Muscle development in the grasshopper embryo. III. Sequential origin of the flexor tibiae muscle pioneers

The flexor (FlTi) and extensor (ETi) tibiae are antagonist muscles located in the femur of the metathoracic leg of the grasshopper. Both are complex, consisting of an array of bundles of muscle fibers connecting the ectoderm of the wall of the femur with their respective apodemes. In the previous paper (E. E. Ball and C. S. Goodman, 1985, Dev. Biol. 111, 399-416) we described the embryonic development of the ETi muscle, focusing in particular on its syncytial origin from a giant supramuscle pioneer which later divides into an array of individual muscle pioneers. Here we describe the embryonic development of the FlTi muscle. In contrast to the development of the ETi muscle, the array of individual muscle pioneers for the FlTi does not have a syncytial origin but rather arises by sequential recruitment from the mass of smaller, undifferentiated mesoderm cells. The FlTi MPs first appear as two cells symmetrically placed on the corners of the FlTi apodeme at around 37%. A third MP is then added between these two; this third MP later dies. Subsequent growth occurs by symmetrical addition of MPs distally along the sides of the developing apodeme and by enlargement of the individual MPs. Initially each MP contains only a single nucleus; by about 50% there are at least two to three nuclei per MP and each is surrounded by a cluster of smaller, undifferentiated mesoderm cells. Each MP develops into a bundle of muscle fibers by a cycle of fusion and division. The individual mesoderm cells surrounding each MP fuse with it starting at about 60%. At the same time, the large MP begins to divide into smaller muscle fibers.     

Axon growth from limb motorneurons in the locust embryo: the effect of target limb removal on the pattern of axon branching in the periphery

Metathoracic limb buds have been unilaterally ablated from locust embryos at 25 to 30% of embryonic development and the effect of this operation on the axon morphology of the motorneuron fast extensor tibiae (FETi) observed at later embryonic stages. In control embryos this neuron sends a single axon out the main leg nerve, nerve 5, to the extensor tibiae muscle in the femur. In limb ablated embryos the axon of FETi is found in a wide variety of aberrant peripheral nerve pathways and projects to a wide range of foreign muscles. There is a degree of apparent selectivity, but no rigid hierarchy, in the choice of pathway and muscle made by FETi. A high degree of variability is found between one embryo and another in the extent and pattern of axon branching. The axon of FETi is generally found in pathways that correspond to nerves in control embryos but on occasion grows along novel routes. An anteriorly directed dendritic branch, seldom seen in control FETi neurons, is frequently seen in experimental FETis. These findings are discussed in terms of the rules for specific axon growth in normal development.     

Muscle development in the grasshopper embryo. II. Syncytial origin of the extensor tibiae muscle pioneers

The extensor tibiae muscle (ETi) in the metathoracic leg of the grasshopper, which powers the jump, is among the most studied insect muscles. In contrast to many insect muscles which are simple (consisting of only a single bundle of muscle fibers), the ETi is a complex muscle which consists of an array of bundles of muscle fibers, each with a separate site of insertion on the body wall ectoderm and on the ETi apodeme ectoderm. Here we describe the embryonic development of this complex muscle. The ETi muscle develops from a single muscle pioneer (MP) which connects the initial invagination of the ETi apodeme to the wall of the femur. This MP then dramatically expands around the developing apodeme to form a large horseshoe-shaped, multinucleate cell, called the supramuscle pioneer (supra-MP); the number of nuclei in the supra-MP increases by cell fusion rather than by nuclear division. The arms of the supra-MP grow steadily longer and their outer edges begin to appear scalloped, certain areas remaining tightly apposed to the ectoderm of the wall of the leg while adjacent areas lose their adhesion and are pulled away. By about 50% of embryonic development the ETi supra-MP consists of a periodic series of bridges (cytoplasmic extensions) connecting the leg wall ectoderm with the apodeme, and linked into a giant syncytium near their inner, apodeme surface by a thin layer of cytoplasm containing hundreds of nuclei. Each bridge is surrounded by a cluster of many smaller mesoderm cells. Next the syncytium begins to divide such that by 60% the periodic bridges of the supra-MP have lost syncytial contact with each other and now themselves form an array of smaller, individual, multinucleate MPs connecting the body wall to the apodeme, each surrounded by a mass of undifferentiated mesoderm cells. This initial cycle of fusion and division is followed by a second similar cycle in which the individual mesoderm cells surrounding each MP fuse with the MP. At the same time, the MP divides into the initial bundle of smaller muscle fibers. Coincident with this division into muscle fibers is the further development of thick and thin filaments and the T-tubule system.     

