the cell cycle, myoblast differentiation and prostaglandin as a developmental signal

DEVELOPMENTAL BIOLOGY 71.274-266 (1979)

The Cell Cycle, Myoblast Differentiation and Prostaglandin as a Developmental Signal

ROSALIND ZALIN

Department of Biochemistry, University of Sussex, Falmer, Brighton, Sussex BNl9QG, United Kingdom

Received July 7, 1978;; accepted January 30, 1979

The duration of the cell cycle in primary cultures of proliferating chick myoblasts was determined. The myoblasts were found to have a mean G1 of 2.7 hr, S of 6.0 hr, G2 of 2.8 hr, and M of 0.8 hr. lo-’ M Prostaglandin El (PGEI) produces a transient rise in the intracellular level of cyclic AMP and provokes a burst of precocious fusion 5-7 hr later in the myoblast cultures. Use was made of this finding to examine the possibility that the subpopulation of cells which responds to the rise in intracellular cyclic AMP is in a specific part of its cell cycle. [3H]Thymidine was added to the myoblast cultures at discrete intervals before exposure to the PG 34 hr after plating. Six and seven hours later (i.e., at 40-41 hr) the cultures were futed and autoradiographed and the appearance of labeled nuclei in myotubes was assessed. Only a basal level of labeled nuclei appeared in myotubes in control cultures and in those exposed to r3H]thymidine for less than 4 hr before PGEl addition. With increases in the time of exposure to [sH]thymidine above the minimum of 4 hr, increasing numbers of labeled nuclei appeared in myotubes. Taking into account the duration of the phases of the myoblast’s cell cycle, it is concluded that the cells which respond to PGEI by fusing 5-7 hr later are in the G1 phase of their cell cycle. From these and related results, a model of cellular decision making in myoblast differentiation is proposed.

INTRODUCTION

Considerable attention has been devoted to the study of cell differentiation. A prob- lem less well understood and equally important is the nature of the decisions that cells make between alternate pathways of differentiation. To arrive at a biology of cellular decision making, we need initially to know two things: When does the cell make decisions and how (in molecular terms) are the decisions made?

Embryonic skeletal muscle cells cultured in vitro offer a convenient system for the dissection of key processes in cell develop- ment. The myoblast population undergoes the same changes in morphology and protein synthesis characteristic of the differentiation process in vivo but separated from the complex interactions present in the whole organism and with a far higher degree of synchrony. The process in vitro is characterized by an initial phase of proliferation followed by cell fusion to form syncytia and the synthesis of a number of

2

0012-1606/79/08000274-15$02.00/O Copyright 0 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.

muscle specific proteins. Once inside a myotube, the myoblast nucleus does not undergo further DNA synthesis or division (Firket, 1958; Okazaki and Holtzer, 1956). Thus the differentiation of muscle in vitro involves a sharp transition from a population of rapidly growing single cells to large nondividing multinucleate units. This transition provides an excellent model for the study of cellular decision making.

The majority of myoblasts appears to be unable to fuse with each other upon contact in vitro until they have passed through one or more proliferative cycles. The length of the proliferative phase depends upon the conditions of culture. This behaviour suggests that the myoblasts have to undergo a covert change(s) in order to acquire the competence to fuse and that the timing of these changes is in some way dependent upon information from the cells’ environment.

The transition from the cycling to the fusing state in myoblasts must involve at

74

ROSALINII ZALIN The Cell Cycle 275

least two types of processes. First, each cell must undergo the changes necessary to in- itiate fusion; second, since fusion, by defi- nition, involves more than one cell, the population of fusing cells must achieve synchrony in their intracellular changes. There is evidence in other systems that decision making may occur concurrently in more than one cell (Gehring, 1967; Garcia-Belhdo et al., 1976).

In relation to the preparation of individual myoblasts for fusion, there is evidence to suggest that a transient rise in intracellular cyclic AMP is the signal within the cell which brings about the covert changes which result in fusion. We have reported previously the presence of a transient rise in intracellular AMP in chick myoblast cultures which occurs 5-6 hr before the onset of cell fusion (Zalin and Montague, 1974). Evidence for a causal connection between this change in cyclic AMP and myoblast fusion was then obtained’by making use of the ability of prostaglandin E1 (PGEI) to produce a similar transient increase in intracellular cyclic AMP. We found that the addition of PGE, (10-5-10-1” J4) to chick myoblasts not only produced a precocious rise in intracellular cyclic AMP but also caused a correspondingly precocious burst of cell fusion (Zalin and Leaver, 1975; Zalin, 1977).

These findings argue for a possible in- volvement of prostaglandin (PG) in the syn- chronization process. Aspirin (3 x 1O-4M) and indomethacin (lO-‘j M), inhibitors of PG synthesis, were added to myoblast cultures to determine whether PG is the physiological agent involved in coordinating the fusion decision. Both inhibitors produced a complete block in fusion which was totally reversed by the addition of 1O-5 M PGE (Zalin, 1977). Fusion occurred with a 5-hr lag after PGEl administration. There is am- ple evidence of the ability of myoblast populations to alter their culture medium in such a way as to promote their subsequent differentiation (Hauschka and White, 1971; Konigsberg, 1971). Prostaglandin produc-

tion and action offer an explanation at the molecular level for this phenomenon.

Knowing something of the molecular basis of the inter- and intracellular events associated with the decision to fuse, it is now possible to begin probing the decision- making process itself. We can ask: What state does the cell have to be in to be able to make this decision? One important as- pect of cellular state is the position of the cell in its division cycle. This question has already received considerable attention. However, in the absence of any information concerning the molecular nature of the myoblast’s decision to differentiate, definitive experiments to distinguish between different hypotheses have been difficult to achieve.

