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PubMed2005

A dual fate of the hindlimb muscle mass: cloacal/perineal musculature develops from leg muscle cells.

The cloaca serves as a common opening to the urinary and digestive systems. In most mammals, the cloaca is present only during embryogenesis, after which it undergoes a series of septation events leading to the formation of the anal canal and parts of the urogenital tract. During embryogenesis it is surrounded by skeletal muscle. The origin and the mechanisms regulating the development of these muscles have never been determined. Here, we show that the cloacal muscles of the chick originate from somites 30-34, which overlap the domain that gives rise to leg muscles (somites 26-33). Using molecular and cell labelling protocols, we have determined the aetiology of cloacal muscles. Surprisingly, we found that chick cloacal myoblasts first migrate into the developing leg bud and then extend out of the ventral muscle mass towards the cloacal tubercle. The development of homologous cloacal/perineal muscles was also examined in the mouse. Concordant with the results in birds, we found that perineal muscles in mammals also develop from the ventral muscle mass of the hindlimb. We provide genetic evidence that the perineal muscles are migratory, like limb muscles, by showing that they are absent in metd/d mutants. Using experimental embryological procedures (in chick) and genetic models (in chick and mouse), we show that the development of the cloacal musculature is dependent on proximal leg field formation. Thus, we have discovered a novel developmental mechanism in vertebrates whereby muscle cells first migrate from axially located somites to the pelvic limb, then extend towards the midline and only then differentiate into the single cloacal/perineal muscles.

Animals↗

PubMed2000

Genetics of muscle determination and development.

Skeletal muscles in vertebrates develop from somites as the result of patterning and cell type specification events. Here, we review the current knowledge of genes and signals implicated in these processes. We discuss in particular the role of the myogenic determination genes as deduced from targeted gene disruptions in mice and how their expression may be controlled. We also refer to other transcription factors which collaborate with the myogenic regulators in positive or negative ways to control myogenesis. Moreover, we review experiments that demonstrate the influence of tissues surrounding the somites on the process of muscle formation and provide model views on the underlying mechanisms. Finally, we present recent evidence on genes that play a role in regeneration of muscle in adult organisms.

Animals↗

PubMed1987

Primary, secondary and tertiary myotubes in developing skeletal muscle: a new approach to the analysis of human myogenesis.

Monoclonal antibodies to myosins have been used to describe and define the appearance and maturation of 3 different classes of myotube in developing human quadriceps muscle. Five monoclonal antibodies were used: (i) MAb A against human slow myosin heavy chain; (ii) MAb B against a myosin heavy chain present in most adult Type 2 fibres; (iii) MAb C against myosin heavy chain present in all mature and immature Type 2 fibres; (iv) MAb D, with similar reactivity to MAb C; (v) MAb E against human embryonic myosin. The combined use of two of these antibodies (A and B) enables the confident early identification of each of 3 classes (primary, secondary, tertiary) of myotubes, which appear sequentially during myogenesis. Our results show that induction of slow myosin heavy chain synthesis is a biphasic phenomenon in developing human skeletal muscle. Slow myosin heavy chain was present in all the earliest (9 weeks gestation) primary myotubes, but was not detected in secondary or tertiary myotubes until about 29 weeks gestation. Each stage of fetal muscle development has a characteristic immunocytochemical pattern which reveals cellular heterogeneity not evident on myosin ATPase histochemistry. Myosin immunocytochemistry may usefully be applied to assess the gestational age of fetuses. A new interpretation of human skeletal muscle development is proposed, based on the separate programming of 3 different kinds of myotube. This may be important in the analysis of diseased muscle in which developmental abnormalities or regeneration are present.

Antibodies, Monoclonal↗

PubMed2006

PDGF and microvessel wall remodeling in adult rat lung: imaging PDGF-AA and PDGF-Ralpha molecules in progenitor smooth muscle cells developing in experimental pulmonary hypertension.

