- Email: [email protected]
Skeletal myogenesis
Printed in Sweden Copyright @ 1977 by Academic Press, Inc. All rights of reproduction in any form reserved ISSA’ WI44827
Experimental Cell Research lOl(l977)
SKELETAL
MYOGENESIS
Control of Proliferation S. R. DIENSTMAN’ Departments
355-364
in a Normal Cell Lineage and H. HOLTZER
of Biology and Anatomy, University Philadelphia, PA 19174, USA
of Pennsylvania,
SUMMARY The fates of IO-day chick embryo myogenic cell populations cultured in conditions that maximize or suppress proliferation or fusion were examined. The repression of the cell cycle, prevalence of fusible cells, or expression of myofibril formation were monitored by autoradiography, counts of myotube nuclei, staining with fluorescein-labelled antibody against skeletal myosin heavy chain, or electron microscopy. Using a calcium chelator (EGTA) to block cell fusion did not prevent the accumulation of myoblasts blocked in Gl of the cell cycle that initiated skeletal myosin synthesis and myofibril formation. Trypsinization, dilution, and daily feeding with fresh medium and serum did not reverse this cell cycle block. Using cytochalasin B (CB) as an alternative fusion block confirmed these results. Using fluorodeoxyuridine (FUdR) to prevent the cycling of myogenic cells that normally would have multiplied in vitro did not prompt these cells to fuse. Muscle-conditioned medium could not prompt a switch in the commitment of replicating cells when in Gl to terminal differentiation. The indications are that myogenic cell populations contain definite mixtures of precursor phenotypes. The terminal phenotype is initiated coordinately with a relatively stable cue for the repression of the cell cycle. Differentiation-specified growth control is discussed within the context of a set of growth control mechanisms known to operate in culture.
The propagation in vitro of various normal and transformed animal cells has emphasized two mechanisms of cellular growth control: (1) long-range growth control, thought to represent “aging” on the cellular level [14], and, (2) interim growth control, which is a function of the nutrition or environment of the cells in question and is keyed into the cell cycle [15, 30, 351. Both mechanisms halt proliferation. In the first case, this occurs through a slow dying-out of the culture. By contrast, in the second case, the transition from growing to resting states occurs more synchronously, can be 1 Present address: Department of Pathology, New York University, School of Medicine, New York, NY 10016,USA.
reversed, and involves cell types that never permanently repress the cell cycle. Holtzer et al. [21] and Dienstman & Holtzer [12] have proposed another case of cellular growth control. Counting time from the establishment of the zygote, this third mechanism is a relatively short-range case of growth control. For example, within the chick embryo, proliferation ceases among subpopulations of skeletal muscle and nerve cells within the first 48-72 h of incubation and by the 5th day, there are in addition large numbers of post-mitotic red blood cells, skin cells, and pancreatic cells. Overlapping the halt in proliferation is the appearance of new cell products, marking the acquisition of new differentiated states for the cells in question. This type of represExp
Cell
Res
107 (1977)
356
Dienstman and Holtzer
sion of the cell cycle, specified in differentiation, is generally irreversible. Owing to its adaptibility to culture, the existence of well-defined markers for the differentiated state, and the derivation of cell lines, skeletal myogenesis has lent itself to studies of growth and differentiation. Many laboratories have documented the following: (1) The differentiation of skeletal muscle in vivo or in vitro entails a transition from a population of mononucleated cells to a population of multinucleated myotubes; (2) the multinucleated condition results from the fusion of mononucleated cells; (3) in any inoculum, some mononucleated cells in Gl fuse directly, while the rest replicate and contribute to myotube formation through their progeny; (4) the nuclei in myotubes are post-mitotic [3, 7, 16, 17,411. What governs the transition from the growing to the fixed Gl state, however, has remained a matter of controversy. Konigsberg [22-241 and Buckley & Konigsberg [6] have inferred from the available data that (1) all the mononucleated myogenic cells are capable of continuous proliferation; but (2) high cell density, high concentration of conditioning factors, or prolonging the time spent in Gl can induce these cells to fuse; and (3) only as a consequence of cell fusion do the muscle cells become fixed in a post-mitotic state and initiate the synthesis of the definitive contractile proteins. Essentially similar conclusions have been reached by O’Neill & Stockdale [28, 371, Doering & Fischman [13], and others [5, 31, 33, 34, 401. For a critical review of these experiments see Dienstman & Holtzer [ 121. The experiments to be reported here demonstrate that (1) fusion is not the cause of the repression of the cell cycle in myogenesis; and (2) the post-mitotic state is initiated coordinately with other differenExp Cell Res 107 (1977)
tiated functions in a relatively inflexible program. Additional experiments show that precursor myogenic cells, which are normally committed to replicate, cannot undergo precocious differentiation by inhibiting DNA synthesis or by applying conditioned medium for 48 h. These experiments have in part been reported preliminarily [9, lo].
MATERIALS
AND METHODS
Mononucleated myogenic cell populations were iso_ lated from lO-day embryonic chick breasts and cultured similarly to the methods of Bischoff & Holtzer [2]. However; inocula were plated at a concentration of 2x 10” cells/35 mm dish in collagen-coated dishes and were fed daily with fresh medium. Fresh medium (8 : 1: 1) consisted of 8 parts Eagle’s minimum essential medium, 1 part horse serum: 1 part chick embryo extract, and 1% antibiotics. Under these conditions, control cultures sustained 3-4 doublings during the first 5 days in vitro. These cultures contained nonmyogenic, Bbrogenic cells as well, which accounted for most of the population increase after the third culture day [ 1,9]. Eorsome cultures, conditioned medium (CM) was used instead of fresh medium. CM was recovered from muscle cultures (5X 105cells plated in 60 mm dishes with 3 ml of medium) after 48 h, grown without a change of medium at 24 h; at this time, the fusion period was already underway. 1.75 mM EGTA was found to prevent muscle cell fusion, but would not cause discernable cell death or inhibit mitosis. EGTA was added to cultures from the time of plating [lo]. Cytochalasin B (CB) (5-10 pg/ml) also was used as a fusion inhibitor [lo]. In order to identify the subpopulation of cells in the inoculum which synthesized DNA and proliferated, 0.25 &i/ [3H]TdR (40-60 Cilmmole) was added to each culture at the outset and/or upon the change of the medium. Autoradiography was performed to detect labeled nuclei in mononucleated cells and multinucleated myotubes. FUdR (1O-BM) was added to cultures from the outset in those experiments designed to inhibit proliferation from the outset [4, 81. By this means, up to 90% of the surviving myogenic cells can be stalled at the Gl/S interface [25]. For following fusion, the standard approach of counting the number of nuclei in myotubes versus nuclei of all cells was used. However. for the analvses described in this report, a more informative approach was obtained by combining rSH]TdR labelling with counts of myotube nuclei, both labelled and unlabelled, in autoradionraohs at one fixed time. This procedure allowed th; perception of cell cycle activities in myogenesis taking place early in culture, although the processes of differentiation could be allowed to mature up to 4 or 5 days more. Thus comparisons always were made between populations of
Controls on proliferation
1. Cultures were grown for 4 days with daily feeding of fresh medium containing EGTA at a level to inhibit fusion but not replication. Multinucleated myotubes did not form; highly elongate mononucleated cells accumulated that tended to pack side by side in parallel fashion. Phase contrast, x 10 obj. Bar, 100pm.
