shifting from cytoplasm during activation to nuclear translocation upon differentiation.

There are two main issues with this manuscript:

1A) The authors revealed a role for p57kip2 during post-natal muscle growth and adult skeletal muscle regeneration relying on the germline p57kip2 mutant mice. However, compensatory upregulation of other cell cycle inhibitors from the Cip/Kip family (p18, p27), as shown previously in HSC (Zou et al., 2011), may diminish the effect of p57 in MuSC. An inducible knockout may give a more profound phenotype than that observed in straight KO mice, as suggested from severe MuSC differentiation defect derived from Pax7^CreERT2;p57^flox mice (Figure 3J-L) compared to more modest reduced differentiation from p57 mutant myoblasts, shown in Figure 3—figure supplement 1C. In addition, any proliferative phenotype during embryogenesis may negatively impact the adult SC in a regenerative context.

1B) In an attempt to use inducible mutant mice, the authors make a cursory comment that they were not able to achieve high efficiency recombination. This is a technical problem that should be rectified. The analysis of myogenic cell fate should include staining for Pax7, Ki67 and MyoD, at different time points after regeneration (d4, d7 and d30). These additional immunostains would further strengthen the conclusion that loss of p57 results in expansion of progenitors and increased self-renew at expense of differentiation.

1C) in vitro experiments were performed with rare recombined cells, this may be problematic if there is a selection process. Due to these potential artefacts, it is important the authors perform the above experiment.

2A) Despite their attempt to demonstrate that subcellular localization of p57kip2 directly affects the cycling status of myoblasts, they did not fully characterize the molecular mechanisms underlying p57kip2's cytoplasm-nuclear shuttling.

In particular, the authors failed to 1) Demonstrate the cellular phenotype of p57kip2 localization specifically in Pax7+ MuSCs and their progeny; 2) Fully understand the mechanism behind p57kip2 cytoplasm-nuclear shuttling and cell cycle/myogenic state.

2B) The authors generated retroviruses encoding the full length p57 (p57FL) or p57 lacking the nuclear localization signal (p57NLS). They showed that overexpression of p57-FL correlates with nuclear localization of the p57 protein and growth arrest based on the absence of Ki67 staining. NO phenotype with P57 lacking NLS. The authors should assess what is the fate of the arrested cells by immunostaining. Are they differentiating (Myogenin)?

In this experimental design, the authors exploit an overexpression system. The authors should perform retrovirus studies in the null background. Finally, can the authors use P57NLS to drive nuclear expression only?

2C) The manuscript lacks mechanistic detail of how P57 functions in a spatially- dependent manner. The authors speculate in the discussion to four potential mechanisms. It is important that some attempt is made to provide the readers with some mechanistic insight-even data that helps to exclude certain mechanisms that may be more tractable, would support the functional role of P57 localization.

Reviewer #3:

The present work by Mademtzoglou et al. investigates the role of the cell cycle inhibitor p57 on muscle stem cell function. The authors have previously reported that p57 deletion during embryonic development leads to defects in muscle progenitors' cell fate decisions. Here they extend these findings to adult muscle stem cells by showing that in a mixed background a small percentage of p57 knockout mice survived to adulthood and exhibited reduced body weight and myofiber cross-sectional area and increased fibrosis. These mice also exhibited a slight increase in muscle stem cell number, which on myofibers ex vivo or 30 days after tissue injury in vivo are more biased towards self-renewal at the expenses of myogenic commitment. They complement these studies by conditionally inducing p57 deletion in adult muscle stem cells, and upon isolation and culture they observed consistent results. They report that p57 is initially localized in the cytoplasm during early activation of muscle stem cells, and its progressive nuclear localization correlates with growth arrest and myogenic differentiation. Finally, overexpression of a full length, but not a mutant p57 lacking its nuclear localization signal, has a negative effect on myoblast proliferation. They conclude that p57 does not regulate muscle stem cell quiescence but rather their self-renewal vs. commitment cell fate choices.

Overall, the findings in this well written manuscript are interesting and novel, particularly the subcellular localization of p57 in muscle stem cells, although they could be strengthened by increasing the depth of the mechanism underlying the regulation of this process. Also, additional experiments are required in order to fully support the authors' interpretation.

