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

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

Myosatellite cells,growth, and regeneration in murine dystrophic muscle: A quantitative study

Patterns of growth and regeneration in 2-, 4-, 8-, and 17-week-old murine dystrophic (129 ReJ dy/dy) extensor digitorum longus muscles have been determined. Necrosis and myofiber loss, hypertrophy, and regeneration result in a reduced population of myofibers whose diameter distribution is more extensive than that found in the extensor digitorum longus muscles of age-matched normal mice. At the onset of dystrophic symptoms (2 weeks postnatal), the ratio of myosatellite cell nuclei to the total sublaminal nuclear population (myonuclei + myosatellite cells) is similar to that found in 2-week-old control muscles. The frequency of finding myosatellite cells decreases with age in both control and dystrophic muscles. Myosatellite cells account for 11%, 6%, 5%, and 3% of the total sublaminal nuclear population in control muscle and 12%, 8%, 6%, and 5% of the total sublaminal nuclear population in dystrophic muscle at 2, 4, 8, and 17 weeks, respectively. No preferential association of myosatellite cells with myofibers of a particular diameter is found in control muscle or in the two youngest dystrophic groups. At 8 and 17 weeks, myosatellite cells are less frequently encountered on small-diameter, regenerating myofibers of dystrophic muscle, and they are preferentially associated with large diameter, hypertrophied myofibers. The labeling index of myosatellite cells decreases with age in both normal and dystrophic muscle. At all ages the myosatellite cell labeling index is higher in dystrophic muscle (23%, 7%, 5%, and 2% at 2, 4, 8, and 17 weeks, respectively) than in normal muscle (5%, < 1% at 2 and 4 weeks, respectively), with no labeled myosatellite cells being found in 8- and 17-week-old normal muscles. It is suggested that the magnitude of the regenerative response of dystrophic murine muscle decreases with age and that this factor may be responsible for the inability of the regenerative response of dystrophic muscle to keep pace with the rapid muscle deterioration.     

Branched myofibers in long-term whole muscle transplants: a quantitative study

Orthotopic transplants of whole extensor digitorum longus muscles were performed on six 4-6-week-old 129 ReJ mice. One hundred days posttransplantation, the animals were killed and the regenerated muscles were processed for electron microscopy. The grafts contained polygonal-shaped myofibers with persistent central nuclei, organized into discrete muscle fascicles. No central area of fatty infiltration or fibrosis was observed. The mean number of myofibers in a regenerating transplanted muscle, as determined from an ultrathin section taken from the graft's widest girth, was 631 (SEM = +/- 59), a reduction of approximately 32% from that found in age-matched control muscle (Ontell et al., 1983). By following the myofibers in spaced, serial ultrathin sections along their length, it was found that the branched, regenerating myofibers found in immature grafts of normal muscle (Ontell et al., 1982) persisted in stabilized, long-term transplanted muscle. The frequency of branching was determined by following each fiber found at the widest girths of four of the grafts in spaced, serial ultrathin sections (15-micron intervals) for approximately 2% of the total length of the grafts. Over this distance, 6.6% of the fibers were involved in the branching phenomenon. The persistence of branched fibers in long-term grafts and the frequency with which the branching phenomenon was found to occur may have physiological consequences and should be investigated.     

Muscular dystrophy and muscle regeneration

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

Effects of early immobilization on the functional capacity of dystrophic (ReJ 129 dy/dy) mouse leg muscles.

Hindleg muscles of dystrophic (ReJ 129 dy/dy) mice were immobilized during the second post-natal week. Two months after remobilization histopathological features and isometric force characteristics of the m. extensor digitorum longus (EDL) and the m. tibialis anterior (TA) were studied. As a result of early transient immobilization significant differences were observed in muscle morphology and isometric force compared with untreated dystrophic muscles. Restriction of dynamic use of the muscles during this second post-natal week largely prevents the muscles of dystrophic mice from becoming affected by the disease process. Even after two months of remobilization pathology appears to be reduced.     

Long term functional improvement of dystrophic mouse leg muscles upon early immobilization

The long term effects of immobilization of one hindleg, during the second postnatal week, were investigated in dystrophic ReJ 129 dy/dy strain of mice. The muscles of the immobilized limb were compared with those of the contralateral, non-treated side and with those of naturally dystrophic age-mates, after 1, 2 and 3 months of remobilization. It appeared that the experimental animals made better use of their remobilized leg than of the contralateral leg for locomotion. The remobilized muscles were significantly less atrophic than the contralateral muscles and they also contained more muscle fibres. It is concluded that during postnatal growth and differentiation the dystrophic muscle fibres pass through a sensitive period. Immobilization during this period prevents the majority of the muscle fibres from becoming affected. Remobilization induces pathological features in the muscles, but they remain less damaged than the non-immobilized muscles.     

