Absence Of Innervation Research Articles

Local signals in the chick limb bud can override myoblast lineage commitment: induction of slow myosin heavy chain in fast myoblasts

Patterning of fast and slow muscle fibres in limbs is regulated by signals from non-muscle cells. Myoblast lineage has, however, also been implicated in fibre type patterning. Here we test a founder cell hypothesis for the role of myoblast lineage, by implanting characterized fast and slow mouse myoblast clones into chick limb buds. In culture, late foetal mouse myoblast clones are committed to a probability (range 0–0.92) of slow myosin heavy chain (MyHC) expression. In contrast, when implanted into chick limbs, fast mouse myoblast clones express myosin characteristic of their new environment, without fusion to chick muscle cells and in the absence of innervation. Therefore, local signals exist within the chick limb bud during primary myogenesis that can override intrinsic commitment of at least some myoblasts, and induce slow MyHC.

Cellular changes in denervated tissue during wound healing in a rat model.

There is increasing evidence that innervation, possibly mediated via neuropeptides, promotes wound healing. This study presents data on the early cellular events during healing in denervated tissue. Free oblique groin flaps were raised on 25 adult Sprague-Dawley rats. Excisional wounds were placed within the flap and in two control sites, the contralateral inguinal region and the thorax. The absence of innervation in the free flap wounds was confirmed 10 days after surgery by indirect immunofluorescence with a pan-neuronal marker. The cellular infiltrate of the wounds was analysed immunohistochemically with a panel of antibodies to rat macrophages and monocytes (ED1), rat B lymphocytes (CD45R) and T lymphocytes (CD2). The immunostained cellular infiltrate was quantified at 2, 4, 7 and 10 days postoperatively. Our results show that denervated wounds have a significantly lower macrophage and T-lymphocyte count at day 4 of wound healing (P < 0.05). Inflammatory cells, particularly macrophages, are known to play an important part in wound healing and their reduced chemotaxis in denervated tissue may be related to the observed delay in wound closure.

In vivo observations of terminal Schwann cells at normal, denervated, and reinnervated mouse neuromuscular junctions

We found a low-molecular-mass, fluorescent dye, Calcein blue am ester (CB), that labels terminal Schwann cells at neuromuscular junctions in vivo without damaging them. This dye was used to follow terminal Schwann cells at neuromuscular junctions in the mouse sternomastoid muscle over periods of days to months. Terminal Schwann cell bodies and processes were stable in their spatial distribution over these intervals, with processes that in most junctions were precisely aligned with motor nerve terminal branches. Three days after nerve cut, the extensive processes elaborated by terminal Schwann cells in denervated muscle were labeled by CB. The number and length of CB-labeled terminal Schwann cell processes decreased between 3 days and 1 month after denervation, suggesting that terminal Schwann cell processes are only transiently maintained in the absence of innervation. During reinnervation after nerve crush, however, terminal Schwann cell processes were extended in advance of axon sprouts, and these processes persisted until reinnervation was completed. By viewing the same junctions twice during reinnervation, we directly observed that axon sprouts used existing Schwann cell processes and chains of cell bodies as substrates for outgrowth. Thus, CB can be used to monitor the dynamic behavior of terminal Schwann cells, whose interactions with motor axons and their terminals are important for junction homeostasis and repair.

Development of muscarinic receptors and regulation of secretory responsiveness in rodent sweat glands

Sweat glands are innervated by sympathetic neurons which undergo a change in transmitter phenotype from noradrenergic to cholinergic during development. As soon as the glands begin to differentiate, M3 muscarinic receptor mRNA and binding sites are detectable. Receptor expression appears in the absence of innervation and is maintained after denervation. While receptor expression is not regulated by innervation, secretory responsiveness is. Muscarinic blockade during development or in adult animals results in the loss of responsiveness and its reappearance requires several days. Cholinergic muscarinic activation is most likely to regulate one or more steps in the signalling cascade that are downstream of calcium mobilization. The anterograde regulation of sweat gland responsiveness is one facet of the reciprocal interactions are required to establish a functional synapse in this system.

Vertebrate taste-bud development: are salamanders the model?