Programmed death of peripheral pioneer neurons in the grasshopper embryo

The Ti1 pioneer neurons arise at the distal tip of the metathoracic leg in the grasshopper embryo, and are the first neurons in the limb bud to extend axons to the central nervous system (C. M. Bate (1976) Nature (London) 260, 54-56; H. Keshishian (1980) Dev. Biol. 80, 388-397). By providing a neural pathway along which growth cones of later arising neurons migrate, these pioneer axons establish the route of one of the major nerve trunks in the leg (Keshishian, 1980; R. K. Ho and C. S. Goodman (1982) Nature (London) 297, 404-406; D. Bentley and H. Keshishian (1982) Science 218, 1082-1088). Here, we demonstrate that at the 55-59% stage of development, the two Ti1 pioneer neurons undergo programmed death. The role which these pioneers serve in establishing a nerve route appears to be their only function, and may be important for the normal development of the peripheral nervous system. The Ti1 pioneers provide an example of a previously hypothesized class (J. W. Truman (1984) Annu. Rev. Neurosci. 7, 171-188) of programmed neuron death: obsolete neurons whose function was developmental rather than behavioral.     

Regeneration of motorneurones to a cricket muscle after partial or complete denervation

The metathoracic extensor tibiae muscle of the cricket Teleogryllus oceanicus is innervated by two excitatory axons; one of which leaves the metathoracic ganglion through nerve 5, the other through nerve 3. Axons in nerve 5 frequently regenerate to reinnervate the extensor tibiae if the nerve is sectioned in a late nymphal stage; functional reinnervation is rare if the nerve is sectioned in young adults. The muscle may become reinnervated by several axons regenerating through nerve 5, and individual muscle fibres may receive inputs from two regenerated axons. Axons regrowing through nerve 5 to a partially-denervated extensor tibiae preferentially innervate fibres in the central portion of the muscle, which is the normal innervation field of nerve 5. If the muscle is totally denervated by transection of both nerve 5 and nerve 3b, reinnervation is less specific and fibres throughout the muscle may be reinnervated by axons in either nerve. Reinnervation by regenerating axons is progressive. The proportion of muscles which are functionally reinnervated by regenerated axons increases with survival time as does the proportion of fibres within a muscle with reinnervation. The amplitude of excitatory junctional potentials and of muscle contraction evoked by regenerated axons both increase with survival time.     

Embryogenesis of peripheral nerve pathways in grasshopper legs. I. The initial nerve pathway to the CNS

The founding of the first nerve path of the grasshopper metathoracic leg was examined at the level of identified neurons, using intracellular dye fills, immunohistochemistry, Nomarski optics, and scanning and transmission electron microscopy. The embryonic nerve is established by the axonal trajectory of a pair of afferent pioneer neurons, the tibial 1 (Ti1) cells. Following a period of profuse filopodial sprouting, the Ti1 axonal growth cones, possessing 75- to 100-microns-long filopodia, navigate a stereotyped path across the limb bud epithelium to the base of the appendage and into the CNS. The Ti1 axons grow from cell to cell along a chain of preaxonogenesis neurons spaced at intervals along the pathway, forming dye-passing junctions with them. The contacted neurons subsequently undergo axonogenesis and follow the pioneer axons into the CNS. Later arising neurons project their axons onto the cell bodies of the chain, thereby establishing the principal branch points of the nerve. Among the later arising afferents are the sensory neurons of the femoral chordotonal and subgenual organs. The morphology of the adult nerve appears to be determined by the stereotyped positioning of neurons in the differentiating limb bud and by the resultant axonal trajectories established during the first 10% of peripheral neurogenesis.     

