Peg3/Pw1 is an imprinted gene involved in the TNF-NFkappaB signal transduction pathway.

Tumor necrosis factor (TNF) mediates a variety of biological activities including cell proliferation, differentiation and programmed cell death. The specific response to TNF depends upon cell type and reflects the presence of specific regulatory proteins that participate in the TNF response pathway. TNF signal transduction is mediated by TRAF2 which binds the TNF Receptor2 (TNFR2) and activates NFkappaB. We previously identified a gene Pw1, which encodes a large zinc-finger containing protein. We have determined that Pw1 is identical to Peg3, a paternally expressed gene of unknown function (and will therefore be referred to as Peg3 throughout this text). We report here that Peg3 associates specifically with TRAF2 but not with other TRAF family members. Peg3 expression activates NFkappaB via IkappaB-NFkappaB dissociation and acts synergistically with TRAF2. Transfection of a truncated Peg3 containing the TRAF2 interaction site, abolishes NFkappaB activation by TRAF2 and/or TNF. We conclude that Peg3 is a regulator of the TNF response. These data reveal the involvement of an imprinted gene in this pathway.

CDO, a robo-related cell surface protein that mediates myogenic differentiation.

CDO, a member of the Ig/fibronectin type III repeat subfamily of transmembrane proteins that includes the axon guidance receptor Robo, was identified by virtue of its down-regulation by the ras oncogene. We report here that one prominent site of cdo mRNA expression during murine embryogenesis is the early myogenic compartment (newly formed somites, dermomyotome and myotome). CDO is expressed in proliferating and differentiating C2C12 myoblasts and in myoblast lines derived by treating 10T1/2 fibroblasts with 5-azacytidine, but not in parental 10T1/2 cells. Overexpression of CDO in C2C12 cells accelerates differentiation, while expression of secreted soluble extracellular regions of CDO inhibits this process. Oncogenic Ras is known to block differentiation of C2C12 cells via downregulation of MyoD. Reexpression of CDO in C2C12/Ras cells induces MyoD; conversely, MyoD induces CDO. Reexpression of either CDO or MyoD rescues differentiation of C2C12/Ras cells without altering anchorage-independent growth or morphological transformation. CDO and MyoD are therefore involved in a positive feedback loop that is central to the inverse relationship between cell differentiation and transformation. It is proposed that CDO mediates, at least in part, the effects of cell-cell interactions between muscle precursors that are critical in myogenesis.

Differential expression patterns of Wnt genes in the murine female reproductive tract during development and the estrous cycle.

The murine female reproductive tract differentiates during postnatal development. This process of cytodifferentiation and morphogenesis is dependent upon specific mesenchymal-epithelial interactions as well as circulating steroid hormones (Cunha, G.R., 1976. Int. Rev. Cytol. 47, 137-194; Pavlova, A. et al., 1994. Development 120, 335-346). Members of the Wnt family of signaling molecules have been recently identified in this system (Pavlova, A. et al., 1994. Development 120, 335-346; Bui, T.D. et al., 1997. Br. J. Cancer 75, 1131-1136; Miller, C., Sassoon, D.A., 1998. Development, in press). We describe the expression patterns of Wnt genes in the developing and adult female reproductive tract. Additionally, we note that changes in the levels of expression occur during the estrous cycle. Wnt gene expression patterns are regulated by the presence of epithelium in tissue graft experiments, suggesting that Wnt genes may indeed play roles in the mesenchymal-epithelial interactions critical for female reproductive tract development and function.

Perinatal outcome in triplet versus twin gestations.

The present study was conducted to determine whether triplet pregnancies are associated with a significantly worse perinatal outcome than twin pregnancies. Maternal and neonatal outcome was evaluated in 15 triplet and twin pregnancies that were matched for maternal age, race, type of medical insurance, delivery mode, parity, and history of previous preterm delivery. Preterm labor occurred significantly more often in triplet than in twin gestations (80 versus 40%), as did preterm delivery (87 versus 26.7%). Triplets had a significantly lower mean birth weight (1720 versus 2475 g) and gestational age at delivery (33 versus 36.6 weeks). In addition, 53.3% of triplet pregnancies but only 6.7% of twin pregnancies had one or more neonates with intrauterine growth retardation. Discordancy also occurred more frequently in triplets than in twins (66.7 versus 13.3%). The mean averaged neonatal hospital stay was significantly higher in triplets (29 versus 8.5 days), and triplets had a fivefold increased risk of requiring neonatal intensive care as compared with twins. However, there were no significant differences between the groups in maternal morbidity or major neonatal complications such as respiratory distress syndrome or intraventricular hemorrhage. We believe that these data will be useful in counseling patients with respect to the anticipated perinatal outcome of triplet pregnancies.

