Centrioles are lost as embryonic myoblasts fuse into myotubes in vitro

Embryonic chick myoblasts possess an extensive network of cytoplasmic microtubules which emanate from a single, perinuclear centrosome containing a microtubule-organizing center (MTOC) and the centrioles. However, after myoblasts fuse into myotubes the centrosome is no longer apparent, and instead long parallel arrays of microtubules are seen. From ultrastructural studies on developing muscle tissue, it has been proposed that centrioles are present in myoblasts but are absent from fused muscle fibers. We have examined this hypothesis in vitro in cultures of chick embryonic muscle cells using sera which specifically label centrioles. Almost all (90-97%) mononucleated cells in these cultures, including myoblasts aligned just prior to fusion, contain a pair of centrioles in close proximity to the nucleus. However, in newly fused multinucleated myotubes as well as in older myotubes that had developed myofibrils, centrioles were rarely found (1-10% positive cells). This study thus provides direct evidence for a loss of centrioles from muscle cells soon after they fuse to form myotubes.     

DISEASED MUSCLE CELLS IN CULTURE

(1) Cultures of differentiated muscle cells have been grown from diseased human, mouse and chick skeletal muscle, and from cardiac muscle of the myopathic hamster. (2) Methods of culture established for normal embryonic and adult skeletal muscle cells have proved suitable for cultures of diseased muscle cells. (3) Myoblasts obtained from dy^2J mouse muscle crushed in vivo before explanting fuse in culture and form morphologically normal myotubes. Studies of the effects of innervation by dy^2J spinal cord neurones on the differentiation of normal, dy^2J and dy myotubes have been inconclusive but it is probable that innervation does not play a part in the pathogenesis of this disorder. (4) Myoblasts prepared by trypsinization of embryonic dy muscle behave normally in culture and fuse to form myotubes that appear normal. It is not clear if myoblasts that migrate from explants of adult muscle in vitro fuse. Aggregates of non-fusing cells have been described, but under other culture conditions normal and abnormal forms of myotube have been observed. dy muscle fibres fail to regenerate even when cultured with normal spinal cord explants and dy nerves are without effect on regenerating normal muscle fibres. These tissue-culture studies suggest that the dy mouse mutation is a myopathic disorder. (5) Embryonic mdg myoblasts have a normal cell cycle in vitro and fuse to form well-differentiated myotubes with cross-striations. mdg myotubes have normal electro-physiological properties but do not contract spontaneously or on depolarization. The defect in the muscle of the mdg mutant appears to be a failure of excitation-contraction coupling. (6) Cells migrate earlier from explants of adult dystrophic chick muscle than from normal muscle but dystrophic chick myotubes appear morphologically normal. Myotubes prepared from embryonic dystrophic chick muscle become vacuolated and degenerate, changes that can be prevented by anti-proteases such as antipain. Lactic dehydrogenase isozyme subunit M4 is absent from dystrophic muscle in vivo but reappears in cultured myotubes. Dystrophic myotubes innervated in culture by either normal or dystrophic neurones exhibit bi-directional lcoupling and multiple innervation. These results suggest that there are changes in dystrophic myotubes and that chick muscular dystrophy is a myopathy. (7) Cardiac muscle cells from the cardiomyopathic hamster synthesize less actin and myosin than normal cells, and Z lines in dystrophic cells are irregularly arranged. The beat frequency of myopathic cardiac cells is lower than that of normal cells and declines more rapidly. Tissue-culture studies have not been made of hamster skeletal muscle. (8) Human dystrophic myotubes do not show degenerative changes in culture and have normal histochemical reactions. RNA synthesis appears normal in dystrophic myotubes but there may be changes in adenyl-cyclase activity and protein synthesis in dystrophic cells. Morphological and biochemical changes have been found in muscle cells cultured from a case of acid-maltase deficiency but phosphorylase activity re-appeared in myotubes cultured from biopsies of phosphorylase-deficient muscle. Innervation by normal mouse nerves does not induce degenerative changes in dystrophic myotubes. (9) Studies on the origins of myoblasts in explants of muscle fibres in culture suggest that in these conditions myoblasts are derived only from satellite cells and that this process may be the same in normal and diseased muscle.     

