Isolation of myogenic progenitor populations from Pax7-deficient skeletal muscle based on adhesion characteristics.

In an attempt to determine whether muscle-derived stem cells are distinct from satellite cells, we investigated whether muscle-derived stem cells could be isolated from the skeletal muscle of Pax7-deficient mice, which have been shown to be devoid of or to contain only a minimal number of satellite cells. Utilizing a technique that separates cells based on their adhesion characteristics (the preplate technique), several distinct populations of muscle-derived cells were isolated. In these mice, the Pax7 gene was knocked out with the insertion of the LacZ gene. One population was both rapidly adhering, LacZ-positive, and displayed a high myogenic index, but was rapidly lost to terminal differentiation when continuously replated. A second population, which persisted over 50 passages, was LacZ-negative and displayed a low myogenic index. Although Pax3 may have acted as a compensatory mechanism for the myogenic commitment of the LacZ-positive cells, the LacZ-negative cells, despite expressing Pax3, required Pax7 transduction to restore their myogenic capacity. We believe that these two populations of myogenic progenitor cells, each endowed with different adhesion characteristics, may help explain the discrepancy in the literature concerning the presence of myogenic cells found in Pax7-deficient mice.

The molecular regulation of muscle stem cell function.

Muscle satellite cells are responsible for the postnatal growth and robust regeneration capacity of adult skeletal muscle. A subset of satellite cells purified from adult skeletal muscle is capable of repopulating the satellite cell pool, suggesting that it has direct therapeutic potential for treating degenerative muscle disease. Satellite cells uniformly express the transcription factor Pax7, and Pax7 is required for satellite cell viability and to give rise to myogenic precursors that express the basic helix-loop-helic (bHLH) transcription factors Myf5 and MyoD. Pax7 activates expression of target genes such as Myf5 and MyoD through recruitment of the Wdr5/Ash2L/MLL2 histone methyltransferase complex. Extensive genetic analysis has revealed that Myf5 and MyoD are required for myogenic determination, whereas myogenin and MRF4 have roles in terminal differentiation. Using a Myf5-Cre knockin allele and an R26R-YFP Cre reporter, we observed that in vivo about 10% of satellite cells only express Pax7 and have never expressed Myf5. Moreover, we found that Pax7(+)/Myf5(-) satellite cells give rise to Pax7(+)/Myf5(+) satellite cells through basal-apical asymmetric cell divisions. Therefore, satellite cells in skeletal muscle are a heterogeneous population composed of satellite stem cells (Pax7(+)/Myf5(-)) and satellite myogenic cells (Pax7(+)/Myf5(+)). Evidence is accumulating that indicates that satellite stem cells represent a true stem cell reservoir, and targeting mechanisms that regulate their function represents an important therapeutic strategy for the treatment of neuromuscular disease.

Filter selection for five color flow cytometric analysis with a single laser.

Flow cytometry has evolved from single- and two-color analysis to the current use of 11-16 colors. The relatively bright excitation spectra of most fluorochromes have made color compensation a challenge especially when performed manually. We describe how by choosing filters with narrower bandwidths results in the color compensation values between FITC, PE, PE-TxR (ECD), PE-Cy5, and PE-Cy7 that range from 0 % to 50% depending on the combination of fluorochromes. Peripheral blood mononuclear cells were stained with alpha-CD4-FITC, alpha-CD27-PE, alpha-CD62L-ECD, alpha-CD45RA-PE-Cy5 and alpha-CD3-PE-Cy7. The samples were acquired on a MO Flo. The initial (first) and second filter sets for our experiments consisted of 530/30 or 519/20 for FITC, 580/30 or 575/20for PE, 630/30 or 630/22 for PE-TxR (ECD), 670/30 or 675/20 for PE-Cy5 and 740LP or 780/40 for PE-Cy7. Nonstained cells were used to adjust the threshold values of detection for each photo multiplier tube (PMT) for each filter set. The mean fluorescent intensity (MFI) of each fluorochrome was not reduced to any great extent by either filter set. However, the compensation value between PE and PE-TxR (ECD) with the first filter selection ranged from 84% to 89% and with the second set of filters it was 25-36%. In addition, the compensation between PE-TxR (ECD) and PE-Cy5 were reduced to 30.2% from 44.2% with the second filter set. The reduction of filter bandwidths that results in minimizing spectral overlaps without lost of signal provides a method by which discrimination of signals between PE containing fluorochromes can be achieved.

