Dissecting dual roles of MyoD during lineage conversion to mature myocytes and myogenic stem cells.

The generation of myotubes from fibroblasts upon forced MyoD expression is a classic example of transcription factor-induced reprogramming. We recently discovered that additional modulation of signaling pathways with small molecules facilitates reprogramming to more primitive induced myogenic progenitor cells (iMPCs). Here, we dissected the transcriptional and epigenetic dynamics of mouse fibroblasts undergoing reprogramming to either myotubes or iMPCs using a MyoD-inducible transgenic model. Induction of MyoD in fibroblasts combined with small molecules generated Pax7+ iMPCs with high similarity to primary muscle stem cells. Analysis of intermediate stages of iMPC induction revealed that extinction of the fibroblast program preceded induction of the stem cell program. Moreover, key stem cell genes gained chromatin accessibility prior to their transcriptional activation, and these regions exhibited a marked loss of DNA methylation dependent on the Tet enzymes. In contrast, myotube generation was associated with few methylation changes, incomplete and unstable reprogramming, and an insensitivity to Tet depletion. Finally, we showed that MyoD's ability to bind to unique bHLH targets was crucial for generating iMPCs but dispensable for generating myotubes. Collectively, our analyses elucidate the role of MyoD in myogenic reprogramming and derive general principles by which transcription factors and signaling pathways cooperate to rewire cell identity.

Nfix regulates fetal-specific transcription in developing skeletal muscle.

Skeletal myogenesis, like hematopoiesis, occurs in successive developmental stages that involve different cell populations and expression of different genes. We show here that the transcription factor nuclear factor one X (Nfix), whose expression is activated by Pax7 in fetal muscle, in turn activates the transcription of fetal specific genes such as MCK and beta-enolase while repressing embryonic genes such as slow myosin. In the case of the MCK promoter, Nfix forms a complex with PKC theta that binds, phosphorylates, and activates MEF2A. Premature expression of Nfix activates fetal and suppresses embryonic genes in embryonic muscle, whereas muscle-specific ablation of Nfix prevents fetal and maintains embryonic gene expression in the fetus. Therefore, Nfix acts as a transcriptional switch from embryonic to fetal myogenesis.

Development and patterning of rib primordia are dependent on associated musculature.

The importance of skeletal muscle for rib development and patterning in the mouse embryo has not been resolved, largely because different experimental approaches have yielded disparate results. In this study, we utilize both gene knockouts and muscle cell ablation approaches to re-visit the extent to which rib growth and patterning are dependent on developing musculature. Consistent with previous studies, we show that rib formation is highly dependent on the MYOD family of myogenic regulatory factors (MRFs), and demonstrate that the extent of rib formation is gene-, allele-, and dosage-dependent. In the absence of Myf5 and MyoD, one allele of Mrf4 is sufficient for extensive rib growth, although patterning is abnormal. Under conditions of limiting MRF dosage, MyoD is identified as a positive regulator of rib patterning, presumably due to improved intercostal muscle development. In contrast to previous muscle ablation studies, we show that diphtheria toxin subunit A (DTA)-mediated ablation of muscle progenitors or differentiated muscle, using MyoDiCre or HSA-Cre drivers, respectively, profoundly disrupts rib development. Further, a comparison of three independently derived Rosa26-based DTA knockin alleles demonstrates that the degree of rib perturbations in MyoDiCre/+/DTA embryos is markedly dependent on the DTA allele used, and may in part explain discrepancies with previous findings. The results support the conclusion that the extent and quality of rib formation is largely dependent on the dosage of Myf5 and Mrf4, and that both early myotome-sclerotome interactions, as well as later muscle-rib interactions, are important for proper rib growth and patterning.

Disruption of ATP-sensitive potassium channel function in skeletal muscles promotes production and secretion of musclin.

