Differentiation of fiber types in wing muscles during embryonic development: effect of neural tube removal

The embryonic precursors of the avian slow (type I and III) and fast (type II) fibers can be distinguished from each other early in muscle formation (stage 28, V. Hamburger and H. L. Hamilton, J. Morphol, 88, 49-92, 1951) on the basis of the differential sensitivity of their myosin ATPases. To test the neural dependence of fiber type differentiation, the source of motor innervation was eliminated by excision of the brachial neural tube at stages 16-18 before muscles are innervated. Removal of the brachial neural tube did not affect the number of primary myotubes in a sample muscle of the forelimb (ulnimetacarpalis dorsalis, UMD) up until stage 36. Myosin ATPase staining at a variety of pHs revealed the typical patterns of fiber types in muscles of neural-tube free embryos in stages 35-37. These muscles included the anterior latissimus dorsi, brachialis, and UMD which showed presumptive type III staining (type IIIEMB), the pronator superficialis and flexor carpi ulnaris which showed embryonic type II staining (type IIEMB), and the triceps brachii muscles which showed characteristic arrangements of both type IEMB and type IIEMB fibers. The normal patterns of type IEMB and type IIEMB myotubes were also seen in muscles containing a heterogeneous mixture of fiber types such as the biceps brachii, extensor metacarpi radialis, and adductor indicis muscles, although the intensity of acid-stable ATPase staining of the type IEMB myotubes in these muscles was lower than in innervated muscles. It is concluded that the earliest differentiation of muscle fiber types is independent of the nervous system.     

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.     

Differentiation of muscle fiber types in aneurogenic brachial muscles of the chick embryo

Cross-reinnervation studies performed ex ovo with newly hatched chicks demonstrate that peripheral motor neurons control the phenotypic characteristics of avian muscles. The present experiments were designed to determine whether or not nerves play a similar role during the initial expression of muscle fiber types. Previous experiments indicated that differentiation of specific fiber types occurs during the first week of embryogenesis, temporally coincident with the penetration of nerves within muscle masses. These observations suggested that peripheral nerves may be associated with the initial differentiation of fiber types. To test this hypothesis directly, anterior limb buds of the chick embryo were rendered aneurogenic by deletion of the brachial segment of the neural tube. To ensure a completely aneurogenic environment for developing brachial muscles, surgery was performed at day 2 in ovo before the exit of ventral root fibers. Experimental and control embryos from Stage (St) 25 (4.5 d) through St 45 (19d) were analyzed histochemically by a silver-cholinesterase reaction to detect nerves and by the myosin ATPase reaction, following alkali and acid preincubation, to determine the fiber type composition of the muscles. In addition, the total volume of aneurogenic and control muscles was compared. Results demonstrate that the characteristic myosin ATPase profiles of individual aneurogenic and innervated (control) muscles were identical throughout the entire period analyzed. Therefore, we conclude that these enzymic profiles are endogenously expressed and are not under neuronal control during early embryogenesis. Furthermore, the entire sequence of events from the migration of myogenic cells to the anterior limb bud through the division of the primary muscle masses to form individual brachial muscles proceeded on schedule in the absence of nerves. Since the growth of aneurogenic muscles was impaired, we conclude that during embryogenesis peripheral motor nerves are necessary initially for the proper growth of muscles and ultimately, for their survival. They are not involved, however, with either the initial formation or initial differentiation of individual brachial muscles.     

Motoneuron projection patterns in chick embryonic limbs with a double complement of dorsal thigh musculature

During the normal development of the chick, lateral motoneurons within the lumbosacral motor column of the spinal cord consistently project to muscles of dorsal origin within the limb while medial motoneurons project to muscles of ventral origin. To determine if specific cues arising from each type of target are the dominant guidance cues used by lateral and medial motoneurons to create this pattern, I examined motoneuron projections in embryonic chick limbs with a double complement of dorsal thigh musculature and no ventral musculature. Results indicate that cues associated with muscles of a specific developmental origin do not invariably dominate. Before and after the major period of motoneuron death, all muscles in dorsal limb regions (host) were innervated by lateral or dorsal pool neurons. Most ventrally positioned (donor) muscles were innervated by medial or ventral pool neurons. Only the donor iliofibularis, a muscle located very near to its original source of innervation, received projections from some lateral neurons. Within the limb proper, medial or ventral pool neurons projected to donor muscles in a patterned manner suggesting that they were following nonspecific regional cues and perhaps also responding to the availability of uninnervated target tissue. I conclude that axon sorting into distinct lateral and medial classes is independent of limb target complement and that subsequent pathway choice is a separate event governed by both specific target cues and other guidance mechanisms.     

