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Pax7 is back

Pax7 is back Two recent studies have reinvigorated the conversation regarding the role of Pax7 in adult satellite. Studies by Gunther et al (Cell Stem Cell 13:590? 601, 2013) and Von Maltzhen et al (Proc Natl Acad Sci U S A 110:16474? 16479) show that Pax7 is critical for adult satellite cell function and their contribution to muscle repair. Previously, Lepper et al (Nature 460:627? 631, 2009) demonstrated that Pax7 was dispensable for adult muscle repair. In this commentary I have summarized the results from these studies, focusing on the differences in experimental paradigms that led the authors to different conclusions. I also take this opportunity to discuss the potential limitations and hurdles of Cre-lox technology that are responsible for the discrepant results. Keywords: Pax7, Satellite cell, Skeletal muscle, Regeneration, Lineage tracing, Cre Background Main text Adult skeletal muscle has a remarkable regenerative Since the first description of gene targeted mice in 1989, capacity due to a rare population of cells termed satellite which led to the Nobel Prize being given to Cappeci, cells that are wedged between the basal lamina and Evans, and Smithies in 2007, there has been an exponential sarcolemma, interspersed along each adult muscle fiber use of genetic mutant mouse models to study mammalian [1]. Since the original characterization of Pax7 as a specific gene function. Today, sophisticated genetic strategies that marker of adult satellite cells there has been a concerted allow biologists to delete a gene of interest from a cell effort among skeletal muscle biologists to ascertain its role and its descendants in a temporally controlled manner in satellite cell function [2]. Germline mutants and in vitro have now become almost commonplace. The tamoxifen knockdown studies have all convincingly shown a key role (TMX) inducible Cre lox system is used most frequently for Pax7 in survival and proliferative potential of satellite in mouse models. A modifed Cre recombinase fused with cells and their committed progenitors, called myoblasts an estrogen receptor is expressed under the control of a [2-5]. Until recently, a definitive demonstration for the specific promoter, selected due to its tissue or cell type functional role of Pax7 in adult satellite cells in vivo was restricted expression. Active Cre will recombine loxP sites, lacking. Over the past 5 years, three laboratories have engineered to flank a gene of interest, resulting in a null examined the requirement for Pax7 in adult satellite cells allele. This approach has been used to indelibly mark and using an inducible mouse strain that allowed Pax7 to be track a cell and its descendants. In such cases, loxP sites normally expressed during development and subsequently flank a stop sequence upstream of a fluorescent gene deleted in adult mice [6-8]. While the mouse genetics reporter inserted into the accessible ROSA26 locus. used by the labs were identical, the regimens employed to The muscle scientific community is fortunate to have delete Pax7 were not, resulting in very different outcomes has four different inducible Pax7 Cre alleles at its disposal and interpretations. [6,7,9,10]. All four lines are knockin alleles, three contain- ing an internal ribsome entry site (IRES) to retain Pax7 translation [7,9,10], and one with increased Cre sensitivity ERT2 (Cre ) [10]. To test the requirement for Pax7 in adult satellite cells, that is, delete Pax7 from Pax7 expressing satellite cells, Correspondence: brack.andrew@mgh.harvard.edu the laboratories of Fan, Rudnicki, and Braun used a mouse Center for Regenerative Medicine, Massachusetts General Hospital, Boston, strain that harbored the same Pax7-loxP and TMX- MA 02114, USA inducible Pax7Cre allele, developed by Lepper et al.TMX Harvard Stem Cell Institute (HSCI), Boston, MA 02114, USA Harvard Medical School, Boston, MA 02115, USA ? 2014 Brack; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Brack Skeletal Muscle 2014, 4:24 Page 2 of 5 http://www.skeletalmusclejournal.com/content/4/1/24 was administered to adult mice, either immediately prior muscle fibers. In support of their in vivo findings, Lepper to injury, 5 days prior to injury, immediately prior to et al.reportedthat freshly isolated β-gal + satellite cells injury and during repair, and prior to injury with a long appeared to be capable of normal replication and differen- chase period, in the range of 1 to 8 months (see Figure 1). tiation in cultured conditions. The results clearly show that satellite cell function and Two compounding factors are likely responsible for regeneration capacity is significantly impacted when TMX the different outcomes of Pax7 deletion on muscle repair: is chased for long periods prior to injury or administered (1) limitations in Cre lox technology, and (2) the insatiable during repair. In response to muscle injury, there was a contribution of optimally functioning satellite cells to the reduction in the number of Pax7-expressing cells, progres- muscle repair process. Successful application of inducible sive loss of muscle tissue, increased myofibroblasts, and Cre lox technology relies on many individual elements an accumulation of fatty infiltrate [7,8]. Importantly, tar- working optimally, including uniform Cre activation geting Pax7 in satellite cells and their progeny in a cell in all cells within a target population, efficient gene culture model reduced satellite cell proliferative capacity, recombination, and loss of the gene product (protein). due to precocious differentiation. In stark contrast, regen- In the presence of suboptimal gene targeting, resulting eration was unaffected if a short chase (5 days) followed cellular phenotypes can be misinterpreted as evidence for TMX administration prior to muscle injury [6]. Despite redundant gene function. the normal regenerative output, assessment of recom- The potency of Pax7 expressing satellite cells to prolifer- bination based on β-galactosidase (β-gal) activity driven ate and repair tissue is unquestionable. Therefore, a