DNA synthesis and the cell cycle in cultures of normal and mutant (mdg/mdg) embryonic mouse muscle cells.

The relationship between cell fusion, DNA synthesis and the cell cycle in cultured embryonic normal and dysgenic ($\text{mdg}\text{mdg}$) mouse muscle cells has been determined by autoradiography. The experimental evidence shows that the homozygous mutant myotubes form by a process of cell fusion and that nuclei within the myotubes do not synthesize DNA or undergo mitotic or amitotic division. The duration of the total cell cycle and its component phases was statistically the same in 2-day normal and mutant ($\text{mdg}\text{mdg}$) myogenic cultures with the approximate values: T, 21.5 hr; G₁, 10.5 hr; S, 7.5 hr; and G₂, 2.5 hr. In both kinds of cultures, labeled nuclei appeared in myotubes 15–16 hr after mononucleated cells were exposed to [³H]thymidine, and the rate of incorporation of labeled nuclei into multinucleated muscle cells was comparable in control and dysgenic cultures. Thus, homozygous $\text{mdg}\text{mdg}$ muscle cells in culture are similar to control cells with respect to their mechanism of myotube formation and the coordinate regulation of DNA synthesis and the cell cycle during myogenesis.     

MITOSIS AND THE PROCESSES OF DIFFERENTIATION OF MYOGENIC CELLS IN VITRO

The relation between the mitotic cycle and myoblast fusion has been studied in chick skeletal muscle in vitro. The duration of the cell cycle phases was the same in both early and late cultures. By tracing a cohort of pulse-labeled cells, it was found that myoblast fusion does not occur in S, G₂, or M. Cell surface alterations required for fusion are dependent upon the position of the cell in the division cycle. In early cultures, fusion takes place only after a minimum delay of 5 hr from the time the cell has entered G₁. The mitosis preceding fusion may condition the cell for the abrupt shift in synthetic activity that occurs in the subsequent G₁. In older cultures fusion of labeled cells is diminished. Two factors account for the cessation of fusion in older cultures. First, the number of myogenic stem cells declines, but these cells do not disappear as the cultures mature. Their persistence was demonstrated by labeling dividing mononucleated cells in older cultures and challenging them with nascent myotubes. Some of these labeled cells were incorporated into the forming myotubes. Second, a block to fusion develops during myotube maturation. Well developed myotubes challenged with labeled competent myogenic cells failed to incorporate the labeled nuclei.     

Vitellogenin synthesis by the fat body of the mosquito Aedes aegypti: evidence of transcriptional control

The acetylcholinesterase (AChE) activity of cultures from 11-day-old chick embryo muscle cells was studied for up to 4 weeks in vitro. AChE activity was found in mononucleated cells and multinucleated myotubes. The activity increased greatly after fusion. Maximum AChE levels were reached after 7–10 days of incubation and tended to decline thereafter. Multiple forms of AChE found in embryo muscle in situ were present in cultures before and after fusion. Selective inhibitors and substrates were used to show that AChE was released by the cells into their medium. Within a 2-day period the AChE that accumulated in the medium averaged over 6 times that remaining in the cells. Release of AChE from the cells was inhibited by cycloheximide, and AChE levels in cells and medium were much reduced when differentiation was inhibited by bromodeoxyuridine. Little AChE was present in subcultures of fibroblasts from muscle cultures. Acetyl-β-methylcholine and, to a lesser degree, choline itself, prevented the decrease in AChE levels of 2- to 3-week-old muscle cultures.     

Creatine kinase and aldolase isoenzyme transitions in cultures of chick skeletal muscle cells

