Muscle satellite cells are the primary source of stem cells for postnatal skeletal muscle growth and regeneration. Understanding genetic control of satellite cell formation, maintenance, and acquisition of their stem cell properties is on-going, and we have identified SOXF (SOX7, SOX17, SOX18) transcriptional factors as being induced during satellite cell specification. We demonstrate that SOXF factors regulate satellite cell quiescence, self-renewal and differentiation. Moreover, ablation of Sox17 in the muscle lineage impairs postnatal muscle growth and regeneration. We further determine that activities of SOX7, SOX17 and SOX18 overlap during muscle regeneration, with SOXF transcriptional activity requisite. Finally, we show that SOXF factors also control satellite cell expansion and renewal by directly inhibiting the output of β-catenin activity, including inhibition of Ccnd1 and Axin2. Together, our findings identify a key regulatory function of SoxF genes in muscle stem cells via direct transcriptional control and interaction with canonical Wnt/β-catenin signaling.https://doi.org/10.7554/eLife.26039.001
Maintenance, repair, and regeneration of adult tissues rely on a small population of stem cells, which are maintained by self-renewal and generate tissue-specific differentiated cell types (Weissman, 2000). Most adult stem cells are quiescent within their niche, dividing infrequently to generate both a copy of the stem cell and a rapidly cycling cell (Barker et al., 2010). These features make adult stem cells essential for either normal tissue homeostasis or repair/regeneration following damage (Slack, 2000). Hence, identification and manipulation of stem cells, including understanding mechanisms of cell fate decision and self-renewal, are essential to develop stem cell-based therapeutic strategies (Relaix, 2006).
Skeletal muscle contains a population of resident stem cells - termed satellite cells (Katz, 1961; Mauro, 1961). Around birth, fetal muscle progenitor cells adopt a satellite cell position, becoming embedded within the basal lamina in close contact to the muscle fibers (Ontell and Kozeka, 1984; Relaix et al., 2005). Importantly, during postnatal growth, the emerging satellite cells progressively enter quiescence, a molecular state poorly characterized in vivo. However, in response to injury or disruption of the basal lamina, satellite cells are activated and proliferate to form myoblasts that either fuse to existing myofibers to repair, or fuse together to form multinucleated de novo myotubes for regeneration. Alternatively, a subset of satellite cells self-renews to maintain a residual pool of quiescent stem cells that has the capability of supporting additional rounds of growth and regeneration (Zammit et al., 2006). Satellite cells are indispensable for muscle recovery after injury, confirming their pivotal and non-redundant role as skeletal muscle stem cells (reviewed in Relaix and Zammit, 2012).
Many studies have demonstrated a balance between extrinsic cues and intracellular signaling pathways to preserve stem cell function, with Notch and Wnt signaling being of particular importance (Brack and Rando, 2012; Dumont et al., 2015). Wnt signaling has been extensively studied in satellite cells (Brack et al., 2008; Kuang et al., 2008). Whereas canonical Wnt signaling, implying β-catenin/TCF activation, is upregulated upon muscle regeneration and regulates satellite cell differentiation (Otto et al., 2008; von Maltzahn et al., 2012), non-canonical Wnt signaling (independent of β-catenin), mediates satellite cell self-renewal and muscle fiber growth (Le Grand et al., 2009; von Maltzahn et al., 2012). However, how Wnt signaling pathways interact with intrinsic transcriptional regulators remains unclear. Therefore, identifying the transcriptomic changes in muscle progenitors and satellite cells through development, growth and maturity is fundamental in order to build a comprehensive model of satellite cell formation and function (Alonso-Martin et al., 2016). Focusing on the important transition from developmental to postnatal myogenesis, we identified the SOXF family (SOX7, SOX17, SOX18) as potentially having a pivotal role in muscle stem cell function (Alonso-Martin et al., 2016).
SOX factors belong to the high mobility group (HMG) superfamily of transcription factors (Bernard and Harley, 2010), and act in the specification of stem cells in a number of tissues during development (Irie et al., 2015; Lizama et al., 2015). SOX17 plays important roles in development, particularly in embryonic stem cells (Sarkar and Hochedlinger, 2013; Séguin et al., 2008) and endoderm formation (Hudson et al., 1997; Kanai et al., 1996), and is critical for spermatogenesis (Kanai et al., 1996) and specification of human primordial germ cell fate (Irie et al., 2015). SOX17 is also implicated in stem cell homeostasis in adult hematopoietic tissues and in cancer (Corada et al., 2013; He et al., 2011; Lange et al., 2009; Ye et al., 2011). SOX7 shares a role in endoderm formation with SOX17, and interestingly, genetic interaction of Sox7 with Sox17 has been recently reported in developmental angiogenesis (Kim et al., 2016; Shiozawa et al., 1996; Takash et al., 2001). Finally, loss of SOX18 leads to cardiovascular and hair follicle defects (Pennisi et al., 2000). Moreover, SOX18 together with SOX7 and SOX17 regulates vascular development in the mouse retina (Zhou et al., 2015).
While SoxF genes play key functions in different stem cell systems, little is known of their role in myogenesis. Here, using a set of ex vivo and in vivo experiments including genetic ablation and regeneration studies, we demonstrate that these factors regulate skeletal muscle stem cell self-renewal as well as satellite cell-driven postnatal growth and muscle regeneration. Moreover, we show that SOXF factors operate via interaction with β-catenin in myogenic cells to modulate the output of Wnt canonical signaling during postnatal myogenesis.
To characterize the formation, establishment and maintenance of satellite cells, we performed a chronological global transcriptomic profiling in embryonic, fetal, and postnatal muscle progenitors and satellite cells (Alonso-Martin et al., 2016). These cells were prospectively isolated from a Pax3GFP/+ population, with minimal contamination of endothelial cells, as previously reported (Alonso-Martin et al., 2016) (Figure 1—figure supplement 1). Focusing on establishment of satellite cells, we identified the SOXF family (SOX7, SOX17, SOX18) of transcriptional regulators as likely key regulators of satellite cell function.
Strikingly, SoxF genes are barely detectable during embryonic and fetal stages (Figure 1A–B) but are induced at onset of the emergence of satellite cells and robustly expressed in postnatal satellite cells at the transcript and protein level (Figure 1A–C).
To examine whether SOXF factors were present specifically in quiescent satellite cells, we performed primary culture experiments in proliferation and differentiation conditions. We isolated freshly FACS-sorted quiescent satellite cells and compared their expression profile to those undergoing culture (Figure 1D). Whereas activation (Myod), proliferation (Ki67), and differentiation (Myog, Myh1) transcripts were all induced in culture conditions, SoxF were predominately detectable in quiescent (Pax7) satellite cells (Figure 1D).
