Epiblast cells that express MyoD recruit pluripotent cells to the skeletal muscle lineage

Embryonic stem cells are derived from the epiblast. A subpopulation of epiblast cells expresses MyoD mRNA and the G8 antigen in vivo. G8 positive (G8pos) and G8 negative (G8neg) populations were isolated by magnetic cell sorting. Nearly all G8pos cells switched from E- to N-cadherin and differentiated into skeletal muscle in culture. G8neg cells were impaired in their ability to switch cadherins and few formed skeletal muscle. Medium conditioned by G8pos cells stimulated skeletal myogenesis and N-cadherin synthesis in G8neg cultures. The effect of conditioned medium from G8pos cultures was inhibited by bone morphogenetic protein (BMP) 4. Treatment of G8neg cells with a soluble form of the BMP receptor-IA or Noggin promoted N-cadherin synthesis and skeletal myogenesis. These results demonstrate that MyoD-positive epiblast cells recruit pluripotent cells to the skeletal muscle lineage. The mechanism of recruitment involves blocking the BMP signaling pathway.     

Myo/Nog cell regulation of bone morphogenetic protein signaling in the blastocyst is essential for normal morphogenesis and striated muscle lineage specification

Cells that express MyoD mRNA, the G8 antigen and the bone morphogenetic protein (BMP) inhibitor noggin (Nog) are present in the epiblast before gastrulation. Ablation of “Myo/Nog” cells in the blastocyst results in an expansion of canonical BMP signaling and prevents the expression of noggin and follistatin before and after the onset of gastrulation. Once eliminated in the epiblast, they are neither replaced nor compensated for as development progresses. Older embryos lacking Myo/Nog cells exhibit severe axial malformations. Although Wnts and Sonic hedgehog are expressed in ablated embryos, skeletal muscle progenitors expressing Pax3 are missing in the somites. Pax3+ cells do emerge adjacent to Wnt3a+ cells in vitro; however, few undergo skeletal myogenesis. Ablation of Myo/Nog cells also results in ectopically placed cardiac progenitors and cardiomyocytes in the somites. Reintroduction of Myo/Nog cells into the epiblast of ablated embryos restores normal patterns of BMP signaling, morphogenesis and skeletal myogenesis, and inhibits the expression of cardiac markers in the somites. This study demonstrates that Myo/Nog cells are essential regulators of BMP signaling in the early epiblast and are indispensable for normal morphogenesis and striated muscle lineage specification.     

MyoD-positive myoblasts are present in mature fetal organs lacking skeletal muscle.

The epiblast of the chick embryo gives rise to the ectoderm, mesoderm, and endoderm during gastrulation. Previous studies revealed that MyoD-positive cells were present throughout the epiblast, suggesting that skeletal muscle precursors would become incorporated into all three germ layers. The focus of the present study was to examine a variety of organs from the chicken fetus for the presence of myogenic cells. RT-PCR and in situ hybridizations demonstrated that MyoD-positive cells were present in the brain, lung, intestine, kidney, spleen, heart, and liver. When these organs were dissociated and placed in culture, a subpopulation of cells differentiated into skeletal muscle. The G8 antibody was used to label those cells that expressed MyoD in vivo and to follow their fate in vitro. Most, if not all, of the muscle that formed in culture arose from cells that expressed MyoD and G8 in vivo. Practically all of the G8-positive cells from the intestine differentiated after purification by FACS. This population of ectopically located cells appears to be distinct from multipotential stem cells and myofibroblasts. They closely resemble quiescent, stably programmed skeletal myoblasts with the capacity to differentiate when placed in a permissive environment.     

MyoD cannot compensate for the absence of myogenin during skeletal muscle differentiation in murine embryonic stem cells