Changes in the isoenzyme patterns and activities of the two enzymes creatine kinase (CPK) and fructose diphosphate aldolase have been followed during the course of differentiation of chick skeletal muscle cells in vitro. The characteristic isoenzyme transitions of both of these enzymes known to occur in developing muscle in situ can be demonstrated in extracts of cultured myogenic cells by cellulose polyacetate electrophoresis followed by specific enzymatic staining: MM-CPK replaces the embryonic BB-CPK, while aldolase isoenzymes containing A subunits replace the C-containing forms which predominate at earlier stages. The specific activities of both enzymes increase during in vitro differentiation. Although the major part of these concomitant changes occurs after myoblast fusion has reached a maximum level, analysis of their timing relative to the process of fusion indicates that the increases in the activities of both enzymes, as well as the accumulation of nuclei within myotubes, proceed exponentially from the beginning of the second day in culture. Fusion and enzyme accumulation are unaffected by addition of dibutyryl cyclic AMP (1 × 10^?4M) to the medium. In calcium-deficient medium, or in media containing 5-bromodeoxyuridine (BrdUrd) at concentrations from 0.2 to 7 × 10^?5M, fusion is almost completely blocked, while cell viability is maintained. The CPK and aldolase isoenzyme transitions fail to occur normally in both fusion-preventing media. This blockage of the normal differentiative changes is, however, less complete in the calcium-deficient cultures, which, in contrast to the BrdUrd containing cultures, contained a number of long bipolar cells thought to be able to differentiate without fusion. These results are interpreted as indicating that for most, but possibly not for all, myogenic cells in typical primary muscle cell cultures, fusion is a prerequisite for the parallel differentiative changes in CPK and aldolase isoenzymes. The possibility is discussed that a “cluster” of proteins, including CPK and aldolase, may be coordinately regulated during myogenesis.     

A KINETIC ANALYSIS OF MYOGENESIS IN VITRO

Conditions which yielded reproducible growth kinetics with extensive, relatively synchronous differentiation are described for chick muscle cultures. The effects of cell density and medium changes on the timing of cell fusion were examined. Low-density cultures which received a change of medium at 24 hr after plating show the highest rate of cell fusion, increasing from 15 to 80% fused cells in a 10 hr period. These optimal culture conditions were employed to reexamine two questions from the earlier literature on muscle culture: (a) can cells which normally would fuse at the end of one cell cycle be forced to go through another cell cycle before fusion; and (b) how soon after its final S period can a cell complete fusion? In answer to the first question, it was found that if the medium is changed, many cells which would otherwise fuse can be made to undergo another cell cycle before fusion. In the second case, radioautographs were made from cultures incubated with tritiated thymidine for various times at the beginning of the fusion period. These show labeled nuclei in myotubes as early as 3 hr after the beginning of the incubation period. This indicates that cells can fuse as early as the beginning of the G₁ period, and suggests that there is not an obligatory exit from the cell cycle or a prolonged G₁ period before cell fusion and differentiation during myogenesis.     

THE EFFECT OF MITOTIC INHIBITORS ON MYOGENESIS IN VITRO

The effect of colchicine and other antimitotic drugs was studied in cultures of 11-day chick embryo breast muscle. Exposure of such cultures to 10^-6 M colchicine results in fragmentation of the elongate myotubes into rounded, cytoplasmic sacs (myosacs) containing various numbers of nuclei. Comparison of the dose-response relation between myotube fragmentation and metaphase arrest suggests that the underlying mechanism may be similar in both cases. Low temperature does not duplicate the effects of colchicine. Glycerinated myotubes are not affected by the mitotic inhibitors. The effect of colchicine on myotubes is reversible. Myosacs elongate within several days after removal from colchicine. However, the regenerated myotubes fail to incorporate additional mononucleated cells. Colchicine does not interfere with the process of fusion itself, but the metaphase block prevents cells from entering that phase of the cell cycle during which fusion can occur. Cells arrested in mitosis by colchicine do not recover when incubated in normal medium. Colcemid-induced arrest is reversible and does not prevent subsequent fusion of the cells.     

Autoradiographic labeling of spinal cord neurons with high affinity choline uptake in cell culture

