Distribution of N-CAM in synaptic and extrasynaptic portions of developing and adult skeletal muscle

Previous studies of denervated and cultured muscle have shown that the expression of the neural cell adhesion molecule (N-CAM) in muscle is regulated by the muscle's state of innervation and that N-CAM might mediate some developmentally important nerve-muscle interactions. As a first step in learning whether N-CAM might regulate or be regulated by nerve-muscle interactions during normal development, we have used light and electron microscopic immunohistochemical methods to study its distribution in embryonic, perinatal, and adult rat muscle. In embryonic muscle, N-CAM is uniformly present on the surface of myotubes and in intramuscular nerves; N-CAM is also present on myoblasts, both in vivo and in cultures of embryonic muscle. N-CAM is lost from the nerves as myelination proceeds, and from myotubes as they mature. The loss of N-CAM from extrasynaptic portions of the myotube is a complex process, comprising a rapid rearrangement as secondary myotubes form, a phase of decline late in embryogenesis, a transient reappearance perinatally, and a more gradual disappearance during the first two postnatal weeks. Throughout embryonic and perinatal life, N-CAM is present at similar levels in synaptic and extrasynaptic regions of the myotube surface. However, N-CAM becomes concentrated in synaptic regions postnatally: it is present in postsynaptic and perisynaptic areas of the muscle fiber, both on the surface and intracellularly (in T-tubules), but undetectable in portions of muscle fibers distant from synapses. In addition, N-CAM is present on the surfaces of motor nerve terminals and of Schwann cells that cap nerve terminals, but absent from myelinated portions of motor axons and from myelinating Schwann cells. Thus, in the adult, N-CAM is present in synaptic but not extrasynaptic portions of all three cell types that comprise the neuromuscular junction. The times and places at which N-CAM appears are consistent with its playing several distinct roles in myogenesis, synaptogenesis, and synaptic maintenance, including alignment of secondary along primary myotubes, early interactions of axons with myotubes, and adhesion of Schwann cells to nerve terminals.     

Role of nerve and muscle factors in the development of rat muscle spindles

The soleus muscles of fetal rats were examined by electron microscopy to determine whether the early differentiation of muscle spindles is dependent upon sensory innervation, motor innervation, or both. Simple unencapsulated afferent-muscle contacts were observed on the primary myotubes at 17 and 18 days of gestation. Spindles, encapsulations of muscle fibers innervated by afferents, could be recognized early on day 18 of gestation. The full complement of spindles in the soleus muscle was present at day 19, in the region of the neuromuscular hilum. More afferents innervated spindles at days 18 and 19 of gestation than at subsequent developmental stages, or in adult rats; hence, competition for available myotubes may exist among afferents early in development. Some of the myotubes that gave rise to the first intrafusal (bag2) fiber had been innervated by skeletomotor (alpha) axons prior to their incorporation into spindles. However, encapsulated intrafusal fibers received no motor innervation until fusimotor (gamma) axons innervated spindles 3 days after the arrival of afferents and formation of spindles, at day 20. The second (bag1) intrafusal fiber was already formed when gamma axons arrived. Thus, the assembly of bag1 and bag2 intrafusal fibers occurs in the presence of sensory but not gamma motor innervation. However, transient innervation of future bag2 fibers by alpha axons suggests that both sensory and alpha motor neurons may influence the initial stages of bag2 fiber assembly. The confinement of nascent spindles to a localized region of the developing muscle and the limited number of spindles in developing muscles in spite of an abundance of afferents raise the possibility that afferents interact with a special population of undifferentiated myotubes to form intrafusal fibers.     