rare off a ROSA26.LacZ reporter revealed a robust contribu- non-recombined satellite cell with presumed normal func- tion from recombined Pax7+ satellite cells to regenerating tion (? escaper? cell) will propagate under the stimulus of Figure 1 Strategies to target Pax7 cells using inducible Cre recombination and its implications for defining the cellular dynamics during muscle regeneration. Upper box: In response to injury, Pax7+ satellite cells exit from quiescence (QSC) (grey cells) to produce Pax7+ rapidly dividing transit amplifying progenitor cells (TAC, blue) that differentiate (myogenic progenitor cell, MPC), coupled to a decrease in Pax7 levels (-) (red) before forming new muscle. Proliferating Pax7+ SCs return to quiescence (grey cells) and reoccupy the niche. After injury, facultative cells (cyan) that express Pax7 transcript transiently (+/-) enter the muscle and contribute to the repair process. Lower box: Different TMX strategies (blue box) employed to target Pax7+ cells, before (followed by a chase: dashed line) and during injury. TMX half-life is estimated at around 10 days in vivo, therefore recombination of Pax7+ cells is possible following the TMX administration regimen (green box and triangle). If TMX is administered shortly before or during injury, Cre has the potential to recombine escaper Pax7+ QSCs as they proliferate (TAC). Other facultative cells that transiently express Pax7 transcript as part of the regeneration process and SCs that return back to quiescence can also be targeted. In all of these aforementioned scenarios, deletion of Pax7 gene if functionally important, will exacerbate the muscle phenotype, but at the expense of identifying the cell state that requires the gene. Targeting with TMX months prior to injury allows a definitive conclusion to be made against the QSC. This is critical since Pax7 levels differ across the QSC compartment and change dynamically during lineage progression. Brack Skeletal Muscle 2014, 4:24 Page 3 of 5 http://www.skeletalmusclejournal.com/content/4/1/24 injury and contribute robustly to tissue repair. Lepper cell diminution. Based on the sum of the data, it is et al. demonstrated extensive contribution of recombined appropriate to conclude that Pax7 is critical for normal (β-gal+) Pax7 derived cells to regenerated muscle fibers. adult satellite cell function and in its absence skeletal However, strict conclusions on recombination efficiency muscle repair is severely compromised. cannot be drawn in a multinucleated syncytium such as While it is becoming clear that high levels of recombin- skeletal muscle; it cannot be ruled out that ? escaper? cells ation should not be assumed and strategies to maximize contributed extensively to regenerated muscle fibers. recombination are desired, caution is also advised. It was To analyze recombination at the level of Pax7, Lepper recently demonstrated that TMX promoted apoptosis in et al. isolated whole muscle or small numbers of cells the intestine and biased the contribution of one stem cell from muscle tissue, with both methods showing Pax7 subset over another without impacting tissue morphology was not expressed after TMX administration. In con- [13]. In the same vein, long-term TMX delivered to mice in trast, Van Maltzhan et al. demonstrated that when a their chow, while increasing recombination efficiency may population of TMX treated satellite cells is contaminated have unintentional consequences that influence satellite cell with rare escaper cells, this small fraction of wild type contributions. Neither Gunther et al. nor Maltzahn et al. cells could proliferate and out-compete the majority of reported detrimental effects of long-term TMX on muscle recombined cells, achieving a repopulation of the cell pool regeneration, however its effects on the satellite cell popu- with wild type function. This warns against performing lation and muscle repair warrants further scrutiny. There- analysis on low cell numbers and from whole tissue when fore, it is imperative that non-specific effects of TMX making interpretations about rare cell populations. More- administration and Cre lox recombination that could in- over, during the course of our experiments using mice directly alter cell function are considered and minimized. harboring inducible Cre alleles, we have noticed variable On one hand, long-term administration of TMX leads recombination efficiency between mice, even within the to greater deletion efficiency but at the same time masks same litter and of the same gender. Therefore, assessment whether the cellular phenotype is borne from quiescent of recombination in every experimental animal is strongly cells specifically and/or their Pax7+ progeny. This issue advised. is particularly relevant if downstream progenitors have The persistence of active TMX increases the likelihood different levels of Pax7 and are biased towards differen- that a gene locus will undergo successful recombination. tiation or self-renewal, as reported [14]. In such a sce- In vivo estimates of TMX half-life is 7 to 11 days [11,12], nario, cells expressing low levels of Pax7 are less likely therefore, after a 24 h chase or TMX administration recombined and retain wild type function. In addition, it during repair, as performed by Van Maltzahn et al. and has become apparent that muscle resident non-satellite Gunther et al., residual TMX and hence Cre activity cells participate in muscle repair, of which some cell could act on proliferating satellite cells and their down- types may transiently activate the Pax7 promoter [15-17]. stream Pax7 expressing progeny, thus increasing the If facultative cells are identified in adult muscle that likelihood of recombination in escaper cells. This may express and require Pax7 for muscle contribution, admi- explain, in part, why chasing TMX for 1 day instead nistration of TMX immediately before or during repair of 5 days prior to injury led to a regenerative decline. will induce recombination in these cells. In contrast, In support of the escaper cell hypothesis, proliferative administering TMX prior to injury and with