Clonal analysis of vertebrate myogenesis. 3. Developmental changes in the muscle-colony-forming cells of the human fetal limb

The cell lineage of developing human limb muscle has been investigated by means of an in vitro clonal assay. Single cells capable of forming differentiated muscle colonies have been detected within the prospective leg muscle region as early as the 36th day of human development (Streeter's Horizon XVI). At this stage clonable myoblasts account for 14% of the total colony-forming cells. The relative proportion of clonable myoblasts increases rapidly during subsequent fetal development and attains a plateau level of approximately 90% by the 100th day of development. The 90% plateau level persists at least until day 172. By correlating the percent muscle colony differentiation with clonal plating efficiency and with the number of single cells derived from the total limb muscle region of fetuses of different ages, an estimate of the actual number of clonable myoblasts within the developing limb musculature is obtained.Sequential changes within the muscle cell lineage have been further dissected by the temporal analysis of age-dependent medium effects on muscle colony differentiation. The analysis indicates that clonable myoblasts derived from early fetuses are sensitive to medium conditions to which older muscle-colony-forming cells are relatively insensitive. In addition, fusing and nonfusing colonies have been classified into recognizable morphological types whose relative proportions are observed to change during limb development. The results are correlated with human limb morphogenesis and skeletal muscle histogenesis and an operational model of muscle cell lineage is proposed.     

Differentiation of chick embryo myoblasts is transiently sensitive to functional denervation

Clonal analysis of myoblast differentiation has been used to assess effects of denervation on developing skeletal muscle: chick embryo legs denervated by spinal cord cautery yield reduced proportions of clonable myoblasts (P. H. Bonner, 1978, Develop. Biol., 66, 207–219). The present work examines the effects on clonable myoblasts of functional denervation by d-tubocurarine. Curare treatment during the third or fourth days of embryonic development had no effect on clonable myoblasts later in development, treatment during the fifth or sixth days resulted in reduced proportions of clonable myoblasts, and treatment during the eighth or ninth days again had no effect. Clonal analysis of treated and control embryo leg muscle cells was performed between Days 10 and 18. Embryos were also permanently denervated by spinal cord cautery late in the sixth day. These embryos showed no effect of denervation on clonable myoblast proportion. It is concluded that the differentiation of skeletal muscle myoblasts is affected by interference with normal nerve-muscle relationships only during a “window” of sensitivity and that this “window” extends approximately from Hamburger and Hamilton stage 27 to stage 30.     

Nerve-dependent changes in clonable myoblast populations

Between the 3rd and 12th days of development at least four distinct classes of clonable myoblast are present in chick embryo leg skeletal muscle (N. K. White, P. H. Bonner, D. R. Nelson, and S. D. Hauschka, 1975, Develop. Biol.44, 346–361). In the present study, the behavior of each class has been examined quantitatively by clonal growth and differentiation of cells derived from denervated and normal embryos. Embryos functionally denervated by cauterization of the posterior spinal cord or by injection of the neuromuscular blocking agent d-tubocurarine exhibit changes in the two broad categories of clonable myoblast—fresh medium-sufficient (FMS) and conditioned medium-requiring (CMR) muscle cells. Clonal analysis of cells derived from leg muscle tissue of 10- to 12-day-old embryos denervated early in development (Days 3 to 6) has shown that the proportion of FMS clonable myoblasts is reduced to about 60% of the level found in normally innervated leg muscle. The CMR class is composed of three subclasses: CMR-I, CMR-II, and CMR-III (White et al., 1975). Clonal analysis of cells from denervated muscle has shown that, while the total CMR clone proportion is unchanged, the three subclasses are rearranged. The proportions of CMR-I and CMR-II muscle clones are greatly increased and the CMR-III subclass is severely reduced or absent when compared to clones derived from normally innervated leg muscle tissue.     