Stem cell based therapies to treat muscular dystrophy.

Muscular dystrophies comprise a heterogeneous group of neuromuscular disorders, characterized by progressive muscle wasting, for which no satisfactory treatment exists. Multiple stem cell populations, both of adult or embryonic origin, display myogenic potential and have been assayed for their ability to correct the dystrophic phenotype. To date, many of these described methods have failed, underlying the need to identify the mechanisms controlling myogenic potential, homing of donor populations to the musculature, and avoidance of the immune response. Recent results focus on the fresh isolation of satellite cells and the use of multiple growth factors to promote mesangioblast migration, both of which promote muscle regeneration. Throughout this chapter, various stem cell based therapies will be introduced and evaluated based on their potential to treat muscular dystrophy in an effective and efficient manner.

The potential use of myogenic stem cells in regenerative medicine.

More than a century after the initial description of muscular dystrophy, no curative treatment is currently available. To date, clinical trials with myogenic stem cell transplantation have met with only modest success. There are multiple factors behind these failures, yet they provide powerful insights for improvement. In this chapter, we review the different myogenic stem cell populations that have been reported to be potential vectors for the treatment of myopathies in a context of regenerative medicine.

MyoD enhances BMP7-induced osteogenic differentiation of myogenic cell cultures.

The muscle-specific, basic helix-loop-helix transcription factor MyoD can induce cells from other mesenchymal lineages to express a skeletal muscle phenotype. Interestingly, MyoD is initially upregulated in myogenic cells incubated with bone morphogenetic proteins (BMPs), a treatment that induces osteogenic differentiation, suggesting that MyoD has a role in BMP-induced osteogenesis of myogenic cells. This possibility is supported by our observations that muscle satellite cells derived from adult MyoD(-/-) mice show severely impaired osteogenic induction by BMP-7 (osteogenic protein 1; OP-1) as indicated by the decreased gene expression of the bone markers alkaline phosphatase, osteocalcin, Runx2/Cbfa1, and Osterix. Ectopic expression of MyoD increased alkaline phosphatase activity and Osterix mRNA expression in response to BMP treatment. Similarly, ectopic expression of MyoD in the pluripotent mesenchymal cell line C3H10T1/2 increased alkaline phosphatase activity induced by BMP-7. Transcription assays showed that transfection with a MyoD-expression vector, but not other myogenic basic helix-loop-helix transcription factors (Myf5, myogenin) increased Runx2/Cbfa1 transactivation of a reporter gene construct containing either six OSE sequences in tandem or a single OSE site. This effect was enhanced by BMP treatment. These studies, therefore, demonstrate that the muscle transcription factor MyoD is required for efficient BMP-induced osteogenesis of myogenic cells and indicate that MyoD might exert its effects through co-operative interactions with Runx2/Cbfa1.

Cloning, genomic organization and expression pattern of a novel Drosophila gene, the disco-interacting protein 2 (dip2), and its murine homolog.

We report the cloning and initial characterization of a novel gene encoding the Disco interacting protein 2 (Dip2). dip2 DNA complementary to RNA (cDNA) showed a high degree of sequence similarity to cDNAs of unknown function previously identified in humans and Caenorhabditis elegans. We have cloned the mouse homolog of the dip2 cDNA and characterized the expression of this gene by Northern blotting analysis and in situ hybridization to whole mount embryos. Our observations demonstrate that there is a remarkable degree of sequence conservation at the dip2 locus that is reflected in the nervous system-specific expression of both the Drosophila and mouse homologs.

