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Mouse Anti-Chicken PAX7 Monoclonal Antibody, Unconjugated

RRID:AB_528428

Skeletal Muscle Stem Cells from PSC-Derived Teratomas Have Functional Regenerative Capacity.

  • Chan SS
  • Cell Stem Cell
  • 2018 Jul 5

Literature context:


Abstract:

Derivation of functional skeletal muscle stem cells from pluripotent cells without genetic modification has proven elusive. Here we show that teratomas formed in adult skeletal muscle differentiate in vivo to produce large numbers of α7-Integrin+ VCAM-1+ myogenic progenitors. When FACS-purified and transplanted into diseased muscles, mouse teratoma-derived myogenic progenitors demonstrate very high engraftment potential. As few as 40,000 cells can reconstitute ∼80% of the tibialis anterior muscle volume. Newly generated fibers are innervated, express adult myosins, and ameliorate dystrophy-related force deficit and fatigability. Teratoma-derived myogenic progenitors also contribute quiescent PAX7+ muscle stem cells, enabling long-term maintenance of regenerated muscle and allowing muscle regeneration in response to subsequent injuries. Transcriptional profiling reveals that teratoma-derived myogenic progenitors undergo embryonic-to-adult maturation when they contribute to the stem cell compartment of regenerated muscle. Thus, teratomas are a rich and accessible source of potent transplantable skeletal muscle stem cells. VIDEO ABSTRACT.

Funding information:
  • Wellcome Trust - 098330(United Kingdom)

Metabolic Maturation during Muscle Stem Cell Differentiation Is Achieved by miR-1/133a-Mediated Inhibition of the Dlk1-Dio3 Mega Gene Cluster.

  • Wüst S
  • Cell Metab.
  • 2018 May 1

Literature context:


Abstract:

Muscle stem cells undergo a dramatic metabolic switch to oxidative phosphorylation during differentiation, which is achieved by massively increased mitochondrial activity. Since expression of the muscle-specific miR-1/133a gene cluster correlates with increased mitochondrial activity during muscle stem cell (MuSC) differentiation, we examined the potential role of miR-1/133a in metabolic maturation of skeletal muscles in mice. We found that miR-1/133a downregulate Mef2A in differentiated myocytes, thereby suppressing the Dlk1-Dio3 gene cluster, which encodes multiple microRNAs inhibiting expression of mitochondrial genes. Loss of miR-1/133a in skeletal muscles or increased Mef2A expression causes continuous high-level expression of the Dlk1-Dio3 gene cluster, compromising mitochondrial function. Failure to terminate the stem cell-like metabolic program characterized by high-level Dlk1-Dio3 gene cluster expression initiates profound changes in muscle physiology, essentially abrogating endurance running. Our results suggest a major role of miR-1/133a in metabolic maturation of skeletal muscles but exclude major functions in muscle development and MuSC maintenance.

Funding information:
  • Wellcome Trust - EGA16107(United Kingdom)

The Dystrophin Glycoprotein Complex Regulates the Epigenetic Activation of Muscle Stem Cell Commitment.

  • Chang NC
  • Cell Stem Cell
  • 2018 May 3

Literature context:


Abstract:

Asymmetrically dividing muscle stem cells in skeletal muscle give rise to committed cells, where the myogenic determination factor Myf5 is transcriptionally activated by Pax7. This activation is dependent on Carm1, which methylates Pax7 on multiple arginine residues, to recruit the ASH2L:MLL1/2:WDR5:RBBP5 histone methyltransferase complex to the proximal promoter of Myf5. Here, we found that Carm1 is a specific substrate of p38γ/MAPK12 and that phosphorylation of Carm1 prevents its nuclear translocation. Basal localization of the p38γ/p-Carm1 complex in muscle stem cells occurs via binding to the dystrophin-glycoprotein complex (DGC) through β1-syntrophin. In dystrophin-deficient muscle stem cells undergoing asymmetric division, p38γ/β1-syntrophin interactions are abrogated, resulting in enhanced Carm1 phosphorylation. The resulting progenitors exhibit reduced Carm1 binding to Pax7, reduced H3K4-methylation of chromatin, and reduced transcription of Myf5 and other Pax7 target genes. Therefore, our experiments suggest that dysregulation of p38γ/Carm1 results in altered epigenetic gene regulation in Duchenne muscular dystrophy.

Funding information:
  • NCI NIH HHS - CA32317(United States)

A Potent and Specific CD38 Inhibitor Ameliorates Age-Related Metabolic Dysfunction by Reversing Tissue NAD+ Decline.

  • Tarragó MG
  • Cell Metab.
  • 2018 May 1

Literature context:


Abstract:

Aging is characterized by the development of metabolic dysfunction and frailty. Recent studies show that a reduction in nicotinamide adenine dinucleotide (NAD+) is a key factor for the development of age-associated metabolic decline. We recently demonstrated that the NADase CD38 has a central role in age-related NAD+ decline. Here we show that a highly potent and specific thiazoloquin(az)olin(on)e CD38 inhibitor, 78c, reverses age-related NAD+ decline and improves several physiological and metabolic parameters of aging, including glucose tolerance, muscle function, exercise capacity, and cardiac function in mouse models of natural and accelerated aging. The physiological effects of 78c depend on tissue NAD+ levels and were reversed by inhibition of NAD+ synthesis. 78c increased NAD+ levels, resulting in activation of pro-longevity and health span-related factors, including sirtuins, AMPK, and PARPs. Furthermore, in animals treated with 78c we observed inhibition of pathways that negatively affect health span, such as mTOR-S6K and ERK, and attenuation of telomere-associated DNA damage, a marker of cellular aging. Together, our results detail a novel pharmacological strategy for prevention and/or reversal of age-related NAD+ decline and subsequent metabolic dysfunction.

Funding information:
  • NHLBI NIH HHS - N01-HV-28186(United States)

Inhibition of Methyltransferase Setd7 Allows the In Vitro Expansion of Myogenic Stem Cells with Improved Therapeutic Potential.

  • Judson RN
  • Cell Stem Cell
  • 2018 Feb 1

Literature context:


Abstract:

The development of cell therapy for repairing damaged or diseased skeletal muscle has been hindered by the inability to significantly expand immature, transplantable myogenic stem cells (MuSCs) in culture. To overcome this limitation, a deeper understanding of the mechanisms regulating the transition between activated, proliferating MuSCs and differentiation-primed, poorly engrafting progenitors is needed. Here, we show that methyltransferase Setd7 facilitates such transition by regulating the nuclear accumulation of β-catenin in proliferating MuSCs. Genetic or pharmacological inhibition of Setd7 promotes in vitro expansion of MuSCs and increases the yield of primary myogenic cell cultures. Upon transplantation, both mouse and human MuSCs expanded with a Setd7 small-molecule inhibitor are better able to repopulate the satellite cell niche, and treated mouse MuSCs show enhanced therapeutic potential in preclinical models of muscular dystrophy. Thus, Setd7 inhibition may help bypass a key obstacle in the translation of cell therapy for muscle disease.

Funding information:
  • BLRD VA - I01 BX002324()
  • NCI NIH HHS - R01 CA073808(United States)
  • NIA NIH HHS - P01 AG036695()
  • NIAMS NIH HHS - R21 AR071039()
  • RRD VA - I01 RX001222()

A defective dNTP pool hinders DNA replication in cell cycle-reactivated terminally differentiated muscle cells.

  • Pajalunga D
  • Cell Death Differ.
  • 2018 Feb 15

Literature context:


Abstract:

Terminally differentiated cells are defined by their inability to proliferate. When forced to re-enter the cell cycle, they generally cannot undergo long-term replication. Our previous work with myotubes has shown that these cells fail to proliferate because of their intrinsic inability to complete DNA replication. Moreover, we have reported pronounced modifications of deoxynucleotide metabolism during myogenesis. Here we investigate the causes of incomplete DNA duplication in cell cycle-reactivated myotubes (rMt). We find that rMt possess extremely low levels of thymidine triphosphate (dTTP), resulting in very slow replication fork rates. Exogenous administration of thymidine or forced expression of thymidine kinase increases deoxynucleotide availability, allowing extended and faster DNA replication. Inadequate dTTP levels are caused by selective, differentiation-dependent, cell cycle-resistant suppression of genes encoding critical synthetic enzymes, chief among which is thymidine kinase 1. We conclude that lack of dTTP is at least partially responsible for the inability of myotubes to proliferate and speculate that it constitutes an emergency barrier against unwarranted DNA replication in terminally differentiated cells.

Isolation, Cryosection and Immunostaining of Skeletal Muscle.

  • Ortuste Quiroga HP
  • Methods Mol. Biol.
  • 2018 Jan 12

Literature context:


Abstract:

Adult skeletal muscle is maintained and repaired by resident stem cells called satellite cells, located between the plasmalemma of a muscle fiber, and the surrounding basal lamina. When needed, satellite cells are activated to form proliferative myoblasts, that then differentiate and fuse to existing muscle fibers, or fuse together to form replacement myofibers. In parallel, a proportion of satellite cells self-renew, to maintain the stem cell pool. To date, Pax7 is the marker of choice for identifying quiescent satellite cells. Co-immunostaining of skeletal muscle with Pax7 and laminin allows both identification of satellite cells, and the myofiber that they are associated with. Furthermore, satellite cells can be followed through the early stages of the myogenic program by co-immunostaining with myogenic regulatory factors such as MyoD. To test genetically modified mice for satellite cell expression, co-immunostaining can be performed for Pax7 and reporter genes such as eGFP. Here, we describe a method for identification of satellite cells in skeletal muscle sections, including muscle isolation, cryosectioning and co-immunostaining for Pax7 and laminin.

mTORC1 Activation during Repeated Regeneration Impairs Somatic Stem Cell Maintenance.

  • Haller S
  • Cell Stem Cell
  • 2017 Dec 7

Literature context:


Abstract:

The balance between self-renewal and differentiation ensures long-term maintenance of stem cell (SC) pools in regenerating epithelial tissues. This balance is challenged during periods of high regenerative pressure and is often compromised in aged animals. Here, we show that target of rapamycin (TOR) signaling is a key regulator of SC loss during repeated regenerative episodes. In response to regenerative stimuli, SCs in the intestinal epithelium of the fly and in the tracheal epithelium of mice exhibit transient activation of TOR signaling. Although this activation is required for SCs to rapidly proliferate in response to damage, repeated rounds of damage lead to SC loss. Consistently, age-related SC loss in the mouse trachea and in muscle can be prevented by pharmacologic or genetic inhibition, respectively, of mammalian target of rapamycin complex 1 (mTORC1) signaling. These findings highlight an evolutionarily conserved role of TOR signaling in SC function and identify repeated rounds of mTORC1 activation as a driver of age-related SC decline.

Funding information:
  • BLRD VA - I01 BX002324()
  • NCRR NIH HHS - UL1 RR024989(United States)
  • NHLBI NIH HHS - R01 HL132996()
  • NIA NIH HHS - K99 AG041764()
  • NIA NIH HHS - P01 AG036695()
  • NIA NIH HHS - R00 AG041764()
  • NIA NIH HHS - R01 AG047497()
  • NIA NIH HHS - R01 AG047820()
  • NIA NIH HHS - R37 AG023806()
  • NIDDK NIH HHS - R01 DK100342()
  • NIDDK NIH HHS - R01 DK113144()

Myotome adaptability confers developmental robustness to somitic myogenesis in response to fibre number alteration.