Fig.
in muscle
357
large numbers of highly elongated mononucleated cells but no myotubes (fig. 1). In control cultures about 60% of all nuclei were in myotubes and thereafter there was little increase in numbers of myotubes or their nuclei. Accordingly, on day 4 the fusion block in the experimental cultures was broken by removing the EGTA and adding fresh medium containing calcium as well as r3H]TdR. Cultures were maintained for an additional 5 days, then fixed and autoradiographed. All the nuclei in the myotubes which formed subsequent to the removal of EGTA were unlabelled (fig. 2). Thus, the cells that fused between days 4 and 9 must have been held in Gl of the cell
myotubes which were more mature and more uniform in shape and age. The criteria of Bischoff & Holtzer [3] were used to score nuclei in myotubes and each experiment was repeated at least six times. Given the biological and stochastic variation between experiments, the data presented are from individual repetitions. Conclusions drawn from any one were representative of the others. Surveys of nuclear labelling and fusion were done on 48 microscopic fields in duplicate cultures to obtain a mean and variance for each data point. The properties of the fluorescein labelled antibody against skeletal myosin heavy chains have been described elsewhere [ 19,201. This antibody is specific for myosin heavy chains only from skeletal muscle; it does not cross react in Ouchterlony double diffusion chambers or histochemically with the myosins from smooth muscle myoblasts, fibroblasts, nerve cells or presumptive myoblasts. This antibody does not crossreact with C-protein [27]. The nomenclature used in regard to cell types is the same as described previously [l, 11, 121. (1) Mb cells are the post-mitotic mononucleated myoblasts that have initiated the synthesis of the definitive myosin heavy and light chains; (2) PMb cells are of the replicating precursors to the myoblast; (3) Fb cells, the associated tibroblasts; and (4) PPMbFb cells, the common precursor to these myogenic and fibrogenic cells.
RESULTS To determine whether the repression of the cell cycle in myogenic cells is dependent upon fusion, or whether it occurs in the mononucleated cell, cultures were set up in the presence of EGTA (1.75 mM). Cultures grown in EGTA for 4 days contained
Fig. 2. After inhibiting fusion for 4 days the EGTA
block was broken and cultures were kept an additional 5 days in the presence of [3H]TdR, then fixed and autoradiographed. Here, such a 9-day culture reveals that by 4 days in vitro the myogenic population was made up of post-mitotic cells which fused to form myotubes having unlabelled nuclei. (a) Phase contrast; (b) bright field; x 16 obj. Bar, 50 pm. Exp CellRes 107(1977)
358
Dienstman
and Holtzer
appear in subculture after 48 h. Subcultures were maintained for 4 days, fixed, and autoradiographed. Virtually all (94 %) the nuclei in myotubes that formed in subculture were unlabelled (fig. 3, III). The repression of the cell cycle in those cells was stable to the effects of trypsinization, to the lowering of cell density, and to daily application of fresh medium. At the same time under these conditions, the operationally non-myogenic cells did replicate. Fig. 3 shows that the Fig. 3. Ordinate: %. 0, Total nuclei; 0, labelled nu- change of conditions so favored the growth clei. PPMbFb-PMb-Mb transitions occur over a 4-dav of replicating cells that they eventually period in vitro without fusion or density requirements: primary cultures grown in EGTA for 2 (I), 3 (II), or dominated the subcultures. 4 (III) days were suspended and replated in fresh meUsing EGTA-blocked primary cultures, dium with calcium and lSH]TdR and grown for 4 more days. The subpopulation of unlabelled myotube nuclei other diluted subcultures were established is equivalent to the mononucleated myogenic fraction in the inoculum which fuses directly, i.e., the un- at times earlier than 4 days. As early as day striped compartments in the “myotube” columns com- 2, about 35 % of the post-mitotic myoblasts prise the fraction of muscle contributed by Mb cells had been generated (fig. 3, I). The progresalready generated in primary culture prior to replating and introduction of label. sive course of proliferation and terminal difLyIbelled myotube nuclei]/[All myotube nuclei] ferentiation was such that the frequency of atsuvxudDfblat smcu~ture +[Mb]genera,ec, ,nsu,,culture: for I, 35%; II, 70%; III, 94 %. The frequency of Mb replicating myogenic cells diminished concells increased while the PPMbFb or PMb declined. siderably by day 3, and by day 4 relatively few new replicating myogenic cells were formed. The transition from a replicating to cycle since day 4. Clearly, repression of a non-replicating myogenic cell population the cell cycle occurs prior to and inde- occurred steadily but asynchronously. pendently of fusion. That the myogenic cells that accumulate Equally important in the above experi- in EGTA-treated cultures are Mb cells and ment, the autoradiography showed that can initiate the synthesis of definitive skelemost mononucleated cells did incorporate tal myosin heavy chains is shown in fig. 4. the label. Hence those cells that had the Using fluorescein-labelled antibody against skeletal myosin heavy chains mononucapacity to proliferate could do so. It might be argued that the halt in pro- cleated cells with cross-striated myofibrils liferation in the muscle cells occurred be- were first evident on day 3 of culture and cause after 4 days in vitro, the myogenic such cells increased in numbers with time. population had reached some critically high Mb cells usually were highly elongated. The cell density that favored fusion over pro- more angular cells, the myogenic preliferation. Accordingly, additional cultures cursors and fibroblasts, did not bind the kept in EGTA for 4 days were trypsinized, fluorescein-labelled antimyosin. On day 2, diluted, replated, and grown in fresh me- very few elongate cells bound the antibody. dium along with [3H]TdR. Extensive DNA Post-mitotic myoblasts also should acsynthesis and cell division occurred, and cumulate with any other agent that blocks the first oligonucleated myotubes began to fusion per se. Also, these cells should disExp Cell Res 107 (1977)
Controls on proliferation in muscle
359
Fig. 4. Low density muscle cultures grown on cover-
slips were glycerinated and stained with fluorescent anti-myosin to detect the synthesis and distribution of skeletal myosin. (a) High power view of a crossstriated myotube in an uninhibited .5-day culture, positions of nuclei are the unstained oval regions; (b) after 3 days in EGTA-blocked cultures mononucleated myoblasts are present which have delicate, apparently single myotibrils; (c, a’) low power view of aCB-treated culture in which round cells specifically bound the antibody. Glycerination and subsequent washing of (B-treated cultures tended to easily detach many round and arborized cells. (a, b, d) Fluorescence microscopy; (c) phase contrast; high power, x40 obj.; bar, 10 pm; low power, x 16 obj.; bars, 50 Wm.
play the capacity to synthesize and organize the definitive contractile proteins into interdigitating thick and thin filaments. Cytochalasin B (CB) is an alternative agent known to block fusion [18, 361. CB was added to a series of cultures between days 2 and 3 to “trap” unfused mononucleated cells and to study their properties. Cells in primary cultures respond to CB in two ways: replicating fibroblasts and presumptive myoblasts retract and form “arborized” cells, whereas post-mitotic myoblasts form rounded cells, many of which detach from the substrate and float into the medium (fig. 5). These round myoblasts have been (1) treated with labelled antibodies against skeletal myosin; (2) inspected by electron microscopy. After 2 days in CB, round cells could be
observed to contract or “beat” spontaneously. Fig. 4 shows that many round cells strongly bound the antibody to skeletal myosin heavy chains. At this time, about half of the adherent population were rounded, and of these cells, most if not all were antibody-positive. Fig. 6 shows that in these cells, although they are rounded, there are interdigitating arrays of thick and thin filaments. Arborized cells were not observed to contract; they did not bind the antibody nor produce thick and thin filament arrays. Each round cell contained only one nucleus, although bi-, tri-, and oligo-nucleate arborized cells did occur. Experiments were designed to disclose the number of myogenic cells (Mb) in control cultures which fuse without any rounds of DNA synthesis in vitro. [3H]TdR was ExpCellRes 107(1977)
360
Dienstman and Holtzer
5. Cultures 2 days old were placed in CB for an additional 2 days. Differential responses to the drug result in cells of distinctly different morphologies: round cells sitting above the substrate and attached to it at apparently one point and arborized cells adhering closely to the substrate with flat arms around a seemingly compressed cell body. Phase contrast, x 16 obj. Bar, 50 pm.
