Ultrastructural and morphometric analysis of the separation of two thigh muscles in the chick

Limb muscles separate from one another in a complex but highly stereotyped sequence and spatial pattern. The process of separation is characterized by the progression of a region of increased extracellular space, the cleavage zone, along the proximodistal axis between the individual muscle anlagen. We analyzed ultrastructurally the muscles and cleavage zone during the separation of two representative muscles, the developing sartorius and iliotibialis in the chick thigh, to establish an accurate baseline for an analysis of the mechanisms of separation. Comparisons of the morphology and distribution of cells before and after separation show no evidence that muscles became separated by the massive influx of an exterior cell population; if populations invade the cleavage zone, they are small. We do find characteristic transitions within the cell population of the cleavage zone in situ that could accomplish cleavage without invoking massive cell movements. These progressive transitions within the cleavage zone include a loss of close cell-cell interactions, an increase in extracellular space, the assumption of a more stellate morphology by mesenchyme cells, and a gradual alteration in the composition of the extracellular matrix from one typical of early muscle to one typical of loose connective tissue. Myotubes do differentiate between the incipient muscles, ruling out the possibility that the location where muscles will separate is defined by sites where myotubes fail to differentiate. Instead, the myotubes in the cleavage zone gradually diminish in number and appear to be specifically recognized and removed from the cleavage zone by phagocytes. We suggest that the transitions within the cleavage zone, including the loss of muscle cells, are a result of the progressive differentiation of loose connective tissue. If so, then the spatial pattern and process of cleavage is a consequence of spatially programmed cell differentiation.     

The influence of presumptive limb connective tissue on motoneuron axon guidance

During embryogenesis in the chick, the lumbosacral (LS) somatopleure gives rise to the connective tissue and the epidermis of the limb. We wished to determine if the LS somatopleure was a primary source of guidance cues for motoneuron pathway choices along the anteroposterior axis of the limb. At stage (st) 15, prior to its population by muscle cell precursors and the neural crest, the LS somatopleure was shifted anteriorly. This surgery resulted in the development of limbs that were shifted one to four segments into the thoracic region. Muscles within the anterior thigh of the shifted limb were normally patterned and of composite origin: connective tissues were of LS origin, while muscle cells were of LS and thoracic origin. Retrograde HRP labeling at st 35-37 indicated that motoneuron pools to these anterior thigh muscles were located within LS rather than thoracic cord segments. Pools to individual muscles were smaller than normal but occupied segmental and transverse positions in the LS cord that generally matched those of normal embryos. These findings suggest that individual muscles within somatopleure-shifted limbs are innervated specifically and are in accord with their connective tissue (and epidermal) level of origin. Reconstructions of nerve patterns at st 28-31 suggested that LS motoneurons corrected for the shift by altering their pathways at midthigh regions. We conclude that the somatopleure, and most likely its connective tissue component, contains the information for setting up a specific axon guidance system in the developing limb.     

Comparison of hind limb muscle mass in neonate and adult prosimian primates

Little ontogenetic data exist to indicate whether muscular organization of neonates reflects adult locomotion (e.g., leaping) or infant activities like clinging or the initial quadrupedal phase of locomotion that typifies most infant primates. In the present study, five species of primates with contrasting modes of locomotion were examined. Twenty-eight preserved neonatal and adult cadavers were studied by careful dissection of the hip, thigh, and leg muscles. Wet weights were taken of limb muscles after removal, and the muscles were combined into major functional groups (e.g., flexors, extensors) of each limb segment. Results demonstrate that the distribution of muscle mass within the thigh and within the leg are similar between neonates and adults for all species, with major groups varying by 5% or less in all but two age comparisons. Crural indices of the neonates are nearly identical to those of the adults, but leg/thigh muscle mass ratios were higher in the neonates. Species vary greatly in the percentage of adult limb segment muscle mass present in neonates, with Tarsius syrichta having the greatest percentage for all segments and two lemurids showing the least. These results primarily track differences in relative body mass at birth rather than developmental differences. The adaptive distribution of muscle, as discussed previously for adult prosimians, appears to be established at birth. Neonates of leaping species already have much larger quadriceps muscles than quadrupeds. Differences between large- and small-bodied leapers (e.g., pronounced superficial plantarflexor masses in tarsiers and pronounced deep plantarflexor masses in sifakas) also are present in neonates. Ratios of muscle mass over body mass are smaller in all neonates than in their adult counterparts, suggesting that the neonates are relatively poorly muscled, and that muscle mass must increase with positive allometry during growth.     