  • Roy SD
  • Dev. Biol.
  • 2017 Nov 15

Literature context:


Abstract:

Balancing the number of stem cells and their progeny is crucial for tissue development and repair. Here we examine how cell numbers and overall muscle size are tightly regulated during zebrafish somitic muscle development. Muscle stem/precursor cell (MPCs) expressing Pax7 are initially located in the dermomyotome (DM) external cell layer, adopt a highly stereotypical distribution and thereafter a proportion of MPCs migrate into the myotome. Regional variations in the proliferation and terminal differentiation of MPCs contribute to growth of the myotome. To probe the robustness of muscle size control and spatiotemporal regulation of MPCs, we compared the behaviour of wild type (wt) MPCs with those in mutant zebrafish that lack the muscle regulatory factor Myod. Myodfh261 mutants form one third fewer multinucleate fast muscle fibres than wt and show a significant expansion of the Pax7+ MPC population in the DM. Subsequently, myodfh261 mutant fibres generate more cytoplasm per nucleus, leading to recovery of muscle bulk. In addition, relative to wt siblings, there is an increased number of MPCs in myodfh261 mutants and these migrate prematurely into the myotome, differentiate and contribute to the hypertrophy of existing fibres. Thus, homeostatic reduction of the excess MPCs returns their number to normal levels, but fibre numbers remain low. The GSK3 antagonist BIO prevents MPC migration into the deep myotome, suggesting that canonical Wnt pathway activation maintains the DM in zebrafish, as in amniotes. BIO does not, however, block recovery of the myodfh261 mutant myotome, indicating that homeostasis acts on fibre intrinsic growth to maintain muscle bulk. The findings suggest the existence of a critical window for early fast fibre formation followed by a period in which homeostatic mechanisms regulate myotome growth by controlling fibre size. The feedback controls we reveal in muscle help explain the extremely precise grading of myotome size along the body axis irrespective of fish size, nutrition and genetic variation and may form a paradigm for wider matching of organ size.

Funding information:
  • Biotechnology and Biological Sciences Research Council - BB/K010115/1()
  • Biotechnology and Biological Sciences Research Council - BB/I025883/1()
  • Medical Research Council - G1001029()
  • Medical Research Council - MR/N021231/1()
  • Wellcome Trust - 101529/Z/13/Z()

Spin infection enables efficient gene delivery to muscle stem cells.

  • Kodaka Y
  • BioTechniques
  • 2017 Aug 1

Literature context:


Abstract:

Viral vector-mediated foreign gene expression in cultured cells has been extensively used in stem cell studies to explore gene function. However, it is difficult to obtain high-quality stem cells and primary cells after viral vector infection. Here, we describe a new protocol for high-efficiency retroviral infection of primary muscle stem cell (satellite cell) cultures. We compared multiple commercially available transfection reagents to determine which was optimal for retroviral infections of primary myoblasts. Centrifugation force was also tested, and a spin infection protocol with centrifugation at 2800 × g for 90 min had the highest infection efficiency for primary myoblasts. We confirmed that infected muscle stem cells maintain cell proliferation and the capacity for in vitro and in vivo myogenic differentiation. Our new, efficient retroviral infection protocol for muscle stem cells can be applied to molecular biology experiments as well as translational studies.

Funding information:
  • NIAMS NIH HHS - R01 AR062142()
  • NIAMS NIH HHS - R21 AR070319()

The PERK arm of the unfolded protein response regulates satellite cell-mediated skeletal muscle regeneration.

  • Xiong G
  • Elife
  • 2017 Mar 23

Literature context:


Abstract:

Regeneration of skeletal muscle in adults is mediated by satellite stem cells. Accumulation of misfolded proteins triggers endoplasmic reticulum stress that leads to unfolded protein response (UPR). The UPR is relayed to the cell through the activation of PERK, IRE1/XBP1, and ATF6. Here, we demonstrate that levels of PERK and IRE1 are increased in satellite cells upon muscle injury. Inhibition of PERK, but not the IRE1 arm of the UPR in satellite cells inhibits myofiber regeneration in adult mice. PERK is essential for the survival and differentiation of activated satellite cells into the myogenic lineage. Deletion of PERK causes hyper-activation of p38 MAPK during myogenesis. Blocking p38 MAPK activity improves the survival and differentiation of PERK-deficient satellite cells in vitro and muscle formation in vivo. Collectively, our results suggest that the PERK arm of the UPR plays a pivotal role in the regulation of satellite cell homeostasis during regenerative myogenesis.

Funding information:
  • NIA NIH HHS - R01 AG029623()
  • NIAMS NIH HHS - R01 AR059810()
  • NIAMS NIH HHS - R01 AR068313()

Tridimensional Visualization and Analysis of Early Human Development.

  • Belle M
  • Cell
  • 2017 Mar 23

Literature context:


Abstract:

Generating a precise cellular and molecular cartography of the human embryo is essential to our understanding of the mechanisms of organogenesis in normal and pathological conditions. Here, we have combined whole-mount immunostaining, 3DISCO clearing, and light-sheet imaging to start building a 3D cellular map of the human development during the first trimester of gestation. We provide high-resolution 3D images of the developing peripheral nervous, muscular, vascular, cardiopulmonary, and urogenital systems. We found that the adult-like pattern of skin innervation is established before the end of the first trimester, showing important intra- and inter-individual variations in nerve branches. We also present evidence for a differential vascularization of the male and female genital tracts concomitant with sex determination. This work paves the way for a cellular and molecular reference atlas of human cells, which will be of paramount importance to understanding human development in health and disease. PAPERCLIP.

Angiotensin-II-induced Muscle Wasting is Mediated by 25-Hydroxycholesterol via GSK3β Signaling Pathway.

  • Shen C
  • EBioMedicine
  • 2017 Feb 5

Literature context:


Abstract:

While angiotensin II (ang II) has been implicated in the pathogenesis of cardiac cachexia (CC), the molecules that mediate ang II's wasting effect have not been identified. It is known TNF-α level is increased in patients with CC, and TNF-α release is triggered by ang II. We therefore hypothesized that ang II induced muscle wasting is mediated by TNF-α. Ang II infusion led to skeletal muscle wasting in wild type (WT) but not in TNF alpha type 1 receptor knockout (TNFR1KO) mice, suggesting that ang II induced muscle loss is mediated by TNF-α through its type 1 receptor. Microarray analysis identified cholesterol 25-hydroxylase (Ch25h) as the down stream target of TNF-α. Intraperitoneal injection of 25-hydroxycholesterol (25-OHC), the product of Ch25h, resulted in muscle loss in C57BL/6 mice, accompanied by increased expression of atrogin-1, MuRF1 and suppression of IGF-1/Akt signaling pathway. The identification of 25-OHC as an inducer of muscle wasting has implications for the development of specific treatment strategies in preventing muscle loss.

In Vivo Amelioration of Age-Associated Hallmarks by Partial Reprogramming.

  • Ocampo A
  • Cell
  • 2016 Dec 15

Literature context:


Abstract:

Aging is the major risk factor for many human diseases. In vitro studies have demonstrated that cellular reprogramming to pluripotency reverses cellular age, but alteration of the aging process through reprogramming has not been directly demonstrated in vivo. Here, we report that partial reprogramming by short-term cyclic expression of Oct4, Sox2, Klf4, and c-Myc (OSKM) ameliorates cellular and physiological hallmarks of aging and prolongs lifespan in a mouse model of premature aging. Similarly, expression of OSKM in vivo improves recovery from metabolic disease and muscle injury in older wild-type mice. The amelioration of age-associated phenotypes by epigenetic remodeling during cellular reprogramming highlights the role of epigenetic dysregulation as a driver of mammalian aging. Establishing in vivo platforms to modulate age-associated epigenetic marks may provide further insights into the biology of aging.

Myofiber-specific TEAD1 overexpression drives satellite cell hyperplasia and counters pathological effects of dystrophin deficiency.

  • Southard S
  • Elife
  • 2016 Oct 11

Literature context:


Abstract:

When unperturbed, somatic stem cells are poised to affect immediate tissue restoration upon trauma. Yet, little is known regarding the mechanistic basis controlling initial and homeostatic 'scaling' of stem cell pool sizes relative to their target tissues for effective regeneration. Here, we show that TEAD1-expressing skeletal muscle of transgenic mice features a dramatic hyperplasia of muscle stem cells (i.e. satellite cells, SCs) but surprisingly without affecting muscle tissue size. Super-numeral SCs attain a 'normal' quiescent state, accelerate regeneration, and maintain regenerative capacity over several injury-induced regeneration bouts. In dystrophic muscle, the TEAD1 transgene also ameliorated the pathology. We further demonstrate that hyperplastic SCs accumulate non-cell-autonomously via signal(s) from the TEAD1-expressing myofiber, suggesting that myofiber-specific TEAD1 overexpression activates a physiological signaling pathway(s) that determines initial and homeostatic SC pool size. We propose that TEAD1 and its downstream effectors are medically relevant targets for enhancing muscle regeneration and ameliorating muscle pathology.

Patched1 and Patched2 inhibit Smoothened non-cell autonomously.

  • Roberts B
  • Elife
  • 2016 Aug 23

Literature context:


Abstract:

Smoothened (Smo) inhibition by Patched (Ptch) is central to Hedgehog (Hh) signaling. Ptch, a proton driven antiporter, is required for Smo inhibition via an unknown mechanism. Hh ligand binding to Ptch reverses this inhibition and activated Smo initiates the Hh response. To determine whether Ptch inhibits Smo strictly in the same cell or also mediates non-cell-autonomous Smo inhibition, we generated genetically mosaic neuralized embryoid bodies (nEBs) from mouse embryonic stem cells (mESCs). These experiments utilized novel mESC lines in which Ptch1, Ptch2, Smo, Shh and 7dhcr were inactivated via gene editing in multiple combinations, allowing us to measure non-cell autonomous interactions between cells with differing Ptch1/2 status. In several independent assays, the Hh response was repressed by Ptch1/2 in nearby cells. When 7dhcr was targeted, cells displayed elevated non-cell autonomous inhibition. These findings support a model in which Ptch1/2 mediate secretion of a Smo-inhibitory cholesterol precursor.

Pax3 and Pax7 interact reciprocally and regulate the expression of cadherin-7 through inducing neuron differentiation in the developing chicken spinal cord.

  • Lin J
  • J. Comp. Neurol.
  • 2016 Apr 1

Literature context:


Abstract:

Pax3 and Pax7 are closely related transcription factors that are widely expressed in the developing nervous system and somites. In the CNS, both genes are expressed in the dorsal part of the neural tube during development. Pax3 and Pax7 are involved in the sonic hedgehog (Shh) signaling pathway and are inhibited by Shh overexpression. The present study confirms in vivo that Pax3 overexpression represses the expression of Pax7, whereas Pax7 overexpression endogenously enhances and ectopically induces the expression of Pax3 in the developing chicken spinal cord. Overexpression of Pax3 and Pax7 represses the endogenous expression of cadherin-7, a member of the cadherin family of morphogenetic genes, and induces its ectopic expression. The present study also shows that overexpression of Pax3 and Pax7 changes the fate and morphology of cells in the neuroepithelial layer and induces the expression of postmitotic neuronal markers. We show that both Pax3 and Pax7 promote the differentiation of neural progenitor cells into neurons. Furthermore, the downregulation of Pax3 and Pax7 with specific shRNAs results in apoptosis in the developing spinal cord. Collectively, these results suggest that the transcription factors Pax3 and Pax7 play important roles in regulating morphogenesis and cell differentiation in the developing spinal cord.

FHL1 reduces dystrophy in transgenic mice overexpressing FSHD muscular dystrophy region gene 1 (FRG1).