Long term functional improvement of dystrophic mouse leg muscles upon early immobilization.

The long term effects of immobilization of one hindleg, during the second postnatal week, were investigated in dystrophic ReJ 129 dy/dy strain of mice. The muscles of the immobilized limb were compared with those of the contralateral, non-treated side and with those of naturally dystrophic age-mates, after 1, 2 and 3 months of remobilization. It appeared that the experimental animals made better use of their remobilized leg than of the contralateral leg for locomotion. The remobilized muscles were significantly less atrophic than the contralateral muscles and they also contained more muscle fibres. It is concluded that during postnatal growth and differentiation the dystrophic muscle fibres pass through a sensitive period. Immobilization during this period prevents the majority of the muscle fibres from becoming affected. Remobilization induces pathological features in the muscles, but they remain less damaged than the non-immobilized muscles.     

The study of (Methyl-3H)decamethonium dichloride incorporation into normal and dystrophic mouse muscle.

1. (Methyl-3H)decamethonium dichloride was injected intravenously into the tail vein of dystrophic and normal mice of the Bar Harbour strain 129 ReJ/dy in paralysing doses. 2. Scintillation counts were made of 1 mm sections of diaphragm and tibialis anterior which showed a normal distribution of tritiated decamethonium in the dystrophic animals. 3. Intraperitoneal injections of L-(gamma-3H)leucine were made into dystrophic and normal mice. Examination of diaphragm and tibialis anterior by scintillation counting showed the abnormal uptake typical of dystrophic involvement of murine muscle. 4. The normal distribution of (methyl-3H)decamethonium dichloride uptake by the dystrophic muscle does not support the concept of active denervation in this disease.     

Adeno-associated virus-mediated vascular endothelial growth factor gene therapy in skeletal muscle before transplantation promotes revascularization of regenerating muscle

Successful clinical transplantation of whole skeletal muscles can be limited by impaired muscle revascularization and regeneration. The aim of this study was to enhance the revascularization (and hence speed of regeneration) of transplanted whole muscles by transducing muscles with the vascular endothelial growth factor (VEGF) gene before transplantation, using a recombinant adeno-associated virus (rAAV). The rAAV encoding VEGF and green fluorescent protein (GFP) (rAAV.VEGF.GFP) was injected into the tibialis anterior muscles of adult BALB/c mice. One month after injection whole muscle autotransplantation was performed. Muscles were sampled 7 days after autografting. GFP expression was examined as an indicator of persistent transgene expression after grafting, and immunohistochemistry was used to identify VEGF, blood vessels, and newly formed myotubes. After grafting, GFP expression persisted only in a few surviving myofibers in the periphery of rAAV.VEGF.GFP-pretreated muscles, although abundant VEGF expression was seen in myogenic cells in all grafted muscles. Quantitative analysis demonstrated that, although only small numbers of rAAV.VEGF.GFP-transduced myofibers were present, whole muscle grafts preinjected with rAAV.VEGF.GFP were significantly more vascular than saline-injected and uninjected control muscle grafts. Furthermore, rAAV.VEGF.GFP-injected whole muscle transplants were further advanced in terms of regeneration (myotube formation) compared with the uninjected control muscle transplants. This study clearly shows that rAAV-mediated VEGF expression persists only in myofibers that survive the necrosis induced by muscle transplantation; however, this amount of VEGF results in significantly increased revascularization and regeneration of whole muscle transplants.     

Neuromuscular transmission in dystrophic mice.

1. Neuromuscular transmission was studied in the extensor digitorum-longus muscle of dystrophic mice (strain 129/ReJ) by means of intracellular recording techniques. 2. In a large population of normal and dystrophic muscle fibers tested, the incidence of transmission failure was about 2% and showed no significant difference between the two groups. 3. Quantal size and quantum content of dystrophic junctions were found to be normal. This was true even of nerve terminal on apparently atrophied muscle fibers. 4. The facilitation ratio at dystrophic junctions was not significantly different from normal. 5. Dystrophic neuromuscular junctions exhibited an abnormality high frequency of giant spontaneous potentials. Application of tetrodotoxin (10(-6) M) and curare (10(-6) M) indicated that these potentials were caused by impulse-independent release of acetylcholine. 6. Neuromuscular transmission in dystrophic mice was found functionally normal and unrelated to the degenerative state of the muscle.     