Clonal analysis of vertebrate myogenesis: IV. Medium-dependent classification of colony-forming cells

The cell lineage of chick leg muscle between 3 and 12 days of development has been studied by use of an in vitro clonal assay. The assay permits distinctions to be made among various types of muscle-colony-forming cells (MCF cells) on the basis of their medium requirements and clonal morphology. Results suggest the sequential occurrence of at least four types of MCF cells, three of which require conditioned medium for their differentiation and one of which can form differentiated colonies in fresh medium.The nature of the “conditioned medium effect” was further investigated by the use of medium-switch experiments. By this process it was shown that the same populations of colony-forming cells attach and grow in fresh and conditioned medium and that the differentiation of colonies derived from conditioned-medium-requiring myoblasts is permitted by brief exposure to conditioned medium followed by culture in fresh medium. Further investigation indicated that during brief exposure to conditioned medium the gelatin-coated petri plate surface is altered such that differentiation of conditioned-medium-requiring colonies is allowed. We conclude that the conditioned medium effect involves a surface-mediated interaction between myoblasts and one or more conditioned medium components.     

Clonal analysis of vertebrate myogenesis. V. Nerve-muscle interaction in chick limb bud chorio-allantoic membrane grafts.

The clonable cell populations of innervated and noninnervated chorio-allantoic membrane (CAM) grafts of early chick embryo leg buds have been compared. Co-grafts of stage 21 or 22 leg buds with stage 22–26 spinal cord segments exhibit a near twofold greater proportion of clonable muscle cells than noninnervated control grafts (leg bud alone). A statistically significant increase is apparent by the 8th graft day. Grafts constructed of stage 17–20 leg buds plus spinal cord and grown for up to 11 days do not exhibit a proportion of myoblasts greater than noninnervated controls. This stimulation of relative muscle cell content suggests a role of motor nerves in regulation of the single-cell population of developing skeletal muscle.     

Isolation and characterization of terminally differentiated chicken and rat skeletal muscle myoblasts

The ability of skeletal muscle myoblasts to differentiate in the absence of spontaneous fusion was studied in cultures derived from chicken embryo leg muscle, rat myoblast lines L6 and L8, and the mouse myoblast line G8. Following 48–96 hr of culture in a low-Ca²⁺ (25 μm), Mg²⁺-depleted medium, chicken myoblasts exhibited only 3–5% fusion whereas up to 64% of the cells fused in control cultures. Depletion of Mg²⁺ led to preferential elimination of fibroblasts, with the result that 97% of the mononucleated cells remaining at 120 hr exhibited a bipolar morphology and stained with antibodies directed against M-creatine kinase, skeletal muscle myosin, and desmin. Mononucleated myoblasts rarely showed visible cross-striations or M-line staining with anti-myomesin unless the medium was supplemented with 0.81 mM Mg²⁺, suggesting that Mg²⁺ plays a role in sarcomere assembly. Conditions of Ca²⁺ and Mg²⁺ depletion inhibited myoblast fusion in the rodent cell lines as well, but mononucleated myoblasts failed to differentiate under these conditions. Differentiated individual myoblasts from rat cell lines and from chicken cell cultures were obtained when fusion was inhibited by growth in cytochalasin B (CB). CB-treated rat myoblast cultures accumulated MM-CK to nearly twice the specific activity found in extensively fused control cultures of comparable age. Spherical cells which accumulated during CB treatment were isolated and shown to contain nearly eight times the CK specific activity present in nonspherical cells from the same cultures. Approximately 90% of these cells exhibited immunofluorescent staining with antibodies to skeletal muscle myosin, failed to incorporate [³H]thymidine or to form colonies in clonal subculture, and thus represent terminally differentiated rat myoblasts. Quantitative microfluorometric DNA measurements on individual nuclei demonstrated that the terminally differentiated myoblasts obtained in these experiments from both chicken and rat contain 2cDNA levels, suggesting arrest in the G₀ stage of the cell cycle.     

The enhancement of in vitro survival and chondrogenesis of limb bud cells by cartilage conditioned medium

Dissociated stage 21–28 chick embryo limb bud cells showed an increasing ability to produce cartilage colonies in vitro with in vivo maturation. In addition dissociated stage 21–28 chick embryo limb bud cells exposed to cartilage conditioned medium continuously or only for 48 hr prior to subculture showed an enhanced (as much as 15-fold) ability to form differentiated cartilage colonies. By this criterion, cells were more responsive to conditioned medium prior to stage 25. Conditioned medium from fibroblast cultures caused an inhibition of cartilage colony formation, suggesting that the effect is cell-type specific. Besides increasing cartilage colony formation by enhanced cell survival, the incorporation of S³⁵O₄ into isolated glycosaminoglycans is also stimulated when limb bud cells are exposed to cartilage conditioned medium. The results support a model for cell differentiation which involves the enhancement of a particular differentiated capacity by a diffusible cell-type-specific macromolecule.     