Sarcolemmal ATP-sensitive potassium (KATP) channels control skeletal muscle energy use through their ability to adjust membrane excitability and related cell functions in accordance with cellular metabolic status. Mice with disrupted skeletal muscle KATP channels exhibit reduced adipocyte size and increased fatty acid release into the circulation. As yet, the molecular mechanisms underlying this link between skeletal muscle KATP channel function and adipose mobilization have not been established. Here, we demonstrate that skeletal muscle-specific disruption of KATP channel function in transgenic (TG) mice promotes production and secretion of musclin. Musclin is a myokine with high homology to atrial natriuretic peptide (ANP) that enhances ANP signaling by competing for elimination. Augmented musclin production in TG mice is driven by a molecular cascade resulting in enhanced acetylation and nuclear exclusion of the transcription factor forkhead box O1 (FOXO1) - an inhibitor of transcription of the musclin encoding gene. Musclin production/secretion in TG is paired with increased mobilization of fatty acids and a clear trend toward increased circulating ANP, an activator of lipolysis. These data establish KATP channel-dependent musclin production as a potential mechanistic link coupling "local" skeletal muscle energy consumption with mobilization of bodily resources from fat. Understanding such mechanisms is an important step toward designing interventions to manage metabolic disorders including those related to excess body fat and associated co-morbidities.

MyoD-expressing progenitors are essential for skeletal myogenesis and satellite cell development.

Skeletal myogenesis in the embryo is regulated by the coordinated expression of the MyoD family of muscle regulatory factors (MRFs). MyoD and Myf-5, which are the primary muscle lineage-determining factors, function in a partially redundant manner to establish muscle progenitor cell identity. Previous diphtheria toxin (DTA)-mediated ablation studies showed that MyoD+ progenitors rescue myogenesis in embryos in which Myf-5-expressing cells were targeted for ablation, raising the possibility that the regulative behavior of distinct, MRF-expressing populations explains the functional compensatory activities of these MRFs. Using MyoD(iCre) mice, we show that DTA-mediated ablation of MyoD-expressing cells results in the cessation of myogenesis by embryonic day 12.5 (E12.5), as assayed by myosin heavy chain (MyHC) and Myogenin staining. Importantly, MyoD(iCre/+);R26(DTA/+) embryos exhibited a concomitant loss of Myf-5+ progenitors, indicating that the vast majority of Myf-5+ progenitors express MyoD, a conclusion consistent with immunofluorescence analysis of Myf-5 protein expression in MyoD(iCre) lineage-labeled embryos. Surprisingly, staining for the paired box transcription factor, Pax7, which functions genetically upstream of MyoD in the trunk and is a marker for fetal myoblasts and satellite cell progenitors, was also lost by E12.5. Specific ablation of differentiating skeletal muscle in ACTA1Cre;R26(DTA/+) embryos resulted in comparatively minor effects on MyoD+, Myf-5+ and Pax7+ progenitors, indicating that cell non-autonomous effects are unlikely to explain the rapid loss of myogenic progenitors in MyoD(iCre/+);R26(DTA/+) embryos. We conclude that the vast majority of myogenic cells transit through a MyoD+ state, and that MyoD+ progenitors are essential for myogenesis and stem cell development.

Tissue-specific role of the Na,K-ATPase α2 isozyme in skeletal muscle.