Acetylcholine-sensitivity in fast and slow twitch muscle of normal and dystrophic (C57 BL/6J dy2J/dy2J) mice

Small bundles of muscle fibres were isolated from diaphragm, extensor digitorum longus (EDL) and soleus (SOL) muscles of normal and dystrophic (C57 BL/6J dy2J/dy2J) mice, and their isometric tension developed in response to acetylcholine (ACh) was recorded. For each type of muscle the relationship between the maximum amplitude of the ACh-contracture and log [ACh] was similar in normal and dystrophic animals. However, this relationship was steeper for normal and dystrophic SOL than for EDL and diaphragm muscles. Dystrophy did not induce changes in the time course of the ACh-contractures, except a significant 'speeding' of dystrophic SOL that appeared in the time to peak of the contractile response. The amplitude of ACh-contractures of both normal and dystrophic diaphragm preparations increased by about 50% after perfusion for 80-90 min in physiological solution containing phospholipase C 5 mU/ml. ACh-sensitivity was measured in normal and dystrophic diaphragm preparations by iontophoretic application of ACh from high resistance pipettes. ACh-potentials were similar in time course in the two types of muscle fibres, and there was also no significant difference in the length of sensitive fibre segments and maximum sensitivity values. Extrajunctional ACh-sensitivity was absent in normal as well as in dystrophic fibres. It is concluded that the absence in dystrophic muscles of stronger ACh-contractures and of extrajunctional sensitivity can be considered as evidence against a primary neuronal involvement in murine dystrophy of the dy2J strain.     

Morphometric analysis of the developing mouse soleus muscle

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

A behavioral profile of two dystrophic mouse strains

Samples of the spontaneous activity of individual mice of two dystrophic strains (129/ReJ dy/dy and C57BL/6J dy/dy), their littermates (heterozygous or normal), and parent-strain animals were intermittently recorded on video over a 24-h period. Twelve behavioral categories were derived from all behaviors observed. Dystrophic mice showed significantly higher numbers of grooming-related seizures and instances of feeding than their unaffected controls. The frequency of each of these behaviors increased during the dark portion of the laboratory light cycle. Hard and moist food consumption was also measured in dystrophic and control animals of both strains. Young dystrophic animals ate considerably more hard food (relative to body weight) than controls, while older, severely dystrophic mice ate less. However, when given moist food, the latter animals increased their food intake over that of controls. Spontaneous seizures, observed only in affected mice, appeared to be a neurological component of murine muscular dystrophy. In addition, the 129/Re strain showed potentially lethal audiogenic seizures that appeared to be a strain-related phenomenon not directly associated with the disease.     

Platelet-derived growth factor and its receptors are related to the progression of human muscular dystrophy: an immunohistochemical study

This study has examined the immunological localization of platelet-derived growth factor (PDGF)-A, PDGF-B, and PDGF receptor (PDGFR) alpha and beta to clarify their role in the progression of muscular dystrophy. Biopsied frozen muscles from patients with Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMD), and congenital muscular dystrophy (CMD) were analysed immunohistochemically using antibodies raised against PDGF-A, PDGF-B, and PDGFR alpha and beta. Muscles from two dystrophic mouse models (dy and mdx mice) were also immunostained with antibodies raised against PDGFR alpha and beta. In normal human control muscle, neuromuscular junctions and vessels were positively stained with antibodies against PDGF-A, PDGF-B, PDGFR alpha and PDGFR beta. In human dystrophic muscles, PDGF-A, PDGF-B, PDGFR alpha and PDGFR beta were strongly immunolocalized in regenerating muscle fibres and infiltrating macrophages. PDGFR alpha was also immunolocalized to the muscle fibre sarcolemma and necrotic fibres. The most significant finding in this study was a remarkable overexpression of PDGFR beta and, to a lesser extent, PDGFR alpha in the endomysium of DMD and CMD muscles. PDGFR was also overexpressed in the interstitium of muscles from dystrophic mice, particularly dy mice. Double immunolabelling revealed that activated interstitial fibroblasts were clearly positive for PDGFR alpha and beta. However, DMD and CMD muscles with advanced fibrosis showed very poor reactivity against PDGF and PDGFR. Those findings were confirmed by immunoblotting with PDGFR beta. These findings indicate that PDGF and its receptors are significantly involved in the active stage of tissue destruction and are associated with the initiation or promotion of muscle fibrosis. They also have roles in muscle fibre regeneration and signalling at neuromuscular junctions in both normal and diseased muscle.     