Some decades ago, Stone and associates reported that newt taste buds would develop in the absence of gustatory innervation[1]and could subsequently survive in the absence of any innervation[2]. More recently Northcutt and Barlow[3]examined axolotl taste-bud development and, with embryonic transplantation and dye injection, confirmed and extended these classic observations. Rather than migrating in from either the neural crest or placodes, axolotl taste-cell precursors originate in local epithelial tissue where their progeny can differentiate into taste buds even in the absence of innervation. Does the well-established nerve-independence of salamander taste buds also apply to mammalian taste buds, as Northcutt and Barlow now propose?

Fiber Apoptosis in Developing Rat Muscles Is Regulated by Activity, Neuregulin

Motor neurons are thought to critically regulate the survival of a subset of developing muscle fibers. In muscles that develop in the absence of innervation, primary muscle fibers appear to form in normal numbers, but their long-term survival is thought to require innervation-induced suprathreshold activity. Here I examine interactions between motor neurons and muscle fibers in newborn rats. I report that a small fraction of muscle fibers in developing muscles undergo apoptosis. Many, if not all, of these fibers appear to have lost innervation owing to the retraction of nerve terminals. That the apoptosis is initiated by the loss of functional innervation is suggested by a severalfold increase in the magnitude of fiber apoptosis following denervation and muscle paralysis. Finally, both naturally occurring and denervation-induced muscle fiber apoptosis can be prevented by exogenous administration of neuregulin. These results argue that the survival of all or a subset of developing muscle fibers is ultimately regulated by neuregulin. Furthermore, these results suggest a model in which innervation-induced suprathreshold activity indirectly regulates muscle fiber survival by modulating the levels of neuregulin at developing neuromuscular junctions.

The aminergic and peptidergic innervation of insect salivary glands

Insect salivary glands are glands associated with nutrient intake whose secretions are generally involved in the digestion and lubrication of food. They are under the control of neuroactive substances and may be innervated from several sources including the suboesophageal ganglion, the stomatogastric nervous system and the unpaired median nerves. Both amines and peptides have been suggested to play roles in the control of insect salivation, as indicated by their association with terminals on salivary glands, their effects in salivary gland bioassays and their ability to alter second messenger levels and ion channel conformations. Serotonin and dopamine appear to be the most prominent amines associated with insect salivary glands. Either one or both of these amines are found associated with the salivary glands of the locust, stick insect, cockroach, cricket, dragonfly, mosquito, adult moth and kissing bug. Their roles, although not fully elucidated, appear to be in the control of salivary secretion. Several peptides, including members of the FMRFamide-related family of peptides, are also found associated with insect salivary glands. Sources of peptidergic innervation are as varied as those for aminergic innervation, but information regarding the physiological role of these peptides is lacking. The relevance of the different levels of complexity of salivary gland innervation, which range from the absence of innervation in some species (blowfly) to the presence of several distinct sources in others (locust, cockroach), is not well understood. This review serves to consolidate what is known of the phenotype of salivary neurones in relation to the control of salivation.

Changes in luminal fluid protein composition in the rat cauda epididymidis following partial sympathetic denervation.

Sympathetic denervation of the rat cauda epididymidis by surgical removal of the inferior mesenteric ganglion (IMG) results in an excessive accumulation of sperm in the cauda epididymidis as well as significant changes in cauda sperm motility and cauda epididymal gross histology. The objective of the present study was to determine if the cauda-specific changes in sperm storage, sperm motility, and epididymal histology following the loss of sympathetic innervation were accompanied by changes in the protein composition of epididymal fluid. One and 4 weeks after surgical IMG removal or sham operations, luminal fluid obtained from the caput and cauda epididymidis and cauda epididymal sperm-associated proteins were subjected to two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) and silver-stained proteins were quantitated. One week after IMG removal, two cauda epididymal fluid (CEF) proteins (2 and 13) had increased 43% and 49%, respectively, whereas four CEF proteins (5, 8, 9, and 19) had decreased between 30% and 73% compared to controls. Four weeks after IMG removal, changes in CEF proteins observed 1 week following surgery were no longer present, but the staining intensities of three additional CEF proteins (11, 12, and 18) were reduced an average of 70% compared to control CEF proteins. By obstructing the cauda epididymidis, we confirmed that the changes in CEF protein composition observed following IMG removal were not the result of sperm accumulation but were due directly to the loss of innervation; the staining intensity of CEF protein 2 increased as a result of excessive sperm accumulation in the cauda epididymidis both in the presence and absence of innervation from the IMG. No significant changes in caput epididymal fluid proteins or cauda epididymal sperm-associated proteins were detected following IMG removal. These data show that the protein composition of rat CEF is significantly affected by the loss of sympathetic innervation and suggest that neuronal input may play an important role in the maintenance of epididymal function.

