Conditional Cripto overexpression in satellite cells promotes myogenic commitment and enhances early regeneration

Skeletal muscle regeneration mainly depends on satellite cells, a population of resident muscle stem cells. Despite extensive studies, knowledge of the molecular mechanisms underlying the early events associated with satellite cell activation and myogenic commitment in muscle regeneration remains still incomplete. Cripto is a novel regulator of postnatal skeletal muscle regeneration and a promising target for future therapy. Indeed, Cripto is expressed both in myogenic and inflammatory cells in skeletal muscle after acute injury and it is required in the satellite cell compartment to achieve effective muscle regeneration. A critical requirement to further explore the in vivo cellular contribution of Cripto in regulating skeletal muscle regeneration is the possibility to overexpress Cripto in its endogenous configuration and in a cell and time-specific manner. Here we report the generation and the functional characterization of a novel mouse model for conditional expression of Cripto, i.e., the Tg:DsRedloxP/loxPCripto-eGFP mice. Moreover, by using a satellite cell specific Cre-driver line we investigated the biological effect of Cripto overexpression in vivo, and provided evidence that overexpression of Cripto in the adult satellite cell compartment promotes myogenic commitment and differentiation, and enhances early regeneration in a mouse model of acute injury.


Introduction
The responses of skeletal muscle tissue following acute or chronic damages are highly complex and coordinated processes, involving many different cell populations that interact each other to promote muscle regeneration, inflammation and angiogenesis, until full regeneration of the tissue and its functional recovery. This process is tightly controlled by signals released by the damaged fibers, which lead to the activation of the quiescent satellite cells, i.e., the myogenic stem cell population that is among the major players responsible for the regeneration of skeletal muscle (Collins et al., 2005). Following an injury, the satellite cells are activated and proliferate as myogenic progenitors that migrate to the damaged site, differentiate and fuse each other to form new myofibers (Hawke and Garry, 2001;Chargé and Rudnicki, 2004). This sequential process is correlated with the finely regulated expression of the Myogenic Regulatory Factors (MRFs). Indeed, quiescent satellite cells express Pax7 and, upon activation, upregulate the myogenic transcription factor MyoD. Upon commitment to differentiation these transient amplifying cells (Pax7 + /MyoD + ) downregulate Pax7 and upregulate differentiation genes, as Myogenin and MRF4, while a subset of these cells downregulate MyoD retaining Pax7 expression to replenish the pool of quiescent satellite cells (Zammit et al., 2004;Olguin et al., 2007;Tajbakhsh, 2009). Beside satellite cells, regeneration process is also orchestrated by the crosstalk between heterogeneous cell populations that are recruited/activated after damage, such as the inflammatory cells that are always associated with tissue regeneration, supporting myogenic progression by interacting with satellite cells (Kharraz et al., 2013;Saclier et al., 2013). Besides extensive studies, knowledge of the mechanisms underlying this highly orchestrated process and how the different cell populations establish their fate is still far to be fully elucidated, and much less is known on the extrinsic regulation of this process (Bentzinger et al., 2010). We have recently demonstrated that Cripto, a key regulator of early vertebrate embryogenesis (Shen and Schier, 2000;Minchiotti et al., 2001), is a new player in this process . Cripto is a GPI-anchored protein (Minchiotti et al., 2001) and a developmental factor required for correct orientation of the anterior-posterior axis in the vertebrate embryos (Chu et al., 2005;Minchiotti, 2005). Despite its well-characterized role in embryo development and embryonic stem cell differentiation (Minchiotti, 2005), the role of Cripto in adult life has been poorly investigated also due to the fact that Cripto knockout mice are embryonic lethal (Ding et al., 1998;Xu et al., 1999), and that its expression is almost absent in adult physiological conditions. Indeed, Cripto expression is undetectable in skeletal muscles under baseline conditions. However, it becomes rapidly and transiently re-expressed after acute injury, both in myogenic and inflammatory cells, and it is required in the myogenic compartment to achieve an efficient regeneration . Interestingly, a soluble form of the protein (sCripto) is able to rescue the effect of genetic inactivation of Cripto, thus recapitulating the function of endogenous membrane form of the protein ). Yet, the cellular contribution of Cripto in skeletal muscle repair remains to be further clarified, and its knowledge is limited also by the absence of mouse models for conditional and tissue-specific Cripto expression. Here we report the generation and characterization of novel transgenic mice for conditional expression of Cripto in its endogenous configuration, which allowed us to study the biological effect of satellite cell-specific Cripto overexpression on muscle regeneration and myogenic cell fate determination.