Formation of primary and secondary myotubes in rat lumbrical muscles

Numbers of myoblasts, primary myotubes and secondary myotubes in developing rat embryo hindlimb IVth lumbrical muscles were counted at daily intervals up until the time of birth, using electron microscopy. Motoneurone death at the spinal cord level supplying the lumbricals was assessed by counting axons in the 4th lumbar ventral root. Death of the motoneurones that supply the intrinsic muscles of the hindfoot was monitored by comparing the timecourse of development of total muscle choline acetyltransferase activity in control embryos with that in embryos where motoneurone death was inhibited by chronic paralysis with TTX, and by counting axons in the mixed nerve trunks at the level of the ankle at daily intervals. Condensations of undifferentiated cells marking the site of formation of the muscle were seen on embryonic day 15 (E15). Primary myotubes began to appear on E16 and reached a stable number (102 +/- 4) by E17. Secondary myotubes first appeared two days later, on E19, and numbered 280 at the time of birth (E22). The adult total of about 1000 muscle fibres, derived from both primary and secondary myotubes, was reached at postnatal day 7 (PN7) so considerable generation of secondary myotubes occurred after birth. There was a linear correlation between the number of undifferentiated mononucleate cells in a muscle and the rate of formation of secondary myotubes. The major period of motoneurone death in lumbar spinal cord was during E16-E17, when axon numbers in the L4 ventral root fell from 12,000 to 4000, but a discontinuity in the curve of muscle ChAT activity versus time indicated that death in the lumbrical motor pool occurred during E17-E19, after all primary myotubes had formed and before generation of secondary myotubes began. We suggest that motoneurone death, by regulating the final size of the motoneurone pool, regulates the ratio of secondary to primary myotube numbers in a muscle.     

Neural determination of muscle fibre numbers in embryonic rat lumbrical muscles

The generation and development of muscle cells in the IVth hindlimb lumbrical muscle of the rat was studied following total or partial denervation. Denervation was carried out by injection of beta-bungarotoxin (beta-BTX), a neurotoxin which binds to and destroys peripheral nerves. Primary myotubes were generated in denervated muscles and reached their normal stable number on embryonic day 17 (E17). This number was not maintained and denervated muscles examined on E19 or E21 contained many degenerating primary myotubes. Embryos injected with beta-bungarotoxin (beta-BTX) on E12 or E13 suffered a partial loss of motoneurones, resulting in a reduced number of axons in the L4 ventral root (the IVth lumbrical muscle is supplied by axons in L4, L5 and L6 ventral roots) and reduced numbers of nerve terminals in the intrinsic muscles of the hindfoot. Twitch tension measurements showed that all myotubes in partly innervated muscles examined on E21 contracted in response to nerve stimulation. Primary myotubes were formed and maintained at normal numbers in muscles with innervation reduced throughout development, but a diminished number of secondary myotubes formed by E21. The latter was correlated with a reduction in number of mononucleate cells within the muscles. If beta-BTX was injected on E18 to denervate muscles after primary myotube formation was complete, E21 embryo muscles contained degenerating primary myotubes. After injection to denervate muscles on E19, the day secondary myotubes begin to form, E21 muscles possessed normal numbers of primary myotubes. In both cases, secondary myotube formation had stopped about 1 day after the injection and the number of mononucleate cells was greatly reduced, indicating that cessation of secondary myotube generation was most probably due to exhaustion of the supply of competent myoblasts. We conclude that nerve terminals regulate the number of secondary myotubes by stimulating mitosis in a nerve-dependent population of myoblasts and that activation of these myoblasts requires the physical presence of nerve terminals as well as activation of contraction in primary myotubes.     

Sound production by a recoiling system in the pempheridae and terapontidae

Sound‐producing mechanisms in fishes are extraordinarily diversified. We report here original mechanisms of three species from two families: the pempherid Pempheris oualensis, and the terapontids Terapon jarbua and Pelates quadrilineatus. All sonic mechanisms are built on the same structures. The rostral part of the swimbladder is connected to a pair of large sonic muscles from the head whereas the posterior part is fused with bony widenings of vertebral bodies. Two bladder regions are separated by a stretchable fenestra that allows forward extension of the anterior bladder during muscle contraction. A recoiling apparatus runs between the inner face of the anterior swimbladder and a vertebral body expansion. The elastic nature of the recoiling apparatus supports its role in helping the swimbladder to recover its initial position during sonic muscle relaxation. This system should aid fast contraction (between 100 and 250Hz) of sonic muscles. There are many differences between species in terms of the swimbladder and its attachments to the vertebral column, muscle origins, and morphology of the recoiling apparatus. The recoiling apparatus found in the phylogenetically‐related families (Glaucosomatidae, Pempheridae, Terapontidae) could indicate a new character within the Percomorpharia. J. Morphol. 277:717–724, 2016. © 2016 Wiley Periodicals, Inc.     