The suppression of myogenic functions in heterokaryons formed by fusing chick myocytes to diploid rat fibroblasts

Heterokaryons were formed by fusing differentiated chick skeletal myocytes to fibroblasts derived from skin, lung or heart cultures. The heterokaryons were analyzed for the synthesis of skeletal myosin light chains, acetylcholine receptor, total CPK activity and the ability to spontaneously fuse to form myotubes. Whereas all of the above myogenic functions were expressed in control heterokaryons formed between myocytes and myoblasts, all were extinguished in the crosses between myocytes and fibroblasts. These results confirm that the suppression of myogenic functions previously observed in cell hybrids involving fibroblastoid tumor cells also occurs in heterokaryons isolated using biochemical inhibitors between diploid fibroblasts and chick skeletal myocytes.     

Myoblast differentiation in vitro: morphological differentiation of mononucleated myoblasts.

Phospholipase C from Clostridium perfringens has been shown previously to inhibit the fusion of cultured chick myoblasts without affecting recognition or cell cycle parameters. In this paper we report that the mononucleated myoblasts, in phospholipase C, synthesize thick and thin filaments and organize them into myofibrils, and that T-tubules and sarcoplasmic reticulum differentiate and join in morphologically typical junctions. The structurally differentiated myoblasts can then fuse with one another to form myotubes. We conclude that cell fusion is not necessary for muscle differentiation.     

Immunofluorescent visualization of 100 A filaments in different cultured chick embryo cell types.

Antibody prepared against the 55,000 dalton subunit of reconstituted chick gizzard 100 A filaments (anti-G55K) bound to the 100 A filaments of chick smooth muscle, cardiac muscle, and skeletal muscle cells, and to the 100 A filaments of Schwann cells and satellite glial cells of the peripheral nervous system. Anti-G55K did not bind to replicating presumptive myoblasts, fibroblasts, chondroblasts, pigment cells, neurons, or to central nervous system glial cells. This contrasted with the wider range of binding of antibody to the 58,000 dalton subunit of chick fibroblast 100 A filaments (anti-F58K) which bound to the 100 A filaments of all cell types examined except hepatocytes and skin epithelial cells. Anti-G55K) staining revealed a morphologically distinct distribution of 100 A filaments in the three types of muscle cells. Spindle shaped smooth muscle cells exhibited dense fluorescent staining near the poles of the cells, and also exhibited unique patches of fluorescent material after cytochalasin B and Colcemid treatment. In myotubes, the fluorescence was limited to longitudinal bundles of filaments between the striated myofibrils. Cardiac cells contained uniformly distributed fine filaments. Lastly, smooth muscle cells in various phases of mitosis bound the anti-G55K, whereas replicating presumptive skeletal myoblasts failed to bind the anti-G55K.     

Characterization of heterokaryons between skeletal myoblasts and somatic cells formed by fusion with HVJ (Sendai virus); effects on myogenic differentiation

In skeletal myogenic differentiation, myoblasts fuse with myogenic cells spontaneously, but do not fuse with non-myogenic cells either in vivo or in vitro, suggesting that the fusion of myoblasts with non-myogenic cells is unsuitable for differentiation. To understand the inevitability of the fusion among myoblasts, we prepared heterokaryons in crosses between quail myoblasts transformed with a temperature-sensitive mutant of Rous sarcoma virus (QM-RSV cells) and rodent non-myogenic cells, such as tumor cells, fibroblasts, or neurogenic cells by HVJ (Sendai virus) and examined how myogenic differentiation was influenced in the prepared heterokaryons, focusing on myogenin expression and myofibril formation as markers of differentiation. When presumptive QM-RSV cells were fused with non-myogenic cells by HVJ and induced to differentiate, both myogenin expression and myofibril formation were suppressed. When myotubes of QM-RSV cells that had already expressed myogenin and formed myofibrils were fused with non-myogenic cells, both myogenin and myofibrils disappeared. Especially, fibrous structures of myofibrils were significantly lost and dots or aggregations of F-actin were formed within 24 hr after formation of heterokaryons. However, the fusion of presumptive or differentiated QM-RSV cells with rodent myoblasts did not disturb myogenin expression or myofibril formation. These results suggest that mutual fusion of myoblasts is indispensable for normal myogenic differentiation irrespective of the species, and that some factors inhibiting myogenic differentiation exist in the cytoplasm of non-myogenic cells, but not in myoblasts.     

