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The fate of cells and nuclei in chick embryo muscle grown in tissue culture
149
Journal of the neurological Sciences
Elsevier Publishing Company, Amsterdam - Printed in the Netherlands
The Fate of Cells and Nuclei in Chick Embryo Muscle Grown in Tissue Culture R. PARSONS Muscular Dystrophy Group Research Laboratories, Newcastle General Hospital, Newcastle upon Tyne (Great Britain)
(Received 12 June, 1970)
INTRODUCTION
Chick embryo muscle has been used as a source of rapidly multiplying myogenic cells for some years (HOLTZER et al. 1958 ; MOSCONA 1961 ; OKAZAKI AND HOLTZER 1966 ; SHIMADA et al. 1969; LARSON et al. 1970; KEMP 1970), without any knowledge being available to indicate the exact origin of such cells. OKAZAKI AND HOLTZER (1965) pointed out that trypsinization of 11-day-old chick embryo breast muscle would give a population of mononucleate cells, most of which probably originated from the mononucleate cells seen in the uncultured muscle. These are the "stem cells" of muscle and connective tissue which, in vivo, produce the large number of cells needed for the development of adult muscle. The other problem posed, and left unanswered, by OKAZAKI AND HOLTZER (1965) is the fate of the multinucleate myotubes, which have developed in vivo, when the muscle is trypsinized. It is hoped this short communication will throw some light on these two problems.
MATERIALSAND METHODS Muscle tissue from the proximal muscles of the hind limbs of 10-12-day-old chick embryos was examined firstly by conventional histological methods, and the results were noted. Explants, usually of 12-day embryo muscle, were grown in a thin film of cock plasma on a coverslip mounted on a Pulvertaft ring (LARSON et al. 1970). The culture medium consisted of Morton, Morgan and Parker's "199" or Eagles M E M with 10% chick embryo extract and 10% horse serum. Successive stages of growth of the explants were followed with a Reichert differential interference microscope and time-lapse cinemicrography. Most films were taken at a speed of 2 frames per min with a 1 sec exposure using Kodachrome II A film. The This work was supported by grants from the Muscular Dystrophy Group of Great Britain, the Medica! Research Council and the Muscular Dystrophy Associations of America, Inc. The author is a recipi: .~ of a Scholarship for Training in Research Methods from the Medical Research Council. J. n~L.rol. Sci., 1971, 13:149-159
150
R. PARSONS
relatively new differential interference system has appreciable advantages over phase contrast or ordinary interference systems in examining living cells lying in a rather thick preparation such as an explant of muscle. ALLEN et al. (1966) showed on theoretical grounds that with this interference system, the images of sharply focused material could be expected to be much less affected by material lying above or below the plane of focus. This is borne out in practice: the morphology and movements of cells in an explant can be discerned in locations where with any other system the image would be unintelligible, because of optical artifacts. It is believed that this is the first study in which this optical system has been used to follow the successive events which occur in myogenesis. Some cultures were grown on coverslips which had been coated with celloidin to facilitate preparation for electron microscopy (JENKISOY 1965). Sections of these were cut with a Reichert OMU2 ultratome and examined with a Zeiss EM 9A electron microscope. Another group of cultures were grown on coverslips previously coated with a carbon film to enable preparation for histology (PIPER 1962). Photographs of all histological preparations were taken with a Zeiss photomicroscope. RESULTS
The histological survey of uncultured limb muscle gave results which closely paralleled
Fig. 1. A longitudinal section of uncultured 10-day chick embryo muscle stained with haematoxylin and eosin. Myotubes (M) are surrounded by many other nuclei (N), × 320. J. neurol. Sci., 1971, 13:149-159
CELLS AND NUCLEI IN CHICK EMBRYO MUSCLE GROWN IN TISSUE CULTURE
151
the findings of OKAZAKIAND HOLTZER(1965). Ten-day-old chick embryo limb muscle (Figs. 1 and 2) consists of large myotubes with central nuclei surrounded by a very large number of nuclei with indistinct cellular boundaries. Limb muscles of 11- and 12-day-old embryos differ only slightly from those of 10-day embryos with respect to the thickness of the periphery of the myotubes. Electron-microscopic pictures (Fig. 3) show quite clearly that the myotubes are encircled by a large number of mononucleate cells, a few of which contain myosin and actin filaments. After 22 h in culture media the muscle explant has quite a different structure (Figs. 4 and 5). Myotubes can still be seen, but are reduced in diameter and contain very few central nuclei. Between them can be found the mononucleate cells. In some cases multinucleate cells have been seen near the edge of the explant (Fig. 6). They appear to have been formed since the muscle had been cultured. By continual addition of single cells, these structures could form the multinucleate myotubes visible outside the explant in living culture preparations after 36 to 72 h. Alternatively, the whole structure
Fig. 2. A transverse section of uncultured 10-day chick embryo muscle stained with haematoxylin and eosin. Pale central nuclei (CN) seen in the myotubes (M), × 800.