The biophysical profile in labor.

To determine whether the biophysical profile would be a valuable intrapartum addition to fetal heart rate monitoring in predicting umbilical arterial acid-base status at delivery, 95 patients at term had serial studies during labor and umbilical artery blood gas analysis. There was no significant association between biophysical profile score and cord blood pH, nor was there a difference in scores between the acidemic and nonacidemic groups. Of the five components of the initial biophysical profile, only a nonreactive nonstress test (NST) was associated with both pH 7.20 or less (P = .019) and metabolic acidemia (P = .016). None of the individual variables of the final examination correlated with a pH of 7.20 or less. However, a nonreactive NST was associated with metabolic acidemia (P = .03), as was the presence of breathing (P = .03). Of the ten infants with pH 7.20 or less, eight had an initial and five had a final biophysical profile score of 8 or higher. Of the five whose pH was less than 7.15, four had an initial and three a final score of 8 or more. Finally, of the five with metabolic acidemia, four had an initial and two a final score of 8 or higher. Half of the acidemic fetuses had final biophysical profile scores of 8 or higher, suggesting that this score in labor is not reliable to rule out acidemia at delivery.(ABSTRACT TRUNCATED AT 250 WORDS)

Myogenic regulatory factors: dissecting their role and regulation during vertebrate embryogenesis.

The elucidation of the myogenic regulatory factors (MRFs) has resulted in a surge of research activity that promises to bridge molecular and embryological approaches to muscle research. In this review, we consider the arguments that would place MRFs in a key determination role for the skeletal muscle lineage in the vertebrate embryo. Amphibian, avian, and murine systems are compared and recent developments in the molecular embryology of myogenesis are reviewed. A model is proposed to account for the apparent discrepancies in conclusions drawn from studies using cultured cells that indicate a role for the MRFs in determination, and studies of MRF and muscle gene expression in vivo that indicate a role in differentiation but not in lineage determination.

A role for Engrailed-2 in determination of skeletal muscle physiologic properties.

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.

Wnt-7a maintains appropriate uterine patterning during the development of the mouse female reproductive tract.

The murine female reproductive tract differentiates along the anteroposterior axis during postnatal development. This process is marked by the emergence of distinct cell types in the oviduct, uterus, cervix and vagina and is dependent upon specific mesenchymal-epithelial interactions as demonstrated by earlier heterografting experiments. Members of the Wnt family of signaling molecules have been recently identified in this system and an early functional role in reproductive tract development has been demonstrated. Mice were generated using ES-mediated homologous recombination for the Wnt-7a gene (Parr, B. A. and McMahon, A. P. (1995) Nature 374, 350-353). Since Wnt-7a is expressed in the female reproductive tract, we examined the developmental consequences of lack of Wnt-7a in the female reproductive tract. We observe that the oviduct lacks a clear demarcation from the anterior uterus, and acquires several cellular and molecular characteristics of the uterine horn. The uterus acquires cellular and molecular characteristics that represent an intermediate state between normal uterus and vagina. Normal vaginas have stratified epithelium and normal uteri have simple columnar epithelium, however, mutant uteri have stratified epithelium. Additionally, Wnt-7a mutant uteri do not form glands. The changes observed in the oviduct and uterus are accompanied by a postnatal loss of hoxa-10 and hoxa-11 expression, revealing that Wnt-7a is not required for early hoxa gene expression, but is required for maintenance of expression. These clustered hox genes have been shown to play a role in anteroposterior patterning in the female reproductive tract. In addition to this global posterior shift in the female reproductive tract, we note that the uterine smooth muscle is disorganized, indicating development along the radial axis is affected. Changes in the boundaries and levels of other Wnt genes are detectable at birth, prior to changes in morphologies. These results suggest that a mechanism whereby Wnt-7a signaling from the epithelium maintains the molecular and morphological boundaries of distinct cellular populations along the anteroposterior and radial axes of the female reproductive tract.

Androgen regulation of muscle fiber type in the sexually dimorphic larynx of Xenopus laevis.