The somitic level of origin of embryonic chick hindlimb muscles

Studies of avian chimeras made by transplanting groups of quail somites into chick embryos have consistently shown that the muscle cells of the hindlimb are derived from the adjacent somites, however, the pattern of cell distribution from individual somites to individual hindlimb muscles has not been characterized. I have mapped quail cell distribution in the chick hindlimb after single somite transplantation to determine if cells from an individual somite populate discrete limb muscle regions and if there is a spatial correspondence between a muscle's somitic level of origin and the known spinal cord position of its motoneuron pool. At stages 15-18 single chick somites or equivalent lengths of unsegmented somitic mesoderm adjacent to the prospective hindlimb region were replaced with the corresponding tissue from quail embryos. At stages 28-34, quail cell distribution was mapped within individual thigh muscles and shank muscle regions. A quail-specific antiserum and Feulgen staining were used to identify quail cells. Transplants from somite levels 26-33 each gave rise to consistent quail cell labeling in a unique subset of limb muscles. The anteroposterior positions of these subsets corresponded to that of the transplanted somitic tissue. For example, more anterior or anteromedial thigh muscles contained quail cells when more anterior somitic tissue had been transplanted. For the majority of thigh muscles studied and for shank muscle groups, there was also a clear correlation between somitic level of origin and motoneuron pool position. These data are compatible with the hypothesis that motoneurons and the muscle cells of their targets share axial position labels. The question of whether motoneurons from a specific spinal cord segment recognize and consequently innervate muscle cells derived from the same axial level during early axon outgrowth is addressed in the accompanying paper (C. Lance-Jones, 1988, Dev. Biol. 126, 408-419). Quail cell distribution was also mapped in chick embryos in which quail somites or unsegmented mesoderm had been placed 2-3 somites away from their position of origin. In all cases donor somitic tissues contributed to muscles in accord with their host position. These results indicate that muscle cell precursors within the somites are not specified to migrate to a predetermined target region.     

Involvement of vessels and PDGFB in muscle splitting during chick limb development

Muscle formation and vascular assembly during embryonic development are usually considered separately. In this paper, we investigate the relationship between the vasculature and muscles during limb bud development. We show that endothelial cells are detected in limb regions before muscle cells and can organize themselves in space in the absence of muscles. In chick limbs, endothelial cells are detected in the future zones of muscle cleavage, delineating the cleavage pattern of muscle masses. We therefore perturbed vascular assembly in chick limbs by overexpressing VEGFA and demonstrated that ectopic blood vessels inhibit muscle formation, while promoting connective tissue. Conversely, local inhibition of vessel formation using a soluble form of VEGFR1 leads to muscle fusion. The endogenous location of endothelial cells in the future muscle cleavage zones and the inverse correlation between blood vessels and muscle suggests that vessels are involved in the muscle splitting process. We also identify the secreted factor PDGFB (expressed in endothelial cells) as a putative molecular candidate mediating the muscle-inhibiting and connective tissue-promoting functions of blood vessels. Finally, we propose that PDGFB promotes the production of extracellular matrix and attracts connective tissue cells to the future splitting site, allowing separation of the muscle masses during the splitting process.     