Embryonic chick spinal cord neurons grown in dissociated cell culture have a high affinity uptake mechanism for choline. We find that, in addition to acetylcholine synthesis, the accumulated choline is used for the synthesis of metabolites such as lipids that are retained in part by conventional fixation techniques. As a result autoradiographic methods can be used to identify the cells that have the uptake mechanism in spinal cord cultures. About 60% of the neurons are labeled by [³H]choline uptake in cultures prepared with spinal cord cells from 4-day-old embryos, and about 40% are labeled in cultures prepared with cord cells from 7-day-old embryos. Neurons that innervate skeletal myotubes in spinal cord-myotube cultures are consistently labeled by [³H]choline uptake. Neurons unlabeled by the procedure are viable: they exclude the dye trypan blue and accumulate ¹⁴C-amino acids for protein synthesis. Most of the neurons unlabeled by [³H]choline uptake can instead be labeled by uptake of γ-[³H]aminobutyric acid, and vice versa. These results suggest that high affinity choline uptake can be used to label cholinergic neurons in cell culture, and that at least some populations of noncholinergic neurons are not labeled by the procedure. It cannot yet be concluded, however, that all labeled neurons are cholinergic since more labeled neurons are obtained per cord than would be expected from the number of neurons making up identified cholinergic populations in vivo. A three- to fourfold increase in the amount of high affinity choline uptake is observed between Days 3 and 15 in culture for spinal cord cells obtained from 4-day-old embryos. The number of [³H]choline-labeled neurons in such cultures decreases slightly during the same period, suggesting that the increase in uptake reflects neuronal growth or development rather than an increase in population size. Both the magnitude of the uptake and the number of [³H]choline-labeled neurons are the same for spinal cord cells grown with and without skeletal myotubes.     

DNA synthesis, mitosis and fusion of myocardial cells

Myocardial cells obtained from embryonic chick ventricles have been used to investigate (1) whether differentiated cells can undergo DNA synthesis and mitosis and, (2) whether heart cells when grown in culture can fuse with each other and with chick skeletal myoblasts to form heterokaryon myotubes. Electron microscopic observations have shown that myocardial cells of day 3 and day 20 chick embryos did contain myofibrils with defined sarcomeres; these cells have been observed in mitosis. Cells obtained by tryptic digestion of day 12 chick ventricles when grown in culture continued to replicate their DNA as shown by thymidine-³H radioautography with DNase controls and were observed in all stages of mitosis. Electron microscopy showed that myofibrils were present in some of the cultured cells. Bi-, tri- and tetranucleate cells were observed in the cultures. Thymidine-³H radioautography showed that these cells were formed by karyokinesis without cytokinesis and by the fusion of uninucleate cells. Since the heart cells could fuse with each other, we tested the possibility that they could fuse with skeletal myoblasts to form heterokaryon myotubes. This was accomplished by co-culturing thymidine-³H labeled ventricular cells and unlabeled skeletal myoblasts. Radioautography with DNase controls showed that some of the myotubes consisted of unlabeled skeletal muscle nuclei and labeled heart nuclei in varied proportions. The factors initiating the formation of these heterokaryons have not been elucidated.     

Analysis of cartilage differentiation from skeletal muscle grown on bone matrix: I. Ultrastructural aspects

Previous studies have demonstrated that embryonic skeletal muscle is competent to form hyaline cartilage when cultured in vitro on demineralized bone matrix (Nogami, H., and Urist, M. R. (1970). Exp. Cell Res.63, 404–410; Nathanson, M. A., et al. (1978). Develop. Biol.64, 99–117). The present experiments were undertaken to determine the nature of the morphological alterations which attend this phenotypic transformation and to investigate the ultrastructural characteristics of the myoblasts and fibroblasts of skeletal muscle during the transformation. Nineteen-day embryonic rat limb muscles were minced and the tissue fragments explanted to bone matrix or collagen gels. The trauma of excision and mincing causes syncytial myotubes to degenerate and the nuclei of mononucleate cells to enter a heterochromatic “resting stage.” In culture, nuclei of mononucleate cells rapidly regain euchromasia. No myoblast or fibroblast cell death can be detected. On bone matrix, the entire mononucleate population transforms into fibroblast-like cells. Myoblasts are the major contributor to this population; they dissociate from the degenerate myotubes and begin to acquire endoplasmic reticulum by 24 h in vitro. The fibroblast-like morphology persists through 4 days in vitro. By 6 days in vitro some of these fibroblast-like cells acquire the phenotypic characteristics of chondrocytes, and by 10 days masses of hyaline cartilage are found. In control explants of skeletal muscle onto collagen gels, the heterochromatic nuclei of the mononucleated cells expand after 24 hr in vitro, but the mononucleated cells remain as myoblasts and fibroblasts and begin to regenerate skeletal muscle by 4 days in vitro. No cartilage forms. The results indicate that both myoblasts and fibroblasts have chondrogenic potential when grown on demineralized bone. It is tempting to conclude that the embryonic mesenchymal cells which give rise to skeletal muscle, cartilage, and other connective tissue of the limb have similar developmental potentials and that local influences, rather than separate cell lineages, account for the final pattern of differentiation.     