Local signals in the chick limb bud can override myoblast lineage commitment: induction of slow myosin heavy chain in fast myoblasts.

Patterning of fast and slow muscle fibres in limbs is regulated by signals from non-muscle cells. Myoblast lineage has, however, also been implicated in fibre type patterning. Here we test a founder cell hypothesis for the role of myoblast lineage, by implanting characterized fast and slow mouse myoblast clones into chick limb buds. In culture, late foetal mouse myoblast clones are committed to a probability (range 0-0.92) of slow myosin heavy chain (MyHC) expression. In contrast, when implanted into chick limbs, fast mouse myoblast clones express myosin characteristic of their new environment, without fusion to chick muscle cells and in the absence of innervation. Therefore, local signals exist within the chick limb bud during primary myogenesis that can override intrinsic commitment of at least some myoblasts, and induce slow MyHC.     

Clonal analysis of vertebrate myogenesis. II. Environmental influences upon human muscle differentiation

In vitro procedures for obtaining the differentiation of human fetal muscle colonies were developed, and the sensitivity of clonal differentiation to environmental influences was examined. Human muscle colonies are capable of differentiating in the absence of an exogenous collagen substrate. The dependence of clonal diffeentiation upon the addition of chick embryo extract to the culture medium is determined by the serum type used in the medium and by the substrate upon which the colonies are grown. Clonal differentiation also depends upon conditioning of the medium by the colonies. The rate of medium conditioning is affected by clonal density and initial medium composition. The required medium modification is not species specific since medium conditioned by chick muscle cells also permits the early differentiation of human muscle clones. By manipulating the various environmental parameters described above it has been possible to define a number of in vitro conditions which permit a normal rate of cell proliferation but do not permit cell fusion. Results from these experiments are discussed in terms of their developmental implications.     

Temporal appearance of satellite cells during myogenesis.

In this study, differences between fetal and adult myoblasts in clonal and high density culture have been used to determine when adult myoblasts can first be detected during avian development. The results indicate that avian adult myoblasts are apparent as a distinct population of myoblasts during the midfetal stage of development. Three different criteria were used to differentiate fetal and adult myoblasts and demonstrate when adult myoblasts become a major proportion of the myoblast population: (1) differences in slow myosin heavy chain 1 (MHC1) isoform expression, (2) initiation of DNA synthetic activity, and (3) average myoblast length. Fetal chicken (ED10-12) pectoralis muscle (PM) myoblasts form myotubes that express slow MHC1 after prolonged culture, while adult chicken PM myoblasts do not. Fetal avian myoblasts were active in DNA synthesis and large when first isolated, reaching peak rates of synthesis by 24 hr in culture, while adult myoblasts were inactive in DNA synthesis and small when first isolated, only reaching peak rates of DNA synthesis and size at 3 days of incubation. A dramatic decrease in the percentage of muscle colonies with fibers that expressed slow MHC1 was observed between the midfetal stage and hatching in the chicken, along with a corresponding decrease in myoblast DNA synthetic activity and average length during this same period in both the chicken and the quail. Myoblast activity and average length increased again 3-4 days posthatch and a small transient increase in the number of slow MHC1-expressing clones was also associated with the massive growth of muscle that occurs in the neonatal bird. We conclude that adult myoblasts are present as a distinct population of myoblasts at least as early as the midfetal stages of avian development.     