INHIBITION OF MYOBLAST FUSION AFTER ONE ROUND OF DNA SYNTHESIS IN 5-BROMODEOXYURIDINE

The thymidine analogue 5-bromodeoxyuridine (BUdR) has a differential effect on the synthesis of tissue-specific products and molecules required for growth and division. Proliferating myogenic cells cultured in BUdR fail to fuse and fail to initiate the synthesis of contractile protein filaments. Conversely, BUdR has but a minor effect on cell viability and reproductive integrity. Low concentrations of BUdR result in an enhancement of cell number relative to the controls; higher concentrations are cytotoxic. Suppression of myogenesis is reversible after at least 10 cell generations of growth in the analogue. Cells that do not synthesize DNA, such as postmitotic myoblasts and myotubes, are not affected by BUdR. Incorporation of BUdR for one round of DNA synthesis was accomplished by first incubating myogenic cells, prior to fusion, in 5-fluorodeoxyuridine (FUdR) to block DNA synthesis and collect cells in the presynthetic phase. The cells were then allowed to synthesize either normal DNA or BU-DNA for one S period by circumventing the FUdR block with BUdR or BUdR plus thymidine (TdR). The cultures were continued in FUdR to prevent dilution of the incorporated analogue by further division. After 3 days, the cultures from the FUdR-BUdR series showed the typical BUdR effect; the cells were excessively flattened and few multinucleated myotubes formed. Cells in the control cultures were of normal morphology, and multinucleated myotubes were present. These results were confirmed in another experiment in which BUdR-³H was added to 2-day cultures in which myotubes were forming. Fusion of thymidine-³H-labeled cells begins at 8 hr after the preceding S phase. In contrast, cells which incorporate BUdR-³H for one S period do not fuse with normal myotubes.     

Mechanism of multinucleate myotomal muscle fibre formation in Hymenochirus boettgeri (Anura, Pipidae)

During somitogenesis in Hymenochirus boettgeri, somites separate from non-segmented mesoderm. Somite formation involves changes in position of myotomal cells from perpendicular to parallel relative to axial organs; the changes are asynchronous and show a dorsoventral gradient. After the rotation has been completed, the myotomal cells (primary myoblasts) occupy the whole length of the myotomes. MyoD is present in nuclei of non-segmented mesoderm cells, of myotomal cells during their rotation and of myoblasts occupying the whole length of the myotomes. The effect of MyoD which activates muscle-specific genes is confirmed by the appearance of skeletal α-actin in mononucleate myoblasts in which myofibrils and the sarcotubular system develop. Differentiation of primary myoblasts results in development of mononucleate, morphologically mature myotubes. Differentiating myotubes are initially not accompanied by any other cells. In further developmental stages, mesenchymal cells appear in intermyotomal fissures and then in myotomes. Their role depends on their position: mesenchymal cells remaining in the intermyotomal fissures differentiate into fibroblasts while those that have migrated into the myotomes, between the myotubes, transform into secondary myoblasts. Their myogenic function is evidenced by the presence of MyoD in their nuclei. These cells fuse with the already existing mononucleate myotubes, resulting in an increase in their size and number of nuclei. Accepted: 30 January 2001     

Intracellular antigen migration in interspecific myoblast heterokaryons

Intracellular migration of species-specific nuclear antigens was studied in chick-rat heterokaryons. These cells were produced by virus-induced or spontaneous fusion of different chick cells with rat myoblasts or myotubes. Chick erythrocyte nuclei introduced into rat myogenic cells increased in volume and were reactivated to synthesize RNA. As the chick erythrocyte nuclei enlarged, they rapidly accumulated rat nuclear antigens. Rat nucleolar and nucleoplasmic antigens assumed a distribution in the chick nuclei corresponding to that in rat nuclei. In hybrid myotubes formed by the spontaneous fusion of chick myoblasts and rat myoblasts antigen exchange was at a much lower level. Some exchange of both rat and chick nuclear antigens could, however, be detected also in this system. Thus chick nuclear envelope and nucleolar antigens migrated into the rat myoblast nuclei and assumed an intranuclear localization analogous to that in chick nuclei. On the basis of these results it appears that antigenic nuclear macromolecules are constantly exchanged between the rat and chick nuclear compartments and the cytoplasm of the heterokaryon. During the rapid nuclear swelling which occurs when chick erythrocyte nuclei are activated in rat myoblast heterokaryons, the inward migration of rat nuclear antigens into the chick erythrocyte nucleus is more impressive than the migration of chick antigens into the rat nuclei.     