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.     

In Vitro effects of thyroxine and insulin on myoblasts from chick embryo skeletal muscle

The role of insulin and l-thyroxine (L-T₄) in stimulating myoblast proliferation and differentiation was investigated in vitro. A superphysiological concentration of insulin or a physiological concentration of L-T₄ was added to cultures of myoblasts from 11-day-old chick embryo thigh muscle, grown in serum-free DM-153 medium. While the addition of insulin resulted in an increase in the total number of cells, in the extent of fusion, and in the creatine phosphokinase (CPK) activity, myotubes changed into globular structures which tended to degenerate rapidly. On the other hand, while the addition of L-T₄ had less effect on myogenesis, myotubes retained their differentiated state longer. Furthermore, the two hormones exhibited synergistic effects. An increase in the initial cell density resulted in an increase in the amount of protein and CPK activity, irrespective of the presence or absence of the hormones. This suggests that the effect of insulin and L-T₄ on myogenesis is not a differentiation-specific effect, but rather an indirect result of cell proliferation.     

Isolation of myosin-synthesizing polysomes from cultures of embryonic chicken myoblasts before fusion.

Mononucleated myoblasts and multinucleated myotubes were obtained by culturing embryonic chicken skeletal muscle cells. Comparison of total polysomes isolated from these mononucleated and multinucleated cell cultures by density gradient centrifugation and electron microscopy revealed that mononucleated myoblasts contain polysomes similar to those contained by multinucleated myotubes and large enough to synthesize the 200,000-dalton subunit of myosin. When placed in an in vitro protein-synthesizing assay containing [³H]leucine, total polysomes from both mononucleated and multinucleated myogenic cultures were active in synthesizing polypeptides indistinguishable from myosin heavy chains as detected by measurement of radioactivity in slices through the myosin band on sodium dodecyl sulfate (SDS)-polyacrylamide gels. Fractionation of total polysomes on sucrose density gradients showed that myosin-synthesizing polysomes from mononucleated myoblasts may be slightly smaller than myosin-synthesizing polysomes from myotubes. Multinucleated myotubes contain approximately two times more myosin-synthesizing polysomes per unit of DNA than mononucleated myoblasts, and the proportion of total polysomes constituted by myosin polysomes is only 1.2 times higher in multinucleated myotubes than it is in mononucleated myoblasts. The results of this study suggest that mononucleated myoblasts contain significant amounts of myosin messenger RNA before the burst of myosin synthesis that accompanies muscle differentiation and that a portion of this messenger RNA is associated with ribosomes to form polysomes that will actively translate myosin heavy chains in an in vitro protein-synthesizing assay.     

The relationship of polyoma virus-induced tumor (T) antigen to activation of DNA synthesis in rat myotubes

Rat myotubes infected with polyoma virus (PV) introduced into the multinucleated cells by virus-bearing myoblasts at the time of cell fusion incorporate ³H-TdR and exhibit mitotic-type figures. The infected myotubes also produce a viral-specific nuclear antigen, tumor (T) antigen, which appears in groups of adjacent nuclei or in all nuclei of the myotubes. The proportion of myotubes which synthesize DNA, T-antigen and exhibit mitotic-type figures is related to the multiplicity of virus infection.Intact myotubes which are resistant to infection with PV by virus absorption can be infected by microinjection of the virus into the cells. Myotubes thus infected produce T-antigen which appears in multiple nuclei, but do not incorporate ³H-TdR or contain mitotic-type figures. The data suggest that the resistance of myotubes to infection with PV might be due to a change in the cell surface membrane during differentiation so that virus cannot penetrate the cell. The T-antigen apparently has no bearing on the activation of the DNA-synthesizing apparatus in multinucleated muscle cells.     