adequate compensation of satellite cells was prevented after mice chase, would avoid recombination of these transient Pax7 received TMX in their chow during the regeneration expressers, presumably retaining their normal function process [8]. and contribution to muscle tissue repair (see Figure 1). The stability of the targeted protein also needs to be Therefore, prolonged TMX administration will increase considered when designing a TMX strategy. If protein the likelihood of targeting transient cell populations. turnover is longer than the length of chase period, many Technologies that allow inducible and reversible gene cells will have residual protein at sufficient levels to pre- deletion strategies such as doxycycline inducible reverse serve normal function. Using the Lepper et al. allele and tetracycline-controlled transactivator system (rtTA) will chasing TMX for months instead of weeks, Gunther allow examination of the state specific role of Pax7 in et al. observed a requirement for Pax7 in satellite cell satellite cells as they progress along their lineage or other maintenance and function during repair. Pax7 transiently expressed populations as they contribute In addition, Gunther et al. examined a different indu- to muscle repair. ER cible Pax7Cre and Pax7 loxP allele in which Pax7 pro- Due to the caveats of TMX stability, its long-term tein was more rapidly degraded. A shorter TMX chase administration for effective gene recombination and the was required to deplete the satellite cell pool and lose dynamic cell populations within a regenerating muscle, muscle regenerative capacity. Therefore, retention of it would be useful to determine how long TMX can re- Pax7 biological activity altered the kinetics of stem tain recombining activity of satellite cells in vivo. To this Brack Skeletal Muscle 2014, 4:24 Page 4 of 5 http://www.skeletalmusclejournal.com/content/4/1/24 end, one could administer TMX to wild type mice A characteristic morphological feature of quiescent stem that are chased for varying lengths of time and subse- cells is their tightly packed heterochromatic state. Is it quently injected with satellite cells harboring Pax7C- possible that escaper cells have more compact chromatin, ER re -loxP ROSA26 reporter alleles into pre-injured which is relieved during the cell cycle? Gunther et al. muscle. The presence of reporter expression in the re- highlighted an unappreciated role for Pax7 in maintenance generated muscle would provide an estimate of TMX re- of the heterochromatic state; after Pax7 deletion, satellite combining half life. These results would help in the design cells took on a euchromatic morphology, which preceded of experiments that maximize recombination efficiency in their numeric and functional loss. It will be important satellite cells prior to injury, or targeting transit-amplifying to resolve whether subsets of satellite cells have more cells (TAC) cells and facultative cells during injury densely-packed chromatin and whether deregulation of (see Figure 1). chromatin architecture via Pax7-dependent and independ- As the chapter on Pax7?s requirement for adult satellite ent mechanisms lead to changes in satellite cell function. cell function comes to a close, what is next for Pax7 and Gene methylation is a significant roadblock for Cre me- the satellite cell? The phenotypic differences between the diated recombination [18]. Therefore, inaccessible loci will work of Lepper et al. and that of Van Malzhen et al. and more likely avoid recombination, leading to the retention Gunther et al. can be distilled to the presence or absence of normal gene function. To circumvent this limitation, of ? escaper? cells. Which begs the question: are Pax7+ es- due to its open configuration, the ROSA26 locus is often caper cells a unique population or a stochastic anomaly? targeted to insert Cre mediated reporters. While this is a Escape from recombination can arise from many factors, reliable approach for lineage tracing studies, it should be some technical, such as suboptimal TMX, or biological used cautiously as a proxy for gene deletion. Methylation variances, including chromatin compaction and gene status of a target gene at the time of TMX administration methylation or promoter activity. I would argue it unlikely will impact the accuracy with which the ubiquitous re- that TMX dosage played a role. Comparisons between porter reflects deletion of a specific gene. Therefore, direct the different administration strategies employed by Lepper measurement of recombination of the targeted gene locus et al., Van Maltzen et al., and Gunther et al. strongly sug- or protein within the cell of interest is critical. In Table 1 I gest that cell cycle state (quiescence versus proliferating) have outlined TMX strategies for optimal gene recombin- may impose resistance to recombination. It is important ation, measuring recombination efficiency and testing to keep in mind that Lepper et al. observed substantial escaper cell contribution. recombination of the ROSA26 locus in both neonatal and adult muscle, however, only in neonatal muscle, which Conclusions comprises mainly cycling Pax7+ cells, did a phenotype In closing, the studies from Gunther et al. and Van manifest. Maltzen et al. wrap up the debate about the importance Table 1 Strategies for effective gene recombination in adult satellite cells and their progeny during muscle repair TMX delivery Recovery period Gene deletion analysis Escaper cell analysis TMX injection at 3 mg/day for Allow >15 days of TMX chase prior to Isolate purified SCs by FACS, Measure gene or protein levels a c 5 consecutive days muscle injury to target quiescent SCs or from single fibers from over time and minimize lingering TMX activity each experimental animal Five daily injections prior to injury Pros: Temporal identity of cell Measurement of protein In vitro: Measure gene/protein followed by continuous TMX type and state by western blot or immediately after isolation and after feeding delivered in chow immunohistochemistry approximately 10 days in culture (1 mg TMX per day) Minimize recombination of