Effect of nerve section on beta-endorphin and alpha-melanotropin immunoreactivity in motor nerves of normal and dystrophic mice

beta-Endorphin and alpha-melanotropin immunoreactivity was demonstrated in some motor nerves in histological sections of soleus and extensor digitorum longus muscles of the mouse. In two strains (C57BL/6J and 129/ReJ) which can inherit muscular dystrophy, the proportion of immunoreactive nerves was greater in the dystrophic individuals than in their healthy littermates. At 24 h after section of the sciatic nerve in the normal mice the proportion of immunoreactive nerve profiles had increased significantly in the denervated muscles even though most of the nerve axons had degenerated. Similar increases were also seen in nerves in the unoperated contralateral muscles.     

The dystrophic murine skeletal muscle cell plasma membrane is structurally intact but "leaky" to creatine phosphokinase. A freeze-fracture analysis

Skeletal muscle cells of genetically dystrophic mice (dy/dy) of the REJ-129 Bar Harbor strain exhibit reduced cytoplasmic levels of the enzyme creatine phosphokinase (CPK) when compared with normal (+/+) mice following SDS-gel electrophoresis of sarcoplasmic proteins. This observation has been thought to reflect "leakage" of CPK from dystrophic muscle cells through lesions in the sarcolemma. The present study has employed the freeze-fracture method to examine vast expanses of sarcolemma fracture face for determination of whether lesions do exist in the membrane or an alternate route is present for extravasation of CPK from dystrophic muscle cells. Most of the dystrophic cells examined in this study appeared intact and were therefore presumed viable. The intramembrane lipoprotein particles characteristic of PF-fracture face membrane were reduced in dystrophic as compared with normal murine skeletal muscle, and the plasmalemma possessed a greatly amplified population of caveolae as compared with nondiseased sarcolemma. No abnormal structural feature of these dystrophic muscle plasma membranes could be interpreted as a perforating focal "delta" lesion, such as the structures seen in thin plastic sections by other investigators. However, a second group of cells, generally few in number, that exhibited features indicative of necrosis (and loss of viability), were seen in both thin sections and platinum replicas. These moribund cells were usually embedded in dense sheaves of connective tissue along with other dystrophic cells that lacked signs of necrosis. The cytoplasm of the necrotic muscle cells was disorganized, as was the contractile machinery. The sarcolemma showed numerous perforations, through which CPK could escape into the tissue extracellular compartment. We conclude on the basis of our observations that the "focal lesions" reported by other investigators are not a structural feature of viable dystrophic muscle cell plasma membranes and are found only in necrotic or dying cells, and that the elevated serum levels of CPK associated with muscular dystrophy may result either from escape of the enzyme through lesions present in necrotic or dying cells or by extravasation along avenues provided by the hyperplastic mass of membrane caveolae present in dystrophic sarcolemma.     

Activation of the intramyofibral autophagic-lysosomal system in muscular dystrophy.

Skeletal muscles obtained from myopathies with myofiber necrosis, including mdx dystrophic mice, plasmocid-induced myopathy in rats, and patients with Duchenne muscular dystrophy, were examined immunohistochemically with anticathepsin-peroxidase conjugates. Strong reactions for lysosomal cysteine proteinases, which can degrade myofibrillar proteins, were demonstrated in macrophages invading and surrounding the necrotic areas and some degenerative myofibers and also in intramyofibral portions of atrophic fibers of dystrophic mice and humans. Apparently normal and regenerating myofibers did not stain for lysosomal cathepsins. Abnormal increases of cathepsins L and B were seen even in the early stage of plasmocid myopathy and in a 20-day-old young mdx mouse before infiltration of macrophages, suggesting that autodigestion by intramyofibral lysosomal proteinases is an important event before digestion of the necrotic fibers by macrophage proteinases. Activation of the intramyofibral lysosomal system, as in muscular dystrophy, was also observed in distal myopathy with rimmed vacuoles without macrophageal infiltration (Am J Pathol 1986, 122:193-198). Thus, this activation seems to be an important, early response to myocellular damage.     

