The survival of motor neurons is controlled by multiple factors that regulate different aspects of their physiology. The identification of these factors is important because of their relationship to motor neuron disease. We investigate here whether Mullerian Inhibiting Substance (MIS) is a motor neuron survival factor. We find that motor neurons from adult mice synthesize MIS and express its receptors, suggesting that mature motor neurons use MIS in an autocrine fashion or as a way to communicate with each other. MIS was observed to support the survival and differentiation of embryonic motor neurons in vitro. During development, male-specific MIS may have a hormone effect because the blood-brain barrier has yet to form, raising the possibility that MIS participates in generating sex-specific differences in motor neurons.Mullerian Inhibiting Substance type II receptor M otor neurons are particularly prone to age-related deterioration (1-3), which, in the extreme, leads to motor neuron disease and to death by paralysis. The survival of motor neurons is controlled by multiple factors, each of which appears to have a different physiological role. Motor neurons are, for instance, regulated by skeletal muscle fibers and Schwann cells via cardiotrophin-1 (4), TGF-␤2 (5, 6), and glial-cell-line-derived neurotrophic factor (GDNF) (7,8). Motor neurons also receive protection against viral-and hypoxic-induced damage through IL-6 (9) and VEGF (10, 11), respectively. Variations in the VEGF gene cause adult-onset motor-neuron degeneration in some mice and have been linked to ALS in some human populations (10, 11). These findings have renewed interest in identifying nonclassical neuronal survival factors.Mullerian Inhibiting Substance (MIS) is examined herein as a motor-neuron survival factor given that we found high expression of ligand and receptors in motor neurons. MIS is a member of the TGF-␤ superfamily, which includes motor-neuron survival factors, such as GDNF and TGF-␤2. The known physiological actions of MIS are thought to be limited to sexual differentiation of males and to the function of mature reproductive tissues of both sexes (12). These studies introduce a possible function for this interesting molecule and its known signaling pathway.TGF-␤ superfamily members signal through a complex of type I and type II receptors (13). MIS has a unique type II receptor (MISRII) but shares type I receptors with other members of the superfamily (12, 13). Genetic, organ culture, and cellular evidence implicates activin receptor-like kinase 3 (ALK3) (14) and ALK2 (Y. Zhan, D.T.M., and P.K.D., unpublished data) (15) as type I receptors for MIS in murine sexual differentiation, although ALK6 is likely to be involved in other cellular contexts (12, 16).We find that adult motor neurons from male and female mice synthesize MIS and its receptors, with the MIS receptor mRNA in motor neurons being much more abundant than the mRNAs for the GDNF and TGF-␤ receptors. Our experiments show that MIS supports the survival of embryonic motor n...

MyoD belongs to a family of helix-loop-helix proteins that control myogenic differentiation. Transfection of various non-myogenic cell lines with MyoD transforms them into myogenic cells. In normal embryonic development MyoD is upregulated at the time when the hypaxial musculature begins to form, but its role in the function of adult muscle remains to be elucidated. In this study we examined the cellular locations of MyoD protein in normal and abnormal muscles to see whether the presence of MyoD protein is correlated with a particular cellular behaviour and to assess the usefulness of MyoD as a marker for satellite cells. Adult rats were anaesthetised and their tibialis anterior or soleus muscles either denervated, tenotomised, freeze lesioned, lesioned and denervated, or lesioned and tenotomised. At various intervals after the operations the rats were killed and their muscles removed, snap frozen, and sectioned with a cryostat along with muscles from unoperated neonatal and adult rats. The sections were processed for immunohistochemistry using a rabbit affinity-purified antibody to recombinant MyoD. MyoD proved to be an excellent marker for active satellite cells; satellite cells in neohatal and regenerating muscles contained high levels of MyoD protein. MyoD positive cells were not observed in the muscles of old adults, in which the satellite cells are fully quiescent. MyoD immunoreactivity was rapidly lost from satellite cell nuclei after they fused into myotubes and was not detected in either sub-synaptic or non-synaptic nuclei of mature fibers. Denervation, and to a lesser extent tenotomy, of lesioned muscles induced expression of MyoD in myotubal nuclei. Denervation of normal muscles also upregulated MyoD in muscle fiber nuclei, an effect which was maximal after 3 days. We conclude that MyoD protein is neurally regulated in both myotubes and muscle fibers.

Vitamin D may be a positive regulator of AMH production in adults, and vitamin D deficiency may confound clinical decisions based on AMH. Vitamin D deficiency should be considered when serum AMH levels are obtained for diagnosis.

