Development of chicken intrafusal muscle fibers

The first sign of developing intrafusal fibers in chicken leg muscles appeared on embryonic day (E) 13 when sensory axons contacted undifferentiated myotubes. In sections incubated with monoclonal antibodies against myosin heavy chains (MHC) diverse immunostaining was observed within the developing intrafusal fiber bundle. Large primary intrafusal myotubes immunostained moderately to strongly for embryonic and neonatal MHC, but they were unreactive or reacted only weakly with antibodies against slow MHC. Smaller, secondary intrafusal myotubes reacted only weakly to moderately for embryonic and neonatal MHC, but 1–2 days after their formation they reacted strongly for slow and slow-tonic MHC. In contrast to mammals, slow-tonic MHC was also observed in extrafusal fibers. Intrafusal fibers derived from primary myotubes acquired fast MHC and retained at least a moderate level of embryonic MHC. On the other hand, intrafusal fibers developing from secondary myotubes lost the embryonic and neonatal isoforms prior to hatching and became slow. Based on relative amounts of embryonic, neonatal and slow MHC future fast and slow intrafusal fibers could be first identified at E14. At the polar regions of intrafusal fibers positions of nerve endings and acetylcholinesterase activity were seen to match as early as E16. Approximately equal numbers of slow and fast intrafusal fibers formed prenatally; however, in postnatal muscle spindles fast fibers were usually in the majority, suggesting that some fibers transformed from slow to fast.     

Transient expression of a ventricular myosin heavy chain isoform in developing chicken intrafusal muscle fibers

Sections of chicken tibialis anterior and extensor digitorium longus muscles were incubated with monoclonal antibodies against myosin heavy chains (MHC). Ventricular myosin was present in developing secondary intrafusal myotubes when they were first recognized at embryonic days (E) 13–14, and in developing extrafusal fibers prior to that date. The reaction in intrafusal fibers began to fade at E17, and in 2-week-old postnatal and older muscles the isoform was no longer recognized. Only those intrafusal fibers which also reacted with a monoclonal antibody against atrial and slow myosin contained ventricular MHC. Intrafusal myotubes which developed into fast fibers did not express the isoform. Hence, based on the presence or absence of ventricular MHC, two lineages of intrafusal fiber are evident early in development. Strong immunostaining for ventricular MHC was observed in primary extrafusal myotubes at E10, but the isoform was already downregulated at E14, when secondary intrafusal myotubes were still forming and expressed ventricular MHC. Only light to moderate and transient immunostaining was observed in coexisting secondary extrafusal myotubes, most of which developed into fast fibers. Thus at the time when nascent muscle spindles are first recognized, differences in MHC profiles already exist between prospective intrafusal and extrafusal fibers. If intrafusal fibers stem from a pool of primordial muscle cells, which is common to intrafusal and extrafusal myotubes, they diverged from it some time prior to E13.This paper is dedicated to Prof. D. Pette, Konstanz, on the occasion of his 60th birthday     

Proportions of slow myosin heavy chain-positive fibers in muscle spindles and adjoining extrafusal fascicles, and the positioning of spindles relative to these fascicles.

Chicken leg muscles were examined to calculate the percentages of slow myosin heavy chain (MHC)-positive fibers in spindles and in adjacent extrafusal fascicles, and to clarify how the encapsulated portions of muscle spindles are positioned relative to these fascicles. Unlike mammals, in chicken leg muscles slow-twitch MHC and slow-tonic MHC are expressed in intrafusal fibers and in extrafusal fibers, suggesting a close developmental connection between the two fiber populations. In 8-week-old muscles the proportions of slow MHC-positive extrafusal fibers that ringed muscle spindles ranged from 0-100%. In contrast, proportions of slow MHC-positive intrafusal fibers in spindles ranged from 0-57%. Similar proportions in fiber type composition between intrafusal fibers and surrounding extrafusal fibers were apparent at embryonic days 15 and 16, demonstrating early divergence of extrafusal and intrafusal fibers. Muscle spindles were rarely located within single fascicles. Instead, they were commonly placed where several fascicles converged. The frequent extrafascicular location of spindles suggests migration of intrafusal myoblasts from developing clusters of extrafusal fibers toward the interstitium, perhaps along a neurotrophic gradient established by sensory axons that are advancing in the connective tissue matrix that separates adjoining fascicles.     