Generation of Conditional Cripto Gain of Function Transgenic Mice
To get insight into the cellular contribution of Cripto in skeletal muscle regeneration, and to finely modulate Cripto expression in vivo, we generated a novel transgenic mouse line for conditional Cripto expression based on the Cre-loxP strategy. To generate the pDsRed loxP/loxP Cripto targeting vector, a Cripto-IRES-eGFP cassette was inserted downstream of the DsRed gene sequence followed by three termination sequences, and flanked by two LoxP sites (see Materials and Methods for details; Figure 1A). The effectiveness of the pDsRed loxP/loxP Cripto vector was first evaluated in vitro. To this end, HEK-293T cells were transfected with the pCMV-Cre, expressing the Cre recombinase, and the pDsRed loxP/loxP Cripto plasmids, either alone or in combination, and Cripto protein expression was evaluated. We first verified that eGFP expression was induced in cells cotransfected with pDsRed loxP/loxP Cripto and pCMV-Cre ( Figure S1A). Accordingly, Cripto protein was specifically induced ( Figure 1B) and, as expected, it localized at the cell membrane (Minchiotti et al., 2000) of eGFP expressing cells ( Figure S1B). Following the in vitro validation of the targeting vector, transgenic mice were generated by pronuclear injection, and the presence of the transgene in the offspring was assessed by PCR genotyping of tail biopsies (Figures 1C,D). One out of three transgenic mice obtained gave germline transmission and carried two copies of the transgene that segregated independently in the offspring ( Figure 1E). Two founder lines were thus established and bred to FVB/N wild type mice to generate the Tg:DsRed loxP/loxP Cripto-eGFP (A) and Tg:DsRed loxP/loxP Cripto-eGFP (B) colonies (from now onwards named Tg:Cripto (A) and Tg:Cripto (B) , respectively).

Functional Characterization of the Tg:Cripto Transgenic Lines
Different studies have shown that significative differences exist in the expression level of the same transgene between individual founder siblings, due to different integration loci, and the influence of the genomic sequences flanking the integration site (Palmiter et al., 1982;Overbeek et al., 1986). To characterize Tg:Cripto (A) and Tg:Cripto (B) transgenic lines, we first assessed DsRed expression in freshly isolated muscles by direct fluorescence and found a stronger DsRed signal in Tg:Cripto (A) compared to Tg: Cripto (B) muscles ( Figure 1F). We thus evaluated whether Cripto was expressed upon Cremediated recombination in vivo. To this end, TA muscles of both transgenic lines were injected with Adeno Associated Virus 9 (Inagaki et al., 2006) encoding Cre recombinase (AAV-Cre) or empty vector (AAV-Cntl) as control. Ten days after infection, TA muscles were explanted and genotyped by PCR. As expected, a 838-bp band characterizing the recombining allele was specifically detected in the AAV-Cre injected muscles ( Figure 1G), and Cripto protein was induced at different levels as determined by ELISA-based assay (2.36 ± 0.06 ng/mg in Tg:Cripto (A) vs. 0.43 ± 0.09 ng/mg in Tg:Cripto (B) ; * * P = 0.005) ( Figure 1H).
All together these data demonstrate that Cripto expression is regulated upon Cre-mediated recombination and is induced at different levels in the two transgenic lines.