Nerve-dependent accumulation of myosin light chain 3 in developing limb musculature

Myosin alkali light chain accumulation in developing quail limb musculature has been analysed on immunoblots using a monoclonal antibody which recognizes an epitope common to fast myosin light chain 1 (MLC1f) and fast myosin light chain 3 (MLC3f). The limb muscle of early embryos (i.e. up to day 10 in ovo) has a MLC profile similar to that observed in myotubes cultured in vitro; although MLC1f is abundant, MLC3f cannot be detected. MLC3f is first detected in 11-day embryos. To determine whether this alteration in MLC3f accumulation is nerve or hormone dependent, limb buds with and without neural tube were cultured as grafts on the chorioallantoic membrane of chick hosts. Although differentiated muscle develops in both aneural and innervated grafts, innervated grafts contain approximately three times as much myosin as aneural grafts. More significantly, although aneural grafts reproducibly accumulate normal levels of MLC1f, they fail to accumulate detectable levels of MLC3f. In contrast, innervated grafts accumulate both MLC1f and MLC3f, suggesting that the presence of neural tube in the graft promotes the maturation, as well as the growth, of muscle tissue. This is the first positive demonstration that innervation is necessary for the accumulation of MLC3f that occurs during normal limb development in vivo.     

Spinal cord-muscle relations: their role in neuro-muscular development in birds

In the present study we focused our attention on the role of spinal cord-muscle interactions in the development of muscle and spinal cord cells. Four experimental approaches were used: 1) muscle fiber-spinal cord co-culture; 2) chronic spinal cord stimulation in chick embryos; 3) direct electrical stimulation of the denervated chick muscle; 4) skeletal muscle transplantation in close apposition to the spinal cord in chick embryos. The characteristics of mATPase and energetic metabolism enzyme activities and of myosin isoform expression were used as markers for fiber types in two peculiar muscles, the fast-twitch PLD and the slow-tonic ALD. In vitro, in the absence of neurons, myoblasts can express some characteristics of either slow or fast muscle types according to their origin, while in the presence of neurons, muscle fiber differentiation seems to be related to the spontaneous rhythm delivered by the neurons. The in ovo experiments of chronic spinal cord stimulation demonstrate that the differentiation of the fast and slow muscle features appears to be rhythm dependent. In the chick, direct stimulation of denervated muscles shows that the rhythm of the muscle activity is also involved in the control of muscle properties. In chick embryos developing ALD, the changes induced by modifications of muscle tension demonstrate that this factor also influences muscle development. Other experiments show that muscle back-transplantation can alter the early spinal cord development.     

Differentiation of muscle fiber types in the chicken hindlimb

The differentiation of myotubes into fiber types was studied by examining the ATPase staining characteristics of chicken embryo thigh muscles. Two distinct fiber types, designated type IEMB and IIEMB, could be distinguished as early as stage 29. Paralysis of the embryo with d-tubocurarine prevented the differentiation of type IEMB but not type IIEMB characteristics. The two embryonic fiber types differed from each other, and mature type I and II fibers, in the acid and alkali labilities of their ATPases. Myotubes which were type IEMB at stage 29 matured into type I fibers, whereas those which were type IIEMB predominantly but not exclusively developed into type II fibers. The process of maturation involved sequential changes in the staining characteristics of the myotubes. Thus, the ultimate fiber type of a myotube can be detected long before it expresses its mature characteristics.     