To characterize the role of SOXF factors in satellite cell function, we used the myofiber culture model, which maintains a functional niche for skeletal muscle stem cells while allowing their observation (Zammit et al., 2004). We generated retroviruses encoding a bi-cistronic expression for full-length SOX7FL, SOX17FL or SOX18FL, or transactivation defective SOX7ΔCt, SOX17ΔCt or SOX18ΔCt proteins (Figure 2—figure supplement 1A), together with GFP to identify transduced cells. As SOXF proteins share the same consensus DNA binding sequence, any SOXFΔCt is expected to behave as a dominant negative for all three transcription factors (Hou et al., 2017). Retrovirus encoding IRES-GFP only was used as a control (CTRL). Overexpression of any of the SoxF genes (SOXF-FL) induced a similar phenotype in satellite cells, increasing the pool of self-renewing satellite cells (PAX7+/GFP+) (Figure 2A–D), concomitant with less activation (MYOD+/GFP+) (Figure 2E–H), proliferation (KI67+/GFP+) (Figure 2I–L), and differentiation (MYOG+/GFP+) (Figure 2—figure supplement 1C–F). All PAX7+/GFP+ cells underwent at least one division after exiting their quiescent state, as shown by EdU incorporation in transduced GFP+ cells (Figure 2—figure supplement 1B). This SOXF overexpression in satellite cells parallels the effects observed in other stem cell types, such as adult hematopoietic progenitors (He et al., 2011). Conversely, expression of transactivation defective SOXFΔCt caused a decrease in self-renewal (PAX7+/GFP+) (Figure 2A–D) and promoted proliferation (MYOD+/GFP+, KI67+/GFP+) of satellite cells (Figure 2E–H and I–L), but had no measurable effect on differentiation (MYOG+/GFP+) (Figure 2—figure supplement 1C–F). Taken together, these results show that SoxF genes promote self-renewal of adult muscle stem cells and their return to a mitotically quiescent state.
Considering the important role of SOX17 in cell stemness and cell fate decisions (Chhabra and Mikkola, 2011; Irie et al., 2015; McDonald et al., 2014), we chose to investigate its function in postnatal skeletal muscle satellite cells in vivo. Since Sox17 mutant mice die during development (Kim et al., 2007), we combined a null Sox17 reporter allele (Sox17GFP) with a conditional Sox17fl allele to perform tissue-specific genetic ablation of Sox17: intercrossing with Pax3Cre/+ mice to achieve lineage-specific Sox17 deletion during development and consequently postnatally, or Pax7CreERT2/+ mice for an inducible adult satellite-cell-specific deletion. Pax3Cre/+;Sox17GFP/fl mutant mice had no obvious differences in body or muscle weight during postnatal growth or in adulthood (Figure 3—figure supplement 1A–C). Yet, Sox17-knockout Soleus muscle in adult Pax3Cre/+;Sox17GFP/fl mice contained more myofibers, but with reduced cross-sectional area (Figure 3A–D). Myofibers from Pax3Cre/+;Sox17GFP/fl Soleus also had a lower myonuclei density (Figure 3E), suggesting that Sox17-deficient muscles have less satellite cells contributing to postnatal muscle growth (White et al., 2010; Yin et al., 2013; Zammit, 2008). Indeed, direct quantification using PAX7 or MCAD immunolabeling, including reduction of Pax7 transcripts, revealed that there were fewer satellite cells in Pax3Cre/+;Sox17GFP/fl muscles (Figure 4A,B,D and Figure 4—figure supplement 1). Interestingly, this reduction was already evident by two weeks of postnatal growth (Figure 4B), a time when a significant proportion of satellite cells are becoming quiescent, forming the pool of adult muscle stem cells. Finally, consistent with our myofiber culture experiments (Figure 2), we found that the decrease in muscle stem cells in Sox17-knockout mice was associated with a striking decrease of quiescent cells (Figure 4C). Instead, an increased proportion of satellite cells expressed PAX7 and MYOD (18.3% vs. 3.4% in controls) in Sox17-knockout mutants, and thus were activated, and 16.8% even expressed just MYOD (compared to 2.4% in controls), indicating that they were potentially entering the differentiation program (Figure 4C).
Conditional knockout of Sox17 specifically in adult satellite cells caused a similar loss of satellite cells as soon as three weeks after tamoxifen injection in Pax7CreERT2/+;Sox17fl/fl mutant mice (Figure 4E–G). Myofiber content and morphology was not affected in satellite-cell-specific Sox17-conditional knockout (Pax7CreERT2/+;Sox17fl/fl) adult mutant mice though (Figure 3—figure supplement 2), suggesting that the phenotype in Pax3Cre/+;Sox17GFP/fl mice was linked to impaired early postnatal growth and satellite cell-derived myonuclear accretion (White et al., 2010). These results demonstrate that SOX17 plays an important role in induction and maintenance of satellite cell quiescence.
To evaluate the role of SOX17 during satellite cell activation, renewal and differentiation in vivo, we carried out skeletal muscle regeneration assays. Following cardiotoxin (CTX)-induced regeneration in Tibialis anterior (TA) muscle of wild type mice, we first assessed the dynamics of SoxF gene expression by RT-qPCR in total injured muscle. We observed progressive up-regulation of SoxF genes, with distinct peaks at days (d) 4, 6, and 15 following injury (Figure 5—figure supplement 1A). Noticeably, d4 and d6 expression peaks coincided with increased levels of satellite cell markers such as Pax7 and Myf5 (Figure 5—figure supplement 1B), and at d4 with the myogenic regulatory factors Myod and Myog (Figure 5—figure supplement 1C), which mark activated satellite cells in the process of proliferation and differentiation to form new myofibers. Specific isolation of satellite cells using Tg:Pax7-nGFP (Rocheteau et al., 2012) through muscle regeneration depicts an identical behavior of all SoxF transcripts, being downregulated upon injury, and induced as regeneration proceeds (Figure 5A). SoxF genes and Pax7 display a similar profile, contrary to commitment and differentiation markers (Myod and Myog, Figure 5—figure supplement 1D), inferring that SOXF have stem cell specific activity during regenerative myogenesis (Figure 5A).
Regenerating TA muscles in Pax3Cre/+;Sox17GFP/fl mice were strikingly smaller than controls and expressed lower levels of myogenic genes (Figure 5B–C). Furthermore, we observed a loss of quiescence in Sox17-knockout satellite cells after muscle regeneration, likely preventing cells from re-establishing the pool of quiescent satellite cells (Figure 5D–E) so that when regeneration was over, the satellite cell pool was smaller in Sox17-knockout mutants (Figure 5F). Interestingly, when plating fresh FACS-sorted isolated satellite cells in vitro, Sox17-knockout cells proliferated more than control cells, yielding bigger colonies (Figure 5—figure supplement 2A–B). This result mimicked the effect obtained in satellite cells transduced with SOXFΔCt, with increased satellite cell proliferation at the expense of self-renewal (Figure 2). Histological analysis of TA muscles in Pax3Cre/+;Sox17GFP/fl mice at d7 after CTX-induced regeneration revealed cell infiltration, fat accumulation and fibrosis, that were absent in regenerating muscles of control Sox17GFP/fl mice (Figure 5G), suggesting abnormal regeneration and impaired satellite cell function (Mann et al., 2011; Sambasivan et al., 2011). Moreover, this delay in regeneration was still observed at d28, with signs of cell infiltration still evident (Figure 5—figure supplement 2C–D). However, a second injury at d28 did not exacerbate the phenotype seven days later (Figure 5—figure supplement 2C,E).
