437
J. Anat. (1977), 124, 2, pp. 437 444
With 6 figures Printed in Great Britain
Observations on the fusion of chick embryo myoblasts in culture J. FEAR
Department of Anatomy, University of Leeds, Leeds LS2 9NL
(Accepted 10 September 1976) INTRODUCTION
The cultivation of dissociated chick embryonic myoblasts in vitro facilitates detailed study of the formation of skeletal muscle fibres and has shown that multinucleated syncytial myotubes are produced by the fusion of individual bipolar cells (Cooper & Konigsberg, 1961; Yaffe & Feldman, 1965; Bischoff & Holtzer, 1968, 1969). The myotubes, which initially have centrally placed nuclei and no crossstriations, later develop striations and their nuclei move to the periphery. Although hypernucleation and the formation of branching fibres are seen in vitro, it is generally agreed that the histogenesis of skeletal muscle in culture is similar to that occurring in vivo. Little is known of the actual mechanisms of fusion of myogenic cells, but it has been suggested (Fischman, Shimada & Moscona, 1967) that fusion follows the sideto-side alignment of myoblasts. There have been two previous cinemicrographic studies of myogenesis (Capers, 1960; Cooper & Konigsberg, 1961). These were, however, concerned with the origin of nuclei in myotubes; in the present study particular attention was given to the behaviour of cells during fusion in an attempt to clarify this stage of myogenesis. MATERIALS AND METHODS
A suspension of cells was prepared from thigh muscle of 11 day chick embryos, using a method based on that of Moscona (1961). The tissue was incubated at 37 °C for 20 minutes in a 0'05 % solution of collagenase (Sigma Chemicals Type I) in phosphate buffered calcium and magnesium free (C.M.F.) saline (Dulbecco). The tissue was subsequently washed in C.M.F. saline, and it was then dissociated in culture medium to give a final concentration of approximately 5 x 105 cells per culture chamber. The dispersed cells were injected into modified Rose culture chambers (Sharp, 1959) and incubated at 37 °C; the medium was changed after 24 hours and then every 2 days. The culture medium consisted of Eagle's minimal essential medium with 1 % glutamine added (M.E.M. Flow Labs), horse serum (Flow Labs.) and chick embryo extract in the ratio 88:10:2 (O'Neill & Stockdale, 1972). No antibiotics were used. Since collagen has been shown to promote the differentiation of muscle in vitro (Haushka & Konigsberg, 1966), before assembly of the culture chamber the coverslip on which the cells settled had been coated with a solution of collagen, which was subsequently dried under ultraviolet light to ensure sterility. The cultures were maintained and studied for 7 days. Time-lapse cinemicrography was carried out between 40 and 75 hours in vitro, as fusion of cells was found to occur within this period. More than 200 culture chambers were examined; of these, ten were used for filming and a total of 800 feet of film was taken. The film was taken
438
J. FEAR
2 Fig. 1. 40 hours in vitro. A myoblast (my) and a fibroblastic cell (f) can be seen. The myoblast has a bipolar shape and a cytoplasmic expansion can be seen at the pole of the cell. Phase contrast. x 750. Fig. 2. 7 days in vitro. At this stage cross-striations are apparent within the myotube (mt) which resembles a mature skeletal muscle fibre. Phase contrast. x 750.
with a Vinten Scientific 16 mm camera, using a Vickers time-lapse control unit, on Ilford Pan F film. The 16 mm cine and 35 mm still photography was carried out on a Reichert 'Biovert' inverted microscope, fitted with a warm stage to maintain the culture at 37 °C, and equipped with both phase contrast and Nomarski interference contrast optics.
439
Myoblasts in culture
I*
4$' t:,.:..
I-
r
-
t Stspr t
00or
tovI
..
c~ ~ -
t
w~I
,,p
I* ....
.: .::-
?'
t
....... ~-
Fig. 3. (A) 50 hours in vitro. A group of myoblasts (my) aligned in a side-to-side configuration can be seen. (B) 52 hours in vitro. The same group of cells. (C) 55 hours in vitro. The cells, initially aligned side-to-side, have not undergone fusion. Nomarski interference contrast. x 350. Enlarged from 16 mm film.
