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1.
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  相似文献   

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

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Summary The expression of four myosin heavy chain (MHC) isoforms, avian slow-tonic (ATO) or neonatal-twitch (ANT) and mammalian slow-twitch (MST) or fast-twitch (MFT) in intrafusal fibers was examined by immunocytochemistry of spindles in the tenuissimus muscle of adult eats. The predominant MHCs expressed by nuclear bag fibers were ATO and MST, whereas the MHCs prevalent in nuclear chain fibers were ANT and MFT. The expression of these isoforms of MHC was not uniform along the length of intrafusal fibers. In general, both bag and chain fibers expressed avian MHC in the intracapsular region and mammalian MHC in the extracapsular region. The nonuniform expression of MHCs observed along the length of bag and chain fibers implies that different genes are activated in myonuclei located in the intracapsular and extracapsular regions of the same muscle fiber. Regional differences in gene activation might result from a greater effect of afferents on myonuclei located near the equator of intrafusal fibers then on myonuclei outside the spindle capsule.  相似文献   

6.
J Kucera  J M Walro 《Histochemistry》1989,92(4):291-299
The expression of four myosin heavy chain (MHC) isoforms, avian slow-tonic (ATO) or neonatal-twitch (ANT) and mammalian slow-twitch (MST) or fast-twitch (MFT) in intrafusal fibers was examined by immunocytochemistry of spindles in the tenuissimus muscle of adult cats. The predominant MHCs expressed by nuclear bag fibers were ATO and MST, whereas the MHCs prevalent in nuclear chain fibers were ANT and MFT. The expression of these isoforms of MHC was not uniform along the length of intrafusal fibers. In general, both bag and chain fibers expressed avian MHC in the intracapsular region and mammalian MHC in the extracapsular region. The nonuniform expression of MHCs observed along the length of bag and chain fibers implies that different genes are activated in myonuclei located in the intracapsular and extracapsular regions of the same muscle fiber. Regional differences in gene activation might result from a greater effect of afferents on myonuclei located near the equator of intrafusal fibers then on myonuclei outside the spindle capsule.  相似文献   

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J Kucera  J M Walro 《Histochemistry》1988,90(2):151-160
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.  相似文献   

9.
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.  相似文献   

10.
J Kucera  J M Walro 《Histochemistry》1990,93(6):567-580
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.  相似文献   

11.
The effects of neuromuscular block on the pattern of distribution of myosin isozymes in developing skeletal muscle fibers was examined by immunocytochemistry. The homogeneous population of fibers in the anterior latissimus dorsi (ALD) of the 18-day chick embryo was converted by curare to a mosaic of at least two categories of fibers. Normally all fibers in this slow muscle reacted with antibodies against slow myosin (anti-ALD). They also reacted with an antibody specific for the alkali 1 light chain (anti-delta 1) but not the alkali 2 light chain (anti-delta 2) of fast myosin. After treatment with curare, which inhibits neuronal cell death and increases the number of axonal endings, ALD muscle fibers continued to react with anti-delta 1, but many now reacted with anti-delta 2 as well. The same fibers failed to react with anti-ALD. From this it can be concluded that the myosin in this population was converted to a type not normally present. The changes, therefore, are not merely a result of the preferential loss of a slow type of fiber, nor are they a result of delayed maturation. In contrast, curare had no apparent effect on the fast posterior latissimus dorsi (PLD). As in the normal muscle at 18 days, all fibers reacted strongly with anti-delta 1 and to variable degrees with anti-delta 2, and very few fibers reacted with anti-ALD. Our observations suggest that the dual response to antibodies against fast and slow myosin during development is not a necessary consequence of multiple axon terminals. We present evidence that curare induces the expression of a different myosin in the embryonic ALD, and we suggest that the selective transformation of the fiber population may be a manifestation of a change in composition of the motoneuron pool.  相似文献   