Topographical reinnervation of the toad glutaeus muscle by axons of only one spinal nerve.

The toad glutaeus muscle is innervated by axons from two spinal nerves. Following regeneration from nerve lesion, axonal reinnervation from both spinal nerves remains topographically selective. The aim of the present experiments was to determine whether axons from one spinal nerve (nerve 9) would topographically reinnervate the glutaeus muscle in the absence of innervation from the other spinal nerve (nerve 8). Glycogen depletion of single motor units showed that nerve 9 axons are topographically selective when reinnervating the muscle alone. The synapses formed are stronger in topographically correct locations even in the absence of nerve 8 innervation. The distinction between the territory innervated by rostral and caudal nerve 9 axons arises independently of the presence of nerve 8 axons.

Denervation and concomitant amputation of advanced forelimb regenerative outgrowths in adult Xenopus laevis

The current investigation was undertaken to determine whether year-old regenerative outgrowths of adult Xenopus laevis respond to forelimb denervation and concomitant amputation through the distal one-third of the regenerate in the same manner as younger regenerates of postmetamorphic X. laevis froglets. Following limb amputation, our innervated and sham-denervated adult frogs regenerated typical spike-like outgrowths by 63 weeks. However, denervation of 60-week-old regenerates and denervation concomitant with amputation through the regenerative outgrowths of the mature frogs in series III and VI, respectively, resulted in no significant regression (resorption) or further growth of the regenerates 3 weeks later. Therefore, 60-week-old experimental regenerates can be considered mature appendages, as they maintained morphological stability for an additional 3 weeks in the absence of innervation. Our results are in contrast with the regression of regenerate tissues previously reported in delayed-denervated younger forelimb regenerates, as well as concomitantly denervated and amputated regenerates of X. laevis froglets.

Organization and fine structure of a pacemaker derived from fetal rat myocardium.

At an unknown point in mammalian development, cardiac precursor cells become committed to the cardiocyte phenotype. Certain of these are believed to specialize further into pacemaker cardiocytes. By culturing explanted embryonic ventricles into in vivo organ culture (Tucker DC, Snider C, Woods WT Jr: Pediatr Res 23:637-642, 1988), we observed pacemaker cells arising apparently from cardiocytes. We hypothesized that this event can be triggered by intercellular attachments, innervation, vascularization, or other factors. The present study was designed to test the hypothesis that primitive ventricular cells in the tubular heart can organize into an anatomically and electrophysiologically distinct pacemaker structure in the absence of innervation or vascularization from extrinsic sources. Developing ventricles of tubular hearts from 10-d-old rat embryos (n = 22) were excised and incubated in culture dishes. Within each explant, a group of cells organized into a uniform cluster (diameter, 150 +/- 50 microns) after 8 +/- 2 d in culture. These cells resembled immature conduction system cells and had electrophysiologic features similar to those of mature pacemaker cells. Single-cell action potentials and impulse conduction patterns proved that the pacemaker cluster was the dominant pacemaker for the entire explant. These results confirm that, in the absence of extrinsic nerves and blood vessels, culture of the tubular ventricle elicits formation of an organized pacemaker, resembling the mature cardiac pacemaker and suggesting that contact with the culture dish surface or some other factor triggers conversion of cardiocytes to the pacemaker phenotype.

Nerve-dependent recovery of metabolic pathways in regenerating soleus muscles.