440
J. FEAR RESULTS
The two basic cell types, myoblasts and fibroblasts, found in the cultures, could be distinguished by their characteristic morphology and locomotion. The myoblast was bipolar, with a central cytoplasmic expansion containing a phase dark nucleus, and two cytoplasmic processes (Fig. 1). There was an expanded region (subsequently referred to as the pole) at the tip of each cytoplasmic process, and membrane ruffling and the formation of cytoplasmic extensions were limited to this region. It has been shown that these cells give rise to muscle fibres (Konigsberg, 1963). The myoblast maintained its bipolar shape when moving, and it migrated in the direction of its long axis. The fibroblast was triangular or pleomorphic (Fig. 1). The nucleus was less dense, and the cytoplasm was granular. This type of cell was more mobile than the myoblast and moved with a broad leading edge, showing ruffled membranes, leaving a trailing cytoplasmic extension, and did not give rise to muscle fibres (Konigsberg, 1963). Over the first 2 days in culture the myoblasts settled, spread on the collagen coated coverslip, and underwent multiplication, migration and side-to-side as well as endto-end alignment. The formation of myotubes occurred mainly over a 15 hour period between the second and third day in vitro. The myotubes began to show spontaneous contractions between the fourth and fifth days and at the same time cross-striations became apparent within them. Their nuclei, initially centrally placed, migrated to the periphery of the myotube and by day 7 the myotube resembled a mature skeletal muscle fibre with respect to nuclear position and striations (Fig. 2). Particular attention was paid to cultures filmed between 50 and 65 hours in vitro as preliminary experiments had indicated that most fusion occurred during this period. Myoblasts appeared to orient themselves in two ways. On a number of occasions myoblasts were seen to align side-by-side, with their long axes parallel to each other. The cells remained in this configuration for approximately 4 hours, but then tended to migrate past one another, and did not undergo fusion (Fig. 3 A-C). On other occasions myoblasts were seen to align end-to-end with their ruffled membranes in contact; this appeared to be a result of the characteristic mode of migration of the myoblast. It was found that fusion often followed such end-to-end encounters, though it did not occur until about 6 hours after the initial contact between the two cells. Figure 4 (A-E) shows a myoblast approaching a nascent myotube and subsequently fusing. Complete fusion was indicated by the migration of the myoblast nucleus into the myotube. Fusion occurring between two myotubes can be seen in Figure 5 (A-F); the tips of the two myotubes displayed considerable membrane activity in the form of ruffled membranes before fusion occurred. Myoblasts were also observed to align with their long axes parallel to the long axis of a myotube and, with the light microscope, these appeared to fuse. After a period of contact, however, the myoblast tended to migrate further along the edge of the myotube and it was clear that no fusion had, in fact, taken place. The formation of branched myotubes was frequently seen in vitro (Fig. 6). These appeared to result from contact between the end of one myotube and the lateral surface of another, and the tip of the latter did not seem to be involved (Fig. 5 A-F). Branched myotubes were seen to undergo development similar to that of unbranched myotubes in culture.
Myoblasts in culture
441
my1
Fig. 4. (A) 50 hours in vitro. Two myoblasts (my,, my2) are in an end-to-end alignment with a nascent myotube (mt). (B-E) Show the cells at 53 hours, 55 hours, 56 hours and 57 hours in vitro respectively. Both myoblasts undergo fusion with the myotube. Fusion was not completed until about 6 hours after the initial contact. Nomarski interference contrast. x 300. Enlarged from 16 mm film.
442
J. FEAR
Fig. 5. For legend see opposite.
443
Myoblasts in culture
-r
>s ___ OF.
6~
~'_jf
t_w
OW-
Fig. 6. 80 hours in vitro. A branched myotube (mt) can be seen. At this stage the nuclei (n) are centrally placed and cross-striations are not apparent. Phase contrast. x 250. DISCUSSION
It has been suggested that myogenic cells align in a side-to-side configuration prior to undergoing fusion, and such configurations have been examined with electron microscopy to determine the early morphological changes in cell fusion (Fischman et al. 1967; Shimada, 1971). The present cinemicrographic study suggests that myogenic cells which align side-to-side are unlikely to succeed in fusing and that fusion is more likely to result from contact between the tips of myogenic cells. The cell membrane in the region of the pole shows considerable membrane activity in the form of ruffling and the production of cytoplasmic projections. Electron microscopy shows that the cytoplasm in this region contains numerous cytoplasmic vesicles. Some of these appear to arise from the plasma membrane during pinocytosis, while others emerge from the Golgi apparatus and subsequently fuse with the plasma membrane (Lipton & Konigsberg, 1972). These observations suggest that there is considerable plasma membrane activity in this region. The mode of migration of the myogenic cell increases the probability that the advancing pole will be the first part of the cell to contact another myogenic cell. The suggestion that fusion
Fig. 5. (A) 56 hours in vitro. Two growing myotubes (mtl, mt,) and a third (mtt3) which is beginning to form are apparent. (B) 58 hours in vitro. The same cells. (C) 60 hours in vitro. Two myotubes (mtl, mt2) are in close approximation and the third myotube has come into contact with the lateral surface of one of the myotubes (arrow). (D, E) Show the cells at 61 hours and 62 hours in vitro respectively. Fusion between mt, and Mt2 was accompanied by considerable membrane activity (arrows). (F) 66 hours in vitro. The orientation and subsequent fusion of the myotubes result in the formation of a branched myotube (Wt4). Nomarski interference contrast. x 300. Enlarged from 16 mm film.