  • Feeney SJ
  • PLoS ONE
  • 2015 Nov 16

Literature context:


Abstract:

Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal-dominant disease with no effective treatment. The genetic cause of FSHD is complex and the primary pathogenic insult underlying the muscle disease is unknown. Several disease candidate genes have been proposed including DUX4 and FRG1. Expression analysis studies of FSHD report the deregulation of genes which mediate myoblast differentiation and fusion. Transgenic mice overexpressing FRG1 recapitulate the FSHD muscular dystrophy phenotype. Our current study selectively examines how increased expression of FRG1 may contribute to myoblast differentiation defects. We generated stable C2C12 cell lines overexpressing FRG1, which exhibited a myoblast fusion defect upon differentiation. To determine if myoblast fusion defects contribute to the FRG1 mouse dystrophic phenotype, this strain was crossed with skeletal muscle specific FHL1-transgenic mice. We previously reported that FHL1 promotes myoblast fusion in vitro and FHL1-transgenic mice develop skeletal muscle hypertrophy. In the current study, FRG1 mice overexpressing FHL1 showed an improvement in the dystrophic phenotype, including a reduced spinal kyphosis, increased muscle mass and myofiber size, and decreased muscle fibrosis. FHL1 expression in FRG1 mice, did not alter satellite cell number or activation, but enhanced myoblast fusion. Primary myoblasts isolated from FRG1 mice showed a myoblast fusion defect that was rescued by FHL1 expression. Therefore, increased FRG1 expression may contribute to a muscular dystrophy phenotype resembling FSHD by impairing myoblast fusion, a defect that can be rescued by enhanced myoblast fusion via expression of FHL1.

Differentiation of pluripotent stem cells to muscle fiber to model Duchenne muscular dystrophy.

  • Chal J
  • Nat. Biotechnol.
  • 2015 Sep 9

Literature context:


Abstract:

During embryonic development, skeletal muscles arise from somites, which derive from the presomitic mesoderm (PSM). Using PSM development as a guide, we establish conditions for the differentiation of monolayer cultures of mouse embryonic stem (ES) cells into PSM-like cells without the introduction of transgenes or cell sorting. We show that primary and secondary skeletal myogenesis can be recapitulated in vitro from the PSM-like cells, providing an efficient, serum-free protocol for the generation of striated, contractile fibers from mouse and human pluripotent cells. The mouse ES cells also differentiate into Pax7(+) cells with satellite cell characteristics, including the ability to form dystrophin(+) fibers when grafted into muscles of dystrophin-deficient mdx mice, a model of Duchenne muscular dystrophy (DMD). Fibers derived from ES cells of mdx mice exhibit an abnormal branched phenotype resembling that described in vivo, thus providing an attractive model to study the origin of the pathological defects associated with DMD.

Funding information:
  • NCATS NIH HHS - UL1 TR001439(United States)

Transcriptomic analysis of tail regeneration in the lizard Anolis carolinensis reveals activation of conserved vertebrate developmental and repair mechanisms.

  • Hutchins ED
  • PLoS ONE
  • 2015 May 11

Literature context:


Abstract:

Lizards, which are amniote vertebrates like humans, are able to lose and regenerate a functional tail. Understanding the molecular basis of this process would advance regenerative approaches in amniotes, including humans. We have carried out the first transcriptomic analysis of tail regeneration in a lizard, the green anole Anolis carolinensis, which revealed 326 differentially expressed genes activating multiple developmental and repair mechanisms. Specifically, genes involved in wound response, hormonal regulation, musculoskeletal development, and the Wnt and MAPK/FGF pathways were differentially expressed along the regenerating tail axis. Furthermore, we identified 2 microRNA precursor families, 22 unclassified non-coding RNAs, and 3 novel protein-coding genes significantly enriched in the regenerating tail. However, high levels of progenitor/stem cell markers were not observed in any region of the regenerating tail. Furthermore, we observed multiple tissue-type specific clusters of proliferating cells along the regenerating tail, not localized to the tail tip. These findings predict a different mechanism of regeneration in the lizard than the blastema model described in the salamander and the zebrafish, which are anamniote vertebrates. Thus, lizard tail regrowth involves the activation of conserved developmental and wound response pathways, which are potential targets for regenerative medical therapies.

Funding information:
  • NIDCD NIH HHS - R01DC009236(United States)

Granulin knock out zebrafish lack frontotemporal lobar degeneration and neuronal ceroid lipofuscinosis pathology.

  • Solchenberger B
  • PLoS ONE
  • 2015 Mar 19

Literature context:


Abstract:

Loss of function mutations in granulin (GRN) are linked to two distinct neurological disorders, frontotemporal lobar degeneration (FTLD) and neuronal ceroid lipofuscinosis (NCL). It is so far unknown how a complete loss of GRN in NCL and partial loss of GRN in FTLD can result in such distinct diseases. In zebrafish, there are two GRN homologues, Granulin A (Grna) and Granulin B (Grnb). We have generated stable Grna and Grnb loss of function zebrafish mutants by zinc finger nuclease mediated genome editing. Surprisingly, the grna and grnb single and double mutants display neither spinal motor neuron axonopathies nor a reduced number of myogenic progenitor cells as previously reported for Grna and Grnb knock down embryos. Additionally, grna-/-;grnb-/- double mutants have no obvious FTLD- and NCL-related biochemical and neuropathological phenotypes. Taken together, the Grna and Grnb single and double knock out zebrafish lack any obvious morphological, pathological and biochemical phenotypes. Loss of zebrafish Grna and Grnb might therefore either be fully compensated or only become symptomatic upon additional challenge.

Funding information:
  • Biotechnology and Biological Sciences Research Council - BB/E02209X/1(United Kingdom)
  • NCRR NIH HHS - RR-03051(United States)

PAX7 expression defines germline stem cells in the adult testis.

  • Aloisio GM
  • J. Clin. Invest.
  • 2014 Sep 3

Literature context:


Abstract:

Spermatogenesis is a complex, multistep process that maintains male fertility and is sustained by rare germline stem cells. Spermatogenic progression begins with spermatogonia, populations of which express distinct markers. The identity of the spermatogonial stem cell population in the undisturbed testis is controversial due to a lack of reliable and specific markers. Here we identified the transcription factor PAX7 as a specific marker of a rare subpopulation of A(single) spermatogonia in mice. PAX7+ cells were present in the testis at birth. Compared with the adult testis, PAX7+ cells constituted a much higher percentage of neonatal germ cells. Lineage tracing in healthy adult mice revealed that PAX7+ spermatogonia self-maintained and produced expanding clones that gave rise to mature spermatozoa. Interestingly, in mice subjected to chemotherapy and radiotherapy, both of which damage the vast majority of germ cells and can result in sterility, PAX7+ spermatogonia selectively survived, and their subsequent expansion contributed to the recovery of spermatogenesis. Finally, PAX7+ spermatogonia were present in the testes of a diverse set of mammals. Our data indicate that the PAX7+ subset of A(single) spermatogonia functions as robust testis stem cells that maintain fertility in normal spermatogenesis in healthy mice and mediate recovery after severe germline injury, such as occurs after cancer therapy.

Funding information:
  • Intramural NIH HHS - P30 8P30-GM-103507(United States)

Effect of vitamin D status improvement with 25-hydroxycholecalciferol on skeletal muscle growth characteristics and satellite cell activity in broiler chickens.

  • Hutton KC
  • J. Anim. Sci.
  • 2014 Aug 30

Literature context:


Abstract:

Skeletal muscle satellite cells (SC) play a critical role in the hypertrophic growth of postnatal muscle. Increases in breast meat yield have been consistently observed in broiler chickens fed 25-hydroxycholecalciferol (25OHD3), but it is unclear whether this effect is mediated by SC. Thus, our objective was to determine the effect of vitamin D status improvement by replacing the majority of dietary vitamin D3 (D3) with 25OHD3 on SC activity and muscle growth characteristics in the pectoralis major (PM) and the biceps femoris (BF) muscles. Day-old, male Ross 708 broiler chickens (n = 150) were fed 1 of 2 corn and soybean meal-based diets for 49 d. The control diet (CTL) contained 5,000 IU D3 per kg of diet and the experimental diet (25OHD3) contained 2,240 IU D3 per kg of diet + 2,760 IU 25OHD3 per kg of diet. Ten birds per treatment were harvested every 7 d. Two hours before harvest, birds were injected intraperitoneally with 5'-bromo-2'deoxyuridine (BrdU) to label mitotically active cells. Blood was collected from each bird at harvest to measure circulating concentrations of 25OHD3, a marker of vitamin D status. The PM and BF muscles were weighed and processed for cryohistological determination of skeletal muscle fiber cross-sectional area, enumeration of Myf-5+ and Pax7+ SC, and mitotically active (BrdU+) SC using immunofluorescence microscopy. Circulating 25OHD3 concentrations were greater in 25OHD3-fed birds on d 7, 14, 21, 28, 35, 42, and 49 when compared with CTL (P < 0.001). Growth performance and feed efficiency did not differ among dietary treatments (P > 0.10). Improved vitamin D status as a result of feeding 25OHD3 increased the number of mitotically active (Pax7+;BrdU+) SC (P = 0.01) and tended to increase the density of Pax7+ SC (P = 0.07) in the PM muscles of broilers on d 21 and 35, respectively. Broiler chickens fed 25OHD3 also tended to have greater Myf-5+ SC density (P = 0.09) on d 14, greater total nuclear density (P = 0.05) on d 28, and a greater muscle fiber cross-sectional area (P = 0.09) on d 49 in their PM muscles compared with CTL birds. Collectively, these results suggest that improvement of vitamin D status by replacing the majority of D3 in the diet with 25OHD3 can stimulate SC activity in the predominantly fast-twitch PM muscle and provide evidence toward understanding the mechanism behind previously observed increases in breast meat yield in 25OHD3-fed commercial broiler chickens.

Funding information:
  • NIDDK NIH HHS - T32 DK007319(United States)

Myogenic program dysregulation is contributory to disease pathogenesis in spinal muscular atrophy.

  • Boyer JG
  • Hum. Mol. Genet.
  • 2014 Aug 15

Literature context:


Abstract:

Mutations in the survival motor neuron (SMN1) gene lead to the neuromuscular disease spinal muscular atrophy (SMA). Although SMA is primarily considered as a motor neuron disease, the importance of muscle defects in its pathogenesis has not been fully examined. We use both primary cell culture and two different SMA model mice to demonstrate that reduced levels of Smn lead to a profound disruption in the expression of myogenic genes. This disruption was associated with a decrease in myofiber size and an increase in immature myofibers, suggesting that Smn is crucial for myogenic gene regulation and early muscle development. Histone deacetylase inhibitor trichostatin A treatment of SMA model mice increased myofiber size, myofiber maturity and attenuated the disruption of the myogenic program in these mice. Taken together, our work highlights the important contribution of myogenic program dysregulation to the muscle weakness observed in SMA.

Funding information:
  • NINDS NIH HHS - R01 NS045195(United States)

Immunohistochemical analysis of Pax6 and Pax7 expression in the CNS of adult Xenopus laevis.

  • Bandín S
  • J. Chem. Neuroanat.
  • 2014 May 26

Literature context:


Abstract:

Pax6 and Pax7 are transcription factors essential for the development of the CNS. In addition, increasing data, mainly obtained in amniotes, support that they are expressed in subsets of neurons in the adult, likely playing a role in maintaining neuron type identity. In the present study we analyzed the detailed distribution of Pax6 and Pax7 cells in the adult CNS of Xenopus laevis. Immunohistochemistry with antibodies that are required for high-resolution analysis of Pax-expressing cells was conducted. A wide distribution of Pax6 and Pax7 cells throughout the CNS was detected, with distinct patterns that showed only slight overlapping. Only Pax6 was expressed in the telencephalon, including the olfactory bulbs, septum, striatum and amygdaloid complex. In the diencephalon, Pax6 and Pax7 were distinct in the alar and basal parts, respectively, of prosomere 3. Large numbers of Pax6 and Pax7 cells were distributed in the pretectal region (alar plate of prosomere 1) but only Pax6 cells extended into basal plate. Pax7 specifically labeled cells in the optic tectum, including the ventricular zone, and Pax6 cells were the only cells found in the tegmentum. Pax6 was found in most granule cells of the cerebellum and Pax7 expression was found only in the ventricular zone. In the rostral rhombomere 1, Pax7 labeling was detected in cells of the ventricular zone of the alar plate, but numerous migrated cells were located in the basal plate, including the griseum centrale and the interpeduncular nucleus. Pax6 cells also formed a column of scattered neurons in the reticular formation and were found in the octavolateral area. The rhombencephalic ventricular zone of the alar plate expressed Pax7. Dorsal Pax7 cells and ventral Pax6 cells were found along the spinal cord separated from the ventricle, which did not show immunoreactivity. Our results show that the expression of Pax6 and Pax7 is widely maintained in the adult brain of Xenopus, like in urodele amphibians and in contrast to the situation described in amniotes. Therefore, in amphibians these transcription factors seem to be needed to maintain specific entities of subpopulations of neurons in the adult CNS.