The independence of myogenesis and chondrogenesis in micromass cultures of chick wing buds

Micromass cultures prepared from stage 23, 24, or 25 chick wing buds and cultured under identical conditions produce similar numbers of myoblasts. After treatment with the DNA synthesis inhibitor cytosine-1-beta-D-arabinofuranoside, [3H]thymidine labeling and autoradiography of the cultures show that the increase in myoblast number during the first 48 hr of culture is due primarily to cell division. Micromass cultures prepared from proximal and distal portions of stage 23 or 24 wing buds have very different chondrogenic potentials in vitro (B.J. Swalla, E.M. Owens, T.F. Linsenmayer, and M. Solursh (1983). Dev. Biol. 97, 59-69) but a similar myogenic potential under these culture conditions. Medium supplements that significantly enhance chondrogenesis by proximal cell cultures, such as low serum or 1 mM db cyclic AMP, do not affect the number of myoblasts per unit area of culture during the first 3 days. Muscle cells are eventually reduced in number in whole limb micromass cultures, yet persist as long as 6 days in proximal and distal cultures. These results suggest that myogenic cells are already committed in the early limbs but are inhibited from differentiation in situ until a later time. Myogenesis and chondrogenesis occur independently in culture, consistent with the idea that these two differentiated cells are derived from two separate cell populations. Furthermore, treatments which enhance chondrogenesis do not act indirectly by killing the myoblast population in these cultures.     

Developmentally regulated lectin in embryonic chick muscle and a myogenic cell line.

Soluble extracts of embryonic chick pectoral muscle and myoblast clone L6 agglutinated trypsin treated glutaraldehyde fixed rabbit erythrocytes. Agglutination activity was blocked by thiodigalactoside, lactose and related saccharides but not by many other saccharides. Agglutination activity of chick pectoral muscle extracts increased at least one order of magnitude between 8 and 16 days of chick embryo development, as the pectoral muscle differentiated. With L6 myoblasts there was a three-fold increase in activity of the extracts as the myoblasts fused to form multinucleated myotubes.     

Accelerated maturation of limb mesenchyme by the BrachypodH mouse mutation

Abstract. Mesenchyme cell populations prepared from proximal and distal halves of stage 20 mouse forelimb buds are shown to behave under in vitro micromass culture conditions like analogous cell populations obtained from chick embryo limb buds. While the distal cells are spontaneously chondrogenic, the proximal cells make aggregates which are only potentially chondrogenic after treatment with dibutyryl cyclic AMP. In addition, stage 20 mouse whole limb bud cells homozygous for the brachypodism^H ( bp ^H ) mutation are shown to behave similarly to 'normal' proximal cells. Both make fewer aggregates and nodules and both have faster aggregation rates (determined as the rate of disappearance of single cells over time) in rotation cultures than 'normal' distal or whole limb bud cells. These results support the hypothesis that the bp ^H mutation specifically decreases the proportion of spontaneously chondrogenic mesenchyme cells (that is, distal-like cells) present at certain developmental stages in the limb bud, resulting in a prematurely high proportion of proximal-like cells.     

Immunological studies of the embryonic muscle cell surface. Antiserum to the prefusion myoblast

Xenogeneic antisera raised in rabbits have been used to detect compositional changes at the cell surfaces of differentiating embryonic chick skeletal muscle. In this report, we present the serological characterization of antiserum (Anti-M-24) against muscle tissue and developmental stage-specific cell surface antigens of the prefusion myoblast. Cells from primary cultures of 12-d-old embryonic chick hindlimb muscle were injected into rabbits, and the resulting antisera were selectively absorbed to obtain immunological specificity. Cytotoxicity and immunohistochemical assays were used to test this antiserum. Absorption with embryonic or adult chick heart, brain, retina, liver, erythrocytes, or skeletal muscle fibroblasts failed to remove all reactivity of Anti-M-24 for myogenic cells at all stages of development. After absorption with embryonic myotubes, however, Anti-M-24 no longer reacted with differentiated myofibers, but did react with prefusion myoblasts. The myoblast surface antigens detected with Anti-M-24 are components of the muscle cell membrane: (a) these macromolecules are free to diffuse laterally within the myoblast membrane; (b) Anti-M-24, in the presence of complement, induced lysis of the muscle cell membrane; and (c) intact monolayers of viable myoblasts completely absorbed reactivity of Anti-M-24 for myoblasts. These antigens are not loosely adsorbed culture medium components or an artifact of tissue culture because: (a) absorption of Anti-M-24 with homogenized embryonic muscle removed all antibodies to cultured myoblasts; (b) Anti-M-24 reacted with myoblast surfaces in vivo; and (c) absorption of Anti-M-24 with culture media did not affect the titer of this antiserum for myoblasts. We conclude that myogenic cells at all stages of development possess externally exposed antigens which are undetected on other embryonic and adult chick tissues. In addition, myoblasts exhibit surface antigenic determinants that are either masked, absent, or present in very low concentrations on skeletal muscle fibroblasts, embryonic myotubes, or adult myofibers. These antigens are free to diffuse laterally within the myoblast membrane and may be modulated in response to appropriate environmental cues during myodifferentiation.     