myogenin (-/-) mice display severe skeletal muscle defects despite expressing normal levels of MyoD. The failure of MyoD to compensate for myogenin could be explained by distinctions in protein function or by differences in patterns of gene expression. To distinguish between these two possibilities, we compared the abilities of constitutively expressed myogenin and MyoD to support muscle differentiation in embryoid bodies made from myogenin (-/-) ES cells. Differentiated embryoid bodies from wild-type embryonic stem (ES) cells made extensive skeletal muscle, but embryoid bodies from myogenin (-/-) ES cells had greatly attenuated muscle-forming capacity. The inability of myogenin (-/-) ES cells to generate muscle was independent of endogenous MyoD expression. Skeletal muscle was restored in myogenin (-/-) ES cells by constitutive expression of myogenin. In contrast, constitutive expression of MyoD resulted in only marginal enhancement of skeletal muscle, although myocyte numbers greatly increased. The results indicated that constitutive expression of MyoD led to enhanced myogenic commitment of myogenin (-/-) cells but also indicated that committed cells were impaired in their ability to form muscle sheets without myogenin. Thus, despite their relatedness, myogenin's role in muscle formation is distinct from that of MyoD, and the distinction cannot be explained merely by differences in their expression properties.     

MyoD-positive epiblast cells regulate skeletal muscle differentiation in the embryo

MyoD mRNA is expressed in a subpopulation of cells within the embryonic epiblast. Most of these cells are incorporated into somites and synthesize Noggin. Ablation of MyoD-positive cells in the epiblast subsequently results in the herniation of organs through the ventral body wall, a decrease in the expression of Noggin, MyoD, Myf5, and myosin in the somites and limbs, and an increase in Pax-3-positive myogenic precursors. The addition of Noggin lateral to the somites compensates for the loss of MyoD-positive epiblast cells. Skeletal muscle stem cells that arise in the epiblast are utilized in the somites to promote muscle differentiation by serving as a source of Noggin.     

Msx1 antagonizes the myogenic activity of Pax3 in migrating limb muscle precursors.

The migration of myogenic precursors to the vertebrate limb exemplifies a common problem in development - namely, how migratory cells that are committed to a specific lineage postpone terminal differentiation until they reach their destination. Here we show that in chicken embryos, expression of the Msx1 homeobox gene overlaps with Pax3 in migrating limb muscle precursors, which are committed myoblasts that do not express myogenic differentiation genes such as MyoD. We find that ectopic expression of Msx1 in the forelimb and somites of chicken embryos inhibits MyoD expression as well as muscle differentiation. Conversely, ectopic expression of Pax3 activates MyoD expression, while co-ectopic expression of Msx1 and Pax3 neutralizes their effects on MyoD. Moreover, we find that Msx1 represses and Pax3 activates MyoD regulatory elements in cell culture, while in combination, Msx1 and Pax3 oppose each other's trancriptional actions on MyoD. Finally, we show that the Msx1 protein interacts with Pax3 in vitro, thereby inhibiting DNA binding by Pax3. Thus, we propose that Msx1 antagonizes the myogenic activity of Pax3 in migrating limb muscle precursors via direct protein-protein interaction. Our results implicate functional antagonism through competitive protein-protein interactions as a mechanism for regulating the differentiation state of migrating cells.     

Noggin producing, MyoD-positive cells are crucial for eye development

A subpopulation of cells expresses MyoD mRNA and the cell surface G8 antigen in the epiblast prior to the onset of gastrulation. When an antibody to the G8 antigen was applied to the epiblast, labeled cells were later found in the ocular primordia and muscle and non-muscle forming tissues of the eyes. In the lens, retina and periocular mesenchyme, G8-positive cells synthesized MyoD mRNA and the bone morphogenetic protein inhibitor Noggin. MyoD expressing cells were ablated in the epiblast by labeling them with the G8 MAb and lysing them with complement. Their ablation in the epiblast resulted in eye defects, including anopthalmia, micropthalmia, altered pigmentation and malformations of the lens and/or retina. The right eye was more severely affected than the left eye. The asymmetry of the eye defects in ablated embryos correlated with differences in the number of residual Noggin producing, MyoD-positive cells in ocular tissues. Exogenously supplied Noggin compensated for the ablated epiblast cells. This study demonstrates that MyoD expressing cells serve as a Noggin delivery system to regulate the morphogenesis of the lens and optic cup.     

Conditional conversion of ES cells to skeletal muscle by an exogenous MyoD1 gene.