Funding information:
  • NIBIB NIH HHS - R37 EB003320(United States)

Skeletal muscle characterization of Japanese quail line selectively bred for lower body weight as an avian model of delayed muscle growth with hypoplasia.

  • Choi YM
  • PLoS ONE
  • 2014 Apr 25

Literature context:


Abstract:

This study was designed to extensively characterize the skeletal muscle development in the low weight (LW) quail selected from random bred control (RBC) Japanese quail in order to provide a new avian model of impaired and delayed growth in physically normal animals. The LW line had smaller embryo and body weights than the RBC line in all age groups (P<0.05). During 3 to 42 d post-hatch, the LW line exhibited approximately 60% smaller weight of pectoralis major muscle (PM), mainly resulting from lower fiber numbers compared to the RBC line (P<0.05). During early post-hatch period when myotubes are still actively forming, the LW line showed impaired PM growth with prolonged expression of Pax7 and lower expression levels of MyoD, Myf-5, and myogenin (P<0.05), likely leading to impairment of myogenic differentiation and consequently, reduced muscle fiber formation. Additionally, the LW line had delayed transition of neonatal to adult myosin heavy chain isoform, suggesting delayed muscle maturation. This is further supported by the finding that the LW line continued to grow unlike the RBC line; difference in the percentages of PMW to body weights between both quail lines diminished with increasing age from 42 to 75 d post-hatch. This delayed muscle growth in the LW line is accompanied by higher levels of myogenin expression at 42 d (P<0.05), higher percentage of centered nuclei at 42 d (P<0.01), and greater rate of increase in fiber size between 42 and 75 d post-hatch (P<0.001) compared to the RBC line. Analysis of physiological, morphological, and developmental parameters during muscle development of the LW quail line provided a well-characterized avian model for future identification of the responsible genes and for studying mechanisms of hypoplasia and delayed muscle growth.

Funding information:
  • NINDS NIH HHS - R37NS008174(United States)
  • NINDS NIH HHS - U24NS057631(United States)

Accelerated regeneration of the skeletal muscle in RNF13-knockout mice is mediated by macrophage-secreted IL-4/IL-6.

  • Meng J
  • Protein Cell
  • 2014 Mar 27

Literature context:


Abstract:

RING finger protein 13 (RNF13) is a newly identified E3 ligase reported to be functionally significant in the regulation of cancer development, muscle cell growth, and neuronal development. In this study, the function of RNF13 in cardiotoxin-induced skeletal muscle regeneration was investigated using RNF13-knockout mice. RNF13(-/-) mice exhibited enhanced muscle regeneration-characterized by accelerated satellite cell proliferation-compared with wild-type mice. The expression of RNF13 was remarkably induced in macrophages rather than in the satellite cells of wild-type mice at the very early stage of muscle damage. This result indicated that inflammatory cells are important in RNF13-mediated satellite cell functions. The cytokine levels in skeletal muscles were further analyzed and showed that RNF13(-/-) mice produced greater amounts of various cytokines than wild-type mice. Among these, IL-4 and IL-6 levels significantly increased in RNF13(-/-) mice. The accelerated muscle regeneration phenotype was abrogated by inhibiting IL-4/IL-6 action in RNF13(-/-) mice with blocking antibodies. These results indicate that RNF13 deficiency promotes skeletal muscle regeneration via the effects on satellite cell niche mediated by IL-4 and IL-6.

Funding information:
  • NIDDK NIH HHS - R37 DK33165(United States)

Negative auto-regulation of myostatin expression is mediated by Smad3 and microRNA-27.

  • McFarlane C
  • PLoS ONE
  • 2014 Feb 5

Literature context:


Abstract:

Growth factors, such as myostatin (Mstn), play an important role in regulating post-natal myogenesis. In fact, loss of Mstn has been shown to result in increased post-natal muscle growth through enhanced satellite cell functionality; while elevated levels of Mstn result in dramatic skeletal muscle wasting through a mechanism involving reduced protein synthesis and increased ubiquitin-mediated protein degradation. Here we show that miR-27a/b plays an important role in feed back auto-regulation of Mstn and thus regulation of post-natal myogenesis. Sequence analysis of Mstn 3' UTR showed a single highly conserved miR-27a/b binding site and increased expression of miR-27a/b was correlated with decreased expression of Mstn and vice versa both in vitro and in mice in vivo. Moreover, we also show that Mstn gene expression was regulated by miR-27a/b. Treatment with miR-27a/b-specific AntagomiRs resulted in increased Mstn expression, reduced myoblast proliferation, impaired satellite cell activation and induction of skeletal muscle atrophy that was rescued upon either blockade of, or complete absence of, Mstn. Consistent with this, miR-27a over expression resulted in reduced Mstn expression, skeletal muscle hypertrophy and an increase in the number of activated satellite cells, all features consistent with impaired Mstn function. Loss of Smad3 was associated with increased levels of Mstn, concomitant with decreased miR-27a/b expression, which is consistent with impaired satellite cell function and muscular atrophy previously reported in Smad3-null mice. Interestingly, treatment with Mstn resulted in increased miR-27a/b expression, which was shown to be dependent on the activity of Smad3. These data highlight a novel auto-regulatory mechanism in which Mstn, via Smad3 signaling, regulates miR-27a/b and in turn its own expression. In support, Mstn-mediated inhibition of Mstn 3' UTR reporter activity was reversed upon miR-27a/b-specific AntagomiR transfection. Therefore, miR-27a/b, through negatively regulating Mstn, plays a role in promoting satellite cell activation, myoblast proliferation and preventing muscle wasting.

Funding information:
  • NIMH NIH HHS - U01 MH105971(United States)

Sip1 mediates an E-cadherin-to-N-cadherin switch during cranial neural crest EMT.

  • Rogers CD
  • J. Cell Biol.
  • 2013 Dec 9

Literature context:


Abstract:

The neural crest, an embryonic stem cell population, initially resides within the dorsal neural tube but subsequently undergoes an epithelial-to-mesenchymal transition (EMT) to commence migration. Although neural crest and cancer EMTs are morphologically similar, little is known regarding conservation of their underlying molecular mechanisms. We report that Sip1, which is involved in cancer EMT, plays a critical role in promoting the neural crest cell transition to a mesenchymal state. Sip1 transcripts are expressed in premigratory/migrating crest cells. After Sip1 loss, the neural crest specifier gene FoxD3 was abnormally retained in the dorsal neuroepithelium, whereas Sox10, which is normally required for emigration, was diminished. Subsequently, clumps of adherent neural crest cells remained adjacent to the neural tube and aberrantly expressed E-cadherin while lacking N-cadherin. These findings demonstrate two distinct phases of neural crest EMT, detachment and mesenchymalization, with the latter involving a novel requirement for Sip1 in regulation of cadherin expression during completion of neural crest EMT.

Funding information:
  • NCI NIH HHS - T32 CA106209(United States)

Spatiotemporal patterns of Pax3, Pax6, and Pax7 expression in the developing brain of a urodele amphibian, Pleurodeles waltl.

  • Joven A
  • J. Comp. Neurol.
  • 2013 Dec 1

Literature context:


Abstract:

The onset and developmental dynamics of Pax3, Pax6, and Pax7 expressions were analyzed by immunohistochemical techniques in the central nervous system (CNS) of embryos, larvae, and recently metamorphosed juveniles of the urodele amphibian Pleurodeles waltl. During the embryonic period, the Pax proteins start being detectable in neuroepithelial domains. Subsequently, they become restricted to subsets of cells in distinct brain regions, maintaining different degrees of expression in late larvae and juvenile brains. Specifically, Pax6 is broadly expressed all along the urodele CNS (olfactory bulbs, pallium, basal ganglia, diencephalon, mesencephalic tegmentum, rhombencephalon, and spinal cord) and the developing olfactory organ and retina. Pax3 and Pax7 are excluded from the rostral forebrain and were usually observed in overlapping regions during embryonic development, whereas Pax3 expression is highly downregulated as development proceeds. Thus, Pax3 is restricted to the roof plate of prosomere 2, pretectum, optic tectum, rhombencephalon, and spinal cord. Comparatively, Pax7 was more conspicuous in all these regions. Pax7 cells were also found in the paraphysis, intermediate lobe of the hypophysis, and basal plate of prosomere 3. Our data show that the expression patterns of the three Pax genes studied are overall evolutionarily conserved, and therefore could unequivocally be used to identify subdivisions in the urodele brain similar to other vertebrates, which are not clearly discernable with classical techniques. In addition, the spatiotemporal sequences of expression provide indirect evidence of putative migratory routes across neuromeric limits and the alar-basal boundary.

Funding information:
  • NCI NIH HHS - CA126189(United States)

Skeletal muscle cells express ICAM-1 after muscle overload and ICAM-1 contributes to the ensuing hypertrophic response.

  • Dearth CL
  • PLoS ONE
  • 2013 Sep 6

Literature context:


Abstract:

We previously reported that leukocyte specific β2 integrins contribute to hypertrophy after muscle overload in mice. Because intercellular adhesion molecule-1 (ICAM-1) is an important ligand for β2 integrins, we examined ICAM-1 expression by murine skeletal muscle cells after muscle overload and its contribution to the ensuing hypertrophic response. Myofibers in control muscles of wild type mice and cultures of skeletal muscle cells (primary and C2C12) did not express ICAM-1. Overload of wild type plantaris muscles caused myofibers and satellite cells/myoblasts to express ICAM-1. Increased expression of ICAM-1 after muscle overload occurred via a β2 integrin independent mechanism as indicated by similar gene and protein expression of ICAM-1 between wild type and β2 integrin deficient (CD18-/-) mice. ICAM-1 contributed to muscle hypertrophy as demonstrated by greater (p<0.05) overload-induced elevations in muscle protein synthesis, mass, total protein, and myofiber size in wild type compared to ICAM-1-/- mice. Furthermore, expression of ICAM-1 altered (p<0.05) the temporal pattern of Pax7 expression, a marker of satellite cells/myoblasts, and regenerating myofiber formation in overloaded muscles. In conclusion, ICAM-1 expression by myofibers and satellite cells/myoblasts after muscle overload could serve as a mechanism by which ICAM-1 promotes hypertrophy by providing a means for cell-to-cell communication with β2 integrin expressing myeloid cells.

Funding information:
  • Biotechnology and Biological Sciences Research Council - BB/I00016X/1(United Kingdom)
  • NINDS NIH HHS - NS047357(United States)

Improvement of maternal vitamin D status with 25-hydroxycholecalciferol positively impacts porcine fetal skeletal muscle development and myoblast activity.