We used histochemical techniques [assays for adenine triphosphatase (ATPase) and succinate dehydrogenase (SDHase) activity] to identify muscle fiber types in the larynx of Xenopus laevis. Male muscle is made up of one fiber type, medium-sized fibers (approximately 9 microns2) that stain lightly for acid-stable ATPase and intensely for SDHase activity. In contrast, the female has 3 fiber types: small fibers (approximately 6 microns2) that stain intensely for ATPase and SDHase, medium-sized fibers (approximately 13 microns2) with moderate staining for ATPase and dark staining for SDHase, and large fibers (approximately 15 microns2) with little SDHase or ATPase activity. Long-term castration (6 months) has no effect on histochemical staining of adult male fibers. Long-term testosterone treatment (5 months) increases the proportion of medium-sized, moderately staining fibers in adult females, and reduces the proportion of both the small, darkly staining fibers and the large, lightly staining fibers. At metamorphosis, both males and females have 3 fiber types whose ATPase activity is similar to that of the adult female. However, no SDHase activity is observed. Treatment of juveniles for 3 weeks with testosterone results in nearly complete masculinization of muscle fibers, as judged by increased cross-sectional area, homogeneous ATPase staining, and a marked increase in SDHase activity. Thus, juvenile muscle is considerably more responsive to testosterone than is adult female muscle. We propose that the uniform metabolic properties of male laryngeal muscle contribute to the production of the rapid (66 Hz) mate call vocalizations characteristic of this species. Further, our results suggest that androgens direct the masculinization of laryngeal muscle fibers during postmetamorphic development.

Transcripts of alpha-cardiac and alpha-skeletal actins are early markers for myogenesis in the mouse embryo.

Among the first tissues to differentiate in the mammalian embryo are cardiac and subsequently skeletal striated muscle. We have developed specific cRNA probes corresponding to the 5' noncoding regions of alpha-cardiac and alpha-skeletal actin mRNAs in order to investigate myogenesis in the mouse embryo. Transcripts coding for cardiac actin which is the major isoform of the adult heart can first be detected between 7.5 and 7.8 days p.c. in the developing heart and are observed in all somites as they are formed. In addition, alpha-skeletal actin transcripts are accumulated at much lower levels in cardiac tissue and newly formed somites; both heart and skeletal muscle show co-expression of this actin gene pair at all stages of development examined. The fact that cardiac actin transcripts can be observed in the myotomal portion of the somite prior to muscle fibre differentiation indicates that cardiac actin transcripts (and to a lesser extent skeletal actin transcripts) are markers not only of striated muscle tissue, but also of earlier stages of the myogenic programme in vivo.

Myogenin, a factor regulating myogenesis, has a domain homologous to MyoD.

In this report, we describe the isolation, sequence, and initial characterization of the cDNA for the muscle-specific regulatory factor skeletal myogenin. Transfection of myogenin into the mesenchymal cell line C3H10T1/2 produces cells expressing muscle-specific markers. Myogenin is absent in undifferentiated cells, peaks, and then declines following a stimulus to differentiate, and is overexpressed in myoblasts selected with 5-bromodeoxyuridine for the overproduction of factors that regulate the decision to differentiate. High levels of myogenin transcripts are present in the myotomal region of somites at 8.5 days of gestation in the mouse. Although myogenin and MyoD are different genes, they share the myc homology domain. Myogenin and MyoD thus form part of a gene family regulating myogenesis, and together with myd may constitute a set of factors that interact to regulate the determination and differentiation of muscle cells.

Id expression during mouse development: a role in morphogenesis.

We have characterized the spatial and temporal pattern of Id transcription during mouse embryogenesis. The Id gene encodes a helix-loop-helix (HLH) protein which can heterodimerize with the ubiquitously expressed HLH protein products of the E2A gene, and prevent them from binding DNA either alone or as a heterodimer with tissue specific HLH transcription factors such as the muscle determination gene, MyoD1 (Benezra et al., 1990: Cell 61:49-59). Since Id has been shown to be down-regulated during induced differentiation in several cell lines, it has been postulated that Id plays a general inhibitory role in cell differentiation (Benezra et al., 1990). In situ analysis of Id mRNA expression in the mouse embryo was performed in order to determine whether the pattern of Id expression is consistent with this postulate. A detailed study throughout the entirety of mouse postimplantation development reveals that Id is expressed upon gastrulation at very high levels in almost all regions of the mouse embryo and expression declines as embryogenesis proceeds. In skeletal muscle, in which the inhibitory action of Id has been established in tissue culture models (Benezra et al., 1990), Id and the HLH myogenic factors are expressed in a mutually exclusive manner suggesting that myogenic precursors do not express both types of HLH gene products. In addition, Id colocalizes both spatially and temporally with Hox-7.1, a murine homeobox gene which is associated with regions of high cell proliferation and positional fate assignment.

