Biomedical subjects

M Ontell

Publications and source records attributed to M Ontell.

At least 37 records · Page 2Linked to original sources

PubMed1992

Expression of muscle genes in the mouse embryo.

Using isogene specific probes and in situ hybridization on sections, we have examined the expression of structural and regulatory genes in the mouse embryo during the formation of cardiac and skeletal muscle. The temporal and spatial information thus obtained about the onset of expression of muscle genes provides insight into the regulation of myogenesis in vivo. Actin and myosin sequences present in different compartments of the adult heart are initially all co-expressed in the cardiac tube (between 7-8 days). The process of spatial restriction to atrial or ventricular compartments of the heart takes place asynchronously later. In contrast, the onset of expression of actin and myosin genes in the first skeletal muscle, the myotome, which corresponds to the central compartment of the somite, as well as their subsequent down-regulation in different skeletal muscle masses, takes place very asynchronously. One might predict that factor(s) responsible for the transcriptional activation of these genes are present in sufficient quantity in the cardiac tube, whereas in skeletal muscle individual genes are responding to variable levels of factor(s). In fact the four myogenic regulatory sequences present in the mouse - MyoD1, myogenin, myf-5 and myf-6 - do show distinct patterns of expression during the development of skeletal muscle. None of these sequences have been detected in the heart. In the myotome there is no general correlation between the appearance of a particular myogenic sequence and the activation of a particular structural gene. A striking example of this is provided by the muscle isoform of creatine phosphokinase. We would propose that each muscle structural gene has a different threshold of activation, depending on the quantity and nature of the myogenic factor present. We have also examined the onset of expression of the X-linked dystrophin gene known to be expressed in adult heart and skeletal muscle. In the myotome dystrophin transcripts are first detected at the time when myosin heavy chains first accumulate and muscular contraction is initiated. In contrast in the cardiac tube dystrophin transcripts are not detected initially, at a time (from 8 days) when the heart contracts. This observation can be correlated with the pathology of the disease which points to a more essential role of dystrophin in skeletal muscle. No muscle structural gene examined is expressed in the somite prior to myotome formation. If the myogenic regulatory sequences are implicated in muscle cell determination then they should be expressed in the dermomyotome of the immature somite which gives rise to muscle precursor cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Actins↗

PubMed1991

Reinnervation of murine muscle following fetal sciatic nerve transection.

A technique is reported that permits transection of the sciatic nerve of mouse fetuses without interfering with fetal viability. Sciaticotomy was performed on Swiss Webster mice at day 17 of gestation; the contralateral side served as control. Six weeks later the extensor digitorum longus (EDL) muscles on both sides were injected with horseradish peroxidase (HRP). Examination of the lumbar spinal cord revealed that while a substantial number of motor neurons in the region of the spinal cord giving rise to the sciatic nerve died, the EDL muscle did become reinnervated. The size of the EDL motor neuron pool on the denervated-reinnervated side was approximately 43% of that seen on the control side. While the control EDL motor neuron pool was located in lumbar segments L3-L5, the location of the pool to the denervated-reinnervated EDL was shifted cranially to L2-L4. Denervated-reinnervated EDL muscles were analyzed immunohistochemically to study the effect of fetal denervation on the neuronal cell adhesion molecule (N-CAM) expression. At 2 weeks postnatal, N-CAM immunoreactivity in control muscle was segregated to the motor end-plate region, while fetally denervated muscle continued to express N-CAM along the length of the sarcolemma. Thus fetally denervated muscle does not develop the same pattern of N-CAM expression as normal, innervated muscle. By 6 weeks of age, the denervated-reinnervated muscle showed the same level and distribution of N-CAM immunoreactivity as did age-matched control muscle, indicating that most, if not all, of its myofibers had been reinnervated.

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PubMed1991

Emergence of the mature myosin phenotype in the rat diaphragm muscle.