Fig.
present from the outset of culture, and after 4 days, cultures were fixed and autoradiographed. About 4-10% of myotube nuclei were free of label (e.g. 7 %, table 1, ser. I). This corresponds to a frequency of 15-20 %
of cells in the original inoculum which fused directly (i.e., final number of unlabelled myotube nuclei/inoculum size/dish). Table 1, ser. II was designed to test two points. (1) Does merely blocking proliferation and prolonging the time spent prior to DNA synthesis produce greater numbers of cells which fuse directly as compared with the controls? (2) Do precursor cells retain their commitment to replicate and afterward generate differentiated progeny, in spite of the exogenous inhibition on the cell cycle? Cultures were kept in FUdR for the first 48 h in vitro. At 48 h, the FUdR block was broken and culture continued for another 2 days in medium with unlabelled TdR and [3H]TdR. Cultures were fixed after a total of 4 days and autoradiographs made. Table 1 shows that overall fusion was suppressed as a result of the FUdR (ser. II) compared with uninhibited controls (ser. I). However, the number of cells that did express an option to fuse directly in the FUdR-treated cul-
Table 1. Myogenic cells which fuse without replication in vitro Lack of effect of inhibiting DNA synthesis
Days in FUdR
Davs whhout FUdR, in [3H]TdR
0
4
Series IIc
2
2
Series IIId
2
2
Series
1”
Unlabeled total myotube nuclei/dish 29OOOf 6300 415 OOOf55000 32OOOf 8700 36OOOf 9400 31OOOf 8400 37 000+10 000
As % of total myotube nuclei 7 % unlabeled 90 % unlabeled 83 % unlabeled
(1 The subpopulation of unlabelled myotube nuclei is equivalent to the mononucleated myogenic fraction in the inoculum which fuses directly. Values per dish obtained from extrapolating averaged counts (mean f95% confidence interval) of microscopic fields in duplicate cultures. The data then are absolute frequencies of nonreplicating myogenic cellslinoculum of 2x lo5 cells/dish. b Fresh medium. c Fresh medium; lOmEM cold TdR supplement during the release period. d Conditioned medium; then fresh medium+ lOmeM cold TdR during the release period. Exp Cell Res 107 (1977)
Controls on proliferation
Fig. 6. Although Mb cells were unable to fuse in CB,
they tended to occur singly or in clusters of round cells. (a) Electron micrograph of one section through a round cell. Notable in these CB-treated cells is an apparent “segregation” of the cytoplasm with filaments
tures was no greater than it was in controls. No myogenic cells that normally would have replicated were prompted to fuse. During the recovery interval, some cells did generate progeny that fused. However, multiple factors likely combined in table 1, ser. II producing less total fusion even after the recovery interval than was seen in controls. First, there was greater overall proliferation over a 4-day period in controls than occurred over only 2 days in FUdRtreated cultures. Another possible cause was the inability of some precursor cells, which were geared for replication, to survive the externally imposed block in the cell cycle over long intervals [29]. Ser. III was set-up to repeat the experiment of ser. II, but using conditioned medium (CM) plus FUdR. The use of CM would reveal whether it contained any fac-
in muscle
361
peripherally. x9600; (b) enlargement showing the absence of sarcomeres with normal Z disks in CB, but the presence of interdigitating thick and thin filaments and dense bodies instead (arrow).
tors which could prompt any of the myogenie precursor cells to fuse. Comparing the numbers of fusible cells in ser. III with those in ser. I and II shows that no more cells fused with CM than without it; CM did not possess the quality to “switch” FUdR-treated cells from a commitment to replicate to a commitment to fuse in G 1. In conclusion, inocula of myogenic cells taken from the embryo and placed in these various conditions in vitro contain at least two subpopulations: a small group of postmitotic, fusible cells (Mb), and a group(s) of precursors, which only in the normal course of proliferation generate up to 90% of the fusible cells. Prolonging the time prior to DNA synthesis either with FUdR or with CM plus FUdR does not prompt the differentiation of the myogenic precursors. Exp CeURes 107(1977)
362
Dienstman and Holtzer
across a series of cell cycles (see [21] for this type of control during erythrogenesis). These experiments demonstrate that pri- Many investigators have demonstrated the mary muscle cultures consist of a popula- activity of interim growth controls on replition of cells that is heterogeneous with cating cells whose phenotypic activities do not appear to require transition in nor rerespect to its capacities for differentiation and growth control. It is very misleading to pression of the cell cycle. Typically interim analyse such cultures as if they consist of growth control in culture is readily affected pure populations of “myoblasts” and by changes in cell density, large and small “fibroblasts”. Replicating myogenic pre- molecular weight molecules in the ambient cursor cells can neither fuse themselves, medium, trypsinization, insulin-like molesynthesize skeletal myosin, nor assemble cules, etc. [32, 38, 391. Given this multiplicity of growth conmyofibrils. Cloning experiments have demonstrated that, at a minimum, there are two trols, the observation that the relative prosuccessive classes of replicating precursors portion of terminal skeletal muscle generin the myogenic lineage, the PMb and the ated in vitro varies with culture conditions PPMbFb [ 1, 111.Further proliferation, how- does not demonstrate that environmental ever, generates a distinctly different pheno- factors directly and immediately control the type, the post-mitotic, mononucleated myo- basic events of differentiation. For exblast. Myoblasts possess the capacity to ample, in the experiments using FUdR and fuse and to organize the definitive contrac- CM, the percentage of muscle nuclei tile proteins into interdigitating thick and formed by Mb cells fusing directly, seems thin filaments. Their repression of cell cycle greatly enhanced over control levels alactivity, linked to differentiation, is not af- though in actuality neither treatment profected by daily use of fresh medium, by motes any significant increase in the fretrypsinization, by reculture at lowered den- quency of Mb cells (table 1, col. 3 vs 4). sity, or by factors in CM. Similarly, Doering & Fischman [13] have Using myogenesis as a paradigm, it is to reported that the “% fusion” in culture rises be stressed that the outcome for differentiaover control levels following 48 h treattion in vitro or in vivo particularly from ments with cytosine arabinoside. This was mixed populations of precursor cells, rep- interpreted to mean that otherwise repliresents an interplay of variables. Each vari- cating cells were cued to fuse. However, as able singly may affect primarily one of the this agent is known to kill replicating cells, subpopulations in a lineage. The roles of the “% fusion” index must increase in the proliferation, interim growth control and presence of cell cycle inhibitors, though the differentiation-specified growth control inhibitor would have no effect on the state must be separated, identified, and tested in of differentiation of the surviving cells. assessing variation in any developing sysA significant element in the EGTA and tem. The growth controls that act on the CB studies reported here was the use of exreplicating myogenic cells and fibroblasts perimental approaches that tended to maxido not act on the myoblasts. These interim mize proliferation. Paterson & Strohman growth controls appear to operate within [31] had claimed to observe a decline in the each cell cycle, unlike differentiation-speproportion of myogenic cells incorporating cified growth controls which appear to act r3H]TdR in cultures released from EGTA at DISCUSSION