To confirm that muscle regeneration defect in Pax3Cre/+;Sox17GFP/fl mice was due to satellite cell function compromised by loss of SOX17, we also examined regeneration in TA muscles of Pax7CreERT2/+;Sox17fl/fl mice (Figure 6A). Analysis of regeneration at d7 in Sox17-conditional knockout mutants revealed that satellite cell numbers were reduced, with fewer in quiescence (Figure 6B–D). At d28, diminution of the satellite cell pool was confirmed in regenerating muscle of adult conditional Pax7CreERT2/+;Sox17fl/fl mutant mice (Figure 6E–G) as observed with Pax3Cre/+;Sox17GFP/fl mice. Again, consistent with the phenotype in Pax3Cre/+;Sox17GFP/fl mice, histological analysis of regenerated Sox17-conditional knockout TA muscles revealed cell infiltration, fat and fibrosis deposition, that were absent in regenerating muscles of control Sox17fl/fl mice (Figure 6H–L), confirming abnormal regeneration and impaired satellite cell function in the absence of SOX17.
Both myofiber culture and in vivo experiments suggested that SOXF factors are involved in satellite cell self-renewal. Alterations of SoxF gene function in myofiber culture experiments yielded stronger phenotypes than in vivo genetic ablation of just Sox17, suggesting a compensatory mechanism between SOX17 and other SOXF proteins. To study such a possible compensatory effect between SOXF members, we performed myofiber culture experiments in control Sox17GFP/fl and Pax3Cre/+;Sox17GFP/fl mutant mice, and analyzed the effect of expressing each of the SOXF factors (Figure 7A–C). Consistent with the data shown in Figure 4, Sox17 mutant satellite cells displayed reduced self-renewal (PAX7+/GFP+) (Figure 7A, CTRL vs. KO), associated with increased activation (MYOD+/GFP+) (Figure 7B, CTRL vs. KO), and little effect on differentiation (MYOG+/GFP+) (Figure 7C, CTRL vs. KO). Interestingly, transduction with retrovirus encoding either SOX7 or SOX17 rescued this defect in self-renewal, whereas expression of SOX18 was unable to revert this effect (Figure 7A). Moreover, overexpression of SOX7 or SOX17 strongly decreased the number of activated satellite cells, to even lower levels compared to control animals (Figure 7B). Expression of SOX18, however, did not modify the activation status of the cells. Finally, overexpression of each SOXF proteins induced a strong decrease in differentiation (Figure 7C), as previously observed in wild type cells (Figure 2—figure supplement 1C–F). These results demonstrate that overexpression of SOX7 or SOX17, but not SOX18, rescues the quiescence and activation phenotype of Sox17-knockout satellite cells.
To further characterize the redundant activity of SoxF genes in vivo, we took advantage of the dominant negative effect of SOX17∆Ct (Figure 2) to carry out electroporation into regenerating muscle (Figure 7D–F). Two days after CTX injection of wild type TA muscles, we electroporated a bi-cistronic construct co-expressing SOX17ΔCt and GFP (Figure 7D and Figure 7—figure supplement 1), together with a TdTomato reporter that revealed efficient electroporation along the regenerating muscle (Figure 7—figure supplement 1). Post-electroporation, we observed many areas of regenerating muscle devoid of fibers, with accumulation of fat and fibrosis, compared to control, indicating a general failure of muscles to regenerate (Figure 7E). A dramatic reduction in Pax7 expression was associated with the exacerbated phenotype of SOX17ΔCt electroporated into muscle, compared to regeneration in Sox17-knockouts (Figures 5C,G and and 7E–F). These results are consistent with SOXF activity being required for skeletal muscle regeneration and confirm the overlapping role of SOXF members, as previously reported in other tissues (Matsui et al., 2006; Sakamoto et al., 2007; Sarkar and Hochedlinger, 2013).
SOXF and β-catenin (CTNNB1) interact through a site located in the C-terminus of SOXF proteins (Figure 2—figure supplement 1A) and that deletion of this region is sufficient to ablate SOXF - β-catenin interaction (Guo et al., 2008; Sinner et al., 2007; Sinner et al., 2004; Zhang et al., 2005). Moreover, expression of constitutively active β-catenin in satellite cells in vivo leads to reduced myofiber size (Hutcheson et al., 2009; Kuroda et al., 2013), a phenotype similar to that we observe with the ablation of SOX17 in these cells (Figure 3). This suggests that SOXF inhibition of β-catenin activity could be required for muscle homeostasis. Upon activation of Wnt signaling, non-phosphorylated β-catenin is stabilized and translocates to the nucleus where it associates with TCF/LEF transcription factors to regulate target gene expression (MacDonald et al., 2009).
We designed two transcriptional reporter assays in C2C12 myoblasts to further characterize the SOXF - β-catenin interaction following β-catenin canonical signaling activation by LiCl (Figure 8A–B). All SOXF proteins individually, strongly activated our novel SoxF reporter, SoxF-B-TKnLacZ (containing five multimerized SOXF consensus binding motifs), demonstrating binding to the same consensus sequence (Figure 8A). Upon β-catenin co-expression with SOXF proteins, SoxF-B-TKnLacZ transactivation was further increased (Figure 8A). Conversely, we explored the role of SOXF proteins on LEF/TCF-β-catenin transcriptional activity (Figure 8B). In this system, β-catenin expression led to a four-fold increase in β-catenin reporter pTOP-TKnLacZ activity, while co-expression of SOXF impaired β-catenin-mediated induction of this reporter (Figure 8B). These functional assays indicate that while β-catenin enhances the transactivation activity of SOXF members, SOXF proteins hinder β-catenin-mediated activation of a TCF/LEF reporter in myogenic cells. Hence, our results imply that SoxF genes modulate β-catenin signaling during myogenesis. Strikingly, expression levels of known target genes of the canonical β-catenin pathway appear modified in Sox17-knockout muscles (Figure 8C). Indeed, Jun, Ccnd1, and Axin2 expression were all increased two- to ten-fold in Sox17 mutant muscles (Figure 8C).
In agreement with previous reports (Otto et al., 2008; Rudolf et al., 2016), we observed nuclear β-catenin expression in activated, but not quiescent, satellite cells indicating that induction of canonical signaling is synchronous with the activation of satellite cells (Figure 8D). To assess the functional significance of β-catenin binding to SOXF proteins, retroviral constructs of SOXF lacking β-catenin binding domain (SOXFΔBCAT) were generated (Figure 2—figure supplement 1A). Expression of SOXFΔBCAT in wild type satellite cells ex vivo caused a significant decrease in self-renewal capacity and increased activation (Figure 8E–J). These results mirrored those obtained with SOXFΔCt (Figure 2 and Figure 2—figure supplement 1C–F), demonstrating that this motif is required for normal muscle stem cell function. Importantly, transactivation ability of SOXF∆BCAT mutant constructs on SOXF target genes was retained, as shown using the SoxF-B-TKnLacZ reporter (Figure 8—figure supplement 1A), whereas β-catenin transactivation of pTOP-TKnLacZ was partially restored when compared to SOXF-FL constructs (Figure 8—figure supplement 1B). Thus, interaction between SOXF proteins and β-catenin regulates muscle stem cell behavior following activation.