Slow and Fast Muscle Fibers Are Preferentially Derived from Myoblasts Migrating into the Chick Limb Bud at Different Developmental Times

Avian limb myoblasts originate from somites and migrate into the periphery during limb bud formation. It is not known how these precursors become arranged into a stereotyped pattern of muscles and primary fiber types. We used in vivo surgical transplantation and anatomical analyses of thigh muscle patterns to ask whether myoblasts migrating into the limb bud at different developmental times adopt different fates. When myoblast migration was interrupted by transplanting limb bud tissue to the coelomic cavity of a host embryo early in the migratory period (stages 16-early 17), few thigh muscles were found at stages 30-33. Primordia that were present corresponded to muscles that normally contain a majority of slow myotubes. In limbs transplanted slightly later (stages late 17-18), the only missing muscles were those that normally contain the highest numbers of fast myotubes. Parallel results were obtained in chimeric limbs made by transplanting a quail limb bud to a chick host at different times during the migratory period, an experimental situation in which the limbs were not depleted of muscle precursors or nerves. These findings suggest that the earliest myoblast migrants give rise mainly to slow primary myotubes, the later migrants to fast myotubes. To determine whether the early limb bud environment defines the fate of migrating myoblasts, we assessed fiber type patterns in limbs that developed from young limb bud tissue (stages 15-early 16) transplanted to older hosts (stage 17). A significant depletion of slow myosin-positive profiles was found within slow muscles. Fast muscles were generally normal in size. These results provide in vivo evidence that limb myoblast diversity arises prior to the entry of myoblasts into the limb. We suggest that there is a gradual change in the proportions of myoblasts capable of forming slow and fast fiber types, a change which may begin in the somites or early in the migratory period.     

Distribution of fiber types in locomotory muscles of dogs

The distribution of Type I and Type II fibers, as determined from histochemical estimation of myofibrillar ATPase activity, was studied within and among the locomotory muscles of the forelimb, trunk, and hindlimb of three mongrel dogs. All Type II fibers had high oxidative capacities as estimated from the histochemical assay for reduced nicotinamide adenine dinucleotide tetrazolium reductase, so they were not further divided into subpopulations. Furthermore, Type I and Type II fibers had similar oxidative potentials as indicated by both histochemistry and biochemistry. Type I fiber populations ranged between 14% and 100% in the muscles sampled. The highest percentages of Type I fibers were found in deep muscles of physiological extensor groups in the arm and thigh that serve to resist gravity (antigravity muscles) when the dog is in the quadrupedal standing position. More superficial muscles in these same groups had fewer Type I fibers. The patterns of Type I fiber distribution among muscles in the antigravity groups of the forearm and leg were the opposite of those in the arm and thigh, with the more superficial muscles of the distal limb segments having more Type I fibers than the deeper muscles. In all limb segments, muscle groups that do not serve to resist gravity did not show as much intermuscular variation. Type I fiber populations in these muscles did not exceed 50%. A stratification of fiber types also existed within muscles, both in extensor and flexor groups, with the deeper portions of the muscles having more Type I fibers than the more superficial portions.     

Reduction of neuromuscular redundancy for postural force generation using an intrinsic stability criterion

Postural control requires the coordination of multiple muscles to achieve both endpoint force production and postural stability. Multiple muscle activation patterns can produce the required force for standing, but the mechanical stability associated with any given pattern may vary, and has implications for the degree of delayed neural feedback necessary for postural stability. We hypothesized that muscular redundancy is reduced when muscle activation patterns are chosen with respect to intrinsic musculoskeletal stability as well as endpoint force production. We used a three-dimensional musculoskeletal model of the cat hindlimb with 31 muscles to determine the possible contributions of intrinsic muscle properties to limb stability during isometric force generation. Using dynamic stability analysis we demonstrate that within the large set of activation patterns that satisfy the force requirement for posture, only a reduced subset produce a mechanically stable limb configuration. Greater stability in the frontal-plane suggests that neural control mechanisms are more highly active for sagittal-plane and for ankle joint control. Even when the limb was unstable, the time-constants of instability were sufficiently great to allow long-latency neural feedback mechanisms to intervene, which may be preferential for movements requiring maneuverability versus stability. Local joint stiffness of muscles was determined by the stabilizing or destabilizing effects of moment-arm versus joint angle relationships. By preferentially activating muscles with high local stiffness, muscle activation patterns with feedforward stabilizing properties could be selected. Such a strategy may increase intrinsic postural stability without co-contraction, and may be useful criteria in the force-sharing problem.     