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.     

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.     

The development of the pattern of innervation in chicken hindlimb muscles: evidence for specification of nerve-muscle connections

The pattern of innervation in 13 chicken hindlimb muscles was studied at various stages of development in order to examine the mechanisms which regulate its formation. The pattern of innervation was visualized by examining the distribution of fiber types within each muscle. It was found that the fiber type which a myotube acquired was influenced by both its time of formation and its position within a muscle. The earliest generation of myotubes (primary) had a marked tendency to become type I fibers, whereas, in contrast, the later generation of myotubes (secondary) tended to differentiate into type II fibers. There were regions of muscle, however, in which primary myotubes differentiated into type II fibers and other regions in which secondary myotubes acquired type I characteristics. During the development of some muscles the pattern of fiber types changed as a result of either a selective loss of type I fibers or, in other cases, a rearrangement of some of the initial neuromuscular contacts. These observations are consistent with the pattern of innervation of a muscle being established as a result of differential projection patterns of fast and slow motoneurons and the existence of some type of chemoaffinity where particular myotubes are preferentially innervated by particular motoneurons.     

Cellular insertion of primary and secondary myotubes in embryonic rat muscles

Mammalian muscles develop from two populations of myotubes; primary myotubes appear first and are few in number; secondary myotubes appear later and form most of the muscle fibres. We have made an ultrastructural study to investigate how primary and secondary myotubes in embryonic rat muscles transmit tension during the period of their development. Primary myotubes extend from end to end of the muscle from the earliest times, and attach directly to the tendon. In contrast, newly formed secondary myotubes are short cells which insert solely into the primary myotubes by a series of complex interdigitating folds along which adhering junctions occur. As the secondary myotubes lengthen and mature, their insertion is progressively transferred from the primary myotube to the tendon proper. We suggest that this variable insertion of immature secondary myotubes, combined with complex patterns of innervation and electrical coupling in developing muscle, makes it difficult to predict the overall contribution of secondary myotubes to muscle tension development. This work extends other studies showing the unique relationship between a primary myotube and its associated secondary myotubes, indicating that these may constitute a developmental compartment.     

Absence of a seasonal cycle in the sonic neuromuscular system of the oyster toadfish

No seasonal pattern was found in total swimbladder weight, sonic muscle weight, or spinal sonic motor nucleus neuron soma size of the oyster toadfish Opsanus tau , indicating that additional nonsteroidal factors are also involved in the development of the toadfish sonic neuromuscular system.     

Neuromuscular synaptogenesis in wild-type and mutant zebrafish

Genetic screens for synaptogenesis mutants have been performed in many organisms, but few if any have simultaneously screened for defects in pre- and postsynaptic specializations. Here, we report the results of a small-scale genetic screen, the first in vertebrates, for defects in synaptogenesis. Using zebrafish as a model system, we identified seven mutants that affect different aspects of neuromuscular synapse formation. Many of these mutant phenotypes have not been previously reported in zebrafish and are distinct from those described in other organisms. Characterization of mutant and wild-type zebrafish, from the time that motor axons first arrive at target muscles through adulthood, has provided the new information about the cellular events that occur during neuromuscular synaptogenesis. These include insights into the formation and dispersal of prepatterned AChR clusters, the relationship between motor axon elongation and synapse size, and the development of precise appositions between presynaptic clusters of synaptic vesicles in nerve terminals and postsynaptic receptor clusters. In addition, we show that the mechanisms underlying synapse formation within the myotomal muscle itself are largely independent of those that underlie synapse formation at myotendinous junctions and that the outgrowth of secondary motor axons requires at least one cue not necessary for the outgrowth of primary motor axons, while other cues are required for both. One-third of the mutants identified in this screen did not have impaired motility, suggesting that many genes involved in neuromuscular synaptogenesis were missed in large scale motility-based screens. Identification of the underlying genetic defects in these mutants will extend our understanding of the cellular and molecular mechanisms that underlie the formation and function of neuromuscular and other synapses.     