The Na,K-ATPase α2 isozyme is the major Na,K-ATPase of mammalian skeletal muscle. This distribution is unique compared with most other cells, which express mainly the Na,K-ATPase α1 isoform, but its functional significance is not known. We developed a gene-targeted mouse (skα2(-/-)) in which the α2 gene (Atp1a2) is knocked out in the skeletal muscles, and examined the consequences for exercise performance, membrane potentials, contractility, and muscle fatigue. Targeted knockout was confirmed by genotyping, Western blot, and immunohistochemistry. Skeletal muscle cells of skα2(-/-) mice completely lack α2 protein and have no α2 in the transverse tubules, where its expression is normally enhanced. The α1 isoform, which is normally enhanced on the outer sarcolemma, is up-regulated 2.5-fold without change in subcellular targeting. skα2(-/-) mice are apparently normal under basal conditions but show significantly reduced exercise capacity when challenged to run. Their skeletal muscles produce less force, are unable to increase force to match demand, and show significantly increased susceptibility to fatigue. The impairments affect both fast and slow muscle types. The subcellular targeting of α2 to the transverse tubules is important for this role. Increasing Na,K-ATPase α1 content cannot fully compensate for the loss of α2. The increased fatigability of skα2(-/-) muscles is reproduced in control extensor digitorum longus muscles by selectively inhibiting α2 enzyme activity with ouabain. These results demonstrate that the Na,K-ATPase α2 isoform performs an acute, isoform-specific role in skeletal muscle. Its activity is regulated by muscle use and enables working muscles to maintain contraction and resist fatigue.

A non-canonical E-box within the MyoD core enhancer is necessary for circadian expression in skeletal muscle.

The myogenic differentiation 1 (MyoD) gene is a master regulator of myogenesis. We previously reported that the expression of MyoD mRNA oscillates over 24 h in skeletal muscle and that the circadian clock transcription factors, BMAL1 (brain and muscle ARNT-like 1) and CLOCK (circadian locomotor output cycles kaput), were bound to the core enhancer (CE) of the MyoD gene in vivo. In this study, we provide in vivo and in vitro evidence that the CE is necessary for circadian expression of MyoD in adult muscle. Gel shift assays identified a conserved non-canonical E-box within the CE that is bound by CLOCK and BMAL1. Functional analysis revealed that this E-box was required for full activation by BMAL1/CLOCK and for in vitro circadian oscillation. Expression profiling of muscle of CE(loxP/loxP) mice found approximately 1300 genes mis-expressed relative to wild-type. Based on the informatics results, we analyzed the respiratory function of mitochondria isolated from wild-type and CE(loxP/loxP) mice. These assays determined that State 5 respiration was significantly reduced in CE(loxP/loxP) muscle. The results of this work identify a novel element in the MyoD enhancer that confers circadian regulation to MyoD in skeletal muscle and suggest that loss of circadian regulation leads to changes in myogenic expression and downstream mitochondrial function.

Sex as a Critical Variable in Basic and Pre-Clinical Studies of Fibrodysplasia Ossificans Progressiva.

Heterotopic ossification (HO) is most dramatically manifested in the rare and severely debilitating disease, fibrodysplasia ossificans progressiva (FOP), in which heterotopic bone progressively accumulates in skeletal muscles and associated soft tissues. The great majority of FOP cases are caused by a single amino acid substitution in the type 1 bone morphogenetic protein (BMP) receptor ACVR1, a mutation that imparts responsiveness to activin A. Although it is well-established that biological sex is a critical variable in a range of physiological and disease processes, the impact of sex on HO in animal models of FOP has not been explored. We show that female FOP mice exhibit both significantly greater and more variable HO responses after muscle injury. Additionally, the incidence of spontaneous HO was significantly greater in female mice. This sex dimorphism is not dependent on gonadally derived sex hormones, and reciprocal cell transplantations indicate that apparent differences in osteogenic activity are intrinsic to the sex of the transplanted cells. By circumventing the absolute requirement for activin A using an agonist of mutant ACVR1, we show that the female-specific response to muscle injury or BMP2 implantation is dependent on activin A. These data identify sex as a critical variable in basic and pre-clinical studies of FOP.

Survival motor neuron protein in motor neurons determines synaptic integrity in spinal muscular atrophy.