J. neurol. ScL, 1971,13:149-159
152
R. PARSONS
could move out of the explant and become visible even before the mononucleate ceils appeared (Fig. 7). Figs. 8 and 9 give an indication of the explant structure after 48 h in culture. The " o l d " myotubes, i.e. the myotubes formed in the embryo muscle before being put into culture, are bunched together and surrounded by large numbers of single cells which appear to be making their way out of the explant. At greater magnification (Fig. 10) the degenerating myotubes are seen to contain numerous inclusions, but never any nuclei. Surrounding the myotubes are mononucleate cells.
Fig. 3. An electron micrograph of uncultured 10-day chick embryo muscle. Section fixed with glutaraldehyde, and osmium tetroxide; stained with uranyl acetate. Small mononucleate cells(MC)surrounding large myotube (M), myofibrils (mf), mitochondria (m), × 4950. J. neurol. Sci.,1971, 13:149-159
CELLS AND NUCLEI IN CHICK EMBRYO MUSCLE GROWN IN TISSUE CULTURE
153
Sixteen cine films have been made to record the explant changes continuously over a period of time. The areas filmed were selected as showing most clearly the same kind of organization as that observed in the histological survey already described. They were typical of the explant structure as seen in over 180 cultures. Filming was started within 1 h of the cultures being prepared, and continued for between 20 and 30 h. Preliminary films showed quite clearly that the optical system of the differential interference microscope had quite distinct advantages over any other system. The very shallow depth of focus and the virtual disappearance of anything which is out of focus allows critical examination of the explants. However, this shallow depth of focus is a disadvantage when explants of muscle are continuously changing their organization. Some structures or activities are often removed from the preset plane of focus and as a result are lost to view. Despite this difficulty the films have proved that the mononucleate cells move out of the explant to form the typical fringe of emerging cells. The " o l d " myotubes exhibit neither internal structural changes, nor cytoplasmic or nuclear movements. Such movements are common occurrences in normal single cells and multinucleate myotubes. " O l d " myotube nuclei are lightly stained in sections of
Fig. 4. A transverse section of 10-day chick embryo muscle fixed after 22 h in culture. Haematoxylin and eosin. Shrunken myotubes (M) without central nuclei. Mononucleate cells (MC), × 720. £neuroI. Sei., 1971, 13:149-159
i
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2 2 h in culture. H a e m a t ~ i n and eosin. Shrunken myotubes (M), periphery of exp!ant (P), × 320.
Fig. 6. An enlargement of part of Fig. 5 to show multinucleatc cells (MUC) at the periphery of explant, ~,, 800.
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Fig. 7. A multinucleate cell growing from 12-day chick embryo muscle explant. Enlargements from two 16 m m cine frames using a differential interference microscope. A: at 24 h after setting up in culture; B: 2 h after, A, × 390.
Fig. 8. A section of an explant of 12-day chick embryo muscle after 48 h in culture. Haematoxylin and eosin. Remnants of pre-existing myotubes (RM) uninucleate cells (UC), × 320.