A variety of differentiated cell types can be converted to skeletal muscle cells following transfection with the myogenic regulatory gene MyoD1. To determine whether multipotent embryonic stem (ES) cells respond similarly, cultures of two ES cell lines were electroporated with a MyoD1 cDNA driven by the beta-actin promoter. All transfected clones, carrying a single copy of the exogenous gene, expressed high levels of MyoD1 mRNA. Surprisingly, although maintained in mitogen-rich medium, this ectopic expression was associated with a transactivation of the endogenous myogenin and myosin light chain 2 gene but not the endogenous MyoD1, MRF4, Myf5, the skeletal muscle actin, or the myosin heavy chain genes. Preferential myogenesis and the appearance of contracting skeletal muscle fibers were observed only when the transfected cells were allowed to differentiate in vitro, via embryoid bodies, in low-mitogen-containing medium. Myogenesis was associated with the activation of MRF4 and Myf5 genes and resulted in a significant increase in the level of myogenin mRNA. Not all cells were converted to skeletal muscle cells, indicating that only a subset of stem cells can respond to MyoD1. Moreover, the continued expression of the introduced gene was not required for myogenesis. These results show that ES cells can respond to MyoD1, but environmental factors control the expression of its myogenic differentiation function, that MyoD1 functions in ES cells even under environmental conditions that favor differentiation is not dominant (incomplete penetrance), that MyoD1 expression is required for the establishment of the myogenic program but not for its maintenance, and that the exogenous MyoD1 gene can trans-activate the endogenous myogenin and MLC2 genes in undifferentiated ES cells.     

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

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

Myf-5 and myoD genes are activated in distinct mesenchymal stem cells and determine different skeletal muscle cell lineages.

Targeted inactivation of the myogenic determination genes myf-5 and myoD in mice resulted in moderate (Myf-5) or no muscle phenotypes (MyoD) and double knock-out mutants lacking both genes failed to develop any skeletal muscle. In order to determine the mechanism of this apparent genetic redundancy we investigated the basis of functional overlap between the two genes. Here we demonstrate that Myf-5 and MyoD are not expressed within the same muscle precursor cell, but rather determine different muscle cell lineages arising from independently committed stem cell populations. Selective ablation of Myf-5-expressing muscle precursors from differentiating ES cells does not prevent Myo-D-dependent muscle differentiation. The early muscle progenitor cells which normally express Myf-5 do not develop into later appearing MyoD cells, even when the myf-5 gene has been inactivated. Thus skeletal musculature in vertebrates develops from two separate cell lineages and complementation may occur at the cellular level, but not between different myogenic factor genes within one cell.     

Skeletal muscle phenotypes initiated by ectopic MyoD in transgenic mouse heart.

Forced expression of the myogenic regulatory gene MyoD in many types of cultured cells initiates their conversion into skeletal muscle. It is not known, however, if MyoD expression serves to activate all or part of the skeletal muscle program in vivo during animal development, nor is it known how limiting the influences of cellular environment may be on the regulatory effects of MyoD. To begin to address these issues, we have produced transgenic mice which express MyoD in developing heart, where neither MyoD nor its three close relatives--myogenin, Myf-5, and MRF4/herculin/Myf-6--are normally expressed. The resulting gross phenotype in offspring from multiple, independent transgenic founders includes abnormal heart morphology and ultimately leads to death. At the molecular level, affected hearts exhibit activation of skeletal muscle-specific regulatory as well as structural genes. We conclude that MyoD is able to initiate the program that leads to skeletal muscle differentiation during mouse development, even in the presence of the ongoing cardiac differentiation program. Thus, targeted misexpression of this tissue-specific regulator during mammalian embryogenesis can activate, either directly or indirectly, a diverse set of genes normally restricted to a different cell lineage and a different cellular environment.     

Different autonomous myogenic cell populations revealed by ablation of Myf5-expressing cells during mouse embryogenesis

The development of myogenic cells is mainly determined by expression of two myogenic factors, Myf5 and Myod1 (MyoD), which genetically compensate for each other during embryogenesis. Here, we demonstrate by conditional cell ablation in mice that Myf5 determines a distinct myogenic cell population, which also contains some Myod1-positive cells. Ablation of this lineage uncovers the presence of a second autonomous myogenic lineage, which superseded Myf5-dependent myogenic cells and expressed Myod1. By contrast, ablation of myogenin-expressing cells erased virtually all differentiated muscle cells, indicating that some aspects of the myogenic program are shared by most skeletal muscle cells. We conclude that Myf5 and Myod1 define different cell lineages with distinct contributions to muscle precursor cells and differentiated myotubes. Individual myogenic cell lineages seem to substitute for each other within the developing embryo.