It is known that myoblasts fuse only in the G1 phase of their cycle, since all nuclei found in myotubes have a diploid amount of DNA (Strehler et al., 1963). Bischoff and Holtzer (1969), using conditions which shortened the average G1 period of the proliferating myoblast population, found that cells did not fuse sooner than 8 hr after the end of DNA synthesis, indicating that these fusing cells spent a longer time in G1 prior to fusion than the 2-hr average obtained for cells reentering the DNA synthetic phase (S) of the division cycle. These findings led Holtzer (1970) to postulate that the emer- gence of a presumptive muscle cell capable of differentiating is coupled in an obligatory fashion to a “critical” of “quantal” cell division. According to this view, a critical event occurs during the DNA synthetic phase or mitosis of a particular cell cycle which gives rise to a “postmitotic” competent myoblast. The longer duration of the final G1 before fusion is interpreted as the overt sign that the cell has passed through its critical cycle and is in Go. In contrast, on the basis of the marked effects that changes in the myoblasts’ culture medium have on both the timing and the extent of myoblast differentiation, other investigators stress the importance of environmental cues in regulating the transition from a proliferat-

276 DEVELOPMENTAL BIOLOGY VOLUME 71,1979

ing “stem” cell to a differentiated multinucleate (Konigsberg, 1971; O’Neill and Stockdale, 1972 a, b). The latter investigators have also suggested that cells do not withdraw from the mitotic cycle before entry into myotubes but rather as a result of the transition to the multinucleate state. In support of this interpretation are the more recent findings of Konigsberg and co-workers (Buckley and Konigsberg, 1974 a, b; Konigsberg, 1978) which demonstrate first, that the onset of differentiation is associated with an increase in the duration of the G1 phase of the myoblasts’ division cycle. Second, the increase in the length of G1 and the initiation of differentiation can be provoked in proliferating cultures by exposure of the cells to “conditioned medium” (i.e., culture medium taken from differentiating myoblasts). Third, all the single cells in the differentiating cultures are cycling, albeit more slowly than during the earlier proliferative phase of culture.

The aim of the present investigation is to reexamine the relationship between the myoblast’s cell cycle and its decision to differentiate and, specifically, to ask the question: Where is the myoblast in its division cycle when it responds to the presence of an environmental “cue” such as PGEl by fusing 5 hr later? The experimental approach adopted involved the addition of [3H]thymidine to myoblast cultures at discrete intervals before and after the addition of 10m7 M PGE1. Sufficient time was then allowed for the responsive cells to fuse, and the numbers of labeled nuclei in myotubes were assessed. The results reported in this paper demonstrate that G1, not S, Gz, or mitosis (M), is the decision-making period of the cell cycle.

mm-diameter Falcon tissue culture dishes coated with rat tail collagen. The medium used (Medium 199) was supplemented with 10% horse serum (Gibco Biocult Lts.), 2.0% chick embryo extract, glutamine (1 mJ4), penicillin (50 units/ml), and streptomycin (50 pg/ml), and the pH was adjusted by the addition of 5% NaHC02. In all the experiments described, the culture medium was changed only once at 24 hr. In the experiments in which the cell cycle parameters were assessed, the “conditioned” medium used was obtained from dishes containing cells cultured as described above. The medium was removed from cultures at the times corresponding to the [3H]thymidine pulse in the experimental group, then pooled, and cold thymidine was added in Medium 199 to give a final concentration of 5 x low7 M. This conditioned medium was then added to the experimental group to replace the medium removed from the latter at the end of the 30-min [3H]thymidine pulse.

r3H]Thymidine Labeling and Autoradiography

MATERIALS AND METHODS

Skeletal Muscle Cell Cultures

[3H]Thymidine of specific activity 16 Ci/ mmole was added in 0.1 ml of Medium 199 to each culture dish to give an activity of 0.5 @i/ml. At the end of the 30-min incu- bation the medium was aspirated off the dishes and rinsed twice with physiological saline, care being taken not to knock or shake dishes in order to minimize loss of mitotic cells. The cells were then fixed in methanol and air-dried. Photographic emulsion (Kodak Type NTB2) diluted 1:l with distilled water at 45°C was added to the dishes and poured out again and the plates were stacked vertically at room temperature until the emulsion was dry. The plates were then transferred to a light-tight box and stored at 4°C for 2 weeks. At the end of the exposure period, the plates were developed in Kodak DlOB, diluted 1:4 with water, and kept at 16°C. The plates were then washed with water and treated with

Cells were prepared from ll- or 12-day embryonic chick thigh muscle using the method of Konigsberg (1967). The cells ,. were seeded at a density of 2 X 10” in 90- Kodak fixer.

HOSALIND ZALIN The Cell Cycle 277

Staining and Microscopic Examination

(a) Labeled Mitoses. To determine the mitoses labeled, the autoradiographed plates were stained with Erhlich acid-hematoxylin for approximately twice the time required under control conditions, air- dried, and then coated with 5% polyvinyl alcohol in distilled water. The addition of the polyvinyl coat reduced the refractory properties of the cell membranes and em- phasized the color produced by the stain, allowing easier identification of mitotic cells. Depending upon the resulting optics, examination was carried out either directly on the dishes or after removal of the cells on the polyvinyl coat and transfer to glass slides (Konigsberg, 1974b). Duplicate plates were taken at each time point, and using an eye-piece grid at a magnification of 250 X, a minimum of 200 mitotic nuclei was counted on each dish in random fields, making a total of 400 for each time point.

(b) Labeled nuclei in single and multinucleate cells. The autoradiographed plates were stained as above and the unlabeled and labeled nuclei in single cells and multinucleates assessed directly using an eye-piece grid at a magnification of 250 X. Total counts per dish of between 500 and 1000 nuclei were obtained by assessing 16 separate fields spaced evenly over the dish.

