THE ANATOMICAL RECORD 204:199-207 (1982) Secondary Myogenesis of Normal Muscle Produces Abnormal Myotu bes MARCIA ONTELL, DONNA HUGHES, AND DIANNA BOURKE Department of Anatomy and Cell Biology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261 ABSTRACT The extensor digitorum longus muscles of 2-, 4-, and 12-weekold 129-ReJ mice were subjected to homotopic, whole-muscle transplantation. Subsequent to myofiber necrosis and phagocytosis, a new population of myotubes was produced. The three-dimensional cytoarchitecture of these newly formed myotubes was determined in spaced, serial, ultrathin sections. Myotubes, which for long distances along their length appeared to be separate and discrete, were found to branch and recombine, forming a complex syncytium. It has been well established that mammalian striated muscle is capable of regeneration in response to injury or disease. The regeneration may involve repair of existing fibers, or, when the trauma to the muscle is sufficient to cause whole-myofiber necrosis, de novo formation of myotubes may occur. This wave of secondary myogenesis occurs, as does myogenesis during fetal development, by the fusion of mononucleated cells (Carlson, 1973; Lipton and Schultz, 1979).It is generally believed that the cells responsible for this regenerative response are myosatellite cells, mononucleated cells found sandwiched between the basal lamina and the sarcolemma of intact muscle fibers (Mauro, 1961). However, the possibility that other types of cells may also participate in secondary myogenesis has not been entirely excluded (Carlson, 1973; Snow, 1977a). One of the most widely studied models of secondary myogenesis is the whole-muscle transplantation system, first described by Studitsky and Bosova (1960) and Bosova (1962) and modified by Carlson and Gutmann (1974). In this system the entire muscle, cut free of its tendons, nerves, and vascular supply, is placed into a suitably prepared bed. The tendons of the transplanted muscle are attached to rigid structures (bones or other tendons), restoring the muscle to its original resting length. No microsurgical procedures are performed to anastomose the blood vessels or nerves of the grafted muscle to the surrounding vasculature or nerves. With the exception of a few fibers a t the periphery of the implant, all of the myofibers become ischemic and undergo total fiber necrosis (Carlson and Gutmann, 1975;Lischka et al., 1977).Macrophages remove the necrotic 0003-276W82/2043-0199$03.00 1982 ALAN R LISS, INC. fibers (Snow, 1977a1, leaving only their relatively intact basal lamina (Carlson et al., 1979; Hansen-Smith and Carlson, 1979). New myotubes are formed, within the old basal lamina1 tubes, by a secondary wave of myogenesis (Hansen-Smith and Carlson, 1979; Carlson et al., 1979). Ultimately, each new myotube becomes enclosed in its own basal lamina, and the old basal lamina is lost. Despite the large number of morphological (cf. Carlson, 1978), physiological (cf. Faulkner et al., 1980; Hakelius et al., 19751, and histochemical (Maxwell et al., 1978; Hakelius et al., 1975; Carlson and Gutmann, 1975) studies of whole-muscletransplants, little is known about the three-dimensional cytoarchitecture of the regenerating myotubes formed in this system. In the present study, spaced serial ultrathin sections have been used to define the myotubes’ cytoarchitecture. MATERIALS AND METHODS Orthotopic whole muscle transplants (Carlson and Gutmann, 1974) were performed on the extensor digitorum longus muscles of Metafane (Pitmann-Moore)-anesthetized2-, 4-, and 12-week-old 129-ReJ mice (obtained from a normal colony maintained at Jackson Laboratory). The muscle was removed by severing its tendons, being careful not to leave any muscle attached to the tendon stumps. After soaking the muscle at room temperature for 15-20 minutes in 0.75% Marcaine (Breon Laboratories), a known myotoxic agent (Benoit and Belt, 1970;Libelius et al., 1970)which prevents surReceived March 18,1982;accepted July 9,1982 200 M. ONTELL, D. HUGHES, AND D. BOURKE viva1 of the peripheral myofibers (Carlson, 19761, the muscle was replaced into its original muscle bed and sutured to the tendon stumps. Operated mice were allowed to move freely about the cage and were given fresh tetracycline (400 mg/liter) in their water daily for up to 5 days following surgery. Fig. 1. Light micrograph of a transverse section through the widest girth of a control extensor digitorurn longus muscle taken from a 2-week-old mouse. The polygonal-shaped fibers are arranged into discrete fascicles. Toluidine blue. x 90. At