Time -Dependent Effect Of Cripto Overexpression in Adult Satellite Cells on Skeletal Muscle Regeneration
We have recently shown that adenovirus-mediated soluble Cripto (sCripto) overexpression accelerates muscle  regeneration induced by acute injury . Nevertheless, viral mediated Cripto expression does not allow us to discriminate between the inflammatory and satellite cell contribution of Cripto overexpression.
To overcome this limitation, we crossed Tg:Cripto (A) and Tg:Cripto (B) mice with the tamoxifen-inducible Tg:Pax7CreER T2 mice  and obtained the Tg:Pax7CreER T2 ::DsRed loxP/loxP Cripto-eGFP (A) and Tg:Pax7CreER T2 ::DsRed loxP/loxP Cripto-eGFP (B) trangenic lines (from now onwards named Tg:Pax7CT2::Cripto (A) and Tg:Pax7CT2::Cripto (B) ) ( Figure 2A). We first assessed the effects of satellite cell-specific Cripto overexpression on skeletal muscle regeneration. To this end, Tg:Pax7CT2::Cripto (A) and Tg:Pax7CT2::Cripto (B) adult mice and their control littermates were treated with tamoxifen once a day for 5 days; at day 4, muscle regeneration was triggered in TA muscles by local injection of cardiotoxin (CTX; Figure 2A). Genetic recombination was first confirmed by PCR analysis on TA muscle genomic DNA ( Figure 2B, Figure S2A), and Cripto protein levels were quantified by ELISA assay on total protein extracts at different time points after injury. Increased Cripto protein levels were detected in both Tg:Pax7CT2::Cripto (A) and Tg:Pax7CT2::Cripto (B) mice compared to control, with Tg:Pax7CT2::Cripto (A) showing the highest levels of Cripto upon Cre-mediated recombination ( Figure 2C, Figure S2B). To assess if Cripto overexpression might influence muscle regeneration, we first evaluated the expression of the embryonic Myosin Heavy Chain (eMyHC), which is a marker of newly regenerated fibers, at day 8 after injury. Both immunofluorescence and Western blot analysis clearly showed increased eMyHC protein levels in Tg:Pax7CT2::Cripto (A) mice compared to their control littermates (Figures 2D,E). In line with these findings, expression of both neonatal Myosin Heavy Chain (nMyHC) and the early muscle differentiation marker Myogenin (Myog) similarly increased in the overexpressing mice (Figures 2F,G). Furthermore, expression of Myostatin (Mstn), which is a negative regulator of muscle growth (Thomas et al., 2000), was significantly reduced in Tg:Pax7CT2::Cripto (A) mice compared to control ( Figure 2H). Of note, similar results were obtained from the analysis of the other transgenic line (Tg:Pax7CT2::Cripto (B) ; Figures S2C-F). Morphological analysis of H&E-stained TA muscle sections at day 8 showed that the number of myofibers containing more than one nucleus (n >1) significantly increased in Cripto overexpressing mice compared to control (0.12 ± 0.02 for Tg:Pax7CT2::Cripto (A) vs. 0.06 ± 0.01 for Tg:Cripto (A) of fibers; * P ≤ 0.05; Figures 3A,B). Interestingly, while there was no significant difference in Cross Sectional Area (CSA) between the two groups at day 8 ( Figures 3A,C), later on (i.e., at day 15) both CSA distribution and the relative average values significantly increased in the Tg:Pax7CT2::Cripto (A) mice compared to control (Figures 3A,D,E). To assess if the positive effect of Cripto overexpression on muscle regeneration was either transient or persistent, we extended the analysis to a later time point (i.e., at day 30). Morphometric analysis of the CSA showed no difference in the distribution of muscle fibers in the two groups at day 30 (Figures 3A,F)

Conditional Overexpression of Cripto in Satellite Cells Enhances Myogenic Differentiation
To determine how Cripto overexpression in the satellite cells impact on skeletal muscle regeneration, we first evaluated its effect on early stages of myoblast differentiation in vitro.
Taken together our results provide the first in vivo evidence that Cripto overexpression in myogenic compartment accelerates the early stages of regeneration process promoting satellite cell myogenic commitment.