444
J. FEAR
involves the pole of the myogenic cell is supported by in vivo observations of muscle development; thus, myoblasts are oriented with their long axes parallel to the long axis of the embryo during myogenesis in the chick embryo somite (Przybylski & Blumberg, 1966). This alignment of myoblasts is also seen in myogenesis occurring in the regeneration of the salamander tail (Hey, 1963). When myotubes form, these too are oriented parallel to the long axis of the embryo. Branched myotubes are frequently seen in vitro (Konigsberg, 1963) but have not been reported in vivo. The branching of myotubes appears to occur because nascent myotubes are not aligned parallel to each other in vitro. If, as this study indicates, the pole of the myogenic cell is primarily involved in the initiation of cell fusion, then the chance of myotubes making end-to-side contacts leading to the formation of branched myotubes is much greater in vitro than in vivo. SUMMARY
Chick embryo myoblasts were grown as monolayers in culture. The formation of myotubes from myoblasts occurred over a 15 hour period in vitro. Time-lapse cinemicrography was used to study the behaviour of myoblasts during fusion and particular attention was paid to the way in which myoblasts aligned themselves. It was found that fusion tended to follow end-to-end rather than side-to-side alignment of myoblasts. This observation suggests that cell orientation is an important factor in myogenesis. Branched myotubes were frequently observed in culture; an-explanation of this is offered. REFEREN CES
BISCHOFF, R. & HOLTZER, H. (1968). Effect of mitotic inhibitors on myogenesis in vitro. Journal of Cell Biology 36, 111-127. BISCHOFF, R. & HOLTZER, H. (1969). Mitosis and the process of differentiation of myogenic cells in vitro. Journal of Cell Biology 41, 188-200. CAPERS, C. R. (1960). Multinucleation of skeletal muscle in vitro. Journal of Biophysical and Biochemical Cytology 7, 559-567. COOPER, W. G. & KONIGSBERG, I. R. (1961). Dynamics of myogenesis in vitro. Anatomical Record 140, 195-205. FISCHMAN, D. A., SHIMADA, Y. & MOSCONA, A. (1967). Myogenesis in vitro: an electron microscopic study. Journal of Cell Biology 35, 445-452. HAUSHKA, S. & KONIGSBERG, I. R. (1966). The influence of collagen on developing muscle clones. Proceedings of the National Academy of Sciences 55, 119-126. HEY, E. D. (1963). Fine structure of muscle in regenerating salamander tail. Zeitschrift fpr Zellforschung und mikroscopische Anatomie 59, 6-34. KONIGSBERG, I. R. (1963). Clonal analysis of myogenesis. Science 140, 1273-1284. LIPTON, B. & KONIGSBERG, I. R. (1972). A fine structural analysis of the fusion of myogenic cells. Journal of Cell Biology 53, 348-364. MOSCONA, A. (1961). Rotation mediated histogenetic aggregation of dissociated cells. Experimental Cell Research 22, 455-475. O'NEILL, M. C. & STOCKDALE, F. E. (1972). A kinetic analysis of myogenesis in vitro. Journal of Cell Biology 52, 52-65. PRZYBYLSKI, R. & BLUMBERG, J. (1966). Ultrastructural aspects of myogenesis in the chick. Laboratory Investigation 15, 836-863. SHARP, J. A. (1959). A modification of the Rose perfusion chamber. Experimental Cell Research 17, 519-521. SHIMADA, Y. (1971). E.M. observations of fusion of chick myoblasts in vitro. Journal of Cell Biology 48, 128-142. YAFFE, D. & FELDMAN, G. (1965). The formation of hybrid multinucleate fibres from myoblasts of different genetic origins. Developmental Biology 4, 37-52.