The effect of neonatal deafferentation or defferentation on myosin heavy chain expression in intrafusal muscle fibers of the rat

Muscle spindles were either deafferented or deefferented by selectively severing the sensory or motor nerve supply to neonatal soleus muscles of rats at a time when spindles are formed but when intrafusal muscle fibers are structurally and immunocytochemically immature. Experimental muscles were excised two months after nerve section. Control and experimental spindles were examined using monoclonal antibodies specific for myosin heavy chains of slow-tonic (ALD58) and fast-twitch (MF30) chicken muscles. Only intrafusal fibers bound these antibodies in intact soleus muscles. The deefferented spindles exhibited a pattern of ALD58 and MF30 binding similar to that of normal adult intrafusal fibers, whereas deafferented intrafusal fibers were unreactive with the two antibodies. Thus intact sensory innervation is essential for myosin heavy chain expression in intrafusal muscle fibers during postnatal development of rat spindles.     

The effect of neonatal deafferentation or deefferentation on myosin heavy chain expression in intrafusal muscle fibers of the rat

Summary Muscle spindles were either deafferented or deefferented by selectively severing the sensory or motor nerve supply to neonatal soleus muscles of rats at a time when spindles are formed but when intrafusal muscle fibers are structurally and immunocytochemically immature. Experimental muscles wereexcised two months after nerve section. Control and experimental spindles were examined using monoclonal antibodies specific for myosin heavy chains of slow-tonic (ALD58) and fast-twitch (MF30) chicken muscles. Only intrafusal fibers bound these antibodies in intact soleus muscles. The deefferented spindles exhibited a pattern of ALD58 and MF30 binding similar to that of normal adult intrafusal fibers, whereas deafferented intrafusal fibers were unreactive with the two antibodies. Thus intact sensory innervation is essential for myosin heavy chain expression in intrafusal muscle fibers during postnatal development of rat spindles.     

Slow-tonic MHC expression in paralyzed hindlimbs of fetal rats.

Whether nerve activity and active contraction of myotubes are essential for the assembly and initial differentiation of muscle spindles was investigated by paralyzing fetal rats with tetrodotoxin (TTX) from embryonic day 16 (E16) to E21, prior to and during the period when spindles typically form. TTX-treated soleus muscles were examined by light and electron microscopy for the presence of spindles and expression of myosin heavy chain (MHC) isoforms by the intrafusal fibers. Treatment with TTX did not inhibit the formation of a spindle capsule or the expression of a slow-tonic MHC isoform characteristic of intrafusal fibers, but did retard development of spindles. Spindles of TTX-treated E21 muscles usually consisted of one intrafusal fiber (bag2) only rather than two fibers (bag1 and bag2) typically present in untreated (control) E21 spindles. Intrafusal fibers of TTX-treated spindles also had only one sensory region supplied by multiple afferents, and were devoid of motor innervation. These features are characteristic of spindles in normal E18-E19 muscles. Thus, nerve and/or muscle activity is not essential for the assembly of muscle spindles, formation of a spindle capsule, and transformation of undifferentiated myotubes into the intrafusal fibers containing spindle-specific myosin isoforms. However, activity may promote the maturation of intrafusal bundles, as well as the maturation of afferent and efferent nerve supplies to intrafusal fibers.     

Slow-tonic MHC expression in paralyzed hindlimbs of fetal rats

Summary Whether nerve activity and active contraction of myotubes are essential for the assembly and initial differentiation of muscle spindles was investigated by paralyzing fetal rats with tetrodotoxin (TTX) from embryonic day 16 (E16) to E21, prior to and during the period when spindles typically form. TTX-treated soleus muscles were examined by light and electron microscopy for the presence of spindles and expression of myosin heavy chain (MHC) isoforms by the intrafusal fibers. Treatment with TTX did not inhibit the formation of a spindle capsule or the expression of a slow-tonic MHC isoform characteristic of intrafusal fibers, but did retard development of spindles. Spindles of TTX-treated E21 muscles usually consisted of one intrafusal fiber (bag₂) only rather than two fibers (bag₁ and bag₂) typically present in untreated (control) E21 spindles. Intrafusal fibers of TTX-treated spindles also had only one sensory region supplied by multiple afferents, and were devoid of motor innervation. These features are characteristic of spindles in normal E18–E19 muscles. Thus, nerve and/or muscle activity is not essential for the assembly of muscle spindles, formation of a spindle capsule, and transformation of undifferentiated myotubes into the intrafusal fibers containing spindle-specific myosin isoforms. However, activity may promote the maturation of intrafusal bundles, as well as the maturation of afferent and efferent nerve supplies to intrafusal fibers.     