DNA Estimation

The DNA content of the cultures was measured using a modification of the method of Kissane and Robins (1958). Plates were rapidly rinsed twice at room temperature with 0.85% NaCl and the cells removed using a silicone rubber policeman before homogenizing in 1 ml of ice-cold 0.85% NaCl. Five-hundredths milliliter in triplicate was taken from the pooled ho- mogenates of three dishes and transferred to glass-distilled Hz0 rinsed tubes. The samples were then evaporated to dryness, incubated at 60°C for 45 min with 0.1 ml 3,5-diaminobenzoic acid previously decolor-

ized with charcoal, and filtered through a Millipore filter of pore size 0.45 pm. The reaction was stopped by the addition of 2 ml of 1 N HCl (Hinegardner, 1971) and the fluorescence then measured with an Amino-Bowman spectrofluorimeter (exci- tation, 405 nm; emission, 520 nm); 0.5-4.0 pg of calf thymus DNS (Sigma) was used to produce a linear standard curve.

RESULTS

Under the culture conditions adopted in the present study in which primary chick myoblasts were seeded at 2 x 10” cells in go-mm dishes and the culture medium was renewed after 24 hr, the majority of the myoblast population undergoes cell division prior to the onset of differentiation. Exper- iments were carried out to determine the cell cycle parameters of this proliferating population of myoblasts and the proportion of single cells undergoing cell division. These measurements were made during the period of culture after medium renewal at 24 hr and before the main onset of fusion which occurs at approximately 44 hr. This period of culture was chosen because it corresponds to that used in subsequent experiments to examine the myoblasts’ response to PGE,. The duration of the phases of the cell cycle were determined using the pulse-chase technique of Quastler and Sherman (1959). This procedure involves exposing cells to [“Hlthymidine for a short time to label those cells in the S period of their cell cycle and then following the pas- sage of this cohort of cells through its subsequent mitosis.

A number of precautions were taken with respect to these measurements. It has been demonstrated that the cell cycle time of quail myoblasts is sensitive to changes in the composition of their culture medium (Buckley and Konigsberg, 1974b). Of particular relevance to the present study is the finding that the addition of fresh medium at the end of the pulse produces a substan- tially shorter cell cycle time than medium that has been previously in contact with


the myoblasts. To minimize such effects in the present study, the medium used to chase the [3H]thymidine was taken from cultures of equivalent age and cell density. Second, both the specific activity of the r3H]thymidine used as a pulse and the concentration of excess nonradioactive thymidine added immediately afterward to dilute out the intracellular pool of r3H]thymidine have been found to be critical in avoiding an inhibition of cell growth (Buckley and Konigsberg, 1974b). [3H]Thymidine (specific activity, 6 Ci/mmole) was used in the present study at a final activity of 0.5 &i/ ml, since this level of activity gave a sufficient number of grains in the nucleus after a 0.5~hr exposure but had no effect upon cell growth. This concentration of r3H]thymidine requires 5 x lo-’ M “cold” thymidine to achieve a sufficiently rapid chase of the radioactive precursor pools (Buckley and Konigsberg, 1974b). To test the effect of 5 x 10e7 M thymidine on cell growth, the nucleotide at this concentration was added to equivalent myoblast cultures at 32 hr and the total DNA content of these and control cultures assessed at 4-hr intervals over the subsequent 16-hr period. No significant difference was obtained between the DNA increases in control (8.5 to 21.0 pg DNA/plate) and treated cultures (8.5 to 20.0 pg DNA/plate) between 32 and 48 hr. This concentration of cold r3H]thymidine was therefore adopted for the pulse-chase experiments. Finally, to eliminate the effects of any temperature change upon the myoblast cultures, all manipulations prior to fixation of the plates, including those outside the incubator, were carried out at 37°C.

To determine the average durations of the myoblast cell cycle phases, cultures were pulsed at 28 hr for 30 min with r3H]- thymidine at 0.5 &i/ml and chased with medium of equivalent age containing 5 X

lo-' M cold thymidine. Plates in duplicate were then f=ed at 2-hr intervals over the subsequent 20-hr period. After autoradiographic processing and Erhlich acid-he-

matoxylin staining, the mitotic figures, both labeled and unlabeled, were scored and the time course of the percentage labeled mitoses present was plotted (Fig. 1). The durations of the total cell cycle and of S were evaluated using the time points corresponding to the values of one-half the maximum of each peak; these estimates reflect the median cell cycle times. The time required to reach the midpoint of the first ascending limb of the labeled mitoses curve is a meas- ure of Gz + % M. The duration of mitosis (0.8 hr) was taken from the value obtained by Bishoff and Holtzer (1969) for primary chick myoblasts. G1 was then calculated from the values of the other cell cycle phases. The values obtained are given in Table 1, together with those obtained by Bischoff and Holtzer (1969) for primary chick myoblasts and Buckley and Konigs- berg (1974) for quail myoblasts. The average cell cycle time obtained in the present study is slightly longer than those obtained by the other investigators. The longer cell cycle may reflect a response to medium changes and preparations for the onset of differentiation (Buckley and Konigsberg, 1974b). The shorter cell cycle time of the quail myoblast population may be due to the fact that the determination of the cell cycle parameters in these cultures was carried out more than 24 hr earlier with respect

hours after pulse

FIG. 1. The percentage labeled mitoses present in the chick myoblast cultures with increasing time after a 30-min pulse of [3H]thymidine at 28 hr of culture. Labeled mitoses under control conditions (O--4); labeled mitoses after the addition of 10m7 A4 PGE, at time 0 (0- - -0).