Evidence for expression of a common myosin heavy chain phenotype in future fast and slow skeletal muscle during initial stages of avian embryogenesis

We have utilized a key biochemical determinant of muscle fiber type, myosin isoform expression, to investigate the initial developmental program of future fast and slow skeletal muscle fibers. We examined myosin heavy chain (HC) phenotype from the onset of myogenesis in the limb bud muscle masses of the chick embryo through the differentiation of individual fast and slow muscle masses, as well as in newly formed myotubes generated in adult muscle by weight overload. Myosin HC isoform expression was analyzed by immunofluorescence localization with a battery of anti-myosin antibodies and by electrophoretic separation with SDS-PAGE. Results showed that the initial myosin phenotype in all skeletal muscle cells formed during the embryonic period (until at least 8 days in ovo) consisted of expression of a myosin HC which shares antigenic and electrophoretic migratory properties with ventricular myosin and a distinct myosin HC which shares antigenic and electrophoretic migratory properties with fast skeletal isomyosin. Similar results were observed in newly formed myotubes in adult muscle. Future fast and slow muscle fibers could only be discriminated from each other in developing limb bud muscles by the onset of expression of slow skeletal myosin HC at 6 days in ovo. Slow skeletal myosin HC was expressed only in myotubes which became slow fibers. These findings suggest that the initial commitment of skeletal muscle progenitor cells is to a common skeletal muscle lineage and that commitment to a fiber-specific lineage may not occur until after localization of myogenic cells in appropriate premuscle masses. Thus, the process of localization, or events which occur soon thereafter, may be involved in determining fiber type.     

Slow and Fast Muscle Fibers Are Preferentially Derived from Myoblasts Migrating into the Chick Limb Bud at Different Developmental Times

Avian limb myoblasts originate from somites and migrate into the periphery during limb bud formation. It is not known how these precursors become arranged into a stereotyped pattern of muscles and primary fiber types. We used in vivo surgical transplantation and anatomical analyses of thigh muscle patterns to ask whether myoblasts migrating into the limb bud at different developmental times adopt different fates. When myoblast migration was interrupted by transplanting limb bud tissue to the coelomic cavity of a host embryo early in the migratory period (stages 16-early 17), few thigh muscles were found at stages 30-33. Primordia that were present corresponded to muscles that normally contain a majority of slow myotubes. In limbs transplanted slightly later (stages late 17-18), the only missing muscles were those that normally contain the highest numbers of fast myotubes. Parallel results were obtained in chimeric limbs made by transplanting a quail limb bud to a chick host at different times during the migratory period, an experimental situation in which the limbs were not depleted of muscle precursors or nerves. These findings suggest that the earliest myoblast migrants give rise mainly to slow primary myotubes, the later migrants to fast myotubes. To determine whether the early limb bud environment defines the fate of migrating myoblasts, we assessed fiber type patterns in limbs that developed from young limb bud tissue (stages 15-early 16) transplanted to older hosts (stage 17). A significant depletion of slow myosin-positive profiles was found within slow muscles. Fast muscles were generally normal in size. These results provide in vivo evidence that limb myoblast diversity arises prior to the entry of myoblasts into the limb. We suggest that there is a gradual change in the proportions of myoblasts capable of forming slow and fast fiber types, a change which may begin in the somites or early in the migratory period.     

Muscle type-specific myosin isoforms in crustacean muscles

Differential expression of multiple myosin heavy chain (MyHC) genes largely determines the diversity of critical physiological, histochemical, and enzymatic properties characteristic of skeletal muscle. Hypotheses to explain myofiber diversity range from intrinsic control of expression based on myoblast lineage to extrinsic control by innervation, hormones, and usage. The unique innervation and specialized function of crayfish (Procambarus clarkii) appendicular and abdominal musculature provide a model to test these hypotheses. The leg opener and superficial abdominal extensor muscles are innervated by tonic excitatory motoneurons. High resolution SDS-PAGE revealed that these two muscles express the same MyHC profile. In contrast, the deep abdominal extensor muscles, innervated by phasic motoneurons, express MyHC profiles different from the tonic profiles. The claw closer muscles are dually innervated by tonic and phasic motoneurons and a mixed phenotype was observed, albeit biased toward the phasic profile seen in the closer muscle. These results indicate that multiple MyHC isoforms are present in the crayfish and that differential expression is associated with diversity of muscle type and function.     