Distribution of fiber types in embryonic chick limb muscles innervated by foreign motoneurons

The differentiation of distinct myotube fiber types in chick limb muscle development is coincident with innervation. The role of motoneurons in influencing fiber type differentiation was analyzed by causing chick hind limb muscles to be innervated by inappropriate motoneurons and then examining experimental muscles for changes in the distribution of myosin ATPase fiber types. Motoneuron innervation of limb muscles was altered by performing either limb shifts, limb reversals, or large spinal cord reversals on early neural tube or limb bud stage chick embryos. The distribution of fiber types was then analyzed in muscles from stage 36 (E10) to stage 45 (E20) embryos after processing hind limb sections for myosin ATPase histochemistry. In the majority of experimental muscles examined (267/312), the distribution of myosin ATPase fiber types was unaltered. In the remaining experimental muscles (14%), alterations in the distribution of myosin ATPase fiber types occurred, indicating that in some cases, foreign innervation may alter the developmental program of differentiating myotubes. The results suggest that myotubes differentiate myosin ATPase staining characteristics according to an intrinsic program and that these differentiating myotubes are selectively innervated by motoneurons of the appropriate type under most conditions including normal development. Under exceptional circumstances of motoneuron-muscle fiber type mismatch, embryonic motoneurons can alter fiber type expression.     

Embryogenesis of peripheral nerve pathways in grasshopper legs. II. The major nerve routes

In the preceding paper (H. Keshishian and D. Bentley, 1983a, Dev. Biol. 96, 89-102) the events leading to the morphogenesis of nerve 5B1 in the grasshopper embryonic metathoracic leg were presented. Here the role of later differentiating peripheral neurons in establishing the other major nerves of the leg is examined. In addition to the (tibial 1) (Ti1) pioneer neuron cell pairs that establish nerve 5B1 in the tibia femur, and coxa-trochanter, six later differentiating cells and/or cell pairs were identified and examined with respect to their role in peripheral nerve ontogeny. Nerve path pioneering was observed in two cell pairs of the distal tarsus (Ta1 and Ta2), by neurons of the posterior proximal tibia (Ti2), the posterior midfemur (neurons F3 and F4), and by an additional cell pair in the anterior coxal-trochanteral region of the limb bud (cell pair, CT2). In addition, efferent projections onto limb and epithelia played an important role in establishing nerve branches. In two nerves the axonal trajectory from the periphery to the CNS is established by afferent and efferent pathfinding axons meeting halfway and overgrowing each other's established projections. For each nerve branch examined it was found that axons projected initially to the cell bodies of previously arising neurons along the trajectory. The location along the limb bud ectoderm where neurons arise, and hence their ultimate cell body positions, played an important role in organizing the fasciculation of follower axons and establishing branch points.     

Local innervation patterns of the metathoracic flexor and extensor tibiae motor neurons in the cricket Gryllus bimaculatus

To elucidate neural mechanisms underlying walking and jumping in insects, motor neurons supplying femoral muscles have been identified mainly in locusts and katydids, but not in crickets. In this study, the motor innervation patterns of the metathoracic flexor and extensor tibiae muscles in the cricket, Gryllus bimaculatus were investigated by differential back-fills and nerve recordings. Whereas the extensor tibiae muscle has an innervation pattern similar to that of other orthopterans, the flexor has an innervation unique to this species. The main body of the flexor muscle is divided into the proximal, middle and distal regions, which receive morphologically unique terminations from almost non-overlapping sets of motor neurons. The proximal region is innervated by about 12 moderate-sized excitatory motor neurons and two inhibitory neurons while the middle and distal regions are innervated by three and four large excitatory motor neurons, respectively. The most-distally located accessory flexor muscle, inserting on a common flexor apodeme with the main muscle, is innervated by at least four small excitatory (slow-type) and two common inhibitory motor neurons. The two excitatory and two inhibitory motor neurons that innervate the accessory flexor muscle also innervate the proximal bundles of the main flexor muscle. This suggests that the most proximal and distal parts of the flexor muscle participate synergistically in fine motor control while the rest participates in powerful drive of tibial flexion movement.     

Transition of myosin isozymes during development of human masseter muscle. Persistence of developmental isoforms during postnatal stage

Previous results have shown that the adult human masseter muscle contains myosin isoforms that are specific to early stages of development in trunk and limb muscles, i.e. embryonic and fetal (neonatal) myosin heavy chains (MHC) and embryonic myosin light chain (MLC1emb). We wanted to know if this specific pattern is the result of a late maturation or of a distinct evolution during development. We show here that the embryonic and the fetal MHC and the MLC1emb are expressed throughout perinatal and postnatal masseter development. Our results also demonstrate that MLC1emb accumulation increases considerably during the postnatal period. In addition, both the slow MLCs and the slow isoform of tropomyosin are expressed later in the masseter than quadriceps and the fast skeletal muscle isoform MLC3 is not detected during fetal and early postnatal development in the masseter whereas it is expressed throughout fetal development in the quadriceps. Our results thus confirm previous histochemical data and demonstrate that the masseter muscle displays a pattern of myosin and tropomyosin isoform transitions different to that previously described in trunk and limb muscles. This suggests that control of masseter muscle development involves mechanisms distinct from other body muscles, possibly as a result of either its craniofacial innervation or of a possibly different embryonic origin.     