1) Increased self-renewal or failure to reentry into quiescence: In Figure 2 the authors show that p57 knockout mice exhibit a mild increase in the number of muscle stem cells 30 days after injury, but no effect on MyoD expression. The authors conclude that p57 counteracts self-renewal. Are these cells proliferating or they efficiently went back to quiescence? Considering this gene has been previously implicated in stem cell quiescence, it might play a role here in promoting the reentry to this state after tissue repair, even if it is not required for the developmental entry in quiescence. The authors should control for proliferation by EdU labeling in vivo at later stage after tissue repair (30 days), as well as their anatomical location and expression of quiescent genes. In addition, analysis of muscle stem cells should be performed not only at 30 days but also at earlier stages after tissue injury, by immunostaining for Pax7, MyoD and EdU in vivo, in order to evaluate the dynamics of activation and commitment in the native tissue. This would strengthen the interpretation of the findings.

2) Efficiency of p57 floxing in muscle stem cells: The efficiency of the floxing in Figure 3 should be indicated, in order to evaluate whether the FACS isolation of labeled cells yields sufficient numbers to be representative of the stem cell pool, or a very low percentage that may hold an intrinsic bias in population heterogeneity. This should be discussed in the text.

3) Validating p57 expression dynamics upon in vivo activation: Data could be strengthened by reinforcing the observation of p57 subcellular localization in activated muscle stem cells on cultured myofibers, with analysis of p57 protein localization upon in vivo activation, i.e. analysis could be done on muscle stem cells at different early time points after tissue injury, either in tissues or immediately fixed after FACS isolation.

4) Effect of p57 loss on myogenic commitment: In Figure 5 the authors show that overexpression of a full length, but not a mutant p57 lacking its nuclear localization signal, has a negative effect on myoblast proliferation. It would be interesting to test whether there is a similar effect also on myogenic commitment.

[Editors' note: further revisions were requested prior to acceptance, as described below.]

Thank you for resubmitting your work entitled "Cellular localization of the cell cycle inhibitor Cdkn1c controls growth arrest of adult skeletal muscle stem cells" for further consideration at eLife. Your revised article has been favorably evaluated by Didier Stainier (Senior Editor), and a Reviewing Editor and the original reviewers, one of whom is a member of our Board of Reviewing Editors.

The manuscript has been improved but there are some remaining issues that need to be addressed before acceptance, as outlined below:

The authors have satisfactorily addressed most reviewers' comments and as a result the revised manuscript is substantially strengthened. There are some remaining concerns, detailed here below, that the authors should address.

1) Figure 3. Cdkn1c expression is abolished in ~ 100% of the Cdkn1c KO myoblast cultures (Figure 3B-D) and all the subsequent experiments reported in Figure 3 were conducted with these cultures. However, the myoblasts appear to be derived from MuSCs FACS-selected for Cdkn1c deletion. The authors should report the overall deletion efficiency (percentage of Cdkn1c-deleted MuSCs in Pax7^CreERT2; Cdkn1c^Flox), as this information is relevant to the interpretation of subsequent experiments.

2) New Figure 4. The authors have chosen to employ a Pax7^CreERT2 strain expressing only one Pax7 allele. As such, Pax7^CreERT2 mice have reduced Pax7 cells and display increased cell infiltration and reduced regenerating myofibers upon muscle injury. Further regeneration worsening in Pax7^CreERT2; Cdkn1c^Flox is thus to be ascribed to the compounding effect of inactivating both Pax7 and Cdkn1c, with Pax7 inactivation being the major contributor of reduced Pax7+cells (Figure 4H).

3) New Figure 4. It is possible that the number of MuSCs is not decreased but rather Pax7 at the level of the gene-protein is decreased. It is important to quantify MuSCs number using a different antibody (syn-4, or m-cadherin). The answer to this question would help to determine the severity of the Pax7 haplo-insufficiency phenotype. In addition, the images representing the figure need to be accompanied by quantification, for instance, the number and size of regenerating fibers across the 3 groups.

4) New Figure 4. The observed reduced number of Pax7+ cells should also be discussed in more detail, as in most of the other assays the authors show that deletion of Cdkn1c leads to increased self-renewal and impaired myogenic differentiation. It would be useful if the authors could comment on this phenotypic difference in the different conditions. A more thorough analysis of the in vivo phenotype may resolve these contradictions.