J. Anat. (1977), 124, 2, pp. 437 444
With 6 figures Printed in Great Britain
Observations on the fusion of chick embryo myoblasts in culture J. FEAR
Department of Anatomy, University of Leeds, Leeds LS2 9NL
(Accepted 10 September 1976) INTRODUCTION
The cultivation of dissociated chick embryonic myoblasts in vitro facilitates detailed study of the formation of skeletal muscle fibres and has shown that multinucleated syncytial myotubes are produced by the fusion of individual bipolar cells (Cooper & Konigsberg, 1961; Yaffe & Feldman, 1965; Bischoff & Holtzer, 1968, 1969). The myotubes, which initially have centrally placed nuclei and no crossstriations, later develop striations and their nuclei move to the periphery. Although hypernucleation and the formation of branching fibres are seen in vitro, it is generally agreed that the histogenesis of skeletal muscle in culture is similar to that occurring in vivo. Little is known of the actual mechanisms of fusion of myogenic cells, but it has been suggested (Fischman, Shimada & Moscona, 1967) that fusion follows the sideto-side alignment of myoblasts. There have been two previous cinemicrographic studies of myogenesis (Capers, 1960; Cooper & Konigsberg, 1961). These were, however, concerned with the origin of nuclei in myotubes; in the present study particular attention was given to the behaviour of cells during fusion in an attempt to clarify this stage of myogenesis. MATERIALS AND METHODS
A suspension of cells was prepared from thigh muscle of 11 day chick embryos, using a method based on that of Moscona (1961). The tissue was incubated at 37 °C for 20 minutes in a 0'05 % solution of collagenase (Sigma Chemicals Type I) in phosphate buffered calcium and magnesium free (C.M.F.) saline (Dulbecco). The tissue was subsequently washed in C.M.F. saline, and it was then dissociated in culture medium to give a final concentration of approximately 5 x 105 cells per culture chamber. The dispersed cells were injected into modified Rose culture chambers (Sharp, 1959) and incubated at 37 °C; the medium was changed after 24 hours and then every 2 days. The culture medium consisted of Eagle's minimal essential medium with 1 % glutamine added (M.E.M. Flow Labs), horse serum (Flow Labs.) and chick embryo extract in the ratio 88:10:2 (O'Neill & Stockdale, 1972). No antibiotics were used. Since collagen has been shown to promote the differentiation of muscle in vitro (Haushka & Konigsberg, 1966), before assembly of the culture chamber the coverslip on which the cells settled had been coated with a solution of collagen, which was subsequently dried under ultraviolet light to ensure sterility. The cultures were maintained and studied for 7 days. Time-lapse cinemicrography was carried out between 40 and 75 hours in vitro, as fusion of cells was found to occur within this period. More than 200 culture chambers were examined; of these, ten were used for filming and a total of 800 feet of film was taken. The film was taken
438
J. FEAR
2 Fig. 1. 40 hours in vitro. A myoblast (my) and a fibroblastic cell (f) can be seen. The myoblast has a bipolar shape and a cytoplasmic expansion can be seen at the pole of the cell. Phase contrast. x 750. Fig. 2. 7 days in vitro. At this stage cross-striations are apparent within the myotube (mt) which resembles a mature skeletal muscle fibre. Phase contrast. x 750.
with a Vinten Scientific 16 mm camera, using a Vickers time-lapse control unit, on Ilford Pan F film. The 16 mm cine and 35 mm still photography was carried out on a Reichert 'Biovert' inverted microscope, fitted with a warm stage to maintain the culture at 37 °C, and equipped with both phase contrast and Nomarski interference contrast optics.
439
Myoblasts in culture
I*
4$' t:,.:..
I-
r
-
t Stspr t
00or
tovI
..
c~ ~ -
t
w~I
,,p
I* ....
.: .::-
?'
t
....... ~-
Fig. 3. (A) 50 hours in vitro. A group of myoblasts (my) aligned in a side-to-side configuration can be seen. (B) 52 hours in vitro. The same group of cells. (C) 55 hours in vitro. The cells, initially aligned side-to-side, have not undergone fusion. Nomarski interference contrast. x 350. Enlarged from 16 mm film.