The metabolic recovery potential of muscle was studied in regenerating soleus muscles of young adult rats. Degeneration was induced by subfascial injection of a myotoxic snake venom. After regeneration for selected periods up to 2 weeks, samples of whole muscle were analysed for hexokinase (EC 2.7.1.1), phosphofructokinase (EC 2.7.1.11), lactate dehydrogenase (EC 1.1.11.27), adenylokinase (EC 2.7.4.3), creatine kinase (EC 2.7.3.2), malate dehydrogenase (EC 1.1.11.37), citrate synthase (EC 4.1.3.7) and beta-hydroxyacyl CoA dehydrogenase (EC 1.1.1.35). Lactate dehydrogenase, adenylokinase, malate dehydrogenase and beta-hydroxyacyl CoA dehydrogenase were also measured in individual fibres of muscle regenerating up to 4 weeks. We found that in the presence of nerve there was complete recovery of muscle metabolic capacity. However, there were differences in the rate of recovery of the activity of enzymes belonging to different energy-generating pathways. Lactate dehydrogenase, an enzyme representing glycolytic metabolism, reached normal activity immediately upon myofibre formation, only 3 days after venom injection, while oxidative enzymes required a week or more to reach normal activity levels. The delay in oxidative enzyme recovery coincided with physiological parameters of reinnervation. Therefore, to further test the role of nerve on the metabolic recovery process, muscle regeneration was studied following venom-induced degeneration coupled with denervation. In the absence of innervation, most enzymes failed to recover to normal activity levels. Lactate dehydrogenase was the only enzyme to achieve normal levels, and it did so as rapidly as in innervated-regenerating soleus muscles. The remainder of the glycolytic enzymes and the high energy phosphate enzymes recovered only partially. Oxidative enzymes showed no recovery and were severely reduced in the absence of reinnervation.(ABSTRACT TRUNCATED AT 250 WORDS)

Development of fungiform papillae, taste buds, and their innervation in the hamster.

Fungiform taste buds in mature hamsters are less subject to neurotrophic influences than those of other species. This study evaluates taste-bud neurotrophism during development in hamsters by examining the relation between growing nerves and differentiating fungiform papillae. Chorda tympani (CT) or lingual (trigeminal) nerve (LN) fibers were labelled with Lucifer Yellow as they grew into (CT fibers) or around (LN fibers) developing taste buds. Developing fungiform papillae and taste pores were counted with the aid of a topical tongue stain. The tongue forms on embryonic days (E) 10.5-11 and contains deeply placed CT and LN fibers but no papillae. By E12, the tongue epithelium develops scattered elevations. These "eminences" selectively become innervated by LN fibers that grow to the epithelium earlier and in larger numbers than CT fibers. Definitive fungiform papillae form rapidly during E13-14 and become heavily innervated by LN fibers. Intraepithelial CT fibers, rare at E13, invariably innervate fungiform papillae containing nascent taste buds at E14. During E14-15 (birth = E15-16), most papillae contain taste buds with pores, extensive perigemmal LN innervation, and extensive intragemmal CT innervation. At birth, numbers of fungiform papillae and taste pores are adultlike. The results show that fungiform eminences begin forming in the absence of innervation. The subsequent differentiation of definitive fungiform papillae and their innervation by LN fibers occur synchronously, prior to the differentiation of taste buds and their CT innervation. The hamster is precocious (e.g., compared to rat) in terms of LN development and the structural maturity of the anterior tongue at birth.

Sodium channels aggregate at former synaptic sites in innervated and denervated regenerating muscles

The role of innervation in the establishment and regulation of the synaptic density of voltage-activated Na channels (NaChs) was investigated at regenerating neuromuscular junctions. Rat muscles were induced to degenerate after injection of the Australian tiger snake toxin, notexin. The loose-patch voltage clamp technique was used to measure the density and distribution of NaChs on muscle fibers regenerating with or without innervation. In either case, new myofibers formed within the original basal lamina sheaths, and, NaChs became concentrated at regenerating endplates nearly as soon as they formed. The subsequent increase in synaptic NaCh density followed a time course similar to postnatal muscles. Neuromuscular endplates regenerating after denervation, with no nerve terminals present, had NaCh densities not significantly different from endplates regenerating in the presence of nerve terminals. The results show that the nerve terminal is not required for the development of an enriched NaCh density at regenerating neuromuscular synapses and implicate Schwann cells or basal lamina as the origin of the signal for NaCh aggregation. In contrast, the change in expression from the immature to the mature form of the NaCh isoform that normally accompanies development occurred only partially on muscles regenerating in the absence of innervation. This aspect of NaCh regulation is thus dependent upon innervation.