Quantify DNA directly at the In vivo: Measure gene/protein transient cell populations gene locus using site-specific from SCs isolated from uninjured primers flanking the loxP sites and regenerated muscle (>30 days Cons: Escaper cell contribution after injury) No recovery period Pros: Maximize targeting of both stable and transient cell populations Minimize escaper cell contribution Cons: Lose cell type and state resolution Recommendation based on 30-gram adult mouse. Due to variable recombination efficiency between mice, assessment of recombination in every mouse is strongly advised. In addition, I caution the use of R26R-based fluorescent reporters as a surrogate to assess recombination of a gene of interest. In the presence of escaper cells, gene/protein levels of the targeted allele will increase over time if the recombined gene has a loss-of-function phenotype. If recombination results in a gain-of-function mutant or acts redundantly, then gene/protein levels will decrease or not change, respectively. In the absence of escaper cells, the gene/protein of interest will not be detectable. Brack Skeletal Muscle 2014, 4:24 Page 5 of 5 http://www.skeletalmusclejournal.com/content/4/1/24 of Pax7 in adult muscle regeneration. However, the mech- muscle resident progenitor during postnatal development. Nat Cell Biol 2010, 12:257? 266. anisms that preserve satellite cell homeostasis in the 16. Pannerec A, Formicola L, Besson V, Marazzi G, Sassoon DA: Defining absenceofinjury, theroleofPax7asamodifier of skeletal muscle resident progenitors and their cell fate potentials. chromatin architecture, and the requirement of Pax7 Development 2013, 140:2879? 2891. 17. Dellavalle A, Maroli G, Covarello D, Azzoni E, Innocenzi A, Perani L, Antonini S, in muscle resident facultative cells are not yet solved. Sambasivan R, Brunelli S, Tajbakhsh S, Cossu G: Pericytes resident in postnatal In addition, while we critically try to minimize, or at skeletal muscle differentiate into muscle fibres and generate satellite cells. least control for, the limitations of inducible Cre lox Nat Commun 2011, 2:499. 18. Long MA, Rossi FM: Silencing inhibits Cre-mediated recombination of the technology, it is also worth considering the possibility Z/Ap and Z/Eg reporters in adult cells. Plos One 2009, 4:E5435. that such limitations, if understood, may be exploited to help define cellular and molecular heterogeneity doi:10.1186/s13395-014-0024-4 Cite this article as: Brack: Pax7 is back. Skeletal Muscle 2014 4:24. within the satellite cell compartment. Competing interests The author declares that he has no competing interests. Acknowledgements The author would like to thank Susan Eliazer of the Brack lab for reading the commentary. This work was supported by grants from MGH start up funds and NIH grants (RO1 AR060868, R01 AR061002) to ASB. Received: 23 October 2014 Accepted: 27 November 2014 References 1. Mauro A: Satellite cell of skeletal muscle fibers. J Biophys Biochem Cytol 1961, 9:493? 495. 2. Seale P, Sabourin LA, Girgis-Gabardo A, Mansouri A, Gruss P, Rudnicki MA: Pax7 is required for the specification of myogenic satellite cells. Cell 2000, 102:777? 786. 3. Oustanina S, Hause G, Braun T: Pax7 directs postnatal renewal and propagation of myogenic satellite cells but Not their specification. Embo J 2004, 23:3430? 3439. 4. Relaix F, Rocancourt D, Mansouri A, Buckingham M: A Pax3/Pax7- dependent population of skeletal muscle progenitor cells. Nature 2005, 435:948? 953. 5. Kuang S, Charge SB, Seale P, Huh M, Rudnicki MA: Distinct roles for Pax7 and Pax3 in adult regenerative myogenesis. J Cell Biol 2006, 172:103? 113. 6. Lepper C, Conway SJ, Fan CM: Adult satellite cells and embryonic muscle progenitors have distinct genetic requirements. Nature 2009, 460:627? 631. 7. Gunther S, Kim J, Kostin S, Lepper C, Fan CM, Braun T: Myf5-positive satellite cells contribute to Pax7-dependent long-term maintenance of adult muscle stem cells. Cell Stem Cell 2013, 13:590? 601. 8. Von Maltzahn J, Jones AE, Parks RJ, Rudnicki MA: Pax7 is critical for the normal function of satellite cells in adult skeletal muscle. Proc Natl Acad Sci U S A 2013, 110:16474? 16479. 9. Nishijo K, Hosoyama T, Bjornson CR, Schaffer BS, Prajapati SI, Bahadur AN, Hansen MS, Blandford MC, Mccleish AT, Rubin BP, Epstein JA, Rando TA, Capecchi MR, Keller C: Biomarker system for studying muscle, stem cells, and cancer in vivo. FASEB J 2009, 23:2681? 2690. 10. Mm M, Ja L, Sj M, Da H, Kardon G: Satellite cells, connective tissue fibroblasts and their interactions are crucial for muscle regeneration. Development 2011, 138:3625? 3637. 11. Mw D, Coronado E, Osborne CK: Tumor and serum tamoxifen Submit your next manuscript to BioMed Central concentrations in the athymic nude mouse. Cancer Chemother Pharmacol 1989, 23:68? 70. and take full advantage of: 12. Ea L, Solheim E, Lea OA, Lundgren S, Kvinnsland S, Ueland PM: Distribution of 4-hydroxy-N-desmethyltamoxifen and other tamoxifen metabolites in ? Convenient online submission human biological fluids during tamoxifen treatment. Cancer Res 1989, ? Thorough peer review 49:2175? 2183. 13. Zhu Y, Huang YF, Kek C, Bulavin DV: Apoptosis differently affects lineage ? No space constraints or color ?gure charges tracing of Lgr5 and Bmi1 intestinal stem cell populations. Cell Stem Cell ? Immediate publication on acceptance 2013, 12:298? 303. ? Inclusion in PubMed, CAS, Scopus and Google Scholar 14. Rocheteau P, Gayraud-Morel B, Siegl-Cachedenier I, Blasco MA, Tajbakhsh S: A subpopulation of adult skeletal muscle stem cells retains all template ? Research which is freely available for redistribution DNA strands after cell division. Cell 2012, 148:112? 125. 15. Kj M, Pannerec A, Cadot B, Parlakian A, Besson V, Gomes ER, Marazzi G, Submit your manuscript at Sassoon DA: Identification and characterization of a non-satellite cell www.biomedcentral.com/submit http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Skeletal Muscle Springer Journals

Pax7 is back

Skeletal Muscle , Volume 4 (1) – Dec 14, 2014

