Adult Leg Muscles Research Articles

Insulin-like growth factor 1 regulation of proliferation and differentiation of Xenopus laevis myogenic cells in vitro.

To understand the mechanism of muscle remodeling during Xenopus laevis metamorphosis, we examined the in vitro effect of insulin-like growth factor 1 (IGF-1) on growth and differentiation of three different-fate myogenic cell populations: tadpole tail, tadpole dorsal, and young adult leg muscle. IGF-1 promoted growth and differentiation of both tail and leg myogenic cells only under conditions where these cells could proliferate. Inhibition of cell proliferation by DNA synthesis inhibitor cytosine arabinoside completely canceled the IGF-1's cell differentiation promotion, suggesting the possibility that IGF-1's differentiation-promotion effect is an indirect effect via IGF-1's cell proliferation promotion. IGF-1 promoted differentiation dose dependently with maximum effect at 100-500ng/ml. RT-PCR analysis revealed the upregulation (11-fold) of ifg1 mRNA expression in developing limbs, suggesting that IGF-1 plays a role in promoting muscle differentiation during limb development. The combined effect of triiodo-L-thyronine (T3) and IGF-1 was also examined. In adult leg cells, IGF-1 promoted growth and differentiation irrespective of the presence of T3. In larval tail cells, cell count was 76% lower in the presence of T3, and IGF-1 did not promote proliferation and differentiation in T3-containing medium. In larval dorsal cells, cell count was also lower in the presence of T3, but IGF-1 enhanced proliferation and differentiation in T3-containing medium. This result is likely due to the presence among dorsal cells of both adult and larval types (1:1). Thus, IGF-1 affects only adult-type myogenic cells in the presence of T3 and helps accelerate dorsal muscle remodeling during metamorphosis.

Body size and the neural control of movement

Summary Nervous systems, muscles, and motor organs (biomechanics) generate movement cooperatively and should therefore change coordinately as body size changes. Proving this hypothesis requires a description of the nervous system, muscle, and biomechanics of individual species and comparison of these properties across a collection of species spanning a large size range. Obtaining these data in even one species is daunting, and we are far from reaching a general theory of how nervous system, muscle, and biomechanics depend on body size. Sufficient cross-species data nonetheless exist to identify some consequences of body size on the neural control of movement, which form the focus of this Primer: for example, locomotor cycle period depends on leg length; large animals should devote more neural resources to hazard avoidance and stride correction; the importance of gravity and momentum increases, and of passive muscle properties decreases, with limb size; and the medium in which movements occur affects these size dependencies. These considerations suggest that, in applying biological data to robotic design, proper ‘size' should be maintained across all levels (i.e., if small-animal neural mechanisms are used, so should small-animal muscle and biomechanical properties).

Remodeling of motor terminals during metamorphosis of the moth Manduca sexta: expression patterns of two distinct isoforms of Manduca fasciclin II.

During metamorphosis of the moth Manduca sexta, the neuromuscular system of the thoracic legs is reorganized dramatically. Larval leg muscles degenerate at the end of larval life, and new adult leg muscles develop during the ensuing pupal stage. Larval leg motoneurons persist, but undergo substantial remodeling of central and peripheral processes. As part of our on-going investigation of mechanisms underlying the remodeling of motor terminals, we have used antisera generated against Manduca-specific isoforms of the homophilic adhesion molecule fasciclin II (MFas II) to label motor terminals during metamorphosis. Antisera generated against the glycosyl-phosphatidylinositol (GPI) -linked isoform of MFas II (GPI-MFas II) labeled the motor nerves at all stages and seemed to be associated with glial cells ensheathing the peripheral nerves. In addition, the anti-GPI-MFas II antisera labeled regions associated with synaptic boutons at both larval and adult stages. In contrast, antisera generated against a transmembrane isoform of MFas II (TM-MFas II) only labeled specific neuronal processes at discrete intervals during remodeling. Identified leg motoneurons (such as the femoral depressor motoneuron) expressed detectable levels of TM-MFas II in their peripheral processes only during phases of motor-terminal retraction and initial stages of motor-terminal re-growth. Putative modulatory neurons (such as the unpaired median neurons), however, expressed TM-MFas II in their processes during larval stages as well as during remodeling. Use of the isoform-specific anti-MFas II antisera provided a novel method for visualizing remodeling of motor terminals during metamorphosis and helped distinguish different components of the motor nerves and neuromuscular junction.