Reduced levels of acetylcholine receptor expression in chick ciliary ganglion neurons developing in the absence of innervation

Chick ciliary ganglion neurons receive innervation from a single source, the accessory oculomotor nucleus (AON), and nicotinic ACh receptors (AChRs) mediate chemical synaptic transmission through the ganglion. Previous experiments examining the developmental expression of AChRs in embryonic chick ciliary ganglion neurons in situ have shown that AChR levels increase substantially in the neurons at the time of innervation. Prior to synapse formation, few AChRs are detected in the neurons. In the present experiments, the role of presynaptic inputs in inducing an increase in AChRs was established by examining AChR levels in ciliary ganglion neurons that have been deprived of innervation by surgical ablation of the AON prior to synapse formation. AChR levels were dramatically reduced in neurons of input-deprived ganglia as compared to control innervated neurons at all developmental stages examined from embryonic day (ED) 5 to ED 12 as determined by indirect immunocytochemical labeling of frozen ganglion sections with the anti-AChR monoclonal antibody mAb 35, and light microscopy. In contrast, neuronal somata of input-deprived and control ganglia had equivalent levels of immunolabeling for three other components, a transmembrane glycoprotein of synaptic vesicles, SV2, and two microtubule-associated proteins, MAP 1B and MAP 2, from ED 5 up to ED 10. The results demonstrate that presynaptic inputs specifically increase the levels of AChR expression in developing neurons. In addition, changes in the levels of immunolabeling for AChRs, SV2, MAP 1B, and MAP 2 in neuronal somata after ED 10 demonstrate that other major developmental events also influence the levels of these components in neurons. Declines in the intensity of AChR, SV2, MAP 1B, and MAP 2 immunolabeling within a subset of neuronal somata in both operated and control ganglia at ED 10 and 12 coincide with the period of neuronal cell death. Increases in AChR labeling in the rest of the neuronal population of input-deprived ganglia at ED 12 suggest that, in addition to innervation, synapse formation with the peripheral target tissue influences AChR levels in developing neurons in situ.

Formation of Myotubes in Aneural Rat Muscles

Skeletal muscles formed in the absence of innervation are deficient in number of fibers, atrophic, and prone to degeneration. We and others have suggested that primary myotubes form autonomously, and the major effect of embryonic denervation is to halt the formation of secondary myotubes by removing the mitotic stimulus for generation of their precursor myoblasts. This hypothesis has recently been put into question by the results of work by Condon et al . (Dev. Biol., 138, 275-295, 1990) and by B. J. Fredette and L. T. Landmesser (Dev. Biol. 143, 19-35, 1991) who propose that lowered secondary myotube numbers are at least in part due to absence of the primary myotubes which would normally serve as scaffold for their formation. We distinguish between these hypotheses by studying generation and degeneration of primary myotubes and formation of secondary myotubes in fetal rat muscles denervated by injection of β-bungarotoxin at different times between Embryonic Days (E) 14 and 17. Denervation at any of these times reduced primary myotube numbers on E18 to about 80% of control. Despite this modest decrease in primary myotube numbers, secondary myotube generation fell to below 3% of normal in extensor digitorum longus (EDL) muscles denervated on E14 or E15 and to below 12% of control in soleus muscles denervated on E14-E16. Secondary myotubes were associated with degenerating as well as intact primary myotubes. More secondary myotubes were generated only if nerves had been present up until E16 for EDL or E17 for soleus. We suggest that these observations of a critical period until E16 (EDL) or E17 (soleus) during which innervation is an absolute prerequisite for secondary myotube formation reflect the presence of a nerve-dependent population of myoblasts which must be activated before secondary myotube formation can commence.

Changes in mitochondrial calmitine and calcium in rat denervated skeletal muscle after injection of a myotoxic drug, chlorpromazine

1. Calmitine and mitochondrial calcium were studied after injection of chlorpromazine into control and denervated gastrocnemius muscle in rat. 2. Calmitine decreased under the effect of chlorpromazine and then increased again. Regenerative capacity was more marked for denervated than control muscle. Calcium increased and then returned to its normal level in control muscle while remaining elevated in denervated muscle. 3. Thus, it would appear that calmitine synthesis can occur in the absence of innervation and that denervation, which probably causes disturbances in mitochondrial calcium regulation systems, may prevent total regeneration of muscle after an injury.

Synapse-specific expression of acetylcholine receptor genes and their products at original synaptic sites in rat soleus muscle fibres regenerating in the absence of innervation.