Smooth muscle cells are mostly absent from the walls of microvessels in the adult lung but develop in large numbers as part of the pathology of human and experimental pulmonary hypertensions (PHs). We have previously shown, in an in vivo model of experimental PH, that mesenchymal (interstitial) fibroblasts and intermediate cells are the progenitors of these cells. Although smooth muscle cell development is a defining pathophysiological feature of human PH, little is known about the angiogenic signaling molecules responsible. Here, we report data for platelet-derived growth factor AA (PDGF-AA) and PDGF-Ralpha, two components of an important signaling pathway for fibroblast and myofibroblast proliferation and migration. Using antibodies linked to protein-A gold and high-resolution imaging techniques, we analyzed the expression of these molecules as smooth muscle cells developed from progenitor cell populations and in endothelial cells of the same microvessels. PDGF-AA was highly expressed by each cell type in control lung. As PH developed, the number of antigenic sites for PDGF-AA decreased with time. PDGF-Ralpha expression levels in the control lung were low, relative to the ligand, and fell in PH. These data show, for the first time, a marked phenotypic shift in expression levels of the PDGF-AA isoform and its receptor tyrosine kinase in the progenitor smooth muscle cells developing in the microvessels of the adult hypertensive lung.

Animals↗

PubMedDate not supplied

PDGF and microvessel wall remodeling in adult lung: imaging PDGF-Rbeta and PDGF-BB molecules in progenitor smooth muscle cells developing in pulmonary hypertension.

Smooth muscle cells are relatively rare cells in the microvessels of the normal adult lung but develop in high numbers in the clinical pulmonary hypertensions (PHs). Understanding this cellular response has profound implications for determining the pathogenesis of PH, and for the development of therapeutic strategies, yet little is known of the angiogenic molecules responsible. The authors have previously shown that interstitial fibroblasts, and intermediate cells, are the progenitors of smooth muscle cells developing in adult lung microvessels in an in vivo model of experimental PH. The present study evaluates PDGF-Rbeta/PDGF-BB, an important angiogenic signaling pathway, using antibodies linked to protein A-gold (pA-AU) and quantitative high-resolution imaging techniques to detect expression by these cells. Each progenitor cell type in the control lung expressed PDGF-Rbeta and PDGF-BB. In the hypertensive lung, PDGF-Rbeta was highly expressed by fibroblasts developing as perivascular cells, the mean number of pA-AU labeled antigenic sites per cell profile, and their density (microm(-2)), increasing with time: in intermediate cells the mean number of sites per cell profile, although not their density (microm(-2)), also increased with time but less so than in the fibroblasts. In clear contrast to the RTK, constitutive expression levels of PDGF-BB were low in each progenitor cell type and remained restricted in the hypertensive lung.

Animals↗

PubMed1993

mRNA for cardiac calcium channel is expressed during development of skeletal muscle.

During the early development of skeletal muscle, cardiac isotypes of several contractile proteins are known to be transiently expressed. We report here that skeletal muscle developing in vivo, as well as primary cultures derived from skeletal muscle, express mRNA encoding the cardiac dihydropyridine-sensitive calcium channel. The mRNA is detectable at high concentration at the earliest stage tested in vivo and diminishes rapidly in concentration as myofibers mature. The concentration of the cardiac calcium channel mRNA also diminishes during the in vivo development of skeletal muscle in a genetically paralyzed mouse (mdg), indicating that muscle contractile activity is not necessary for the down-regulation. In contrast, mRNA for the skeletal muscle-specific calcium channel accumulates gradually in developing skeletal muscle. A similar temporal pattern of expression is also seen in primary cultures of skeletal myotubes. These results raise the question of whether the cardiac calcium channel may be functionally important during the early development of skeletal myofibers.

3T3 Cells↗

PubMed1995

Monitoring development and pathology of Drosophila indirect flight muscles using green fluorescent protein.

We describe the use of a green fluorescent protein (GFP) reporter construct to monitor indirect flight muscle development in normal and mutant Drosophila melanogaster strains. We used polymerase chain reaction to amplify a portion of the Act88F actin gene that includes 1420 nucleotides of flanking DNA, the transcription start, first intron, and initiator codon, incorporating the fragment into the Drosophila germ line transformation vector pCaSpeR. We fused the fragment to the gene encoding green fluorescent protein of the bioluminescent jellyfish Aequorea victoria. We could detect GFP protein in transgenic strains and found that its accumulation, conveniently visualized in living flies using epifluorescence microscopy, was limited to the indirect flight muscles. GFP fluorescence can be used to visualize all stages of flight muscle development subsequent to myoblast fusion nad facilitates the detection of morphological changes in fibers caused by particular mutations.

Actins↗

PubMed1985

Spatial distribution and size of acetylcholine receptor clusters determined by motor nerves in developing chick muscles.

