Gabrielle Kardon

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.

Embryonic and fetal limb myogenic cells are derived from developmentally distinct progenitors and have different requirements for β-catenin

Vertebrate muscle arises sequentially from embryonic, fetal, and adult myoblasts. Although functionally distinct, it is unclear whether these myoblast classes develop from common or different progenitors. Pax3 and Pax7 are expressed by somitic myogenic progenitors and are critical myogenic determinants. To test the developmental origin of embryonic and fetal myogenic cells in the limb, we genetically labeled and ablated Pax3(+) and Pax7(+) cells. Pax3(+)Pax7(-) cells contribute to muscle and endothelium, establish and are required for embryonic myogenesis, and give rise to Pax7(+) cells. Subsequently, Pax7(+) cells give rise to and are required for fetal myogenesis. Thus, Pax3(+) and Pax7(+) cells contribute differentially to embryonic and fetal limb myogenesis. To investigate whether embryonic and fetal limb myogenic cells have different genetic requirements we conditionally inactivated or activated beta-catenin, an important regulator of myogenesis, in Pax3- or Pax7-derived cells. beta-Catenin is necessary within the somite for dermomyotome and myotome formation and delamination of limb myogenic progenitors. In the limb, beta-catenin is not required for embryonic myoblast specification or myofiber differentiation but is critical for determining fetal progenitor number and myofiber number and type. Together, these studies demonstrate that limb embryonic and fetal myogenic cells develop from distinct, but related progenitors and have different cell-autonomous requirements for beta-catenin.

Local Extrinsic Signals Determine Muscle and Endothelial Cell Fate and Patterning in the Vertebrate Limb

Both the muscle and endothelium of the vertebrate limb derive from somites. We have used replication-defective retroviral vectors to analyze the lineage relationships of these somite-derived cells in the chick. We find that myogenic precursors in the somites or proximal limb are not committed to forming slow or fast muscle fibers, particular anatomical muscles, or muscles within specific proximal/distal or dorsal/ventral limb regions. Somitic endothelial precursors are uncommitted to forming endothelium in particular proximal/distal or dorsal/ventral limb regions. Surprisingly, we also find that myogenic and endothelial cells are derived from a common somitic precursor. Thus, local extrinsic signals are critical for determining muscle and endothelial patterning as well as cell fate in the limb.

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.

A Tcf4-Positive Mesodermal Population Provides a Prepattern for Vertebrate Limb Muscle Patterning

Nai;ve myogenic cells migrate from the somites into the developing vertebrate limb, where they simultaneously differentiate into myotubes and form distinct anatomical muscles. Limb signals have been hypothesized to direct the pattern of muscles formed, but the molecular nature of these signals and the identity of the cells that produce them have remained unclear. We have identified a population of lateral plate-derived limb mesodermal cells in both chick and mouse that expresses the transcription factor Tcf4 in a muscle-specific pattern independently of the muscle cells themselves. Functional experiments in the chick demonstrate that TCF4 and the Wnt-beta-catenin pathway in these limb mesodermal cells are critical for muscle patterning. We propose that Tcf4-expressing cells establish a prepattern in the limb mesoderm that determines the sites of myogenic differentiation and thus establishes the basic pattern of limb muscles.

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.

Origin of Vertebrate Limb Muscle: The Role of Progenitor and Myoblast Populations

Muscle development, growth, and regeneration take place throughout vertebrate life. In amniotes, myogenesis takes place in four successive, temporally distinct, although overlapping phases. Understanding how embryonic, fetal, neonatal, and adult muscle are formed from muscle progenitors and committed myoblasts is an area of active research. In this review we examine recent expression, genetic loss-of-function, and genetic lineage studies that have been conducted in the mouse, with a particular focus on limb myogenesis. We synthesize these studies to present a current model of how embryonic, fetal, neonatal, and adult muscle are formed in the limb.

Muscle connective tissue controls development of the diaphragm and is a source of congenital diaphragmatic hernias

The diaphragm is an essential mammalian skeletal muscle, and defects in diaphragm development are the cause of congenital diaphragmatic hernias (CDHs), a common and often lethal birth defect. The diaphragm is derived from multiple embryonic sources, but how these give rise to the diaphragm is unknown, and, despite the identification of many CDH-associated genes, the etiology of CDH is incompletely understood. Using mouse genetics, we show that the pleuroperitoneal folds (PPFs), which are transient embryonic structures, are the source of the diaphragms muscle connective tissue and regulate muscle development, and we show that the striking migration of PPF cells controls diaphragm morphogenesis. Furthermore, Gata4 mosaic mutations in PPF-derived muscle connective tissue fibroblasts result in the development of localized amuscular regions that are biomechanically weaker and more compliant, leading to CDH. Thus, the PPFs and muscle connective tissue are critical for diaphragm development, and mutations in PPF-derived fibroblasts are a source of CDH.

Development of the diaphragm, a skeletal muscle essential for mammalian respiration

The mammalian diaphragm muscle is essential for respiration, and thus is one of the most critical skeletal muscles in the human body. Defects in diaphragm development leading to congenital diaphragmatic hernias (CDH) are common birth defects and result in severe morbidity or mortality. Given its functional importance and the frequency of congenital defects, an understanding of diaphragm development, both normally and during herniation, is important. We review current knowledge of the embryological origins of the diaphragm, diaphragm development and morphogenesis, as well as the genetic and developmental aetiology of diaphragm birth defects.

Autonomous and nonautonomous roles of Hedgehog signaling in regulating limb muscle formation

Muscle progenitor cells migrate from the lateral somites into the developing vertebrate limb, where they undergo patterning and differentiation in response to local signals. Sonic hedgehog (Shh) is a secreted molecule made in the posterior limb bud that affects patterning and development of multiple tissues, including skeletal muscles. However, the cell-autonomous and non-cell-autonomous functions of Shh during limb muscle formation have remained unclear. We found that Shh affects the pattern of limb musculature non-cell-autonomously, acting through adjacent nonmuscle mesenchyme. However, Shh plays a cell-autonomous role in maintaining cell survival in the dermomyotome and initiating early activation of the myogenic program in the ventral limb. At later stages, Shh promotes slow muscle differentiation cell-autonomously. In addition, Shh signaling is required cell-autonomously to regulate directional muscle cell migration in the distal limb. We identify neuroepithelial cell transforming gene 1 (Net1) as a downstream target and effector of Shh signaling in that context.