Expression of fascin-1, the gene encoding the actin-bundling protein fascin-1, during mouse embryogenesis

Fascin-1 is an actin-bundling protein that contributes to the architecture and function of cell protrusions and microfilaments in cell adhesion, interactions and motility. Fascin-1 has been studied in cultured cells and by biophysical methods, but little is known about its distribution and functions in vertebrate development. As a first step to understanding the role of fascin-1 in embryogenesis, we have characterised the expression pattern of fascin-1 by in situ hybridisation on whole-mount and sectioned mouse embryos from embryonic day (E)8.0-E16.5. Fascin-1 was widely expressed throughout the embryo and the developing nervous system and mesenchymal tissues represented major sites of expression. Intense signals were observed in different regions of the brain, in the spinal cord and retina, and the cranial and dorsal root ganglia (DRG) appeared strongly positive. This neural expression remained strong throughout development. Fascin-1 was also present in the developing somites. High expression was detected in branchial arches and limb bud mesenchyme. At later stages, fascin-1 was expressed in different muscles of the face, skeletal muscles of the body, and in smooth muscle layers of several organs. Limb tendons appeared strongly positive. There was weak expression in heart ventricles. These results show that fascin-1 is principally expressed in neural and mesenchymal derivatives during embryonic development.     

Homonomies within the ventral muscle system and the associated motoneurons in the locust,Schistocerca gregaria (Insecta,Caelifera)

To understand the segmental reiteration of an insect, the serially arranged neuromuscular system of the locust, Schistocerca gregaria, is studied. The ventral muscle system is chosen and its motoneuronal supply is described in the thoracic and pregenital segments. In general, repetitively arranged, similar sets of motoneurons (MNs) supply the ventral muscles of these segments. Common criteria of both topology of muscles and neural features (nerve branches and motoneuronal supply) suggest possible homonomies of the ventral longitudinal muscles and ventral diaphragm of the thoracic and abdominal system. Based on a segment-by-segment analysis, muscle topology and motor supply match, in most instances. There are, however, cases where such a parallelism is missing. In a particular cases the supply of apparently homonomous muscles shifts from one set of MNs to another. In another case, putatively equivalent MNs of different ganglia supply morphologically different muscle structures in the adult animal. Therefore, it becomes apparent that muscles and their supplying MNs are, in principle, independent elements which might be subjected autonomously to ontogenetic processes. As a consequence, in the search for the basic segmental Bauplan depending on homonomous structures, muscles and MNs have to be regarded as separate entities.Abbreviations A1–6 abdominal ganglion (or neuromere A1–3) - AS1–6 abdominal segment 1–6 - DUM doisal unpaired median - M muscle (number) - MN motoneuron - N nerve (number) - PMN paramedian nerve - T1–3 pro-, meso-, metathoracic ganglion - TS1–3 pro-, meso-, metathoracic segment - VD ventral diaphragm - VM ventral muscle     

The structure and function of serially homologous leg motor neurons in the locust. I. Anatomy

Twenty-one prothoracic and 17 mesothoracic motor neurons innervating leg muscles have been identified physiologically and subsequently injected with dye from a microelectrode. A tract containing the primary neurites of motor neurons innervating the retractor unquis, levator and depressor tarsus, flexor tibiae, and reductor femora is described. All motor neurons studied have regions in which their dendritic branches overlap with those of other leg motor neurons. Identified, serially homologous motor neurons in the three thoracic ganglia were found to have: (1) cell bodies at similar locations and morphologically similar primary neurites (e.g., flexor tibiae motor neurons), (2) cell bodies at different locations in each ganglion and morphologically different primary neurites in each ganglion (e.g., fast retractor unguis motor neurons), or (3) cell bodies at similar locations and morphologically similar primary neurites but with a functional switch in one ganglion relative to the function of the neurons in the other two ganglia. As an example of the latter, the morphology of the metathoracic slow extensor tibiae (SETi) motor neurons was similar to that of pro- and mesothoracic fast extensor tibiae (FETi) motor neurons. Similarly the metathoracic FETi bears a striking resemblance to the pro- and the mesothoracic SETi. It is proposed that in the metathoracic ganglion the two extensor tibiae motor neurons have switched functions while retaining similar morphologies relative to the structure and function of their pro- and mesothoracic serial homologues.     