440
J. FEAR RESULTS
The two basic cell types, myoblasts and fibroblasts, found in the cultures, could be distinguished by their characteristic morphology and locomotion. The myoblast was bipolar, with a central cytoplasmic expansion containing a phase dark nucleus, and two cytoplasmic processes (Fig. 1). There was an expanded region (subsequently referred to as the pole) at the tip of each cytoplasmic process, and membrane ruffling and the formation of cytoplasmic extensions were limited to this region. It has been shown that these cells give rise to muscle fibres (Konigsberg, 1963). The myoblast maintained its bipolar shape when moving, and it migrated in the direction of its long axis. The fibroblast was triangular or pleomorphic (Fig. 1). The nucleus was less dense, and the cytoplasm was granular. This type of cell was more mobile than the myoblast and moved with a broad leading edge, showing ruffled membranes, leaving a trailing cytoplasmic extension, and did not give rise to muscle fibres (Konigsberg, 1963). Over the first 2 days in culture the myoblasts settled, spread on the collagen coated coverslip, and underwent multiplication, migration and side-to-side as well as endto-end alignment. The formation of myotubes occurred mainly over a 15 hour period between the second and third day in vitro. The myotubes began to show spontaneous contractions between the fourth and fifth days and at the same time cross-striations became apparent within them. Their nuclei, initially centrally placed, migrated to the periphery of the myotube and by day 7 the myotube resembled a mature skeletal muscle fibre with respect to nuclear position and striations (Fig. 2). Particular attention was paid to cultures filmed between 50 and 65 hours in vitro as preliminary experiments had indicated that most fusion occurred during this period. Myoblasts appeared to orient themselves in two ways. On a number of occasions myoblasts were seen to align side-by-side, with their long axes parallel to each other. The cells remained in this configuration for approximately 4 hours, but then tended to migrate past one another, and did not undergo fusion (Fig. 3 A-C). On other occasions myoblasts were seen to align end-to-end with their ruffled membranes in contact; this appeared to be a result of the characteristic mode of migration of the myoblast. It was found that fusion often followed such end-to-end encounters, though it did not occur until about 6 hours after the initial contact between the two cells. Figure 4 (A-E) shows a myoblast approaching a nascent myotube and subsequently fusing. Complete fusion was indicated by the migration of the myoblast nucleus into the myotube. Fusion occurring between two myotubes can be seen in Figure 5 (A-F); the tips of the two myotubes displayed considerable membrane activity in the form of ruffled membranes before fusion occurred. Myoblasts were also observed to align with their long axes parallel to the long axis of a myotube and, with the light microscope, these appeared to fuse. After a period of contact, however, the myoblast tended to migrate further along the edge of the myotube and it was clear that no fusion had, in fact, taken place. The formation of branched myotubes was frequently seen in vitro (Fig. 6). These appeared to result from contact between the end of one myotube and the lateral surface of another, and the tip of the latter did not seem to be involved (Fig. 5 A-F). Branched myotubes were seen to undergo development similar to that of unbranched myotubes in culture.
Myoblasts in culture
441
my1
Fig. 4. (A) 50 hours in vitro. Two myoblasts (my,, my2) are in an end-to-end alignment with a nascent myotube (mt). (B-E) Show the cells at 53 hours, 55 hours, 56 hours and 57 hours in vitro respectively. Both myoblasts undergo fusion with the myotube. Fusion was not completed until about 6 hours after the initial contact. Nomarski interference contrast. x 300. Enlarged from 16 mm film.
442
J. FEAR
Fig. 5. For legend see opposite.
443
Myoblasts in culture
-r
>s ___ OF.
6~
~'_jf
t_w
OW-
Fig. 6. 80 hours in vitro. A branched myotube (mt) can be seen. At this stage the nuclei (n) are centrally placed and cross-striations are not apparent. Phase contrast. x 250. DISCUSSION
It has been suggested that myogenic cells align in a side-to-side configuration prior to undergoing fusion, and such configurations have been examined with electron microscopy to determine the early morphological changes in cell fusion (Fischman et al. 1967; Shimada, 1971). The present cinemicrographic study suggests that myogenic cells which align side-to-side are unlikely to succeed in fusing and that fusion is more likely to result from contact between the tips of myogenic cells. The cell membrane in the region of the pole shows considerable membrane activity in the form of ruffling and the production of cytoplasmic projections. Electron microscopy shows that the cytoplasm in this region contains numerous cytoplasmic vesicles. Some of these appear to arise from the plasma membrane during pinocytosis, while others emerge from the Golgi apparatus and subsequently fuse with the plasma membrane (Lipton & Konigsberg, 1972). These observations suggest that there is considerable plasma membrane activity in this region. The mode of migration of the myogenic cell increases the probability that the advancing pole will be the first part of the cell to contact another myogenic cell. The suggestion that fusion
Fig. 5. (A) 56 hours in vitro. Two growing myotubes (mtl, mt,) and a third (mtt3) which is beginning to form are apparent. (B) 58 hours in vitro. The same cells. (C) 60 hours in vitro. Two myotubes (mtl, mt2) are in close approximation and the third myotube has come into contact with the lateral surface of one of the myotubes (arrow). (D, E) Show the cells at 61 hours and 62 hours in vitro respectively. Fusion between mt, and Mt2 was accompanied by considerable membrane activity (arrows). (F) 66 hours in vitro. The orientation and subsequent fusion of the myotubes result in the formation of a branched myotube (Wt4). Nomarski interference contrast. x 300. Enlarged from 16 mm film.