Diversity in expression of myosin heavy chain isoforms and M-band proteins in rat muscle spindles

The composition of adult rat soleus muscle spindles, with respect to myosin heavy chain isoforms and M-band proteins, was studied by light-microscope immunohistochemistry. Serial sections were labelled with antibodies against slow tonic, slow twitch, fast twitch and neonatal myosin isoforms as well as against myomesin, M-protein and the MM form of creatine kinase. Intrafusal fiber types were distinguished according to the pattern of ATPase activity following acid and alkaline preincubations. Nuclear bag1 fibers were always strongly stained throughout with anti-slow tonic myosin, were positive for anti-slow twitch myosin towards and in the C-region but were unstained with anti-fast twitch and anti-neonatal myosins. The staining of nuclear bag2 fibers was in general highly variable. However, they were most often strongly stained by anti-slow tonic myosin in the A-region and gradually lost this reactivity towards the poles, whereas a positive reaction with anti-slow twitch myosins was found along the whole fiber. Regional staining variability with anti-neonatal and anti-fast myosins was apparent, often with decreasing intensity towards the polar regions. Nuclear chain fibers showed strong transient reactivity with anti-slow tonic myosin in the equatorial region, did not react with anti-slow twitch and were always evenly stained by anti-fast twitch and anti-neonatal myosins. All three intrafusal fiber types were stained with anti-myomesin. Nuclear bag1 fibers lacked staining for M-protein, whereas bag2 fibers displayed intermediate staining, with regional variability, often increasing in reactivity towards the polar regions. Chain fibers were always strongly stained by anti-M-protein. The MM form of creatine kinase was present in all three fiber types, but bag1 fibers were less reactive and clear striations were not observed, in contrast to bag2 and chain fibers. Out of 38 cross sectioned spindles two were found to have an atypical fiber composition (lack of chain fibers) and a rather diverse staining pattern for the different antibodies tested. Taken together, the data show that in adult rat soleus, slow tonic and neonatal myosin heavy chain isoforms are only expressed in the muscle spindle fibers and that each intrafusal fiber type has a unique, although variable, composition of myosin heavy chain isoforms and M-band proteins. We propose that both motor and sensory innervation might be the determining factors regulating the variable expression of myosin heavy chain isoforms and M-band proteins in intrafusal fibers of rat muscle spindles.     

Diversity in expression of myosin heavy chain isoforms and M-band proteins in rat muscle spindles

Summary The composition of adult rat soleus muscle spindles, with respect to myosin heavy chain isoforms and M-band proteins, was studied by light-microscope immunohistochemistry. Serial sections were labelled with antibodies against slow tonic, slow twitch, fast twitch and neonatal myosin isoforms as well as against myomesin, M-protein and the MM form of creatine kinase. Intrafusal fiber types were distinguished according to the pattern of ATPase activity following acid and alkaline preincubations.Nuclear bag₁ fibers were always strongly stained throughout with anti-slow tonic myosin, were positive for anti-slow twitch myosin towards and in the C-region but were unstained with anti-fast twitch and anti-neonatal myosins. The staining of nuclear bag₂ fibers was in general highly variable. However, they were most often strongly stained by anti-slow tonic myosin in the A-region and gradually lost this reactivity towards the poles, whereas a positive reaction with anti-slow twitch myosins was found along the whole fiber. Regional staining variability with antineonatal and anti-fast myosins was apparent, often with decreasing intensity towards the polar regions. Nuclear chain fibers showed strong transient reactivity with anti-slow tonic myosin in the equatorial region, did not react with anti-slow twitch and were always evenly stained by anti-fast twitch and anti-neonatal myosins. All three intrafusal fiber types were stained with anti-myomesin. Nuclear bag₁ fibers lacked staining for M-protein, whereas bag₂ fibers displayed intermediate staining, with regional variability, often increasing in reactivity towards the polar regions. Chain fibers were always strongly stained by anti-M-protein. The MM form of creatine kinase was present in all three fiber types, but bag₁ fibers were less reactive and clear striations were not observed, in contrast to bag₂ and chain fibers. Out of 38 cross sectioned spindles two were found to have an atypical fiber composition, (lack of chain fibers) and a rather diverse staining pattern for the different antibodies tested.Taken together, the data show that in adult rat solcus, slow tonic and neonatal myosin heavy, chain isoforms are only expressed in the muscle spindle fibers and that each intrafusal fiber type has a unique, although variable, composition of myosin heavy chain isoforms and M-band proteins. We propose that both motor and sensory innervation might be the determining factors regulating the variable expression of myosin heavy chain isoforms and M-band proteins in intrafusal fibers of rat muscle spindles.     