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Life Sciences; Cell Biology; Developmental Biology; Biochemistry, general; Systems Biology; Biotechnology
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Abstract

Two recent studies have reinvigorated the conversation regarding the role of Pax7 in adult satellite. Studies by Gunther et al (Cell Stem Cell 13:590? 601, 2013) and Von Maltzhen et al (Proc Natl Acad Sci U S A 110:16474? 16479) show that Pax7 is critical for adult satellite cell function and their contribution to muscle repair. Previously, Lepper et al (Nature 460:627? 631, 2009) demonstrated that Pax7 was dispensable for adult muscle repair. In this commentary I have summarized the results from these studies, focusing on the differences in experimental paradigms that led the authors to different conclusions. I also take this opportunity to discuss the potential limitations and hurdles of Cre-lox technology that are responsible for the discrepant results. Keywords: Pax7, Satellite cell, Skeletal muscle, Regeneration, Lineage tracing, Cre Background Main text Adult skeletal muscle has a remarkable regenerative Since the first description of gene targeted mice in 1989, capacity due to a rare population of cells termed satellite which led to the Nobel Prize being given to Cappeci, cells that are wedged between the basal lamina and Evans, and Smithies in 2007, there has been an exponential sarcolemma, interspersed along each adult muscle fiber use of genetic mutant mouse models to study mammalian [1]. Since the original characterization of Pax7 as a specific gene function. Today, sophisticated genetic strategies that marker of adult satellite cells there has been a concerted allow biologists to delete a gene of interest from a cell effort among skeletal muscle biologists to ascertain its role and its descendants in a temporally controlled manner in satellite cell function [2]. Germline mutants and in vitro have now become almost commonplace. The tamoxifen knockdown studies have all convincingly shown a key role (TMX) inducible Cre lox system is used most frequently for Pax7 in survival and proliferative potential of satellite in mouse models. A modifed Cre recombinase fused with cells and their committed progenitors, called myoblasts an estrogen receptor is expressed under the control of a [2-5]. Until recently, a definitive demonstration for the specific promoter, selected due to its tissue or cell type functional role of Pax7 in adult satellite cells in vivo was restricted expression. Active Cre will recombine loxP sites, lacking. Over the past 5 years, three laboratories have engineered to flank a gene of interest, resulting in a null examined the requirement for Pax7 in adult satellite cells allele. This approach has been used to indelibly mark and using an inducible mouse strain that allowed Pax7 to be track a cell and its descendants. In such cases, loxP sites normally expressed during development and subsequently flank a stop sequence upstream of a fluorescent gene deleted in adult mice [6-8]. While the mouse genetics reporter inserted into the accessible ROSA26 locus. used by the labs were identical, the regimens employed to The muscle scientific community is fortunate to have delete Pax7 were not, resulting in very different outcomes has four different inducible Pax7 Cre alleles at its disposal and interpretations. [6,7,9,10]. All four lines are knockin alleles, three contain- ing an internal ribsome entry site (IRES) to retain Pax7 translation [7,9,10], and one with increased Cre sensitivity ERT2 (Cre ) [10]. To test the requirement for Pax7 in adult satellite cells, that is, delete Pax7 from Pax7 expressing satellite cells, Correspondence: brack.andrew@mgh.harvard.edu the laboratories of Fan, Rudnicki, and Braun used a mouse Center for Regenerative Medicine, Massachusetts General Hospital, Boston, strain that harbored the same Pax7-loxP and TMX- MA 02114, USA inducible Pax7Cre allele, developed by Lepper et al.TMX Harvard Stem Cell Institute (HSCI), Boston, MA 02114, USA Harvard Medical School, Boston, MA 02115, USA ? 2014 Brack; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Brack Skeletal Muscle 2014, 4:24 Page 2 of 5 http://www.skeletalmusclejournal.com/content/4/1/24 was administered to adult mice, either immediately prior muscle fibers. In support of their in vivo findings, Lepper to injury, 5 days prior to injury, immediately prior to et al.reportedthat freshly isolated β-gal + satellite cells injury and during repair, and prior to injury with a long appeared to be capable of normal replication and differen- chase period, in the range of 1 to 8 months (see Figure 1). tiation in cultured conditions. The results clearly show that satellite cell function and Two compounding factors are likely responsible for regeneration capacity is significantly impacted when TMX the different outcomes of Pax7 deletion on muscle repair: is chased for long periods prior to injury or administered (1) limitations in Cre lox technology, and (2) the insatiable during repair. In response to muscle injury, there was a contribution of optimally functioning satellite cells to the reduction in the number of Pax7-expressing cells, progres- muscle repair process. Successful application of inducible sive loss of muscle tissue, increased myofibroblasts, and Cre lox technology relies on many individual elements an accumulation of fatty infiltrate [7,8]. Importantly, tar- working optimally, including uniform Cre activation geting Pax7 in satellite cells and their progeny in a cell in all cells within a target population, efficient gene culture model reduced satellite cell proliferative capacity, recombination, and loss of the gene product (protein). due to precocious differentiation. In stark contrast, regen- In the presence of suboptimal gene targeting, resulting eration was unaffected if a short chase (5 days) followed cellular phenotypes can be misinterpreted as evidence for TMX administration prior to muscle injury [6]. Despite redundant gene function. the normal regenerative output, assessment of recom- The potency of Pax7 expressing satellite cells to prolifer- bination based on β-galactosidase (β-gal) activity driven ate and repair tissue is unquestionable. Therefore, a rare off a ROSA26.LacZ reporter revealed a robust contribu- non-recombined satellite cell with presumed normal func- tion from recombined Pax7+ satellite cells to regenerating tion (? escaper? cell) will propagate under the stimulus of Figure 1 Strategies to target Pax7 cells using inducible Cre recombination and its implications for defining the cellular dynamics during muscle regeneration. Upper box: In response to injury, Pax7+ satellite cells exit from quiescence (QSC) (grey cells) to produce Pax7+ rapidly dividing transit amplifying progenitor cells (TAC, blue) that differentiate (myogenic progenitor cell, MPC), coupled to a decrease in Pax7 levels (-) (red) before forming new muscle. Proliferating Pax7+ SCs return to quiescence (grey cells) and reoccupy the niche. After injury, facultative cells (cyan) that express Pax7 transcript transiently (+/-) enter the muscle and contribute to the repair process. Lower box: Different TMX strategies (blue box) employed to target Pax7+ cells, before (followed by a chase: dashed line) and during injury. TMX half-life is estimated at around 10 days in vivo, therefore recombination of Pax7+ cells is possible following the TMX administration regimen (green box and triangle). If TMX is administered shortly before or during injury, Cre has the potential to recombine escaper Pax7+ QSCs as they proliferate (TAC). Other facultative cells that transiently express Pax7 transcript as part of the regeneration process and SCs that return back to quiescence can also be targeted. In all of these aforementioned scenarios, deletion of Pax7 gene if functionally important, will exacerbate the muscle phenotype, but at the expense of identifying the cell state that requires the gene. Targeting with TMX months prior to injury allows a definitive conclusion to be made against the QSC. This is critical since Pax7 levels differ across the QSC compartment and change dynamically during lineage progression. Brack Skeletal Muscle 2014, 4:24 Page 3 of 5 http://www.skeletalmusclejournal.com/content/4/1/24 injury and contribute robustly to tissue repair. Lepper cell diminution. Based on the sum of the data, it is et al. demonstrated extensive contribution of recombined appropriate to conclude that Pax7 is critical for normal (β-gal+) Pax7 derived cells to regenerated muscle fibers. adult satellite cell function and in its absence skeletal However, strict conclusions on recombination efficiency muscle repair is severely compromised. cannot be drawn in a multinucleated syncytium such as While it is becoming clear that high levels of recombin- skeletal muscle; it cannot be ruled out that ? escaper? cells ation should not be assumed and strategies to maximize contributed extensively to regenerated muscle fibers. recombination are desired, caution is also advised. It was To analyze recombination at the level of Pax7, Lepper recently demonstrated that TMX promoted apoptosis in et al. isolated whole muscle or small numbers of cells the intestine and biased the contribution of one stem cell from muscle tissue, with both methods showing Pax7 subset over another without impacting tissue morphology was not expressed after TMX administration. In con- [13]. In the same vein, long-term TMX delivered to mice in trast, Van Maltzhan et al. demonstrated that when a their chow, while increasing recombination efficiency may population of TMX treated satellite cells is contaminated have unintentional consequences that influence satellite cell with rare escaper cells, this small fraction of wild type contributions. Neither Gunther et al. nor Maltzahn et al. cells could proliferate and out-compete the majority of reported detrimental effects of long-term TMX on muscle recombined cells, achieving a repopulation of the cell pool regeneration, however its effects on the satellite cell popu- with wild type function. This warns against performing lation and muscle repair warrants further scrutiny. There- analysis on low cell numbers and from whole tissue when fore, it is imperative that non-specific effects of TMX making interpretations about rare cell populations. More- administration and Cre lox recombination that could in- over, during the course of our experiments using mice directly alter cell function are considered and minimized. harboring inducible Cre alleles, we have noticed variable On one hand, long-term administration of TMX leads recombination efficiency between mice, even within the to greater deletion efficiency but at the same time masks same litter and of the same gender. Therefore, assessment whether the cellular phenotype is borne from quiescent of recombination in every experimental animal is strongly cells specifically and/or their Pax7+ progeny. This issue advised. is particularly relevant if downstream progenitors have The persistence of active TMX increases the likelihood different levels of Pax7 and are biased towards differen- that a gene locus will undergo successful recombination. tiation or self-renewal, as reported [14]. In such a sce- In vivo estimates of TMX half-life is 7 to 11 days [11,12], nario, cells expressing low levels of Pax7 are less likely therefore, after a 24 h chase or TMX administration recombined and retain wild type function. In addition, it during repair, as performed by Van Maltzahn et al. and has become apparent that muscle resident non-satellite Gunther et al., residual TMX and hence Cre activity cells participate in muscle repair, of which some cell could act on proliferating satellite cells and their down- types may transiently activate the Pax7 promoter [15-17]. stream Pax7 expressing progeny, thus increasing the If facultative cells are identified in adult muscle that likelihood of recombination in escaper cells. This may express and require Pax7 for muscle contribution, admi- explain, in part, why chasing TMX for 1 day instead nistration of TMX immediately before or during repair of 5 days prior to injury led to a regenerative decline. will induce recombination in these cells. In contrast, In support of the escaper cell