To further demonstrate the functional interplay between SOX17 and β-catenin transcriptional activity in myogenic stem cells, single myofiber-associated satellite cells were treated with LiCl. This induction of β-catenin signaling yielded an expansion of the activated satellite cell pool (CTRL, Figure 9A). Overexpression of Sox17 (SOX17FL) abolished the expansion of satellite cells (Figure 9A), while SOX17ΔCt did not affect the enhanced LiCl-driven expansion. Similar results were obtained when using CHIR9902, a specific inhibitor of the Glycogen synthase kinase-3 (GSK3B), which targets β-catenin for degradation (data not shown) (Ying et al., 2008). Our findings point to modulation of cell cycle by SOXF activity: satellite cells fail to acquire quiescence when SOXF function is impaired in vivo and ex vivo. In accord with these observations, the cell cycle regulator Ccnd1 (Cyclin-D1) was up-regulated in Sox17-knockout satellite cells but absent in wild type cells (Figure 8C and Figure 9B). We next investigated how SOXF proteins affect the β-catenin transcriptional regulation of two target genes found increased in Sox17-knockouts, Ccnd1 [also a SOX17 target (Lange et al., 2009)] and Axin2. We designed a cell-based transcriptional reporter assay using either 1 kb of the 5’UTR of Ccnd1 (Ccnd1-nLacZ), encompassing binding motifs for TCF/LEF and SOXF proteins, or 5.6 kb of the proximal Axin2 promoter (Axin2-nLacZ) (Figure 9C–D). β-catenin expression increased activity of both Ccnd1-nLacZ and Axin2-nLacZ reporters following LiCl treatment, while co-expression of SOX17 impaired β-catenin-mediated induction of these two reporters in a dose-dependent manner (Figure 9C–D). SOX7ΔBCAT, lacking the β-catenin binding site, however, was unable to influence activation of either the Ccnd1-nLacZ or Axin2-nLacZ reporters. Accordingly, Axin2 expression levels appeared to be progressively down-regulated at the onset of satellite cells emergence, thus displaying general inverse dynamics to SoxF genes (Figure 9E) (Alonso-Martin et al., 2016).
Together, our data demonstrate that SOXF factors control expansion and self-renewal of adult muscle stem cells, associated with an inhibition of TCF/LEF-β-catenin target genes.
We previously performed a global transcriptomic analysis of the changes in gene expression in murine muscle stem cells throughout life (Alonso-Martin et al., 2016). Focusing on the signature associated with establishment and maintenance of satellite cells from their developmental progenitors, we identified SoxF genes, Sox7, Sox17, and Sox18 as of interest. SoxF transcripts become expressed at the time of satellite cell emergence, with a maximum expression in the quiescent adult state, highlighting their role in establishment, maintenance and function of muscle stem cells. Of relevance, SOX17 is involved in cell fate decisions in human primordial germ cells and embryo-derived stem cells (Irie et al., 2015; McDonald et al., 2014).
Absence of SOX17 leads to impaired postnatal muscle development, with an increase of smaller fibers. Postnatal muscle fiber hypertrophy depends on the total number of muscle fibers within a muscle; thus, the postnatal growth rate of the individual muscle fiber would be lower when there are more myofibers (Rehfeldt et al., 2000). In addition, the reduction of myonuclei per myofiber suggests that myofiber growth impairment may be due to a reduced contribution of satellite cell fusion (White et al., 2010). Consistent with these findings, we observed fewer satellite cells in Sox17-knockout mice, associated with a loss of quiescence and a reduced stem cell pool in postnatal muscles. Moreover, when SOXF function is impaired in satellite cells, self-renewal capacity is reduced and both activation and proliferation are increased. Satellite cell self-renewal is critical to maintain the pool of the satellite cells, so impairment of this process translates into reduced cell numbers, resulting in defective muscle regeneration in both Pax3Cre/+;Sox17GFP/fl and Pax7CreERT2/+;Sox17fl/fl mutant mice, highlighting the specific relevance of SoxF genes postnatally and specifically in adult satellite cells. Moreover, we show that SOXF overexpression in satellite cells inhibits proliferation and differentiation and promotes self-renewal, with SOX17 promoting self-renewal in other stem cell types, such as adult hematopoietic progenitors (Chhabra and Mikkola, 2011; He et al., 2011).
Specific genetic ablation of Sox17 leads to milder phenotypes than when dominant negative constructs are used, which suppress transcriptional activation through all SOXF proteins, in myofiber cultures (ex vivo) or injured muscle electroporation (in vivo). Yet, despite apparently normal expression of Sox7 and Sox18 in Sox17 mutant mice (Figure 4D), there is a general loss of quiescence in satellite cells. SoxF genes have been reported to act with redundant functions, as versatile regulators of embryonic development and determination of different stem and progenitor cell fate (Matsui et al., 2006; Sakamoto et al., 2007; Sarkar and Hochedlinger, 2013). However, our data suggest that in muscle stem cells, redundancy between SoxF genes is more complex. For instance, overexpression of SOX7 or SOX17 but not SOX18 is sufficient to rescue the phenotype in Sox17 mutant mice. Recently, a Sox7fl mutant mouse has been reported, revealing the genetic interaction of SOX7 with SOX17 in developmental angiogenesis (Kim et al., 2016). Furthermore, during revisions for this study, a muscle-specific ablation of Sox7 (Pax3Cre/+;Sox7fl/fl) was reported, showing upregulation of Sox17 and Sox18 in the absence of Sox7 (Rajgara et al., 2017). Nevertheless, Sox7-deficient muscles demonstrated severe phenotypes in homeostatic and regeneration conditions (Rajgara et al., 2017), similar to Sox17 ablation in myogenic cells (Figures 3–6). Future studies analyzing the impact of ablating both SOX7 and SOX17 for muscle stem cell function will be of interest.
Finally, our data link SOXF regulation of satellite cell self-renewal with control of β-catenin activity in satellite cells. Interaction between SOXF and β-catenin has been reported in other cell types, i.e. repression of β-catenin-stimulated expression of dorsal genes (Zorn et al., 1999), regulation of endodermal genes (Sinner et al., 2004), or acting as tumor suppressors antagonizing Wnt/β-catenin signaling (Liu et al., 2016; Sinner et al., 2007; Takash et al., 2001), as well as regulators of this pathway in oligodendrocyte progenitor cells (Chew et al., 2011; Ming et al., 2013). More importantly, our data provide a molecular mechanism for previous reports which demonstrate that a tight regulation of the Wnt/β-catenin canonical signaling output is required to ensure skeletal muscle regeneration (Brack et al., 2008; Brack et al., 2007; Figeac and Zammit, 2015; Murphy et al., 2014; Otto et al., 2008; Parisi et al., 2015; Rudolf et al., 2016; Seale et al., 2003; von Maltzahn et al., 2012). Hence, SOXF factors display a dual activity as both intrinsic regulators of muscle stem cell quiescence and interacting with extrinsic signaling pathways to regulate the expansion of activated muscle stem cells. Moreover, recent findings demonstrate that old satellite cells are incapable of maintaining their normal quiescent state in muscle homeostatic conditions, by switching to an irreversible pre-senescence state (Sousa-Victor et al., 2014). Satellite cells fail to regulate their quiescence with aging, leading to depletion of the pool of stem cells (Blau et al., 2015). Interestingly, satellite cell functional impairment is associated with up-regulation of canonical Wnt/β-catenin (Brack et al., 2008; Brack et al., 2007). Our data therefore points to a potential role of SOXF-β-catenin interaction in this context.