Expression of the muscle regulatory factor MRF4 during somite and skeletal myofiber development.

The muscle regulatory factors MRF4, myogenin, myf-5, and MyoD constitute a family of proteins that can function as muscle-specific transcriptional activators. Although this gene family has been extensively studied, a specific role for each factor during myogenesis remains to be determined. Understanding how these factors function requires a detailed analysis of their expression patterns during development. Toward this goal, we examined the temporal pattern of expression of MRF4 and the other factors in the rat myogenic cell line L6J1-C, in newborn rat primary muscle cell cultures and in fetal and postnatal rat limb muscle. Our results demonstrate that MyoD, myogenin, and myf-5 transcripts accumulate maximally at various stages of myoblast differentiation and decline to low expression levels in adult muscle tissue. In contrast, MRF4 transcript accumulation is restricted to cell cultures containing multinucleate myofibers, and its expression in vivo increases sharply during late fetal muscle development. This level of MRF4 expression is maintained in the adult which, together with decreased expression of the other three muscle regulatory factors, makes MRF4 the predominant factor in adult muscle. In situ hybridization of mouse embryo tissue sections indicates that MRF4 transcripts accumulate in the limb beginning 13.5 days post coitum, which is 2 days later than the initial appearance of myogenin and MyoD transcripts. Hybridization to earlier stages of development reveals, however, that MRF4 mRNA initially is present in the myotomal compartment of the somites, just after myogenin but 2 days prior to MyoD expression. Unlike myogenin and MyoD, MRF4 expression declines in the myotomes at the time that multinucleate axial muscles begin to form in this region, although during later development MRF4 is expressed in the myofibers of axial muscles at levels comparable to those in the limb. Differences in the expression patterns for MRF4, myogenin, myf-5 and MyoD between myotomal and other skeletal muscle development suggest that the relative timing of expression for each muscle regulatory factor may control the distinct phenotypes associated with myotomal myocytes and multinucleate myofibers.

Myogenesis in the mouse.

The first striated muscle to form during mouse embryogenesis is the heart followed by skeletal muscle which is derived from the somites. The expression of genes encoding muscle structural proteins and myogenic regulatory sequences of the MyoD1 family has been examined using 35S-labelled riboprobes. In the cardiac tube, actin and myosin genes are expressed together from an early stage, whereas in the myotome, the earliest skeletal muscle, they are activated asynchronously over days. They are not expressed in the somite prior to myotome formation. One potential muscle marker, carbonic anhydrase III, is expressed in early mesoderm and subsequently in the notochord, similarly to the Brachyury gene. The myogenic sequences are not detectable in the heart. In the myotome they show distinct patterns of expression; this is discussed in the context of their role as muscle transcription factors. myf-5 is the only myogenic factor sequence present in the somite prior to muscle formation and thus is potentially involved in an earlier step of muscle determination. It is also present in the early limb bud, but the status of myogenic precursor cells in the limb in this context is less clear.

Loss of N-myc function results in embryonic lethality and failure of the epithelial component of the embryo to develop.

myc genes are thought to function in the processes of cellular proliferation and differentiation. To gain insight into the role of the N-myc gene during embryogenesis, we examined its expression in embryos during postimplantation development using RNA in situ hybridization. Tissue- and cell-specific patterns of expression unique to N-myc as compared with the related c-myc gene were observed. N-myc transcripts become progressively restricted to specific cell types, primarily to epithelial tissues including those of the developing nervous system and those in developing organs characterized by epithelio-mesenchymal interaction. In contrast, c-myc transcripts were confined to the mesenchymal compartments. These data suggest that c-myc and N-myc proteins may interact with different substrates in performing their function during embryogenesis and suggest further that there are linked regulatory mechanisms for normal expression in the embryo. We have mutated the N-myc locus via homologous recombination in embryonic stem (ES) cells and introduced the mutated allele into the mouse germ line. Live-born heterozygotes are under-represented but appear normal. Homozygous mutant embryos die prenatally at approximately 11.5 days of gestation. Histologic examination of homozygous mutant embryos indicates that several developing organs are affected. These include the central and peripheral nervous systems, mesonephros, lung, and gut. Thus, N-myc function is required during embryogenesis, and the pathology observed is consistent with the normal pattern of N-myc expression. Examination of c-myc expression in mutant embryos indicates the existence of coordinate regulation of myc genes during mouse embryogenesis.

MHox: a mesodermally restricted homeodomain protein that binds an essential site in the muscle creatine kinase enhancer.