Immunohistochemical analysis of myosin heavy chain (MHC) isoform expression in perinatal and adult rat diaphragm muscles was performed with antibodies which permitted the identification of all known MHC isoforms found in typical rat muscles. Isoform switching, leading to the emergence of the adult phenotype, was more complex than had been previously described. As many as four isoforms could be coexpressed in a single myofiber. Elimination of developmental isoforms did not usually result in the myofiber immediately achieving its adult phenotype. Activation of genes for specific adult isoforms might be delayed to puberty. For example, two of the three fast MHCs, MHC2X and MHC2A appeared perinatally, while MHC2B did not appear until 30 days postnatal. By Day 60 this isoform was present in approximately 27% of the myofibers, but in most myofibers expression of this isoform was transient (i.e., at Day greater than or equal to 115, less than 4% of the myofibers expressed MHC2B). Fibers which contained MHC beta/slow during the late fetal and early neonatal period coexpressed MHCemb. A marked increase in the frequency of fibers containing MHC beta/slow occurred between 4 and 21 days postnatal. These slow fibers arose from a population of myofibers which expressed MHCemb and MHCneo during their development, and they accounted for the majority of slow fibers found in the adult diaphragm. The adult myosin phenotype of the diaphragm myofibers (as determined with immunocytochemistry, and 5% SDS-PAGE) was not achieved until the rat was greater than or equal to 115 days old.

Age Factors↗

PubMed1991

Effects of perinatal undernutrition on elimination of immature myosin isoforms in the rat diaphragm.

The effect of perinatal undernutrition on the postnatal elimination of immature myosin isoforms in rat diaphragm muscle was examined using electrophoretic and immunocytochemical techniques. Electrophoresis of native myosin showed that neonatal bands were present in diaphragm muscles of both control and undernourished rats on day 4. By day 21, the neonatal bands were diminished in the control diaphragm compared with the diaphragm of the undernourished rats. Neonatal bands persisted on postnatal day 30 in the diaphragm of the undernourished rats but not in the diaphragm of control rats. No significant difference in the time course of elimination of embryonic myosin light chain (LCemb) was observed between the diaphragm muscles of control and undernourished rats with two-dimensional gel electrophoresis. Immunocytochemical analysis demonstrated embryonic myosin heavy chain (MHCemb) in all myofibers of the diaphragm muscle of both groups at day 4, but this isoform was not detected in either group by day 14. Reactivity with anti-neonatal myosin heavy chain (MHCneo) indicated that rate of elimination of the MHCneo was delayed in the undernourished state as compared with the normal rats (P less than 0.001). Serum triiodothyronine levels were measured at 14, 21, and 30 days and were significantly lower in the undernourished rats compared with age-matched controls. These data demonstrate that the normal postnatal decrease in MHCneo, but not MHCemb or LCemb, is affected by the nutritional state of the animal. We speculate that these alterations in myosin isoform transitions are induced by hypothyroidism associated with undernutrition.

Aging↗

PubMed1990

Electrophoretic separation and immunological identification of type 2X myosin heavy chain in rat skeletal muscle.

One slow and three fast myosin heavy chains have been described in typical skeletal muscles of the adult rat using immunocytochemical analysis. Electrophoretic isolation and immunochemical identification of these four isoforms has not been achieved. An electrophoretic procedure is described which, by altering the cross-linkage and polymerization kinetics of 5% polyacrylamide gels, allows resolution of these four distinct myosin heavy chains. Using specific monoclonal antibodies and double immunoblotting analysis, the identity and electrophoretic migration order of the myosin heavy chains was established to be: 2A less than 2X less than 2B less than beta/slow.

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PubMed1990

The expression of myosin genes in developing skeletal muscle in the mouse embryo.

Using in situ hybridization, we have investigated the temporal sequence of myosin gene expression in the developing skeletal muscle masses of mouse embryos. The probes used were isoform-specific, 35S-labeled antisense cRNAs to the known sarcomeric myosin heavy chain and myosin alkali light chain gene transcripts. Results showed that both cardiac and skeletal myosin heavy chain and myosin light chain mRNAs were first detected between 9 and 10 d post coitum (p.c.) in the myotomes of the most rostral somites. Myosin transcripts appeared in more caudal somites at later stages in a developmental gradient. The earliest myosin heavy chain transcripts detected code for the embryonic skeletal (MHCemb) and beta-cardiac (MHC beta) isoforms. Perinatal myosin heavy chain (MHCpn) transcripts begin to accumulate at 10.5 d p.c., which is much earlier than previously reported. At this stage, MHCemb is the major MHC transcript. By 12.5 d p.c., MHCpn and MHCemb mRNAs are present to an equal extent, and by 15.5 d p.c. the MHCpn transcript is the major MHC mRNA detected. Cardiac MHC beta transcripts are always present as a minor component. In contrast, the cardiac MLC1A mRNA is initially more abundant than that encoding the skeletal MLC1F isoform. By 12.5 d p.c. the two MLC mRNAs are present at similar levels, and by 15.5 d p.c., MLC1F is the predominant MLC transcript detected. Transcripts for the ventricular/slow (MLC1V) and another fast skeletal myosin light chain (MLC3F) are not detected in skeletal muscle before 15 d p.c., which marks the beginning of the fetal stage of muscle development. This is the first stage at which we can detect differences in expression of myosin genes between developing muscle fibers. We conclude that, during the development of the myotome and body wall muscles, different myosin genes follow independent patterns of activation and accumulation. The data presented are the first detailed study of myosin gene expression at these early stages of skeletal muscle development.