Essential revisions:

1) Beside causing perinatal lethality, germline p57 ablation induces gross developmental defects owing to its role in controlling quiescence of hematopoietic and neural stem cells. Thus, the combination of very few (~4%) p57 knock-out surviving animals and the extensive non-cell specific function of p57 make the results of the presented experiments difficult to interpret.

In the revised manuscript, we added data on satellite-cell-specific p57 deletion in adult MuSCs, using the tamoxifen-inducible Pax7-CreERT2 recombinase. These data are presented in (new) Figure 4. Inclusion of this figure changed the numbering of subsequent figures and figure supplements.

2) To circumvent the limitations related to germline deletion, the authors have attempted to specifically delete p57 in MuSCs using the Pax7-CreERT2 driver. This approach has been proven unsuccessful in ablating p57 with high frequency. This is a technical issue that should be rectified. Without deleting p57 in the majority of MuSCs, in vivo experiments aimed at evaluating the role of p57 in homeostatic as well as regenerative conditions are simply not possible and the major tenet of this study, i.e., the function of p57 in adult MuSCs, cannot be conclusively determined.

In order to provide more conclusive evidence of p57 function in adult MuSCs, we treated Pax7^CreERT2; p57^flox animals with tamoxifen diet for a total of five weeks. This approach allowed the ablation of p57 in adult muscle satellite cells and their post-regeneration progeny, in contrast to our previous approaches of 3-5 i.p. tamoxifen injections or 1-2 weeks of tamoxifen diet. Ablation of p57 in MuSC led to impaired muscle repair after injury. We also provide relevant controls. The new data are included in new Figure 4.

3) Because of the low recombination of the p57 allele, in vitro experiments were presumably conducted with rare recombined MuSCs. Were there a selection process, the results may be difficult to interpret.

We agree with the reviewers remark and in the revised manuscript, we strengthened these in vitroresults by in vivoanalysis of MuSC-specific p57 ablation (in Pax7^CreERT2; p57^flox animals) during muscle repair (Figure 4). Under these conditions, recombined and non-recombined cells were equally challenged by cardiotoxin-induced muscle degeneration and regeneration and showed a severely compromised potential to reconstitute normal muscle tissue.

Of note, we conducted all in vitroexperiments with freshly isolated 70-80% (recombined) MuSCs, avoiding passages that potentially increase the presumed selection process.

4) Based on the results presented in Figure 2, the authors conclude that p57 counteracts self-renewal. It remains unclear whether Pax7+ p57-null MuSCs proliferate or return to quiescence 30 days after injury. For this, it is necessary to evaluate cell proliferation by EdU labeling in vivo at later stages after tissue repair (30 days), as well as to determine cell anatomical position and expression of quiescent genes.

In our new data shown in Figure 2—figure supplement 1D, PAX7+ p57-null MuSCs located underneath the LAMININ+ basal lamina returned to quiescent state 30 days after injury confirmed by EdU staining.

5) The cytoplasmic-nuclear shuttling of p57 should be documented upon MuScs in vivo activation. P57 subcellular localization could be determined in activated MuSCs on cultured myofibers with analysis of p57 protein localization upon in vivo activation. Analysis could be done on muscle stem cells at different early time points after tissue injury, either in tissues or on fixed cells after FACS isolation.

We performed muscle injury and monitored p57 subcellular presence at early and late time points following regeneration in muscle sections. We observed cytoplasmic p57 at D3 post-injury, while later on p57 was present in the central nuclei of the newly-formed, regenerated myofibers. We present data on D3 and D13 postinjury in updated Figure 5.

6) The fate of cells in which p57 has been overexpressed should be investigated to determine whether they are differentiating (Myogenin staining). Moreover, p57 overexpression experiments should be conducted in p57-null MuSCs.

In the revised manuscript we analyzed the effects of p57 overexpression on differentiation, by quantifying the Myogenin+ cells at early and late time points of Myogenin expression in single myofiber culture. Due to the lack of significant difference, we further investigated whether p57 overexpression acts earlier during myogenic differentiation, by analyzing the MyoD+ cells after 72h of single myofiber culture. At that time point, MyoD labels not only activated myoblasts but also those that will proceed to differentiation. Furthermore, we performed overexpression experiments in p57-deficient MuSCs after FACS isolation as requested. These data are now shown in new Figure 6—figure supplement 2.