Treatment with beta bungarotoxin blocks muscle spindle formation in fetal rats

Sensory and motor fibers of peripheral nerves were irreversibly destroyed in fetal rats by administering beta bungarotoxin (BTX) on embryonic day 16 or 17, after assembly of primary myotubes, but before the formation of muscle spindles. Soleus muscles of toxin-treated fetuses and their untreated littermates were removed just prior to birth and were examined by light microscopy of serial transverse sections for the presence of spindles and immunocytochemical expression of several isoforms of myosin heavy chains (MHC). Untreated muscles exhibited numerous spindles that were innervated by branches of intramuscular nerves and contained muscle fibers expressing a slow-tonic MHC isoform characteristic of the intrafusal but not extrafusal fibers. Toxin-treated muscles were devoid of intramuscular nerve bundles and perineurial structures. Encapsulations of muscle fibers resembling spindles were absent and no myotubes expressed the slow-tonic MHC isoform associated with intrafusal fibers in beta BTX-treated muscles. Thus, the assembly of muscle spindles, formation of the spindle capsule, and transformation of undifferentiated myotubes into the intrafusal fibers that contain spindle-specific myosin isoforms all depend on the presence of innervation in prenatal rat muscles.     

Regenerating adult chicken skeletal muscle and satellite cell cultures express embryonic patterns of myosin and tropomyosin isoforms

Regenerating areas of adult chicken fast muscle (pectoralis major) and slow muscle (anterior latissimus dorsi) were examined in order to determine synthesis patterns of myosin light chains, heavy chains and tropomyosin. In addition, these patterns were also examined in muscle cultures derived from satellite cells of adult fast and slow muscle. One week after cold-injury the regenerating fast muscle showed a pattern of synthesis that was predominately embryonic. These muscles synthesized the embryonic myosin heavy chain, beta-tropomyosin and reduced amounts of myosin fast light chain-3 which are characteristic of embryonic fast muscle but synthesized very little myosin slow light chains. The regenerating slow muscle, however, showed a nearly complete array of embryonic peptides including embryonic myosin heavy chain, fast and slow myosin light chains and both alpha-fast and slow tropomyosins. Peptide map analysis of the embryonic myosin heavy chains synthesized by regenerating fast and slow muscles showed them to be identical. Thus, in both muscles there is a return to embryonic patterns during regeneration but this return appears to be incomplete in the pectoralis major. By 4 weeks postinjury both regenerating fast and slow muscles had stopped synthesizing embryonic isoforms of myosin and tropomyosin and had returned to a normal adult pattern of synthesis. Adult fast and slow muscles yielded a satellite cell population that formed muscle fibers in culture. Fibers derived from either population synthesized the embryonic myosin heavy chain in addition to alpha-fast and beta-tropomyosin. Thus, muscle fibers derived in culture from satellite cells of fast and slow muscles synthesized a predominately embryonic pattern of myosin heavy chains and tropomyosin. In addition, however, the satellite cell-derived myotubes from fast muscle synthesized only fast myosin light chains while the myotubes derived from slow muscle satellite cells synthesized both fast and slow myosin light chains. Thus, while both kinds of satellite cells produced embryonic type myotubes in culture the overall patterns were not identical. Satellite cells of fast and slow muscle appear therefore to have diverged from each other in their commitment during maturation in vivo.     

Muscle spindles in the deep muscles of the human neck: a morphological and immunocytochemical study.

Muscle spindle density is extremely high in the deep muscles of the human neck. However, there is a paucity of information regarding the morphology and immunoreactivity of these muscle spindles. The objective of this study was to investigate the intrafusal fiber content and to assess the myosin heavy chain (MyHC) composition of muscle spindles from human deep neck muscles. In addition to the conventional spindles containing bag(1), bag(2), and chain fibers (b(1)b(2)c spindle), we observed a number of spindles lacking bag(1) (b(2)c spindle) or bag(2) (b(1)c spindle) fibers. Both bag(1) and bag(2) fibers contained slow tonic MyHCs along their entire fiber length and MyHCI, MyHCIIa, embryonic, and alpha-cardiac MyHC isoforms along a variable length of the fibers. Fetal MyHC was present in bag(2) fibers but not in bag(1) fibers. Nuclear chain fibers contained MyHCIIa, embryonic, and fetal isoforms with regional variations. We also compared the present data with our previous results obtained from muscle spindles in human biceps brachii and the first lumbrical muscles. The allotment of numbers of intrafusal fibers and the MyHC composition showed some muscle-related differences, suggesting functional specialization in the control of movement among different human muscles.     