Acetylcholine synthesis by chick spinal cord neurons in dissociated cell culture

Acetylcholine (ACh) synthesis was examined in cultures of chick spinal cord cells to follow the development of the cholinergic neurons. The cells, prepared from 4-day-old embryonic chick spinal cords, were grown either alone in dissociated cell cultures (SC cultures) or with chick myotubes (SC-M cultures). ACh synthesis was measured by incubating the cultures in [³Hcholine and using high-voltage paper electrophoresis to quantitate the amount of [³H]ACh present in cell extracts prepared from the labeled cultures. The amount of [³H]ACh synthesized in SC-M cultures was strictly proportional to the number of spinal cord cells used to prepare the cultures, and was linear with the time of incubation in [³H]choline for periods up to 1 hr. Maximal rates of synthesis were observed with [³H]choline concentrations in excess of 100 μM. Such rates for 1-week-old SC-M cultures were approximately 10–20 pmoles of [³H]ACh/hr/10⁵ spinal cord cells. Studies on the stability of the intracellular [³H]ACh revealed the presence of a major pool with a half-time of 20–30 min. A second, small pool decayed more rapidly. No detectable [³H]ACh was spontaneously released from the cells, suggesting that most of the decay represented intracellular degradation. Development of cholinergic neurons as monitored by [³H]ACh synthesis continued over a 2-week period in SC-M cultures and paralleled general cell growth. When examined at 1 week, SC-M cultures had about a 50% greater capacity for [³H]ACh synthesis and 60% more choline acetyltransferase activity than did SC cultures. No difference was observed in the stability of the [³H]ACh formed for the two types of cultures at 1 week, and no further difference was observed in the rates of [³H]ACh synthesis at 2 weeks. Growth of SC cultures in medium containing different amounts of chick embryo extract (2–10%) or in medium with fetal calf serum (10%) instead of extract produced only small differences in the measured rates of [³H]ACh synthesis. Thus chick spinal cord cells can undergo some of the early stages of cholinergic development in cell culture without sustained contact with skeletal myotubes, one of the normal postsynaptic target cells for the cholinergic neuron population. No absolute requirement for muscle factors was revealed under these conditions, although such factors may have been provided by other cell types in the spinal cord population or may have been present in other additions to the culture medium.     

The distribution of acetylcholine sensitivity over uninnervated and innervated muscle fibers grown in cell culture

This paper describes the physiological and pharmacological parameters of the response of mature muscle fibers that develop from myoblasts in vitro to iontophoretically applied acetylcholine (ACh) and the distribution of ACh sensitivity over fibers innervated in vitro by spinal cord cells and uninnervated (control) fibers. Peaks of sensitivity were detected near nerve terminals on functionally innervated fibers, but the “extrasynaptic” chemosensitivity remained high. The distribution of chemosensitivity over uninnervated fibers is not uniform: peaks or “hot spots” were detected over most fibers. Autoradiography of cultures exposed to ¹²⁵I-α-bungarotoxin is consistent with the uneven distribution detected by iontophoresis. Sensitivity peaks were usually located in the immediate vicinity of obvious muscle nuclei and conversely the membrane near most nuclei was more sensitive than that over other regions along the same cell. The relation between innervation and distribution of ACh sensitivity is discussed.     

Theileria parva: Effects of irradiation on a culture of parasitized bovine lymphoid cells

Aliquots of a culture of Theileria parva-infected bovine lymphoid cells were irradiated at 0, 300, 600, 900, and 1200 rads. The short-term effects of irradiation were evaluated on examination of Giemsa-stained smears and on autoradiography of cells labeled with [³H]thymidine. Irradiation inhibited cell division but parasite division did not appear to be inhibited and macroschizont nuclear particles increased in number, frequently to several hundred per schizont. There was no evidence of an increased percentage switch from macro- to microschizont. Apparently viable cells were still present in all cultures 4 days after irradiation.     

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.     