The inherited motor neuron disease spinal muscular atrophy (SMA) is caused by deficient expression of survival motor neuron (SMN) protein and results in severe muscle weakness. In SMA mice, synaptic dysfunction of both neuromuscular junctions (NMJs) and central sensorimotor synapses precedes motor neuron cell death. To address whether this synaptic dysfunction is due to SMN deficiency in motor neurons, muscle, or both, we generated three lines of conditional SMA mice with tissue-specific increases in SMN expression. All three lines of mice showed increased survival, weights, and improved motor behavior. While increased SMN expression in motor neurons prevented synaptic dysfunction at the NMJ and restored motor neuron somal synapses, increased SMN expression in muscle did not affect synaptic function although it did improve myofiber size. Together these data indicate that both peripheral and central synaptic integrity are dependent on motor neurons in SMA, but SMN may have variable roles in the maintenance of these different synapses. At the NMJ, it functions at the presynaptic terminal in a cell-autonomous fashion, but may be necessary for retrograde trophic signaling to presynaptic inputs onto motor neurons. Importantly, SMN also appears to function in muscle growth and/or maintenance independent of motor neurons. Our data suggest that SMN plays distinct roles in muscle, NMJs, and motor neuron somal synapses and that restored function of SMN at all three sites will be necessary for full recovery of muscle power.

Overexpression of Wild-Type ACVR1 in Fibrodysplasia Ossificans Progressiva Mice Rescues Perinatal Lethality and Inhibits Heterotopic Ossification.

Fibrodysplasia ossificans progressiva (FOP) is a devastating disease of progressive heterotopic bone formation for which effective treatments are currently unavailable. FOP is caused by dominant gain-of-function mutations in the receptor ACVR1 (also known as ALK2), which render the receptor inappropriately responsive to activin ligands. In previous studies, we developed a genetic mouse model of FOP that recapitulates most clinical aspects of the disease. In this model, genetic loss of the wild-type Acvr1 allele profoundly exacerbated heterotopic ossification, suggesting the hypothesis that the stoichiometry of wild-type and mutant receptors dictates disease severity. Here, we tested this model by producing FOP mice that conditionally overexpress human wild-type ACVR1. Injury-induced heterotopic ossification (HO) was completely blocked in FOP mice when expression of both the mutant and wild-type receptor were targeted to Tie2-positive cells, which includes fibro/adipogenic progenitors (FAPs). Perinatal lethality of Acvr1R206H/+ mice was rescued by constitutive ACVR1 overexpression, and these mice survived to adulthood at predicted Mendelian frequencies. Constitutive overexpression of ACVR1 also provided protection from spontaneous abnormal skeletogenesis, and the incidence and severity of injury-induced HO in these mice was dramatically reduced. Analysis of pSMAD1/5/8 signaling both in cultured cells and in vivo indicates that ACVR1 overexpression functions cell-autonomously by reducing osteogenic signaling in response to activin A. We propose that ACVR1 overexpression inhibits HO by decreasing the abundance of ACVR1(R206H)-containing signaling complexes at the cell surface while increasing the representation of activin-A-bound non-signaling complexes comprised of wild-type ACVR1. © 2022 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

An anti-ACVR1 antibody exacerbates heterotopic ossification by fibro-adipogenic progenitors in fibrodysplasia ossificans progressiva mice.

Fibrodysplasia ossificans progressiva (FOP) is a rare genetic disease characterized by progressive and catastrophic heterotopic ossification (HO) of skeletal muscle and associated soft tissues. FOP is caused by dominantly acting mutations in the gene encoding the bone morphogenetic protein (BMP) type I receptor, ACVR1 (ALK2), the most prevalent of which results in an arginine to histidine substitution at position 206 (ACVR1[R206H]). The fundamental pathological consequence of FOP-causing ACVR1 receptor mutations is to enable activin A to initiate canonical BMP signaling in fibro-adipogenic progenitors (FAPs), which drives HO. We developed a monoclonal blocking antibody (JAB0505) against the extracellular domain of ACVR1 and tested its effect on HO in 2 independent FOP mouse models. Although JAB0505 inhibited BMP-dependent gene expression in wild-type and ACVR1(R206H)-overexpressing cell lines, JAB0505 treatment profoundly exacerbated injury-induced HO. JAB0505-treated mice exhibited multiple, distinct foci of heterotopic lesions, suggesting an atypically broad anatomical domain of FAP recruitment to endochondral ossification. This was accompanied by dysregulated FAP population growth and an abnormally sustained immunological reaction following muscle injury. JAB0505 drove injury-induced HO in the absence of activin A, indicating that JAB0505 has receptor agonist activity. These data raise serious safety and efficacy concerns for the use of bivalent anti-ACVR1 antibodies to treat patients with FOP.