Fluctuation in the development of various skeletal muscles in the chick embryo,with special reference to AChE activity and the formation of neuromuscular junctions

Acetylcholinesterase (AChE)-rich cytoplasmic granules in the developing myofibers increased remarkably until the establishment of neuromuscular junctions and thereafter decreased rapidly, whereas junctional AChE activities continued to increase (K. Wake, 1976, Cell Tissue Res. 173, 383–400). In the present paper, during the developmental course of the chick embryo, the temporal and regional gradients in differentiation of skeletal muscles at various sites were examined with special reference to the fluctuation of intracellular AChE activity. AChE-rich granules in each muscle throughout the whole body of chick embryos were observed. Since the distribution pattern of these granules changed regularly in the course of the muscle fiber development, advances of muscle differentiation in various sites of the body were compared. (1) The process of muscle development is more advanced in the trunk muscles than in the limb muscles. (2) The dorsal trunk muscles differentiate one day earlier than the ventral ones. (3) Within the same limb, proximal muscles differentiate approximately 24 hr ahead of distal ones. (4) The development of posterior limb muscles advances faster than that of anterior limb muscles. (5) Within the thigh muscles, the flexor muscles tend to differentiate earlier than the extensor muscles.     

The distribution of motoneurons supplying hind limb muscles in the clawed toad, Xenopus laevis

The distribution of motoneurons in the lumbar spinal cord (spinal segments 8-10) of the clawed toad, Xenopus laevis, was studied with the horseradish peroxidase technique. In a total of 13 different hind limb muscles this tracer was applied in a slow-release gel. Motoneurons innervating a particular hind limb muscle were clustered in longitudinally arranged motor pools. Motor pools of different muscles did show considerable overlap both in the rostrocaudal and transverse plane. But, the various motor pools clearly show a somatotopic organization of motoneurons even in such a condensed lumbar spinal cord as in Xenopus laevis. Motoneurons innervating more distally positioned muscles are generally found in more caudal segments, while proximal muscles (with the exception of the m. adductor magnus) are supplied by motoneurons more or less throughout the lumbar enlargement. Flexor muscles usually are innervated by motoneurons situated ventrolaterally in the ventral horn, extensor muscles by dorsomedially found motoneurons. This pattern is particularly apparent for proximal (thigh) muscles, less so for more distal (shank and foot) muscles. The present data are in keeping with those obtained with the retrograde cell degeneration technique in ranid frogs and are consistent with observations in other tetrapods, although a more clear separation of motor pools is evident in "higher" vertebrates such as birds and mammals.     

Thyroid hormone induces synthesis and accumulation of tropomyosin and myosin heavy chain in limb buds of premetamorphic tadpoles

Myosin heavy chain (MHC) and tropomyosin (Tm) have been isolated from limb muscles of the North American bullfrog, Rana catesbeiana, and injected into rabbits to raise monospecific antibodies. These antibodies were used to study the localization and synthesis of myosin heavy chain and tropomyosin in the limb buds of premetamorphic (stage VI-VII) tadpoles treated with triiodothyronine (T3) to induce metamorphosis. Indirect immunofluorescence localization detects the accumulation of both MHC and Tm in the developing thigh region within 24 h of T3 treatment. During the subsequent 48 h, the accumulation of these proteins is enhanced in the thigh and progresses from thigh to the distal regions of the limb. Quantitative immunochemical determinations indicate that within 24 h of T3 treatment, synthesis of Tm and MHC are increased 23-fold and 6-fold, respectively. Following 5 days of T3 treatment, the synthetic rates of Tm and MHC are 266 and 70 times the control values, respectively. Both methods suggest that Tm is synthesized and accumulated at a greater rate than myosin heavy chain. These observations suggest that T3 promotes the differentiation of muscle in the limb buds of premetamorphic tadpoles and that limb development promoted by T3 in tadpoles is similar to that described during the embryonic development of higher vertebrates.     