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Fig. 9. An enlargement of part of Fig. 8. Myotube remnants (RM), ,i 720.
uncultured muscle (Fig. 2), and visible only as thin-edged circles in culture. During filming the nuclei of the "old" myotubes disappear. A series of 16 mm frame enlargements (Fig. 11) outline the events taking place in a 28 h sequence. DISCUSSION It was presumed by HOLTZERet al. (1958) that trypsinization of 6-9-day-old myotubes produced mononucleate cells by fragmentation of the myotubes. Later OKAZAKI AND HOLTZER 0965) assumed that trypsinization yielded only the mononucleate cells which surrounded the myotubes, but they could form no clear conclusions. It is possible that trypsin may have a different effect on chick embryo muscle when compared with the results of culturing untrypsinized muscle as explants. However, the population of cells obtained by both methods seems to be the same. Electron microscopy of cultured muscle reveals that the great majority of mononucleate cells, both in the explant and outside it, are "presumptive myoblasts" devoid of actin and myosin, and presumptive connective tissue cells. Experiments involving fluorescin-labelled antimyosin by OKAZAKI AND HOLTZER (1966) showed that myosin was absent from the majority of the mononucleate cells obtained by trypsinization of ll-day chick embryo muscle. If this is sufficient evidence to conclude that trypsinization and culturJ. neurol. Sci.,
1971, 13:149-159
CELLS AND NUCLEI IN CHI{?KEMBRYOMUSCLEGROWN IN TISSUE CULTURE
157
ing have the same effect on chick embryo muscle, then it can be assumed that the way in which cells are produced by culturing would be similar to the way in which cells would be produced by trypsinization. It has been stated that " o l d " myotube nuclei disappear in culture. They could become the nuclei of new cells (BETz 1951; ALLBROOK 1962; REZNIK 1969). I f this were true then cinemicrography would almost certainly have shown migration of these nuclei in the " o l d " myotubes, and a marked activity in their cytoplasm during the necessary reorganization. This was not found. Cytoplasmic activity in these myotubes, typically indicated by the movement of intracellular particles, was nil. Even if the mononucleate cells were formed passively, migration of such cells toward the periphery of the explant would have been seen, but it was not. Therefore, it seems
Fig. 10. An electron micrograph of 12-day chick embryo muscle fixed after 48 h in culture. Fixative: 5 ~ glutaraldehyde and Palade's fixative; staining: uranyl acetate. Degenerating myotube (DM), myotube inclusions (mi), degenerating myofilaments (df), mononucleate cells (UC), × 14,400. J. neurol. Sei., 197I, 13:149-159
Fig. 11. A series of 16 mm framed enlargements from a cine film taken with a differential interference microscope, of a living culture of 12-day chick embryo muscle at several time intervals after culture preparation. A: 1 h 45 rain, B: 5 h 36 rain, C: 10 h 18 rain, D: 15 h42 rain, E: 21 h, F: 28 h 18 rain. Note that the myotube (M) throughout the sequence remains unchanged. Particles (PI equivalent to myotube inclusions in Fig. 10, do not move, ~<680.
J. neuro[. Sci., 1971, 13:149-159
CELLS AND NUCLEI IN CHII~K EMBRYO MUSCLE GROWN IN TISSUE CULTURE
159
most likely that the "old" myotube nuclei simply degenerate: and that the cells obtained by trypsinizing chick embryo muscle do not include any myotube nuclei, or indeed any part of the old myotubes. ACKNOWLEDGEMENTS
i wish to thank Dr. K. F. A. Ross for his guidance and for his advice in the preparation of the manuscript. I would also like to thank Miss A. Brown, Mr. C. Wallace and Mr. G. Henderson for their technical assistance; and Miss E. Treece-Birch for typing the manuscript. SUMMARY