Assembly of different isoforms of actin and tropomyosin into the skeletal tropomyosin-enriched microfilaments during differentiation of muscle cells in vitro

We have used a monoclonal antibody (CL2) directed against striated muscle isoforms of tropomyosin to selectively isolate a class of microfilaments (skeletal tropomyosin-enriched microfilaments) from differentiating muscle cells. This class of microfilaments differed from the one (tropomyosin-enriched microfilaments) isolated from the same cells by a monoclonal antibody (LCK16) recognizing all isoforms of muscle and nonmuscle tropomyosin. In myoblasts, the skeletal tropomyosin-enriched microfilaments had a higher content of alpha-actin and phosphorylated isoforms of tropomyosin as compared with the tropomyosin-enriched microfilaments. Moreover, besides muscle isoforms of actin and tropomyosin, significant amounts of nonmuscle isoforms of actin and tropomyosin were found in the skeletal tropomyosin-enriched microfilaments of myoblasts and myotubes. These results suggest that different isoforms of actin and tropomyosin can assemble into the same set of microfilaments, presumably pre-existing microfilaments, to form the skeletal tropomyosin-enriched microfilaments, which will eventually become the thin filaments of myofibrils. Therefore, the skeletal tropomyosin-enriched microfilaments detected here may represent an intermediate class of microfilaments formed during thin filament maturation. Electron microscopic studies of the isolated microfilaments from myoblasts and myotubes showed periodic localization of tropomyosin molecules along the microfilaments. The tropomyosin periodicity in the microfilaments of myoblasts and myotubes was 35 and 37 nm, respectively, whereas the nonmuscle tropomyosin along chicken embryo fibroblast microfilaments had a 34-nm repeat.     

A new technique to identify hybrid myotubes in vitro without culture fixation.

Fluorescent latex microspheres (FLMs) were used to label myoblasts and to permit the observation of hybrid myotubes before culture fixation. This type of labeling did not affect survival, development, or fusion of these cells. The FLMs were retained for several weeks. Labeled mouse myoblasts were co-cultured with unlabeled rat myoblasts to verify whether the marker was released and spread from labeled to unlabeled cells. The nuclear stain Hoechst 33258 was used to distinguish the myoblasts from both species and permitted the demonstration that there was virtually no re-uptake. Hybrid myotubes were also obtained by co-culturing mouse myoblasts containing rhodamine FLMs and rat myoblasts containing green FLMs. These mixed cultures were observed repeatedly with a fluorescent microscope without any cytotoxic effect. Several myotubes were observed before fixation of the cultures to contain both types of fluorescent labels. Subsequent fixation and staining with Hoechst dye confirmed that these myotubes were hybrids.     

Mitosis and intermediate-sized filaments in developing skeletal muscle

A new class of filaments intermediate in diameter between actin and myosin filaments has been demonstrated in skeletal muscle cells cultured from chick embryos. These filaments, which account for the majority of free filaments, average 100 A in diameter. They may run for more than 2 µ in a single section and can be distinguished in size and appearance from the thick and thin filaments assembled into myofibrils. The 100-A filaments are seen scattered throughout the sarcoplasm at all stages of development and show no obvious association with the myofibrils. The 100-A filaments are particularly conspicuous in myotubes fragmented by the mitotic inhibitors, colchicine and Colcemid. In addition, filaments similar in size and appearance to those found in myotubes are present in fibroblasts, chondrocytes, and proliferating mononucleated myoblasts. The 100-A filaments are present in cells arrested in metaphase by mitotic inhibitors. Definitive thick (about 150 A) or thin (about 60 A) myofilaments are not found in skeletal myogenic cells arrested in metaphase. Myogenic cells arrested in metaphase do not bind fluorescein-labeled antibody directed against myosin or actin. For these reasons, it is concluded that not all "thin" filaments in myogenic cells are uniquely associated with myogenesis.     