In conclusion, we demonstrate that SOXF transcription factors play a key role in stem cell quiescence and myogenesis through both direct transcriptional control and by modulation of the output of β-catenin activity to affect canonical Wnt signaling.
Pax3GFP/+ mouse strain was previously generated (Relaix et al., 2005). Pax3Cre/+, Pax7CreERT2/+ (Pax7+/CE), Tg:Pax7-nGFP, and Sox17 (Sox17GFP/+ and Sox17fl/+) mutant mice were kindly provided by Jonathan A. Epstein, Chen-Ming Fan, Shahragim Tajbakhsh and Sean J. Morrison, respectively (Engleka et al., 2005; Kim et al., 2007; Lepper et al., 2009; Rocheteau et al., 2012). All mice were maintained in a C56BL/6J background.
Sox17fl/+ was inter-crossed to generate Sox17fl/fl. Sox17GFP/+ mice were bred with Pax3Cre/+ in order to produce Pax3Cre/+;Sox17GFP/+ mutants, and the latter with Sox17fl/fl mice to obtain the ablation of Sox17 in the muscle lineage (Pax3Cre/+;Sox17GFP/fl). For specific deletion of Sox17 in satellite cells (Pax7CreERT2/+;Sox17fl/fl) Sox17fl/fl and Pax7CreERT2/+ mice were crossbred. For recombination induction with the Pax7CreERT2 allele, mice were fed in tamoxifen diet (TD.55125.I, Envigo) or intraperitoneally injected for four consecutive days in the adulthood (Roche-Sigma-Aldrich, St. Quentin Fallavier, France). Littermate Sox17GFP/fl or Sox17fl/fl were used as control animals (CTRL).
For FACS, muscle samples were isolated from adult mice (forelimb, hindlimb, and trunk muscles). Following dissection, all muscles were minced and incubated in digestion buffer [HBSS (Life Technologies, Saint-Aubin, France), 0.2% BSA (Sigma-Aldrich, St. Quentin Fallavier, France), 2 μg/ml Collagenase A (Roche-Sigma-Aldrich, St. Quentin Fallavier, France), 2.4 U/ml Dispase II (Roche-Sigma-Aldrich, St. Quentin Fallavier, France), 10 ng/mL DNaseI (Roche-Sigma-Aldrich, St. Quentin Fallavier, France), 0.4 mM CaCl2, and 5 mM MgCl2], and purified by filtration using 100 µm and 40 µm cell strainers (BD Falcon, Le Pont de Claix, France). For labeling extracellular antigens, 10 ng/ml of the following antibodies were used: rat anti-mouse CD45-PE-Cy7 (BD, Le Pont de Claix, France), rat anti-mouse Ter119-PE-Cy7 (BD, Le Pont de Claix, France), rat anti-mouse CD34-BV421 (BD, Le Pont de Claix, France), rat anti-mouse integrin-α7-A700 (R and D Systems, Abingdon, UK), rat anti-mouse Sca1-FITC (BD, Le Pont de Claix, France), rat anti-mouse CD31-PE (BD, Le Pont de Claix, France). Muscle cells were stained using LIVE/DEAD® Fixable Blue Dead Cell Stain Kit (Life Technologies, Saint-Aubin, France) to exclude dead cells and purified via FACS Aria II based on TER119 (LY76)-, CD45 (PTPRC, LY5)-, CD34+, SCA1- and gating on the cell fraction integrin-α7+. Satellite cells isolated from either Pax3GFP/+ or Tg:Pax7-nGFP were obtained using the FITC channel to recover the GFP+ population.
Purified satellite cells were plated on 0.1% gelatin-coated dishes at low density for clonal analysis (500 cells/well in four-well plates). The remaining sorted cells were either frozen (quiescent) or plated for RNA extraction (proliferation or differentiation conditions). Cells were allowed to grow in proliferation medium: DMEM Glutamax containing 20% fetal bovine serum, 10% horse serum, 1% penicillin–streptomycin, 1% HEPES, 1% sodium pyruvate (Life Technologies, Saint-Aubin, France), 1/4000 bFGF (20 ng/ml Peprotech, Neuilly-sur-Seine, France) for one week at a density of 1000 cells/cm2, and then switched into differentiation medium (5% HS) for four extra days.
Total RNA from FACS-sorted satellite cells was extracted from independent experiments according to the RNasy Micro Kit (QIAGEN, Courtaboeuf, France) RNA extraction protocol. For whole muscle total RNA, RNeasy Fibrous Tissue Midi Kit (QIAGEN, Courtaboeuf, France) was used. cDNA synthesis was performed using Transcriptor First Strand cDNA Synthesis Kit (Roche-Sigma-Aldrich, St. Quentin Fallavier, France). RNA quality was assessed by spectrophotometry (Nanodrop ND-1000).
qPCR reactions were carried out in triplicate using LightCycler 480 SYBR Green I Master (Roche-Sigma-Aldrich, St. Quentin Fallavier, France). Expression of each gene was normalized to that of Hypoxanthine Phosphoribosyltransferase 1 (Hprt1) for total muscle, or TATA Box Protein (TBP) for cultured cells. Results are given as mean ± standard error. The single (*), double (**), triple (***), and quadruple (****) asterisks represent p-values p<0.05, p<0.01 and p<0.001, respectively, for Student’s unpaired t-test. The oligonucleotides used in this study are listed in table 1.
Muscles were dissected and snap-frozen in liquid nitrogen-cooled isopentane. Eight µm cryosections were fixed in 4% paraformaldehyde (PFA) and immunofluorescence was carried out as previously described (Mitchell et al., 2010). Primary antibodies and used dilutions are summarized in table 2.
Secondary antibodies were Alexa 488 goat anti-mouse IgG (H + L), Alexa 546 goat anti-mouse IgG (H + L), Alexa 555 goat anti-mouse IgG (H + L), Alexa 594 goat anti-mouse IgG (H + L), Alexa 488 goat anti-rabbit IgG (H + L), Alexa 594 goat anti-rabbit IgG (H + L), Alexa 594 donkey anti-goat IgG (H + L), Alexa 488 goat anti-Chicken IgY (H + L) (Life Technologies, Saint-Aubin, France), and Cy5-goat anti-rabbit IgG (H + L) (Jackson ImmunoResearch, Suffolk, UK). Nuclei were counterstained with DAPI (Life Technologies, Saint-Aubin, France).
