Research Accomplishments
• 1992: As a post-doctoral fellow in the laboratory of Dr. Rudolf Jaenisch, Dr. Michael Rudnicki generated mice carrying targeted null mutations in the myogenic bHLH transcription factors MyoD and Myf-5. Surprisingly, mice lacking MyoD appear healthy and normal, whereas mice lacking Myf-5 die at birth with rib defects and seemingly normal muscle. Cell 71: 383-390; Cell 71: 369-382.
• 1993: We make the seminal contribution that Myf-5 and MyoD are required for myogenic determination as compound mutant mice lacking both Myf-5 and MyoD are entirely ablated of all skeletal muscle. This work is a citation classic, was the subject of many reviews, and is included in several textbooks (e.g. Developmental Biology, S.F. Gilbert). Dr. Michael Rudnicki was the corresponding author on this publication and all experimental analysis was performed in my laboratory at McMaster. Cell 75: 1351-1359.
• 1996: We describe a hitherto unappreciated role for MyoD in regulating the function of satellite cells during regeneration of adult muscle. Under conditions leading to repetitive regeneration of muscle, myogenic stem cells in the absence of MyoD increase in number due to an increased propensity for self-renewal rather than progress to become proliferative myogenic precursor cells that are capable of efficient differentiation. This work was featured on the cover. Moreover the study provides the first molecular evidence that satellite cells represent a distinct compartment of adult myogenic stem cells. Genes Dev. 10: 1173-1183.
• 1996: A discussion with Frank Graham leads to a collaboration resulting in the development of a novel helper-dependent adenovirus vector system in which removal of helper virus is achieved by Cre-mediated excision of the viral packaging signal. This "breakthrough" was the subject of a review in Trends. Gen. Proc. Nat'l. Acad. Sci. USA 93: 13565-13570.
• 1997: The differing temporal-spatial patterns of myogenesis between Myf-5 and MyoD deficient embryos provides the first molecular evidence that Myf-5 and MyoD expression defines the origins of distinct myogenic lineages. This work has lead to a reinterpretation of phenotypes of compound mutant mice lacking myogenic factors. Moreover, this paper was the subject of an extensive review in Bioessays. Development 124: 4729-4738.
• 1998: We show that targeted mutations in the Rb-related p107 and p130 genes in a Balb/c genetic background results in severe phenotypes that are completely strain dependent. Mice lacking p107 display reduced growth, develop myeloid metaplasia, and fibroblasts exhibit a 2-fold acceleration in cell cycle kinetics. Mice lacking p130 die in utero around E11. Both mutants revert to normal phenotypes following a single backcross to C57/B6. This work provides the first evidence that modifier genes exist that have potentially epistatic relationships with Rb-family members. This work has been mentioned in reviews in Science and Trends Gen.. Mol. Cell. Biol. 18: 7455-7465; Development 125: 4669-4679.
• 1999: Unlike Duchenne muscular dystrophy patients, mdx mice (lacking dystrophin) rarely display cardiac abnormalities and do not develop extensive dystrophy in skeletal muscle. Histological and molecular analysis indicated that mdx:MyoD-/- mice develop a progressive cardiac myopathy with areas of necrosis associated with hypertrophied myocytes. Therefore, these results suggest that the progression of dystrophic damage in skeletal muscle is a hitherto unappreciated contributing factor leading to development of cardiomyopathy. This work was the subject of an article in Bioworld. Proc. Nat'l. Acad. Sci. USA 96: 220-225.
• 1999: Analysis of primary MyoD-/- myogenic cells reveals relatively primitive characteristics together with a marked reduction in differentiation potential. These observations led us to suggest the hypothesis that expression of Myf-5 alone may define an intermediate developmental stage between quiescent satellite cell and proliferating myogenic precursor cells that facilitates stem cell self renewal. J. Cell Biol. 144: 631-643.
• 2000: Using a PCR-based cloning protocol termed representational difference analysis (RDA), we identified the paired box transcription factor Pax7 as being expressed in satellite cells. In situ hybridization revealed that Pax7 was also expressed in satellite cells residing in adult muscle. Cell culture and electron microscopic analysis revealed a complete absence of satellite cells in Pax7-/- skeletal muscle. Surprisingly, FACS analysis indicated that the proportion of muscle-derived stem cells was unaffected. Importantly, stem cells from Pax7-/- muscle displayed almost a 10-fold increase in their ability to form hematopoietic colonies. These results demonstrate that satellite cells and muscle-derived stem cells represent distinct cell populations. Together these studies suggest that induction of Pax7 in muscle-derived stem cells induces satellite cell specification by restricting alternative developmental programs. Cell 102: 777-786 (2000).