Exp CellRes 107(1977)
Controls on proliferation in muscle successively later times. However, this was seen under conditions chosen to minimize proliferation. The decline in cumulative labelling in their cultures correlated to incubations in [3H]TdR of shorter and shorter length. Thus, the minimum incorporation of labelled nuclei into myotubes was obtained after allowing only a period of 10 h for both labelling and then fusion. Under any circumstances, 10 h is the minimal interval for such an event to occur [3]. In the experiments reported here, proliferation was maximized while the decrease in labelled myotube nuclei was found after sufficiently long and uniform time intervals. It is notable that in their conditions, Moss & Strohman [26] do not find myofibril formation in any cells concurrent with the proliferative decline they observe. It has been shown that FUdR or FUdR and CM did not prompt fusion. Using FUdR alone, aside from the cells trapped in S phase at the time of application, the majority of cells should have accumulated in Gl at the Gl/S interface. It can still be argued that replicating myogenic cells may pass through an option in Gl, albeit unexpressed, for fusion. However, it would also have to be argued that this option is passed early in Gl , certainly earlier than the GllS interface and probably earlier than some CM-sensitive restriction point in the cell cycle. This is also supported by Bischoff & Holtzer’s finding [3] that fusion occurs late in Gl, later than the time most replicating cells become committed to DNA synthesis. Medium conditioning may act on myogenesis in other ways aside from effects on interim growth controls for one or more subpopulations of replicating cells. CM may mimic the effect of collagen by simply promoting the attachment of cells to the substrate. Hypothesized molecules in CM might promote migration or fusion, the fre-
363
quency of collisions, or the actual melding of myoblast surfaces, once these cells have been generated. One hypothetical possibility which will merit future investigation is that CM may affect the frequency with which PMb yield Mb cells rather than more PMb cells. It may be expected that CM obtained through different protocols should differ in some ways. Eagle’s medium has been combined with horse serum and embryo extract in the proportions of 8 : 1: 1 (this report), 8 : 1.5 : 1 [6, 231, or 8 : 1: 0.5 [13] and collected after 42 [6, 231, 45 [13], or 48 (this report) h to prepare CM. It is likely that the differences among CM may be only of minimal degree. In summary, the evidence from chick skeletal myogenesis demonstrates a mechanism for short-range growth control which is relatively insensitive to environmental conditions. Repression of cell cycle function is coordinated with the initiation of a divergent set of synthetic activities characteristic of the terminal muscle phenotype. This fact, coupled with the observations that (1) the transition from proliferating to post-mitotic states occurs asynchronously in culture; (2) that this transition is not mimicked by imposing an external cell cycle block; and that (3) medium factors cannot be demonstrated which actually change the commitment of a particular cell, allow the conclusion that this form of growth control is uniquely part of a compartmentalized program for differentiation. Further, the extent of differentiation observed in vitro results from the cooperative effects of both short-range and interim growth controls. One application of this concept will lie in the realization that cases of uncontrolled or neoplastic growth, particularly those that generate recognizable cell types, may result from different lesions Exp CellRes 107(1977)
364
Dienstman
and Holtzer
in very different mechanisms of growth control. This work was supported by the USPHS (HD-00189), the Muscular Dystrophy Association of America, and the ACS (VC 45A).
REFERENCES 1. Abbott, J, Schiltz, J, Dienstman, S & Holtzer, H, Proc natl acad sci US 71 (1974) 1506. 2. Bischoff, R & Holtzer, H, J cell bio136 (1%8) 111. 3. - Ibid 41 (1969) 188. 4. - Ibid 44 (1970) 134. 5. Buckingham, M, Caput, D, Cohen, A, Whalen, R & Gros, F, Proc natl acad sci US 71 (1974) 1466. 6. Buckley, P & Konigsberg, I, Dev biol 37 (1974) 193. 7. Capers, C J, Biophys biochem cytol7 (1960) 559. 8. Coleman. J & Coleman. A. J cell __ nhvsiol72. suuul. __ i (1968) is. 9. Dienstman. S R, Ph.D. thesis, Universitv of Pennsylvania, Philadelphia (1974): 10. - J cell bio163 (1974) 83. 11. Dienstman, S R,.Biehl, J, Holtzer, S & Holtzer, H, Dev bio139 (1974) 83. 12. Dienstman, S R & Holtzer, H, Results and problems in cell differentiation (ed J Reinert & H Holtzer) vol. 7, p. 1. Springer-Verlag, Heidelberg (1975). 13. Doering, M & Fischman, D, Dev biol 36 (1974) 225. 14. Hayflick, L, Exp cell res 37 (1%5) 614. 15. Holley, R & Kieman, J, Proc natl acad sci US 60 (1968) 300. 16. Holtzer, H, Cell differentiation (ed 0 Scheijde & J DeVillio). Van Nostrand & Reinhold. New York (1970). ’ 17. Holtzer, H, Abbott, J & Lash, J, Anat ret 131 (1957) 576. 18. Holtzer, H, Croop, J, Dienstman, S, Ishikawa, H & Somlyo, A, Proc natl acad sci US 72 (1975) 513. 19. Holtzer, H, Rubinstein, N, Chi, J, Dienstman, S R
Exp Cell Res 107 (1977)
& Biehl, J, Exploratory concepts in muscular dystrophy (ed A Milhorat). Excerpta Medica, Amsterdam (1975). Holtzer, H, Strahs, K, Biehl, J, Somlyo, A & 20’ Ishikawa, H, Science 188 (1975) 943. 21. Holtzer, H, Weintraub, H, Mayne, R & Mochan, B, Current topics in developmental biology (ed A Moscona & A Monroy) vol. 6, p. 229. Academic Press, New York (1972). 22. Konigsberg, I, Organogenesis (ed R DeHaan & H Ursprung) p. 337. Holt, Rinehart & Winston (1%5). 23. - Dev bio126 (1971) 133. 24. - Concepts of development (ed J Lash & R Whittaker) p. 179. Sinauer Associates, Stamford (1974). 25. Lough, J & Bischoff, R, Dev biol50 (1976) 457. 26. Moss, P & Strohman, R, Dev bio148 (1976) 43 1. Offer, G, Proc roy sot, ser. B 192 (1976)439. LG. O’Neill, M & Stockdale, F, Dev bio129 (1972)410. 29: Pardee, A, Proc natl acad sci US 72 (1974) 1286. 30. Pardee, A, Jimenes de Asua, L & Rozengart, E, Cold SorinprHarbor conf in cell proliferation (ed B Clark& g R Baserga) vol. 1, p. 547. Cold Spring Harbor (1947). 31. Paterson, B & Strohman, R, Dev biol 29 (1972) 113. 32. Pierson, R & Temin, H, J cell physiol 79 (1972) 319. 33. Prives, J & Paterson, B, Proc natl acad sci US 71 (1974) 3208. 34. Przvbvla. A & Strohman., R., Proc natl acad sci US 71 (?9?4)‘664. 35. Rubin. H. Cell communication (ed R Cox) D. 127. Wiley; New York (1974). 1 ’ a 36. 727, J, Holtzer, H, Nature new bio1229 (1971)
_-_.