444
J. FEAR
involves the pole of the myogenic cell is supported by in vivo observations of muscle development; thus, myoblasts are oriented with their long axes parallel to the long axis of the embryo during myogenesis in the chick embryo somite (Przybylski & Blumberg, 1966). This alignment of myoblasts is also seen in myogenesis occurring in the regeneration of the salamander tail (Hey, 1963). When myotubes form, these too are oriented parallel to the long axis of the embryo. Branched myotubes are frequently seen in vitro (Konigsberg, 1963) but have not been reported in vivo. The branching of myotubes appears to occur because nascent myotubes are not aligned parallel to each other in vitro. If, as this study indicates, the pole of the myogenic cell is primarily involved in the initiation of cell fusion, then the chance of myotubes making end-to-side contacts leading to the formation of branched myotubes is much greater in vitro than in vivo. SUMMARY
Chick embryo myoblasts were grown as monolayers in culture. The formation of myotubes from myoblasts occurred over a 15 hour period in vitro. Time-lapse cinemicrography was used to study the behaviour of myoblasts during fusion and particular attention was paid to the way in which myoblasts aligned themselves. It was found that fusion tended to follow end-to-end rather than side-to-side alignment of myoblasts. This observation suggests that cell orientation is an important factor in myogenesis. Branched myotubes were frequently observed in culture; an-explanation of this is offered. REFEREN CES
BISCHOFF, R. & HOLTZER, H. (1968). Effect of mitotic inhibitors on myogenesis in vitro. Journal of Cell Biology 36, 111-127. BISCHOFF, R. & HOLTZER, H. (1969). Mitosis and the process of differentiation of myogenic cells in vitro. Journal of Cell Biology 41, 188-200. CAPERS, C. R. (1960). Multinucleation of skeletal muscle in vitro. Journal of Biophysical and Biochemical Cytology 7, 559-567. COOPER, W. G. & KONIGSBERG, I. R. (1961). Dynamics of myogenesis in vitro. Anatomical Record 140, 195-205. FISCHMAN, D. A., SHIMADA, Y. & MOSCONA, A. (1967). Myogenesis in vitro: an electron microscopic study. Journal of Cell Biology 35, 445-452. HAUSHKA, S. & KONIGSBERG, I. R. (1966). The influence of collagen on developing muscle clones. Proceedings of the National Academy of Sciences 55, 119-126. HEY, E. D. (1963). Fine structure of muscle in regenerating salamander tail. Zeitschrift fpr Zellforschung und mikroscopische Anatomie 59, 6-34. KONIGSBERG, I. R. (1963). Clonal analysis of myogenesis. Science 140, 1273-1284. LIPTON, B. & KONIGSBERG, I. R. (1972). A fine structural analysis of the fusion of myogenic cells. Journal of Cell Biology 53, 348-364. MOSCONA, A. (1961). Rotation mediated histogenetic aggregation of dissociated cells. Experimental Cell Research 22, 455-475. O'NEILL, M. C. & STOCKDALE, F. E. (1972). A kinetic analysis of myogenesis in vitro. Journal of Cell Biology 52, 52-65. PRZYBYLSKI, R. & BLUMBERG, J. (1966). Ultrastructural aspects of myogenesis in the chick. Laboratory Investigation 15, 836-863. SHARP, J. A. (1959). A modification of the Rose perfusion chamber. Experimental Cell Research 17, 519-521. SHIMADA, Y. (1971). E.M. observations of fusion of chick myoblasts in vitro. Journal of Cell Biology 48, 128-142. YAFFE, D. & FELDMAN, G. (1965). The formation of hybrid multinucleate fibres from myoblasts of different genetic origins. Developmental Biology 4, 37-52.