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PubMed1990

Myosin isoforms in neonatal rat extensor digitorum longus, diaphragm, and soleus muscles.

The postnatal elimination of embryonic and neonatal myosin isoforms in the rat extensor digitorum longus, diaphragm, and soleus muscles was compared using electrophoresis and immunohistochemical techniques. Electrophoresis of native myosin showed that neonatal bands were present in all three muscles on day 4 but were absent from the day 21 extensor digitorum longus muscle that exhibited its adult electrophoretic pattern. Mature electrophoretic banding patterns were present on days 60 and 125 in the diaphragm and soleus muscles, respectively. Immunohistochemical analysis indicated that embryonic myosin heavy chain was present in all day 4 samples but absent by day 21. Quantitative evaluation determined that the rate of elimination of neonatal myosin heavy chain (MHCneo) was faster in the extensor digitorum longus muscle than in the diaphragm, with the soleus muscle having the slowest rate of elimination of this isoform. Embryonic myosin light chain was detected by two-dimensional electrophoresis through day 8 in each of the muscles. These data indicate that postnatal elimination of MHCneo is tissue specific and time dependent but not governed by either activity level or rostral-caudal position.

Aging↗

PubMed1988

Spontaneous regeneration of older dystrophic muscle does not reflect its regenerative capacity.

Young dystrophic (dy) murine muscle is capable of "spontaneous" regeneration (i.e., regeneration in the absence of external trauma); however, by the time the mice are 8 weeks old, this regeneration ceases. It has been suggested that the cessation of regeneration in dystrophic muscle may be due to exhaustion of the mitotic capability of myosatellite cells during the early stages of the disease. To test this hypothesis, orthotopic transplantation of bupivacaine treated, whole extensor digitorum longus muscles has been performed on 14 to 16-week-old 129 ReJ/++ and 129 ReJ/dydy mice. The grafted dystrophic muscle is able to produce and maintain for 100 days post-transplantation 356 +/- 22 myofibers, a number similar to that found in age-matched dystrophic muscle. The ability of old dystrophic muscle to regenerate subsequent to extreme trauma indicates that the cessation of "spontaneous" regeneration is due to factor(s) other than the exhaustion of mitotic capability of myosatellite cells. Moreover, there is no significant difference in myosatellite cell frequencies between grafted normal and dystrophic muscles (100 days post-transplantation). Myosatellite cell frequencies in grafted muscles are similar to those in age-matched, untraumatized muscles. While grafting of young dystrophic muscle modifies the phenotypic expression of histopathological changes usually associated with murine dystrophy, grafts of older dystrophic muscle show extensive connective-tissue infiltration and significantly fewer myofibers than do grafts of age-matched normal muscle. As early as 14 days post-transplantation, it is possible to distinguish between grafts of old, normal and dystrophic muscles. It is suggested that the connective tissue stroma, present in the dystrophic muscle at the time of transplantation, may survive the grafting procedure.

Age Factors↗

PubMed1988

Cytoarchitecture of the fetal murine soleus muscle.