Chick embryo muscle was studied in tissue culture by cinemicrography, electron microscopy and in fixed and stained preparations. The uncultured muscle consisted of large numbers of mononucleate cells surrounding multinucleate myotubes. When cultured, the mononucleate cells moved out of the explant and the myotubes degenerated. These observations, and the importance of differential interference microscopy in this type of study are discussed. REFERENCES
ALLEN, R. D., G. B. DAVID AND L. F. Hmsit (1966) The sources of contrast in different types of transmitted light polarizing microscopes. Theoretical considerations about the image-duplicating and differential instruments, Proc. roy. microsc. Soc., l : 141. ALLBROOK, D. (1962) An electronmicroscopic study of regenerating skeletal muscle, J. Anat. (Lond.), 96: 137. BETZ, E. H. (1951) Contribution a 1'6rude de la d6g6n6rescence et de la r6gdn6ration musculaire, Arch. Anat. Micr. morph, exp., 40: 115. HOLTZER, H., J. AnBOT AND J. LASH (1958) On the formation of multinucleated myotubes, Anat. Rec., 131 : 567. JENKISON, M. (1965) Unpublished observations. KFMP, R. B. (1970) The effect of neuraminidase (3:2:1 :l 8) on the aggregation of cells dissociated from embryonic chick muscle tissue, J. cell. Sci., 6: 751. LARSON, P. F., M. JENKISON AND P. HUDGSON 0970) The morphological development of chick embryo skeletal muscle grown in tissue culture as studied by electron microscopy, J. neurol. Sci., 10: 385. MOSCONA, A. A. (1961) Rotation mediated histogenic aggregation of dissociated cells, Exp. Cell Res., 22 : 455. OKAZAKI, K. AND H. HOLTZER (1965) An analysis of myogenesis in vitro using fluorescin-labelled antimyosin, J. Histochem. Cytochem., 13: 726. OKAZAKI,K. AND H. HOLTZER (1966) Myogenesis: myosin synthesis, and the mitotic cycle, Proc. nat. Acad. Sci. (Wash.), 56: 1484. PIPER, R. (1962) Microtomy techniques and the problem of relating the cell outgrowth to events within the explants, J. reed. Lab. TechnoL, 19: 1. REZN1K, M. (1969) Origin of myoblasts during skeletal muscle regeneration, Lab. Int:est., 20: 353. SHIMADA, Y., D. A. FISCHMANAND A. A. MOSCONA(1969) The development of nerve-muscle junctions in monolayer cultures of embryonic spinal cord and skeletal muscle cells, J. Cell. Biol., 44: 436.
J. neurol. Sci., 1971, 13:149-159
Journal of the neurological Sciences
Elsevier Publishing Company, Amsterdam - Printed in the Netherlands
The Fate of Cells and Nuclei in Chick Embryo Muscle Grown in Tissue Culture R. PARSONS Muscular Dystrophy Group Research Laboratories, Newcastle General Hospital, Newcastle upon Tyne (Great Britain)
(Received 12 June, 1970)
INTRODUCTION
Chick embryo muscle has been used as a source of rapidly multiplying myogenic cells for some years (HOLTZER et al. 1958 ; MOSCONA 1961 ; OKAZAKI AND HOLTZER 1966 ; SHIMADA et al. 1969; LARSON et al. 1970; KEMP 1970), without any knowledge being available to indicate the exact origin of such cells. OKAZAKI AND HOLTZER (1965) pointed out that trypsinization of 11-day-old chick embryo breast muscle would give a population of mononucleate cells, most of which probably originated from the mononucleate cells seen in the uncultured muscle. These are the "stem cells" of muscle and connective tissue which, in vivo, produce the large number of cells needed for the development of adult muscle. The other problem posed, and left unanswered, by OKAZAKI AND HOLTZER (1965) is the fate of the multinucleate myotubes, which have developed in vivo, when the muscle is trypsinized. It is hoped this short communication will throw some light on these two problems.