7) No mechanistic insight of how p57 regulate different function in the cytoplasm and the nucleus is offered. P57 has been reported to associate with MyoD. Does nuclear p57 colocalize with MyoD at muscle regulatory regions? Even data that helps to exclude certain mechanisms would be useful in determining the subcellular functional role of p57.

We thank the reviewers for this comment. To gain insight into the molecular mechanisms of p57 action, we used data generated by a MyoD ChIP sequencing experiment (Cao et al., 2010) to identify MyoD binding sites at muscle regulatory regions. We then performed ChIP experiments with an anti-p57 antibody to test whether p57 shares these binding sites with MyoD. The results are shown in new Figure 6—figure supplement 1.

Cao Y, Yao Z, Sarkar D, Lawrence M, Sanchez GJ, Parker MH, MacQuarrie KL, Davison J, Morgan MT, Ruzzo WL, Gentleman RC, Tapscott SJ. (2010). Genome-wide MyoD binding in skeletal muscle cells: a potential for broad cellular reprogramming. Dev Cell 18 (4): 662-674.

[Editors' note: further revisions were requested prior to acceptance, as described below.]

The manuscript has been improved but there are some remaining issues that need to be addressed before acceptance, as outlined below:

The authors have satisfactorily addressed most reviewers' comments and as a result the revised manuscript is substantially strengthened. There are some remaining concerns, detailed here below, that the authors should address.

1) Figure 3. Cdkn1c expression is abolished in ~ 100% of the Cdkn1c KO myoblast cultures (Figure 3B-D) and all the subsequent experiments reported in Figure 3 were conducted with these cultures. However, the myoblasts appear to be derived from MuSCs FACS-selected for Cdkn1c deletion. The authors should report the overall deletion efficiency (percentage of Cdkn1c-deleted MuSCs in Pax7^CreERT2; Cdkn1c^Flox), as this information is relevant to the interpretation of subsequent experiments.

We added data on the recombination efficiency in Figure 3—figure supplement 1. The lack of Cdkn1c expression in quiescent MuSCs prevented us from evaluating the percentage of Cdkn1c‐deleted MuSCs in Pax7^CreERT2;Cdkn1c^Flox mice. Instead, we used Pax7^CreERT2;Rosa^mTmG mice, which express GFP as a result of recombination, and we found that one-week of tamoxifen administration leads to 84.5% recombination (percentage of Pax7+GFP+ cells among Pax7+ satellite cells).

Furthermore, we quantified the recombination efficiency for the experiments presented in Figure 4 and added these data in Figure 4—figure supplement 1 of the revised manuscript.

2) New Figure 4. The authors have chosen to employ a Pax7^CreERT2 strain expressing only one Pax7 allele. As such, Pax7^CreERT2 mice have reduced Pax7 cells and display increased cell infiltration and reduced regenerating myofibers upon muscle injury. Further regeneration worsening in Pax7^CreERT2; Cdkn1c^Flox is thus to be ascribed to the compounding effect of inactivating both Pax7 and Cdkn1c, with Pax7 inactivation being the major contributor of reduced Pax7+cells (Figure 4H).

We agree with the reviewers' remark and we added relevant discussion in the revised manuscript.

3) New Figure 4. It is possible that the number of MuSCs is not decreased but rather Pax7 at the level of the gene-protein is decreased. It is important to quantify MuSCs number using a different antibody (syn-4, or m-cadherin). The answer to this question would help to determine the severity of the Pax7 haplo-insufficiency phenotype. In addition, the images representing the figure need to be accompanied by quantification, for instance, the number and size of regenerating fibers across the 3 groups.

When quantified using M-cadherin, MuSCs were reduced from wt to Cre control and further from Cre control to Cdkn1c cKO (Author response image 1), as originally reported in Figure 4.

Author response image 1

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The post-injury fiber size distributions were added in the revised manuscript, in Figure 4—figure supplement 1.