Spatial analysis of limb bud myogenesis: A proximodistal gradient of muscle colony-forming cells in chick embryo leg buds

The regional distribution of myogenic cells in developing chick leg buds has been investigated using an in vitro clonal assay. Leg buds were embedded in gelatin and sectioned at intervals of 100–300 μm utilizing a vibratome, and cells dissected from prospective myogenic areas were analyzed for their ability to form colonies containing multinucleated myotubes. The results show that muscle colony-forming (MCF) cells from stage 23 ( to 4-day incubation) are exclusively of the early morphological type, and are found in the proximal two-thirds of the bud. Late-type MCF cells are first obtained from the proximal sections of stage 24–25 (4- to day) buds; in succeeding stages (26–29), late MCF cells supercede the early MCF cell type in the proximal regions, and extend into progressively more distal sections in a graded fashion. Results from sequential sections suggest that early and late MCF cells are located within the same muscle groups. The proportion of late MCF cells continues to increase throughout this period, until by stage 31 (7 days) only the most distal myogenic regions (the toe muscle regions) have an appreciable proportion of early MCF cells. Clonal plating efficiencies increase throughout the period of analysis, and by stage 31 precisely dissected myogenic regions yield plating efficiencies as high as 36% with greater than 95% of these colonies differentiating as muscle.     

Clonal analysis of vertebrate myogenesis. VI. Acetylcholinesterase and acetylcholine receptor in myogenic and nonmyogenic clones from chick embryo leg cells.

Myogenic clones grown in vitro from cells of 4-, 6-, and 12-day chick embryo leg buds demonstrate reproducible stage-specific characteristics of morphology, extent of myotube formation, and culture medium requirements for differentiation, suggesting heterogeneity in the myogenic cell populations of the developing limb. To determine whether there is heterogeneity in the cytodifferentiation of different muscle colony types, clones have been examined for the appearance of two muscle-specific gene products—acetylcholinesterase (AChE) and acetylcholine receptor (AChR). AChE (detected by cytochemical reaction) and AChR (detected by autoradiography of [¹²⁵I]α-bungarotoxin binding) appeared in myotubes of all muscle colony types, and also appeared in about 5% of the mononucleated cells of all muscle colonies; but neither were detectable in cells of nonfused clones (colonies containing no myotubes). The results suggest that all muscle colony-forming cell types have equivalent capacities to elaborate muscle-specific gene products once the process of differentiation is initiated. However, when putative muscle colony-forming cells are grown under certain conditions that do not permit cell fusion (e.g., conditioned medium-requiring clones grown in fresh medium), mononucleated cells do not accumulate AChE or AChR. Conditioned medium-dependent differentiation thus differs from the fusion-specific processes affected by Ca²⁺ deprivation and phospholipase C treatment, since in these cases mononucleated cells exhibit differentiated functions. The apparent cytodifferentiation (without fusion) of some mononucleated cells within muscle colonies in which most mononucleated cells continue to proliferate raises questions concerning the control of myoblast differentiation and its relationship to the cell cycle and to fusion.     

Lack of evidence for the involvement of a β-D-galactosyl-specific lectin in the fusion of chick myoblasts

The role of a β-D-galactosyl-specific lectin, first reported by Teichberg et al., in the fusion of myoblasts in vitro was investigated. The concentration of this lectin in embryonic chick skeletal muscle was found to reach maximal levels at the time of myoblast fusion in vivo. β-D-Galactosyl-β-thiogalactopyranoside and lactose are potent inhibitors of agglutination of trypsinized rabbit erythrocytes caused by the lectin. However, at concentrations of 50 mM these compounds had no effect on either nonsynchronous fusion of myoblasts or on the release of synchronized myoblast cultures from EGTA fusion block. The presence of the agglutinin in the external membranes of chick myoblasts and myotubes could not be demonstrated. It is, therefore, concluded that the involvement of the lectin in the fusion of chick myoblasts remains questionable.     