MATERIALSAND METHODS Muscle tissue from the proximal muscles of the hind limbs of 10-12-day-old chick embryos was examined firstly by conventional histological methods, and the results were noted. Explants, usually of 12-day embryo muscle, were grown in a thin film of cock plasma on a coverslip mounted on a Pulvertaft ring (LARSON et al. 1970). The culture medium consisted of Morton, Morgan and Parker's "199" or Eagles M E M with 10% chick embryo extract and 10% horse serum. Successive stages of growth of the explants were followed with a Reichert differential interference microscope and time-lapse cinemicrography. Most films were taken at a speed of 2 frames per min with a 1 sec exposure using Kodachrome II A film. The This work was supported by grants from the Muscular Dystrophy Group of Great Britain, the Medica! Research Council and the Muscular Dystrophy Associations of America, Inc. The author is a recipi: .~ of a Scholarship for Training in Research Methods from the Medical Research Council. J. n~L.rol. Sci., 1971, 13:149-159
150
R. PARSONS
relatively new differential interference system has appreciable advantages over phase contrast or ordinary interference systems in examining living cells lying in a rather thick preparation such as an explant of muscle. ALLEN et al. (1966) showed on theoretical grounds that with this interference system, the images of sharply focused material could be expected to be much less affected by material lying above or below the plane of focus. This is borne out in practice: the morphology and movements of cells in an explant can be discerned in locations where with any other system the image would be unintelligible, because of optical artifacts. It is believed that this is the first study in which this optical system has been used to follow the successive events which occur in myogenesis. Some cultures were grown on coverslips which had been coated with celloidin to facilitate preparation for electron microscopy (JENKISOY 1965). Sections of these were cut with a Reichert OMU2 ultratome and examined with a Zeiss EM 9A electron microscope. Another group of cultures were grown on coverslips previously coated with a carbon film to enable preparation for histology (PIPER 1962). Photographs of all histological preparations were taken with a Zeiss photomicroscope. RESULTS
The histological survey of uncultured limb muscle gave results which closely paralleled
Fig. 1. A longitudinal section of uncultured 10-day chick embryo muscle stained with haematoxylin and eosin. Myotubes (M) are surrounded by many other nuclei (N), × 320. J. neurol. Sci., 1971, 13:149-159
CELLS AND NUCLEI IN CHICK EMBRYO MUSCLE GROWN IN TISSUE CULTURE
151
the findings of OKAZAKIAND HOLTZER(1965). Ten-day-old chick embryo limb muscle (Figs. 1 and 2) consists of large myotubes with central nuclei surrounded by a very large number of nuclei with indistinct cellular boundaries. Limb muscles of 11- and 12-day-old embryos differ only slightly from those of 10-day embryos with respect to the thickness of the periphery of the myotubes. Electron-microscopic pictures (Fig. 3) show quite clearly that the myotubes are encircled by a large number of mononucleate cells, a few of which contain myosin and actin filaments. After 22 h in culture media the muscle explant has quite a different structure (Figs. 4 and 5). Myotubes can still be seen, but are reduced in diameter and contain very few central nuclei. Between them can be found the mononucleate cells. In some cases multinucleate cells have been seen near the edge of the explant (Fig. 6). They appear to have been formed since the muscle had been cultured. By continual addition of single cells, these structures could form the multinucleate myotubes visible outside the explant in living culture preparations after 36 to 72 h. Alternatively, the whole structure
Fig. 2. A transverse section of uncultured 10-day chick embryo muscle stained with haematoxylin and eosin. Pale central nuclei (CN) seen in the myotubes (M), × 800.
J. neurol. ScL, 1971,13:149-159
152
R. PARSONS
could move out of the explant and become visible even before the mononucleate ceils appeared (Fig. 7). Figs. 8 and 9 give an indication of the explant structure after 48 h in culture. The " o l d " myotubes, i.e. the myotubes formed in the embryo muscle before being put into culture, are bunched together and surrounded by large numbers of single cells which appear to be making their way out of the explant. At greater magnification (Fig. 10) the degenerating myotubes are seen to contain numerous inclusions, but never any nuclei. Surrounding the myotubes are mononucleate cells.