  • Hines EA
  • J. Anim. Sci.
  • 2013 Sep 27

Literature context:


Abstract:

There is little information available regarding the influence of maternal vitamin D status on fetal skeletal muscle development. Therefore, we investigated the effect of improved vitamin D status resulting from 25-hydroxycholecalciferol (25OHD3) supplementation of dams on fetal skeletal muscle developmental characteristics and myoblast activity using Camborough 22 gilts (n = 40) randomly assigned to 1 of 2 corn-soybean meal-based diets. The control diet (CTL) contained 2,500 IU cholecalciferol (D3)/kg diet, whereas the experimental diet contained 500 IU D3/kg diet plus 50 µg 25OHD3/kg diet. Gilts were fed 2.7 kg of their assigned diet once daily beginning 43 d before breeding through d 90 of gestation. On gestational d 90 (± 1), fetal LM and semitendinosus muscle samples were collected for analysis of developmental characteristics and myoblast activity, respectively. No treatment difference was observed in fetal LM cross-sectional area (P = 0.25). Fetuses from 25OHD3-supplemented gilts had more LM fibers (P = 0.04) that tended to be smaller in cross-sectional area compared with CTL fetuses (P = 0.11). A numerical increase in the total number of Pax7+ myoblasts was also observed in fetuses from 25OHD3-supplemented gilts (P = 0.12). Myoblasts derived from the muscles of fetuses from 25OHD3-fed dams displayed an extended proliferative phase in culture compared with those from fetuses of dams fed only D3 (P < 0.0001). The combination of additional muscle fibers and Pax7+ myoblasts with prolonged proliferative capacity could enhance the postnatal skeletal muscle growth potential of fetuses from 25OHD3-supplemented gilts. These data highlight the importance of maternal vitamin D status on the development of fetal skeletal muscle.

Funding information:
  • NHLBI NIH HHS - HL64541(United States)
  • NICHD NIH HHS - R03HD061853(United States)

Myf5 expression during fetal myogenesis defines the developmental progenitors of adult satellite cells.

  • Biressi S
  • Dev. Biol.
  • 2013 Jul 15

Literature context:


Abstract:

Myf5 is a member of the muscle-specific determination genes and plays a critical role in skeletal muscle development. Whereas the expression of Myf5 during embryonic and fetal myogenesis has been extensively studied, its expression in progenitors that will ultimately give rise to adult satellite cells, the stem cells responsible for muscle repair, is still largely unexplored. To investigate this aspect, we have generated a mouse strain carrying a CreER coding sequence in the Myf5 locus. In this strain, Tamoxifen-inducible Cre activity parallels endogenous Myf5 expression. Combining Myf5(CreER) and Cre reporter alleles, we were able to evaluate the contribution of cells expressing Myf5 at distinct developmental stages to the pool of satellite cells in adult hindlimb muscles. Although it was possible to trace back the origin of some rare satellite cells to a subpopulation of Myf5(+ve) progenitors in the limb buds at the late embryonic stage (∼E12), a significant number of satellite cells arise from cells which expressed Myf5 for the first time at the fetal stage (∼E15). These studies provide direct evidence that adult satellite cells derive from progenitors that first express the myogenic determination gene Myf5 during fetal stages of myogenesis.

Funding information:
  • NHGRI NIH HHS - R01 HG003469(United States)

Ultrasound modulates the inflammatory response and promotes muscle regeneration in injured muscles.

  • Nagata K
  • Ann Biomed Eng
  • 2013 Jun 29

Literature context:


Abstract:

The purpose of this study was to examine the effect of ultrasound on inflammatory skeletal muscle in vitro and in vivo. C2C12 cells were cultured in medium with or without TNF-α or IL-1β. After stimulation with cytokines, the cells received ultrasound or sham exposure. Furthermore, the tibialis anterior (TA) muscle in C57BL/6 mice injured by cardiotoxin (CTX) were dissected after a series of ultrasound treatments and examined. Exposure of C2C12 cells to ultrasound resulted in down-regulation of cyclooxygenase-2 (COX-2) mRNA and protein expression induced by TNF-α or IL-1β, and up-regulated myogenin mRNA and protein depressed by TNF-α or IL-1β. In injured TA muscle induced by CTX, ultrasound caused increase of COX-2 mRNA at 1 day after ultrasound treatment, however, at day 5, reduction of COX-2 mRNA and protein. At day 5, ultrasound caused increase of myogenin mRNA and protein, increase of fast myosin protein, and increase of paired-box transcription factor 7 positive cells. At day 7, the cross-sectional area of myofibers in the ultrasound exposure side was significantly larger than that on the control side. In conclusion, ultrasound stimulation may be a better candidate as a medical remedy to promote myogenesis in inflammatory muscle states, such as muscle injury.

Funding information:
  • Biotechnology and Biological Sciences Research Council - BBS/E/B/000C0419(United Kingdom)
  • Wellcome Trust - (United Kingdom)

Sulf1 modulates BMP signaling and is required for somite morphogenesis and development of the horizontal myoseptum.

  • Meyers JR
  • Dev. Biol.
  • 2013 Jun 15

Literature context:


Abstract:

Heparan sulfate proteoglycans (HSPGs) are glycosylated extracellular or membrane-associated proteins. Their unbranched heparan sulfate (HS) disaccharide chains interact with many growth factors and receptors, modifying their activity or diffusion. The pattern of HS sulfation can be altered by the enzymes Sulf1 and Sulf2, secreted extracellular 6-O endosulfatases, which remove specific sulfate groups from HS. Modification by Sulf enzymes changes the binding affinity of HS for protein such as ligands and receptors, affecting growth factor gradients and activities. The precise expression of these sulfatases are thought to be necessary for normal development. We have examined the role of the sulf1 gene in trunk development of zebrafish embryos. sulf1 is expressed in the developing trunk musculature and as well as in midline structures such as the notochord, floorplate and hypochord. Knockdown of sulf1 with antisense morpholinos results in poor differentiation of the somitic trunk muscle, loss of the horizontal myoseptum, lack of pigmentation along the mediolateral stripe, and improper migration of the lateral line primordium. sulf1 knockdown results in a decrease in the number of Pax7-expressing dermomyotome cells, particularly along the midline where the horizontal myoseptum develops. It also leads to decreased sdf1/cxcl12 expression along the mediolateral trunk musculature. Both the Pax7 and cxcl12 expression can be restored by inhibition pharmacological inhibition of BMP signaling, which also restores formation of the myoseptum, fast muscle development, and pigmentation patterning. Lateral line migration and neuromast deposition depend on sdf1/cxcl12 and FGF signaling respectively, both of which are disrupted in sulf1 morphants. Pharmacological activation of FGF signaling can rescue the spacing of neuromast deposition in these fish. Together this data indicate that sulf1 plays a crucial role in modulating both BMP and FGF signaling along the developing myoseptum to coordinate the morphogenesis of trunk musculature, associated pigment cells, and lateral line neuromasts.

Funding information:
  • NHGRI NIH HHS - HG004695(United States)

Elevated SOCS3 and altered IL-6 signaling is associated with age-related human muscle stem cell dysfunction.

  • McKay BR
  • Am. J. Physiol., Cell Physiol.
  • 2013 Apr 15

Literature context:


Abstract:

Aging is associated with increased circulating interleukin-6 (IL-6) and a reduced myogenic capacity, marked by reduced muscle stem cell [satellite cell (SC)] activity. Although IL-6 is important for normal SC function, it is unclear whether elevated IL-6 associated with aging alters SC function. We hypothesized that mild chronically elevated IL-6 would be associated with a blunted SC response through altered IL-6 signaling and elevated suppressor of cytokine signaling-3 (SOCS3) in the elderly. Nine healthy older adult men (OA; 69.6 ± 3.9 yr) and 9 young male controls (YC; 21. 3 ± 3.1 yr) completed 4 sets of 10 repetitions of unilateral leg press and knee extension (75% of 1-RM). Muscle biopsies and blood were obtained before and 3, 24, and 48 h after exercise. Basal SC number was 33% lower in OA vs. YC, and the response was blunted in OA. IL-6(+)/Pax7(+) cells demonstrated a divergent response in OA, with YC increasing to 69% at 3 h and peaking at 24 h (72%), while IL-6(+)/Pax7(+) cells were not increased until 48 h in OA (61%). Type II fiber-associated phosphorylated signal transducer and activator of transcription (pSTAT3)(+)/Pax7(+) cells demonstrated a similar delay in OA, not increasing until 48 h (vs. 3 h in YC). SOCS3 protein was 86% higher in OA. These data demonstrate an age-related impairment in normal SC function that appears to be influenced by SOCS3 protein and delayed induction of IL-6 and pSTAT3 in the SCs of OA. Collectively, these data suggest dysregulated IL-6 signaling as a consequence of aging contributes to the blunted muscle stem cell response.

Funding information:
  • NIGMS NIH HHS - GM061603(United States)
  • NIGMS NIH HHS - R01 GM061987(United States)

Angiotensin 1-7 as means to prevent the metabolic syndrome: lessons from the fructose-fed rat model.

  • Marcus Y
  • Diabetes
  • 2013 Apr 22

Literature context:


Abstract:

We studied the effects of chronic angiotensin 1-7 (Ang 1-7) treatment in an experimental model of the metabolic syndrome, i.e., rats given high-fructose/low-magnesium diet (HFrD). Rats were fed on HFrD for 24 weeks with and without Ang 1-7 (576 µg/kg/day, s.c., Alzet pumps). After 6 months, Ang 1-7-treated animals had lower body weight (-9.5%), total fat mass (detected by magnetic resonance imaging), and serum triglycerides (-51%), improved glucose tolerance, and better insulin sensitivity. Similar metabolic effects were also evident, albeit in the absence of weight loss, in rats first exposed to HFrD for 5 months and then subjected to short-term (4 weeks) treatment with Ang 1-7. Six months of Ang 1-7 treatment were associated with lower plasma renin activity (-40%) and serum aldosterone (-48%), less hepatosteatatitis, and a reduction in epididymal adipocyte volume. The marked attenuation of macrophage infiltration in white adipose tissue (WAT) was associated with reduced levels of the pP65 protein in the epididymal fat tissue, suggesting less activation of the nuclear factor-κB (NFκB) pathway in Ang 1-7-treated rats. WAT from Ang 1-7-treated rats showed reduced NADPH-stimulated superoxide production. In single muscle fibers (myofibers) harvested and grown ex vivo for 10 days, myofibers from HFrD rats gave rise to 20% less myogenic cells than the Ang 1-7-treated rats. Fully developed adipocytes were present in most HFrD myofiber cultures but entirely absent in cultures from Ang 1-7-treated rats. In summary, Ang 1-7 had an ameliorating effect on insulin resistance, hypertriglyceridemia, fatty liver, obesity, adipositis, and myogenic and adipogenic differentiation in muscle tissue in the HFrD rats.

Funding information:
  • NINDS NIH HHS - P30 NS055077(United States)
  • Wellcome Trust - 085982(United Kingdom)

Delta-like 1 homolog (dlk1): a marker for rhabdomyosarcomas implicated in skeletal muscle regeneration.

  • Jørgensen LH
  • PLoS ONE
  • 2013 Apr 11

Literature context:


Abstract:

Dlk1, a member of the Epidermal Growth Factor family, is expressed in multiple tissues during development, and has been detected in carcinomas and neuroendocrine tumors. Dlk1 is paternally expressed and belongs to a group of imprinted genes associated with rhabdomyosarcomas but not with other primitive childhood tumors to date. Here, we investigate the possible roles of Dlk1 in skeletal muscle tumor formation. We analyzed tumors of different mesenchymal origin for expression of Dlk1 and various myogenic markers and found that Dlk1 was present consistently in myogenic tumors. The coincident observation of Dlk1 with a highly proliferative state in myogenic tumors led us to subsequently investigate the involvement of Dlk1 in the control of the adult myogenic programme. We performed an injury study in Dlk1 transgenic mice, ectopically expressing ovine Dlk1 (membrane bound C2 variant) under control of the myosin light chain promotor, and detected an early, enhanced formation of myotubes in Dlk1 transgenic mice. We then stably transfected the mouse myoblast cell line, C2C12, with full-length Dlk1 (soluble A variant) and detected an inhibition of myotube formation, which could be reversed by adding Dlk1 antibody to the culture supernatant. These results suggest that Dlk1 is involved in controlling the myogenic programme and that the various splice forms may exert different effects. Interestingly, both in the Dlk1 transgenic mice and the DLK1-C2C12 cells, we detected reduced myostatin expression, suggesting that the effect of Dlk1 on the myogenic programme might involve the myostatin signaling pathway. In support of a relationship between Dlk1 and myostatin we detected reciprocal expression of these two transcripts during different cell cycle stages of human myoblasts. Together our results suggest that Dlk1 is a candidate marker for skeletal muscle tumors and might be involved directly in skeletal muscle tumor formation through a modulatory effect on the myogenic programme.