Variability of neural activation during walking in humans: short heels and big calves

People come in different shapes and sizes. In particular, calf muscle size in humans varies considerably. One possible cause for the different shapes of calf muscles is the inherent difference in neural signals sent to these muscles during walking. In sedentary adults, the variability in neural control of the calf muscles was examined with muscle size, walking kinematics and limb morphometrics. Half the subjects walked while activating their medial gastrocnemius (MG) muscles more strongly than their lateral gastrocnemius (LG) muscles during most walking speeds ('MG-biased'). The other subjects walked while activating their MG and LG muscles nearly equally ('unbiased'). Those who walked with an MG-biased recruitment pattern also had thicker MG muscles and shorter heel lengths, or MG muscle moment arms, than unbiased walkers, but were similar in height, weight, lower limb length, foot length, and exhibited similar walking kinematics. The relatively less plastic skeletal system may drive calf muscle size and motor recruitment patterns of walking in humans.     

Regional differences in hyoid muscle activity and length dynamics during mammalian head shaking

The sternohyoid (SH) and geniohyoid (GH) are antagonist strap muscles that are active during a number of different behaviors, including sucking, intraoral transport, swallowing, breathing, and extension/flexion of the neck. Because these muscles have served different functions through the evolutionary history of vertebrates, it is quite likely they will have complex patterns of electrical activity and muscle fiber contraction. Different regions of the SH exhibit different contraction and activity patterns during a swallow. We examined the dynamics of the SH and GH muscles during an unrestrained, and vigorous head shaking behavior in an animal model of human head, neck, and hyolingual movement. A gentle touch to infant pig ears elicited a head shake of several revolutions. Using sonomicrometry and intramuscular EMG, we measured regional (within) muscle strain and activity in SH and GH. We found that EMG was consistent across three regions (anterior, belly, and posterior) of each muscle. Changes in muscle length, however, were more complex. In the SH, mid-belly length-change occurred out-of-phase with the anterior and posterior end regions, but with a zero lag timing; the anterior region shortened before the posterior. In the GH, the anterior region shortened before and out-of-phase with the mid-belly and posterior regions. Head shaking is a relatively simple reflex behavior, yet the underlying patterns of muscle length dynamics and EMG activity are not. The regional complexity in SH and GH, similar to regionalization of SH during swallowing, suggests that these anatomically simple hyoid strap muscles have more complex function than textbooks often suggest.     

Regeneration studies on a crayfish neuromuscular system. I. Connectivity changes after intersegmental nerve transplants

The superficial flexor muscles of the crayfish are a neuromuscular system of a few muscle cells innervated by six neurons in a precise position-dependent pattern. The neurons are capable of regenerating their normal connectivity patterns within a short span of time when conditions are favorable. The superficial flexor muscles of the second and third segments, despite their similarities in neuronal and muscle cell size and number, have distinctive connectivity patterns; some homologous neurons form similar patterns but other homologous neurons form patterns that are reversed between segments. We transplanted each segment's nerve into each other's muscle in order to observe regeneration of the nerves into a target area that differed in connectivity patterns from their original muscle. During the first weeks of regeneration all neurons formed a connectivity pattern with more connections medially and declining connections laterally, a pattern determined by the medial location of the nerve transplant. This pattern is maintained for most of the neurons, but for some there is an eventual reduction in medial connections as maximum synapse formation shifts to the lateral muscle fibers. Three of the eight neurons studied were able to regenerate connectivity patterns that corresponded to their segment of origin and not to their host muscle. This suggests that intersegmental muscle differences are not influencing the formation of these connectivity patterns, so the neurons will follow their inherent synaptogenesis program.     