37. Stockdale, F & G’Neill, M, In vitro 8 (1972) 212. Temin, H, J cell physio169 (1%7) 377. 3 -Ibid 78 (1971) 161. 40: Yaffe, D & Dym, H, Cold Spring Harbor symp quant bio137 (1973) 543. 41. Yaffe, D & Feldman, M, Dev biol 11 (1%5) 300. Received March 1, 1976 Accepted February 18, 1977
Experimental Cell Research lOl(l977)
SKELETAL
MYOGENESIS
Control of Proliferation S. R. DIENSTMAN’ Departments
355-364
in a Normal Cell Lineage and H. HOLTZER
of Biology and Anatomy, University Philadelphia, PA 19174, USA
of Pennsylvania,
SUMMARY The fates of IO-day chick embryo myogenic cell populations cultured in conditions that maximize or suppress proliferation or fusion were examined. The repression of the cell cycle, prevalence of fusible cells, or expression of myofibril formation were monitored by autoradiography, counts of myotube nuclei, staining with fluorescein-labelled antibody against skeletal myosin heavy chain, or electron microscopy. Using a calcium chelator (EGTA) to block cell fusion did not prevent the accumulation of myoblasts blocked in Gl of the cell cycle that initiated skeletal myosin synthesis and myofibril formation. Trypsinization, dilution, and daily feeding with fresh medium and serum did not reverse this cell cycle block. Using cytochalasin B (CB) as an alternative fusion block confirmed these results. Using fluorodeoxyuridine (FUdR) to prevent the cycling of myogenic cells that normally would have multiplied in vitro did not prompt these cells to fuse. Muscle-conditioned medium could not prompt a switch in the commitment of replicating cells when in Gl to terminal differentiation. The indications are that myogenic cell populations contain definite mixtures of precursor phenotypes. The terminal phenotype is initiated coordinately with a relatively stable cue for the repression of the cell cycle. Differentiation-specified growth control is discussed within the context of a set of growth control mechanisms known to operate in culture.
The propagation in vitro of various normal and transformed animal cells has emphasized two mechanisms of cellular growth control: (1) long-range growth control, thought to represent “aging” on the cellular level [14], and, (2) interim growth control, which is a function of the nutrition or environment of the cells in question and is keyed into the cell cycle [15, 30, 351. Both mechanisms halt proliferation. In the first case, this occurs through a slow dying-out of the culture. By contrast, in the second case, the transition from growing to resting states occurs more synchronously, can be 1 Present address: Department of Pathology, New York University, School of Medicine, New York, NY 10016,USA.
reversed, and involves cell types that never permanently repress the cell cycle. Holtzer et al. [21] and Dienstman & Holtzer [12] have proposed another case of cellular growth control. Counting time from the establishment of the zygote, this third mechanism is a relatively short-range case of growth control. For example, within the chick embryo, proliferation ceases among subpopulations of skeletal muscle and nerve cells within the first 48-72 h of incubation and by the 5th day, there are in addition large numbers of post-mitotic red blood cells, skin cells, and pancreatic cells. Overlapping the halt in proliferation is the appearance of new cell products, marking the acquisition of new differentiated states for the cells in question. This type of represExp
Cell
Res
107 (1977)
356
Dienstman and Holtzer
sion of the cell cycle, specified in differentiation, is generally irreversible. Owing to its adaptibility to culture, the existence of well-defined markers for the differentiated state, and the derivation of cell lines, skeletal myogenesis has lent itself to studies of growth and differentiation. Many laboratories have documented the following: (1) The differentiation of skeletal muscle in vivo or in vitro entails a transition from a population of mononucleated cells to a population of multinucleated myotubes; (2) the multinucleated condition results from the fusion of mononucleated cells; (3) in any inoculum, some mononucleated cells in Gl fuse directly, while the rest replicate and contribute to myotube formation through their progeny; (4) the nuclei in myotubes are post-mitotic [3, 7, 16, 17,411. What governs the transition from the growing to the fixed Gl state, however, has remained a matter of controversy. Konigsberg [22-241 and Buckley & Konigsberg [6] have inferred from the available data that (1) all the mononucleated myogenic cells are capable of continuous proliferation; but (2) high cell density, high concentration of conditioning factors, or prolonging the time spent in Gl can induce these cells to fuse; and (3) only as a consequence of cell fusion do the muscle cells become fixed in a post-mitotic state and initiate the synthesis of the definitive contractile proteins. Essentially similar conclusions have been reached by O’Neill & Stockdale [28, 371, Doering & Fischman [13], and others [5, 31, 33, 34, 401. For a critical review of these experiments see Dienstman & Holtzer [ 121. The experiments to be reported here demonstrate that (1) fusion is not the cause of the repression of the cell cycle in myogenesis; and (2) the post-mitotic state is initiated coordinately with other differenExp Cell Res 107 (1977)
tiated functions in a relatively inflexible program. Additional experiments show that precursor myogenic cells, which are normally committed to replicate, cannot undergo precocious differentiation by inhibiting DNA synthesis or by applying conditioned medium for 48 h. These experiments have in part been reported preliminarily [9, lo].
MATERIALS
AND METHODS
Mononucleated myogenic cell populations were iso_ lated from lO-day embryonic chick breasts and cultured similarly to the methods of Bischoff & Holtzer [2]. However; inocula were plated at a concentration of 2x 10” cells/35 mm dish in collagen-coated dishes and were fed daily with fresh medium. Fresh medium (8 : 1: 1) consisted of 8 parts Eagle’s minimum essential medium, 1 part horse serum: 1 part chick embryo extract, and 1% antibiotics. Under these conditions, control cultures sustained 3-4 doublings during the first 5 days in vitro. These cultures contained nonmyogenic, Bbrogenic cells as well, which accounted for most of the population increase after the third culture day [ 1,9]. Eorsome cultures, conditioned medium (CM) was used instead of fresh medium. CM was recovered from muscle cultures (5X 105cells plated in 60 mm dishes with 3 ml of medium) after 48 h, grown without a change of medium at 24 h; at this time, the fusion period was already underway. 1.75 mM EGTA was found to prevent muscle cell fusion, but would not cause discernable cell death or inhibit mitosis. EGTA was added to cultures from the time of plating [lo]. Cytochalasin B (CB) (5-10 pg/ml) also was used as a fusion inhibitor [lo]. In order to identify the subpopulation of cells in the inoculum which synthesized DNA and proliferated, 0.25 &i/ [3H]TdR (40-60 Cilmmole) was added to each culture at the outset and/or upon the change of the medium. Autoradiography was performed to detect labeled nuclei in mononucleated cells and multinucleated myotubes. FUdR (1O-BM) was added to cultures from the outset in those experiments designed to inhibit proliferation from the outset [4, 81. By this means, up to 90% of the surviving myogenic cells can be stalled at the Gl/S interface [25]. For following fusion, the standard approach of counting the number of nuclei in myotubes versus nuclei of all cells was used. However. for the analvses described in this report, a more informative approach was obtained by combining rSH]TdR labelling with counts of myotube nuclei, both labelled and unlabelled, in autoradionraohs at one fixed time. This procedure allowed th; perception of cell cycle activities in myogenesis taking place early in culture, although the processes of differentiation could be allowed to mature up to 4 or 5 days more. Thus comparisons always were made between populations of
Controls on proliferation
1. Cultures were grown for 4 days with daily feeding of fresh medium containing EGTA at a level to inhibit fusion but not replication. Multinucleated myotubes did not form; highly elongate mononucleated cells accumulated that tended to pack side by side in parallel fashion. Phase contrast, x 10 obj. Bar, 100pm.