Signals regulating muscle formation in the limb during embryonic development

Classically, somites have been the preparation of choice for the study of muscle development, while the limb bud is the preferred model of axis formation. Nevertheless, the limb bud offers some experimental advantages for muscle studies. This review describes the successive events involved in limb muscle formation during embryonic development, the properties of the key marker molecules and resumes our current knowledge of the signalling pathways involved.     

Peptidergic innervation of leg muscles of the cockroach,Periplaneta americana (L.), and a possible role in modulation of muscle contraction

FMRFamide-related peptides of insects are particularly important because of their possible function as neurohormones and neuromodulators on a wide variety of tissues. Part of this study was an investigation of the immunofluorescent staining of motor nerves which arise in the metathoracic ganglion, examined in wholemount using an antiserum that recognizes extended -RFamide peptides (generally recognized to be of the FMRFamide family). This antiserum revealed immunochemical staining of numerous cell bodies in the metathoracic ganglion and of axons in peripheral nerve 5, a large nerve which contains both motor and sensory fibres. Axons staining positive for FMRFamide-related peptides were traced in nerve 5 as far as the femur-tibia joint, and into the first (sensory-motor) and third (motor only) ramus of nerve 5. Reverse-phase HPLC with radioimmunoassay revealed a peak of FMRFamide-related peptide activity in nerve 5 that was coincident with a peak found when thoracic ganglia were processed in the same fashion. A physiological assay was devised to test the ability of various non-native peptides to alter the characteristics of contraction of skeletal muscles of the legs. Using neurally evoked contractions of coxal depressor muscles of the metathoracic leg it was determined that several non-native peptides could potentiate muscle contractions.The results of this study suggest that muscles of the legs receive innervation by identifiable, FMRFamide-related peptide-containing neurons and that the release of peptide(s) at the muscle may be yet another method of modulating the mechanics of muscle contraction.Abbreviations D _f fast depressor motor neuron - D _s slow depressor motor neuron - DU M dorsal unpaired median - FaRPs FMRFamide related peptides - FEFe fast extensor of the femur - FFFe fast flexor of the femur - FITC fluorescein isothiocyanate - FPC fast promotor of the coxa - FPT fast flexor of the pretarsus - I _1–3 inhibitory motor neurons - LMS leucomyosuppressin, N5 nerve 5 - N5r1 first ramus of nerve 5 - PBS phosphate buffered saline - PLCl posterior lateral cluster - RIA radioimmunoassay - SETi slow extensor of the tibia - SFTi slow flexor of the tibia - TFA trifluoroacetic acid - VMCl ventral median cluster     

Combinations of monoclonal antibodies distinguish mesenchymal, myogenic, and chondrogenic precursors of the developing chick embryo

Monoclonal antibodies (MAbs) were used as probes for molecular differences in the surfaces of nonterminally differentiated cells of the developing chick limb. The specificity of the MAbs was determined by immunofluorescent localization performed on cultured breast muscle and limb bud cells and cryosections of a variety of embryonic (stages 15-37) and neonatal tissues. Subpopulations of MAb-positive and -negative cells were isolated by fluorescence-activated cell sorting and their developmental potential was assessed in vitro. Cells of the compacted somite, lateral plate mesoderm, and early limb bud were labeled with the CSAT MAb. Myogenic precursors of the dermatome and limb bud were labeled with the CSAT and L4 MAbs. Chondrogenic precursors of the sclerotome and limb bud were labeled with the CSAT, L4, and C5 MAbs. These precursors were distinguished from fibroblasts which were labeled with the CSAT and C1 MAbs. The differentiation and maturation of muscle and cartilage were accompanied by alterations in the labeling patterns of the MAbs. These results indicate that combinations of these MAbs can be used to distinguish mesenchymal, myogenic, and chondrogenic precursors, identify their site of origin during development, and isolate subpopulations of embryonic cells.     