Progenitors of skeletal muscle satellite cells express the muscle determination gene, MyoD.

Satellite cells are tissue-specific stem cells responsible for skeletal muscle growth and regeneration. Although satellite cells were identified almost 50 years ago, the identity of progenitor populations from which they derive remains controversial. We developed MyoD(iCre) knockin mice, and used Cre/lox lineage analysis to determine whether satellite cell progenitors express MyoD, a marker of myogenic commitment. Recombination status of satellite cells was determined by confocal microscopy of isolated muscle fibers and by electron microscopic observation of muscle tissue fixed immediately following isolation, using R26R-EYFP and R26R (beta-gal) reporter mice, respectively. We show that essentially all adult satellite cells associated with limb and body wall musculature, as well as the diaphragm and extraocular muscles, originate from MyoD+ progenitors. Neonatal satellite cells were Cre-recombined, but only a small minority exhibited ongoing Cre expression, indicating that most satellite cells had expressed MyoD prenatally. We also show that satellite cell development in MyoD-null mice is not due to functional compensation by MyoD non-expressing lineages. The results suggest that satellite cells are derived from committed myogenic progenitors, irrespective of the anatomical location, embryological origin, or physiological properties of associated musculature.

Mutant ACVR1 Arrests Glial Cell Differentiation to Drive Tumorigenesis in Pediatric Gliomas.

Diffuse intrinsic pontine gliomas (DIPGs) are aggressive pediatric brain tumors for which there is currently no effective treatment. Some of these tumors combine gain-of-function mutations in ACVR1, PIK3CA, and histone H3-encoding genes. The oncogenic mechanisms of action of ACVR1 mutations are currently unknown. Using mouse models, we demonstrate that Acvr1G328V arrests the differentiation of oligodendroglial lineage cells, and cooperates with Hist1h3bK27M and Pik3caH1047R to generate high-grade diffuse gliomas. Mechanistically, Acvr1G328V upregulates transcription factors which control differentiation and DIPG cell fitness. Furthermore, we characterize E6201 as a dual inhibitor of ACVR1 and MEK1/2, and demonstrate its efficacy toward tumor cells in vivo. Collectively, our results describe an oncogenic mechanism of action for ACVR1 mutations, and suggest therapeutic strategies for DIPGs.

A multifunctional reporter mouse line for Cre- and FLP-dependent lineage analysis.

The Cre/lox and FLP/FRT recombination systems have been used extensively for both conditional knockout and cell lineage analysis in mice. Here we report a new multifunctional Cre/FLP dual reporter allele (R26(NZG)) that exhibits strong and apparently ubiquitous marker expression in embryos and adults. The reporter construct, which is driven by the CAG promoter, was knocked into the ROSA26 locus providing an open chromatin domain for consistent expression and avoiding site-of-integration effects often observed with transgenic reporters. R26(NZG) directs Cre-dependent nuclear-localized beta-galactosidase (beta-gal) expression, and can be converted into a Cre-dependent EGFP reporter (R26(NG)) by germline excision of the FRT-flanked nlslacZ cassette. Alternatively, germline excision of the floxed PGKNEO cassette in R26(NZG) generates an FLP-dependent EGFP reporter (R26(ZG)) that expresses beta-gal in FLP-nonexpressing cells. Finally, by the simultaneous use of both Cre and FLP deleters, R26(NZG) allows lineage relationships to be interrogated with greater refinement than is possible with single recombinase reporter systems.

Response to comment on 'Palovarotene reduces heterotopic ossification in juvenile FOP mice but exhibits pronounced skeletal toxicity'.