Fig.
in muscle
357
large numbers of highly elongated mononucleated cells but no myotubes (fig. 1). In control cultures about 60% of all nuclei were in myotubes and thereafter there was little increase in numbers of myotubes or their nuclei. Accordingly, on day 4 the fusion block in the experimental cultures was broken by removing the EGTA and adding fresh medium containing calcium as well as r3H]TdR. Cultures were maintained for an additional 5 days, then fixed and autoradiographed. All the nuclei in the myotubes which formed subsequent to the removal of EGTA were unlabelled (fig. 2). Thus, the cells that fused between days 4 and 9 must have been held in Gl of the cell
myotubes which were more mature and more uniform in shape and age. The criteria of Bischoff & Holtzer [3] were used to score nuclei in myotubes and each experiment was repeated at least six times. Given the biological and stochastic variation between experiments, the data presented are from individual repetitions. Conclusions drawn from any one were representative of the others. Surveys of nuclear labelling and fusion were done on 48 microscopic fields in duplicate cultures to obtain a mean and variance for each data point. The properties of the fluorescein labelled antibody against skeletal myosin heavy chains have been described elsewhere [ 19,201. This antibody is specific for myosin heavy chains only from skeletal muscle; it does not cross react in Ouchterlony double diffusion chambers or histochemically with the myosins from smooth muscle myoblasts, fibroblasts, nerve cells or presumptive myoblasts. This antibody does not crossreact with C-protein [27]. The nomenclature used in regard to cell types is the same as described previously [l, 11, 121. (1) Mb cells are the post-mitotic mononucleated myoblasts that have initiated the synthesis of the definitive myosin heavy and light chains; (2) PMb cells are of the replicating precursors to the myoblast; (3) Fb cells, the associated tibroblasts; and (4) PPMbFb cells, the common precursor to these myogenic and fibrogenic cells.
RESULTS To determine whether the repression of the cell cycle in myogenic cells is dependent upon fusion, or whether it occurs in the mononucleated cell, cultures were set up in the presence of EGTA (1.75 mM). Cultures grown in EGTA for 4 days contained
Fig. 2. After inhibiting fusion for 4 days the EGTA
block was broken and cultures were kept an additional 5 days in the presence of [3H]TdR, then fixed and autoradiographed. Here, such a 9-day culture reveals that by 4 days in vitro the myogenic population was made up of post-mitotic cells which fused to form myotubes having unlabelled nuclei. (a) Phase contrast; (b) bright field; x 16 obj. Bar, 50 pm. Exp CellRes 107(1977)
358
Dienstman
and Holtzer
appear in subculture after 48 h. Subcultures were maintained for 4 days, fixed, and autoradiographed. Virtually all (94 %) the nuclei in myotubes that formed in subculture were unlabelled (fig. 3, III). The repression of the cell cycle in those cells was stable to the effects of trypsinization, to the lowering of cell density, and to daily application of fresh medium. At the same time under these conditions, the operationally non-myogenic cells did replicate. Fig. 3 shows that the Fig. 3. Ordinate: %. 0, Total nuclei; 0, labelled nu- change of conditions so favored the growth clei. PPMbFb-PMb-Mb transitions occur over a 4-dav of replicating cells that they eventually period in vitro without fusion or density requirements: primary cultures grown in EGTA for 2 (I), 3 (II), or dominated the subcultures. 4 (III) days were suspended and replated in fresh meUsing EGTA-blocked primary cultures, dium with calcium and lSH]TdR and grown for 4 more days. The subpopulation of unlabelled myotube nuclei other diluted subcultures were established is equivalent to the mononucleated myogenic fraction in the inoculum which fuses directly, i.e., the un- at times earlier than 4 days. As early as day striped compartments in the “myotube” columns com- 2, about 35 % of the post-mitotic myoblasts prise the fraction of muscle contributed by Mb cells had been generated (fig. 3, I). The progresalready generated in primary culture prior to replating and introduction of label. sive course of proliferation and terminal difLyIbelled myotube nuclei]/[All myotube nuclei] ferentiation was such that the frequency of atsuvxudDfblat smcu~ture +[Mb]genera,ec, ,nsu,,culture: for I, 35%; II, 70%; III, 94 %. The frequency of Mb replicating myogenic cells diminished concells increased while the PPMbFb or PMb declined. siderably by day 3, and by day 4 relatively few new replicating myogenic cells were formed. The transition from a replicating to cycle since day 4. Clearly, repression of a non-replicating myogenic cell population the cell cycle occurs prior to and inde- occurred steadily but asynchronously. pendently of fusion. That the myogenic cells that accumulate Equally important in the above experi- in EGTA-treated cultures are Mb cells and ment, the autoradiography showed that can initiate the synthesis of definitive skelemost mononucleated cells did incorporate tal myosin heavy chains is shown in fig. 4. the label. Hence those cells that had the Using fluorescein-labelled antibody against skeletal myosin heavy chains mononucapacity to proliferate could do so. It might be argued that the halt in pro- cleated cells with cross-striated myofibrils liferation in the muscle cells occurred be- were first evident on day 3 of culture and cause after 4 days in vitro, the myogenic such cells increased in numbers with time. population had reached some critically high Mb cells usually were highly elongated. The cell density that favored fusion over pro- more angular cells, the myogenic preliferation. Accordingly, additional cultures cursors and fibroblasts, did not bind the kept in EGTA for 4 days were trypsinized, fluorescein-labelled antimyosin. On day 2, diluted, replated, and grown in fresh me- very few elongate cells bound the antibody. dium along with [3H]TdR. Extensive DNA Post-mitotic myoblasts also should acsynthesis and cell division occurred, and cumulate with any other agent that blocks the first oligonucleated myotubes began to fusion per se. Also, these cells should disExp Cell Res 107 (1977)
Controls on proliferation in muscle
359
Fig. 4. Low density muscle cultures grown on cover-
slips were glycerinated and stained with fluorescent anti-myosin to detect the synthesis and distribution of skeletal myosin. (a) High power view of a crossstriated myotube in an uninhibited .5-day culture, positions of nuclei are the unstained oval regions; (b) after 3 days in EGTA-blocked cultures mononucleated myoblasts are present which have delicate, apparently single myotibrils; (c, a’) low power view of aCB-treated culture in which round cells specifically bound the antibody. Glycerination and subsequent washing of (B-treated cultures tended to easily detach many round and arborized cells. (a, b, d) Fluorescence microscopy; (c) phase contrast; high power, x40 obj.; bar, 10 pm; low power, x 16 obj.; bars, 50 Wm.
play the capacity to synthesize and organize the definitive contractile proteins into interdigitating thick and thin filaments. Cytochalasin B (CB) is an alternative agent known to block fusion [18, 361. CB was added to a series of cultures between days 2 and 3 to “trap” unfused mononucleated cells and to study their properties. Cells in primary cultures respond to CB in two ways: replicating fibroblasts and presumptive myoblasts retract and form “arborized” cells, whereas post-mitotic myoblasts form rounded cells, many of which detach from the substrate and float into the medium (fig. 5). These round myoblasts have been (1) treated with labelled antibodies against skeletal myosin; (2) inspected by electron microscopy. After 2 days in CB, round cells could be
observed to contract or “beat” spontaneously. Fig. 4 shows that many round cells strongly bound the antibody to skeletal myosin heavy chains. At this time, about half of the adherent population were rounded, and of these cells, most if not all were antibody-positive. Fig. 6 shows that in these cells, although they are rounded, there are interdigitating arrays of thick and thin filaments. Arborized cells were not observed to contract; they did not bind the antibody nor produce thick and thin filament arrays. Each round cell contained only one nucleus, although bi-, tri-, and oligo-nucleate arborized cells did occur. Experiments were designed to disclose the number of myogenic cells (Mb) in control cultures which fuse without any rounds of DNA synthesis in vitro. [3H]TdR was ExpCellRes 107(1977)
360
Dienstman and Holtzer
5. Cultures 2 days old were placed in CB for an additional 2 days. Differential responses to the drug result in cells of distinctly different morphologies: round cells sitting above the substrate and attached to it at apparently one point and arborized cells adhering closely to the substrate with flat arms around a seemingly compressed cell body. Phase contrast, x 16 obj. Bar, 50 pm.