Analysis was carried out using either a Leica TCS SPE confocal microscope or a Zeiss AxioImager.Z1 ApoTome (for scanning of whole Soleus cryosections). Images were processed with either Adobe Photoshop CS5 software (Adobe Systems) or MetaMorph 7.5 Software (Molecular Devices). Counting was performed using ImageJ (version 1.47 v; National Institutes of Health, USA, https://imagej.nih.gov/ij/). Transduced satellite cells in myofiber cultures were directly counted under a Leica fluorescent microscope at 40x magnification. Mean ± standard error (s.e.m.) was given. The single (*), double (**), and triple (***) asterisks represent p-values p<0.05, p<0.01, and p<0.001 respectively by Student’s unpaired t-test. All experiments have been performed on at least three independent experiments for each condition. For the characterization of Sox17 mutant mice, 2–5 whole scanned cryosections in at least three different animals (controls and mutants) were analyzed.
Single myofiber procedure was performed as previously described (Moyle and Zammit, 2014). Briefly, both Extensor digitorum longus (EDL) muscles were dissected and digested in Collagenase type I (Sigma-Aldrich, St. Quentin Fallavier, France) solution for 1.5 hr. Flushing medium against the digested muscle, myofibers detached from whole muscle and were placed into another culture dish. Fibers were taken at different time points, freshly isolated (T0), and 24 (T24), 48 (T48), and 72 (T72) hours after culture in activation medium [DMEM High Glucose (Life Technologies, Saint-Aubin, France), 10% horse serum (Life Technologies, Saint-Aubin, France) and 0.5% chicken embryo extract (MP-Biomedical, Illkirch-Graffenstaden, France)] at 37°C in 5% CO2. Retroviral expression vectors and transduction were carried out as previously reported (Zammit et al., 2006). To transduce myofiber-associated satellite cells, 1:10 dilution of the retroviral supernatant was used 24 hr after fiber isolation. Satellite cells were transduced for 48 hr and then recovered for fixation and immunostaining. EdU (2 μM; C10340, Thermo Fisher Scientific, Montigny-le-Bretonneux, France) chase was performed for 72 hr (last 48 hr together with retroviral transduction). EdU-incorporating cells were detected according to the manufacturer’s protocol.
Sox7 and Sox18 cDNAs were amplified by PCR from IMAGE clones 40131228 and 3967084 respectively; Sox17 cDNA was cloned by PCR from mouse kidney cDNA (gift of Dr. J. Hadchouel). All were subcloned in pCig mammalian bi-cistronic expression vector and pMSCV-IRES-eGFP (MIG) retroviral packaging vector using XhoI and EcoRI added to cloning primers (Megason and McMahon, 2002; Pear et al., 1998).
Control and mutant mice were injected with 40 µL of cardiotoxin (CTX; 10 µM, Latoxan, Portes-lès-Valence, France) in Tibialis anterior (TA) muscles following general anesthesia. Muscles were recovered 7, 10, and 28 days later, to compare control vs. mutant mice; for regeneration expression profile, all days from day 0 up to day 7, and then days 10, 15, 21, and 28. Second injury was performed as above, 28 days after first injury. Muscle electroporation was performed using an Electro Square-Porator ECM 830 (BTX®, Genetronics Inc., Holliston, MA). According to (Sousa-Victor et al., 2014), 40 µg of DNA solutions were injected and TA muscles were electroporated using external plate electrodes two days after CTX injection. TAs were examined five, seven, or ten days later. Seven and 28 days after injury, TA muscles were processed for histology analysis by Hematoxylin and eosin , Oil red O, and Sirius red staining as previously described (Sambasivan et al., 2011).
C2C12 cells were grown in DMEM High Glucose (Life Technologies, Saint-Aubin, France) supplemented with 10% FBS (Bio West). A total of 1.2 µg DNA was transfected in 105 cells using lipofectamine LTX PLUS reagent (Life Technologies, Saint-Aubin, France). Generated reporters were as follows: SoxF-B-TKnLacZ, five multimerized SOXF consensus binding motifs (annealed oligonucleotides 5'-CAACAATCATCATTGTTGGGGCCAACAATCTACATTGTTCAGA-3' and 5'-TCTGAACAATGTAGATTGTTGGCCCCAACAATGATGATTGTTG-3') (Kanai et al., 1996); β-catenin TOP pTOP-TKnLacZ, six tandem repeats of the TCF/LEF Transcriptional Response Element (Molenaar et al., 1996); Ccnd1-nLacZ, 1 kb of the 5’UTR region, encompassing binding motifs for TCF/LEF and SOXF proteins, was amplified from C57BL/6J genomic DNA (Lange et al., 2009); and Axin2-nLacZ, 5.6 kb of the proximal promoter fragment was excised from Ax2-Luc (gift of Dr. J. Briscoe) and subcloned (Jho et al., 2002). Fixed concentrations of all reporters (0.6 µg) were used. 48 hr after transfection, cells were lysed in 100 µl RIPA buffer supplemented with protease inhibitors (Complete Mini, Roche-Sigma-Aldrich, St. Quentin Fallavier, France). β-galactosidase assays were performed with 10 µl lysates based on 2-Nitrophenyl β-D-galactopyranoside (ONPG) substrate hydrolysis. When indicated, 1 mM LiCl treatment was performed 24 hr post-transfection and carried for 24 hr. Individual transfections were repeated at least three times; measurements are expressed as mean of the amount of ONPG hydrolyzed normalized to control. Error bars correspond to the standard error of the mean (s.e.m.). The single (*), double (**), triple (***), and quadruple (****) asterisks represent p-values p<0.05, p<0.01, p<0.001 and p<0.0001, respectively, for Mann-Whitney statistical test.
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Randy SchekmanReviewing Editor; Howard Hughes Medical Institute, University of California, Berkeley, United States
In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.
Thank you for submitting your article "SOXF factors regulate satellite cell self-renewal and function through inhibition of β-catenin activity" for consideration by eLife. Your article has been evaluated by Fiona Watt (Senior Editor) and three reviewers, one of whom, Amy J Wagers (Reviewer #1), is a member of our Board of Reviewing Editors. The following individual involved in review of your submission has also agreed to reveal their identity: Pier Lorenzo Puri (Reviewer #3).
The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted the following summary and review.
This manuscript identifies SOXF (SOX7, SOX17, and SOX18) members as highly expressed transcription factors in adult Pax3GFP+ cells and evaluates the role of SOX17 in regulating satellite cell fate through overexpression and loss of function studies using Pax3Cre-Sox17null mice and electroporation of Sox17 dominant negative constructs. The authors also demonstrate potential interactions between SOXF family members and an effector of canonical Wnt signaling, b-catenin.
This paper investigates the role of Sox7, Sox17 and Sox18 (cumulatively, SoxF) in adult muscle satellite cell biology. The authors report developmentally regulated expression of SoxF in muscle and, and further assess the impact of SoxF gain- and loss-of-function on muscle satellite cells in vivo and in vitro using transfection studies and gene-modified mice. They conclude from these studies that SoxF regulate satellite cell "quiescence, self-renewal and differentiation". They further demonstrate significant impairment of muscle regenerative function in animals lacking SoxF (Sox17) function in muscle. Finally, they perform a series of reporter assays that suggest an interaction of SoxF with Wnt signaling (β-catenin).