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The regulation of vertebrate muscle contraction with respect to the role of the different subunits of myosin remains somewhat uncertain. One approach to gaining a better understanding of the molecular basis of contraction is to study developing muscle which undergoes changes in myosin isozyme composition and contractile properties during the normal course of maturation. The present study utilizes single fibers from psoas muscles of rabbits at several ages as a model system for fast-twitch muscle development. This approach eliminates the inherent problems of interpreting results from studies on whole muscles which usually contain heterogeneous fiber types with respect to contractile properties and isoenzyme composition. Maximum velocity of shortening and tension-generating ability of individual fibers were measured and the myosin heavy chain composition of the same fibers was examined using an ultrasensitive sodium dodecyl sulfate-polyacrylamide gel system. The results indicate that 1) with regard to contractile properties, there is a transitional period from slow to fast shortening velocities within the first postnatal month; 2) a strong, positive correlation exists between the speed of shortening and tension-generating ability of individual postnatal day 7 fibers, suggesting that as more myosin is incorporated in these developing fibers it is of the fast type; and 3) there is a wide variation in maximum velocity of shortening among postnatal day 7 psoas fibers which is also a time when a mixture of heavy chain isoforms characterizes the myosin composition of single muscle fibers.  相似文献   

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Myosin was localized in situ in the posthatch chicken pectoralis using isoform-specific mAbs. The distribution among myofibrils was demonstrated by immunofluorescence and by immunogold EM. Fluorescein- or rhodamine-labeled antibody (12C5) specific for the head region (S1) of myosin was used as a marker to identify "embryonic" myosin. In longitudinal semithin frozen sections, a minority population of myofibrils stained intensely with 12C5. All other myofibrils in the same cell stained only weakly. Similarly, in Lowicryl-embedded ultrathin sections prepared for EM, a minority population reacted preferentially with gold-labeled 12C5. An antibody (5B4) specific for the rod portion of "neonatal" myosin reacted strongly with nearly all myofibrils, and this was evident by light and electron microscopy. A few of the fibrils that reacted strongly with 12C5 reacted weakly with 5B4. These observations demonstrate that an epitope reacting with 12C5 is more abundant in some myofibrils than in others within the same cell. Three categories of myofibrils can be identified by their relative proportions of embryonic and neonatal forms of myosin: in nearly all fibrils, a neonatal isoform predominates; in a minority population, embryonic and neonatal isoforms are both abundant; and in a few fibrils, an embryonic isoform predominates. It is concluded that there are distinct populations of myofibrils in which specific isoforms are segregated within an individual cell.  相似文献   

17.
Isozymes of myosin have been localized with respect to individual fibers in differentiating skeletal muscles of the rat and chicken using immunocytochemistry. The myosin light chain pattern has been analyzed in the same muscles by two-dimensional PAGE. In the muscles of both species, the response to antibodies against fast and slow adult myosin is consistent with the speed of contraction of the muscle. During early development, when speed of contraction is slow in future fast and slow muscles, all the fibers react strongly with anti-slow as well as with anti-fast myosin. As adult contractile properties are acquired, the fibers react with antibodies specific for either fast or slow myosin, but few fibers react with both antibodies. The myosin light chain pattern slow shows a change with development: the initial light chains (LC) are principally of the fast type, LC1(f), and LC2(f), independent of whether the embryonic muscle is destined to become a fast or a slow muscle in the adult. The LC3(f), light chain does not appear in significant amounts until after birth, in agreement with earlier reports. The predominance of fast light chains during early stages of development is especially evident in the rat soleus and chicken ALD, both slow muscles, in which LC1(f), is gradually replaced by the slow light chain, LC1(s), as development proceeds. Other features of the light chain pattern include an "embryonic" light chain in fetal and neonatal muscles of the rat, as originally demonstrated by R.G. Whalen, G.S. Butler- Browne, and F. Gros. (1978. J. Mol. Biol. 126:415-431.); and the presence of approximately 10 percent slow light chains in embryonic pectoralis, a fast white muscle in the adult chicken. The response of differentiating muscle fibers to anti-slow myosin antibody cannot, however, be ascribed solely to the presence of slow light chains, since antibody specific for the slow heavy chain continues to react with all the fibers. We conclude that during early development, the myosin consists of a population of molecules in which the heavy chain can be associated with a fast, slow, or embryonic light chain. Biochemical analysis has shown that this embryonic heavy chain (or chains) is distinct from adult fast or slow myosin (R.G. Whalen, K. Schwartz, P. Bouveret, S.M. Sell, and F. Gros. 1979. Proc. Natl. Acad. Sci. U.S.A. 76:5197-5201. J.I. Rushbrook, and A. Stracher. 1979. Proc Natl. Acad. Sci. U.S.A. 76:4331-4334. P.A. Benfield, S. Lowey, and D.D. LeBlanc. 1981. Biophys. J. 33(2, Pt. 2):243a[Abstr.]). Embryonic myosin, therefore, constitutes a unique class of molecules, whose synthesis ceases before the muscle differentiates into an adult pattern of fiber types.  相似文献   