Funding information:
  • NCI NIH HHS - 1R15CA141519-01(United States)
  • NIMH NIH HHS - R01 MH083094(United States)

Smooth muscle fascicular reorientation is required for esophageal morphogenesis and dependent on Cdo.

  • Romer AI
  • J. Cell Biol.
  • 2013 Apr 15

Literature context:


Abstract:

Postnatal maturation of esophageal musculature involves proximal-to-distal replacement of smooth muscle with skeletal muscle by elusive mechanisms. We report that this process is impaired in mice lacking the cell surface receptor Cdo and identify the underlying developmental mechanism. A myogenic transition zone containing proliferative skeletal muscle precursor cells migrated in a proximal-distal direction, leaving differentiated myofibers in its wake. Distal to the transition zone, smooth muscle fascicles underwent a morphogenetic process whereby they changed their orientation relative to each other and to the lumen. Consequently, a path was cleared for the transition zone, and smooth muscle ultimately occupied only the distal-most esophagus; there was no loss of smooth muscle. Cdo(-/-) mice were specifically defective in fascicular reorientation, resulting in an aberrantly proximal skeletal-smooth muscle boundary. Furthermore, Cdo(-/-) mice displayed megaesophagus and achalasia, and their lower esophageal sphincter was resistant to nitric oxide-induced relaxation, suggesting a developmental linkage between patterning and sphincter function. Collectively, these results illuminate mechanisms of esophageal morphogenesis and motility disorders.

Funding information:
  • NCI NIH HHS - 1RC2CA148222-01(United States)

A rat model for muscle regeneration in the soft palate.

  • Carvajal Monroy PL
  • PLoS ONE
  • 2013 Apr 4

Literature context:


Abstract:

BACKGROUND: Children with a cleft in the soft palate have difficulties with speech, swallowing, and sucking. Despite successful surgical repositioning of the muscles, optimal function is often not achieved. Scar formation and defective regeneration may hamper the functional recovery of the muscles after cleft palate repair. Therefore, the aim of this study is to investigate the anatomy and histology of the soft palate in rats, and to establish an in vivo model for muscle regeneration after surgical injury. METHODS: Fourteen adult male Sprague Dawley rats were divided into four groups. Groups 1 (n = 4) and 2 (n = 2) were used to investigate the anatomy and histology of the soft palate, respectively. Group 3 (n = 6) was used for surgical wounding of the soft palate, and group 4 (n = 2) was used as unwounded control group. The wounds (1 mm) were evaluated by (immuno)histochemistry (AZAN staining, Pax7, MyoD, MyoG, MyHC, and ASMA) after 7 days. RESULTS: The present study shows that the anatomy and histology of the soft palate muscles of the rat is largely comparable with that in humans. All wounds showed clinical evidence of healing after 7 days. AZAN staining demonstrated extensive collagen deposition in the wound area, and initial regeneration of muscle fibers and salivary glands. Proliferating and differentiating satellite cells were identified in the wound area by antibody staining. CONCLUSIONS: This model is the first, suitable for studying muscle regeneration in the rat soft palate, and allows the development of novel adjuvant strategies to promote muscle regeneration after cleft palate surgery.

LMO4 is an essential cofactor in the Snail2-mediated epithelial-to-mesenchymal transition of neuroblastoma and neural crest cells.

  • Ferronha T
  • J. Neurosci.
  • 2013 Feb 13

Literature context:


Abstract:

Neuroblastoma is an embryonic tumor derived from cells of the neural crest. Taking advantage of a newly developed neural crest lineage tracer and based on the hypothesis that the molecular mechanisms that mediate neural crest delamination are also likely to be involved in the spread of neuroblastoma, we were able to identify genes that are active both in neural crest development and neuroblastoma tumor formation. A subsequent search of the neuroblastoma gene server for human orthologues of genes differentially expressed in the chick embryo neural crest screen retrieved the LIM domain only protein 4 (LMO4), which was expressed in both cell types analyzed. Functional experiments in these two model systems revealed that LMO4 activity is required for neuroblastoma cell invasion and neural crest delamination. Moreover, we identified LMO4 as an essential cofactor in Snail2-mediated cadherin repression and in the epithelial-to-mesenchymal transition of both neural crest and neuroblastoma cells. Together, our results suggest that the association of high levels of LMO4 with aggressive neuroblastomas is dependent on LMO4 regulation of cadherin expression and hence, tumor invasiveness.

Funding information:
  • NHGRI NIH HHS - P41HG003619-01(United States)
  • NINDS NIH HHS - NS078530(United States)

Elk3 is essential for the progression from progenitor to definitive neural crest cell.

  • Rogers CD
  • Dev. Biol.
  • 2013 Feb 15

Literature context:


Abstract:

Elk3/Net/Sap2 (here referred to as Elk3) is an Ets ternary complex transcriptional repressor known for its involvement in angiogenesis during embryonic development. Although Elk3 is expressed in various tissues, additional roles for the protein outside of vasculature development have yet to be reported. Here, we characterize the early spatiotemporal expression pattern of Elk3 in the avian embryo using whole mount in situ hybridization and quantitative RT-PCR and examine the effects of its loss of function on neural crest development. At early stages, Elk3 is expressed in the head folds, head mesenchyme, intersomitic vessels, and migratory cranial neural crest (NC) cells. Loss of the Elk3 protein results in the retention of Pax7+ precursors in the dorsal neural tube that fail to upregulate neural crest specifier genes, FoxD3, Sox10 and Snail2, resulting in embryos with severe migration defects. The results putatively place Elk3 downstream of neural plate border genes, but upstream of neural crest specifier genes in the neural crest gene regulatory network (NC-GRN), suggesting that it is critical for the progression from progenitor to definitive neural crest cell.

Funding information:
  • NIMH NIH HHS - P30 MH062261-01(United States)

MEF2A regulates the Gtl2-Dio3 microRNA mega-cluster to modulate WNT signaling in skeletal muscle regeneration.

  • Snyder CM
  • Development
  • 2013 Jan 1

Literature context:


Abstract:

Understanding the molecular mechanisms of skeletal muscle regeneration is crucial to exploiting this pathway for use in tissue repair. Our data demonstrate that the MEF2A transcription factor plays an essential role in skeletal muscle regeneration in adult mice. Injured Mef2a knockout mice display widespread necrosis and impaired myofiber formation. MEF2A controls this process through its direct regulation of the largest known mammalian microRNA (miRNA) cluster, the Gtl2-Dio3 locus. A subset of the Gtl2-Dio3 miRNAs represses secreted Frizzled-related proteins (sFRPs), inhibitors of WNT signaling. Consistent with these data, Gtl2-Dio3-encoded miRNAs are downregulated in regenerating Mef2a knockout muscle, resulting in upregulated sFRP expression and attenuated WNT activity. Furthermore, myogenic differentiation in Mef2a-deficient myoblasts is rescued by overexpression of miR-410 and miR-433, two miRNAs in the Gtl2-Dio3 locus that repress sFRP2, or by treatment with recombinant WNT3A and WNT5A. Thus, miRNA-mediated modulation of WNT signaling by MEF2A is a requisite step for proper muscle regeneration, and represents an attractive pathway for enhancing regeneration of diseased muscle.

Compromised genomic integrity impedes muscle growth after Atrx inactivation.

  • Huh MS
  • J. Clin. Invest.
  • 2012 Dec 3

Literature context:


Abstract:

ATR-X syndrome is a severe intellectual disability disorder caused by mutations in the ATRX gene. Many ancillary clinical features are attributed to CNS deficiencies, yet most patients have muscle hypotonia, delayed ambulation, or kyphosis, pointing to an underlying skeletal muscle defect. Here, we identified a cell-intrinsic requirement for Atrx in postnatal muscle growth and regeneration in mice. Mice with skeletal muscle-specific Atrx conditional knockout (Atrx cKO mice) were viable, but by 3 weeks of age presented hallmarks of underdeveloped musculature, including kyphosis, 20% reduction in body mass, and 34% reduction in muscle fiber caliber. Atrx cKO mice also demonstrated a marked regeneration deficit that was not due to fewer resident satellite cells or their inability to terminally differentiate. However, activation of Atrx-null satellite cells from isolated muscle fibers resulted in a 9-fold reduction in myoblast expansion, caused by delayed progression through mid to late S phase. While in S phase, Atrx colocalized specifically to late-replicating chromatin, and its loss resulted in rampant signs of genomic instability. These observations support a model in which Atrx maintains chromatin integrity during the rapid developmental growth of a tissue.

Funding information:
  • NIDA NIH HHS - 1U54DA021519-01A1(United States)

The ubiquitin ligase Nedd4-1 participates in denervation-induced skeletal muscle atrophy in mice.

  • Nagpal P
  • PLoS ONE
  • 2012 Oct 30

Literature context:


Abstract:

Skeletal muscle atrophy is a consequence of muscle inactivity resulting from denervation, unloading and immobility. It accompanies many chronic disease states and also occurs as a pathophysiologic consequence of normal aging. In all these conditions, ubiquitin-dependent proteolysis is a key regulator of the loss of muscle mass, and ubiquitin ligases confer specificity to this process by interacting with, and linking ubiquitin moieties to target substrates through protein:protein interaction domains. Our previous work suggested that the ubiquitin-protein ligase Nedd4-1 is a potential mediator of skeletal muscle atrophy associated with inactivity (denervation, unloading and immobility). Here we generated a novel tool, the Nedd4-1 skeletal muscle-specific knockout mouse (myo(Cre);Nedd4-1(flox/flox)) and subjected it to a well validated model of denervation induced skeletal muscle atrophy. The absence of Nedd4-1 resulted in increased weights and cross-sectional area of type II fast twitch fibres of denervated gastrocnemius muscle compared with wild type littermates controls, at seven and fourteen days following tibial nerve transection. These effects are not mediated by the Nedd4-1 substrates MTMR4, FGFR1 and Notch-1. These results demonstrate that Nedd4-1 plays an important role in mediating denervation-induced skeletal muscle atrophy in vivo.

Funding information:
  • Canadian Institutes of Health Research - 200910GSD-226209-172830(Canada)

Hypoxia promotes satellite cell self-renewal and enhances the efficiency of myoblast transplantation.

  • Liu W
  • Development
  • 2012 Aug 26

Literature context:


Abstract:

Microenvironmental oxygen (O(2)) regulates stem cell activity, and a hypoxic niche with low oxygen levels has been reported in multiple stem cell types. Satellite cells are muscle-resident stem cells that maintain the homeostasis and mediate the regeneration of skeletal muscles. We demonstrate here that hypoxic culture conditions favor the quiescence of satellite cell-derived primary myoblasts by upregulating Pax7, a key regulator of satellite cell self-renewal, and downregulating MyoD and myogenin. During myoblast division, hypoxia promotes asymmetric self-renewal divisions and inhibits asymmetric differentiation divisions without affecting the overall rate of proliferation. Mechanistic studies reveal that hypoxia activates the Notch signaling pathway, which subsequently represses the expression of miR-1 and miR-206 through canonical Hes/Hey proteins, leading to increased levels of Pax7. More importantly, hypoxia conditioning enhances the efficiency of myoblast transplantation and the self-renewal of implanted cells. Given the robust effects of hypoxia on maintaining the quiescence and promoting the self-renewal of cultured myoblasts, we predict that oxygen levels in the satellite cell niche play a central role in precisely balancing quiescence versus activation, and self-renewal versus differentiation, in muscle stem cells in vivo.