Overall, the work is intriguing for its potential identification of a new class of muscle satellite cell regulators, and particularly the suggestion that SoxF's regulate satellite cell quiescence, a state whose regulation is poorly understood. Still, there are a number of places where the reviewers raised concerns that authors' conclusions may overreach the available data. These are outlined below, and would need to be addressed via new experimentation and revision of the manuscript text.
1) The data in Figure 1 were obtained from sorted Pax3GFP cells, where GFP is a reporter for Pax3 expression, not a lineage tracer (Relaix et al., 2006). For adult satellite cell collection this is a problem since, based on the authors' previously published work, Pax3 expression is rare, except in the diaphragm, in adult satellite cells. Furthermore, Pax3GFP expression is observed in major adult blood vessels in the limbs (Goupille et al., 2011). Based on the Materials and methods (subsection “Cell sorting and culture”, first paragraph), CD31 is not used for negative selection. Since hindlimb, forelimb and trunk muscles were used one cannot be sure what fraction of the cells in Figure 1 are CD31+/Pax3GFP+ blood vessel derived cells. Due to the questions with regards to Pax3GFP expression and lack of CD31 use, sort profiles with all gates and populations need to be shown to demonstrate how Pax3GFP+ cells were prospectively isolated. Also, they need to report what proportion of the isolated Pax3GFP+ cells used for their studies express Pax7 and what proportion express Sca1 (by immunostaining of isolated cells and FACS), clarify if they used CD31 counterselection in their sorts, and assess the anatomical localization of the cells (sublaminar vs. interstitial) by staining in tissue sections.
2) Throughout the manuscript, the authors often inappropriately conflate self-renewal and quiescence with Pax7 positivity. For example, in reporting the results of retroviral transductions in Figure 2, they claim that overexpression of SoxF promotes "self-renewal", but their data show changes in the frequency of Pax7+ and MyoD+ cells and does not assay self-renewal itself (which would require tracking of either individual cell divisions or minimally of input cell number versus output cell number). Similarly, in Figure 3, the authors have not directly evaluated quiescence, and in Figures 5 and 6, they have not evaluated self-renewal (just Pax7 expression). Finally, the authors' conclusion that "Our findings point to modulation of cell cycle by SOXF activity: satellite cells fail to acquire quiescence when SOXF function is impaired in vivo and ex vivo" is problematic, as in several studies the authors have not directly assessed proliferation or quiescence states, and have only inferred these from pax7/myod expression. For renewal, the authors need to evaluate absolute numbers of Pax7+, MyoD+, and Myog+ cells per fiber. EdU pulse experiments would be helpful as well. An alternative explanation for the Sox7FL effects could be decreased activation as opposed to supporting renewal. Based on the Sox7DN data, are the authors suggesting that loss of Pax7 is coupled to premature differentiation or apoptosis? At 72 hours Myog+ cells are observed yet their proportion does not increase. Depending on the absolute number count per fiber the authors will probably also need to assess apoptosis.
3) A major limitation of the authors' conclusions, which center entirely around the notion that SoxF's exert their influence on muscle repair capacity by regulating transcriptional events in satellite cells themselves is that neither of the models they use to evaluate the in vivo regenerative phenotypes caused by loss of SoxF's are satellite cell specific. For example, the data in Figure 3 are generated from Pax3Cre/Sox17fl(Sox17 KO) mice. Although Sox17 is not abundantly expressed in embryonic Pax3GFP+ cells (Figure 1A and 1B), the Cre is and therefore all cells derived from the Pax3 lineage will be disrupted for Sox17. Such Sox17 disrupted cells in addition to satellite cells and derived myogenic progenitors include myonuclei and blood vessel cells(Goupille et al., 2011; Relaix et al., 2006). Therefore, the muscle fiber disruptions such as fiber type transitions and atrophy could reflect Sox17 roles in muscle fibers and blood vessels. Thus, the authors cannot exclude potential influences of SoxF loss in other cell types, and should address this issue using an inducible satellite cell specific model (e.g., Pax7CreER).
4) The manner of myonuclei count (Figure 3F) is not adequate, the authors should obtain dissociated single fibers and count myonuclei along their length (Brack et al., 2005). The assumption is that myonuclear loss reflects loss of satellite cells and derived fusion competent myogenic progenitors. Also, no assessment of myogenic progenitor number or activity (BrdU/EdU) is done at any stage up to adult skeletal muscle ages. The reduction in Pax7 numbers in Figure 3H and loss of quiescence in Figure 3I could reflect disruptions in the muscle fiber niche (loss of Sox17 in myonuclei or blood vessel cells).
5) It is unclear whether the fiber count and sizing of soleus sections in this figure are valid given the number of fibers cut longitudinally in the representative images. It seems that the results could be skewed if interpreting sections with these artifacts. Also, TA and EDL muscle data should also be included or mentioned since they use these muscles for regeneration experiments and culture.
6) Figure 4 should include analysis by immunofluorescence with appropriate fate markers and/or FACs of satellite cell and derived myogenic progenitor numbers at stages of regeneration where fate decisions are readily apparent (~3-7 days after injury). To assess renewal and proliferation, BrdU or EdU pulse experiments with appropriate fate markers should be conducted. Considering Sox17 would be lost in Pax3 derived cells (myonculei and blood vessels), it is difficult to comprehend the conclusion that the phenotypes strictly reflect satellite cell autonomous fate decisions.
7) For the in vivo electroporation studies (Figure 5D-F), it is important to evaluate GFP expression at early and late time points to assess the transfection efficiency and document the cell types in which the dominant negative SoxF is expressed. Also, pockets of ORO+ and Sirius red+ reactivity seem regional in these tissues. How were regions chosen for the quantification shown in this figure?
8) A physical interaction needs to be demonstrated between b-catenin and SOXF family members to support the authors' conclusions. Also, whether these interactions are lost upon removal of the b-catenin interaction site in the SOXF family needs to be tested. Considering the timing of canonical Wnt activity and SOXF family member expression during regeneration, it is not immediately clear as to the relevance of SoxF factors with b-catenin in the context of this manuscript (Brack et al., 2008; Murphy et al., 2014; Rudolf et al., 2016). Although controversial, b-catenin activity is highest during stages of active fate decisions in satellite cells and myogenic progenitors during regeneration (days 3-7 after injury). Yet in Figure 4, SoxF expression peaks at later stages of regeneration. Also, these transcripts are measured in whole TA muscle, whereas they should be measured in sorted satellite cells and myogenic progenitors. Alternatively, SoxF family members along with myogenic fate markers Pax7, MyoD, and Myog could be tested with immunofluorescence at days 3-7 during injury. Since based on the literature and the data in this study, b-catenin activity is associated with myogenic progression, and SoxF family members are proposed to interfere with b-catenin activity; the authors should test localization of SoxF members (Sox17) with myogenic fate regulators at days 3-7 of muscle regeneration. Another possibility is b-catenin activity should be higher in Sox17 null mice, which could explain some of the phenotypes observed in this manuscript (Murphy et al., 2014).