ROSALIND ZALIN The Cell Cycle 279

TABLE 1 oblasts (Konigsberg, 1978). DURATION OF THE CELL CYCLE PHASES OF

DIVIDING CELLS FROM MYOCENIC CULTURES PGEl at 10m7 M, when added to the cul-

tures 34 hr after plating, produces discrete bursts of fusion 5 hr later which appear to last for approximately 2 hr (Fig. 3). How- ever, it is possible that cells, albeit at a slower rate, are continuing to fuse outside the 7-hr period. If this is the case, then an increased rate of fusion over the control is to be expected in the treated cultures during the 41- to 48-hr period (i.e., 7-14 hr after administration of PGEJ. In order to examine this possibility, the rates of fusion were determined by linear regression anal-

Phase Duration (hr)

Present studv

Bischoff and

Holtzer (1969)

Buckley and

S 6.0 4.3 4.8 G1 2.8 2.4 M 0.8” 0.8 2.0

G1 2.7 2.0 3.2 Total cycle 12.3 9.5 10.0

” This value was taken from Bischoff and Holtzer (1969).

to the onset of differentiation. A very sim- ysis of the experimental and control cul-

ilar time of culture was examined by Bis- tures between 41 and 48 hr. No significant

chaff and Holtzer, however, the use of fresh difference (at the 1% level) was obtained

medium to chase the [3H]thymidine may explain the shorter cell cycle time obtained by them.

The proportion of the total cell population cycling was assessed over the prefusion period of culture by the addition of [3H]- thymidine (0.5 @i/ml) at 32 hr. Duplicate culture dishes were then fixed at 2-hr intervals, put through autoradiographic processing, and stained with hematoxylin. Counts of total unlabeled and labeled nuclei (Fig. 2) reveal 85% of the total cell population labeled at 12.5 hr, the experimentally determined mean cell cycle time (Table 1). Over a 24-hr period, a further 10% of the cells entered S, giving a final percentage of 95. This result demonstrates the presence of a substantial number of cells with cell cycle times considerably longer than the mean value. The nonlinearity of the graph is also consistent with an increase in cell cycle times over the period examined. The cultures were not sufficiently dense to explain the slower rate of growth in terms of density inhibition. The period of culture examined spans the transition from rapidly growing single cells to nondividing syncytia. Thus the increase in cell cycle times may reflect an increase in the time that cells spend in the G1 phase of their cycle before differentiating, as is the case for quail my-

hcus of cdture

FIG. 2. The percentage of cells with labeled nuclei present in the chick myoblast cultures with increasing time after the addition of [“Hlthymidine at 32 hr.

I / 3s 42 4s

hours of cultve

FIG. 3. The effect of lo-’ M PGE, on myoblast fusion. No additions (O--O); +lO~’ M PGE, (M). Each point represents the mean value obtained from six separate plates. The fusion levels in the presence of the PG were significantly different from the control values (at the 0.054, level) 8-16 hr after its addition.


between the rates of fusion in the control and PG-treated cultures. Thus, if there are cells which take longer than 7 hr to respond to PGE1, they are insufficient in number to be detected by the method employed. Since the rate of fusion over the period of culture examined (i.e., 34-42 hr) is very low under control conditions, the subpopulation of cells which responds to PGEl by fusing can be studied. Use was made of this effect of PGE, to examine the possibility that the subpopulation of responsive myoblasts is in a specific phase of its cell cycle. Since interpretation of the results of such an experiment requires an accurate assessment of the duration of the cell cycle phases, it was necessary to examine the effect of lop7 M PGEl upon the cell cycle parameters. It has been shown from studies on a number of cell types (Millis et al., 1972; Nose and Katsuta, 1975; Willingham et al., 1972) that an increase in intracellular cyclic AMP in S or Gz can prolong the Gz phase of the cell cycle. To examine the effect of PG upon the appearance of labeled mitoses, cultures were pulsed at 32 hr with [3H]thymidine (0.5 $X/ml) as described above and then chased with medium containing 10e7 M PGE1. Duplicate dishes were then fixed at 2-hr intervals and the percentage of labeled mitoses was scored as described above. The rate of appearance of this cohort of labeled cells in mitosis is given in Fig. 1, represented by the broken line. PGEl delayed the entry of the cohort of labeled cells into mitosis by approximately 0.66 hr, giving a Ga value of 3.46 hr and a total mean time for cells to pass through GP and M of 4.26 hr. The percentages of labeled mitoses 10 and 12 hr after PGEl exposure are very similar to those under control conditions. The tran- sience of the PGEl effect upon entry of cells into mitosis presumably reflects the short duration of the rise in cyclic AMP it produces in the myoblast. Since the length of the S period is the same as under control conditions (Fig. l), it follows that the delay into mitosis reflects an increased duration

of Gz. The position in the cell cycle at which

the cells respond to 1O-7 M PGEl by fusing was monitored by adding [3H]thymidine (0.5 @i/ml) to the myoblast cultures at discrete intervals before and after their exposure to PGE, at 34 hr. Carrier was added with the [3H]thymidine to ensure that there was sufficient label until the termination of culture by methanol fixation. Duplicate plates for each time of [3H]thymidine addition were fixed 6 and 7 hr after PGE, exposure (i.e., at 40 and 41 hr). An outline of this experimental procedure with the times of all the additions is given in Fig. 4. After autoradiographic preparation and hematoxylin staining, the numbers of labeled and unlabeled nuclei present in myotubes were assessed. The same procedure was carried out on a control group not exposed to PGEI, in which r3H]thymidine was added at 26 and 30 hr, 8 and 4 hr, respectively, before the time of PGE, addition to the experimental group. The average cell numbers and percentage fusing cells obtained under both experimental and control conditions are given in Table 2. 10m7 M PGEl provoked an average increase in fused cells of 6.9% of the total cell population.

Total cell counts obtained from the 40-

FIG. 4. The experimental procedure adopted to examine the relationship between the myoblast’s cell cycle and its response to PGEI. (Asterisks) Times at which [3H]thymidine was added to control cultures not exposed to PGE,. (Arrows) Times of [“Hlthymi- dine addition.