18.
The effect of thyroid hormone on the expression of ventricular isomyosins V1, V2, and V3 was studied in fetal and neonatal rats. Between 15 and 21 days gestation, V3 accounts for 80-90% of fetal ventricular myosin. After birth, there is a rapid transition from the fetal V3 isotype to an equal mixture of V1 and V3 at 3 days, and to 100% V1 at 3 weeks of age. The endogenous serum levels of thyroxine (T4) and triiodothyronine (T3) increase from trace amounts in the fetus to adult levels at 2-3 weeks of age; this increase correlates with the maximal expression of V1 during the same period. Expression of the V1 isomyosin can be eliminated in the neonatal rat if endogenous thyroid hormone synthesis is suppressed by propylthiouracil (PTU) treatment. In the PTU-treated rats, V3 is the only isomyosin synthesized between 1 and 30 days of age. In fetal ventricle, the amount of V1 is also decreased but not completely eliminated by PTU treatment. Conversely, the relative amount of V1 can be increased in the fetal ventricle by increasing the fetal serum concentrations of T4 and T3 to adult physiological levels. In these fetal ventricles, V1 represents greater than 85% of the total myosin. Likewise, the expression and accumulation of V1 could be stimulated in ventricles of PTU-treated, 12-day-old rats by administration of pharmacological or physiological doses of T3. Within 4 to 8 h after an initial dose of T3, V1 accumulates to 5-10% of the ventricular myosin, and by 72 h comprises 60-80% of the myosin. These results indicate that endogenous thyroid hormone induces the synthesis of ventricular heavy chain alpha, which as a dimer forms the V1 isomyosin, or plays a permissive role for the continued synthesis of heavy chain alpha in ventricles of fetal and neonatal rats.  相似文献   

19.
When rat soleus muscles fibers regenerated after notexin-induced damage, AChRs were present at high density on the surface of the new muscle fibers at the sites of the original NMJs, even if the intact motor axons were not present during regeneration. Some AChR molecules which were labelled with R-BgTx before notexin-induced damage persisted for some days at junctional sites after new muscle fibres had regenerated. During muscle fiber degeneration, components of the muscle fiber plasma membrane appeared to remain longer in the junctional region than elsewhere. When muscles on which new "ectopic" NMJs had been forming for at least 2 weeks were damaged, AChR clusters together with sites of high AChE activity were present 2 weeks later on the regenerated muscles in the region of new NMJ formation, even if the "foreign" nerve was not intact during the period of regeneration. If ectopic NMJs had been forming for only 4 days at the time of muscle and nerve damage, neither AChR clusters nor AChE activity were detected on the regenerated muscle fibers.  相似文献   

20.
The dependence of adenosine-triphosphatase (ATPase) and succinic dehydrogenase (SDH) histochemical reactions on the pH of the preincubation medium was studied in serial cross sections of 1- to 6-month-old rat extensor digitorum longus (EDL) and soleus (SOL) muscles. The use of a wide spectrum of pH values confirmed the previous results showing that: (1) according to their ATPase and SDH reactions 3 types of extrafusal muscle fibres, i.e., fast-twitch glycolytic (FG), fast-twitch oxidative-glycolytic (FOG) and slow-twitch oxidative (SO) and 3 types of intrafusal muscle fibres, i.e. typical and intermediate nuclear bag fibres and nuclear chain fibres were observed; (2) only acid preincubation (pH 4.35) is necessary to demonstrate the reversal of the ATPase reaction; while (3) alkali preincubation (pH 10.4) does not provide any new important information as compared with ATPase without preincubation. Furthermore, it was shown that: (4) fast-twitch muscle fibres exhibited high ATPase activity on preincubations at pH 4.9 to 10.4, slow-twitch fibres had very high ATPase activity on preincubation at pH 4.3 and 4.5; (5) after preincubation at pH 4.5 two types of FOG fibres were observed, differing in their ATPase activity; (6) in both muscles there were fibres with intermediate ATPase activity both after acid and/or alkali preincubations; (7) the intrafusal muscle fibres exhibited some specific characteristics when compared with extrafusal fibres. In contrast to the ATPase reactions, SDH activity was decreased equally, in both extra- and intrafusal fibres, with increasing acidity and alkality of the preincubation medium.  相似文献   

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