Myogenic helix-loop-helix (HLH) proteins, such as myogenin and MyoD, can activate muscle-specific transcription when introduced into a variety of nonmuscle cell types. Whereas cells of mesodermal origin are especially permissive to the actions of these myogenic regulators, many other cell types are refractory to myogenic conversion by them. Here we describe a novel homeodomain protein, MHox, that binds an A+T-rich element in the muscle creatine kinase (MCK) enhancer that is essential for muscle-specific transcription and trans-activation by myogenic HLH proteins. MHox is completely restricted to mesodermally derived cell types during embryogenesis and to established cell lines of mesodermal origin. In contrast to most other homeobox genes, MHox expression is excluded from the nervous system, with the highest levels observed in limb bud and visceral arches. In adult mice, MHox is expressed at high levels in skeletal muscle, heart and uterus. The DNA-binding properties and pattern of MHox expression are unique among homeobox genes and suggest a role for MHox as a transcriptional regulator that participates in the establishment of diverse mesodermal cell types.

Developmental expression pattern of the cdo gene.

CDO is a cell-surface protein of the immunoglobulin/fibronectin type III repeat family that positively regulates myogenic differentiation in vitro. To gain a better understanding of the role of cdo during vertebrate development, we carried out an extensive in situ hybridization study to characterize its expression pattern from postimplantation to late stages of mouse embryogenesis and in rat brain from E13 to adult. Our results show a broad pattern of cdo expression that is spatially and temporally restricted during embryogenesis. In the central nervous system (CNS), cdo expression is detected as early as E7.5 and maintained in the dorsal ventricular zones of the brain and spinal cord, becoming increasingly restricted in the adult. High levels of cdo are detected in developing sensory organs, such as the eye and ear. Outside the CNS, cdo is expressed mainly in neural crest and mesodermal derivatives, including skeletal muscle precursors. Overall, the highest levels of cdo expression are seen from E9.0 to E15.5. The temporal onset and restricted expression of cdo suggest that cdo plays a role in the determination and/or differentiation of a number of cell types during embryogenesis.

Msh homeobox genes regulate cadherin-mediated cell adhesion and cell-cell sorting.

Msx-1 and Msx-2 are two closely related homeobox genes expressed in cephalic neural crest tooth buds, the optic cup endocardial cushions, and the developing limb [Hill and Davidson, 1991; Monaghan et al., 1991; Robert et al., 1991]. These sites correspond to regions of active cell segregation and proliferation under the influence of epithelial-mesenchymal cell interactions [Brown et al., 1993; Davidson et al., 1991], suggesting that Msx-1 and Msx-2 regulate cell-cell interactions. We have investigated the potential relationship between expression of the Msh homeobox genes (Msx-1 and Msx-2) and cadherin-mediated cell adhesion and cell sorting. We report that cell lines stably expressing Msx-1 or Msx-2 differentially sort on the basis of Msh gene expression. We demonstrate in vitro that initial cell aggregation involves calcium-dependent adhesion molecules (cadherins) and that Msh genes regulate cadherin-mediated adhesion. These results support the hypothesis that Msh genes play a role in the regulation of cell-cell adhesion and provide a link between the genetic phenomena of homeobox gene expression and cellular events involved in morphogenesis, including cell sorting and proliferation.

Perinatal factors influencing atrial natriuretic peptide levels in umbilical arterial plasma at the time of delivery.

Little is known about atrial natriuretic peptide metabolism or secretion in the human fetus. The purpose of this study was to determine if both the placenta and umbilical vessels are possible sites of atrial natriuretic peptide metabolism and to evaluate the effects that labor, route of delivery, prolonged pregnancy, preeclampsia, and fetal distress have on umbilical arterial atrial natriuretic peptide levels. We found that plasma atrial natriuretic peptide levels in the umbilical artery are significantly greater than those in the vein (p less than 0.001). Umbilical arterial and umbilical venous atrial natriuretic peptide levels were higher in plasma samples collected immediately at delivery when compared with those obtained 10 minutes later (p less than 0.001). Umbilical arterial atrial natriuretic peptide levels were elevated in pregnancies complicated by preeclampsia and fetal distress (p less than 0.01). Labor, route of delivery, and prolonged pregnancy had no effect on umbilical arterial atrial natriuretic peptide levels. We propose that both the placenta and umbilical vessels contain atrial natriuretic peptide receptors that are involved in the clearance or metabolism of atrial natriuretic peptide. The increased umbilical arterial atrial natriuretic peptide levels present in preeclampsia and fetal distress may reflect an attempt by the fetus to regulate blood flow.