Lack of myoblasts migration between transplanted and host muscles of mdx and normal mice

Summary Extensor digitorum longus muscles of normal mice (C57BL/10ScSn hereafter called C57) were orthotopically transplanted into dystrophin-deficient mice (mdx) and reciprocally, mdx Extensor digitorum longus muscles were transplanted into C57 mice. After an initial phase of degeneration, transplanted muscles regenerate nearly completely, as evaluated from the maximum isometric force of muscles isolated 60 days after the surgery. In other similar experiments, instead of isolating the grafted muscles, we excised the antero-external muscles of the leg, including the grafted muscle. Cryostat cross-sections at three levels along the muscles were immunostained with an anti-dystrophin antibody. No muscle cells of dystrophin-deficient muscles grafted into normal mice took the antibody except a few revertant fibres, while all the muscle cells of the normal host were immunostained. Reciprocally, all the muscles cells of normal grafts were stained, whilst no antibody stained the cells of the surrounding muscles of the dystrophin-deficient host. These experiments show that very few if any of the myoblasts or muscle precursor cells, active during the regeneration of grafted muscle, migrate into the adjacent muscles. These results could be explained by the absence, in our work, of injuries of the grafted and adjacent host muscles epimysium and the absence of extensive inflammatory reactions. This lack of myoblast mobility suggest that when myoblast transfer is applied to muscle therapy, it will be necessary to inject myoblasts within each muscle to obtain an efficient treatment.     

Electrophysiological properties of spinal motoneurones of normal and dystrophic mice.

1. The properties of spinal motoneurones of normal and dystrophic mice (129/ReJ) were examined with intracellular electrodes. 2. The following parameters of spinal motoneurones showed no significant differences between normal and dystrophic mice: resting and action potentials, the amplitude and duration of after-hyperpolarization, rheobasic current for excitation, threshold for excitation of the somadendritic membrane (IS-SD inflexion) and input resistance. 3. The changes in motoneurone properties observed 13-16 days after section of the sciatic nerve (axotomy) were similar in both normal and dystrophic mice. 4. The axonal conduction velocity of motoneurones in dystrophic mice was about ten times slower than that in normal mice. The conduction velocity of the sciatic nerve was only about 25% slower in dystrophic mice than in the normal animal. The estimated ventral root conduction velocity as well as the observed dorsal root conduction velocity in dystrophic mice was at least twenty times slower than that in normal mice. 5. In dystrophic mice, spinal motoneurones often showed multiple discharges in response to single, antidromic stimuli. The site of initiation of multiple discharge was located in the motor axon rather than in the motoneurone cell body. 6. In dystrophic mice, nerve impulses were transmitted from fibre to fibre ('cross-talk'). The site of impulse transmission among nerve fibres was near the distal portion of the spinal roots. 7. Synaptic potentials and peripheral reflex discharges evoked by stimulation of the dorsal roots showed a longer latency and were more prolonged in dystrophic mice than in the control mice. 8. The motoneurone properties of dystrophic mice showed no tendency of progressive changes with age ranging from 63 to 148 days. 9. It is concluded that the properties of motoneurone cell bodies examined in dystrophic mice are indistinguishable from those in normal mice and that the only abnormality in motoneurones of the former residues in the motor axon. 10. It is suggested that integrity of the discharge pattern of spinal motoneurones in dystrophic mice is interfered by anomalous impluse transmission in the motor axons and that the motoneurones in dystrophic mice are a homogeneous group rather than a mixture of "normal" and "abnormal" neurones.     

Ablation of the fetal mouse spinal cord: the effect on soleus muscle cytoarchitecture.

A technique is described whereby it is possible to surgically ablate the lumbosacral spinal cord of a developing mouse fetus without interfering with fetal viability. The lumbosacral spinal cords of 14-day in utero, 129ReJ mice were ablated with a Cooper Nd-YAG laser, and the fetuses, enclosed in their membranes and attached to the uterus by their placentae, were allowed to develop in the abdominal cavity of the dam. The cytoarchitecture and the temporal pattern of organogenesis of aneural soleus muscles were studied in spaced, serial, transverse, ultrathin sections of muscles of 16- and 18-day gestation and newborn (20-day gestation) mice. At the time of surgery, the soleus muscle was a discrete mass consisting of primary myotubes and a pleomorphic population of mononucleated cells. Axon bundles and blood vessels were found at the muscle's periphery, but had not penetrated throughout the muscle mass. The organogenesis of the aneural muscle was remarkably similar to that of the innervated soleus muscle (Ontell et al., Am J Anat 181:267-278, 1988). In the aneural muscle, as in the innervated muscle, significant numbers of secondary myotubes formed all along the lengths of primary myotubes. Moreover, the time course of myotube formation, the dynamics of cluster formation and cluster dispersal, and the ultrastructural appearance of the myotubes mimicked that observed in innervated muscle. The frequency of necrotic myotubes was no greater in the aneural muscle than in the innervated soleus muscle. Myotube maturation was similar in aneural and innervated soleus muscles until 18 days gestation. However, at birth, aneural myotubes appeared to be slightly less mature than innervated myotubes. Thus, the major morphogenic phenomena that characterize the development of the soleus muscle appear to be independent of innervation.