TABLE 2

TOTALCELLNUMBERSANDPERCENTACEFUSION PRESENTIN CONTROLCULTURES~OAND~~HOURS AFTERPLATINGANDINCULTURESTREATEDWITH

10e7 MPGE AT 34 HOURS" Time of Mean cell numbers at [?J~- 40and41 hr Percentage fusion

_____

addition +PGE, -PGE, +PGE, -PGE, (hr of cul-

ture)

26 865 + 88 726 -t 134 19.5 + 3.5 7.6 k 1.6 28 668 + 75 13.1 f 0.8 30 804 it 155 709 + 164 16.2 f 2.9 8.9 + 1.2 32 738 + 46 13.0 k 2.8 34 686 rfr 72 15.5 + 0.6 36 675 -t 162 13.2 r 2.5

Mean 752 + 123 717 -c 139 15.1 -t 3.2 8.2 f 1.5 value

“Mean values -C SD were generated from four individual cultures. The cell numbers are the total present in 16 randomly selected fields.

and 41-hr control and PGE1-treated cultures were not significantly different (at the 10% level) (Table 2). This result makes it very improbable that the increased percentage fusion obtained in the PGEI- treated cultures is due to either cell death or an altered growth pattern. The basal level of fusion in the controls is the result of a slow rate of fusion which begins early in the cultures (unpublished finding). This is reflected in the very small percentage of labeled nuclei (mean value of 0.54% of the total nuclei in the cultures +- 0.37 SD of six individual measurements) incorporated into myotubes in the control cultures (Fig. 5). Numbers of labeled nuclei in myotubes significantly higher than those of the controls are seen in cultures exposed to r3H]- thymidine at 26, 28, and 30 hr (at the 1, 1, and 5% levels, respectively). Plotting the percentage labeled nuclei in myotubes after different times of [“Hlthymidine exposure reveals a steady increase in labeled nuclei in multinucleates after a minimum time interval of approximately 4 hr between [“Hlthymidine addition and the subsequent PGE, treatment (Fig. 5). These results demonstrate that a minimum gap of 4 hr is

required between the incorporation of [3H]thymidine and PG addition to see labeled nuclei in myotubes above control values. From these results we can conclude that there is a nonrandom response to PGE1, with respect to the cell cycle. The addition of [“Hlthymidine at the same time of culture as PGE, (i.e., at 34 hr) ensures that, of the cells responding to the PG, only those in S will be labeled. Since there were no labeled nuclei above the basal level in myotubes after this treatment, we can conclude that cells in S are unable to respond to the PG by fusing. Similarly, when [“HI- thymidine is added 2 hr before exposure to the PG (i.e., at 32 hr), the cells labeled at the time of PG addition will be those in S plus a smaller number of cells which were at the end of S when the label was added and have subsequently entered Gz. Again it was found that the nuclei present in myotubes (i.e., that responded to the PG) were unlabeled. This result confirms that cells in S do not respond to the PG and, in addition, demonstrates that the GZ cells are also un- responsive. A 4-hr preincubation with the [“Hlthymidine (i.e., addition of the label at 30 hr) will yield, at the time of PG exposure,

li t i

26 time of addition of [3fl-thymidine

FIG. 5. The labeled nuclei in myotubes expressed as the percentage of total nuclei in the cultures after different periods of exposure to [“Hlthymidine. lOmY M PGE, was added at 34 hr of culture and all the cultures were fixed in preparation for autoradiography and staining at 40 hr. Each point represents the mean of four separate plates + the standard error.

282 DEVELOPMENTAL BIOLOGY VOLUME ?'I,1979

labeled cells in S and Gg and, given the duration of Gz + M (3.6 hr), a small number of cells which have now reached G1. The 4- hr exposure to r3H]thymidine was found to be sufficient to give labeled nuclei in myotubes above the background level (Fig. 5). Since we know from the 34- and 32-h r3H]thymidine additions that cells in S and Gz do not respond, it follows that the labeled cells which have fused in response to the PG after the 4-hr preincubation are those which have just reached G1. Thus, the earliest point at which the cells are competent to respond is early G1; this is illustrated in Fig. 6.

A further minimum of 5 hr is required for the PGE1-sensitive cells to fuse (Fig. 3). Assuming that myoblasts fuse only in G1 (Strehler et al., 1963), it follows that these cells remain in G1 for a minimum of 5 hr. This is a considerably longer time than the mean G1 (2.7 hr) found for cycling cells. A similar conclusion was also reached by Bis- chaff and Holtzer (1969), on the basis of the time they observed between the incorporation of [3H]thymidine by a chick myoblast nucleus and its appearance in a myotube. From the present study, the total predicted minimum time required for the nucleus of a cell in S to enter a myotube is 8.6 hr, that is, the time it takes the cell to traverse Gz and M (i.e., 3.6 hr) plus the time to respond to PGEl (5 hr). This prediction was tested experimentally using the same procedure as described previously. [3H]- Thymidine at 0.5 #X/ml was added at hourly intervals over a 12-hr period to control cultures in which fusion was underway. All cultures (i.e., duplicates at each time of r3H]thymidine addition) were then fixed simultaneously and the distribution of labeled and unlabeled nuclei was assessed as described above. An increase in the number of labeled nuclei over a basal level was first seen after approximately 8 hr of exposure to [3H]thymidine (Fig. 7). Eight hours is therefore the minimum actual time required for a cell at the end of S to enter a

fused myotube and corresponds very closely with the calculated time required for a cell to pass through Gz and M and to respond to the PGEl signal. Further increases in time between the [3H]thymidine pulse and fixation resulted in larger numbers of labeled nuclei in myotubes. In both this experiment and that in which PGEl was used to elicit precocious fusion, only a few labeled nuclei appeared in myotubes at the minimum time required. If cells are able to respond to PGEl for only a short time after entry into G1, then it is to be expected that all nuclei entering myotubes will be labeled after a time delay equal to Gz (2.8 hr) + M (0.8 hr) plus the maximum time it takes cells to respond to PGEl (7.0 hr), i.e.,

FIG. 6. The myoblast cell cycle illustrating the duration of its phases and the times involved in its response to PGEI.

houm after addition of [31-&thymidine

FIG. 7. The numbers of labeled nuclei in myotubes with increasing time after the addition of r3H]thymidine to a fusing culture. Each point represents the number of labeled nuclei in myotubes present in 16 randomly distributed fields.