various time periods after surgery (1, 3, 5, 7, and 20 days), the mice were killed by cardiac puncture. Extensor digitorurn longus muscles were exposed and bathed, in situ, for 30 minutes in 2.0% gluteraldehyde in 0.125 M cacodylate buffer (pH 7.25). The transplant was excised in toto, placed in fresh fixitive for 2 hours, postfixed in 2.0% osmium tetroxide in cacodylate buffer, dehydrated in ethanol and embedded in Epon 812. Epon blocks, containing entire muscles, were placed on a sliding microtome and oriented for transverse sectioning. Sets were cut consisting of ten 15-pm-thick sections and one 6-pm-thick section. The 6-pmthick sections were mounted on glass slides and studied with a phase-contrast microscope. All 15-pm-thick sections were cleared in Epon between two layers of polystyrenefilm and cured in an oven a t 60°C (Davidowitz et al., 1976). Selected 15-pm-thick sections were adhered to a preformed Epon block, and semithin and ultrathin sections were cut using an ultramicrotome. Semithin sections were stained with toluidine blue. Ultrathin sections were collected on slot copper grids, stained with uranyl acetate and lead citrate (Reynolds, 1963), and observed using a Philips 300 electron microscope. This permitted the study of chosen fascicles of regenerating myotubes a t known intervals along their length. Similar studies were performed on normal muscles taken from 2-, 4-, and 12-week-old mice. Fig. 2. Light micrograph of a transverse section through the widest girth of a 1-day-old,Marcaine-treated, orthotopically transplanted extensor digitomm longus muscle, performed on a 2-week-old mouse. The muscle fibers are rounded and swollen, and appear necrotic. Toluidine blue. x 90. Inset X 180. 201 SECONDARY MYOGENESIS The cytoarchitecture of regenerating fibers was initially examined in early transplants (7 days postoperative) of young (2- and 4-weekold) animals and was subsequently examined in 20-day transplants. In order to determine whether the cytoarchitecture of the regenerated fibers was related to the young age of the animals at the time of transplantation, similar studies were performed on 12-week-old mice. RESULTS The sequence of degeneration and regeneration of transplanted normal mouse muscle was essentially similar to what has been described Fig. 3. Light micrograph of a transverse section through the widest girth of a T-day-old, Marcaine-treated, orthotopically grafted extensor digitorurn longus muscle, performed on a 2-week-old mouse. The regenerating fibers are found throughout the width of the graft. The myotubes in the graft are arranged into discrete fascicles. They are smaller than the myofibers in the control muscle (Fig. 1).Toluidine blue. Fig. 5. Electron micrograph showing a necrotic fiber (N) found in the central core of the 7-day-old transplanted muscle Seen in Figure 4. The necrotic fiber displays marked coagulation necrosis, and both phagocytic cells (arrow) and between the cOaWlurn and myotubes (M) are found the Persistent basal lamina (arrowhead). N, necrotic myofiher. Uranyl acetate and lead citrate. 59200. x 90. Fig. 4. Phase micrograph of a transverse section through the widest girth of a typical 7-day-old orthotopic, whole-extensor digitorurn longus transplant performed on a 12-week-old mouse. A peripheral ring of regenerating myotubes surrounds the central core of necrotic fibers. x 90. 202 M. ONTELL, D. HUGHES, AND D. BOURKE Figs. 615. A typical group of regenerating myotubes found in the 7-day-old graft, seen in Figure 3,is followed in spaced, serial, ultrathin sections. Figure 7 is 30 pm distal to Figure 6 and 15 pm proximal to Figure 8.Figure 9 is 15 pm distal to Figure 8 and 15 pm proximal to Figure 10. Figure 11 is 60 pm distal to Figure 10 and 80 km proximal to Figure 12.Figure 13 is 140 km distal to Figure 12 and 15 pm proximal to Figure 14.Figure 15 is 15 pm distal to Figure 14. Over a distances of 400 pm, three branching points are seen, interconnecting what appear to be, in single three independent myotubes sections (Figs.6,10,11,12,13), (1,2, 3). Close to the branching point, one diameter of the myotube elongates (Figs. 8,15).Subsequently a constriction (arrows)occurs perpendicular to the long diameter (Figs. 7, 9,14).Ultimately, two myotubes are formed, each in its own basal lamina (Figs. 6,10,13). Uranyl acetate and lead citrate. x 1,900. SECONDARY MYOGENESIS in the rat (Carlson et al., 1979); however, in the mouse both processes occurred more rapidly. Within 24 hours after transplantation, rounded, swollen myofibers were seen throughout the muscle. (Compare control muscle in Fig. 