Fibre operating lengths of human lower limb muscles during walking

Muscles actuate movement by generating forces. The forces generated by muscles are highly dependent on their fibre lengths, yet it is difficult to measure the lengths over which muscle fibres operate during movement. We combined experimental measurements of joint angles and muscle activation patterns during walking with a musculoskeletal model that captures the relationships between muscle fibre lengths, joint angles and muscle activations for muscles of the lower limb. We used this musculoskeletal model to produce a simulation of muscle-tendon dynamics during walking and calculated fibre operating lengths (i.e. the length of muscle fibres relative to their optimal fibre length) for 17 lower limb muscles. Our results indicate that when musculotendon compliance is low, the muscle fibre operating length is determined predominantly by the joint angles and muscle moment arms. If musculotendon compliance is high, muscle fibre operating length is more dependent on activation level and force-length-velocity effects. We found that muscles operate on multiple limbs of the force-length curve (i.e. ascending, plateau and descending limbs) during the gait cycle, but are active within a smaller portion of their total operating range.     

Clinical and Muscle Imaging Findings in 14 Mainland Chinese Patients with Oculopharyngodistal Myopathy

Oculopharyngodistal myopathy (OPDM) is an extremely rare, adult-onset hereditary muscular disease characterized by progressive external ocular, pharyngeal, and distal muscle weakness and myopathological rimmed vacuole changes. The causative gene is currently unknown; therefore, diagnosis of OPDM is based on clinical and histopathological features and genetic exclusion of similar conditions. Moreover, variable manifestations of this disorder are reported in terms of muscle involvement and severity. We present the clinical profile and magnetic resonance imaging (MRI) changes of lower limb muscles in 14 mainland Chinese patients with OPDM, emphasizing the role of muscle MRI in disease identification and differential diagnosis. The patients came from 10 unrelated families and presented with progressive external ocular, laryngopharyngeal, facial, distal limb muscle weakness that had been present since early adulthood. Serum creatine kinase was mildly to moderately elevated. Electromyography revealed myogenic changes with inconsistent myotonic discharge. The respiratory function test revealed subclinical respiratory muscle involvement. Myopathological findings showed rimmed vacuoles with varying degrees of muscular dystrophic changes. All known genes responsible for distal and myofibrillar myopathies, vacuolar myopathies, and muscular dystrophies were excluded by PCR or targeted next-generation sequencing. Muscle MRI revealed that the distal lower legs had more severe fatty replacement than the thigh muscles. Serious involvement of the soleus and long head of the biceps femoris was observed in all patients, whereas the popliteus, gracilis and short head of biceps femoris were almost completely spared, even in advanced stages. Not only does our study widen the spectrum of OPDM in China, but it also demonstrates that OPDM has a specific pattern of muscle involvement that may provide valuable information for its differential diagnosis and show further evidence supporting the conclusion that OPDM is a unique disease phenotype.     

Anatomy of raccoon (Procyon lotor) and coati (Nasua narica and N. nasua) forearm and leg muscles: relations between fiber length, moment-arm length, and joint-angle excursion

Muscle architecture, moment arms, and locomotor movements in the distal limb segments of the procyonids Nasua (coati) and Procyon (raccoon) are analyzed with reference to patterns of muscle fiber length. This study addresses the hypothesis that relative fiber lengths among muscles in a muscle group can be predicted on the basis of correlates of muscle tension. The results include the following: consistent patterns of fiber length of muscles in a muscle group exist within and between the two genera. Differences in fiber length between muscles can be accounted for by two principal correlates of muscle excursion--length of a muscle's moment arm about a joint and joint-angle excursion. Muscle fiber pinnation permits increased tendon excursion, but this effect is relatively small in comparison to the effects of moment-arm length and joint-angle excursion. Corollary action between two or more joints (or lack thereof) is an important factor in determination of fiber lengths.