hypothesis, proliferative administering TMX prior to injury and with adequate compensation of satellite cells was prevented after mice chase, would avoid recombination of these transient Pax7 received TMX in their chow during the regeneration expressers, presumably retaining their normal function process [8]. and contribution to muscle tissue repair (see Figure 1). The stability of the targeted protein also needs to be Therefore, prolonged TMX administration will increase considered when designing a TMX strategy. If protein the likelihood of targeting transient cell populations. turnover is longer than the length of chase period, many Technologies that allow inducible and reversible gene cells will have residual protein at sufficient levels to pre- deletion strategies such as doxycycline inducible reverse serve normal function. Using the Lepper et al. allele and tetracycline-controlled transactivator system (rtTA) will chasing TMX for months instead of weeks, Gunther allow examination of the state specific role of Pax7 in et al. observed a requirement for Pax7 in satellite cell satellite cells as they progress along their lineage or other maintenance and function during repair. Pax7 transiently expressed populations as they contribute In addition, Gunther et al. examined a different indu- to muscle repair. ER cible Pax7Cre and Pax7 loxP allele in which Pax7 pro- Due to the caveats of TMX stability, its long-term tein was more rapidly degraded. A shorter TMX chase administration for effective gene recombination and the was required to deplete the satellite cell pool and lose dynamic cell populations within a regenerating muscle, muscle regenerative capacity. Therefore, retention of it would be useful to determine how long TMX can re- Pax7 biological activity altered the kinetics of stem tain recombining activity of satellite cells in vivo. To this Brack Skeletal Muscle 2014, 4:24 Page 4 of 5 http://www.skeletalmusclejournal.com/content/4/1/24 end, one could administer TMX to wild type mice A characteristic morphological feature of quiescent stem that are chased for varying lengths of time and subse- cells is their tightly packed heterochromatic state. Is it quently injected with satellite cells harboring Pax7C- possible that escaper cells have more compact chromatin, ER re -loxP ROSA26 reporter alleles into pre-injured which is relieved during the cell cycle? Gunther et al. muscle. The presence of reporter expression in the re- highlighted an unappreciated role for Pax7 in maintenance generated muscle would provide an estimate of TMX re- of the heterochromatic state; after Pax7 deletion, satellite combining half life. These results would help in the design cells took on a euchromatic morphology, which preceded of experiments that maximize recombination efficiency in their numeric and functional loss. It will be important satellite cells prior to injury, or targeting transit-amplifying to resolve whether subsets of satellite cells have more cells (TAC) cells and facultative cells during injury densely-packed chromatin and whether deregulation of (see Figure 1). chromatin architecture via Pax7-dependent and independ- As the chapter on Pax7?s requirement for adult satellite ent mechanisms lead to changes in satellite cell function. cell function comes to a close, what is next for Pax7 and Gene methylation is a significant roadblock for Cre me- the satellite cell? The phenotypic differences between the diated recombination [18]. Therefore, inaccessible loci will work of Lepper et al. and that of Van Malzhen et al. and more likely avoid recombination, leading to the retention Gunther et al. can be distilled to the presence or absence of normal gene function. To circumvent this limitation, of ? escaper? cells. Which begs the question: are Pax7+ es- due to its open configuration, the ROSA26 locus is often caper cells a unique population or a stochastic anomaly? targeted to insert Cre mediated reporters. While this is a Escape from recombination can arise from many factors, reliable approach for lineage tracing studies, it should be some technical, such as suboptimal TMX, or biological used cautiously as a proxy for gene deletion. Methylation variances, including chromatin compaction and gene status of a target gene at the time of TMX administration methylation or promoter activity. I would argue it unlikely will impact the accuracy with which the ubiquitous re- that TMX dosage played a role. Comparisons between porter reflects deletion of a specific gene. Therefore, direct the different administration strategies employed by Lepper measurement of recombination of the targeted gene locus et al., Van Maltzen et al., and Gunther et al. strongly sug- or protein within the cell of interest is critical. In Table 1 I gest that cell cycle state (quiescence versus proliferating) have outlined TMX strategies for optimal gene recombin- may impose resistance to recombination. It is important ation, measuring recombination efficiency and testing to keep in mind that Lepper et al. observed substantial escaper cell contribution. recombination of the ROSA26 locus in both neonatal and adult muscle, however, only in neonatal muscle, which Conclusions comprises mainly cycling Pax7+ cells, did a phenotype In closing, the studies from Gunther et al. and Van manifest. Maltzen et al. wrap up the debate about the importance Table 1 Strategies for effective gene recombination in adult satellite cells and their progeny during muscle repair TMX delivery Recovery period Gene deletion analysis Escaper cell analysis TMX injection at 3 mg/day for Allow >15 days of TMX chase prior to Isolate purified SCs by FACS, Measure gene or protein levels a c 5 consecutive days muscle injury to target quiescent SCs or from single fibers from over time and minimize lingering TMX activity each experimental animal Five daily injections prior to injury Pros: Temporal identity of cell Measurement of protein In vitro: Measure gene/protein followed by continuous TMX type and state by western blot or immediately after isolation and after feeding delivered in chow immunohistochemistry approximately 