The potential of muscle stem cells.

Skeletal muscle contains two types of stem cells: satellite cells, which function as myogenic precursors, and a population of multipotent adult stem cells. Satellite cells are believed to form a stable, self-renewing pool of stem cells in adult muscle where they function in tissue growth and repair. An additional stem cell population in adult muscle displays a remarkable capacity to differentiate into hematopoietic cells as well as muscle following transplantation. This article discusses the characteristics and properties of these cell populations, the relationship between them, and the potential for stem cell-based muscle therapeutics.

Activated MEK1 binds the nuclear MyoD transcriptional complex to repress transactivation.

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 that is 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.

Activation of JNK1 contributes to dystrophic muscle pathogenesis.

Duchenne Muscular Dystrophy (DMD) originates from deleterious mutations in the dystrophin gene, with a complete loss of the protein product. Subsequently, the disease is manifested in severe striated muscle wasting and death in early adulthood. Dystrophin provides a structural base for the assembly of an integral membrane protein complex. As such, dystrophin deficiency leads to an altered mechanical integrity of the myofiber and a predisposition to contraction-induced damage. However, the development of myofiber degeneration prior to an observed mechanical defect has been documented in various dystrophic models. Although activation of a detrimental signal transduction pathway has been suggested as a probable cause, a specific cellular cascade has yet to be defined. Here, it is shown that murine models of DMD displayed a muscle-specific activation of JNK1. Independent activation of JNK1 resulted in defects in myotube viability and integrity in vitro, similar to a dystrophic phenotype. In addition, direct muscle injection of an adenoviral construct containing the JNK1 inhibitory protein, JIP1, dramatically attenuated the progression of dystrophic myofiber destruction. Taken together, these results suggest that a JNK1-mediated signal cascade is a conserved feature of dystrophic muscle and contributes to the progression of the disease pathogenesis.

The adipose tissue phenotype of hormone-sensitive lipase deficiency in mice.

OBJECTIVE: To directly ascertain the physiological roles in adipocytes of hormone-sensitive lipase (HSL; E.C. 3.1.1.3), a multifunctional hydrolase that can mediate triacylglycerol cleavage in adipocytes. RESEARCH METHODS AND PROCEDURES: We performed constitutive gene targeting of the mouse HSL gene (Lipe), subsequently studied the adipose tissue phenotype clinically and histologically, and measured lipolysis in isolated adipocytes. RESULTS: Homozygous HSL-/- mice have no detectable HSL peptide or cholesteryl esterase activity in adipose tissue, and heterozygous mice have intermediate levels with respect to wild-type and deficient littermates. HSL-deficient mice have normal body weight but reduced abdominal fat mass compared with normal littermates. Histologically, both white and brown adipose tissues in HSL-/- mice show marked heterogeneity in cell size, with markedly enlarged adipocytes juxtaposed to cells of normal morphology. In isolated HSL-/- adipocytes, lipolysis is not significantly increased by beta3-adrenergic stimulation, but under basal conditions in the absence of added catecholamines, the lipolytic rate of isolated HSL-/- adipocytes is at least as high as that of cells from normal controls. Cold tolerance during a 48-hour period at 4 degrees C was similar in HSL-/- mice and controls. Overnight fasting was well-tolerated clinically by HSL-/- mice, but after fasting, liver triglyceride content was significantly lower in HSL-/- mice compared with wild-type controls. CONCLUSIONS: In isolated fat cells, the lipolytic rate after beta-adrenergic stimulation is mainly dependent on HSL. However, the observation of a normal rate of lipolysis in unstimulated HSL-/- adipocytes suggests that HSL-independent lipolytic pathway(s) exist in fat. Physiologically, HSL deficiency in mice has a modest effect under normal fed conditions and is compatible with normal maintenance of core body temperature during cold stress. However, the lipolytic response to overnight fasting is subnormal.

bHLH transcription factor MyoD affects myosin heavy chain expression pattern in a muscle-specific fashion.