Discussion
Growing evidence link adult tissue repair to reactivation of developmental gene program; nevertheless, our knowledge on the role of key regulators of early vertebrate embryogenesis in tissue regeneration is still limited also by the lack of adequate in vivo genetic tools. Indeed, transcription levels and timing of expression of key developmental genes are finely tuned, and experimental strategies to generate constitutive gain-offunction and loss-of-function mouse mutants often result in embryonic lethality. In this scenario, we have focused our attention on the developmental gene Cripto, which is a key regulator of vertebrate embryogenesis. Here we report the generation and functional characterization of a conditional Cripto gain-of-function mouse model Tg:DsRed loxP/loxP Cripto-eGFP as a complementary strategy to investigate the effects of Cripto in postnatal and adult life, and potentially in all cell types. Specifically, by crossing Tg:DsRed loxP/loxP Cripto-eGFP mice with a Tg:Pax7CreER T2 mouse line, we have investigated the effect of satellite cell-Cripto overexpression on skeletal muscle regeneration and overcome the limitation of the viralmediated approach . Consistent with our previous data on isolated myofibers in culture, we provide in vivo evidence that satellite cell overexpression of membrane Cripto promotes myogenic commitment and differentiation. However, unlike the viral-mediated overexpression of soluble Cripto (sCripto; Guardiola et al., 2012), the positive effect of satellite cell Cripto overexpression on skeletal muscle regeneration is timedependent, being restricted to the early phases of regeneration. It is unlikely that the discrepancy in the two approaches is due to protein configuration, i.e., membrane vs. soluble Cripto; indeed, sCripto is able to rescue muscle regeneration defects caused by the genetic ablation of Cripto in the satellite cells, indicating that it can recapitulate the role of the endogenous protein . Yet, it is important to consider that Cripto is expressed both in myogenic and inflammatory cells during regeneration. Thus, the different timing of regeneration in the two models might be explained by the fact that while the genetic overexpression is restricted to the satellite cells, the viral-mediated sCripto overexpression has a broader effect and likely affects both the myogenic and the inflammatory cells. Further studies will be necessary to test this hypothesis by deeply investigating the specific contribution of Cripto in inflammatory cells, as well as the autocrine and/or paracrine effects of the endogenous protein.
The positive effect of Cripto overexpression on skeletal muscle regeneration indicated by the increased CSA at day 15 is the expected consequence of its earliest effect on the satellite cell compartment. First, the number of centrally located nuclei is higher in Cripto overexpressing myofibers at day 8 after injury; thus suggesting accelerated differentiation. Accordingly, the myoblast fusion index is increased in postnatal MPCs isolated from Cripto overexpressing mice. Finally, an in depth analysis of Pax7 ± /MyoD ± cell distribution shows that the number of Pax7 − /MyoD + cells committed to differentiation increases both in vivo and in vitro upon Cripto overexpression. We thus conclude that not only the addition of sCripto protein but also its overexpression in a more physiological context, promotes/accelerates satellite cells commitment to differentiation both in vitro and in vivo. Taken together our data provide evidence that a timely overexpression of Cripto in the satellite cells promotes myogenic commitment and fusion/differentiation and transiently accelerates regeneration after acute injury. It will be interesting to investigate in future studies the effect of sustained Cripto overexpression on satellite cell self renewal and regeneration after repeated muscle injury.
Finally, in line with the idea that early regeneration is enhanced upon Cripto overexpression, we found that the expression of Myostatin, a member of the TGFβ superfamily which inhibits myoblasts differentiation and acts as a negative regulator of skeletal muscle growth (Amthor et al., 2002;Langley et al., 2002) is downregulated in Cripto overexpressing mice. Interestingly, this Cripto/Myostatin inverse correlation is consistent with previous findings on isolated myofibers in culture showing that sCripto antagonizes Myostatin leading to increased number of myoblast population committed to differentiation (Pax7 − /MyoD + ) .
In conclusion, all together these findings broaden our knowledge on the role of Cripto in skeletal muscle regeneration, and add novel insights into its activity in the satellite cell compartment. We believe that these novel mouse models, which also offer the unique opportunity to study the effect of Cripto in the regulation of satellite cell quiescence, activation and selfrenewal in vivo, will be instrumental to get fundamental insights into the role of Cripto in skeletal muscle homeostasis and regeneration, as an important step toward the therapeutic use of this molecule.