10 days in culture (1 mg TMX per day) Minimize recombination of Quantify DNA directly at the In vivo: Measure gene/protein transient cell populations gene locus using site-specific from SCs isolated from uninjured primers flanking the loxP sites and regenerated muscle (>30 days Cons: Escaper cell contribution after injury) No recovery period Pros: Maximize targeting of both stable and transient cell populations Minimize escaper cell contribution Cons: Lose cell type and state resolution Recommendation based on 30-gram adult mouse. Due to variable recombination efficiency between mice, assessment of recombination in every mouse is strongly advised. In addition, I caution the use of R26R-based fluorescent reporters as a surrogate to assess recombination of a gene of interest. In the presence of escaper cells, gene/protein levels of the targeted allele will increase over time if the recombined gene has a loss-of-function phenotype. If recombination results in a gain-of-function mutant or acts redundantly, then gene/protein levels will decrease or not change, respectively. In the absence of escaper cells, the gene/protein of interest will not be detectable. Brack Skeletal Muscle 2014, 4:24 Page 5 of 5 http://www.skeletalmusclejournal.com/content/4/1/24 of Pax7 in adult muscle regeneration. However, the mech- muscle resident progenitor during postnatal development. Nat Cell Biol 2010, 12:257? 266. anisms that preserve satellite cell homeostasis in the 16. Pannerec A, Formicola L, Besson V, Marazzi G, Sassoon DA: Defining absenceofinjury, theroleofPax7asamodifier of skeletal muscle resident progenitors and their cell fate potentials. chromatin architecture, and the requirement of Pax7 Development 2013, 140:2879? 2891. 17. Dellavalle A, Maroli G, Covarello D, Azzoni E, Innocenzi A, Perani L, Antonini S, in muscle resident facultative cells are not yet solved. Sambasivan R, Brunelli S, Tajbakhsh S, Cossu G: Pericytes resident in postnatal In addition, while we critically try to minimize, or at skeletal muscle differentiate into muscle fibres and generate satellite cells. least control for, the limitations of inducible Cre lox Nat Commun 2011, 2:499. 18. Long MA, Rossi FM: Silencing inhibits Cre-mediated recombination of the technology, it is also worth considering the possibility Z/Ap and Z/Eg reporters in adult cells. Plos One 2009, 4:E5435. that such limitations, if understood, may be exploited to help define cellular and molecular heterogeneity doi:10.1186/s13395-014-0024-4 Cite this article as: Brack: Pax7 is back. Skeletal Muscle 2014 4:24. within the satellite cell compartment. Competing interests The author declares that he has no competing interests. Acknowledgements The author would like to thank Susan Eliazer of the Brack lab for reading the commentary. This work was supported by grants from MGH start up funds and NIH grants (RO1 AR060868, R01 AR061002) to ASB. Received: 23 October 2014 Accepted: 27 November 2014 References 1. Mauro A: Satellite cell of skeletal muscle fibers. J Biophys Biochem Cytol 1961, 9:493? 495. 2. Seale P, Sabourin LA, Girgis-Gabardo A, Mansouri A, Gruss P, Rudnicki MA: Pax7 is required for the specification of myogenic satellite cells. Cell 2000, 102:777? 786. 3. Oustanina S, Hause G, Braun T: Pax7 directs postnatal renewal and propagation of myogenic satellite cells but Not their specification. Embo J 2004, 23:3430? 3439. 4. Relaix F, Rocancourt D, Mansouri A, Buckingham M: A Pax3/Pax7- dependent population of skeletal muscle progenitor cells. Nature 2005, 435:948? 953. 5. Kuang S, Charge SB, Seale P, Huh M, Rudnicki MA: Distinct roles for Pax7 and Pax3 in adult regenerative myogenesis. J Cell Biol 2006, 172:103? 113. 6. Lepper C, Conway SJ, Fan CM: Adult satellite cells and embryonic muscle progenitors have distinct genetic requirements. Nature 2009, 460:627? 631. 7. Gunther S, Kim J, Kostin S, Lepper C, Fan CM, Braun T: Myf5-positive satellite cells contribute to Pax7-dependent long-term maintenance of adult muscle stem cells. Cell Stem Cell 2013, 13:590? 601. 8. Von Maltzahn J, Jones AE, Parks RJ, Rudnicki MA: Pax7 is critical for the normal function of satellite cells in adult skeletal muscle. Proc Natl Acad Sci U S A 2013, 110:16474? 16479. 9. Nishijo K, Hosoyama T, Bjornson CR, Schaffer BS, Prajapati SI, Bahadur AN, Hansen MS, Blandford MC, Mccleish AT, Rubin BP, Epstein JA, Rando TA, Capecchi MR, Keller C: Biomarker system for studying muscle, stem cells, and cancer in vivo. FASEB J 2009, 23:2681? 2690. 10. Mm M, Ja L, Sj M, Da H, Kardon G: Satellite cells, connective tissue fibroblasts and their interactions are crucial for muscle regeneration. Development 2011, 138:3625? 3637. 11. Mw D, Coronado E, Osborne CK: Tumor and serum tamoxifen Submit your next manuscript to BioMed Central concentrations in the athymic nude mouse. Cancer Chemother Pharmacol 1989, 23:68? 70. and take full advantage of: 12. Ea L, Solheim E, Lea OA, Lundgren S, Kvinnsland S, Ueland PM: Distribution of 4-hydroxy-N-desmethyltamoxifen and other tamoxifen metabolites in ? Convenient online submission human biological fluids during tamoxifen treatment. Cancer Res 1989, ? Thorough peer review 49:2175? 2183. 13. Zhu Y, Huang YF, Kek C, Bulavin DV: Apoptosis differently affects lineage ? No space constraints or color ?gure charges tracing of Lgr5 and Bmi1 intestinal stem cell populations. Cell Stem Cell ? Immediate publication on acceptance 2013, 12:298? 303. ? Inclusion in PubMed, CAS, Scopus and Google Scholar 14. Rocheteau P, Gayraud-Morel B, Siegl-Cachedenier I, Blasco MA, Tajbakhsh S: A subpopulation of adult skeletal muscle stem cells retains all template ? Research which is freely available for redistribution DNA strands after cell division. Cell 2012, 148:112? 125. 15. Kj M, Pannerec A, Cadot B, Parlakian A, Besson V, Gomes ER, Marazzi G, Submit your manuscript at Sassoon DA: Identification and characterization of a non-satellite cell www.biomedcentral.com/submit

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Skeletal MuscleSpringer Journals

Published: Dec 14, 2014

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