The Drosophila melanogaster flightless I gene is required for normal cellularization of the syncytial blastoderm. Highly conserved homologues of flightless I are present in Caenorhabditis elegans, mouse, and human. We have disrupted the mouse homologue Fliih by homologous recombination in embryonic stem cells. Heterozygous Fliih mutant mice develop normally, although the level of Fliih protein is reduced. Cultured homozygous Fliih mutant blastocysts hatch, attach, and form an outgrowing trophoblast cell layer, but egg cylinder formation fails and the embryos degenerate. Similarly, Fliih mutant embryos initiate implantation in vivo but then rapidly degenerate. We have constructed a transgenic mouse carrying the complete human FLII gene and shown that the FLII transgene is capable of rescuing the embryonic lethality of the homozygous targeted Fliih mutation. These results confirm the specific inactivation of the Fliih gene and establish that the human FLII gene and its gene product are functional in the mouse. The Fliih mouse mutant phenotype is much more severe than in the case of the related gelsolin family members gelsolin, villin, and CapG, where the homozygous mutant mice are viable and fertile but display alterations in cytoskeletal actin regulation.We are studying the mammalian homologues of a number of Drosophila melanogaster genes concerned with development or behavior, as part of a program aimed at identifying novel mammalian developmental and neurobiological genes. The D. melanogaster flightless I (fliI) gene (4,15,23,24,33) is required for cellularization of the syncytial blastoderm. With severe mutations in fliI, when the contribution of maternal product is eliminated, cellularization is only partial and gastrulation fails (35,44).

Many behavioral traits and most brain disorders are common to males and females but are more evident in one sex than the other. The control of these subtle sex-linked biases is largely unstudied and has been presumed to mirror that of the highly dimorphic reproductive nuclei. Sexual dimorphism in the reproductive tract is a product of Mü llerian inhibiting substance (MIS), as well as the sex steroids. Males with a genetic deficiency in MIS signaling are sexually males, leading to the presumption that MIS is not a neural regulator. We challenge this presumption by reporting that most immature neurons in mice express the MIS-specific receptor (MISRII) and that male Mis ؊/؊ and Misrii ؊/؊ mice exhibit subtle feminization of their spinal motor neurons and of their exploratory behavior. Consequently, MIS may be a broad regulator of the subtle sex-linked biases in the nervous system.anti-Mü llerian hormone ͉ exploratory behavior ͉ motor neuron ͉ sexual dimorphism

Developing muscles contain at least two types of myoblasts. Early myoblasts are the first myoblast to form and are the only myoblasts present during primary myotube formation. By the time secondary myotube formation begins, early myoblasts are rare and late myoblasts are common. The late myoblasts have been postulated to give rise to secondary myotubes. While this is generally accepted, it is unclear whether late myoblasts also contribute to the growth of primary myotubes. One study has produced evidence that myoblasts present during secondary myogenesis selectively fuse with each other or with secondary myotubes, but not with primary myotubes (Harris et al. 11989a1 Development 107:771-784). However, the sizes of primary myotubes increase during secondary myotube formation. We have therefore re-examined the question of whether primary myotubes absorb new nuclei during secondary myotube formation. Pregnant rats were given a single intraperitoneal injection of 5 mg of 5-bromodeoxyuridine (BrdU) on one embryonic day (from El3 to E19) and their embryos removed on E20. The brominated-nuclei were labelled with an antibody to BrdU and the myotubes were marked with anti-myosin antibodies. Double labelled sections from the soleus, tibialis anterior, and extensor digitorum longus muscles were examined with a confocal microscope. The numbers and locations of labelled nuclear profiles in primary and secondary myotubes were counted and recorded. The results show: (1) that primary myotubes absorb nuclei at all stages of development, including the period of secondary myotube formation; (2) that in the early stages of secondary myotube formation, more myoblasts fuse with primary than secondary myotubes whereas this situation is reversed by the end of secondary myotube formation; and (3) that the nuclei added to primary and secondary myotubes during the early stages of their formation are located within the middle of E20 muscles. The nuclei added to growing myotubes are preferentially located at the ends of the muscles. o 1996 Wiley-Liss, Inc.

Two soleus muscles with degenerating muscle fibres were serially sectioned and adjacent sections from various levels of the skeletal muscles were stained with antibodies that react with either monocytes and inflammatory macrophages (ED1) or with the major subpopulations of resident macrophages (ED2 and ED3). ED2+ and ED3+ resident macrophages were abundant throughout the muscles but were not present within the degenerating fibres. ED1+ cells, in contrast, were rarely observed within the undamaged regions of the muscles but were abundant within the degenerating fibres and in the perimysium between arterioles and degenerating fibres. It is concluded that the phagocytosis of damaged muscle fibres is not carried out by the major subpopulations of resident macrophages.

No abstract