1 with 1-day transplant in Fig. 2.) All of the fibers showed necrotic changes along their entire lengths (Fig. 2). In young mice (2-4weeks old), macrophages were found within necrotic fibers located in the center of the graft by 3 days after transplantation, and regenerated myotubes extended into the center of the graft by 5 days postoperative. In older mice, with larger muscles, the core remained necrotic for longer periods, and regeneration in the center of the graft was delayed. At low magnification, in the light microscope, the 7-day grafts (Fig. 3) performed on young mice were virtually indistinguishable from control muscles (Fig. 1) in that they were packed with myofibers arranged into discrete fascicles. However, a t higher magnification, the nuclei of the regenerated fibers, unlike the nuclei in control fibers, were seen to be centrally located. Sevenday grafts performed on older animals contained regenerated myotubes only in their periphery (Fig. 4). Fine structural examination of the center of the grafts, where necrotic fibers persisted, revealed both macrophages and crescent-shaped immature myotubes sandwiched between the basal lamina of the necrotic fiber and the necrotic coagulum (Fig. 5). During the initial phase of this study the cytoarchitecture of the myotubes of 7-day-old grafts performed on young mice was determined. Fascicles composedof mature myotubes (i.e., myotubes packed with myofibrils), surrounded by their own, newly formed, basal laminae, were followed in closely spaced ( s 15 pm), serial, ultrathin sections for distances along their length (Figs. 6-15). Branching and recombination of regenerating myofibers was repeatedly observed (Figs. 7, 9,141 in multiple 203 randomly chosen fascicles across the width of each graft. The branching example chosen for illustration (Figs. 6-15) was taken from a segment of the graft that was approximately 3% of the graft’s total length. It is entirely possible that the branching pattern was even more complex than indicated in the illustrated segment. Approaching the branching region, one diameter of the regenerating fiber would elongate (Figs. 8, 15). Closer to the branch point, a constriction of the myofiber would occur, perpendicular to the fiber’s long diameter (Figs. 7,9,14). The basal lamina followed the contour of the constriction. Gradually, the constricted area became increasingly attenuated, resulting in the fiber’s giving rise to two “daughter” fibers (Figs. 6, 10, 13), each in its own basal lamina. No more than two daughter fibers were seen at each branch point. No accumulations of specialized organelles characterized the region of the branching (Figs. 16, 17). A similar branching and recombination of myofibers was also found in 20-day-old transplants performed on young animals (not shown). The branched regenerating fibers showed no evidence of any degenerative alterations. In order to determine whether the branched regenerating fibers were a function of the immaturity of the muscle at the time of transplantation (2 and 4 weeks postnatal), the cytoarchitecture of the regenerating myotubes found in a 7-day transplant, performed on 12week-old mice, was studied. A similar branching pattern was noted. In order to determine how early in the regenerative process the branching pattern was established, fascicles of necrotic fibers that displayed immature, crescent-shaped, regenerating myotubes wedged between the muscle coagulum and the old basal lamina (Figs. 5, 18)were followed in a similar series of spaced, ultrathin sections. Even at this stage, branch- 204 M. ONTELL, D. HUGHES, AND D. BOURKE DISCUSSION Fig. 16. Higher magnificationofbranchingregion of myotube 1 + 2, seen in Figure 7.A constriction (arrow) occurs in a plane perpendicular to the elongated diameter of the myotube. No specialized organelles are seen at the branching point. Uranyl acetate and lead citrate. x 6,900. Fig. 17. Higher magnificationof branching region of myotube 3 + 2, seen in Figure 14. A constriction (arrow) occurs in a plane perpendicular to the elongated diameter of the myotube. No specialized organelles are seen at the branching point. Uranyl acetate and lead citrate. x 6,900. ing and recombination were observed between the myotubes that shared a single, old, basal lamina1 tube (Figs. 19, 20). No branching myofibers were found in any of the control muscles. Original reports of successful regeneration of muscle fibers following whole-muscle transplantation (Studitsky and Bosova, 1960; Bosova, 1962)have stimulated a renewed interest in skeletal muscle regeneration (for review see Carlson, 