To test the hypothesis that synaptic basal lamina can induce synapse-specific expression of acetylcholine receptor (AChR) genes, we examined the levels mRNA for the alpha- and epsilon-subunits of the AChR in regenerating rat soleus muscles up to 17 days of regeneration. Following destruction of all muscle fibres and their nuclei by exposure to venom of the Australian tiger snake, new fibres regenerated within the original basal lamina sheaths. Northern blots showed that original mRNA was lost during degeneration. Early in regeneration, both alpha- and epsilon-subunit mRNAs were present throughout the muscle fibres but in situ hybridization showed them to be concentrated primarily at original synaptic sites, even when the nerve was absent during regeneration. A similar concentration was seen in denervated regenerating muscles kept active by electrical stimulation and in muscles frozen 41-44 hours after venom injection to destroy all cells in the synaptic region of the muscle. Acetylcholine-gated ion channels with properties similar to those at normal neuromuscular junctions were concentrated at original synaptic sites on denervated stimulated muscles. Taken together, these findings provide strong evidence that factors that induce the synapse-specific expression of AChR genes are stably bound to synaptic basal lamina.

The development of ACh- and GABA-activated currents in embryonic chick ciliary ganglion neurons in the absence of innervation in vivo

Chemical synaptic transmission in the chick ciliary ganglion is mediated by nicotinic ACh receptors. Ciliary ganglion neurons also express GABAA receptors, although there is no known source of GABAergic innervation of the ganglion, and the function of GABA receptors on these neurons is not known. We examined whether ACh and GABA receptors on embryonic chick ciliary ganglion neurons are regulated by presynaptic inputs. Whole-cell currents evoked by ACh or GABA in neurons soon after dissociation were taken to estimate the level of functional receptors in intact ganglia. We destroyed the accessory oculomotor nucleus (AON), the only source of synaptic input to the ganglion, on embryonic day (E) 4. We determined that 80% of the operations resulted in the virtual elimination of synaptic contacts in the ganglion, using P65 immunohistochemistry (a synaptic vesicle antigen) and direct ultrastructural examination. Previous experiments have shown that during normal development, ACh-activated currents increase over sevenfold between E6 and E18; GABA-activated currents increase only twofold, in proportion to cell size. We found that ACh-activated currents of uninnervated neurons at E14 and E18 were as large as control responses. Furthermore, ACh receptor-like molecules, visualized with monoclonal antibody 35 immunofluorescence, were concentrated in high density clusters on the surface of E18 neurons from AON-ablated embryos. GABA-activated currents were also not affected by AON destruction. We conclude that ACh and GABA receptors are not induced in embryonic chick ciliary ganglion neurons during development by contact with or soluble factors released from AON synaptic terminals.

Morphometric analysis of the developing, murine aneural soleus muscle

The pattern of organogenesis of the aneural soleus muscle of the 129ReJ mouse [rendered aneural by laser ablation of the lumbosacral spinal cord at 14 days in utero (during the period of primary myotube formation, but prior to the formation of secondary myotubes)] was evaluated quantitatively with spaced, serial ultrathin sections and computer-assisted morphometric analysis. Aneural muscles from 16- and 18-day gestation and newborn mice were analyzed to determine age-related changes in a number of parameters including: muscles' maximal girths, numbers of myotubes, myotube diameter distributions, and cluster frequency. Data were compared with a similar study of the organogenesis of the normal soleus muscle (Ontell et al: Am J Anat 181:279-288, 1988). Basic patterns of morphogenesis of the soleus muscle were unchanged by spinal cord ablation, and differences in development between the aneural and innervated muscles were quantitative rather than qualitative. At birth, the aneural muscle contained approximately 76% of the myotubes found in the innervated muscle (approximately 840 myotubes in the innervated muscle and approximately 640 in the aneural muscle). Evidence is presented consistent with the hypothesis that primary myotube formation is reduced by approximately 32% in the aneural muscles and that while extensive secondary myotube formation occurs (approximately 78% of the myotube present at birth in these muscles are secondary myotubes), there is a significant reduction in the number of secondary myotubes in aneural muscles. It is suggested that the reduced numbers of secondary myotubes may be related to the reduction in the number of primary myotubes, which are known to act as scaffolds for secondary myotube formation. The time course of secondary myotube formation and of cluster formation and cluster dispersal and the number of cells per cluster are similar in age-matched, innervated and aneural muscles. The absence of innervation has little effect on myotube growth until birth, when comparison of the myotube diameter distributions reveals a slight alteration in myotube diameter distributions of aneural as compared with innervated muscles.