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.     

Slow and fast myosin heavy chain content defines three types of myotubes in early muscle cell cultures

We prepared monoclonal antibodies specific for fast or slow classes of myosin heavy chain isoforms in the chicken and used them to probe myosin expression in cultures of myotubes derived from embryonic chicken myoblasts. Myosin heavy chain expression was assayed by gel electrophoresis and immunoblotting of extracted myosin and by immunostaining of cultures of myotubes. Myotubes that formed from embryonic day 5-6 pectoral myoblasts synthesized both a fast and a slow class of myosin heavy chain, which were electrophoretically and immunologically distinct, but only the fast class of myosin heavy chain was synthesized by myotubes that formed in cultures of embryonic day 8 or older myoblasts. Furthermore, three types of myotubes formed in cultures of embryonic day 5-6 myoblasts: one that contained only a fast myosin heavy chain, a second that contained only a slow myosin heavy chain, and a third that contained both a fast and a slow heavy chain. Myotubes that formed in cultures of embryonic day 8 or older myoblasts, however, were of a single type that synthesized only a fast class of myosin heavy chain. Regardless of whether myoblasts from embryonic day 6 pectoral muscle were cultured alone or mixed with an equal number of myoblasts from embryonic day 12 muscle, the number of myotubes that formed and contained a slow class of myosin was the same. These results demonstrate that the slow class of myosin heavy chain can be synthesized by myotubes formed in cell culture, and that three types of myotubes form in culture from pectoral muscle myoblasts that are isolated early in development, but only one type of myotube forms from older myoblasts; and they suggest that muscle fiber formation probably depends upon different populations of myoblasts that co-exist and remain distinct during myogenesis.     

Presence in chicken tibialis anterior and extensor digitorum longus muscle spindles of reactive and unreactive intrafusal fibers after incubation with monoclonal antibodies against myosin heavy chains

Cross and longitudinal sections from the encapsulated portions of chicken tibialis anterior and extensor digitorum longus muscle spindles were examined to determine whether their intrafusal fibers were a structurally homogeneous or heterogeneous population. The techniques used were the histochemical actomyosin (mATPase) reaction, and fluorescence immunohistochemistry employing two monoclonal antibodies, CA-83 and CCM-52, that are specific for myosin heavy chains. After incubation with antibody CCM-52, intrafusal fibers fluoresced either strongly or weakly to moderately. Antibody CA-83 was even more selective. In addition to identifying the strongly reactive category, it clearly separated the remaining fibers into unreactive and moderately reactive groups. As a whole, after incubation for mATPase, pH 9.6 preincubation, unreactive fibers stained darker than strongly reactive fibers. Moreover, the cross-sectional area of the unreactive fibers was significantly larger than that of the strongly reactive fibers. In the average-size muscle spindle with six intrafusal fibers, there were four unreactive fibers and two strongly reactive fibers. In about one-third of the receptors examined, one moderately reactive fiber was present. Taken together, the data indicate that intrafusal fibers of chicken tibialis anterior and extensor digitorum longus muscles are not structurally homogeneous. The observed variations can be better explained in terms of different fiber types than of continuous gradients within one type of fiber.     

Differential Expression of PTP1D, a Protein Tyrosine Phosphatase with Two SH2 Domains, in Slow and Fast Skeletal Muscle Fibers

We show that PTP1D, a protein tyrosine phosphatase that contains two SH2 domains, is preferentially expressed in slow skeletal muscle fibers. Immunohistochemical staining using polyclonal antibodies against PTP1D demonstrated that PTP1D was expressed in a subpopulation of rodent muscle fibers. These fibers were identified as slow Type I fibers based on histochemical ATPase assays and slow myosin heavy chain expression. Northern and Western analyses showed that PTP1D levels were higher in predominantly slow muscles than in predominantly fast muscles. This differential expression of PTP1D in slow muscle fibers appeared by birth. In cultures of mouse myogenic cells, PTP1D was expressed after MyoD and myogenin and appeared in myotubes derived from embryonic, fetal, and postnatal myoblasts. Remarkably, PTP1D was organized into sarcomeres in a pattern coincident with myosin heavy chain, suggesting that PTP1D associates with a component of the thick filament. These results show that PTP1D is preferentially expressed in slow muscle fibers. We speculate that PTP1D may play a role in slow muscle fiber function and differentiation.     