Fig. 3. An electron micrograph of uncultured 10-day chick embryo muscle. Section fixed with glutaraldehyde, and osmium tetroxide; stained with uranyl acetate. Small mononucleate cells(MC)surrounding large myotube (M), myofibrils (mf), mitochondria (m), × 4950. J. neurol. Sci.,1971, 13:149-159
CELLS AND NUCLEI IN CHICK EMBRYO MUSCLE GROWN IN TISSUE CULTURE
153
Sixteen cine films have been made to record the explant changes continuously over a period of time. The areas filmed were selected as showing most clearly the same kind of organization as that observed in the histological survey already described. They were typical of the explant structure as seen in over 180 cultures. Filming was started within 1 h of the cultures being prepared, and continued for between 20 and 30 h. Preliminary films showed quite clearly that the optical system of the differential interference microscope had quite distinct advantages over any other system. The very shallow depth of focus and the virtual disappearance of anything which is out of focus allows critical examination of the explants. However, this shallow depth of focus is a disadvantage when explants of muscle are continuously changing their organization. Some structures or activities are often removed from the preset plane of focus and as a result are lost to view. Despite this difficulty the films have proved that the mononucleate cells move out of the explant to form the typical fringe of emerging cells. The " o l d " myotubes exhibit neither internal structural changes, nor cytoplasmic or nuclear movements. Such movements are common occurrences in normal single cells and multinucleate myotubes. " O l d " myotube nuclei are lightly stained in sections of
Fig. 4. A transverse section of 10-day chick embryo muscle fixed after 22 h in culture. Haematoxylin and eosin. Shrunken myotubes (M) without central nuclei. Mononucleate cells (MC), × 720. £neuroI. Sei., 1971, 13:149-159
i
7~
..
"-4
2 2 h in culture. H a e m a t ~ i n and eosin. Shrunken myotubes (M), periphery of exp!ant (P), × 320.
Fig. 6. An enlargement of part of Fig. 5 to show multinucleatc cells (MUC) at the periphery of explant, ~,, 800.
7~
4~
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t
°°
7~
Fig. 7. A multinucleate cell growing from 12-day chick embryo muscle explant. Enlargements from two 16 m m cine frames using a differential interference microscope. A: at 24 h after setting up in culture; B: 2 h after, A, × 390.
Fig. 8. A section of an explant of 12-day chick embryo muscle after 48 h in culture. Haematoxylin and eosin. Remnants of pre-existing myotubes (RM) uninucleate cells (UC), × 320.
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Z
©
© ~r
m
:Z
rr~
~7
r.
156
R. PARSONS
Fig. 9. An enlargement of part of Fig. 8. Myotube remnants (RM), ,i 720.
uncultured muscle (Fig. 2), and visible only as thin-edged circles in culture. During filming the nuclei of the "old" myotubes disappear. A series of 16 mm frame enlargements (Fig. 11) outline the events taking place in a 28 h sequence. DISCUSSION It was presumed by HOLTZERet al. (1958) that trypsinization of 6-9-day-old myotubes produced mononucleate cells by fragmentation of the myotubes. Later OKAZAKI AND HOLTZER 0965) assumed that trypsinization yielded only the mononucleate cells which surrounded the myotubes, but they could form no clear conclusions. It is possible that trypsin may have a different effect on chick embryo muscle when compared with the results of culturing untrypsinized muscle as explants. However, the population of cells obtained by both methods seems to be the same. Electron microscopy of cultured muscle reveals that the great majority of mononucleate cells, both in the explant and outside it, are "presumptive myoblasts" devoid of actin and myosin, and presumptive connective tissue cells. Experiments involving fluorescin-labelled antimyosin by OKAZAKI AND HOLTZER (1966) showed that myosin was absent from the majority of the mononucleate cells obtained by trypsinization of ll-day chick embryo muscle. If this is sufficient evidence to conclude that trypsinization and culturJ. neurol. Sci.,
1971, 13:149-159
CELLS AND NUCLEI IN CHI{?KEMBRYOMUSCLEGROWN IN TISSUE CULTURE
157
ing have the same effect on chick embryo muscle, then it can be assumed that the way in which cells are produced by culturing would be similar to the way in which cells would be produced by trypsinization. It has been stated that " o l d " myotube nuclei disappear in culture. They could become the nuclei of new cells (BETz 1951; ALLBROOK 1962; REZNIK 1969). I f this were true then cinemicrography would almost certainly have shown migration of these nuclei in the " o l d " myotubes, and a marked activity in their cytoplasm during the necessary reorganization. This was not found. Cytoplasmic activity in these myotubes, typically indicated by the movement of intracellular particles, was nil. Even if the mononucleate cells were formed passively, migration of such cells toward the periphery of the explant would have been seen, but it was not. Therefore, it seems
Fig. 10. An electron micrograph of 12-day chick embryo muscle fixed after 48 h in culture. Fixative: 5 ~ glutaraldehyde and Palade's fixative; staining: uranyl acetate. Degenerating myotube (DM), myotube inclusions (mi), degenerating myofilaments (df), mononucleate cells (UC), × 14,400. J. neurol. Sei., 197I, 13:149-159
Fig. 11. A series of 16 mm framed enlargements from a cine film taken with a differential interference microscope, of a living culture of 12-day chick embryo muscle at several time intervals after culture preparation. A: 1 h 45 rain, B: 5 h 36 rain, C: 10 h 18 rain, D: 15 h42 rain, E: 21 h, F: 28 h 18 rain. Note that the myotube (M) throughout the sequence remains unchanged. Particles (PI equivalent to myotube inclusions in Fig. 10, do not move, ~<680.