Diversity of fast myosin heavy chain expression during development of gastrocnemius, bicep brachii, and posterior latissimus dorsi muscles in normal and dystrophic chickens

The expression of fast myosin heavy chain (MHC) isoforms was examined in developing bicep brachii, lateral gastrocnemius, and posterior latissimus dorsi (PLD) muscles of inbred normal White Leghorn chickens (Line 03) and genetically related inbred dystrophic White Leghorn chickens (Line 433). Utilizing a highly characterized monoclonal antibody library we employed ELISA, Western blot, immunocytochemical, and MHC epitope mapping techniques to determine which MHCs were present in the fibers of these muscles at different stages of development. The developmental pattern of MHC expression in the normal bicep brachii was uniform with all fibers initially accumulating embryonic MHC similar to that of the pectoralis muscle. At hatching the neonatal isoform was expressed in all fibers; however, unlike in the pectoralis muscle the embryonic MHC isoform did not disappear. With increasing age the neonatal MHC was repressed leaving the embryonic MHC as the only detectable isoform present in the adult bicep brachii muscle. While initially expressing embryonic MHC in ovo, the post-hatch normal gastrocnemius expressed both embryonic and neonatal MHCs. However, unlike the bicep brachii muscle, this pattern of expression continued in the adult muscle. The adult normal gastrocnemius stained heterogeneously with anti-embryonic and anti-neonatal antibodies indicating that mature fibers could contain either isoform or both. Neither the bicep brachii muscle nor the lateral gastrocnemius muscle reacted with the adult specific antibody at any stage of development. In the developing posterior latissimus dorsi muscle (PLD), embryonic, neonatal, and adult isoforms sequentially appeared; however, expression of the embryonic isoform continued throughout development. In the adult PLD, both embryonic and adult MHCs were expressed, with most fibers expressing both isoforms. In dystrophic neonates and adults virtually all fibers of the bicep brachii, gastrocnemius, and PLD muscles were identical and contained embryonic and neonatal MHCs. These results corroborate previous observations that there are alternative programs of fast MHC expression to that found in the pectoralis muscle of the chicken (M.T. Crow and F.E. Stockdale, 1986, Dev. Biol. 118, 333-342), and that diversification into fibers containing specific MHCs fails to occur in the fast muscle fibers of the dystrophic chicken. These results are consistent with the hypothesis that avian muscular dystrophy is a developmental disorder that is associated with alterations in isoform switching during muscle maturation.     

Propagation failure of action potentials at the bifurcation point of the slow axon innervating the extensor tibiae muscle ofDecticus albifrons (Orthoptera)

Summary Failure of conduction of nerve impulses has been observed at the bifurcation point of the metathoracic slow extensor tibiae motor axon (SETi) ofDecticus albifrons. Records from the region proximal and distal to the bifurcation point of the axon showed that during prolonged and repetitive stimulation and after a certain number of stimuli, proportional to the stimulating frequency, some SETi action potentials failed to cross this point (Fig. 1).Cross-sections of the metathoracic extensor motor nerve ofD. albifrons show that at the region of axonal bifurcation, both the neural lamella and the layer of glial cells (the sheath) around the SETi axons became thinner than the region proximal and distal to the bifurcation (Fig. 2).The possible role of the conduction block in the neuronal control of the muscle has been discussed.Abbreviations ETi extensor tibiae - SETi slow extensor tibiae - PE proximal electrode - DE distal electrode - SE stimulating electrode     

Immunochemical analysis of troponin T isoforms in adult, embryonic, regenerating, and denervated chicken fast skeletal muscles

Using monoclonal antibodies (McAbs) which can distinguish between breast- and leg-type troponin T (TnT), we studied the spatial distribution of TnT isoforms in adult chicken fast skeletal muscles. The breast (pectoralis major) and leg (iliotibialis posterior) muscles were composed predominantly of homogeneous fibers containing breast- and leg-type TnT, respectively. The posterior latissimus dorsi muscle was composed of heterogeneous fibers of at least two types, namely breast and leg types. In developing and regenerating fast muscles, only leg-type TnT was expressed at early stages, and later breast-type TnT appeared either transiently or permanently. This led ultimately to several distinct adult fast muscle breast/leg TnT isoform profiles. Since both types of TnT were synthesized in embryonic and regenerating muscles with nerves intact as well as in regenerating muscles with nerves resected, the switching on of their expression during fast muscle development appears to be independent of nerves. However, its full development ("fine tuning" of the protein isoform distribution within the fast fiber types) and the maintenance of the adult state are presumed to be dependent on the nerves, since, although regenerating fibers in denervated muscles could exhibit the early and then the later embryonic stainabilities, they again returned to the early embryonic state; further, the denervation of adult muscles caused the replacement of TnT isoform from the adult to the early embryonic state.