Fig.
present from the outset of culture, and after 4 days, cultures were fixed and autoradiographed. About 4-10% of myotube nuclei were free of label (e.g. 7 %, table 1, ser. I). This corresponds to a frequency of 15-20 %
of cells in the original inoculum which fused directly (i.e., final number of unlabelled myotube nuclei/inoculum size/dish). Table 1, ser. II was designed to test two points. (1) Does merely blocking proliferation and prolonging the time spent prior to DNA synthesis produce greater numbers of cells which fuse directly as compared with the controls? (2) Do precursor cells retain their commitment to replicate and afterward generate differentiated progeny, in spite of the exogenous inhibition on the cell cycle? Cultures were kept in FUdR for the first 48 h in vitro. At 48 h, the FUdR block was broken and culture continued for another 2 days in medium with unlabelled TdR and [3H]TdR. Cultures were fixed after a total of 4 days and autoradiographs made. Table 1 shows that overall fusion was suppressed as a result of the FUdR (ser. II) compared with uninhibited controls (ser. I). However, the number of cells that did express an option to fuse directly in the FUdR-treated cul-
Table 1. Myogenic cells which fuse without replication in vitro Lack of effect of inhibiting DNA synthesis
Days in FUdR
Davs whhout FUdR, in [3H]TdR
0
4
Series IIc
2
2
Series IIId
2
2
Series
1”
Unlabeled total myotube nuclei/dish 29OOOf 6300 415 OOOf55000 32OOOf 8700 36OOOf 9400 31OOOf 8400 37 000+10 000
As % of total myotube nuclei 7 % unlabeled 90 % unlabeled 83 % unlabeled
(1 The subpopulation of unlabelled myotube nuclei is equivalent to the mononucleated myogenic fraction in the inoculum which fuses directly. Values per dish obtained from extrapolating averaged counts (mean f95% confidence interval) of microscopic fields in duplicate cultures. The data then are absolute frequencies of nonreplicating myogenic cellslinoculum of 2x lo5 cells/dish. b Fresh medium. c Fresh medium; lOmEM cold TdR supplement during the release period. d Conditioned medium; then fresh medium+ lOmeM cold TdR during the release period. Exp Cell Res 107 (1977)
Controls on proliferation
Fig. 6. Although Mb cells were unable to fuse in CB,
they tended to occur singly or in clusters of round cells. (a) Electron micrograph of one section through a round cell. Notable in these CB-treated cells is an apparent “segregation” of the cytoplasm with filaments
tures was no greater than it was in controls. No myogenic cells that normally would have replicated were prompted to fuse. During the recovery interval, some cells did generate progeny that fused. However, multiple factors likely combined in table 1, ser. II producing less total fusion even after the recovery interval than was seen in controls. First, there was greater overall proliferation over a 4-day period in controls than occurred over only 2 days in FUdRtreated cultures. Another possible cause was the inability of some precursor cells, which were geared for replication, to survive the externally imposed block in the cell cycle over long intervals [29]. Ser. III was set-up to repeat the experiment of ser. II, but using conditioned medium (CM) plus FUdR. The use of CM would reveal whether it contained any fac-
in muscle
361
peripherally. x9600; (b) enlargement showing the absence of sarcomeres with normal Z disks in CB, but the presence of interdigitating thick and thin filaments and dense bodies instead (arrow).
tors which could prompt any of the myogenie precursor cells to fuse. Comparing the numbers of fusible cells in ser. III with those in ser. I and II shows that no more cells fused with CM than without it; CM did not possess the quality to “switch” FUdR-treated cells from a commitment to replicate to a commitment to fuse in G 1. In conclusion, inocula of myogenic cells taken from the embryo and placed in these various conditions in vitro contain at least two subpopulations: a small group of postmitotic, fusible cells (Mb), and a group(s) of precursors, which only in the normal course of proliferation generate up to 90% of the fusible cells. Prolonging the time prior to DNA synthesis either with FUdR or with CM plus FUdR does not prompt the differentiation of the myogenic precursors. Exp CeURes 107(1977)
362
Dienstman and Holtzer
across a series of cell cycles (see [21] for this type of control during erythrogenesis). These experiments demonstrate that pri- Many investigators have demonstrated the mary muscle cultures consist of a popula- activity of interim growth controls on replition of cells that is heterogeneous with cating cells whose phenotypic activities do not appear to require transition in nor rerespect to its capacities for differentiation and growth control. It is very misleading to pression of the cell cycle. Typically interim analyse such cultures as if they consist of growth control in culture is readily affected pure populations of “myoblasts” and by changes in cell density, large and small “fibroblasts”. Replicating myogenic pre- molecular weight molecules in the ambient cursor cells can neither fuse themselves, medium, trypsinization, insulin-like molesynthesize skeletal myosin, nor assemble cules, etc. [32, 38, 391. Given this multiplicity of growth conmyofibrils. Cloning experiments have demonstrated that, at a minimum, there are two trols, the observation that the relative prosuccessive classes of replicating precursors portion of terminal skeletal muscle generin the myogenic lineage, the PMb and the ated in vitro varies with culture conditions PPMbFb [ 1, 111.Further proliferation, how- does not demonstrate that environmental ever, generates a distinctly different pheno- factors directly and immediately control the type, the post-mitotic, mononucleated myo- basic events of differentiation. For exblast. Myoblasts possess the capacity to ample, in the experiments using FUdR and fuse and to organize the definitive contrac- CM, the percentage of muscle nuclei tile proteins into interdigitating thick and formed by Mb cells fusing directly, seems thin filaments. Their repression of cell cycle greatly enhanced over control levels alactivity, linked to differentiation, is not af- though in actuality neither treatment profected by daily use of fresh medium, by motes any significant increase in the fretrypsinization, by reculture at lowered den- quency of Mb cells (table 1, col. 3 vs 4). sity, or by factors in CM. Similarly, Doering & Fischman [13] have Using myogenesis as a paradigm, it is to reported that the “% fusion” in culture rises be stressed that the outcome for differentiaover control levels following 48 h treattion in vitro or in vivo particularly from ments with cytosine arabinoside. This was mixed populations of precursor cells, rep- interpreted to mean that otherwise repliresents an interplay of variables. Each vari- cating cells were cued to fuse. However, as able singly may affect primarily one of the this agent is known to kill replicating cells, subpopulations in a lineage. The roles of the “% fusion” index must increase in the proliferation, interim growth control and presence of cell cycle inhibitors, though the differentiation-specified growth control inhibitor would have no effect on the state must be separated, identified, and tested in of differentiation of the surviving cells. assessing variation in any developing sysA significant element in the EGTA and tem. The growth controls that act on the CB studies reported here was the use of exreplicating myogenic cells and fibroblasts perimental approaches that tended to maxido not act on the myoblasts. These interim mize proliferation. Paterson & Strohman growth controls appear to operate within [31] had claimed to observe a decline in the each cell cycle, unlike differentiation-speproportion of myogenic cells incorporating cified growth controls which appear to act r3H]TdR in cultures released from EGTA at DISCUSSION
