Myotomal myogenesis in Triturus vulgaris L. (Urodela) with special reference to the role of mesenchymal cells

Two stages can be distinguished in the differentiation of myotomal muscle fibres in Triturus vulgaris. In the first stage only synchronously differentiating myotomal cells are engaged; in the second stage mesenchymal cells also take part in the process. Myotomal cells (primary myoblasts) fuse to form 2-3 nucleate myotubes. Only in the caudal part of the embryo mononucleate myotubes persist. The mononucleate myotubes, like polynucleate ones, occupy the whole length of the myotome. The differentiation of myotubes is accompanied by vitellolysis. At further development stages mesenchymal cells enter the intermyotomal fissure, after which they migrate to the myotomes, between the myotubes. The cells that remain in the intermyotomal fissures retain their fibroblastic potential (they synthesise collagen). Their daughter cells adjoining the myotubes acquire myogenic abilities. Their myoblastic potential is evidenced by their ability to fuse with the myotube. Fusion of secondary myoblasts (of mesenchymal origin) with the myotube results in further growth of the myotubes. In T. vulgaris myotomal myotubes and muscle fibres developing from them are of myotomal-mesenchymal origin.     

Junctional acetylcholine receptor channel open time is not presynaptically regulated in developing muscle

The role of motor innervation in controlling the development of acetylcholine receptor (AChR) channel open time was tested by examining synaptic current durations in transplanted muscles of Xenopus tadpoles. The presumptive lower jaw region, which gives rise to the interhyoideus muscle, was transplanted to the tail, overlying the myotomal muscle cells. The transplanted muscles became innervated, presumably by spinal nerves which normally innervate myotomal muscle. Despite development in the presence of foreign innervation, synaptic currents in the transplanted interhyoideus were predominantly long in duration and resembled those in the normally innervated interhyoideus. They did not resemble those in the myotomal muscle, where synaptic currents are brief. The apparent lack of neural influence on development of AChR function in muscle contrasts with the evidence for presynaptic control of AChR open time in frog sympathetic ganglia. This may reflect a fundamental difference between nerve and muscle in the regulation of postsynaptic function.     

Integrin alpha6beta1-laminin interactions regulate early myotome formation in the mouse embryo

We addressed the potential role of cell-laminin interactions during epaxial myotome formation in the mouse embryo. Assembly of the myotomal laminin matrix occurs as epaxial myogenic precursor cells enter the myotome. Most Myf5-positive and myogenin-negative myogenic precursor cells localise near assembled laminin, while myogenin-expressing cells are located either away from this matrix or in areas where it is being assembled. In Myf5(nlacZ/nlacZ) (Myf5-null) embryos, laminin, collagen type IV and perlecan are present extracellularly near myogenic precursor cells, but do not form a basement membrane and cells are not contained in the myotomal compartment. Unlike wild-type myogenic precursor cells, Myf5-null cells do not express the alpha6beta1 integrin, a laminin receptor, suggesting that integrin alpha6beta1-laminin interactions are required for myotomal laminin matrix assembly. Blocking alpha6beta1-laminin binding in cultured wild-type mouse embryo explants resulted in dispersion of Myf5-positive cells, a phenotype also seen in Myf5(nlacZ/nlacZ) embryos. Furthermore, inhibition of alpha6beta1 resulted in an increase in Myf5 protein and ectopic myogenin expression in dermomyotomal cells, suggesting that alpha6beta1-laminin interactions normally repress myogenesis in the dermomyotome. We conclude that Myf5 is required for maintaining alpha6beta1 expression on myogenic precursor cells, and that alpha6beta1 is necessary for myotomal laminin matrix assembly and cell guidance into the myotome. Engagement of laminin by alpha6beta1 also plays a role in maintaining the undifferentiated state of cells in the dermomyotome prior to their entry into the myotome.     