We respond to concerns expressed by Pacifici and Shore (2019) about a recent paper (Lees-Shepard and Goldhamer, 2018a) in which we reported that the drug palovarotene can have severe side effects in a mouse model of fibrodysplasia ossificans progressiva.

Late-onset megaconial myopathy in mice lacking group I Paks.

BACKGROUND: Group I Paks are serine/threonine kinases that function as major effectors of the small GTPases Rac1 and Cdc42, and they regulate cytoskeletal dynamics, cell polarity, and transcription. We previously demonstrated that Pak1 and Pak2 function redundantly to promote skeletal myoblast differentiation during postnatal development and regeneration in mice. However, the roles of Pak1 and Pak2 in adult muscle homeostasis are unknown. Choline kinase ß (Chk ß) is important for adult muscle homeostasis, as autosomal recessive mutations in CHKß are associated with two human muscle diseases, megaconial congenital muscular dystrophy and proximal myopathy with focal depletion of mitochondria. METHODS: We analyzed mice conditionally lacking Pak1 and Pak2 in the skeletal muscle lineage (double knockout (dKO) mice) over 1 year of age. Muscle integrity in dKO mice was assessed with histological stains, immunofluorescence, electron microscopy, and western blotting. Assays for mitochondrial respiratory complex function were performed, as was mass spectrometric quantification of products of choline kinase. Mice and cultured myoblasts deficient for choline kinase ß (Chk ß) were analyzed for Pak1/2 phosphorylation. RESULTS: dKO mice developed an age-related myopathy. By 10 months of age, dKO mouse muscles displayed centrally-nucleated myofibers, fibrosis, and signs of degeneration. Disease severity occurred in a rostrocaudal gradient, hindlimbs more strongly affected than forelimbs. A distinctive feature of this myopathy was elongated and branched intermyofibrillar (megaconial) mitochondria, accompanied by focal mitochondrial depletion in the central region of the fiber. dKO muscles showed reduced mitochondrial respiratory complex I and II activity. These phenotypes resemble those of rmd mice, which lack Chkß and are a model for human diseases associated with CHKß deficiency. Pak1/2 and Chkß activities were not interdependent in mouse skeletal muscle, suggesting a more complex relationship in regulation of mitochondria and muscle homeostasis. CONCLUSIONS: Conditional loss of Pak1 and Pak2 in mice resulted in an age-dependent myopathy with similarity to mice and humans with CHKß deficiency. Protein kinases are major regulators of most biological processes but few have been implicated in muscle maintenance or disease. Pak1/Pak2 dKO mice offer new insights into these processes.

Myogenic reprogramming of retina-derived cells following their spontaneous fusion with myotubes.

Satellite cells are recognized as the main source for myoblasts in postnatal muscle. The possible participation of other cell types in myofiber maintenance remains a subject of debate. Here, we investigated the potential of vascular preparations from mouse retina to undergo myogenesis when cultured alone or with differentiated primary myogenic cultures. The choice of retina, an organ richly supplied with capillary network and anatomically separated from skeletal muscles, ensures that the vasculature preparation is devoid of satellite cells. We demonstrate that retina-derived cells spontaneously fuse with preexisting myotubes and contribute additional myonuclei, some of which initiate expression of muscle-specific genes after fusion. Myogenic differentiation of retinal cells prior to their fusion with preexisting myotubes was not detected. Although originating from vasculature preparations, nuclei undergoing myogenic reprogramming were contributed by cells that were neither endothelial nor blood borne. Our results suggest smooth muscle/pericytes as the possible source, and that myogenic reprogramming depends on the muscle specific transcription factor MyoD. Our studies provide insights into a novel avenue for myofiber maintenance, relying on nuclei of non-myogenic origin that undergo fusion and subsequent myogenic conversion within host myofibers. This process may support ongoing myofiber maintenance throughout life.