Mice and Genotyping
To generate the pDsRed loxP/loxP Cripto targeting vector, a mouse Cripto cDNA (750 bp) (Minchiotti et al., 2001) -IRES-eGFP cassette was cloned downstream of the Discosoma sp. red fluorescent protein (DsRed) gene into the pGapDsRed loxP/loxP vector, containing the CMV enhancer/chicken beta-actin promoter (CMV/β-Actin) and two loxP sites flanking the DsRed gene upstream of triple STOP-polyA sequences (kindly provided by Dr. Silvia Brunelli). Briefly, a Xho-AflII 2.2 kb DNA fragment spanning the Cripto-IRES-eGFP cassette was excised from the pCripto-IRES-eGFP plasmid (kindly provided by Dr. Giovanna L. Liguori), blunt ended and cloned into the backbone of the pGapDsRed loxP/loxP vector previously linearized with EcoR1 and filled in with Klenow Fragment of DNA polymerase I (New England Biolabs). Proper orientation of the inserted Cripto-IRES-eGFP DNA fragment was verified by restriction digestion and DNA sequencing. To generate the transgenic mice, a SalI DNA fragment spanning the entire CMV/β-Actin/DsRed loxP/loxP CriptoIRES-eGFP transcription unit released from the pDsRed loxP/loxP Cripto targeting vector was gel purified and injected into fertilized oocytes of FVB/N mice, in collaboration with the Core Facility of Conditional Mutagenesis at San Raffaele Hospital, Milan, Italy. The resulting mice were genotyped by PCR-based screening using primers (a-a ′ ) spanning the Cripto gene exon4-exon5 junction for the amplification of both Cripto wt (247 bp) and transgenic (156 bp) alleles. The integrity of the transgene was further evaluated by PCR using primers (b-b ′ ) annealing on the DsRed and eGFP sequences (2700 bp). Three out of sixty mice analyzed (5%) were positive for the transgene and only one of them (mouse 1) gave germline transmission of the transgene. Two copies of the transgene, which segregate independently, were identified in the genome of the founder mouse by Southern blot analysis. After germline transmission, heterozygous Tg:DsRed loxP/loxP Cripto-eGFP (A) and Tg:DsRed loxP/loxP Cripto-eGFP (B) mice colonies were maintained by crossing with wild-type FVB mice and named Tg:Cripto (A) and Tg:Cripto (B) , respectively.
For Southern Blot analysis, 20 ug of genomic DNA was prepared from tail biopsies, digested with EcoRI and blotted on Immobilion-Ny+ (Millipore). The 32 P-labeled probe was a PCR amplified DNA fragment spanning the eGFP insert (546 bp) from the pDsRed loxP/loxP Cripto vector. The heterozygous Tg:Pax7CreER T2 ::DsRed loxP/loxP Cripto-eGFP (A)/(B) mice were generated by crossing Tg:Pax7CreER T2  with Tg:Cripto (A)/(B) animals; the offsprings of these crosses were genotyped by double PCR analysis to identify the Tg:Cripto (213 bp) and the Tg:Pax7CreER T2 (600 bp) alleles (DsRedCripto for/rev and Pax7Cre for/rev primers). The DNA was prepared from tail biopsy samples of breeding animals. To induce genetic recombination, Tg:Pax7CreER T2 ::Cripto and Tg:Cripto adult mice (5 weeks old) were injected intraperitoneally (i.p.) with tamoxifen (60 µg/g of body weight per day, Sigma-Aldrich) once a day for 5 days. The genetic recombination was verified by PCR analysis (primers c-c ′ ) to distinguish the transgenic (1995 bp) from the recombining (838 bp) allele. The DNA was obtained from tibialis anterior (TA) muscle biopsy samples. The primer sequences for PCR screening, genotyping and genetic recombination analysis are reported in Table S1.