Smooth muscle of the dorsal aorta shares a common clonal origin with skeletal muscle of the myotome

We show that cells of the dorsal aorta, an early blood vessel, and of the myotome, the first skeletal muscle to form within the somite, derive from a common progenitor in the mouse embryo. This conclusion is based on a retrospective clonal analysis, using a nlaacZ reporter targeted to the alpha-cardiac actin gene. A rare intragenic recombination event results in a functional nlacZ sequence, giving rise to clones of beta-galactosidase-positive cells. Periendothelial and vascular smooth muscle cells of the dorsal aorta are the main cell types labelled, demonstrating that these are clonally related to the paraxial mesoderm-derived cells of skeletal muscle. Rare endothelial cells are also seen in some clones. In younger clones, arising from a recent recombination event, myotomal labelling is predominantly in the hypaxial somite, adjacent to labelled smooth muscle cells in the aorta. Analysis of Pax3(GFP/+) embryos shows that these cells are Pax3 negative but GFP positive, with fluorescent cells in the intervening region between the aorta and the somite. This is consistent with the direct migration of smooth muscle precursor cells that had expressed Pax3. These results are discussed in terms of the paraxial mesoderm contribution to the aorta and of the mesoangioblast stem cells that derive from it.     

A histochemical and ultrastructural study of the development of the propulsive musculature of the brown trout, Salmo trutta L., in relation to its swimming behaviour

The differentiation of the myotomal muscle types in the propulsive musculature of Salmo trutta has been investigated histochemically and ultrastructurally from late embryonic to free-swimming fish at 5° months post fertilisation and related to observed changes in swimming behaviour. A histochemical and ultrastructural characterisation was also made of the major myotomal muscle fibre types in fingerling and yearling S. trutta . Two distinct populations of muscle cell types can be recognised prior to hatching. The early development of the white fibre population is related to the short, burst-type swimming activity at early stages. The later increase in the development of the red fibre population is directly related to the appearance of sustained swimming activity. The swimming performance of freeswimming alevins has been investigated and the results are discussed in comparison to adult fish.     

Expression of nicotinic acetylcholine receptors in aneural Xenopus embryos

During gastrulation in vertebrate embryos, the mesoderm moves inward and under the ectoderm and these two cell layers subsequently differentiate in close proximity to each other, providing an opportunity for the exchange of inductive signals. This study examines whether the activation of muscle nicotinic acetylcholine receptor (AChR) genes and the subsequent expression of receptors in Xenopus myotomal muscle are dependent on interaction between the ectoderm and the mesoderm, or their derivatives, after the onset of gastrulation. We eliminated such interaction by inducing total exogastrulation of Xenopus embryos. During exogastrulation, the mesoderm moves away from the ectoderm, and the nervous system fails to develop. Single channel recordings from the myotomal muscle of exogastrulated embryos revealed the presence of two major classes of AChRs, which could be distinguished on the basis of channel conductance. The current amplitudes, conductances, reversal potentials, and open times of these channels closely resembled those reported for the two major classes of AChR channels normally expressed in vivo. We conclude that interaction between ectoderm and mesoderm following the onset of gastrulation is not required for the future expression of the major classes of AChRs in myotomal muscle.     

Dual motor innervation in the axial musculature of fishes

A systematic survey of motor innervation in the myotomal muscle fibres in several groups of fishes was conducted to observe the extent of dual innervation, a phenomenon found within those myotomal muscle fibres with terminal innervation. It is concluded that the dually innervated myotomal muscle fibre is a primitive vertebrate feature. Furthermore, it is suggested that this particular form of polyneuronal innervation is retained in the course of neural evolution within the homogeneous white myotomal muscle fibre region in these fish groups in order to insure synchrony of firing during sudden, rapid locomotion. The findings are examined in light of current ideas regarding the direct adrenergic innervation in addition to cholinergic innervation of striated muscle fibres.     

The neural tube/notochord complex is necessary for vertebral but not limb and body wall striated muscle differentiation.