J. neuro[. Sci., 1971, 13:149-159
CELLS AND NUCLEI IN CHII~K EMBRYO MUSCLE GROWN IN TISSUE CULTURE
159
most likely that the "old" myotube nuclei simply degenerate: and that the cells obtained by trypsinizing chick embryo muscle do not include any myotube nuclei, or indeed any part of the old myotubes. ACKNOWLEDGEMENTS
i wish to thank Dr. K. F. A. Ross for his guidance and for his advice in the preparation of the manuscript. I would also like to thank Miss A. Brown, Mr. C. Wallace and Mr. G. Henderson for their technical assistance; and Miss E. Treece-Birch for typing the manuscript. SUMMARY
Chick embryo muscle was studied in tissue culture by cinemicrography, electron microscopy and in fixed and stained preparations. The uncultured muscle consisted of large numbers of mononucleate cells surrounding multinucleate myotubes. When cultured, the mononucleate cells moved out of the explant and the myotubes degenerated. These observations, and the importance of differential interference microscopy in this type of study are discussed. REFERENCES
ALLEN, R. D., G. B. DAVID AND L. F. Hmsit (1966) The sources of contrast in different types of transmitted light polarizing microscopes. Theoretical considerations about the image-duplicating and differential instruments, Proc. roy. microsc. Soc., l : 141. ALLBROOK, D. (1962) An electronmicroscopic study of regenerating skeletal muscle, J. Anat. (Lond.), 96: 137. BETZ, E. H. (1951) Contribution a 1'6rude de la d6g6n6rescence et de la r6gdn6ration musculaire, Arch. Anat. Micr. morph, exp., 40: 115. HOLTZER, H., J. AnBOT AND J. LASH (1958) On the formation of multinucleated myotubes, Anat. Rec., 131 : 567. JENKISON, M. (1965) Unpublished observations. KFMP, R. B. (1970) The effect of neuraminidase (3:2:1 :l 8) on the aggregation of cells dissociated from embryonic chick muscle tissue, J. cell. Sci., 6: 751. LARSON, P. F., M. JENKISON AND P. HUDGSON 0970) The morphological development of chick embryo skeletal muscle grown in tissue culture as studied by electron microscopy, J. neurol. Sci., 10: 385. MOSCONA, A. A. (1961) Rotation mediated histogenic aggregation of dissociated cells, Exp. Cell Res., 22 : 455. OKAZAKI, K. AND H. HOLTZER (1965) An analysis of myogenesis in vitro using fluorescin-labelled antimyosin, J. Histochem. Cytochem., 13: 726. OKAZAKI,K. AND H. HOLTZER (1966) Myogenesis: myosin synthesis, and the mitotic cycle, Proc. nat. Acad. Sci. (Wash.), 56: 1484. PIPER, R. (1962) Microtomy techniques and the problem of relating the cell outgrowth to events within the explants, J. reed. Lab. TechnoL, 19: 1. REZN1K, M. (1969) Origin of myoblasts during skeletal muscle regeneration, Lab. Int:est., 20: 353. SHIMADA, Y., D. A. FISCHMANAND A. A. MOSCONA(1969) The development of nerve-muscle junctions in monolayer cultures of embryonic spinal cord and skeletal muscle cells, J. Cell. Biol., 44: 436.
J. neurol. Sci., 1971, 13:149-159