ROSALIND ZALIN The Cell Cycle

a total of 10.6 hr. That is to say that at the 12- and 14-hr time points in Table 3, the prediction is that the numbers of unlabeled nuclei in myotubes should equal those found in control cultures exposed to [3H]thymidine for the same length of time. However, this is not the case. Significantly larger numbers of unlabeled nuclei (at the 1% level) are found in myotubes in the PGE1-treated cultures at the 12- and 4-hr time points compared to those in the controls (Table 3), suggesting that cells are able to respond to PGE, for some time after entry into G,.

It is not possible from the above experiments to establish the precise time window in G1 during which the cells are responsive to PGE1. However, if cells were able to respond to PGEl at any point during G1, the prediction, assuming that the myoblast population is asynchronous with respect to the cell cycle, is that the proportion of the single cell population responding would correspond to the fraction of the total cell cycle time that is G1. Given a total cell cycle time

TABLE 3

THE APPEARANCE OF LABELED AND UNLABELED NUCLEI IN MYOTUBES WITH DIFFERENT TIME INTERVALS BETWEEN THE ADDITION OF [3H]-

THYMIDINE AND PGE,-PROVOKED FUSION” Total

time be- Numbers of nuclei in myotubes

tween Unlabeled I’Hlthv-

Labeled

‘m&line addition and ap-

pearance m myotubes

(hr)

AWI. % of total Aver. % of total No. in 16 cells in 16 No. in 16 cells in 16

fields fields fields fields

+PGE, 14 12

10

8

4

Control 14 10

135 + 43 15.4 -c 3.4 36f4 4.1 + 0.2 74 f 14 11.1 kO.9 14f5 2.1 +- 0.8

122 + 42 14.9 f. 2.4 11+7 1.3 f 0.6 93 -t 24 12.6 zk 2.6 4 + 2.6 0.5 -c 0.3 97k16 14.5 + 0.5 7f3.1 1.0 -c 0.31 go-c43 12.9 + 2.8 2f 1.4 0.3 f 0.26

53 f 19 7.1+ 1.3 4+3 0.5 f 0.3 42 + 29 9.8 + 3.0 3.7 + 2 0.6 -t 0.5

n Each time point represents the mean value (&SD) obtained from four seDarate elates.

283

of 12.3 hr and a mean G1 of 2.7 hr, one would expect approximately 22% of the population to respond to PGEl at any given time. This does not occur; only an average of 7.5% of the single cell population responds to the PG. It is unlikely that this result can be explained by postulating the presence of a certain degree of cell synchrony, since a similar percentage of cells is provoked to fuse by a second exposure to PGEl 3 hr after the first (Zalin and Leaver, 1976). It appears, therefore, that only a fraction of the cells in G1 responds to PGE, by fusing. The implications of this result are discussed more fully in the next section.

DISCUSSION

From previous work implicating a transient rise in intracellular cyclic AMP in the positive control of myoblast fusion (Zalin and Montague, 1974), we know something of the molecular nature of the intracellular change which brings about the covert changes resulting in fusion. In addition, we have evidence that the production and action of a PG are required in the myoblast cultures to generate the cyclic AMP signal within the myoblast cell (Zalin and Leaver, 1975; Zalin, 1977). It is generally accepted that PGs of the E series affect cell behavior by their ability to raise intracellular cyclic AMP by stimulation of adenylyl cyclase. There is now considerable evidence that this occurs via a specific external receptor (Tomasi, 1976). Thus these previous findings provide a mechansim by which the decision to prepare for the fusion event is made simultaneously by groups of myoblast cells. The present study has been concerned with when this decision is made by the myoblasts. Specifically, the question asked was: Where are the myoblasts in their division cycle when they respond to PGE, by fusing 5 hr later? The results obtained demonstrate that the subpopulation of myoblasts which responds to PGE, by fusing is

in the G1 phase of its cycle. From these and related findings, a model

264 DEVELOPMENTAL BIOLOGY VOLUME 7~1979

can be derived for the formation of the syncytium from single myoblasts. In proposing such a model it will be necessary to discuss the following points: (a) the importance of cyclic nucleotides and Ca” in the regulation of cell behavior; (b) how critical the timing of the signal is in relation to the cell cycle; (c) the proposition that early G1 is an ambivalent state. Finally, the proposed model will be compared with a previous one put forward by Holtzer (1970) with respect to their ability to explain the experimental findings.