Differentiation of fiber types in aneural musculature of the prenatal rat hindlimb

The presynaptic neurotoxin, beta-bungarotoxin, was injected into rat fetuses in utero to destroy the innervation of their hindlimb muscles. These injections were made prior to the invasion of motor axons into the muscles and, in some cases, prior to the cleavage of individual muscles. Examination of the lateral motor column of the spinal cord showed a dramatic reduction (greater than 95%) in the number of motoneuron cell bodies. Staining of sections of the hindlimb with silver and with antibodies to neurofilament proteins and to a synaptic vesicle protein indicated that the muscles were aneural. Anti-myosin antibodies applied to sections of the hindlimb revealed that these aneural muscles by the 20th day of gestation had the same types of fibers as were present in normal muscles of the same age. Moreover, fiber types in most muscles showed their characteristic intramuscular distributions. These findings suggest that fiber types can differentiate in the absence of the nervous system. However, some fibers achieved their ultimate fiber type fate without passing through the normal sequence of myosin expressions. Moreover, some slow fibers lost their slow expression, suggesting that the maintenance of the slow differentiation may require innervation. Muscle growth was dramatically affected by the absence of motoneurons; some muscles were decreased in size and others disappeared completely. In muscles which had not degenerated by the time secondary myogenesis normally begins, secondary muscle fibers were generated indicating that the genesis of these fibers is not strictly nerve dependent. Because fiber types differentiate independently of the nervous system, this study suggests that motoneurons selectively innervate fiber types during normal development.     

Peripheral specification of Ia synaptic input to motoneurons innervating foreign target muscles

Muscle sensory neurons, called Ia afferents, make monosynaptic connections with functionally related sets of motoneurons in the spinal cord. Previous work has suggested that peripheral target muscles play a major role in determining the central connections of Ia afferents with motoneurons. Here, we ask whether motoneurons can also be influenced by their target muscles in terms of the monosynaptic input they receive from Ia afferents, by transplanting thoracic motoneurons into the lumbosacral spinal cord so that they innervate foreign muscles. Three or four segments of thoracic neural tube from stage 14-15 chicken embryos were transplanted to the lumbosacral region of stage 16-17 embryos, and electrophysiological recordings were made from transplanted motoneurons after the embryos had reached stage 38-40. Transplanted thoracic motoneurons innervated limb muscles and received monosynaptic inputs from Ia afferents. These connections were not random: Most of the connections were formed between Ia afferents and motoneurons projecting to the same muscle (homonymous connections). Few aberrant connections were found although the anatomical distribution of afferents in the transplant indicated that they had ample opportunity to contact inappropriate motoneurons. We conclude that although peripheral target cues are not sufficient to respecify an already committed motoneuron (turn a thoracic motoneuron into a lumbosacral motoneuron), they do provide sufficient information for Ia afferent input to be functionally correct.     

Chick and quail limb bud myoblasts, isolated at different times during muscle development, express stage-specific phenotypes when differentiated in culture

Evidence is presented which shows that myoblasts, isolated at different stages during chick and quail limb bud development, will form, in culture, myotubes which can be distinguished with a combination of morphological as well as biochemical criteria. Hind limb bud myoblasts isolated from 5-day-old embryos form very short myotubes which synthesize a myosin, the light chains of which are predominantly LC1F and LC2S. Myoblasts isolated from the limb buds of 7-8-day-old embryos form large myotubes which synthesize a myosin the light chains of which are predominantly LC1F, LC2S and LC2F. Myoblasts isolated from the thigh muscle of embryos older than 10 days form large myotubes which synthesize a myosin the light chains of which are predominantly LC1F and LC2F. These results have been confirmed by hybridization of the cellular mRNA with a molecular probe specific for LC2F. These results lead us to suggest the existence of at least two classes of myoblasts which appear at different times during limb bud development. The first class, or 'early' myoblasts, is present in the limb buds of 5-day-old embryos, whereas the second class, or 'late' myoblasts, is present in the muscles of embryos older than 8 days. This result, however, is also compatible with the hypothesis that all muscle cells are the same at all times during development, and that the different phenotypes simply reflect differences in the environmental conditions.     