Exp CellRes 107(1977)
Controls on proliferation in muscle successively later times. However, this was seen under conditions chosen to minimize proliferation. The decline in cumulative labelling in their cultures correlated to incubations in [3H]TdR of shorter and shorter length. Thus, the minimum incorporation of labelled nuclei into myotubes was obtained after allowing only a period of 10 h for both labelling and then fusion. Under any circumstances, 10 h is the minimal interval for such an event to occur [3]. In the experiments reported here, proliferation was maximized while the decrease in labelled myotube nuclei was found after sufficiently long and uniform time intervals. It is notable that in their conditions, Moss & Strohman [26] do not find myofibril formation in any cells concurrent with the proliferative decline they observe. It has been shown that FUdR or FUdR and CM did not prompt fusion. Using FUdR alone, aside from the cells trapped in S phase at the time of application, the majority of cells should have accumulated in Gl at the Gl/S interface. It can still be argued that replicating myogenic cells may pass through an option in Gl, albeit unexpressed, for fusion. However, it would also have to be argued that this option is passed early in Gl , certainly earlier than the GllS interface and probably earlier than some CM-sensitive restriction point in the cell cycle. This is also supported by Bischoff & Holtzer’s finding [3] that fusion occurs late in Gl, later than the time most replicating cells become committed to DNA synthesis. Medium conditioning may act on myogenesis in other ways aside from effects on interim growth controls for one or more subpopulations of replicating cells. CM may mimic the effect of collagen by simply promoting the attachment of cells to the substrate. Hypothesized molecules in CM might promote migration or fusion, the fre-
363
quency of collisions, or the actual melding of myoblast surfaces, once these cells have been generated. One hypothetical possibility which will merit future investigation is that CM may affect the frequency with which PMb yield Mb cells rather than more PMb cells. It may be expected that CM obtained through different protocols should differ in some ways. Eagle’s medium has been combined with horse serum and embryo extract in the proportions of 8 : 1: 1 (this report), 8 : 1.5 : 1 [6, 231, or 8 : 1: 0.5 [13] and collected after 42 [6, 231, 45 [13], or 48 (this report) h to prepare CM. It is likely that the differences among CM may be only of minimal degree. In summary, the evidence from chick skeletal myogenesis demonstrates a mechanism for short-range growth control which is relatively insensitive to environmental conditions. Repression of cell cycle function is coordinated with the initiation of a divergent set of synthetic activities characteristic of the terminal muscle phenotype. This fact, coupled with the observations that (1) the transition from proliferating to post-mitotic states occurs asynchronously in culture; (2) that this transition is not mimicked by imposing an external cell cycle block; and that (3) medium factors cannot be demonstrated which actually change the commitment of a particular cell, allow the conclusion that this form of growth control is uniquely part of a compartmentalized program for differentiation. Further, the extent of differentiation observed in vitro results from the cooperative effects of both short-range and interim growth controls. One application of this concept will lie in the realization that cases of uncontrolled or neoplastic growth, particularly those that generate recognizable cell types, may result from different lesions Exp CellRes 107(1977)
364
Dienstman
and Holtzer
in very different mechanisms of growth control. This work was supported by the USPHS (HD-00189), the Muscular Dystrophy Association of America, and the ACS (VC 45A).
REFERENCES 1. Abbott, J, Schiltz, J, Dienstman, S & Holtzer, H, Proc natl acad sci US 71 (1974) 1506. 2. Bischoff, R & Holtzer, H, J cell bio136 (1%8) 111. 3. - Ibid 41 (1969) 188. 4. - Ibid 44 (1970) 134. 5. Buckingham, M, Caput, D, Cohen, A, Whalen, R & Gros, F, Proc natl acad sci US 71 (1974) 1466. 6. Buckley, P & Konigsberg, I, Dev biol 37 (1974) 193. 7. Capers, C J, Biophys biochem cytol7 (1960) 559. 8. Coleman. J & Coleman. A. J cell __ nhvsiol72. suuul. __ i (1968) is. 9. Dienstman. S R, Ph.D. thesis, Universitv of Pennsylvania, Philadelphia (1974): 10. - J cell bio163 (1974) 83. 11. Dienstman, S R,.Biehl, J, Holtzer, S & Holtzer, H, Dev bio139 (1974) 83. 12. Dienstman, S R & Holtzer, H, Results and problems in cell differentiation (ed J Reinert & H Holtzer) vol. 7, p. 1. Springer-Verlag, Heidelberg (1975). 13. Doering, M & Fischman, D, Dev biol 36 (1974) 225. 14. Hayflick, L, Exp cell res 37 (1%5) 614. 15. Holley, R & Kieman, J, Proc natl acad sci US 60 (1968) 300. 16. Holtzer, H, Cell differentiation (ed 0 Scheijde & J DeVillio). Van Nostrand & Reinhold. New York (1970). ’ 17. Holtzer, H, Abbott, J & Lash, J, Anat ret 131 (1957) 576. 18. Holtzer, H, Croop, J, Dienstman, S, Ishikawa, H & Somlyo, A, Proc natl acad sci US 72 (1975) 513. 19. Holtzer, H, Rubinstein, N, Chi, J, Dienstman, S R
Exp Cell Res 107 (1977)
& Biehl, J, Exploratory concepts in muscular dystrophy (ed A Milhorat). Excerpta Medica, Amsterdam (1975). Holtzer, H, Strahs, K, Biehl, J, Somlyo, A & 20’ Ishikawa, H, Science 188 (1975) 943. 21. Holtzer, H, Weintraub, H, Mayne, R & Mochan, B, Current topics in developmental biology (ed A Moscona & A Monroy) vol. 6, p. 229. Academic Press, New York (1972). 22. Konigsberg, I, Organogenesis (ed R DeHaan & H Ursprung) p. 337. Holt, Rinehart & Winston (1%5). 23. - Dev bio126 (1971) 133. 24. - Concepts of development (ed J Lash & R Whittaker) p. 179. Sinauer Associates, Stamford (1974). 25. Lough, J & Bischoff, R, Dev biol50 (1976) 457. 26. Moss, P & Strohman, R, Dev bio148 (1976) 43 1. Offer, G, Proc roy sot, ser. B 192 (1976)439. LG. O’Neill, M & Stockdale, F, Dev bio129 (1972)410. 29: Pardee, A, Proc natl acad sci US 72 (1974) 1286. 30. Pardee, A, Jimenes de Asua, L & Rozengart, E, Cold SorinprHarbor conf in cell proliferation (ed B Clark& g R Baserga) vol. 1, p. 547. Cold Spring Harbor (1947). 31. Paterson, B & Strohman, R, Dev biol 29 (1972) 113. 32. Pierson, R & Temin, H, J cell physiol 79 (1972) 319. 33. Prives, J & Paterson, B, Proc natl acad sci US 71 (1974) 3208. 34. Przvbvla. A & Strohman., R., Proc natl acad sci US 71 (?9?4)‘664. 35. Rubin. H. Cell communication (ed R Cox) D. 127. Wiley; New York (1974). 1 ’ a 36. 727, J, Holtzer, H, Nature new bio1229 (1971)
_-_.
37. Stockdale, F & G’Neill, M, In vitro 8 (1972) 212. Temin, H, J cell physio169 (1%7) 377. 3 -Ibid 78 (1971) 161. 40: Yaffe, D & Dym, H, Cold Spring Harbor symp quant bio137 (1973) 543. 41. Yaffe, D & Feldman, M, Dev biol 11 (1%5) 300. Received March 1, 1976 Accepted February 18, 1977