1978). Despite the widespread recognition that it is possible to replace necrotic myofibers by new myotubes, produced by a second wave of myogenesis, there are few electron microscopic studies of regenerating myotubes in whole-muscle transplants (Carlson et al., 1979; Hansen-Smith and Carlson, 1979; Schmalbruch, 1977). While single, ultrathin section studies have indicated that regenerating fibers appear to be identical to normal developing muscle, in the present report the application of a spaced, serial, ultrathin sectioning technique has clearly demonstrated that a substantial percentage of the myotubes formed as a result of secondary myogenesis undergo complex branching and recombination, resulting in cytoplasmic continuity of myotubes that for extensive regions along their length appear to be independent myotubes. Comparison of the cytoarchitecture of these branched myotubes with similar studies of spaced, serial, ultrathin sections of normal developing myotubes reveals that during no stage of fetal (Kozeka and Ontell, unpublished results) or neonatal development (Ontell, 1977) are normal muscle fibers branched, and no evidence of myofiber branching has been found in the control muscles used in this study. In the present study, the whole-muscle transplants performed in younger mice have regenerated at a much more rapid rate than in older mice. It has not been determined whether this difference is the result of the size of the transplanted muscle and/or of an agerelated ability of the transplanted muscle to undergo regeneration. Clearly, the smaller the Fig. 18. Regenerating myotube (M)found closely apposed to a necrotic myofiber (N). The myofilamenta are clearly visible (arrow). Uranyl acetate and lead citrate. X 28,350. Figs. 19,20. Spaced, serial, ultrathin sectionsofa typical group of necrotic myofibers found in the central region of a 7-day-old, orthotopically transplanted extensor digitorum longus muscle, performed on a 12-week-old mouse. Figure 18 is 15 pm proximal to Figure 19. A single crescentshaped myotube (A) is found sandwiched between the basal lamina and the necrotic coagulum of fiber 1(Fig. 18).Fifteen microns distal to Figure 18,myotube A has branched giving rise to two daughter myotubes (B,C) (Fig. 19). Uranyl acetate and lead citrate. X 1,900. - SECONDARY MYOGENESIS 205 graft, the more rapid its revascularization, and it has been demonstrated that revascularization is necessary for secondary myogenesis (Hansen-Smith et al., 1980). However, if satellite cells are the source of new myofibersfound in transplanted muscle, then the younger muscle may have an advantage, because it has been repeatedly demonstrated that the frequency of myosatellite cells in normal muscle decreases with age (Allbrook et al., 1971; Schultz, 1974; Ontell, 1974;Snow, 1977b).Whatever the cause of the differences in rates of regeneration between the younger and older murine muscles used in this study, the branching phenomenon occurs in both groups. Therefore, branching is unrelated to the age of the muscle a t the time of transplantation. The ultimate fate of the branched striated myotubes has not yet been determined. The branched myotubes found in the longest-term studies reported in the present paper (20 days postoperative) show no evidence of degenerative changes. It should be noted that motor end plates are regularly encounted by 14 days postoperative in these transplants (Ontell, unpublished results). The cytoarchitecture and pattern of motor innervation of the regenerating fibers in longer-term transplants (100 days) are currently under investigation. The use of spaced, serial, ultrathin sections to define the cytoarchitecture of myotubes formed by a secondary wave of myogenesis is particularly beneficial, because multiple myotubes are formed in a single, old, basal laminal tube. It is not possible at the light microscopic level of resolution to determine whether the newly formed myotubes exhibit continuity with each other or whether they are merely closely apposed (Schmalbruch, 1976). Whereas branching may be suggested in single ultrathin longitudinal sections, a section through a myotube with an irregular contour may suggest branching when, in fact, the myotube is only indented (cf. Ontell and Feng, 1981). The present study is the first application of a serial ultrathin sectioning technique to the problem of defining the cytoarchitecture of normal regenerating myotubes. Jirmanova and Thesleff (1972), studying regeneration after methyl bupivacaine treatment, and Schmalbruch (1976), studying regeneration subsequent to injection with