ARIA is concentrated in the synaptic basal lamina of the developing chick neuromuscular junction

ARIA is a member of a family of polypeptide growth and differentiation factors that also includes glial growth factor (GGF), neu differentiation factor, and heregulin. ARIA mRNA is expressed in all cholinergic neurons of the central nervous systems of rats and chicks, including spinal cord motor neurons. In vitro, ARIA elevates the rate of acetylcholine receptor incorporation into the plasma membrane of primary cultures of chick myotubes. To study whether ARIA may regulate the synthesis of junctional synaptic acetylcholine receptors in chick embryos, we have developed riboprobes and polyclonal antibody reagents that recognize isoforms of ARIA that include an amino-terminal immunoglobulin C2 domain and examined the expression and distribution of ARIA in motor neurons and at the neuromuscular junction. We detected significant ARIA mRNA expression in motor neurons as early as embryonic day 5, around the time that motor axons are making initial synaptic contacts with their target muscle cells. In older embryos and postnatal animals, we found ARIA protein concentrated in the synaptic cleft at neuromuscular junctions, consistent with transport down motor axons and release at nerve terminals. At high resolution using immunoelectron microscopy, we detected ARIA immunoreactivity exclusively in the synaptic basal lamina in a pattern consistent with binding to synapse specific components on the presynaptic side of the basal lamina. These results support a role for ARIA as a trophic factor released by motor neuron terminals that may regulate the formation of mature neuromuscular synapses.     

Induced neuroma formation and target muscle perturbation in the giant fiber pathway of the Drosophila temperature-sensitive mutant shibire

Summary The temperature-sensitive mutation shibire (shi) in Drosophila melanogaster is thought to disrupt membrane recycling processes, including endocytotic vesicle pinch-off. This mutation can perturb the development of nerves and muscles of the adult escape response. After exposure to a heat pulse (6 h at 30° C) at 20 h of pupal development, adults have abnormal flight muscles. Wing depressor muscles (DLM) are reduced in number from the normal six to one or two fibers, and are composed of enlarged fibers that appear to represent fiber fusion; large spaces devoid of muscle fibers suggested fiber deletion. The normal five motor axons are present in the peripheral nerve PDMN near the ganglion. However, while some motor axons pass dorsally to the extant fibers, other motor axons lacking end targets pass into an abnormal posterior branch and terminate in a neuroma, i.e., a tangle of axons and glia without muscle target tissue. Hemisynapses are common in axons of the proximal PDMN and within the neuroma, but they are rarely seen in control (no heat pulse) shi or wild-type flies. All surviving muscle fibers are innervated; no muscle tissue exists without innervation. Fibrillar fine structure and neuromuscular synapses appear normal. Fused fibers have dual innervation, suggesting correct and specific matching of target tissue and motor axons. Motor axons lacking target fibers do not innervate erroneous targets but instead terminate in the neuroma. These results suggest developmental constraints and rules, which may contribute to the orderly, stereotyped development in the normal flight system. The nature of the anomalies inducible in the flight motor system in shi flies implies that membrane recycling events at about 20 h of pupal development are critical to the formation of the normal adult nerve-muscle pattern for DLM flight muscles.     

Functional morphology of the sonic apparatus in Ophidion barbatum (Teleostei, Ophidiidae)

Most soniferous fishes producing sounds with their swimbladder utilize relatively simple mechanisms: contraction and relaxation of a unique pair of sonic muscles cause rapid movements of the swimbladder resulting in sound production. Here we describe the sonic mechanism for Ophidion barbatum, which includes three pairs of sonic muscles, highly transformed vertebral centra and ribs, a neural arch that pivots and a swimbladder whose anterior end is modified into a bony structure, the rocker bone. The ventral and intermediate muscles cause the rocker bone to swivel inward, compressing the swimbladder, and this action is antagonized by the dorsal muscle. Unlike other sonic systems in which the muscle contraction rate determines sound fundamental frequency, we hypothesize that slow contraction of these antagonistic muscles produces a series of cycles of swimbladder vibration.     