Computerized morphometric analysis of the chick embryo duodenum organogenesis

A computerized morphometric analysis was performed on histological sections of the proximal and distal limbs of the chick embryo duodenum, from the 8th to the 15th day of incubation, with the aim of evaluating the quantitative aspects of organogenesis. A semiautomatic digital system was used. The areas of total section, of the lumen, of the wall and of its components (subserous stratum, muscle layer, lamina propria, epithelium) and the thickness of the epithelium and muscle layer were measured; the mean +/- S.E.M. of the values obtained was calculated. The percentage of shrinkage, due to the histological procedures, was calculated for each day. The values obtained were added to the measurements of each area and thickness thus giving the true value of each parameter measured. The differences between the mean values of the areas of the proximal and the distal tract were statistically evaluated. Exponential curves and r coefficient were determined to evaluate the general growing pattern of the mean area of the components of the duodenal wall, as a function of age. The main results are the following: the areas of the wall components of the proximal limb are always greater than the corresponding areas of the distal limb of the duodenum. There are some differences between the proximal and the distal limbs of the duodenum in the growing pattern of the intestinal wall components. The anlage of the duodenum also has a different developmental pattern compared with that of the chick embryo ileum.     

鸡胚与胎鼠的成纤维细胞条件培养液促进成肌细胞增殖和融合的比较

用培养过鸡胚(来亨鸡)或胎鼠(ICR小鼠)肌组织的成纤维细胞的条件培养液,定量地研究它们对胎鼠或鸡胚的成肌细胞的增殖和融合的影响。所得结果如下:(1) 胎鼠的成纤维细胞条件培养液促进胎鼠或鸡胚成肌细胞增殖,分别为对照组的2.65倍,(P<0.001)或2.35倍,(P<0.01);(2) 鸡胚的成纤维细胞条件培养液促进鸡胚或胎鼠的成肌细胞增殖,分别为对照组的2.66倍,(P<0.01)或2.17倍,(P<0.01);(3) 胎鼠的成纤维细胞条件培养液增加胎鼠或鸡胚的成肌细胞的融合率,分别为对照组的1.9倍或2.6倍;鸡胚的成纤维细胞条件培养液只增加鸡胚成肌细胞的融合率,为对照组的2.1倍,但对胎鼠成肌细胞的融合无明显的影响。 实验结果提示:成纤维细胞条件培养液促进成肌细胞的增殖,两种动物间无明显的差异,但在融合上却有一定的种属特异性。     

Differential inhibition of myoblast fusion.

The aggregation and fusion of myoblasts in the presence of either metabolic inhibitors or alterations in the incubation medium or under conditions which result in structural changes in the cells was studied using previously described assays for the intercellular interactions of myoblasts in suspension [Knudsen, K. A., and Horwitz, A. F. (1977). Develop. Biol.58, 328]. These perturbations inhibit myoblast fusion differently. For example, energy poisons, prior trypsin or glutaraldehyde treatment, and inhibitors of protein or cholesterol synthesis all inhibit the Ca²⁺-mediated myoblast aggregation. In contrast, whereas myoblasts aggregate in the presence of 20 mM Mg²⁺, these aggregates are dispersed, even after 1–2 hr, with EDTA or trypsin. Furthermore, enriching the fatty acyl chains in elaidate or prior incubation of the myoblasts in the presence of cytochalasin B or colchicine results in aggregates which, after 1–2 hr, are dispersed by trypsin but not by EDTA. Aggregates of unaltered, control myoblasts, on the other hand, begin to show resistance to dispersion by trypsin after these times. These observations support the suggestion that multinucleate cell formation results from a sequence of events. The influence of these perturbations on cellular aggregation also provides some initial, tentative insight into the molecular mechanism of myoblast fusion. Recognition (calcium-mediated aggregate formation) appears to be mediated by a protein(s) that is turning over during the period of fusion competence, while membrane union (formation of aggregates resistant to dispersion by trypsin) most likely involves the direct participation of membrane lipid.