hot Ringer’s solution, have reported what appears in single, longitudinal, ultrathin sections to be fusion between adjacent myotubes. In a previous study, using spaced, serial, ultrathin sections, it has been demonstrated that 206 M. ONTELL, D. HUGHES. AND D. BOURKE regenerating myofibers that form spontaneously (i.e., without secondary trauma) in murine dystrophic muscle display a similar pattern of branching and recombination (Ontell and Feng, 1981).At that time, it could not be determined whether branching was unique to dystrophy or whether it was a characteristic of any or all regenerating myofibers. The present study has established that the branched regenerating fibers found in murine dystrophic muscle do not reflect an alteration in the regenerative pattern produced by the disease, since healthy muscle (obtained from mice derived from a normal colony) can produce regenerating myotubes with the same type of branched cytoarchitecture when subjected to whole-muscle transplantation. Interestingly, cultures of normal muscle produce myotubes with a branched pattern (Murray, 1960). Since it is assumed that the myotubes formed during secondary myogenesis are formed, as are the myotubes during primary myogenesis (fetal development), by the fusion of mononucleated cells into a multinucleated syncytium (Snow, 1977a), i t is interesting to speculate about those factors that may be responsible for the cytoarchitectural differences between the two types of myotubes. One factor may be the age of the myoblast at the time of myotube formation. For example, slight alterations in the phenotypic expression of fusion-related factors ke., membrane-associated variations) may occur with age. Thus, the satellite cell may be released from, or subjected to, different fusion limitations from those the embryonic myoblast is. Second, there are different environmental factors brought to bear on secondary myogenesis as compared to primary myogenesis. The necrotic environment alone may be responsible for alterations in myotube structure. One of the most marked differences between secondary myogenesis and fetal development is the presence of an intact basal lamina in regenerating muscle, which is used as a scaffold within which regenerating myotubes are formed (Vracko and Benditt, 1972; Burden et al., 1979). It may be that mechanical forces, exerted by the basal lamina and the necrotizing fiber a t a critical stage of myogenesis, foster the production of abnormal myotubes. In the present study, it has been demonstrated that branching is already present at a time when the immature, crescent-shaped myotubes are wedged between the muscle coagulum and the old basal lamina. An alternate explanation for the branching phenomenon may be related to the basal lamina’s segregation and partial isola- tion of the satellite cells, which were originally associated with a single myofiber. Although it has not been documented whether additional satellite cells or other types of cells migrate into the necrotic fibers and participate, along with the “resident” satellite cells, in de novo myotube formation, it is likely that the “resident” satellite cells play the major role as the source of myogenic cells during regeneration. Could it be that some sort of “self‘ recognition could allow these “resident” satellite cells to fuse in a less regulated manner with all of the myogenic cells in the old basal lamina? The branching pattern observed in the regenerating myotubes in whole-muscle transplants, the branching myotubes observed in the regenerating fibers formed spontaneously in dystrophic muscle (Ontell and Feng, 19811, and the suggestion that branching may also be observed between myotubes formed subsequent t~ injections of myotoxic substance (Jirmanova and Thesleff, 1972;Schmalbruch, 1976) appear to challenge the paradigm that secondary myogenesis produces myotubes that are indistinguishable from the myotubes produced by primary myogenesis. ACKNOWLEDGMENTS Supported by NIH grant NS 13688 and a grant from the Muscular Dystrophy Association of America. The competent technical assistance of Ms. F. Shagas, Ms. G. Diluiso, and Ms. J. Lieberman is gratefully acknowledged. LITERATURE CITED Allbrook, D.B., M.F.Han, and A.E. Hellmuth (1971) Population of muscle satellite cells in relation to age and mitotic activity. Pathology, 3:233-243. Benoit, P.W., and W.D. Belt (1970) Destruction and regeneration of skeletal muscle after treatment with a local anesthetic, bupivacaine (Marcaine). J. Anat., 107.547-556. Bosova, N. (1962) Free autoplastic transplantation of whole muscles. Byull. Exp. Biol. Med., 