Expression of the myosin heavy chain genes in the tail muscle of thyroid hormone-induced metamorphosingRana catesbeiana tadpoles

In tadpoles of the North American bullfrog, Rana catesbeiana, spontaneous and thyroid hormone (T3)-induced metamorphosis is characterized by regression of the tail, which is preceded by a decrease in total protein synthesis in tail tissues. We have demonstrated that thyroid hormone treatment of a tadpole does not affect the synthesis of all proteins equally in the tadpole tail muscle. For example, the synthesis of myosin heavy chains (MHCs) is depressed within 1 day and decreases to 45% of control values after 5 days of T3 treatment, whereas the decreased synthesis of soluble muscle proteins is transient and returns to above control levels by day 5. To determine whether the hormone-induced decrease in MHC synthesis is the result of changes in the transcription of translation of MHC mRNAs, we isolated cDNAs complementary to five different MHC mRNAs from a tail muscle cDNA library and used them to examine the levels of each MHC mRNA in the tail muscle of T3-treated tadpoles. mRNAs that recognize the cDNAs for these five different MHCs are all expressed in the tadpole tail and limb muscles, as well as in the adult leg muscles. MHC mRNAs unique to tadpole tail were not detected. Interestingly, the relative amounts of mRNA for four of the five MHCs increase in tail muscle after T3 treatment of the tadpole, suggesting that repression of MHC gene expression at the protein level does not result from a decrease in the amount of MHC mRNAs. Rather, these results support the contention that the decreased synthesis of MHCs in the tail muscle of T3-treated tadpoles is caused by this hormone, either directly or indirectly, depressing the translation of the MHC mRNAs in this tissue. These results, coupled with the observation that the synthesis of soluble muscle proteins is depressed only in a transient fashion, suggest that T3 may be initiating the expression of a gene(s) that encodes a protein(s) responsible for inhibiting the translation of the MHCs and, perhaps, other structural proteins in the tadpole tail muscle. Whatever the case, the translational regulation of MHC synthesis occurs well before any degradation of the tail tissue is evident and appears to be one of the earliest events in the hormone-induced cell death program of the tadpole tail muscle.

Accumulation and proliferation of adult leg muscle precursors in Manduca are dependent on innervation.

During metamorphosis, the larval thoracic legs of the moth Manduca sexta are replaced by new adult legs. The leg motoneurons do not die after the loss of the larval muscles, but persist to innervate the new adult leg muscles (Kent and Levine, 1988). The adult muscles form from myoblasts that originate in specific production sites within the legs and migrate to the sites of muscle formation, where they accumulate, proliferate, and fuse to form myofibers (Consoulas et al., 1996b). Throughout adult leg muscle development, there is a close association between nerves and the developing muscles, suggesting a role for the nervous system in myogenesis (Consoulas et al., 1996a). This prediction was confirmed and the role of the nervous system clarified in the present study by cutting the larval leg nerves prior to metamorphosis. Although myoblasts were generated and migrated normally in the operated leg, they failed to accumulate in the appropriate regions. The myoblasts did not die, but failed to proliferate and remained in the denervated legs as dispersed cells or as aggregates in inappropriate regions. In about 26% of cases, this resulted in the formation of adult legs that lacked muscles. In the remaining cases, however, delayed regeneration of the leg nerve occurred and small muscles appeared in the more proximal segments of the denervated legs. Each muscle fiber in these operated legs bore motor terminals belonging to axons of the leg nerves which had grown out from the proximal nerve stump and invaded the leg. Following the delayed appearance of motor axons, myoblasts aggregated and underwent proliferation and differentiation into muscle fibers. In a second set of experiments, denervation was performed later, after myoblasts had aggregated to establish anlagen. Myoblast proliferation was reduced but differentiation continued. These observations suggest that motor nerves are essential for both the accumulation of myoblasts into the correct areas of muscle development and the appropriate level of proliferation.

Development of adult thoracic leg muscles during metamorphosis of the hawk moth Manduca sexta

During metamorphosis, the larval thoracic legs of the hawk moth Manduca sexta are replaced by a new set of adult legs. The larval leg motoneurons persist to innervate new adult muscles, and the motor terminals remain within the developing adult legs. Here we describe the fate of the larval leg muscles and the origin of new muscles within the adult legs. During the larval instars, large and small nuclei proliferate within leg muscle fibers. Near the end of the larval stage a subset of the small nuclei undergo a wave of proliferation, as indicated by the incorporation of 5-bromodeoxyuridine, whereas other nuclei die. However, none of the larval leg muscles fibers persist to serve as templates for adult muscle formation, and there was no evidence for persistence of larval myonuclei. Migrating myoblasts that are born within aggregate to form adult muscle anlagen at specific production sites within the developing imaginal legs. Intense nuclear proliferation occurs within the anlagen during the early pupal stage, followed by muscle fiber formation and striation. We conclude that adult leg muscles form mainly, if not exclusively, from migrating myoblasts that without the involvement of larval elements.