Properties of reticulum cell sarcomas in SJL/J mice: II. Fate of labeled tumor cells in normal and irradiated syngeneic mice

Transplantable reticulum cell sarcoma (RCS) cells were labeled with ³H-uridine or ³H-thymidine in vitro and injected intravenously into normal and irradiated syngeneic SJL/J mice. RCS cells exhibited typical B cell migration characteristics in peripheral lymphoid organs in both normal and irradiated recipients, localizing in follicles in a pattern resembling that of labeled normal bone marrow cells. However, over the first 72 hr after transfer, RCS cells diluted their label much less in irradiated than in normal recipients, reflecting their inability to proliferate in the irradiated hosts. The presence of unlabeled tumor cells did not significantly affect the distribution of labeled normal bone marrow or lymph node cells in the recipients. Thus, RCS fails to grow in irradiated recipients in spite of undisturbed homing characteristics and in the absence of any evidence of cytotoxic influences from the host.     

Distinct Effects of Abelson Kinase Mutations on Myocytes and Neurons in Dissociated Drosophila Embryonic Cultures: Mimicking of High Temperature

Abelson tyrosine kinase (Abl) is known to regulate axon guidance, muscle development, and cell-cell interaction in vivo. The Drosophila primary culture system offers advantages in exploring the cellular mechanisms mediated by Abl with utilizing various experimental manipulations. Here we demonstrate that single-embryo cultures exhibit stage-dependent characteristics of cellular differentiation and developmental progression in neurons and myocytes, as well as nerve-muscle contacts. In particular, muscle development critically depends on the stage of dissociated embryos. In wild-type (WT) cultures derived from embryos before stage 12, muscle cells remained within cell clusters and were rarely detected. Interestingly, abundant myocytes were spotted in Abl mutant cultures, exhibiting enhanced myocyte movement and fusion, as well as neuron-muscle contacts even in cultures dissociated from younger, stage 10 embryos. Notably, Abl myocytes frequently displayed well-expanded lamellipodia. Conversely, Abl neurons were characterized with fewer large veil-like lamellipodia, but instead had increased numbers of filopodia and darker nodes along neurites. These distinct phenotypes were equally evident in both homo- and hetero-zygous cultures (Abl/Abl vs. Abl/+) of different alleles (Abl¹ and Abl⁴) indicating dominant mutational effects. Strikingly, in WT cultures derived from stage 10 embryos, high temperature (HT) incubation promoted muscle migration and fusion, partially mimicking the advanced muscle development typical of Abl cultures. However, HT enhanced neuronal growth with increased numbers of enlarged lamellipodia, distinct from the characteristic Abl neuronal morphology. Intriguingly, HT incubation also promoted Abl lamellipodia expansion, with a much greater effect on nerve cells than muscle. Our results suggest that Abl is an essential regulator for myocyte and neuron development and that high-temperature incubation partially mimics the faster muscle development typical of Abl cultures. Despite the extensive alterations by Abl mutations, we observed myocyte fusion events and nerve-muscle contact formation between WT and Abl cells in mixed WT and Abl cultures derived from labeled embryos.     

Image analysis of rat satellite cell proliferation in vitro

Myogenic cells were isolated from adult rat skeletal muscles and cultured in vitro. Cell proliferation was analyzed between days 1 and 14. The cell cycle phases were determined by examining Feulgen-stained cultures with a cell image processor. The nuclei were automatically analyzed by calculating 18 parameters relating to the texture and densitometry of chromatin and the shape of each nucleus. Cell cycle phases were characterized (Moustafa and Brugal, 1984). The recognition methods made it possible to analyse the nuclei of the myogenic cell populations which were either involved in each phase of the mitotic cycle, or left out of the cycle after fusion into myotubes.After 3 hr of culture 10% of the cell population was involved in the cell cycle. In the presence of foetal calf serum, this percentage increased until day 3 after plating. At that time, the DNA content of 28.2% of the cell population was higher than 3C, whereas it is 2C in G1 or G0 nuclei; image analysis showed that 42% of these cells were in S or G2 phase. From day 4, the proliferation rate gradually slowed down until day 8. After day 8, when numerous myotubes differentiated, the percentage of S and G2 phase cells had diminished to between 3 and 8%. The percentage of nuclei in G0 increased when the first myotubes differentiated around day 5. Myotube nuclei were largely in G0. When horse serum was added to the culture medium on day 4 to enhance myotube differentiation, significant cell proliferation was observed before cell fusion.These methods of analysis give the first daily pattern of myogenic cell proliferation and fusion in a cell population isolated from adult muscles.