The aim of this work was to investigate the role played by the axial organs, neural tube and notochord, on the differentiation of muscle cells from the somites in the avian embryo. Two of us have previously shown that neuralectomy and notochordectomy is followed by necrosis of the somites and consecutive absence of vertebrae and of most muscle cells derived from the myotomes while the limbs develop normally with muscles. Here we have focused our attention on muscle cell differentiation by using the 13F4 mAb that recognizes a cytoplasmic antigen specific of all types of muscle cells. We show that differentiation of muscle cells of myotomes can occur in the absence of notochord and neural tube provided that the somites from which they are derived have been in contact with the axial organs for a defined period of time, about 10 hours for the first somites formed at the cervical level, a duration that progressively reduces caudalward (i.e. for thoracic and lumbar somites). Either one or the other of the two axial organs, the neural tube or the notochord can prevent somitic cell death and fulfill the requirements for myotomal muscle cell differentiation. Separation of the neural tube/notochord complex from the somites by a surgical slit on one side of the embryo gave the same results as extirpation of these organs and provided a perfect control on the non-operated side. A striking finding was that limb and body wall muscles, although derived from the somites, differentiated in the absence of the axial organs. However, limb muscles that develop after excision of the neural tube started to degenerate from E10 onward due to lack of innervation. In vitro explantation of somites from different axial levels confirmed and defined precisely the chronology of muscle cell commitment in the myotomes as revealed by the in vivo experiments.     

Resolving Shifting Patterns of Muscle Energy Use in Swimming Fish

Muscle metabolism dominates the energy costs of locomotion. Although in vivo measures of muscle strain, activity and force can indicate mechanical function, similar muscle-level measures of energy use are challenging to obtain. Without this information locomotor systems are essentially a black box in terms of the distribution of metabolic energy. Although in situ measurements of muscle metabolism are not practical in multiple muscles, the rate of blood flow to skeletal muscle tissue can be used as a proxy for aerobic metabolism, allowing the cost of particular muscle functions to be estimated. Axial, undulatory swimming is one of the most common modes of vertebrate locomotion. In fish, segmented myotomal muscles are the primary power source, driving undulations of the body axis that transfer momentum to the water. Multiple fins and the associated fin muscles also contribute to thrust production, and stabilization and control of the swimming trajectory. We have used blood flow tracers in swimming rainbow trout (Oncorhynchus mykiss) to estimate the regional distribution of energy use across the myotomal and fin muscle groups to reveal the functional distribution of metabolic energy use within a swimming animal for the first time. Energy use by the myotomal muscle increased with speed to meet thrust requirements, particularly in posterior myotomes where muscle power outputs are greatest. At low speeds, there was high fin muscle energy use, consistent with active stability control. As speed increased, and fins were adducted, overall fin muscle energy use declined, except in the caudal fin muscles where active fin stiffening is required to maintain power transfer to the wake. The present data were obtained under steady-state conditions which rarely apply in natural, physical environments. This approach also has potential to reveal the mechanical factors that underlie changes in locomotor cost associated with movement through unsteady flow regimes.     

Different immunoreactivities of anti-soluble lactose lectin antisera to tissues from early chick embryos: a histochemical study