• 2001: To elucidate the mechanism through which MAPK signaling regulates the MyoD-family of transcription factors, we investigated the role of the signaling intermediate MEK1 in myogenesis. Transfection of activated MEK1 strongly repressed gene activation and myogenic conversion by the MyoD-family. This repression was not mediated by direct phosphorylation of MyoD, or by changes in MyoD stability or subcellular distribution. Deletion mapping revealed that MEK1-mediated repression required the MyoD amino-terminal transactivation domain. Moreover, activated MEK1 was nuclearly localized and bound a complex containing MyoD in a manner dependent on the presence of the MyoD amino-terminus. Together, these data demonstrate that MEK1 signaling has a strong negative effect on MyoD activity via a novel mechanism involving binding of MEK1 to the nuclear MyoD transcriptional complex. Molecular Cell 8: 291-301 (2001).
• 2003: This study demonstrated a biological role for endogenous CD45+ muscle stem cells in maintaining tissue integrity by participating in regeneration. Moreover, these experiments established that Wnt-signaling is the mechanism by which CD45+ adult stem cells are induced to undergo myogenic specification. Importantly, our study suggested that targeting the Wnt-pathway represents a promising therapeutic approach for the treatment of neuromuscular degenerative disease. Cell 113:841-852.
• 2004: Demonstrated that infection of muscle derived adult stem cells with retroviral Pax7 vectors induced myogenic specification. This work provided conclusive evidence that Pax7 is necessary and sufficient to induce the myogenic specification of adult stem cells resident in skeletal muscle. Moreover, these experiments suggest that viral transduction of Pax7 is a potential therapeutic approach for the treatment of neuromuscular degenerative diseases. PLoS Biol.2(5):E130. Epub May 11.
• 2005: Demonstrate a role for p107 in regulating brown fat determination in the adult. The Rb-family, Rb, p107 and p130 play important roles in cell-cycle control and cellular differentiation and Rb has been suggested to regulate adipocyte differentiation. We find that mice lacking p107 displayed a uniform replacement of white adipose tissue (WAT) with brown adipose tissue (BAT). Mutant WAT depots contained mutilocular adipocytes that expressed the elevated levels of PGC-1α and UCP-1 typical of BAT. WAT from p107-/- mice contained markedly elevated numbers of preadipogenic precursors that displayed down-regulated expression of pRb. Importantly, pRb was observed to bind the PGC-1α promoter and repress transcription. Therefore, p107 and pRb regulate PGC-1α expression to control the switch between white and brown adipocyte differentiation from a common pool of presumptive adult progenitors in fat tissue. Cell Metabolism 2:283-295. (2005).
• 2006: We demonstrate distinct roles for Pax7 and Pax3 in regenerative myogenesis. Viable Pax7-/- mice in 129Sv/J background display reduced growth and marked muscle wasting together with a complete absence of functional satellite cells. Acute injury resulted in an extreme deficit in regeneration. However, a small number of new myofibers were detected suggesting the presence of residual myogenic cells. Rare Pax3+/MyoD+ myoblasts were recovered from muscle interstitial tissues. Finally, we identified Pax3+ cells in the muscle interstitial environment and demonstrated that they co-expressed MyoD during regeneration. Sublaminar satellite cells in hindlimb muscle did not express detectable levels of Pax3 protein or mRNA. Therefore, we conclude that Pax3-expressing cells represent a novel myogenic population that is distinct from the satellite cell lineage and that Pax7 is essential for the formation of satellite cells. Journal of Cell Biology 172:103-113 (2006)
• 2007: We identify and characterize a subpopulation of satellite cells as bona fide stem cells. Whether satellite cells are stem cells, committed progenitors or de-differentiated myoblasts has remained unclear. Using Myf5-Cre and ROSA26-YFP Cre alleles, we observed that in vivo 10% of sub-laminar Pax7-expressing satellite cells have never expressed Myf5. Moreover, we found that Pax7+/Myf5- satellite cells gave rise to Pax7+/Myf5+ satellite cells through apical-basal oriented divisions that asymmetrically generated a basal Pax7+/Myf5- and an apical Pax7+/Myf5+ cells. Prospective isolation and transplantation into muscle revealed that whereas Pax7+/Myf5+ cells exhibited precocious differentiation, Pax7+/Myf5- cells extensively contributed to the satellite cell reservoir throughout the injected muscle. Therefore, we conclude that satellite cells are a heterogeneous population composed of stem cells and committed progenitors. These results provide critical insights into satellite cell biology and open new avenues for therapeutic treatment of muscular diseases. Cell 129:999-1010 (2007).