Redistribution of intermediate filament subunits during skeletal myogenesis and maturation in vitro

The distribution of intermediate filament (IF) subunits during maturation of skeletal myotubes in vitro was examined by immunofluorescence, using antibodies against two different types of chick IF subunits: (a) 58-kdalton subunits of fibroblasts (anti-58K), and (b) 55-kdalton subunits of smooth muscle (anti-55K). Anti-58K bound to a filament network in replicating presumptive myoblasts and fibroblasts, as well as in immature myotubes. The distribution in immature myotubes was in longitudinal filaments throughout the cytoplasm. With maturation, staining of myotubes by anti-58K diminished and eventually disappeared. Anti-55K selectively stained myotubes, and the fluorescence localization underwent a drastic change in distribution with maturation--from dense, longitudinal filaments in immature myotubes to a cross-striated distribution in mature myotubes that was associated with the I--Z region of myofibrils. However, the emergence of a cross-striated anti-55K pattern did not coincide temperally with the emergence of striated myofibrils, but occurred over a period of days thereafter.     

Molecular cloning and expression of cardiac-specific myosin heavy chain gene sequences in chick embryo

A recombinant DNA plasmid, pMHC8, that contains gene sequences for embryonic chick cardiac myosin heavy chain was constructed, identified and characterized. The identity of the clone was established by hybridization with labeled probes that afford screening of MHC²² with high specificity, by inhibition of MHC synthesis in the in vitro hybrid-arrested translation assay, and by tissue-specific hybridization of labeled pMHC8 DNA to MHC messenger RNA.The pMHC8 DNA probe is highly specific for chick heart muscle tissue, since it hybridized poorly to chick skeletal muscle RNA and did not detectably hybridize to adult rat heart RNA. Upon screening the embryonic chick heart cells in culture, no detectable level of MHC mRNA was observed in dividing myoblasts, but the mRNA appeared in differentiated cardiac myocytes paralleling morphogenetic changes in the embryonic cells.     

Incorporation of fluorescently labeled actin and tropomyosin into muscle cells

The two major proteins in the I-bands of skeletal muscle, actin and tropomyosin, were each labeled with fluorescent dyes and microinjected into cultured cardiac myocytes and skeletal muscle myotubes. Actin was incorporated along the entire length of the I-band in both types of muscle cells. In the myotubes, the incorporation was uniform, whereas in cardiac myocytes twice as much actin was incorporated in the Z-bands as in any other area of the I-band. Labeled tropomyosin that had been prepared from skeletal or smooth muscle was incorporated in a doublet in the I-band with an absence of incorporation in the Z-band. Tropomyosin prepared from brain was incorporated in a similar pattern in the I-bands of cardiac myocytes but was not incorporated in myotubes. These results in living muscle cells contrast with the patterns obtained when labeled actin and tropomyosin are added to isolated myofibrils. Labeled tropomyosins do not bind to any region of the isolated myofibrils, and labeled actin binds to A-bands. Thus, only living skeletal and cardiac muscle cells incorporate exogenous actin and tropomyosin in patterns expected from their known myofibrillar localization. These experiments demonstrate that in contrast to the isolated myofibrils, myofibrils in living cells are dynamic structures that are able to exchange actin and tropomyosin molecules for corresponding labeled molecules. The known overlap of actin filaments in cardiac Z-bands but not in skeletal muscle Z-bands accounts for the different patterns of actin incorporation in these cells. The ability of cardiac myocytes and non-muscle cells but not skeletal myotubes to incorporate brain tropomyosin may reflect differences in the relative actin-binding affinities of non-muscle tropomyosin and the respective native tropomyosins. The implications of these results for myofibrillogenesis are presented.     

Regulation of specific developmental fates of larval- and adult-type muscles during metamorphosis of the frog Xenopus

During anuran metamorphosis, larval-type myotubes in both trunk and tail are removed by apoptosis, and only trunk muscles are replaced by newly formed adult-type myotubes. In the present study, we clarified the regulatory mechanisms for specific developmental fates of adult and larval muscles. Two distinct (adult and larval) types of myoblasts were found to exist in the trunk, but no or very few adult myoblasts were found in the tail. Each type of myoblast responded differently to metamorphic trigger, 3,3',5-triiodo-L-thyronine (T(3)) in vitro. T(3)-induced cell death was observed in larval myoblasts but not in adult myoblasts. These results suggest that the fates (life or death) of trunk and tail muscles are determined primarily by the differential distribution of adult myoblasts within the muscles. However, a transplantation study clarified that each larval and adult myoblast was not committed to fuse into particular myotube types, and they could form heterokaryon myotubes in vivo. Cell culture experiments suggested that the following two mechanisms are involved in the specification of myotube fate: (1) Heterokaryon myotubes could escape T(3)-induced death only when the proportion of adult nuclei number was higher than 70% in the myotubes. Apoptosis was not observed in any larval nuclei within the surviving heterokaryon myotubes, suggesting the conversion of larval nuclei fate. (2) Differentiation of adult myoblasts was promoted by the factor(s) released from larval myoblasts in a cell type-specific manner. Taken together, the developmental fate of myotubes is determined by the ratio of nuclei types, and the formation of adult nuclei-rich myotubes was specifically enhanced by larval myoblast factor(s).     