A strong correlative pattern between MyoD gene expression and myosin heavy chain IIB (MHC IIB) gene expression exists. To test whether this correlative relationship is causative, MHC gene expression in muscles from MyoD(-/-) mice was analyzed. The MHC IIB gene was not detectable in the MyoD(-/-) diaphragm, whereas the MHC IIB protein made up 10.0 +/- 1.7% of the MHC protein pool in the wild-type (WT) mouse diaphragm. Furthermore, the MHC IIA protein was not detectable in the MyoD(-/-) biceps brachii, and the MHC IIB protein was overexpressed in the masseter. To examine whether MyoD is required for the upregulation of the MHC IIB gene within slow muscle after disuse, MyoD(-/-) and WT hindlimb musculature was unweighted. MyoD(-/-) exhibited a diminished response in the upregulation of the MHC IIB mRNA within the soleus muscle as a result of the hindlimb unweighting. Collectively, these data suggest that MyoD plays a role in the MHC profile in a muscle-specific fashion.

Mice deficient in methylenetetrahydrofolate reductase exhibit hyperhomocysteinemia and decreased methylation capacity, with neuropathology and aortic lipid deposition.

Hyperhomocysteinemia, a risk factor for cardiovascular disease, is caused by nutritional and/or genetic disruptions in homocysteine metabolism. The most common genetic cause of hyperhomocysteinemia is the 677C-->T mutation in the methylenetetrahydrofolate reductase (MTHFR) gene. This variant, with mild enzymatic deficiency, is associated with an increased risk for neural tube defects and pregnancy complications and with a decreased risk for colon cancer and leukemia. Although many studies have reported that this variant is also a risk factor for vascular disease, this area of investigation is still controversial. Severe MTHFR deficiency results in homocystinuria, an inborn error of metabolism with neurological and vascular complications. To investigate the in vivo pathogenetic mechanisms of MTHFR deficiency, we generated mice with a knockout of MTHFR: Plasma total homocysteine levels in heterozygous and homozygous knockout mice are 1.6- and 10-fold higher than those in wild-type littermates, respectively. Both heterozygous and homozygous knockouts have either significantly decreased S-adenosylmethionine levels or significantly increased S-adenosylhomocysteine levels, or both, with global DNA hypomethylation. The heterozygous knockout mice appear normal, whereas the homozygotes are smaller and show developmental retardation with cerebellar pathology. Abnormal lipid deposition in the proximal portion of the aorta was observed in older heterozygotes and homozygotes, alluding to an atherogenic effect of hyperhomocysteinemia in these mice.

Male sexual dysfunction in mice bearing targeted mutant alleles of the PEA3 ets gene.

PEA3, a member of the Ets family of transcriptional regulatory proteins, is expressed in a unique spatial and temporal pattern during mouse embryogenesis; its overexpression is positively correlated with HER2-mediated breast tumorigenesis in both humans and mice. To determine whether PEA3 plays a part in development and oncogenesis and to uncover its normal physiological role, we generated mice lacking functional PEA3 by gene targeting in embryonic stem cells. PEA3(-/-) mice arose from heterozygous crosses with the expected Mendelian frequency, revealing that PEA3 is dispensable for embryogenesis. PEA3 mutant mice displayed no overt phenotype and lived a normal life span. However, PEA3-deficient males failed to reproduce. PEA3 is expressed in several male sexual organs, but gross and histological analyses of the organs from PEA3(-/-) mice revealed no abnormalities. Spermatogenesis and spermiogenesis also appeared normal in mice homozygous for the PEA3 mutation, and their sperm were capable of fertilizing eggs in vitro. PEA3(-/-) males engaged in normal mating behavior, but they did not set copulatory plugs and sperm could not be detected in the uteri of females that had mated with PEA3(-/-) males. Erections could be evoked by abdominal pressure in PEA3-deficient male mice, and the results of in vitro experiments revealed that the corpus cavernosum isolated from PEA3 mutant males relaxed in response to acetylcholine. Therefore, the infertility of PEA3 mutant males involves either mechanisms proximal to the cavernosal smooth muscle or an ejaculatory dysfunction. However, PEA3 mutant mice are phenotypically distinguishable from other knockout mice with such deficits and thus provide a unique model for further investigation of male sexual dysfunction.