• 2008: We identify the molecular mechanism through which Pax7 activates gene transcription. Using tandem affinity purification (TAP) and mass spectrometry revealed that Pax7 associates with the Wdr5/Ash2L/MLL2 histone methyltransferase (HMT) complex that directs methylation of histone H3 lysine 4 (H3K4). Importantly, binding of the Pax7-HMT complex to Myf5 resulted in H3K4 tri-methylation of surrounding chromatin. Thus, Pax7 induces chromatin modifications that stimulate transcriptional activation of target genes to regulate entry into the myogenic developmental program. Nature Cell Biology 10:77-84 (2008).
• 2008: In a collaborative project with the Speigelman laboratory, we demonstrate that PRDM16 controls a brown fat/skeletal muscle developmental switch. Our contribution was to perform a lineage marking study to genetical demonstrate that brown fat is derived from muscle progenitors in the embryo. This study was named by Science as one of the top ten discoveries in 2008, is the subject of many commentaries and reviews, and is on its way to become a citation classic. Nature 454(7207):961-967 (2008).
• 2009: We make the seminal discovery that Wnt7a/Fzd7 signaling activates the planar cell polarity pathway to drive the symmetric expansion of satellite stem cells during muscle growth and repair. We found that satellite stem cells expressed the Wnt-receptor Fzd7, and that its ligand Wnt7a was upregulated during regeneration. Wnt7a markedly stimulated the symmetric expansion of satellite stem cells but did not affect the growth or differentiation of myoblasts. Silencing of Fzd7 abrogated Wnt7a binding and stimulation of stem cell expansion. Wnt7a signaling induced the polarized distribution of the planar cell polarity effector Vangl2. Silencing of Vangl2 inhibited Wnt7a action on satellite stem cell expansion. Wnt7a overexpression enhanced muscle regeneration and increased both the number of satellite cells and muscle mass. Muscle lacking Wnt7a exhibited a decrease in satellite cell number following regeneration. Therefore, Wnt7a signaling through the planar cell polarity pathway controls the homeostatic level of satellite stem cells and hence regulates the regenerative potential of muscle. Cell Stem Cell. 4(6):535-547 (2009).
• 2012: We discover a new arm of Wnt signalling, a novel non-canonical anabolic pathway for Wnt7a and its receptor Fzd7 in skeletal muscle. We describe a new mechanism involved in the regulation of hypertrophy of differentiated muscle tissue. Thus, Wnt7a acts at two cellular levels in skeletal muscle, through PCP signalling to regulate the homeostatic levels of satellite cells, and through the Akt/mTor pathway to regulate myofiber growth. Nature Cell Biology 14(2):186-191.
• Using gene expression data combined with genome wide binding-site analysis we show that both Pax3 and Pax7 bind identical DNA motifs and jointly activate a large panel of genes involved in muscle stem cell function. Surprisingly, in adult myoblasts Pax3 binds a subset (6.4%) of Pax7 targets. Despite a significant overlap in their transcriptional network, Pax7 regulates unique panels of genes involved in the promotion of proliferation and inhibition of myogenic differentiation. We show that Pax7 has a higher binding affinity to the homeodomain-binding motif relative to Pax3, suggesting that intrinsic differences in DNA binding contribute to the observed functional difference between Pax3 and Pax7 binding in myogenesis. Together, our data demonstrates unique attributes of Pax7 function and provides mechanistic insight into the non-redundancy of Pax3 and Pax7 in muscle development. Developmental Cell 22(6): 1208-20.
• Using ChIP-seq and gene expression analyses, we show that in primary myoblasts, Snail-HDAC1/2 repressive complex bind and exclude MyoD from its targets. Notably, Snail binds E-box motifs that are G/C-rich in their central dinucleotides, and such sites are almost exclusively associated with genes expressed during differentiation. By contrast, Snail does not bind the A/T-rich E-boxes associated with MyoD targets in myoblasts. Thus, Snai1-HDAC1/2 prevents MyoD occupancy on differentiation-specific regulatory elements and the change from Snail- to MyoD-binding often results in enhancer switching during differentiation. Furthermore, we show that a regulatory network involving Myogenic Regulatory Factors (MRFs), Snail/2, miR-30a and miR-206 acts as a molecular switch that controls entry into myogenic differentiation. Together, these results reveal a regulatory paradigm that directs distinct gene expression programs in progenitors versus terminally differentiated cells. Molecular Cell 47(3):457-68.