Brachially innervated ectopic hindlimbs in the chick embryo. I. Limb motility and motor system anatomy during the development of embryonic behavior

The functional status of brachially innervated hindlimbs, produced by transplanting hindlimb buds of chick embryos in place of forelimb buds, was quantified by analyzing the number and temporal distribution of spontaneous limb movements. Brachially innervated hindlimbs exhibited normal motility until E10 but thereafter became significantly less active than normal limbs and the limb movements were more randomly distributed. Contrary to the findings with axolotls and frogs, functional interaction between brachial motoneurons and hindlimb muscles cannot be sustained in the chick embryo. Dysfunction is first detectable at E10 and progresses to near total immobility by E20 and is associated with joint ankylosis and muscular atrophy. Although brachially innervated hindlimbs were virtually immobile by the time of hatching (E21), they produced strong movements in response to electrical stimulation of their spinal nerves, suggesting a central rather than peripheral defect in the motor system. The extent of motoneuron death in the brachial spinal cord was not significantly altered by the substitution of the forelimb bud with the hindlimb bud, but the timing of motoneuron loss was appropriate for the lumbar rather than brachial spinal cord, indicating that the rate of motoneuron death was dictated by the limb. Measurements of nuclear area indicated that motoneuron size was normal during the motoneuron death period (E6-E10) but the nuclei of motoneurons innervating grafted hindlimbs subsequently became significantly larger than those of normal brachial motoneurons. Although the muscle mass of the grafted hindlimb at E18 was significantly less than that of the normal hindlimb (and similar to that of a normal forelimb), electronmicroscopic examination of the grafted hindlimbs and brachial spinal cords of E20 embryos revealed normal myofiber and neuromuscular junction ultrastructure and a small increase in the number of axosomatic synapses on cross-sections of motoneurons innervating grafted hindlimbs compared to motoneurons innervating normal forelimbs. The anatomical data indicate that, rather than being associated with degenerative changes, the motor system of the brachial hindlimb of late-stage embryos is intact, but inactive. © 1993 John Wiley & Sons, Inc.     

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.     

Effect of neonatal denervation on the distribution of fiber types in a mouse fast-twitch skeletal muscle

This study was designed to assess the changes in fiber-type distribution of the extensor digitorum longus (EDL) muscle of the mouse during the first 21 days of age following neonatal sciatic neurectomy. Denervated and normal muscles were compared at 7, 14, and 21 days of age and the normal EDL was also studied at 1 day of age. Frozen sections of the EDL were treated histochemically to detect NADH-tetrazolium reductase and myosin ATPase reactions. Quantitative assessment included measurements of cross-sectional areas and fiber counting. Denervation resulted in muscle atrophy which was due primarily to a decrease in individual fiber area as opposed to fiber loss. Histochemical maturation of the EDL was severely affected by neonatal denervation during the first three postnatal weeks. By 21 days, two extrafusal fiber types which were both oxidative could be distinguished. One type was highly atrophied and resembled an immature fiber exhibiting myosin ATPase staining at both acid and alkaline preincubation conditions, whereas another type was less atrophied and showed myosin ATPase staining resembling fast-twitch (type IIA) fibers. These findings emphasize the importance of an intact nerve supply in determining the phenotypic expression of skeletal muscle, and point to the early postnatal period as a critical stage in fiber type differentiation.     

Effect of neonatal denervation on the distribution of fiber types in a mouse fast-twitch skeletal muscle

Summary This study was designed to assess the changes in fiber-type distribution of the extensor digitorum longus (EDL) muscle of the mouse during the first 21 days of age following neonatal sciatic neurectomy. Denervated and normal muscles were compared at 7, 14, and 21 days of age and the normal EDL was also studied at 1 day of age. Frozen sections of the EDL were treated histochemically to detect NADH-tetrazolium reductase and myosin ATPase reactions. Quantitative assessment included measurements of cross-sectional areas and fiber counting. Denervation resulted in muscle atrophy which was due primarily to a decrease in individual fiber area as opposed to fiber loss. Histochemical maturation of the EDL was severely affected by neonatal denervation during the first three postnatal weeks. By 21 days, two extrafusal fiber types which were both oxidative could be distinguished. One type was highly atrophied and resembled an immature fiber exhibiting myosin ATPase staining at both acid and alkaline preincubation conditions, whereas another type was less atrophied and showed myosin ATPase staining resembling fast-twitch (type HA) fibers. These findings emphasize the importance of an intact nerve supply in determining the phenotypic expression of skeletal muscle, and point to the early postnatal period as a critical stage in fiber type differentiation.     

