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The tumor necrosis factor-[alpha] (TNF[alpha])-mediated inhibition of myogenic differentiation is found to be dependent on p53 through a PW1-mediated pathway. The p53 plays a novel role in mediating muscle stem cell behavior and muscle atrophy, and has pointed towards new targets for the therapeutic treatment of muscle wasting (cachexia).

PW1 is a mediator of p53 and TNF[alpha] signaling pathways previously identified in a screen to isolate muscle stem cell regulators. We generated transgenic mice carrying a C-terminal deleted form of PW1 ([DELTA]PW1) which blocks p53-mediated cell death and TNF[alpha]-mediated NF[kappa]B activation fused to the myogenin promoter. Embryonic/fetal muscle development appears normal during transgene expression, however, postnatal transgenic pups display severe phenotypes including runtism, reduced muscle mass and fiber diameters resembling atrophy. Atrogin-1, a marker of skeletal muscle atrophy, is expressed postnatally in transgenic mice. Electron microscopic analyses of transgenic muscle reveal a marked decrease in quiescent muscle satellite cells suggesting a deregulation of postnatal stem cells. Furthermore, transgenic primary myoblasts show a resistance to the effects of TNF[alpha] upon differentiation. Taken together, our data support a role for PW1 and related stress pathways in mediating skeletal muscle stem cell behavior which in turn is critical for postnatal muscle growth and homeostasis. In addition, these data reveal that postnatal stem cell behavior is likely specified during early muscle development. Keywords: Satellite cells; Stem cells; Skeletal muscle; Development; Mouse; PW1/Peg3; p53; TNF; Atrophy

The differentiation of epithelial cells and fiber cells from the anterior and posterior compartments of the lens vesicle, respectively, give the mammalian lens its distinctive polarity. While much progress has been made in understanding the molecular basis of fiber differentiation, little is known about factors that govern the differentiation of the epithelium. Members of the Wnt growth factor family appear to be key regulators of epithelial differentiation in various organ systems. Wnts are ligands for Frizzled receptors and can activate several signaling pathways, of which the best understood is the Wnt/[beta]-catenin pathway. The presence of LDL-related protein coreceptors (LRPs) 5 or 6 has been shown to be a requirement for Wnt signaling through the [beta]-catenin pathway. To access the role of this signaling pathway in the lens, we analyzed mice with a null mutation of lrp6. These mice had small eyes and aberrant lenses, characterized by an incompletely formed anterior epithelium resulting in extrusion of the lens fibers into the overlying corneal stroma. We also showed that multiple Wnts, including 5a, 5b, 7a, 7b, 8a, 8b, and Frizzled receptors 1, 2, 3, 4, and 6, were detected in the lens. Expression of these molecules was generally present throughout the lens epithelium and extended into the transitional zone, where early fiber elongation occurs. In addition to both LRP5 and LRP6, we also showed the expression of other molecules involved in Wnt signaling and its regulation, including Dishevelleds, Dickkopfs, and secreted Frizzled-related proteins. Taken together, these results indicate a role for Wnt signaling in regulating the differentiation and behavior of lens cells. Keywords: Lens development; Wnt signaling; [beta]-Catenin; LRP6; Lens epithelium

The expression of the mRNA of CDO, an Ig/fibronectin type III repeat subfamily of transmembrane proteins, at high levels during murine embryogenesis in somites, dermomyotome, myotome and in myoblast cell lines was examined. Results show that while CDO overexpression in myoblasts enhances differentiation, the expression of selected soluble forms of CDO block the process. It was also observed that oncogenic Ras suppresses myogenic differentiation in a fashion dependent on loss of CDO expression while other aspects of the transformed phenotype are independent of CDO.

Homeobox-containing genes play an important role in patterning processes that occur during embryogenesis. Programmed cell death is a key process during pattern formation. The mechanisms by which programmed cell death is spatially regulated are not well characterized. Msx1 and Msx2 are two closely related homeobox-containing genes that are expressed at sites where cellular proliferation and programmed cell death occur, including the developing limb and the cephalic neural crest. Tissue interactions are necessary for the maintenance of Msx1 and Msx2 expression and programmed cell death. It has been demonstrated that BMP4 can regulate cell death at these same sites as well as induce Msx expression. These observations lead to the hypothesis that Msx2 is a key regulator of cell death in the BMP-mediated pathway. Embryonic stem cell lines will undergo processes typical of early embryogenesis upon aggregation and have recently been shown to provide a model system for programmed cell death. In contrast to ES cells, we see that P19 cells do not undergo pronounced cell death upon aggregation; however, constitutive ectopic Msx2 expression in P19 cells results in a marked increase in apoptosis induced upon aggregation but has no effect when cells are grown as a monolayer. If aggregates are allowed to interact with a substrate, the process of programmed cell death is completely inhibited. Addition of BMP4 to aggregated P19 cells also results in cell death; however, BMP4 does not increase levels of cell death in Msx2-expressing cells. Addition of BMP4 to P19 cells results in an induction of Msx2 transcription consistent with its proposed role in cell death in the embryo. Our data support a model by which BMP4 induces programmed cell death via an Msx2-mediated pathway and provide direct functional evidence that Msx2 expression is a regulator of this process.