Funding information:
  • NCI NIH HHS - F31 CA180689(United States)

A novel target of microRNA-29, Ring1 and YY1-binding protein (Rybp), negatively regulates skeletal myogenesis.

  • Zhou L
  • J. Biol. Chem.
  • 2012 Jul 20

Literature context:


Abstract:

Skeletal muscle cell differentiation (myogenesis) is a process orchestrated by a complex network involving transcription factors, epigenetic regulators, and microRNAs. Previous studies identified miR-29 as a pro-myogenic factor that interacts with components of Polycomb repressive complex, YY1 and Ezh2. In a genome-wide survey of miR-29-mediated transcriptome changes in C2C12 myoblasts, many epigenetic factors were found to be down-regulated by miR-29. Among them, Rybp was shown to be a direct target of miR-29 through binding to its 3' UTR. Functional studies demonstrated that Rybp is down-regulated during myogenesis and acts as a negative regulator of skeletal myogenesis both in vitro during C2C12 differentiation and in vivo in injury-induced muscle regeneration. Furthermore, we found that Rybp and YY1 co-occupy several myogenic loci, including miR-29 itself, to silence their expression, thus forming a Rybp-miR-29 feedback loop. Rybp overexpression was found to enhance the enrichment of Ezh2 and trimethylation of H3K27 at target loci, suggesting it may facilitate the recruitment or stabilization of the Polycomb repressive complex. Collectively, our results identify Rybp as a novel regulator of myogenesis that co-acts with YY1 to silence miR-29 and other myogenic loci.

Funding information:
  • Canadian Institutes of Health Research - MOP-82862(Canada)
  • NINDS NIH HHS - T32 NS061764(United States)

The actin regulator N-WASp is required for muscle-cell fusion in mice.

  • Gruenbaum-Cohen Y
  • Proc. Natl. Acad. Sci. U.S.A.
  • 2012 Jul 10

Literature context:


Abstract:

A fundamental aspect of skeletal myogenesis involves extensive rounds of cell fusion, in which individual myoblasts are incorporated into growing muscle fibers. Here we demonstrate that N-WASp, a ubiquitous nucleation-promoting factor of branched microfilament arrays, is an essential contributor to skeletal muscle-cell fusion in developing mouse embryos. Analysis both in vivo and in primary satellite-cell cultures, shows that disruption of N-WASp function does not interfere with the program of skeletal myogenic differentiation, and does not affect myoblast motility, morphogenesis and attachment capacity. N-WASp-deficient myoblasts, however, fail to fuse. Furthermore, our analysis suggests that myoblast fusion requires N-WASp activity in both partners of a fusing myoblast pair. These findings reveal a specific role for N-WASp during mammalian myogenesis. WASp-family elements appear therefore to act as universal mediators of the myogenic cell-cell fusion mechanism underlying formation of functional muscle fibers, in both vertebrate and invertebrate species.

Funding information:
  • Canadian Institutes of Health Research - (Canada)
  • NIDCD NIH HHS - 1R01DC012854(United States)

Myostatin is associated with age-related human muscle stem cell dysfunction.

  • McKay BR
  • FASEB J.
  • 2012 Jun 1

Literature context:


Abstract:

Human aging is accompanied by a progressive loss of muscle mass (sarcopenia). We tested the hypothesis that older males (OMs, 70±4 yr, n=9) would have a blunted myogenic response to a physiological stimulus compared to younger controls (21±3 yr, n=9). Subjects completed an acute bout of intense unilateral muscle loading. Young healthy males matched for body mass and activity level served as the control group. Muscle biopsies and blood were obtained before and at 3, 24, and 48 h after muscle loading. The muscle stem cell response was analyzed using flow cytometry, immunofluorescent microscopy, and standard protein and mRNA analysis. OMs had 35% fewer basal stem cells and a type II fiber-specific impairment in stem cell content and proliferation. Myogenic determination factor staining and cell cycle analysis illustrated a severely blunted progression through the myogenic program. Myostatin protein and mRNA were 2-fold higher in OMs. Stem cell-specific myostatin levels were not different at baseline; however, there were 67% more myostatin-positive type II-associated stem cells in OMs at 24 h. These data illustrate an age-related impairment of stem cell function in a fiber type-specific manner. The greater colocalization of myostatin with stem cells provides a mechanism for the impaired myogenic capacity of aged muscle.

Funding information:
  • NIDDK NIH HHS - R37 DK050107(United States)
  • NINDS NIH HHS - R01 NS056359(United States)

Loss of miR-29 in myoblasts contributes to dystrophic muscle pathogenesis.

  • Wang L
  • Mol. Ther.
  • 2012 Jun 1

Literature context:


Abstract:

microRNAs (miRNAs) are noncoding RNAs that regulate gene expression in post-transcriptional fashion, and emerging studies support their importance in a multitude of physiological and pathological processes. Here, we describe the regulation and function of miR-29 in Duchenne muscular dystrophy (DMD) and its potential use as therapeutic target. Our results demonstrate that miR-29 expression is downregulated in dystrophic muscles of mdx mice, a model of DMD. Restoration of its expression by intramuscular and intravenous injection improved dystrophy pathology by both promoting regeneration and inhibiting fibrogenesis. Mechanistic studies revealed that loss of miR-29 in muscle precursor cells (myoblasts) promotes their transdifferentiation into myofibroblasts through targeting extracellular molecules including collagens and microfibrillar-associated protein 5 (Mfap5). We further demonstrated that miR-29 is under negative regulation by transforming growth factor-β (TGF-β) signaling. Together, these results not only identify TGF-β-miR-29 as a novel regulatory axis during myoblasts conversion into myofibroblasts which constitutes a novel contributing route to muscle fibrogenesis of DMD but also implicate miR-29 replacement therapy as a promising treatment approach for DMD.

Funding information:
  • NIGMS NIH HHS - T32 GM08400(United States)
  • NIMHD NIH HHS - 8G12MD 007603-27(United States)

Nitric oxide and voluntary exercise together promote quadriceps hypertrophy and increase vascular density in female 18-mo-old mice.

  • Leiter JR
  • Am. J. Physiol., Cell Physiol.
  • 2012 May 1

Literature context:


Abstract:

Age-related sarcopenia reduces the size, strength, and function of muscle, and the diameter of muscle fibers. It also disrupts the dystrophin-glycoprotein complex, dislocating nitric oxide synthase 1 (NOS-1) and reducing sarcolemmal integrity. This study of quadriceps muscle in 18-mo-old mice showed that NO-donor treatment with isosorbide dinitrate (I) for 6 wk, in combination with voluntary exercise for 3 wk, increased muscle mass by 25% and stimulated cell proliferation. The resulting fiber hypertrophy was accompanied by a lower ratio of protein:DNA, consistent with myogenic-cell hyperplasia. Treatment enhanced the ratio of NOS-1:β-dystroglycan in correlation with fiber diameter, improved sarcolemmal integrity, and increased vascular density after an increase in vascular endothelial growth factor protein at 3 wk. Results demonstrate that age-related muscle refractoriness to exercise can be overcome with NO-donor treatment. Since activation of muscle stem cells and vascular perfusion are limiting factors in the maintenance, regeneration, and growth of aged muscle, results suggest the feasibility of using NO-donor drugs to combat atrophy and muscle ischemia. Improved function and quality of life from the NO-amplified effects of exercise may be useful in aging and other conditions such as disuse, insulin resistance, or microgravity.

Funding information:
  • NINDS NIH HHS - R01 NS070300(United States)

Classical swine fever virus N(pro) limits type I interferon induction in plasmacytoid dendritic cells by interacting with interferon regulatory factor 7.

  • Fiebach AR
  • J. Virol.
  • 2011 Aug 21

Literature context:


Abstract:

Viruses are detected by different classes of pattern recognition receptors that lead to the activation of interferon regulatory factors (IRF) and consequently to the induction of alpha/beta interferon (IFN-α/β). In turn, efficient viral strategies to escape the type I IFN-induced antiviral mechanisms have evolved. Previous studies established that pestivirus N(pro) antagonizes the early innate immune response by targeting the transcription factor IRF3 for proteasomal degradation. Here, we report that N(pro) of classical swine fever virus (CSFV) interacts also with IRF7, another mediator of type I IFN induction. We demonstrate that the Zn-binding domain of N(pro) is essential for the interaction of N(pro) with IRF7. For IRF3 and IRF7, the DNA-binding domain, the central region, and most of the regulatory domain are required for the interaction with N(pro). Importantly, the induction of IRF7-dependent type I IFN responses in plasmacytoid dendritic cells (pDC) is reduced after wild-type CSFV infection compared with infection with virus mutants unable to interact with IRF7. This is associated with lower levels of IRF7 in pDC. Consequently, wild-type but not N(pro) mutant CSFV-infected pDC show reduced responses to other stimuli. Taken together, the results of this study show that CSFV N(pro) is capable of manipulating the function of IRF7 in pDC and provides the virus with an additional strategy to circumvent the innate defense.

Funding information:
  • NHGRI NIH HHS - 1R01HG004908(United States)
  • NINDS NIH HHS - NS020364(United States)

Isolation and transcriptome analysis of adult zebrafish cells enriched for skeletal muscle progenitors.

  • Alexander MS
  • Muscle Nerve
  • 2011 May 12

Literature context:


Abstract:

INTRODUCTION: Over the past 10 years, the use of zebrafish for scientific research in the area of muscle development has increased dramatically. Although several protocols exist for the isolation of adult myoblast progenitors from larger fish, no standardized protocol exists for the isolation of myogenic progenitors from adult zebrafish muscle. METHODS: Using a variant of a mammalian myoblast isolation protocol, zebrafish muscle progenitors have been isolated from the total dorsal myotome. These zebrafish myoblast progenitors can be cultured for several passages and then differentiated into multinucleated, mature myotubes. RESULTS: Transcriptome analysis of these cells during myogenic differentiation revealed a strong downregulation of pluripotency genes, while, conversely, showing an upregulation of myogenic signaling and structural genes. CONCLUSIONS: Together these studies provide a simple, yet detailed method for the isolation and culture of myogenic progenitors from adult zebrafish, while further promoting their therapeutic potential for the study of muscle disease and drug screening.

Funding information:
  • NHGRI NIH HHS - U54 HG006370(United States)

Regulation of Pax7 protein levels by caspase-3 and proteasome activity in differentiating myoblasts.

  • Olguín HC
  • Biol. Res.
  • 2011 Mar 26

Literature context:


Abstract:

The transcription factor Pax7 negatively regulates the activity of the muscle regulatory transcription factor MyoD, preventing muscle precursor cells from undergoing terminal differentiation. In this context, the ratio between Pax7 and MyoD protein levels is thought to be critical in allowing myogenesis to proceed or to maintain the undifferentiated muscle precursor state. We have previously shown that Pax7 is subject to rapid down regulation in differentiating myoblasts, via a proteasome-dependent pathway. Here we present evidence indicating that Pax7 is also subject to caspase-3-dependent regulation. Furthermore, simultaneous inhibition of caspase-3 and proteasome activity induced further accumulation of Pax7 protein in differentiating myoblasts. These results suggest that at early stages of muscle differentiation, Pax7 levels are regulated by at least two independent mechanisms involving caspase-3 and proteasome activity.

Funding information:
  • NIA NIH HHS - R01 AG010668(United States)
  • NIGMS NIH HHS - P50 GM071508-08(United States)

Characterization of harpy/Rca1/emi1 mutants: patterning in the absence of cell division.