The importance of the G1 phase of the cell cycle in cell growth and differentiation is well established. On the basis of a variety of evidence, it has been proposed that Ca2+ and cyclic nucleotide levels in the early part of the G1 phase of the cell’s cycle are critical in determining its subsequent behavior (Goldberg et al., 1974; Berridge, 1975; Whit- field et al., 1976). Although considerable evidence now exists which implicates an influx of Ca2+ into the cell in early G1 as the positive regulator of the cell cycle, there is only sparse evidence to confirm the proposed complementary role for cyclic AMP as a positive regulator of differentiation. Dibutyryl cyclic AMP has been shown to prolong G1 in a number of cell types and to promote the differentiated function of Chinese hamster ovary cells in the G1 phase of their cell cycle (O’Neill et al., 1976). However, the dibutyryl derivative of cyclic AMP can produce chronic and unphysio- logical increases in intracellular cyclic AMP, making it essential that the results obtained be interpreted cautiously. Just how misleading a result can be obtained is illustrated by comparing the effects of dibutyryl cyclic AMP and PGEl on chick myoblast fusion. The addition of dibutyryl cyclic AMP at 0.1 mM produced a marked delay in the onset of myoblast fusion (Zalin, 1973). However, examination of intracellular cyclic AMP levels at the time of onset of cell fusion revealed the presence of a transient rise in the cyclic nucleotide 5 hr

before the start of fusion (Zalin and Montague, 1974). Using PGEl to produce a similar transient rise in intracellular cyclic AMP, we were able to produce the opposite effect to that obtained with dibutyryl cyclic AMP, namely, a precocious onset of cell fusion (Zalin and Leaver, 1975). Thus these findings taken together with the results of the present study are important in provid- ing evidence for the positive regulation of differentiation by a physiological increase in intracellular cyclic AMP acting at a specific point in the cell cycle.

From a number of other findings, the importance of the timing of the Ca2+ or cyclic AMP signal in bringing about a specific change in cell behavior has emerged (Whitfield et al., 1976). For example, there are at least two distinct points in the G1 phase of lymphocytes stimulated to prolif- erate by the plant lectin phytohemaggluti- nin and of liver cells by partial hepatec- tomy, at which the cells’ progress through their cycle is perturbed if the normal transient change in cyclic AMP does not occur. One explanation for the temporal specificity of the action of the cyclic nucleotide within the cell is the presence of distinct cyclic AMP-dependent protein kinases, each being specifically required for a particular cell cycle event (Costa et al., 1976). This implies that, to a large extent, the specificity of the response to a particular environmental cue is determined by the internal state of the cell. Consistent with this idea is the finding that although PGE, produces a lengthening of the G2 phase of the myoblast cell cycle, suggesting that the cell responds to the PG at this point in its cycle by a rise in intracellular cyclic AMP, this does not trigger the intracellular changes which culminate in fusion. The latter response is restricted to the cell’s G1. A possible explanation for this finding is that the ability of the myoblast to respond to the rise in intracellular cyclic AMP by fusing is dependent upon the appearance of one or more cyclic AMP dependent kinases.


The restriction of the response to the G1 phase of the cycle is then explained by proposing that the appearance of the protein kinase(s) necessary for the activation or synthesis of cellular components required for myoblast fusion is obligatorily coupled to the G1 phase. Evidence for specific protein kinase activations during the cell cycle has been obtained by Russell and co-workers (Costa et al., 1976; Byus et al., 1977).

A minimum of a 5-hr delay between the rise in intracellular cyclic AMP and the onset of myoblast fusion provides an explanation for the lengthened myoblast G1 associated with the onset of fusion (Bischoff and Holtzer, 1969; Buckley and Konigsberg, 1974). Bischoff and Holtzer found that a minimum of 8 hr was required for the nucleus of a cell at the end of S to enter G1 and to appear in a multinucleate unit, a finding confirmed in the present study. Eight hours is also the theoretical time required, given the cell cycle parameters under their conditions of culture, for cells at the end of S to enter G1 (approximately 3 hr) where they are sensitive to PGE and then to respond to that signal by fusing (5 hr). The G1 phase of the cell cycle in primary cultures of quail myoblasts increases from a value of 3-4 hr for the undifferen- tiated proliferating population to 12 hr during the differentiation process (Buckley and Konigsberg, 1974b). The 5-7 hr that it takes chick myoblasts in G1 to respond to PGEl by fusing is considerably less than the time that quail myoblasts may spend in G1 before fusing (Konigsberg, 1978). It seems unlikely that this difference can be explained on the basis of the species difference of the cells in the two studies. A more plausible explanation is that an individual myoblast cell enters G1 and spends a variable time in an uncommitted state before interaction with a PG molecules(s) which directs it toward fusion. That this is the case is suggested by the finding in the present study that there were unlabeled nuclei, signifi-

cantly above basal levels, in myotubes exposed to [3H]thymidine for times in excess of the minimal time interval calculated to be necessary for cells at the end of S to traverse Gz and M and to fuse as a result of their response to PGEl in early G1.

Further information concerning the interaction of PG with the Cl myoblast is provided by the present finding that a single exposure to lOA’ M PGE, provokes only 7.5% of the single cell population to fuse. If all the single cells in G, were able to fuse in response to PGE, the expected percentage increase in fusion, assuming an asynchronous culture, is 22. This calculation yields the maximum theoretical value since it does not take into account fibroblast contami- nation or the presence of nonfusing myoblasts. The minimum expected percentage of myoblasts provoked to fuse by PGE, at any one time, calculated on the assumption that only the proportion of cells which fuse under control conditions (i.e., 55% of the total population) is myoblasts, is 11.4. This is still considerably higher than the 7.5% actually found to respond and suggests that only a proportion of the myoblasts in G1 is competent to respond to the PG. There are two possible explanations of this which should be mentioned-(l) a proportion of the myoblasts in G1 at 34 hr may be devel- opmentally too immature to respond and (2) the ability to respond to PGE, may be restricted to a subsection of the G, phase. Evidence that hormonal sensitivity is restricted to a part of G1 has come from studies on the stimulation of quiescent fibroblasts to enter the S phase (.Jiminez de Asua et al., 1977). This study revealed a temporal relationship in the interactions of prostaglandin FZa, insulin and hydrocorti- sone with the G1 fibroblast cell to produce alterations in the rate of entry into S. On the basis of their findings, the authors were able to separate the G, phase into temporal regions during which a particular hormone exerted its specific effect upon the cell’s behavior. It is well established that, in gen-


eral, cells are most responsive to external factors in the early part of G1. The results of the present study demonstrate that myoblasts are sensitive to PGEl at this point in the cell cycle. It therefore seems probable that if the PG response is restricted to a part of G1, then that part will be at the beginning of the G1 phase. A comparison of the two theoretical values (22.0 and 11.4%) with the actual percentage of cells responding to PGEl (7.5) suggests that the sensitive subsection occupies between 0.3 and 0.66 of the total length of G1.