Muscle Injections, Preparation, and Analysis
Tibialis Anterior (TA) muscles of adult Tg:Cripto (A)/(B) transgenic mice were injected with either Cre recombinaseencoding Adeno Associated Virus (AAV-Cre) or the empty vector (AAV-Cntl) at 1 × 10 12 genome copies/ml in 10 µl. Muscles were collected after 10 days and total protein extracts were prepared for further analysis. The AAV vectors were prepared by the AAV Vector Unit at ICGEB Trieste (http:// icgeb.org/avu-core-facility.html) by packaging AAV2 vector genome backbone into AAV capsid serotype 9, as previously described (Arsic et al., 2003). To induce muscle damage, 10 µl of cardiotoxin (CTX, 70 µM in PBS, Sigma-Aldrich) were injected in the TA muscles of adult (5 weeks old) Tg:Pax7CreER T2 ::Cripto and Tg:Cripto mice. For morphometric analysis, muscles were harvested at the indicated time points after damage, and frozen in isopentane-cooled liquid nitrogen for cryosection. Myofiber Cross Sectional Area (CSA) was analyzed on haematoxylin/eosin (H&E) stained muscle sections using ImageJ software (freely available software developed at the National Institutes of Health, Bethesda, Maryland, USA). The number of myofibers having more than one nucleus on total area was evaluated, as previously described (Mittal et al., 2010).
All experiments were conducted in strict accordance with the institutional guidelines for animal research and approved by the Department of Public Health, Animal Health, Nutrition and Food Safety of the Italian Ministry of Health in accordance with the law on animal experimentation.

Isolation of Mouse Satellite Cells and in Vitro Differentiation
Tg:Pax7CreER T2 ::Cripto and Tg:Cripto new-born mice were injected i.p. with tamoxifen (10 µM, 5 µl) every day, from day 1 to day 5 after birth (P1-P5). Preparation of MPCs from new-born mice (P7) was performed as previously described (Rando and Blau, 1994). Briefly the hindlimb and forelimb were removed and separated from the bones. Muscles were mechanically minced with surgical scissors and dissociated by enzymatic digestion [600 U/ml Collagenase Type IV (Gibco) and 1.8 U/ml Dispase II (Roche) in DMEM (Gibco)] at 37 • C for 2 h in the shaker. Digested muscles were spinned at 500 rpm for 5 ′ and satellite cells were spinned down from supernatant (1500 rpm for 10 ′ ). Cells were plated on gelatin-coated dishes in proliferation medium [1% Pen/Strep (EuroClone), 1% Glutamin (Euroclone), 20% FBS (Euroclone), 2% UltraSerum (LifeScience) in DMEM (Gibco),] and myoblast population was enriched by several pre-plating steps (Richler and Yaffe, 1970). For Pax7/MyoD cell counting, MPCs were cytospinned at a density of 50.000 cells/spot after 24 h in proliferating medium. For myogenic differentiation, myoblasts were plated in 48 wells at a density of 2 × 10 4 cells/cm 2 in proliferation medium. After twenty-four hours, proliferation medium was replaced with differentiation medium for 24-72 h

RNA Preparation and qRT-PCR
TA muscles were homogenized with TissueLyserII (Qiagen) in TriReagent (Life Technologies) and total RNA was isolated according to the manufacturer's instructions. One microgram of total RNA was utilized for cDNA synthesis using SuperScript II Reverse Transcriptase and random hexamers (Qiagen). qRT-PCR was performed using SYBR Green PCR master mix (EuroClone). Primers are listed in Table S2.

Statistical Analysis
All values are expressed as mean ± Standard Error of the Mean (SEM). To determine significance between two groups, comparisons were made using unpaired Student t-tests. * P ≤ 0.05, * * P ≤ 0.005, and * * * P ≤ 0.0005 were considered statistically significant.

Author Contributions
CP, design of the work, data acquisition and analysis, data interpretation. SI, data acquisition and analysis, data interpretation. GA, LZ, FI, data acquisition and analysis. OG, conception and design of the work, data analysis, data interpretation and draft of the article. GM, conception and design of the work, data interpretation, writing and revising of the article. All the authors have read and approved the final manuscript.