Immunofluorescent Visualization of 100 Å Filaments in Different Cultured Chick Embryo Cell Types

Antibody prepared against the 55,000 dalton subunit of reconstituted chick gizzard 100 A filaments (anti-G55K) bound to the 100 Å filaments of chick smooth muscle, cardiac muscle, and skeletal muscle cells, and to the 100 Å filaments of Schwann cells and satellite glial cells of the peripheral nervous system. Anti-G55K did not bind to replicating presumptive myoblasts, fibroblasts, chondroblasts, pigment cells, neurons, or to central nervous system glial cells. This contrasted with the wider range of binding of antibody to the 58,000 dalton subunit of chick fibroblast 100 A filaments (anti-F58K) which bound to the 100 Å filaments of all cell types examined except hepatocytes and skin epithelial cells. Anti-G55K staining revealed a morphologically distinct distribution of 100 A filaments in the three types of muscle cells. Spindle shaped smooth muscle cells exhibited dense fluorescent staining near the poles of the cells, and also exhibited unique patches of fluorescent material after cytochalasin B and Colcemid treatment. In myotubes, the fluorescence was limited to longitudinal bundles of filaments between the striated myofibrils. Cardiac cells contained uniformly distributed fine filaments. Lastly, smooth muscle cells in various phases of mitosis bound the anti-G55K, whereas replicating presumptive skeletal myoblasts failed to bind the anti-G55K.     

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.     

WOUND HEALING AND COLLAGEN FORMATION : VI. The Origin of the Wound Fibroblast Studied in Parabiosis

Healing skin wounds were studied in a series of parabiotic rats. The femurs of one parabiont of each pair were shielded whilst both animals were given 800 r from a Co⁶⁰ source. The animals were wounded 3 days after irradiation. Each animal with partially shielded marrow was then given tritiated thymidine intraperitoneally daily while the cross-circulation was arrested by clamping. After the thymidine-³H had cleared the blood, the clamp was released. Animals were sacrificed, and wounds were prepared for radioautography 1, 2, and 6 days after wounding. In the wounds of the shielded animals thymidine-³H was observed in epidermis, endothelium, leukocytes, fibroblasts, and mast cells. Only neutrophilic leukocytes, monocytes, and lymphocytes were labeled, as determined by light and electron microscope radioautography, in the wounds of each nonshielded parabiont. None of the many fibroblasts present were found to contain label in the wounds of the nonshielded parabionts through the 6 day period. These observations provide further evidence that wound fibroblasts do not arise from hematogenous precursors and, therefore, must arise from adjacent connective tissue cells.     

Alignment of myoblasts on ultrafine gratings inhibits fusion in vitro

During development, skeletal muscle precursor cells fuse to form multi-nucleated myotubes. However, it is unclear how this fusion is regulated such that linear myotubes are produced. In a previous study, we found that linear arrays of myoblasts cultured on micropatterns of laminin fused to form linear myotubes of a constant diameter, independent of the width of the laminin track. This suggested that a mechanism exists to prevent myoblasts from fusing laterally [Exp. Cell Res. 230 (1997) 275]. In this study, we have investigated this further by culturing myoblasts on ultrafine grooved surfaces previously shown to align fibroblasts and epithelial cells. We found that all the individual myoblasts were highly aligned along the groove axis, and time-lapse recordings showed that motility was mostly restricted to a direction parallel to the grooves. In contrast to the previous study, however, there was a strong tendency for early differentiating cells to form aggregates either at an angle of approximately 45 degrees or perpendicular to the groove axis. Nevertheless, we rarely saw myotubes formed at those angles, supporting our earlier idea that the ability of cells to fuse laterally is prohibited. Our data strongly suggest that myoblasts are most likely to fuse in an end-to-end configuration, and it is this that enables them to form linear, rather than irregular myotubes.