The location of soluble lactose-binding proteins (S-lac lectins) has been studied by immunohistochemical methods during morphogenesis of the chick embryo, when segregation and early differentiation of organ primordia was occurring. Using a panel of polyclonal antisera raised to various purified lectin preparations, we observed striking differences in the antigenic properties of these antisera, indicating that diverse versions of the lectins may be expressed during development. The antisera referred to as anti-L-16, anti-M-16, anti-S-14 and anti-I-14 were respectively raised to native or denatured 16 kDa lectins from adult liver and embryonic muscle and to 14 kDa lectins from embryonic skin and adult intestine. Having determined the optimal immunohistochemical conditions in the preparation of embryo sections (fixation, embedding, sectioning) we show that anti-L-16, anti-S-14 and anti-I-14 mostly bind the lectins expressed at the cell surface, in the extracellular matrix and in some released secretion. As previously shown, anti-L-16 and anti-S-14 are also able to recognize the cytoplasmic form of some migrative lectin-rich cells (primitive streak, neural crest cells, germ cells). Anti-M-16 was bound exclusively to the cytoplasmic form of the 16 kDa lectin in the same cell lines as above and also in some others, such as in the notochord, the myotomal part of the somites, the pharyngeal endoderm and the cardiac muscle. These different antigenic properties may be applied to the accurate mapping of various lectin isoforms and evaluation of the respective contribution of their intra-and extracellular variants during development and differentiation.     

Channel open time of acetylcholine receptors on Xenopus muscle cells in dissociated cell culture

The kinetics of acetylcholine (ACh) receptor channels on cultured myotomal muscle cells from Xenopus embryos were studied by analyzing focally recorded membrane currents. The mean open time for receptor channels on embryonic muscle cells grown in dissociated cell cultures showed a time-dependent decrease similar to that seen in vivo. The changes in power density spectra are consistent with the hypothesis that the decrease results from the appearance of a class of ACh receptor with a short mean channel open time (0.7 msec) and a decrease in the proportion of receptors with a long mean channel open time (3 msec). The addition of dissociated neural tube cells to muscle cell cultures resulted in an unexpected increase in mean channel open time for ACh receptors in both synaptic and nonsynaptic regions. These studies demonstrate that ACh receptor function may be altered in cultured muscle cells.     

A protein homologous to the Torpedo postsynaptic 58K protein is present at the myotendinous junction

The 58K protein is a peripheral membrane protein enriched in the acetylcholine receptor (AChR)-rich postsynaptic membrane of Torpedo electric organ. Because of its coexistence with AChRs in the postsynaptic membrane in both electrocytes and skeletal muscle, it is thought to be involved in the formation and maintenance of AChR clusters. Using an mAb against the 58K protein of Torpedo electric organ, we have identified a single protein band in SDS-PAGE analysis of Xenopus myotomal muscle with an apparent molecular mass of 48 kD. With this antibody, the distribution of this protein was examined in the myotomal muscle fibers with immunofluorescence techniques. We found that the 48K protein is concentrated at the myotendinous junctions (MTJs) of these muscle fibers. The MTJ is also enriched in talin and vinculin. By double labeling muscle fibers with antibodies against talin and the 48K protein, these two proteins were found to colocalize at the membrane invaginations of the MTJ. In cultured myotomal muscle cells, the 48K protein and talin are also colocalized at sites of membrane-myofibril interaction. The 48K protein is, however, not found at focal adhesion sites in nonmuscle cells, which are enriched in talin. These data suggest that the 48K protein is specifically involved in the interaction of myofibrillar actin filaments with the plasma membrane at the MTJ. In addition to the MTJ localization, 48K protein is also present at AChR clusters both in vivo and in vitro. Thus, this protein is shared by both the MTJ and the neuromuscular junction.     

Cell coherence during production of the presomitic mesoderm and somitogenesis in the mouse embryo