  • Riley BB
  • Dev. Dyn.
  • 2010 Mar 26

Literature context:


Abstract:

We have characterized mutations in the early arrest gene, harpy (hrp), and show that they introduce premature stops in the coding region of early mitotic inhibitor1 (Rca1/emi1). In harpy mutants, cells stop dividing during early gastrulation. Lineage analysis confirms that there is little change in cell number after approximately cycle-14. Gross patterning occurs relatively normally, and many organ primordia are produced on time but with smaller numbers of cells. Despite the lack of cell division, some organ systems continue to increase in cell number, suggesting recruitment from surrounding areas. Analysis of bromodeoxyuridine incorporation shows that endoreduplication continues in many cells well past the first day of development, but cells cease endoreduplication once they begin to differentiate and express cell-type markers. Despite relatively normal gross patterning, harpy mutants show several defects in morphogenesis, cell migration and differentiation resulting directly or indirectly from the arrest of cell division.

Genome-wide discovery of Pax7 target genes during development.

  • White RB
  • Physiol. Genomics
  • 2008 Mar 14

Literature context:


Abstract:

Pax7 plays critical roles in development of brain, spinal cord, neural crest, and skeletal muscle. As a sequence-specific DNA-binding transcription factor, any direct functional role played by Pax7 during development is mediated through target gene selection. Thus, we have sought to identify genes targeted by Pax7 during embryonic development using an unbiased chromatin immunoprecipitation (ChIP) cloning assay to isolate cis-regulatory regions bound by Pax7 in vivo. Sequencing and genomic localization of a library of chromatin-DNA fragments bound by Pax7 has identified 34 candidate Pax7 target genes, with occupancy of a selection confirmed with independent chromatin enrichment tests (ChIP-PCR). To assess the capacity of Pax7 to regulate transcription from these loci, we have cloned alternate transcripts of Pax7 (differing significantly in their DNA binding domain) into expression vectors and transfected cultured cells with these constructs, then analyzed target gene expression levels using RT-PCR. We show that Pax7 directly occupies sites within genes encoding transcription factors Gbx1 and Eya4, the neurogenic cytokine receptor ciliary neurotrophic factor receptor, the neuronal potassium channel Kcnk2, and the signal transduction kinase Camk1d in vivo and regulates the transcriptional state of these genes in cultured cells. This analysis gives us greater insight into the direct functional role played by Pax7 during embryonic development.

Funding information:
  • NIDDK NIH HHS - DK 072473(United States)
  • NINDS NIH HHS - R01 NS057811(United States)

Dynamic somite cell rearrangements lead to distinct waves of myotome growth.

  • Stellabotte F
  • Development
  • 2007 Apr 12

Literature context:


Abstract:

The myogenic precursors responsible for muscle growth in amniotes develop from the dermomyotome, an epithelium at the external surface of the somite. In teleosts, the myogenic precursors responsible for growth have not been identified. We have used single cell lineage labeling in zebrafish to show that anterior border cells of epithelial somites are myogenic precursors responsible for zebrafish myotome growth. These cells move to the external surface of the embryonic myotome and express the transcription factor Pax7. Some remain on the external surface and some incorporate into the fast myotome, apparently by moving between differentiated slow fibres. The posterior cells of the somite, by contrast, elongate into medial muscle fibres. The surprising movement of the anterior somite cells to the external somite surface transforms a segmentally repeated arrangement of myogenic precursors into a medio-lateral arrangement similar to that seen in amniotes.

Funding information:
  • Doris Duke Charitable Foundation - T32 AI007387(United States)

The zebrafish zic2a-zic5 gene pair acts downstream of canonical Wnt signaling to control cell proliferation in the developing tectum.

  • Nyholm MK
  • Development
  • 2007 Feb 29

Literature context:


Abstract:

Wnt growth factors acting through the canonical intracellular signaling cascade play fundamental roles during vertebrate brain development. In particular, canonical Wnt signaling is crucial for normal development of the dorsal midbrain, the future optic tectum. Wnts act both as patterning signals and as regulators of cell growth. In the developing tectum, Wnt signaling is mitogenic; however, the mechanism of Wnt function is not known. As a step towards better understanding this mechanism, we have identified two new Wnt targets, the closely linked zic2a and zic5 genes. Using a combination of in vivo assays, we show that zic2a and zic5 transcription is activated by Tcf/Lef transcription factors in the dorsal midbrain. Zic2a and Zic5, in turn, have essential, cooperative roles in promoting cell proliferation in the tectum, but lack obvious patterning functions. Collectively these findings suggest that Wnts control midbrain proliferation, at least in part, through regulation of two novel target genes, the zic2a-zic5 gene pair.

Funding information:
  • NINDS NIH HHS - R01 NS073875(United States)

Premature myogenic differentiation and depletion of progenitor cells cause severe muscle hypotrophy in Delta1 mutants.

  • Schuster-Gossler K
  • Proc. Natl. Acad. Sci. U.S.A.
  • 2007 Jan 9

Literature context:


Abstract:

In vertebrates, skeletal myogenesis is initiated by the generation of myoblasts followed by their differentiation to myocytes and the formation of myofibers. The determination of myoblasts and their differentiation are controlled by muscle regulatory factors that are activated at specific stages during myogenesis. During late embryonic and fetal stages a distinct population of resident proliferating progenitor cells is the major source of myogenic cells. How the differentiation of myoblasts and progenitor cells is regulated is not clear. We show that in mouse embryos the Notch ligand Delta1 (Dll1) controls both differentiation of early myoblasts and maintenance of myogenic progenitor cells. Early dermomyotome-derived myoblasts are determined normally in Dll1 mutant embryos, but their differentiation is accelerated, leading to a transient excess of myotomal muscle fibers. Similarly, migratory hypaxial myogenic cells colonize the limb buds and activate muscle regulatory factor expression normally, but muscle differentiation progresses more rapidly. Resident progenitor cells defined by Pax3/Pax7 expression are formed initially, but they are progressively lost and virtually absent at embryonic day 14.5. Muscle growth declines beginning around embryonic day 12, leading to subsequent severe muscle hypotrophy in hypomorphic Dll1 fetuses. We suggest that premature and excessive differentiation leads to depletion of progenitor cells and cessation of muscle growth, and we conclude that Dll1 provides essential signals that are required to prevent uncontrolled differentiation early and ensure sustained muscle differentiation during development.

Funding information:
  • NHGRI NIH HHS - R01 HG004069(United States)
  • NIMH NIH HHS - MH067622-01(United States)

A comparative analysis of shotgun-cloning and tagged-random amplification-cloning of chromatin immunoprecipitation-isolated genome fragments.

  • White RB
  • Biochem. Biophys. Res. Commun.
  • 2006 Jul 28

Literature context:


Abstract:

The cloning of transcription factor antibody-immunoprecipitated genomic fragments from chromatin immunoprecipitation (ChIP) experiments is a technically challenging procedure, especially when the input genomic DNA is isolated from whole tissues (in vivo) rather than cultured cells. Here we adapt a technique known as Tagged-Random PCR (T-PCR) to amplify ChIP-immunoprecipitated DNA from mouse embryonic tissue prior to cloning. Importantly, we then compare this technique with tandem shotgun-cloning experiments in terms of its capacity to identify target genes. We find that T-PCR dramatically increases the efficiency of cloning ChIP fragments without distortion of the relative location of cloned fragments to putative target genes. Thus, T-PCR is a simple procedure which greatly enhances the efficiency of cloning tissue-derived ChIP fragments.

Funding information:
  • NIGMS NIH HHS - U24 GM104203(United States)

Zebrafish msxB, msxC and msxE function together to refine the neural-nonneural border and regulate cranial placodes and neural crest development.

  • Phillips BT
  • Dev. Biol.
  • 2006 Jun 15

Literature context:


Abstract:

The zebrafish muscle segment homeobox genes msxB, msxC and msxE are expressed in partially overlapping domains in the neural crest and preplacodal ectoderm. We examined the roles of these msx genes in early development. Disrupting individual msx genes causes modest variable defects, whereas disrupting all three produces a reproducible severe phenotype, suggesting functional redundancy. Neural crest differentiation is blocked at an early stage. Preplacodal development begins normally, but placodes arising from the msx expression domain later show elevated apoptosis and are reduced in size. Cell proliferation is normal in these tissues. Unexpectedly, Msx-deficient embryos become ventralized by late gastrulation whereas misexpression of msxB dorsalizes the embryo. These effects appear to involve Distal-less (Dlx) protein activity, as loss of dlx3b and dlx4b suppresses ventralization in Msx-depleted embryos. At the same time, Msx-depletion restores normal preplacodal gene expression to dlx3b-dlx4b mutants. These data suggest that mutual antagonism between Msx and Dlx proteins achieves a balance of function required for normal preplacodal differentiation and placement of the neural-nonneural border.

Funding information:
  • Cancer Research UK - CA095525(United Kingdom)

The Polycomb Ezh2 methyltransferase regulates muscle gene expression and skeletal muscle differentiation.

  • Caretti G
  • Genes Dev.
  • 2004 Nov 1

Literature context:


Abstract:

The Ezh2 protein endows the Polycomb PRC2 and PRC3 complexes with histone lysine methyltransferase (HKMT) activity that is associated with transcriptional repression. We report that Ezh2 expression was developmentally regulated in the myotome compartment of mouse somites and that its down-regulation coincided with activation of muscle gene expression and differentiation of satellite-cell-derived myoblasts. Increased Ezh2 expression inhibited muscle differentiation, and this property was conferred by its SET domain, required for the HKMT activity. In undifferentiated myoblasts, endogenous Ezh2 was associated with the transcriptional regulator YY1. Both Ezh2 and YY1 were detected, with the deacetylase HDAC1, at genomic regions of silent muscle-specific genes. Their presence correlated with methylation of K27 of histone H3. YY1 was required for Ezh2 binding because RNA interference of YY1 abrogated chromatin recruitment of Ezh2 and prevented H3-K27 methylation. Upon gene activation, Ezh2, HDAC1, and YY1 dissociated from muscle loci, H3-K27 became hypomethylated and MyoD and SRF were recruited to the chromatin. These findings suggest the existence of a two-step activation mechanism whereby removal of H3-K27 methylation, conferred by an active Ezh2-containing protein complex, followed by recruitment of positive transcriptional regulators at discrete genomic loci are required to promote muscle gene expression and cell differentiation.

Funding information:
  • Biotechnology and Biological Sciences Research Council - BB/H018123/2(United Kingdom)

Distributions of PAX6 and PAX7 proteins suggest their involvement in both early and late phases of chick brain development.

  • Kawakami A
  • Mech. Dev.
  • 1997 Nov 12

Literature context:


Abstract:

We describe here the isolation of chicken PAX6 and PAX7 as members of the PAX gene family expressed in late stages of chick nervous system development. By generating monoclonal antibodies against these PAX molecules, we analyzed their protein distributions in the CNS, neural crest cells and muscle precursor cells. Our detailed description of protein distributions revealed several new features of PAX expression patterns. In particular, PAX6 and PAX7 are expressed in restricted regions in the CNS during early stages. In contrast, at later stages the expression becomes localized to subsets of postmitotic cells in further restricted regions. Such a transition from region-specific expression to cell-specific expression suggests two different roles of these PAX molecules in development: the regionalization and subdivision of the nervous system during early stages, and the differentiation of specific cell populations during late stages.

Funding information:
  • NIDDK NIH HHS - U01 DK067861(United States)

Two critical periods of Sonic Hedgehog signaling required for the specification of motor neuron identity.

  • Ericson J
  • Cell
  • 1996 Nov 15

Literature context:


Abstract:

Antibodies that block Sonic Hedgehog (SHH) signaling have been used to show that SHH activity is required for the induction of floor plate differentiation by the notochord and independently for the induction of motor neurons by both the notochord and midline neural cells. Motor neuron generation depends on two critical periods of SHH signaling: an early period during which naive neural plate cells are converted into ventralized progenitors and a late period that extends well into S phase of the final progenitor cell division, during which SHH drives the differentiation of ventralized progenitors into motor neurons. The ambient SHH concentration during the late period determines whether ventralized progenitors differentiate into motor neurons or interneurons, thus defining the pattern of neuronal cell types generated in the neural tube.

Funding information:
  • NHGRI NIH HHS - P41 HG00739(United States)