THE FINE STRUCTURE OF MOTOR ENDPLATE MORPHOGENESIS

The fine structure of the developing neuromuscular junction of rat intercostal muscle has been studied from 16 days in utero to 10 days postpartum. At 16 days, neuromuscular relations consist of close membrane apposition between clusters of axons and groups of myotubes. Focal electron-opaque membrane specializations more intimately connect axon and myotube membranes to each other. What relation these focal contacts bear to future motor endplates is undetermined. The presence of a group of axons lying within a depression in a myotube wall and local thickening of myotube membranes with some overlying basal lamina indicates primitive motor endplate differentiation. At 18 days, large myotubes surrounded by new generations of small muscle cells occur in groups. Clusters of terminal axon sprouts mutually innervate large myotubes and adjacent small muscle cells within the groups. Nerve is separated from muscle plasma membranes by synaptic gaps partially filled by basal lamina. The plasma membranes of large myotubes, where innervated, simulate postsynaptic membranes. At birth, intercostal muscle is composed of separate myofibers. Soleplate nuclei arise coincident with the peripheral migration of myofiber nuclei. A possible source of soleplate nuclei from lateral fusion of small cells' neighboring areas of innervation is suspected but not proven. Adjacent large and small myofibers are mutually innervated by terminal axon networks contained within single Schwann cells. Primary and secondary synaptic clefts are rudimentary. By 10 days, some differentiating motor endplates simulate endplates of mature muscle. Processes of Schwann cells cover primary synaptic clefts. Axon sprouts lie within the primary clefts and are separated from each other. Specific neural control over individual myofibers may occur after neural processes are segregated in this manner.     

Innervation of developing intrafusal muscle fibers in the rat

The chronology of development of spindle neural elements was examined by electron microscopy in fetal and neonatal rats. The three types of intrafusal muscle fiber of spindles from the soleus muscle acquired sensory and motor innervation in the same sequence as they formed--bag2, bag1, and chain. Both the primary and secondary afferents contacted developing spindles before day 20 of gestation. Sensory endings were present on myoblasts, myotubes, and myofibers in all intrafusal bundles regardless of age. The basic features of the sensory innervation--first-order branching of the parent axon, separation of the primary and secondary sensory regions, and location of both primary and secondary endings beneath the basal lamina of the intrafusal fibers--were all established by the fourth postnatal day. Cross-terminals, sensory terminals shared by more than one intrafusal fiber, were more numerous at all developmental stages than in mature spindles. No afferents to immature spindles were supernumerary, and no sensory axons appeared to retract from terminations on intrafusal fibers. The earliest motor axons contacted spindles on the 20th day of gestation or shortly afterward. More motor axons supplied the immature spindles, and a greater number of axon terminals were visible at immature intrafusal motor endings than in adult spindles; hence, retraction of supernumerary motor axons accompanies maturation of the fusimotor system analogous to that observed during the maturation of the skeletomotor system. Motor endings were observed only on the relatively mature myofibers; intrafusal myoblasts and myotubes lacked motor innervation in all age groups. This independence of the early stages of intrafusal fiber assembly from motor innervation may reflect a special inherent myogenic potential of intrafusal myotubes or may stem from the innervation of spindles by sensory axons.     

Motor nerve topology reflects myocyte morphology in hamster retractor and epitrochlearis muscles