Origin of intrafusal muscle fibers in the rat

The expression of several isoforms of myosin heavy chain (MHC) by intrafusal and extrafusal fibers of the rat soleus muscle at different stages of development was compared by immunocytochemistry. The first intrafusal myotube to form, the bag2 fiber, expressed a slow-twitch MHC isoform identical to that expressed by the primary extrafusal myotubes. The second intrafusal myotube to form, the bag1 fiber, expressed a fast-twitch MHC similar to that initially expressed by the secondary extrafusal myotubes. At subsequent stages of development, the equatorial and juxtaequatorial regions of bag2 and bag1 intrafusal myofibers began to express a slow-tonic myosin isoform not expressed by extrafusal fibers, and ceased to express some of the MHC isoforms present initially. Myotubes which eventually matured into chain fibers expressed initially both the slow-twitch and fast-twitch MHC isoforms similar to some secondary extrafusal myotubes. In contrast, adult chain fibers expressed the fast-twitch MHC isoform only. Hence intrafusal myotubes initially expressed no unique MHCs, but rather expressed MHCs similar to those expressed by extrafusal myotubes at the same chronological stage of muscle development. These observations suggest that both intrafusal and extrafusal fibers develop from common pools of bipotential myotubes. Differences in MHC expression observed between intrafusal and extrafusal fibers of rat muscle might then result from a morphogenetic effect of afferent innervation on intrafusal myotubes.     

Differentiation of original and regenerated skeletal muscle fibres in mdx dystrophic muscles

The differentiation of both original muscle fibres and the regenerated muscle fibres following necrosis in mdx muscles was investigated using immunoblotting and immunocytochemical procedures. Before the onset of necrosis, postnatal skeletal muscles in mdx mouse differentiated well with only a slight delay in differentiation indicated by the level of developmental isoforms of troponin T. Prior to the onset of apparent myopathic change, both fast and slow skeletal muscle fibre types in mdx leg muscles also differentiated well when investigated by analysis of specific myosin heavy chain expression pattern. While the original muscle fibres in mdx leg muscles developed well, the differentiation of regenerated myotubes into both slow and distinct fast muscle fibre types, however, was markedly delayed or inhibited as indicated by several clusters of homogeneously staining fibres even at 14 weeks of age. The number of slow myosin heavy chain-positive myotubes amongst the regenerated muscle clusters was quite small even in soleus. This study thus established that while muscle fibres initially develop normally with only a slight delay in the differentiation process, the differentiation of regenerated myotubes in mdx muscles is markedly compromised and consequently delayed.     

Origin of intrafusal muscle fibers in the rat

Summary The expression of several isoforms of myosin heavy chain (MHC) by intrafusal and extrafusal fibers of the rat soleus muscle at different stages of development was compared by immunocytochemistry. The first intrafusal myotube to form, the bag₂ fiber, expressed a slow-twitch MHC isoform identical to that expressed by the primary extrafusal myotubes. The second intrafusal myotube to form, the bag₁ fiber, expressed a fast-twitch MHC similar to that initially expressed by the secondary extrafusal myotubes. At subsequent stages of development, the equatorial and juxtaequatorial regions of bag₂ and bag₁ intrafusal myofibers began to express a slow-tonic myosin isoform not expressed by extrafusal fibers, and ceased to express some of the MHC isoforms present initially. Myotubes which eventually matured into chain fibers expressed initially both the slow-twitch and fast-twitch MHC isoforms similar to some secondary extrafusal myotubes. In contrast, adult chain fibers expressed the fast-twitch MHC isoform only. Hence intrafusal myotubes initially expressed no unique MHCs, but rather expressed MHCs similar to those expressed by extrafusal myotubes at the same chronological stage of muscle development. These observations suggest that both intrafusal and extrafusal fibers develop from common pools of bipotential myotubes. Differences in MHC expression observed between intrafusal and extrafusal fibers of rat muscle might then result from a morphogenetic effect of afferent innervation on intrafusal myotubes.