Sam J. Mathew

Satellite cells, connective tissue fibroblasts and their interactions are crucial for muscle regeneration

Muscle regeneration requires the coordinated interaction of multiple cell types. Satellite cells have been implicated as the primary stem cell responsible for regenerating muscle, yet the necessity of these cells for regeneration has not been tested. Connective tissue fibroblasts also are likely to play a role in regeneration, as connective tissue fibrosis is a hallmark of regenerating muscle. However, the lack of molecular markers for these fibroblasts has precluded an investigation of their role. Using Tcf4, a newly identified fibroblast marker, and Pax7, a satellite cell marker, we found that after injury satellite cells and fibroblasts rapidly proliferate in close proximity to one another. To test the role of satellite cells and fibroblasts in muscle regeneration in vivo, we created Pax7CreERT2 and Tcf4CreERT2 mice and crossed these to R26RDTA mice to genetically ablate satellite cells and fibroblasts. Ablation of satellite cells resulted in a complete loss of regenerated muscle, as well as misregulation of fibroblasts and a dramatic increase in connective tissue. Ablation of fibroblasts altered the dynamics of satellite cells, leading to premature satellite cell differentiation, depletion of the early pool of satellite cells, and smaller regenerated myofibers. Thus, we provide direct, genetic evidence that satellite cells are required for muscle regeneration and also identify resident fibroblasts as a novel and vital component of the niche regulating satellite cell expansion during regeneration. Furthermore, we demonstrate that reciprocal interactions between fibroblasts and satellite cells contribute significantly to efficient, effective muscle regeneration.

Connective tissue fibroblasts and Tcf4 regulate myogenesis

Muscle and its connective tissue are intimately linked in the embryo and in the adult, suggesting that interactions between these tissues are crucial for their development. However, the study of muscle connective tissue has been hindered by the lack of molecular markers and genetic reagents to label connective tissue fibroblasts. Here, we show that the transcription factor Tcf4 (transcription factor 7-like 2; Tcf7l2) is strongly expressed in connective tissue fibroblasts and that Tcf4GFPCre mice allow genetic manipulation of these fibroblasts. Using this new reagent, we find that connective tissue fibroblasts critically regulate two aspects of myogenesis: muscle fiber type development and maturation. Fibroblasts promote (via Tcf4-dependent signals) slow myogenesis by stimulating the expression of slow myosin heavy chain. Also, fibroblasts promote the switch from fetal to adult muscle by repressing (via Tcf4-dependent signals) the expression of developmental embryonic myosin and promoting (via a Tcf4-independent mechanism) the formation of large multinucleate myofibers. In addition, our analysis of Tcf4 function unexpectedly reveals a novel mechanism of intrinsic regulation of muscle fiber type development. Unlike other intrinsic regulators of fiber type, low levels of Tcf4 in myogenic cells promote both slow and fast myogenesis, thereby promoting overall maturation of muscle fiber type. Thus, we have identified novel extrinsic and intrinsic mechanisms regulating myogenesis. Most significantly, our data demonstrate for the first time that connective tissue is important not only for adult muscle structure and function, but is a vital component of the niche within which muscle progenitors reside and is a critical regulator of myogenesis.

Muscle stem cells contribute to myofibres in sedentary adult mice

Skeletal muscle is essential for mobility, stability, and whole body metabolism, and muscle loss, for instance during sarcopenia, has profound consequences. Satellite cells (muscle stem cells) have been hypothesized, but not yet demonstrated, to contribute to muscle homeostasis and a decline in their contribution to myofiber homeostasis to play a part in sarcopenia. To test their role in muscle maintenance, we genetically labeled and ablated satellite cells in adult sedentary mice. We demonstrate via genetic lineage experiments that even in the absence of injury, satellite cells contribute to myofibers in all adult muscles, although the extent and timing differs. However, genetic ablation experiments showed that satellite cells are not globally required to maintain myofiber cross-sectional area of uninjured adult muscle.

Looking beyond death: a morphogenetic role for the TNF signalling pathway

Tumour necrosis factor α (TNFα) is a pro-inflammatory mediator with the capacity to induce apoptosis. An integral part of its apoptotic and inflammatory programmes is the control of cell shape through modulation of the cytoskeleton, but it is now becoming apparent that this morphogenetic function of TNF signalling is also employed outside inflammatory responses and is shared by the signalling pathways of other members of the TNF-receptor superfamily. Some proteins that are homologous to the components of the TNF signalling pathway, such as the adaptor TNF-receptor-associated factor 4 and the ectodysplasin A receptor (and its ligand and adaptors), have dedicated morphogenetic roles. The mechanism by which TNF signalling affects cell shape is not yet fully understood, but Rho-family GTPases have a central role. The fact that the components of the TNF signalling pathway are evolutionarily old suggests that an ancestral cassette from unicellular organisms has diversified its functions into partly overlapping morphogenetic, inflammatory and apoptotic roles in multicellular higher organisms.