The advantage of a model of the cell cycle which divides the cell’s G1 into two parts, an initial uncommitted one in which the cell is responsive to external stimuli and a second during which the cell makes the commitment to undergo either DNA synthesis or differentiation, is the introduction of an element of ambivalence into the cell cycle. This is consistent with the evidence for a critical role of the external environment in determining the timing and extent of myoblast differentiation. There is also a precedent for such a model. On the basis of the variability in the length of G1 of several different cell types under various culture conditions and the effect of hormones upon the rate of entry of these cell populations into S, Smith and Martin (1974) have proposed that the cell cycle of all proliferating cells is divided into two parts. In the first part (A), which corresponds to the first part of G1, they suggest that the cell is uncommitted, while in the latter part (B), the cell prepares for entry into S and completes the division cycle. Their data suggest that the transition from A to B is random, occurring at a given probability determined by the cell type and its environmental conditions. Although it is proposed that cells in the latter part of G1 (B) are preparing for S, there is evidence to suggest that they do not become irreversibly committed to such a path until very close to the Gl-S border (Rubin and Steiner, 1975). It may also be the case that the myoblast does not become

irreversibly committed to differentiate until very late in G1. The two possible extremes of when the commitment to fuse occurs in the terminal G1 are: (a) the time at which the cell receives the cyclic AMP signal, and (b) upon completion of the subsequent preparation for the fusion event and contact with a second competent cell. Thus in the first case, the preparations for fusion and for reentry into S are mutually exclu- sive. In the second case, both “processes” are carried on simultaneously by the cell. Konigsberg’s finding that individual myoblasts spend a variable length of time in G1 before either fusing or reentering S (Kon- igsberg, 1978) suggests that the latter may be the case. However, the present data do not provide information which would allow an evaluation of these two alternatives.

Holtzer (1970), in his “quantal” mitosis theory, stresses the presence of a critical event in the S or M of the cell cycle immediately prior to the G1 in which the myoblast differentiates; this, he says, is necessary to produce a myoblast competent to differentiate. Since this event is considered to be cell autonomous, in his opinion, environmental factors are merely permissive. While the scheme proposed in this paper does not rigorously exclude the possibility of such an event, it does offer an alternative explanation for the two main observations upon which the hypothesis is built. First, the lengthened G1, which he interpreted as the existence of an event in the previous cell cycle giving rise to noncycling cells in Go, is now explained as being, in part, the result of the cells’ response to PGE1. Sec- ond, the effects of inhibitors of DNA synthesis upon differentiation, which have been previously interpreted as demonstrat- ing that there is a critical event which occurs in the S phase of the cell cycle, are now reinterpreted as due to a requirement of these cells to pass through the cell cycle in order to reach the cyclic AMP-sensitive point in early G1. That is to say that the ability of DNA synthesis inhibitors to block

ROSALIND ZALIN

differentiation is not a test of the importance of S per se; rather, it demonstrates that there is something distal to the GI-S border which is essential for differentiation to occur. A similar conclusion has also been reached by Vonderhaar and Topper (1974) in their work on the differentiation of the mammary gland. These investigators found that although explants from mature virgins were prevented from differentiating by the presence of either fluorodeoxyuridine or cy- tosine arabinoside, explants from immature virgins were unaffected. These results show a correlation between embryonic stage and a sensitivity to the inhibitors, but it is not the correlation predicted by Holtzer’s model.

Fusion by its very nature involves the close cooperation of a number of cells. The ability of a PG, at a precise point in the myoblast’s cell cycle, to trigger the prepa- ratory events for fusion is an excellent mechanism for synchronizing groups of cells in the proliferating myoblast population. The question that now arises is: How is the production of the PG regulated within the myoblast population to ensure that the signal is received by a group of cells at a sufficiently similar time? The simplest possible explanation is that a small amount of PG is produced by the cells continuously, or at a specific point in the myoblast’s cell cycle, so that as the cell density increases, a threshold level is reached. Alternatively, a PG may be produced at a particular time in culture as a result of earlier developmental changes or as a result of alterations in the cell’s environment. An obvious can- didate for the latter category of change is the depletion of the growth promoters which are present in both fresh serum and embryo extract. Further work will be needed to distinguish between these alternatives.

SUMMARY

The model here proposes that: (1) Myoblasts make the decision to differentiate in G,; (2) myoblasts may take up one of two states in G1, the cycling state or the prefusion state; the decision between fusion and

The Cell Cycle

reentry into S occurs late in G1 and may be probabal- istic in nature; (3) the signal which causes cells to enter the prefusion state of Gi is PGE; (4) it is the interaction of the PG with the cell’s surface which leads to the transient rise in intracellular cyclic AMP; and (5) the rise in cyclic AMP, via the activation of a protein kinase(s), leads to the initiation of fusion.

I would like to thank Ronald Leaver for valuable technical assistance throughout this project. I also wish to express my gratitude to Dr. Christopher Ford for advice and Drs. Stephen Hauschka, Thomas Link-. hart, and Neil MacDonald for critical discussion of the manuscript. This work was supported by the Muscular Dystrophy Group of Great Britain.

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the cell cycle, myoblast differentiation and prostaglandin as a developmental signal

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