Neuromuscular activation is a primary determinant of metabolic demand and oxygen transport. The m. retractor and m. epitrochlearis are model systems for studying metabolic control and oxygen transport; however, the organization of muscle fibers and motor nerves in these muscles is unknown. We tested whether the topology of motor innervation was related to the morphology of muscle fibers in m. retractor and m. epitrochlearis of male hamsters ( approximately 100 g). Respective muscles averaged 47 and 12 mm in length 100 and 35 mg in mass. Staining for acetylcholinesterase revealed neuromuscular junctions arranged in clusters throughout m. retractor and as a central band across m. epitrochlearis, suggesting differences in fiber morphology. For both muscles, complete cross-sections contained approximately 1,700 fibers. Fiber cross-sectional areas were distributed nearly normal in m. epitrochlearis (mean = 1,559 +/- 17 microm(2)) and skewed left (P < 0.05) in m. retractor (mean = 973 +/- 15 microm(2)). Single fiber length (Lf) spanned muscle length (Lm) in m. epitrochlearis, while fibers tapered to terminate within m. retractor (Lf/Lm = 0.43 +/- 0. 02). With myelin staining, a single branch of ulnar nerve projected axons across the midregion of m. epitrochlearis. For m. retractor, the spinal accessory nerve branched to give rise to proximal and distal regions of innervation, with intermingling of axons between nerve branches. Nerve bundle cross-sections stained for acetylcholinesterase indicate that each motor axon projects to an average of 65 muscle fibers in m. epitrochlearis and 100 in m. retractor. Differences in fiber morphology, innervation topology, and neuromuscular organization may contribute to the heterogeneity of metabolic demand and oxygen supply in skeletal muscle.     

Dissection of the Transversus Abdominis Muscle for Whole-mount Neuromuscular Junction Analysis

Analysis of neuromuscular junction morphology can give important insight into the physiological status of a given motor neuron. Analysis of thin flat muscles can offer significant advantage over traditionally used thicker muscles, such as those from the hind limb (e.g. gastrocnemius). Thin muscles allow for comprehensive overview of the entire innervation pattern for a given muscle, which in turn permits identification of selectively vulnerable pools of motor neurons. These muscles also allow analysis of parameters such as motor unit size, axonal branching, and terminal/nodal sprouting. A common obstacle in using such muscles is gaining the technical expertise to dissect them. In this video, we detail the protocol for dissecting the transversus abdominis (TVA) muscle from young mice and performing immunofluorescence to visualize axons and neuromuscular junctions (NMJs). We demonstrate that this technique gives a complete overview of the innervation pattern of the TVA muscle and can be used to investigate NMJ pathology in a mouse model of the childhood motor neuron disease, spinal muscular atrophy.     

Relationship of primary and secondary myogenesis to fiber type development in embryonic chick muscle.

The formation of fast and slow myotubes was investigated in embryonic chick muscle during primary and secondary myogenesis by immunocytochemistry for myosin heavy chain and Ca2(+)-ATPase. When antibodies to fast or slow isoforms of these two molecules were used to visualize myotubes in the posterior iliotibialis and iliofibularis muscles, one of the isoforms was observed in all primary and secondary myotubes until very late in development. In the case of myosin, the fast antibody stained virtually all myotubes until after stage 40, when fast myosin expression was lost in the slow myotubes of the iliofibularis. In the case of Ca2(+)-ATPase, the slow antibody also stained all myotubes until after stage 40, when staining was lost in secondary myotubes and in the fast primary myotubes of the posterior iliotibialis and the fast region of the iliofibularis. In contrast, the antibodies against slow muscle myosin heavy chain and fast muscle Ca2(+)-ATPase stained mutually exclusive populations of myotubes at all developmental stages investigated. During primary myogenesis, fast Ca2(+)-ATPase staining was restricted to the primary myotubes of the posterior iliotibialis and the fast region of the iliofibularis, whereas slow myosin heavy chain staining was confined to all of the primary myotubes of the slow region of the iliofibularis. During secondary myogenesis, the fast Ca2(+)-ATPase antibody stained nearly all secondary myotubes, while primaries in the slow region of the iliofibularis remained negative. Thus, in the slow region of the iliofibularis muscle, these two antibodies could be used in combination to distinguish primary and secondary myotubes. EM analysis of staining with the fast Ca2(+)-ATPase antibody confirmed that it recognizes only secondary myotubes in this region. This study establishes that antibodies to slow myosin heavy chain and fast Ca2(+)-ATPase are suitable markers for selective labeling of primary and secondary myotubes in the iliofibularis; these markers are used in the following article to describe and quantify the effects that chronic blockade of neuromuscular activity or denervation has on these populations of myotubes.