Essential role for Dicer during skeletal muscle development.

microRNAs (miRNAs) regulate gene expression post-transcriptionally by targeting mRNAs for degradation or by inhibiting translation. Dicer is an RNase III endonuclease which processes miRNA precursors into functional 21-23 nucleotide RNAs that are subsequently incorporated into the RNA-induced silencing complex. miRNA-mediated gene regulation is important for organogenesis of a variety of tissues including limb, lung and skin. To gain insight into the roles of Dicer and miRNAs in mammalian skeletal muscle development, we eliminated Dicer activity specifically in the myogenic compartment during embryogenesis. Dicer activity is essential for normal muscle development during embryogenesis and Dicer muscle mutants have reduced muscle miRNAs, die perinatally and display decreased skeletal muscle mass accompanied by abnormal myofiber morphology. Dicer mutant muscles also show increased apoptosis and Cre-mediated loss of Dicer in Myod-converted myoblasts results in enhanced cell death. These observations demonstrate key roles for Dicer in skeletal muscle and implicate miRNAs as critical components required for embryonic myogenesis.

Cloning and characterization of a novel MyoD enhancer-binding factor.

Glucocorticoid-induced gene-1 (Gig1) was identified in a yeast one-hybrid screen for factors that interact with the MyoD core enhancer. The Gig1 gene encodes a novel C2H2 zinc finger protein that shares a high degree of sequence similarity with two known DNA binding proteins in humans, Glut4 enhancer factor and papillomavirus binding factor (PBF). The mouse ortholog of PBF was also isolated in the screen. The DNA binding domain of Gig1, which contains TCF-E-tail CR1 and CR2 motifs shown to mediate promoter specificity of TCF-E-tail isoforms, was mapped to a C-terminal domain that is highly conserved in Glut4 enhancer factor and PBF. In mouse embryos, in situ hybridization revealed a restricted pattern of expression of Gig1 that overlaps with MyoD expression. A nuclear-localized lacZ knockin null allele of Gig1 was produced to study Gig1 expression with greater resolution and to assess Gig1 functions. X-gal staining of Gig1(nlacZ) heterozygous embryos revealed Gig1 expression in myotomal myocytes, skeletal muscle precursors in the limb, and in nascent muscle fibers of the body wall, head and neck, and limbs through E14.5 (latest stage examined). Gig1 was also expressed in a subset of Scleraxis-positive tendon precursors/rudiments of the limbs, but not in the earliest tendon precursors of the somite (syndetome) defined by Scleraxis expression. Additional regions of Gig1 expression included the apical ectodermal ridge, neural tube roof plate and floor plate, apparent motor neurons in the ventral neural tube, otic vesicles, notochord, and several other tissues representing all three germ layers. Gig1 expression was particularly well represented in epithelial tissues and in a number of cells/tissues of neural crest origin. Expression of both the endogenous MyoD gene and a reporter gene driven by MyoD regulatory elements was similar in wild-type and homozygous null Gig1(nlacZ) embryos, and mutant mice were viable and fertile, indicating that the functions of Gig1 are redundant with other factors.

Stem cells and heterotopic ossification: Lessons from animal models.

Put most simply, heterotopic ossification (HO) is the abnormal formation of bone at extraskeletal sites. HO can be classified into two main subtypes, genetic and acquired. Acquired HO is a common complication of major connective tissue injury, traumatic central nervous system injury, and surgical interventions, where it can cause significant pain and postoperative disability. A particularly devastating form of HO is manifested in the rare genetic disorder, fibrodysplasia ossificans progressiva (FOP), in which progressive heterotopic bone formation occurs throughout life, resulting in painful and disabling cumulative immobility. While the central role of stem/progenitor cell populations in HO is firmly established, the identity of the offending cell type(s) remains to be conclusively determined, and little is known of the mechanisms that direct these progenitor cells to initiate cartilage and bone formation. In this review, we summarize current knowledge of the cells responsible for acquired HO and FOP, highlighting the strengths and weaknesses of animal models used to interrogate the cellular origins of HO.