The organogenesis of the soleus muscle of the 129 ReJ mouse (a mixed muscle, which in the adult contains approximately equal numbers of slow-twitch oxidative and fast-twitch oxidative-glycolytic myofibers) was studied in spaced, serial transverse, and longitudinal sections of muscles of 14-, 16-, and 18-day in utero and 1- and 5-day postnatal mice. A discrete soleus muscle was distinguished by 14 days in utero. It consisted of groups of closely apposed primary myotubes displaying junctional complexes and a pleomorphic population of mononucleated cells. Between 14 and 16 days in utero there was little de novo myotube formation. At 16 days in utero, basal lamina surrounded groups of primary myotubes; and primitive motor endplates were found on these myotubes. At 18 days in utero, the basal-lamina-enclosed groups of primary myotubes were no longer present. At this stage, basal lamina surrounded clusters (consisting of one primary myotube and one or more secondary myotubes) or independent myotubes (single myotubes surrounded by their own basal lamina). Cluster formation and cluster dispersal occurred concurrently, beginning at 18 days in utero and extending until birth. At birth, there was still a substantial population of immature, secondary myotubes that interdigitated with larger, more mature primary myofibers. At this stage, intermuscular axons had begun to myelinate, and postsynaptic specialization of the motor endplates had begun. Cluster dispersal and myonuclear migration was completed during the first 5 days postnatally with the muscle taking on adult characteristics. Beginning at 16 days in utero and extending into the neonatal period, there was evidence of myotube death in the soleus muscle.

Actin Cytoskeleton↗

PubMed1988

Morphometric analysis of the developing mouse soleus muscle.

The pattern of organogenesis of the soleus muscle of the 129 ReJ mouse was evaluated quantitatively using spaced, serial, ultrathin sections and computer-assisted morphometric analysis. Muscles from 14-, 16-, and 18-day in utero mice and muscles of 1- and 5-day-old mice were analyzed to determine age-related alterations in the maximal girth and length of the muscle, number of myotubes, cluster frequency, and the lengths and diameters of myotubes. Primary myotubes are found in the muscle at 14 days in utero. There is little de novo myotube formation between 14 and 16 days in utero, this interval being principally one of primary myotube growth and maturation. The interval between 16 and 18 days in utero is marked by extensive secondary myotube formation, with more myotubes being formed during this period than in any period studied. Morphometric data support the hypothesis that secondary generation myotubes use primary myotubes as a scaffold on which they are formed. Morphometric data also confirm the hypothesis that cluster formation and cluster dispersal occur concurrently during the prenatal period. Secondary myotubes continue to form until birth. At birth, the soleus muscle contains the adult number of myofibers. The first 5 days postnatally are marked by myofiber growth and maturation.

Actin Cytoskeleton↗

PubMed1988

Regeneration of dystrophic muscle following multiple injections of bupivacaine.

Regenerated myofibers formed subsequent to orthotopic transplantation of young, dystrophic mouse muscle fail to display the extensive histopathological changes characteristics of murine dystrophy. In order to determine whether this modification of the phenotypic expression of murine dystrophy is unique to the transplantation system or whether it can be found when other extreme trauma induces dystrophic muscle to regenerate, the extensor digitorum longus muscles of 4-6-week-old normal (129 ReJ +/+) and dystrophic (129 ReJ dy/dy) mice were given two series of injections of the myotoxin bupivacaine, spaced 12 hours apart. These injections resulted in necrosis of approximately 90% of the original myofibers. At 100 days after injection, the regenerated normal muscle appeared "healthy," whereas the regenerated dystrophic muscle displayed histopathological changes. It is suggested that the differences in the time course of innervation of the myotubes in the transplantation system as compared with that in the bupivacaine system may be a factor in determining whether regenerated dystrophic myofibers express a dystrophic morphology.

Animals↗

PubMed1988

Effect of neonatal denervation-reinnervation on the functional capacity of a 129ReJ dy/dy murine dystrophic muscle.

The sciatic nerves of 14-day-old 129 ReJ normal (++) and dystrophic (dy/dy) mice were transected in the mid-thigh region. The cut ends of the nerves were approximated to facilitate regeneration. One hundred days after denervation, contractile properties of denervated-reinnervated, normal and dystrophic extensor digitorum longus (EDL) muscles were compared to age-matched normal and dystrophic muscles. In dystrophic muscle, in vitro twitch and tetanic tensions were reduced, compared to those of normal muscle. The denervation-reinnervation procedure resulted in an increase in these parameters as compared to unoperated dy muscle. These data correlated with increases in total myofiber cross-sectional areas. Twitch contraction time was not significantly affected by the dystrophic condition or by the denervation-reinnervation protocol. Whereas dystrophic muscle had a longer half-relaxation time than normal muscle, denervation-reinnervation of the dystrophic EDL resulted in a significantly faster half-relaxation time. While fatigue resistance was greater in dystrophic muscles than in normal muscle, there was a significant decrease in fatigue resistance in the denervated-reinnervated dystrophic muscle. Transient neonatal denervation results in modification of both the morphological and physiological characteristics of murine dystrophy.