Role for Traf4 in Polarizing Adherens Junctions as a Prerequisite for Efficient Cell Shape Changes

ABSTRACT Apical constriction of epithelial cells is a widely used morphogenetic mechanism. In the Drosophila embryo, the apical constrictions that internalize the mesoderm are controlled by the transcription factor Twist and require intact adherens junctions and a contractile acto-myosin network. We find that adherens junctions in constricting mesodermal cells undergo extensive remodeling. A Twist target gene encoding a member of the tumor necrosis factor (TNF) receptor-associated factor (TRAF) family, Traf4, is involved in this process. While TRAFs are best known for their functions in inflammatory responses, Traf4 appears to have a different role, and its mechanism of action is poorly understood. We show that Traf4 is required for efficient apical constriction during ventral furrow formation and for proper localization of Armadillo to the apical position in constricting cells. Traf4 and Armadillo interact with each other physically and functionally. Traf4 acts in a TNF receptor- and Jun N-terminal protein kinase (JNK)-independent manner to fine-tune the assembly of adherens junctions in the invaginating mesodermal cells.

A Small Genomic Region Containing Several Loci Required for Gastrulation in Drosophila

Genetic screens in Drosophila designed to search for loci involved in gastrulation have identified four regions of the genome that are required zygotically for the formation of the ventral furrow. For three of these, the genes responsible for the mutant phenotypes have been found. We now describe a genetic characterization of the fourth region, which encompasses the cytogenetic interval 24C3-25B, and the mapping of genes involved in gastrulation in this region. We have determined the precise breakpoints of several existing deficiencies and have generated new deficiencies. Our results show that the region contains at least three different loci associated with gastrulation effects. One maternal effect gene involved in ventral furrow formation maps at 24F but could not be identified. For a second maternal effect gene which is required for germ band extension, we identify a candidate gene, CG31660, which encodes a G protein coupled receptor. Finally, one gene acts zygotically in ventral furrow formation and we identify it as Traf4.

The Groucho/Transducin-like enhancer of split protein family in animal development

Corepressors are proteins that cannot bind DNA directly but repress transcription by interacting with partner proteins. The Groucho/Transducin‐Like Enhancer of Split (TLE) are a conserved family of corepressor proteins present in animals ranging from invertebrates such as Drosophila to vertebrates such as mice and humans. Groucho/TLE proteins perform important functions throughout the life span of animals, interacting with several pathways and regulating fundamental processes such as metabolism. However, these proteins have especially crucial functions in animal development, where they are required in multiple tissues in a temporally regulated manner. In this review, we summarize the functions of the Groucho/TLE proteins during animal development, emphasizing on specific tissues where they play essential roles.

InACTIVatINg cancer cachexia

Summary of and comment on a recent Cell paper entitled ‘Reversal of cancer cachexia and muscle wasting by ActRIIB antagonism leads to prolonged survival’ (Zhou et al., 2010).

SPRY2 is a novel MET interactor that regulates metastatic potential and differentiation in rhabdomyosarcoma

Rhabdomyosarcoma (RMS) is a predominantly pediatric soft-tissue cancer where the tumor cells exhibit characteristics of the developing skeletal muscle, and the two most common sub-types are embryonal and alveolar RMS. Elevated activation of the receptor tyrosine kinase (RTK) MET is frequent in RMS and is thought to cause increased tumor metastasis and lack of differentiation. However, the reasons underlying dysregulated MET expression and activation in RMS are not well understood. Therefore, we explored the role of Sprouty 2 (SPRY2), a modulator of RTK signaling, in regulating MET. We identify SPRY2 as a novel MET interactor that colocalizes with and binds MET in both embryonal and alveolar RMS. We find that depletion of SPRY2 leads to MET degradation, resulting in reduced migratory and clonogenic potential, and induction of differentiation in both embryonal and alveolar RMS, outcomes that are identical to depletion of MET. Activation of the ERK/MAPK pathway, known to be crucial for regulating cell migration and whose inhibition is required for myogenic differentiation, was downregulated upon depletion of MET or SPRY2. This provides a direct connection to the decreased migration and induction of differentiation upon depletion of MET or SPRY2. Thus, these data indicate that SPRY2 interacts with MET and stabilizes it in order to maintain signaling downstream of MET, which keeps the ERK/MAPK pathway active, resulting in metastatic potential and inhibition of differentiation in RMS. Our results identify a novel mechanism by which MET signaling is stabilized in RMS, and is a potential target for therapeutic intervention in RMS.

Myosin Heavy Chain-embryonic is a crucial regulator of skeletal muscle development and differentiation.

Myosin heavy chains (MyHCs) are contractile proteins that are part of the thick filaments of the functional unit of the skeletal muscle, the sarcomere. In addition to MyHCs that are part of the adult muscle contractile network, two MyHCs - MyHC-embryonic and -perinatal are expressed during muscle development and are only transiently expressed in the adult during regeneration. The functions performed by these MyHCs has been a long-standing question and using a targeted mouse allele, we have characterized the role of MyHC-embryonic. Analysis of loss-of-function mice reveals that lack of MyHC-embryonic leads to mis-regulation of other MyHCs, alterations in fiber size, fiber number and fiber type at neonatal stages. We also find that loss of MyHC-embryonic leads to mis-regulation of genes involved in muscle differentiation. A broad theme from these studies is that loss of MyHC-embryonic has distinct effects on different muscles, possibly reflecting the unique fiber type composition of different muscles. Most significantly, our results indicate that MyHC-embryonic is required during embryonic and fetal myogenesis to regulate myogenic progenitor and myoblast differentiation in a non-cell autonomous manner via Mitogen Activated Protein Kinase (MAPKinase) and Fibroblast Growth Factor (FGF) signaling. Thus, our results signify that MyHC-embryonic is a key regulator of myogenic differentiation during embryonic, fetal and neonatal myogenesis.