The size and distribution of acetylcholine receptor clusters (AChR-C) on normal and aneural developing muscle fibres of the chick wing were studied by labelling AChR with fluorescent conjugates of alpha-bungarotoxin (alpha-BGT). AChR-C of a size typical of initial synaptic contacts (5 micron long) were present at 7 days incubation, shortly after the appearance of nerves, and were grouped in bands corresponding to muscle nerve branches. A regular distribution of large (approximately equal to 5 micron) AChR-C separated by 100-200 micron had developed by 10-14 days in the slow-tonic anterior latissimus dorsi and ulnimetacarpalis dorsalis muscles. The role of motor innervation in the formation of AChR-C was assessed by removing the brachial neural tube at 2 days incubation in order to prevent nerves entering the wing. Neural-tube removal prevented the appearance of the large AChR-C normally associated with the early synaptic contacts. Small AChR-C (less than 2 micron long) appeared in aneural muscles, but these were not grouped into bands characteristic of the large AChR-C in normal muscles. The results suggest that the formation of junctional AChR-C is dependent on nerves.

Animals↗

PubMed1981

Outward currents in developing Drosophila flight muscle.

The development of two different voltage-sensitive potassium channels was studied in Drosophila flight muscle by voltage clamp techniques. Early in development active channels are not present in the membrane. The first channels to appear are the A current channels, which carry a fast, rapidly inactivating potassium current. The channels for delayed rectification appear later. Channels carrying inward current also appear only after the A current channels. During development, the A current may be easily studied in isolation from other currents and thus provides a desirable system for studying the genetic determinants of this current.

Animals↗

PubMed1994

Comparison of postnatal development of anterior latissimus dorsi (ALD) muscle in heavy- and light-weight strains of turkey (Meleagris gallopavo).

ALD muscle development was studied from day 2 to week 15 in males of two turkey strains. At 15 weeks, the heavy-weight (HW) strain weighted 2.2 times as much as the light-weight strain (LW). Morphometric and immunocytochemical analysis showed the presence of small fibers in HW ALD muscle which simultaneously accumulated ventricular and embryonic fast myosin heavy chain isoforms. The appearance of these nascent myofibers suggests that hyperplasia contributes to the growth of HW ALD muscle.

Age Factors↗

PubMed1991

Reinnervation of developing rat muscle by non-axotomized motoneurons.

To study the ability of developing motoneurons to reinnervate their denervated muscle, axotomized motoneurons in rat neonates and pups were retrogradely labeled with two fluorescent tracers. Fluorogold (FG), a long-lasting fluorescent dye, was injected into intercostal muscle T8 to retrogradely label the motoneurons that innervated it. Two days later intercostal nerves T7-T9 were cut. The intercostal muscle denervated at birth was reinnervated within 10-20 days, as evidenced by nerve-evoked muscle contraction. Three weeks following axotomy, tetramethylrhodamine isothiocyanate (TRITC) was injected into the same muscle to label the motoneurons that reinnervated it. The motoneurons double-labeled with FG and TRITC were, therefore, axotomized motoneurons that regenerated to reinnervate T8. In neonates, axotomy resulted in a significant reduction in the number of FG-labeled motoneurons, which suggests that axon transection at early postnatal days causes a massive motoneuron death. The percentage of double-labeled motoneurons was significantly smaller than that in non-axotomized rats. TRITC-labeled motoneurons constituted the majority of stained motoneurons; these were located in different nuclei than the intercostal motoneurons. These findings suggest that muscle reinnervation is, at least in part, by motoneurons which originally did not innervate intercostal muscle T8. Unlike axotomy at birth, axotomy performed 2-3 weeks after birth did not result in a significant motoneuron loss. The number of stained motoneurons labeled with both FG and TRITC was significantly smaller, however, than in non-axotomized spinal cords. Our data indicate that in pups only a small percentage of axotomized motoneurons reinnervated the denervated muscle.

Animals↗

PubMed1976

Adult motor patterns produced by moth pupae during development.

Muscle potentials were recorded extracellularly from developing pupae and adults of the saturniid moths Antheraea polyphemus and A. pernyi and the sphingid moth Manduca sexta. During the week prior to the terminal ecdysis, developing moths still enclosed within the pupal cuticle produced motor patterns similar to those recorded from adults during flight and shivering. The pupal patterns had a longer cycle time and were more variable than the adult motor patterns. Characteristic inter-family differences in adult motor patterns were apparent in pupal motor patterns. Development of motor patterns was followed over several days by observing individuals with chronically implanted leads. Early in the pupal period potentials were small and infrequent. The amount of activity gradually increased and became more patterned. As development proceeded adult patterns were produced for increasing lengths of time, although the patterns changed quickly and spontaneously. Restricting the wing movements of A. polyphemus adults increased the cycle time, increased the number of spikes per burst in muscles opposing the restraint, and did not alter the interspike interval within a burst. The flight patterns produced by pharate moths, in which the wings are also immobile, also have a longer cycle time than that of adult flight, but the number of spikes per burst the same and the interspike interval is longer than in adult flight. These observations suggest that the differences between pupal and adult patterns are not necessarily due to the confinement of the wings by the pupal cuticle.