Myotube formation is delayed but not prevented in MyoD-deficient skeletal muscle: studies in regenerating whole muscle grafts of adult mice.

We compared the time course of myogenic events in vivo in regenerating whole muscle grafts in MyoD(-/-) and control BALB/c adult mice using immunohistochemistry and electron microscopy. Immunohistochemistry with antibodies to desmin and myosin revealed a striking delay by about 3 days in the formation of myotubes in MyoD(-/-) autografts compared with BALB/c mice. However, myotube formation was not prevented, and autografts in both strains appeared similar by 8 days. Electron microscopy confirmed myotube formation in 8- but not 5-day MyoD(-/-) grafts. This pattern was not influenced by cross-transplantation experiments between strains examined at 5 days. Antibodies to proliferating cell nuclear antigen demonstrated an elevated level of replication by MyoD(-/-) myoblasts in autografts, and replication was sustained for about 3 days compared with controls. These data indicate that the delay in the onset of differentiation and hence fusion is related to extended proliferation of the MyoD(-/-) myoblasts. Overall, although muscle regeneration was delayed it was not impaired in MyoD(-/-) mice in this model.

Pax7 is required for the specification of myogenic satellite cells.

The paired box transcription factor Pax7 was isolated by representational difference analysis as a gene specifically expressed in cultured satellite cell-derived myoblasts. 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, fluorescence-activated cell sorting 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 alternate developmental programs.

MyoD(-/-) satellite cells in single-fiber culture are differentiation defective and MRF4 deficient.

MyoD-deficient mice are without obvious deleterious muscle phenotype during embryogenesis and fetal development, and adults in the laboratory have grossly normal skeletal muscle and life span. However, a previous study showed that in the context of muscle degeneration on a mdx (dystrophin null) genetic background, animals lacking MyoD have a greatly intensified disease phenotype leading to lethality not otherwise seen in mdx mice. Here we have examined MyoD(-/-) adult muscle fibers and their associated satellite cells in single myofiber cultures and describe major phenotypic differences found at the tissue, cellular, and molecular levels. The steady-state number of satellite cells on freshly isolated MyoD(-/-) fibers was elevated and abnormal branched fiber morphologies were observed, the latter suggesting chronic muscle regeneration in vivo. Single-cell RNA coexpression analyses were performed for c-met, m-cadherin, and the four myogenic regulatory factors (MRFs.) Most mutant satellite cells entered the cell cycle and upregulated expression of myf5, both characteristic early steps in satellite cell maturation. However, they later failed to normally upregulate MRF4, displayed a major deficit in m-cadherin expression, and showed a significant diminution in myogenin-positive status compared with wildtype. MyoD(-/-) satellite cells formed unusual aggregate structures, failed to fuse efficiently, and showed greater than 90% reduction in differentiation efficiency relative to wildtype. A further survey of RNAs encoding regulators of growth and differentiation, cell cycle progression, and cell signaling revealed similar or identical expression profiles for most genes as well as several noteworthy differences. Among these, GDF8 and Msx1 were identified as potentially important regulators of the quiescent state whose expression profile differs between mutant and wildtype. Considered together, these data suggest that activated MyoD(-/-) satellite cells assume a phenotype that resembles in some ways a developmentally "stalled" cell compared to wildtype. However, the MyoD(-/-) cells are not merely developmentally immature, as they also display novel molecular and cellular characteristics that differ from any observed in wild-type muscle precursor counterparts of any stage.

Skeletal muscle development in the mouse embryo.