53~88-92. Burden, S.J., P.B. Sargent, and U.J. McMahan (1979) Acetylcholine receptors in regenerating muscle accumulate at original synaptic sites in the absence of nerve. J. Cell Biol., 82:412425. Carlson, B.M. (1973) The regeneration of skeletal muscle: A review. Am. J. Anat., 37:119-150. Carlson, B.M. (1976) A quantitative study of muscle fiber survival and regeneration in normal, predenervated and Marcaine-treated free muscle grafts in the rat. Exp. Neurol., 52:421-432. Carlson, B.M. (1978) A review of muscle transplantation in mammals. Physiol. Boshemoslov.,271:378-400. Carlson, B.M., and E. Gutmann (1974) Transplantationand “cross transplantation” of free muscle grafls in the rat Experientia,30~1292-1294. Carlson, B.M., and E. Gutmann (1975) Regenerationin free grafls of normal and denervated muscles in the r a t Morphology and histochemistry. Anat. Rec., 183~47-62. SECONDARY MYOGENESIS Carlmn, B.M., F. Hansen-Smith, and D. Magnon (1979) The life history of a free muscle graft. In: Muscle Regeneration. A. Mauro, ed. Raven Press, New York, pp. 493408. Davidowitz, J., B.Pachter, and G. Breinin (1976) Clearing steel knife Epon sections in a polystyrene film sandwich. Stain Technol., 51:139-141. Faulkner, J., L. Maxwell, T. White, and J. Nierneyer (1980) Characteristics of autografted mammalian skeletal muscles. In: Muscle Regeneration. A. Mauro, ed. Raven Press, New York, pp. 485600. Hakelius, L.,B. Nystrom, and E. Stalberg (1975) Histochemical and neurophysiological studies of autotransplanted cat muscle. Scand. J. Plast. Reconstr. Surg., 9:15-24. Hansen-Smith, F., and B.M. Carlson (1979) Cellular responses to free grafting of the extensor digitorum longus muscle of the rat. J. Neurol. Sci., 41:149-173. Hansen-Smith, F., B.M. Carlson, and K. Irwin (1980) Revascularization of the freely grafted extensor digitorum longus muscle in the rat. Am. J. Anat., 158:65-82. Jirmanovh, I., and S. T h e s l d (1972) Ultrastlvctural study of experimental muscle degeneration and regeneration in the adult rat. Z. Zellforsch., 131:77-97. Libelius, R., B. Sonesson, B.A. Stamenovic, and S. Thesleff (1970) Denervation-like changes in skeletal muscle after treatment with a local anesthetic (Marcaine). J. Anat., 106:297-309. Lipton, B., and E. Schultz (1979) Developmentalfate of skeletal muscle satellite cells. Science, 205:1292-1294. Lischka, A,, J. Holle, and G. Freilinger (1977) Lichtmikroskopische und elektronenoptische untersuchungen morphologischer veranderungen von muskelfasern bei freier autologer muskeltransplantation. Acta Anat., 97:450458. Mauro, A (1961) Satellite cells of skeletal muscle fibers. J. Biophys. Biochem. Cytol., 9:493-495. Maxwell, L., J. Faulkner, S. Mufti, and A. Turowski (1978) Free autografting of entire limb muscles in the c a t His- 207 tochemistry and biochemistry. J. Appl. Physiol. Respir. Environ. Exercise Physiol., 44:431437. Murray, M.R. (1960) Skeletal muscle tissue in culture. In: Structure and Function of Muscle. G.H. Bourne, ed. Academic Press, New York, pp. 111-136. Ontell, M. (1974) Muscle satellite cells: A validated technique for light microscopic identification and a quantitative study of changes in their population following denervation. Anat. Rec., I78:211-228. Ontell, M. (1977) Neonatal muscle. An electron microscopic study. Anat. Rec., 189:669-690. Ontell, M., and K.C. Feng (1981) The three dimensional cytoarchitecture and pattern of motor innervation of branched striated myotubes. Anat. Rec., 200:11-31. Reynolds, E.S.(1963) The use of lead citrate at high pH as an electron opaque stain in electron microscopy. J. Cell Biol., 17:208-212. Schmalbruch,H. (1976) The morphology of regeneration of skeletal muscles in the rat. Tissue Cell, 8:673492. Schmalbruch,H. (1977) Regeneration of the soleus muscles of rat autografted in toto as studied by electron microscopy. Cell Tissue Res., 177:159-180. Schultz, E. (1974) A quantitative study of the satellite cell population in postnatal mouse lumbrical muscle. Anat. Rec.,180:589-596. Snow,M.H. (1977a)Myogenic cell formation in regenerating rat skeletal muscle injured by mincing. 1. A fine structural study. Anat. Rec., 188:181-200. Snow, M.H. (1977b) The effects of aging on satellite cells in skeletal muscles of mice and rats. Cell Tissue Res., 185:339408. Studitsky, A., and N. Bosova (1960) Development of atrophic muscular tissue in conditions of transplantation in place of mechanically damaged muscle. Arch. Anat. Gist. Embriol., 39:18-32. Vracko, R., and E.P. Benditt (1972) Basal lamina. The scaffold for orderly cell replacement. J. Cell Biol., 55:406-419.