Animals↗

PubMed1987

Transient and chronic neonatal denervation of murine muscle: a procedure to modify the phenotypic expression of muscular dystrophy.

The extensor digitorum longus muscles of 14-d-old normal (129 ReJ ++) and dystrophic (129 ReJ dy/dy) mice were denervated by cutting the sciatic nerve. One denervation protocol was designed to inhibit reinnervation of the shank muscles, the other to promote reinnervation. Chronically denervated muscles (muscles that remained denervated for 100 d after nerve section) exhibited marked atrophy, but the number of myofibers in these muscles (1066 +/- 46 and 931 +/- 62 for the denervated normal and dystrophic muscles, respectively) was similar to the number of myofibers found in age-matched, unoperated normal muscles [922 +/- 28 (Ontell et al., 1984)] and was significantly greater than the number of myofibers found in age-matched dystrophic muscles [547 +/- 45 (Ontell et al., 1984)]. Similar effects on myofiber number were obtained when denervated muscles were allowed to reinnervate. Reinnervation of both normal and dystrophic muscles mitigated the marked atrophy that characterized chronically denervated muscles. The dystrophic reinnervated muscles appeared "healthier" than age-matched, unoperated dystrophic muscles, having 70% more myofibers, less myofiber diameter variability, substantially less connective tissue infiltration, and a greater amount of contractile tissue at their widest girths. The present study demonstrated that it is possible to alter the phenotypic expression of the histopathological changes associated with murine dystrophy, in dystrophic myofibers that are formed during fetal development, by subjecting the muscle to neonatal denervation.

Animals↗

PubMed1986

Modification of the phenotypic expression of murine dystrophy: a morphological study.

The extensor digitorum longus muscles of 4-6-week-old normal mice (129 ReJ) and dystrophic mice (129 ReJ dy/dy) were orthotopically transplanted. Grafted muscles were examined 1, 3, 7, 14, 20, 50, and 100 days post-transplantation. The myofibers of both types of grafts underwent a similar time course of necrosis and regeneration. Other than during the initial necrotic response, no evidence of necrotic myofibers was found in either type of grafted muscle. At 100 days post-transplantation, the grafted normal and dystrophic muscles were essentially similar, except that the dystrophic graft was of smaller size. Based on a comparison of the number of myofibers found at the 100-day grafts' widest girths [631 +/- 59 SEM, for normal grafts (Bourke and Ontell, 1984); 631 +/- 74 SEM, for dystrophic grafts], it is suggested that the regenerative capability of traumatized 4-6-week-old dystrophic muscle is similar to that of traumatized normal muscle. At 100 days post-transplantation, the grafted dystrophic muscle appeared "healthier" than untraumatized muscle from age-matched dystrophic mice, having less variation in myofiber diameter, better fascicular organization, and less connective tissue. The transplantation system demonstrates the possibility of modifying the expression of genetic programming of myopathic disorders using environmental manipulation.

Animals↗

PubMed1986

Muscular dystrophy and muscle regeneration.

An animal model of muscular dystrophy, the dystrophic (129ReJ dy/dy) mutant mouse, was used to evaluate the regenerative phenomenon in dystrophic muscle. The effect of age on "spontaneous" regeneration (i.e., regeneration in the absence of secondary trauma) was assessed by quantitative morphometric analysis and evaluation of myosatellite cell dynamics (i.e., myosatellite cell frequency, proliferative activity, and fusion capability). Spontaneous regeneration ceased by the time the mice were 8 weeks old. The findings suggested that the small "regenerating" myofibers found in older dystrophic muscle had been formed earlier in the time course of the disease and were growth-inhibited. To determine the cause of the cessation of regeneration, dystrophic muscle was subjected to the severe trauma of whole-muscle transplantation, a trauma that results in total myofiber necrosis followed by de novo myotube formation. When young dystrophic muscle (from 4- to 6-week-old dystrophic mice) was orthotopically transplanted, the time course of degeneration-regeneration was similar to that seen in age-matched normal muscle. Moreover, the regenerated dystrophic myofibers were capable of long-term survival (200 days or longer after transplantation), and they failed to show evidence of histologic changes consistent with murine dystrophy. When older dystrophic muscle (from 17-week-old dystrophic mice), muscle that failed to display spontaneous regeneration, was transplanted, it displayed remarkable regenerative capacity. It was suggested that the cessation of spontaneous regeneration in older dystrophic murine muscle is due not to exhaustion of myosatellite cell proliferative capacity, but rather to age-related loss of the mitogenic effect of dystrophy on the myosatellite cells of dystrophic muscle.