In this review we discuss the recent findings concerning the mechanisms that restrict somitic cells to the skeletal muscle fate, the myogenic regulatory factors controlling skeletal muscle differentiation and specification of myogenic cell lineages, the nature of inductive signals and the role of secreted proteins in embryonic patterning of the myotome. More specifically, we review data which strongly support the hypothesis that Myf-5 plays a unique role in development of epaxial muscle, that MyoD plays a unique role in development of hypaxial muscles derived from migratory myogenic precursor cells, and that both genes are responsible for development of intercostal and abdominal muscles (hypaxial muscles that develop from the dermatomal epithelia). In addition, while discussing upstream and post-translational regulation of myogenic regulatory factors (MRFs), we suggest that correct formation of the myotome requires a complex cooperation of DNA binding proteins and cofactors, as well as inhibitory function of non-muscle cells of the forming somite, whose proteins would sequester and suppress the transcription of MRFs. Moreover, in the third part of our review, we discuss embryonic structures, secreted proteins and myogenic induction. However, although different signaling molecules with activity in the process of somite patterning have been identified, not many of them are found to be necessary during in vivo embryonic development. To understand their functions, generation of multiple mutants or conditional/tissue-specific mutants will be necessary.

The molecular regulation of myogenesis.

Over the past years, several studies have unraveled important mechanisms by which the four myogenic regulatory factors (MRFs: MyoD, Myf-5, myogenin, and MRF4) control the specification and the differentiation of the muscle lineage. Early experiments led to the hypothesis that these factors were redundant and could functionally replace one another. However, recent experiments using in vivo and in vitro models have demonstrated that in fact different aspects of the myogenic program are controlled by different factors in vivo, suggesting that these factors play distinct roles during myogenesis. The activity of the MRFs during proliferation and differentiation of muscle precursor cells has clearly been demonstrated to be dependent on specific cell-cycle control mechanisms as well as distinct interactions with other regulatory molecules, such as the ubiquitously expressed E proteins and several other transcription factors. Furthermore, the observation that the MRFs can recruit chromatin remodeling proteins has shed some light on the mechanisms by which the MRFs activate gene expression. Recently, a functional role for MyoD during satellite cell activation and muscle repair has been identified in vivo, which cannot be substituted for by the other MRFs. This has put forward the hypothesis that these factors also play specific biological roles following muscle injury and repair.

Transdifferentiation of esophageal smooth to skeletal muscle is myogenic bHLH factor-dependent.

Previously, coexpression of smooth and skeletal differentiation markers, but not myogenic regulatory factors (MRFs), was observed from E16.5 mouse fetuses in a small percentage of diaphragm level esophageal muscle cells, suggesting that MRFs are not involved in the process of initiation of developmentally programmed transdifferentiation in the esophagus. To investigate smooth-to-skeletal esophageal muscle transition, we analyzed Myf5nlacZ knock-in mice, MyoD-lacZ and myogenin-lacZ transgenic embryos with a panel of the antibodies reactive with myogenic regulatory factors (MRFs) and smooth and skeletal muscle markers. We observed that lacZ-expressing myogenic precursors were not detected in the esophagus before E15.5, arguing against the hypothesis that muscle precursor cells populate the esophagus at an earlier stage of development. Rather, the expression of the MRFs initiated in smooth muscle cells in the upper esophagus of E15.5 mouse embryos and was immediately followed by the expression of skeletal muscle markers. Moreover, transdifferentiation was markedly delayed or absent only in the absence of Myf5, suggesting that appropriate initiation and progression of smooth-to-skeletal muscle transdifferentiation is Myf5-dependent. Accordingly, the esophagus of Myf5(-/-):MyoD(-/-)embryos completely failed to undergo skeletal myogenesis and consisted entirely of smooth muscle. Lastly, extensive proliferation of muscularis precursor cells, without programmed cell death, occurred concomitantly with esophageal smooth-to-skeletal muscle transdifferentiation. Taken together, these results indicate that transdifferentiation is the fate of all smooth muscle cells in the upper esophagus and is normally initiated by Myf5.