The cellular and molecular processes leading to the establishment of the skeletal muscle lineage in the vertebrate are not well understood. The MyoD-related family of myogenic regulatory factors (MRFs) are expressed during somitogenesis although cells with myogenic capacity are present prior to gastrulation. We propose that regulatory genes exist that guide the skeletal muscle lineage during early development. In an effort to identify these regulatory genes, we performed a differential screening to isolate transcripts that are present in myogenic cells and in the embryo prior to MRF expression but absent in nonmyogenic fibroblasts. We report here the identification of Pw1. The Pw1 transcript is [approximately]8.5 kb long and encodes a large protein containing 12 widespread C2H2 zinc fingers and 3 motifs containing periodic prolines and acidic residues. Consistent with the possibility that Pw1 is a transcription factor, we observe nuclear localization of the protein. Pw1 is strongly expressed upon gastrulation and subsequently becomes restricted to skeletal muscle and subregions of the central nervous system. Pw1 is initially expressed in all mesodermal cells early in development; however, its maintained expression in adult differentiated muscle suggests a specific role in the skeletal muscle lineage. Pw1 expression is cell cycle specific with levels highest during late M-phase. The gene is intronless which may facilitate transcription during cell division. At present, the precise function of Pw1 is not understood; however, we note that Pw1 maps to the proximal region of chromosome 7 near the axial segmentation mutant pudgy which shows severe perturbation of axial skeletal and muscle structures.

A study shows that human MSX1 inhibits activation of MyoD expression in primary fibroblast x 10T1/2 cell hybrids and finds that chromosome 4, but not 8, inhibits myoD activation in 10T1/2 cell hybrids. Forced expression of Msx1 represses myoD enhancer activity. Msx1 proteins binds to the myoD enhancer and likely represses myoD transcription directly. However, antisense MSX1 relieves repression mediated by chromosome 4.

The apical ectodermal ridge (AER) is not necessary for the expression of the homeobox gene Msx-1 in the developing mouse limb. FGF-4 can maintain Msx-1 expression in the absence of AER, but cell-cell interactions are still required. A failure in the cell-cell interactions in the limb mesenchyme leads to a decrease in Msx-1 expression.

Use of isoform specific rat-derived antisense RNA probes in in situ hybridization facilitates the detection of cell-specific regulation of Na(+)-K(+)-activated adenosine triphosphatase (ATPase) alpha subunit isoform (alpha 1, alpha2, and alpha3) gene expression in various stages of neonatal rat tissues and post-implantation mouse embryos. Specialized functions of cells and their development are influenced by isoform specific variations in regional and cellular Na(+)-K(+)-ATPase alpha isoform gene expression.

The myogenic regulatory factors ,MRFs, play an important role in the determination of the skeletal muscle lineage in the vertebrate embryo, and this is revealed by an analysis of the amphibian, murine and avian systems. Varying conclusions can be arrived at from analyses using cultured cells that suggest a role for the MRFs in determination, and studies of MRF and expression of muscle gene in vivo revealing a role in differentiation and not in lineage determination.

People with cancer typically experience an overall decline in weight and muscle strength-a state referred to as cachexia. Cachexia is the major contributor to morbidity and premature mortality from cancer [...]

The molecular basis underlying the establishment of the myogenic lineage, subsequent differentiation, and the establishment of specific fiber types (i.e., fast versus slow) is becoming well understood. In contrast, the regulation of the general properties of a specific anatomical muscle group (e.g., leg versus jaw muscles) and the regulation of muscle-fiber properties within a particular group are less well characterized. We have investigated the potential role of the homeobox-containing gene, Engrailed-2 (En-2), in the mouse, which is specifically expressed in myoblasts in the first arch and maintained in the muscles of mastication in the adult. We have generated mice that ectopically express En-2 in all muscles during early development and primarily in fast muscles in the adult. Ectopic En-2 in nonjaw muscles leads to a decrease in fiber size, whereas overexpression in the jaw muscles leads to a shift in fiber metabolic properties as well as a decrease in fiber size. In contrast, loss of En-2 in the jaw leads to a shift in fiber metabolic properties in the jaw of female mice only. Jaw muscles are sexually dimorphic, and we propose that the function of En-2 and mechanisms guiding sexual dimorphism of the jaw muscles are integrated. We conclude that the specific expression of En-2 in the jaw therefore plays a role in specifying muscle-fiber characteristics that contribute to the physiologic properties of specific muscle groups. [C] 2001 Academic Press Key Words: skeletal muscle; engrailed; fiber type; jaw; masseter; sexual dimorphism; murine.