In this study, we investigated (in the early mouse embryo) the clonal properties of precursor cells which contribute to the segmented myotome, a structure derived from the somites. We used the laacZ method of single cell-labelling to visualise clones born before segmentation and bilateralisation. We found that clones which contribute to several segments both unilateral and bilateral were regionalised along the mediolateral axis and that their mediolateral position was maintained in successive adjacent segments. Furthermore, clones contributed to all segments, from their most anterior to their most posterior borders. Therefore, it appears that mediolateral regionalisation of myotomal precursor cells is a property established before bilateralisation of the presomitic mesoderm and that coherent clonal growth accompanies cell dispersion along both the mediolateral and anteroposterior axes. These findings in the mouse correlate well with what is known in the chick, suggesting conservation of the mode of production and distribution of the cells of the presomitic mesoderm. However, in addition, we also found that the mediolateral contribution of a clone is already determined in the pool of self-renewing cells that produces the myotomal precursor cells and thus that this pool is itself regionalised. Finally, we found that bilateral clones exhibit symmetry in right and left sides in the embryo at all levels of the mediolateral axis of the myotome. All these properties indicate synchrony and symmetry of formation of the presomitic mesoderm on both sides of the embryo leading to formation of a static embryonic structure with few cell movements. We suggest that sequential production of groups of cells with an identical clonal origin for both sides of the embryo from a single pool of self-renewing cells, coupled with acquisition of static cell behaviour, could play a role in colinearity of expression of Hox genes and in the segmentation system of higher vertebrates.     

A morphometric analysis of the spinal motor pool in relation to its target muscle during growth in the trout, Salmo gairdneri Richardson

The relation between number and size of spinal motoneurons and the dimension of myotomal muscle has been investigated in trout at different stages of embryonic, larval and postlarval development (body length 1–15 cm). Three spinal segments have been analysed (cervical, trunk and caudal) and the following parameters were determined by means of a Micromeasurements Image Analyzer: (a) mean cross-sectional myotomal area; (b) mean soma size of principal (or dorsomedian, DM) and secondary (or ventrolateral, VL) motoneurons; (c) DM and VL motoneuron density per segment. Myotomal muscle and motor pool growth was evaluated by percent increments of a, b and c parameters at each stage. Their relationships were denned by equations of computed regression lines.
The analysis provided evidence that: (1) a continuous exponential growth of mean myotomal area takes place in the three segments, with the same trend and with the lowest values in the caudal segment; (2) DM and VL motoneuron size and density per segment also increase during development, with the least value in the caudal segment, VL parameters being of lesser value than DM; (3) motoneuron pool and its target myotomal muscle parameters bear a linear relationship as defined by equations of computer regression lines; (4) motoneuron number percent increment at early eleutherembryonic stage precedes myotomal area increment which takes place during late eleutherembryonic stage.
It is apparent that spinal motor pool and target myotomal muscle grow at the same rate in the trout during the considered stages. The discussion links this fact with the hypothesis of a neuronal influence on muscle fibre type differentiation.     

THE APPEARANCE OF ACETYLCHOLINESTERASE IN THE MYOTOME OF THE EMBRYONIC RABBIT : An Electron Microscope Cytochemical and Biochemical Study

Acetylcholinesterase (AChE) activity has been studied in the myoblast of skeletal muscle of the 9–13 day fetal rabbit. Cytochemical activity is present in the nuclear envelope and the endoplasmic reticulum, including its derivatives the subsurface reticulum and the sarcoplasmic reticulum. End product is also found in the Golgi complex of the more differentiated myoblasts. The formation of reticulum-bound acetylcholinesterase in the myoblast appears to be independent of nerve-muscle contact, since the enzyme is present before the outgrowth of the spinal nerve. The nerve lacks cytochemical end product until the myoblast is well differentiated. Possible mechanisms of spontaneous muscle contraction have been discussed. A second type of myotomal cell, which exhibits a poorly localized end product of AChE activity, has been described. The ready solubility of the enzyme or diffusibility of its end product suggests that the enzyme may be a lyoesterase. This cell may be the precursor of the morphologically undifferentiated cell which is closely apposed to the myotubes in later stages of skeletal muscle development. Biochemical studies show a significant increase in AChE activity in the dermomyotome by day 12, when many of the myoblasts are well differentiated and the second type of myotomal cell is prominent. Cytochemical studies have indicated that many of the cells in the sample lack reaction product of enzymic activity, whereas others are very active. Biochemical values, therefore, reflect the amount of enzyme in the dermomyotome as a whole, but give little information on the enzymic content of individual cells.