Remodelling of Neuromuscular Systems During Insect Metamorphosis

During metamorphosis in the hawkmoth, Manduca sexta , the larval thoracic legs are replaced by a new set of adult legs that include new sensory neurons and muscles, and participate in new patterns of locomotor activity. Larval leg motoneurons persist to innervate the new adult leg muscles, but undergo striking changes in dendritic morphology that are regulated by the insect steroid, 20-hydroxyecdysone. In the periphery, the motor terminals regress as larval muscles degenerate, and expand as new adult muscles form from myoblasts. Evidence obtained both in vivo and in vitro suggests that the proliferation of myoblasts during metamorphosis is dependent upon innervation.

Expression of myosin heavy chain transcripts during Xenopus laevis development

In amphibians, the composition and pattern of expression of the myosin heavy chain (myHC) gene family are mostly unknown. To understand better the regulation of this complex family, we screened cDNA libraries from swimming tadpole and adult leg muscle RNA and have isolated and partially sequenced clones for several myHCs. Two of these, E3 and E19, first appear in mesoderm at late gastrula stage and are coexpressed with muscle actin. They increase in abundance in trunk and tail myotome until metamorphosis, when they begin to decline. E3 and E19 are also both expressed in hind leg muscle at the beginning of metamorphosis, but decline to low levels in adult leg muscle. A new transcript, A7, first appears during early metamorphosis in both the tail and hind leg skeletal muscle. A7 transcripts then decline in degenerating tail but persist in hind leg through metamorphosis and in adults. Two other embryonic myHCs, E14 and E15, code for isoforms closely related to E19 and may be alleles or duplications. Although sequence comparisons with myHCs from other vertebrates could not correlate the transcripts with specific protein isoforms, from the pattern of expression E3 and E19 apparently code for embryonic fast skeletal myHC isoforms, whereas A7 codes for an adult skeletal isoform.

Neural control of leg movements in a metamorphic insect: persistence of larval leg motor neurons to innervate the adult legs of Manduca sexta.

During metamorphosis of the hawkmoth Manduca sexta, the larval thoracic legs along with their associated sensory organs and muscles degenerate and new adult legs develop. The larval legs are small and relatively simple structures capable of lateral extension and medial flexion allowing them to grasp the substrate as the caterpillar crawls along. By contrast, the adult legs are used for walking with an alternating gait. They are much larger than the larval legs and articulate such that they are capable of movement in several directions. This change in form and function is accompanied by a reorganization of the neural circuits controlling leg movements. In a previous report (Kent and Levine: J. Comp. Neurol. 271:559-576, '88) we described motor neurons innervating the larval prothoracic legs, and here we describe motor neurons innervating the prothoracic legs of the adult. Using a combination of cobalt staining methods and the persistent fluorescent dye Fluoro-Gold, we have found that some, if not all, larval leg motor neurons are retained and innervate the new adult leg muscles. Moreover, we have been able to discover the fate of individual larval leg motor neurons by marking a single larval neuron with Fluoro-Gold and using a second fluorescent dye to double label the same neuron in the adult. Our results suggest that specific larval leg motor neurons innervate corresponding muscles in the adult stage, although their apparent function is significantly different. In addition, the motor neurons undergo significant remodeling of their dendritic branching patterns during metamorphosis, alterations which doubtless contribute to their new roles in adult behavior.

Acetylcholine sensitivity in replicating satellite cells.

Mononucleate myogenic cells lying within skeletal muscle fiber endomysium, the so-called satellite cells (SC), isolated from adult leg muscle of normal or dystrophic mice were studied by electrophysiological techniques. Normal SC responded to iontophoretic acetylcholine (ACh) by depolarization even during the replicative phase. Acetylcholine sensitivity increased after cell fusion. Replicating SC from dystrophic muscle did not exhibit any sensitivity to ACh, and neither did embryonic myoblasts.

Microelectrophoresis of α-glycerophosphate dehydrogenase isozymes in various tissues of wild-type and mutant Drosophila

The technique of microgel electrophoresis was used to study the isozymes of α-glycerophosphate dehydrogenase in Drosophila melanogaster. In addition to the three known isozymes, three other isozymes were found. All six forms of the enzyme were absent in animals bearing ‘null’ mutations of the Gpdh-l locus. The distribution of the different isozymes in various tissues and body parts was investigated. The gut, brain and fat-body were found to have characteristic isozyme patterns, and the indirect flight muscles had a qualitatively different pattern from that of the larval muscles or the adult leg muscles. Quantitative estimates of the various isozymes in the mutant wingless revealed a lowered specific activity of those isozymes predominantly found in flight muscles. Further examination of wingless flies and of wild-type flies whose flight ability had been impaired indicate that this reduction in enzyme activity in the wingless mutant is one of the pleiotropic effects of the mutation rather than being directly related to flightlessness.