Action Potentials↗

PubMed1997

Skeletal muscle satellite cells cultured in simulated microgravity.

Satellite cells are postnatal myoblasts responsible for providing additional nuclei to growing or regenerating muscle cells. Satellite cells retain the capacity to proliferate and differentiate in vitro and, therefore, provide a useful model to study postnatal muscle development. Most culture systems used to study postnatal muscle development are limited by the two-dimensional (2-D) confines of the culture dish. Limiting proliferation and differentiation of satellite cells in 2-D could potentially limit cell-cell contacts important for developing the level of organization in skeletal muscle obtained in vivo. Culturing satellite cells on microcarrier beads suspended in the High-Aspect-Ratio-Vessel (HARV) designed by NASA provides a low shear, three-dimensional (3-D) environment to study muscle development. Primary cultures established from anterior tibialis muscles of growing rats (approximately 200 gm) were used for all studies and were composed of greater than 75% satellite cells. Different inoculation densities did not affect the proliferative potential of satellite cells in the HARV. Plating efficiency, proliferation, and glucose utilization were compared between 2-D culture and 3-D HARV culture. Plating efficiency (cells attached divided by cells plated x 100) was similar between the two culture systems. Proliferation was reduced in HARV cultures and this reduction was apparent for both satellite cells and nonsatellite cells. Furthermore, reduction in proliferation within the HARV could not be attributed to reduced substrate availability because glucose levels in medium from HARV and 2-D cell culture were similar. Morphologically, microcarrier beads within the HARV were joined together by cells into 3-D aggregates composed of greater than 10 beads/aggregate. Aggregation of beads did not occur in the absence of cells. Myotubes were often seen on individual beads or spanning the surface of two beads. In summary, proliferation and differentiation of satellite cells on microcarrier beads within the HARV bioreactor results in a 3-D level of organization that could provide a more suitable model to study postnatal muscle development than is currently available with standard culture methods.

Animals↗

PubMed1986

The role of innervation in the establishment of the topographical distribution of primary myotube types during development.

Many avian muscles contain a characteristic topographical distribution of fibre types. In order to study the role of nerves in the establishment and distribution of these fibre types, monoclonal antibodies (McAb) to the heavy chain subunit of myosin (MHC) were produced. The anti-fast McAb (2B12) bound to adult fast MHC and cross-reacted with the embryonic isoform of MHC. The anti-slow McAb (3D1) bound specifically to the heavy chain of slow myosin 2. By indirect immunofluorescence, anti-fast (2B12) stained all myotubes in the anterior latissimus dorsi and triceps and biceps muscles at stage 37 (11 days embryonic), whilst anti-slow (3D1) staining was largely restricted to the future slow fibres of these muscles. Brachial levels of the neural tube were surgically removed at stage 12 (2 days embryonic) so that muscles developed aneurally. Muscles at aneural stage 37 were smaller than normal, but the distribution of myotube types was not altered; all myotubes present still stained with anti-fast antibody while anti-slow staining was restricted to the anterior latissimus dorsi and myotubes in the deep parts of the triceps brachii pars scapularis, triceps brachii par humeralis and biceps brachii muscles (the future slow fibres of normal muscles). The results suggest that despite an overall reduction in MHC in aneural muscles, specialized fast and slow primary myotubes arise independently of the nerve in appropriate regions of the muscle.

Animals↗

PubMed1984

Structural differences in the subfragment 1 and rod portions of myosin isozymes from adult and developing rat skeletal muscles.