9) Figure 6C – β-catenin target gene analyses lack statistical assessment. Also, it appears that the b-cat target genes show variable differences in the Sox17 deficient muscles. The analysis is also complicated by the different cellular and fiber type composition of the muscles in the Sox17 deficient animals. These issues should be accounted for in the authors' presentation and interpretation of these results.
10) Figure 7EAxin2 expression is assayed in sorted Pax3GFP+ cells without CD31 negative selection this is problematic based on the authors publications as described above (Goupille et al., 2011; Relaix et al., 2006). Due to the questions with regards to Pax3GFP expression and lack of CD31 use, sort profiles with all gates and populations need to be shown to demonstrate how Pax3GFP+ cells were prospectively isolated.
11) LiCl is a GSK3b inhibitor, and so, as GSK3b has additional activities that are not related to its role in Wnt signaling, LiCl should not be presented as a specific Wnt activator.
12) The authors need to add an important control to the studies comparing SoxF overexpression and ability to rescue – they must assess the level of overexpression of the various Sox7, Sox17 and Sox18 constructs and ensure that they are similarly overexpressed. Similarly, they provide no evidence that endogenous SOXF protein levels parallel the transcript levels of SoxF genes. These points need to be addressed by the authors.
13) The authors described an increase of slow fibers in soleus muscle of Sox17fl/Pax3Cre mice. First, a more complete analysis of fast and slow myosins should be performed in various muscles should be performed. Second, the authors should at least discuss the potential connection between SOXF expression and muscle metabolism.
[Editors' note: further revisions were requested prior to acceptance, as described below.]
Thank you for resubmitting your work entitled "SOXF factors regulate satellite cell self-renewal and function through inhibition of β-catenin activity" for further consideration at eLife. Your revised article has been favorably evaluated by Fiona Watt (Senior Editor), a Reviewing Editor, and two reviewers.
The manuscript has been improved but one of the reviewers has raised a few remaining issues that need to be addressed before acceptance, as outlined below:
In response to the first review the authors submitted a figure demonstrating lack of Pax3Cre-GFP in CD31+ cells. Inclusion of this as a supplement would be helpful.
It is impressive that the authors observe a similar magnitude of Pax7+ SC loss regardless of whether Pax3Cre or Pax7CreER is used. However, there are discrepancies in the regeneration experiments. The Pax3Cre-Sox17 KO mice demonstrate obvious impairments in regeneration (Figure 5) after 28 days of recovery. Figure 6 demonstrates Pax7+ cell loss in Pax7CreER-Sox17 KO mice after only 7 days of recovery. There are no data from 28 day regenerated Pax7CreER-Sox17 KO muscle. Therefore it is not known whether regeneration or satellite cell renewal are impaired after 28 days in the Pax7CreER-Sox17 KO. These data should be provided and compared/discussed with the Pax3Cre data/published studies to determine if differences in regenerative phenotype occur depending on timing of recombination. Also, some regenerative measures should be quantified for example size of regenerated muscle fibers,% Oil red O area, and% Sirius red area.https://doi.org/10.7554/eLife.26039.029
- Sonia Alonso-Martin
- Despoina Mademtzoglou
- Despoina Mademtzoglou
- Peter S Zammit
- Frédéric Relaix
- Peter S Zammit
- Peter S Zammit
- Peter S Zammit
- Peter S Zammit
- Peter S Zammit
- Peter S Zammit
- Frédéric Relaix
- Frédéric Relaix
- Frédéric Relaix
- Frédéric Relaix
- Frédéric Relaix
- Frédéric Relaix
- Frédéric Relaix
- Frédéric Relaix
- Frédéric Relaix
- Frédéric Relaix
- Frédéric Relaix
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
We thank Sean J. Morrison for the Sox17GFP/+ and Sox17fl/+ mice, Jonathan A. Epstein for the Pax3Cre/+ mice, Shahragim Tajbakhsh for the Tg:Pax7-nGFP mice, and Che-Ming Fan for the Pax7CreERT2/+ mice; Edgar R. Gomes and Bruno Cadot for the C2C12 mouse cell line; James Briscoe for the Ax2-Luc construct and Juliette Hadchouel for providing the mouse kidney cDNA. The authors are grateful to Vanessa Ribes, Andrew TV Ho, Piera Smeriglio, and Maria Grazia Biferi for technical assistance and constructive comments; Peggy Lafuste and Zeynab Koumaiha for qPCR primers (Ki67 and Myh1); Nora Butta, Raquel del Toro, Marta Flandez and Alysia vandenBerg for critical pre-submission review; Keren Bismuth, Ted Hung-Tse Chang and Bernadette Drayton for their input and assistance. We thank Catherine Blanc and Benedicte Hoareau (Flow Cytometry Core CyPS, Sorbonne Université, Pitié-Salpétrière Hospital), Adeline Henry and Aurélie Guguin (Plateforme de Cytométrie en flux, Institut Mondor de Recherche Biomédicale), and Serban Morosan and the animal care facility (Centre d'Expérimentation Fonctionnelle, Sorbonne Université). We finally want to thank the Histopathology and Microscopy Units at Centro Nacional de Investigaciones Cardiovasculares (CNIC, Spain). SA-M was recipient of a postdoctoral fellowship from the Basque Community (BF106.177). Funding from the German Research Society (DFG) through MyoGrad International Graduate School for Myology GK 1631 and KFO192 (Sp1152/8-1) and Labex REVIVE (ANR-10-LABX-73) supported DM. This work was further supported by funding to FR from INSERM Avenir Program, Association Française contre les Myopathies (AFM) via TRANSLAMUSCLE (PROJECT 19507), Association Institut de Myologie (AIM), Labex REVIVE (ANR-10-LABX-73), the European Union Sixth and Seventh Framework Program in the project MYORES and ENDOSTEM (Grant # 241440), Fondation pour la Recherche Médicale (FRM; Grant FDT20130928236 and DEQ20130326526), Agence Nationale pour la Recherche (ANR) grant Epimuscle (ANR 11 BSV2 017 02), Bone-muscle-repair (ANR-13-BSV1-0011-02), BMP-biomass (ANR-12-BSV1-0038- 04), Satnet (ANR-15-CE13-0011-01), BMP-MyoStem (ANR-16-CE14-0002-03), MyoStemVasc (ANR-17-CE14-0018-01), and RHU CARMMA (ANR-15-RHUS-0003). The lab of PSZ is supported by Muscular Dystrophy UK (RA3/3052), the Medical Research Council (MR/P023215/1), Association Française contre les Myopathies (AFM 17865 and AFM 16050), FSH Society (FSHS-82013-06 and FSHS-82016-03), and European Union Seventh Framework Program BIODESIGN (262948-2). The authors declare no competing financial interests.
Animal experimentation: All animals were maintained inside a barrier facility and all experiment were performed in accordance with the European and French regulations for animal care and handling (Project No: 01427.03 approved by MESR and File No: 15-018 from the Ethical Committee of Anses/ENVA/UPEC).
- Randy Schekman, Reviewing Editor, Howard Hughes Medical Institute, University of California, Berkeley, United States
© 2018, Alonso-Martin et al.
This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.