• We identify the arginine methyltransferase Carm1 as a Pax7 interacting protein and found that Carm1 specifically methylates multiple arginines in the N-terminus of Pax7. Methylated Pax7 directly binds the C-terminal cleavage forms of the trithorax proteins MLL1/2 resulting in the recruitment of the ASH2L:MLL1/2:WDR5:RBBP5 histone H3K4 methyltransferase complex to regulatory enhancers and the proximal promoter of Myf5. Finally, Carm1 is required for the induction of de novo Myf5 transcription following asymmetric satellite stem cell divisions. We defined the C-terminal MLL region as a novel reader domain for the recognition of arginine methylated proteins such as Pax7. Thus, arginine methylation of Pax7 by Carm1 functions as a molecular switch controlling the epigenetic induction of Myf5 during satellite stem cell asymmetric division and entry into the myogenic program. Cell Stem Cell 11(3):333-45.
• We investigate the therapeutic potential of Wnt7a for focal treatment of dystrophic DMD muscle using the mdx mouse model. We found that Wnt7a treatment efficiently induced satellite cell expansion and myofiber hypertrophy in treated mucles in mdx mice. Importantly, Wnt7a treatment resulted in a significant increase in strength of the muscle as determined by generation of specific force. Furthermore Wnt7a reduced the level of contractile damage likely by inducing a fiber type shift towards slow-twitch. Lastly, we found that in human primary myotubes Wnt7a similarly induced myotube hypertrophy and a shift in fiber type towards slow-twitch. Taken together, our experiments suggest that Wnt7a is a promising candidate for development as an ameliorative treatment for muscular dystrophy. Proc. Nat’l Acad, Sci. USA 109(50):20614-9.
• 2013: We found that Syndecan-4 (Sdc4) and Frizzled-7 (Fzd7) form a co-receptor complex in satellite cells and that binding of the ECM glycoprotein Fibronectin (FN) to Sdc4 stimulates the ability of Wnt7a to induce the symmetric expansion of satellite stem cells. Newly activated satellite cells dynamically remodel their niche by transient high-level expression of FN. Knockdown of FN in prospectively isolated satellite cells severely impaired their ability to repopulate the satellite cell niche. Conversely, in vivo over-expression of FN with Wnt7a dramatically stimulated the expansion of satellite stem cells in regenerating muscle. Therefore, activating satellite cells remodel their niche through autologous expression of FN that provides feedback to stimulate Wnt7a signaling through the Fzd7/Sdc4 co-receptor complex. Thus, FN and Wnt7a together regulate the homeostatic levels of satellite stem cells and satellite myogenic cells during regenerative myogenesis. Cell Stem Cell 12(1):75-87.
• We discover that adult satellite cells give rise to brown adipocytes and that microRNA-133 regulates the choice between myogenic and brown adipose determination by targeting the 3'UTR of Prdm16. Antagonism of microRNA-133 during muscle regeneration increases uncoupled respiration, glucose uptake and thermogenesis in local treated muscle and augments whole-body energy expenditure, improves glucose tolerance and impedes the development of diet-induced obesity. Finally, we demonstrate that miR-133 levels are down regulated in mice exposed to cold resulting in de novo generation of satellite cell derived brown adipocytes. Therefore, microRNA-133 represents an important therapeutic target for the treatment of obesity. Cell Metabolism 17(2):210-24.
• We demonstrate that Pax7 is an absolute requirement for satellite cell function in adult skeletal muscle. Following Pax7 deletion, satellite cells and myoblasts exhibit cell-cycle arrest and dysregulation of myogenic regulatory factors. Maintenance of Pax7 deletion through continuous tamoxifen administration prevented regrowth of Pax7-expressing satellite cells and a profound muscle regeneration deficit that resembles the phenotype of skeletal muscle following genetically engineered ablation of satellite cells. Therefore, we conclude that Pax7 is essential for regulating the expansion and differentiation of satellite cells during both neonatal and adult myogenesis. Proc. Nat’l Acad, Sci. USA. Early Release.