During development of fast contracting skeletal muscle in the rat hindleg, embryonic and neonatal forms of the myosin heavy chain are present prior to the accumulation of the adult fast type ( Whalen , R. G., Sell, S. M., Butler-Browne, G.S., Schwartz, K., Bouveret, P., and Pinset -H arstr öm, I. (1981) Nature (Lond.) 292, 805-809). Polypeptide mapping of the heavy chain subunit using partial proteolysis in the presence of sodium dodecyl sulfate has shown differences in the cleavage patterns for these various heavy chains. Using this technique, we have now examined subfragments, which represent functional domains, from several different myosin isozymes. The heavy chains of the S-1 subfragments containing either light chain 1 or light chain 3 are indistinguishable for the neonatal or fast myosin isozymes. We also isolated the S-1 fragments and the alpha-helical COOH-terminal half of the molecule (rod) from rat embryonic, neonatal, and adult fast and slow myosin, as well as myosin from cardiac ventricles. All of these S-1 and rod fragments were different, indicating that the previously reported differences among these different myosin heavy chain isozymes are located in both the S-1 and rod subfragments for all myosins examined. However, the polypeptide maps of neonatal and adult fast S-1 show clear similarities, as do the maps of slow and cardiac S-1. These similarities in the two pairs of polypeptide maps were confirmed by the results of immunoblotting experiments using antibodies to adult fast and to slow myosin.

Age Factors↗

PubMed1979

Ultrastructure of human intramuscular blood vessels in development.

Muscles from human foetuses of nine weeks to nine months development were examined by electron microscopy. Capillaries, arteries and veins are frequent in the human foetal muscle at all stages of development. At nine weeks the vessels have the appearance of capillaries and basement membrane lies around the endothelial cells forming them. The capillaries are of continuous type which do not have apertures in their walls. Tight junctions are seen at some zones of adjacent endothelial cells of the capillaries. Sometimes, pericytes are also seen. At sixteen weeks vessels having the features of veins and of arteries can be identified between the muscle cells. Regarding the arteries, some of the endothelial cells are united to the smooth muscle cells and the intimal elastic lamina is interrupted where these cells approximate. The significance of this junction may be to anchor the intima to the media.

Arteries↗

PubMed1991

Changes in the protein kinase C activity or rat sternomastoid muscle during development and after denervation.

The relationship between the activity of protein kinase C (PKC) and muscle innervation was explored in the rat sternomastoid muscle (SM) from day 18 of gestation (E18) to adult age. Between E18 and birth, PKC activity rose 5-fold, and during the day after birth, diminished to a level characteristic of the mature muscle. The rise chiefly occurred in the neural part of the muscle, in both the membrane and the cytosol fractions. Between E18 and day 5 after birth, the ratios of membrane to cytosol PKC activity rose from 0.5 to 10 and 3 respectively in the neural and aneural parts of the muscle. Denervation of adult SM reduced PKC activity by half in the membrane fraction of the neural part but did not significantly change it in the membrane or cytosol fractions of the aneural parts. These results suggest that innervation plays an important part in determining the level of PKC activity in muscle.

Aging↗

PubMed1990

[Effects of denervation of the masseter nerve and bite raising on the masseter muscle of developing rats].

The effects of masseteric denervation and bite raising on muscle fiber type differentiation were examined in the masseteric muscle of developing rats by histological and histochemical studies. Three-week-old Wistar rats had of their masseteric nerve at right side dissected, and a part of them were bite raised at anterior region one week after denervation for three weeks. The unoperated side and number of sham animals served as control. The animals were killed 3, 7, 10, 14, 21, 28, 35, 49 days later and then their masseter muscles were removed wholly and weighed them. After freezing of superficial and deep masseter muscles serial sections were made and HE staining carried out, ATPase staining and NADH-TR staining. On the photographs of the HE stained specimens, I measured the diameter of the muscle fibers. Gross findings on the denervated group, revealed the lower incisors shifted to unoperated side and on with the bite raising group, it shifted to the operated side. In denervated animals, the wet weight of the masseter muscle had decreased significantly. The masseteric muscle fibers in three-old-week control rats were undifferentiated on ATPase staining, but it became well differentiated on and after four-old-week. The superficial masseter of control mainly composed of type 2B fibers. In the deep masseter, about 10% type 1 and 2C fibers were found in limited area around the muscle spindles, and surrounded with type 2A and 2B fibers. In superficial masseter muscle of the denervated group, the percentage of type 2A fibers increased, and that of type 2B fibers decreased. The type 2C fibers were found from the 21st days after denervation. In deep masseter muscles, the percentage of type 1 and 2A fibers decreased, and that of type 2B and 2C fibers increased. In the denervated bite raising group, the composition of the muscle fiber type approached the control group. These results as above suggested, masseteric denervation causes degeneration of muscle fiber composition, and the possibility that early preventive treatment like bite raising may recover the muscle fiber composition normally.

Animals↗