Migratory and organogenetic capacities of muscle cells in bird embryos

Summary The migratory and organogenetic capacities of muscle cells at different stages of differentiation were tested in heterospecific chick/quail recombinants. Grafts containing muscle cells were taken from the premuscular masses from 4- to 5-day quail embryos, from the limb or trunk muscles of 12-day embryonic and 4-day post-natal quails, and from experimentally produced bispecific premuscular masses in which the myoblasts are of quail origin and the connective tissue cells of chick origin. Grafts were implanted into 2-day chick embryos in place of the somitic mesoderm at the limb level. Hosts were examined 4 to 7 days after operation.After implantation of a piece of premuscular mass, quail cells were found at and around the site of the graft in the truncal region and within the limb as far as the autopod. Quail cells participated predominantly in the trunk and limb musculature, which contained a number of quail myotubes and of bispecific quail/chick myotubes. Apart from skeletal muscles, quail cells contributed sporadically to nerve envelopes and blood vessel walls in the limb.When the graft was of bispecific constitution, quail nuclei in the limb and the trunk were found exclusively in monospecific and bispecific myotubes.After implantation of differentiated embryonic or post-natal muscle tissue, quail cells in the limb contributed only sporadically to nerve envelopes and blood vessel walls, while in the trunk they also participated in the formation of muscles and tendons.It is concluded that the myogenic cells in 4 to 5-day quail premuscular masses are still able to undergo an extensive migration into the limb buds and there participate in the formation of myotubes and anatomically normal muscles. They display developmental potentialities equivalent to those of the somitic myogenic stem cells. These capacities are lost in 12-day embryonic muscles.     

Myogenic defect in muscular dystrophy of the chicken.

Inherited muscular dystrophy of the chicken is thought to arise from abnormal development of trophic regulation of skeletal muscles by their innervating nerves. To determine whether expression of muscular dystrophy in the chicken is a property of the nerves or of the muscles, wing limb buds were transplanted between normal and dystrophic chick embryos at $\text{3}\text{1}\text{2}$ days of incubation (stage 19–20). Muscles of donor limbs innervated by nerves of the hosts were compared to contralateral unoperated host limb muscles in chicks from 6 to 25 weeks after hatching. Expression of normal or dystrophic phenotype was determined by examination of five different properties which are altered in dystrophic chick muscle: electromyographic evidence of myotonia; fiber diameter; acetylcholinesterase activity, localization, and isozymes; lactic dehydrogenase activity; and succinic dehydrogenase activity. Genetically normal muscle innervated by nerves of normal or dystrophic hosts was phenotypically normal while genetically dystrophic muscle innervated by normal nerves was phenotypically dystrophic. The results suggest that inherited muscular dystrophy of the chicken arises from a defect of muscle rather than from a lesion in the nerves themselves.     

Basal lamina components are concentrated in premuscle masses and at early acetylcholine receptor clusters in chick embryo hindlimb muscles

As an initial step in characterizing the function of basal lamina components during muscle cell differentiation and innervation in vivo, we have determined immunohistochemically the pattern of expression of three components--laminin, proteins related to agrin (an acetylcholine receptor (AChR)-aggregating protein), and a heparan sulfate proteoglycan--during the development of chick embryo hindlimb muscles. Monoclonal antibodies against agrin were used to purify the protein from the Torpedo ray and to characterize agrin-like proteins from embryonic and adult chicken. In early hindlimb buds (stage 19), antibodies against laminin and agrin stained the ectodermal basement membrane and bound to limb mesenchyme with a generalized, punctate distribution. However, as dorsal and ventral premuscle masses condensed (stage 22-23), mesenchymal immunoreactivity for laminin and agrin-like proteins, but not the proteoglycan, became concentrated in these myogenic regions. Significantly, the preferential accumulation of these molecules in myogenic regions of the limb preceded by 1-2 days the appearance of muscle-specific proteins, myoblast fusion, and muscle innervation. All three basal lamina components were preferentially associated with all AChR clusters from the time we first observed them on newly formed myotubes at stage 26. Localization of these antigens in three-dimensional collagen gel cultures of limb mesenchyme, explanted prior to innervation of the limb, paralleled the staining patterns seen during limb development in the embryo. These results indicate that basal lamina molecules intrinsic to limb mesenchyme are early markers for myogenic and synaptic differentiation, and suggest that these components play important roles during the initial phases of myogenesis and synaptogenesis.