4) New Figure 4. The observed reduced number of Pax7+ cells should also be discussed in more detail, as in most of the other assays the authors show that deletion of Cdkn1c leads to increased self-renewal and impaired myogenic differentiation. It would be useful if the authors could comment on this phenotypic difference in the different conditions. A more thorough analysis of the in vivo phenotype may resolve these contradictions.

The difference in MuSC self-renewal (Cdkn1c mutant) or exhaustion (Cdkn1c cKO) could be attributed to the constitutive versus conditional deletion of the gene, respectively, that could activate different compensatory mechanisms. The myogenic differentiation defect is consistent in both mutant and cKO mice, in our in vivo or ex vivo analyses. These clarifications have been added to the text and the phenotypes of germline and conditional KO mice are comparatively discussed in the updated manuscript.

Author details

1. Despoina Mademtzoglou

   1. Inserm, IMRB U955-E10, F-94010, Créteil, France
   2. Ecole Nationale Veterinaire d'Alfort, Faculté de medecine, F-94000, Université Paris-Est Creteil, Maison Alfort, France

   Contribution

   Formal analysis, Investigation, Visualization, Methodology, Writing—original draft, Writing—review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-4494-7234

2. Yoko Asakura

   Stem Cell Institute, Paul and Sheila Wellstone Muscular Dystrophy Center, Department of Neurology, University of Minnesota Medical School, Minneapolis, United States

   Contribution

   Formal analysis, Investigation, Methodology

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-4107-4236

3. Matthew J Borok

   1. Inserm, IMRB U955-E10, F-94010, Créteil, France
   2. Ecole Nationale Veterinaire d'Alfort, Faculté de medecine, F-94000, Université Paris-Est Creteil, Maison Alfort, France

   Contribution

   Formal analysis, Investigation, Methodology, Writing—review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-2951-6265

4. Sonia Alonso-Martin

   1. Inserm, IMRB U955-E10, F-94010, Créteil, France
   2. Ecole Nationale Veterinaire d'Alfort, Faculté de medecine, F-94000, Université Paris-Est Creteil, Maison Alfort, France

   Present address

   Tissue Regeneration Laboratory, Centro Nacional de Investigaciones Cardiovasculares (CNIC), Madrid, Spain

   Contribution

   Methodology, Writing—review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-3254-0365

5. Philippos Mourikis

   1. Inserm, IMRB U955-E10, F-94010, Créteil, France
   2. Ecole Nationale Veterinaire d'Alfort, Faculté de medecine, F-94000, Université Paris-Est Creteil, Maison Alfort, France

   Contribution

   Methodology, Writing—review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-3164-7109

6. Yusaku Kodaka

   Stem Cell Institute, Paul and Sheila Wellstone Muscular Dystrophy Center, Department of Neurology, University of Minnesota Medical School, Minneapolis, United States

   Contribution

   Formal analysis, Investigation, Visualization, Methodology

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-7993-9640

7. Amrudha Mohan

   Stem Cell Institute, Paul and Sheila Wellstone Muscular Dystrophy Center, Department of Neurology, University of Minnesota Medical School, Minneapolis, United States

   Contribution

   Methodology

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-7368-9350

8. Atsushi Asakura

   Stem Cell Institute, Paul and Sheila Wellstone Muscular Dystrophy Center, Department of Neurology, University of Minnesota Medical School, Minneapolis, United States

   Contribution

   Conceptualization, Resources, Supervision, Funding acquisition, Investigation, Visualization, Writing—review and editing

   For correspondence

   asakura@umn.edu

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-8078-1027

9. Frederic Relaix

   1. Inserm, IMRB U955-E10, F-94010, Créteil, France
   2. Ecole Nationale Veterinaire d'Alfort, Faculté de medecine, F-94000, Université Paris-Est Creteil, Maison Alfort, France
   3. Etablissement Français du Sang, Créteil, France
   4. APHP, Hopitaux Universitaires Henri Mondor, DHU Pepsy & Centre de Référence des Maladies Neuromusculaires GNMH, Créteil, France

   Contribution

   Conceptualization, Resources, Supervision, Funding acquisition, Investigation, Writing—original draft, Writing—review and editing

   For correspondence

   frelaix@gmail.com

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-1270-1472

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