Aging↗

PubMed1986

Morphological aspects of muscle fiber regeneration.

Although striated muscle displays remarkable regenerative potential, the three-dimensional cytoarchitecture of the regenerated myofibers is different from that of myofibers formed during fetal development. It has been demonstrated with spaced, serial ultrathin sections that the regenerating myotubes that occur spontaneously (i.e., without secondary trauma) in dystrophic (dy2J) murine muscle and the regenerating fibers found in free whole-muscle transplants of normal, murine extensor digitorum longus muscles branch and recombine, forming a complex syncytium. Multiple motor end-plate regions are observed on the branched syncytia found in dystrophic muscle. Branched fibers persist in long-term grafts and are found with a frequency that indicates that they should be of physiological significance. Although the number of myofibers found in long-term grafts is approximately 68% of that found in control muscle, comparison of the diameter distributions of the regenerated muscle fibers with age-matched control fibers indicates that many of the regenerating fibers fail to achieve normal size. Type IIb fibers appear to be more growth inhibited than type IIa fibers. The size of the motoneuron pool to grafted muscles is smaller than that to control muscles.

Actin Cytoskeleton↗

PubMed1984

Size and location of the motoneuron pool supplying normal and orthotopically transplanted muscles.

Following intramuscular placement of horseradish peroxidase (HRP) into the 129ReJ mouse extensor digitorum longus muscle, 15 +/- 1 (S.E.M.) labeled motoneurons were found in the antero-lateral motor column, between the exits of spinal roots L3 and L4. When HRP was placed in 100-day orthotopic whole muscle transplants of the extensor digitorum longus muscle, the location of the motoneurons supplying the graft was similar to that of control muscle; however, the number of motoneurons innervating the grafts was variable and usually reduced (7 +/- 1 S.E.M.).

Animals↗

PubMed1984

The organogenesis of murine striated muscle: a cytoarchitectural study.

The ultrastructure and the three-dimensional cytoarchitecture of the developing murine extensor digitorum longus muscle has been studied in spaced, serial, transverse and longitudinal ultrathin sections of the muscles of 12-, 14-, 16-, and 18-day in utero, newborn, and 5-day-old 129 ReJ mice. Despite the fact that in vivo myogenesis is asynchronous (i.e., during most of the fetal period, multiple stages of myogenesis can be seen in a single developing muscle mass), a distinct temporal pattern of development can be seen across the entire width and length of the developing muscle. At 12 days in utero, the developing extensor digitorum longus muscle consists of primary myotubes surrounded by a pleomorphic population of mononucleated cells devoid of myofilaments. At this stage, blood vessels and nerves are found peripheral to but not within the developing muscle mass. A delay of 2 days occurs between the time of formation of the primary and secondary myotubes. Clusters (consisting of one primary myotube and secondary myotubes), axon bundles, capillaries, and primitive motor endplates are found in the muscle by 16 days in utero. Evidence is presented consistent with the hypothesis that cluster formation and cluster dispersal occur simultaneously in the developing muscle, beginning as early as 16-days in utero. By 18 days in utero, many of the primary myotubes of the cluster and the independent myotubes (i.e., single myotubes enclosed in their own basal lamina) have begun to acquire the polygonal shape, fascicular arrangement, and ultrastructure characteristic of more mature myofibers. At birth, clusters are infrequently encountered, and intramuscular axons have begun to undergo myelination. At this time, the only undifferentiated, mononucleated cells present in the muscle are myosatellite cells. The first week postnatal was characterized by further maturation of the myofibers.

Animals↗