The ski oncogene induces muscle differentiation in quail embryo cells

Cell, Vol. 59, 293-303,

October 20, 1989, Copyright

0 1989 by Cell Press

The ski Oncogene Induces Muscle Differentiation in Quail Embryo Cells Clemencia Colmenares and Edward Stavnezer Department of Molecular GWIetiC8, Biochemistry, and Microbiology University of Cincinnati Medical Center Cincinnati, Ohio 45287-0524

Summary Quail embryo cells (QECs) am primary cultures of fibroblastoid cells that become myogenic after infection with avian retroviruses expressing the ski oncogene (SKVs). ski also stimulates proliferation of QECs and induces morphological transformation and anchorage-independent growth. Paradoxically, skitransformed clones picked from soft agar are capable of muscle differentiation. ski-induced differentiation is essentially Indistinguishable from that of uninfected myoblasts in culture with regard to musclespecific gene expression, commitment, and inhibition by growth factors or other oncogenes. However, skiinduced myoblasts have less stringent requirements for growth and differentiation. Uninfected QECs cannot differentiate and do not express an early marker for the myogenic lineage. Clonal analysis indicates that at least 40% of QECs are converted by ski to diffemntlating myoblasts. The data suggest that ski induces either the capacity for differentiation in an ‘incompetent” muscle precursor or the determination of nonmyogenic cells to the myogenic lineage. Introduction Myogenic differentiation in vitro has been thoroughly described at the molecular, biochemical, and cellular levels. Myoblasts in culture can proliferate in the presence of growth factors; depletion or removal of these growth factors can initiate a program of terminal differentiation that involves withdrawal from the cell cycle, commitment, fusion, and the expression of muscle-specific genes (Emerson and Beckner, 1975; Nadal-Ginard, 1978; Konigsberg, 1979; Linkhart et al., 1981). These events appear to be independently regulated, and have been separated by identifying mutants or manipulating culture conditions (Emerson and Beckner, 1975; Devlin and Konigsberg, 1983; Nguyen et al., 1983). Activated oncogenes expressed in myoblasts, and growth factors added to myoblast culture media, both have been shown to inhibit terminal myogenic differentiation (Linkhart et al., 1980; Falcone et al., 1985; Lathrop et al., 1985; Massague et al., 1988; Olson et al., 1987; Webster et al., 1988). Although the pathways affected by these growth regulators are not yet known, they have proved useful tools in dissecting the program of terminal differentiation. Some, such as v-myc or v-src, may provide a mitogenie stimulus that keeps myoblasts proliferating and inhibits their exit from the cell cycle (Falcone et al., 1985);

others, like activated H-ras or transforming growth factor 8 (TGF-8) do not prevent growth arrest but still inhibit fusion and the expression of muscle-specific genes (Massague et al., 1988; Olson et al., 1988; Olson et al., 1987). ElA inhibits the transcription of several muscle-specific genes and may suppress muscle differentiation via this mechanism (Webster et al., 1988). The ski oncogene, expressed in avian retroviruses (SKVs), transforms quail embryo cells (QECs) in culture, as indicated by morphological changes and by the ability of the transformed cells to grow in soft agar (Stavnezer et al., 1981). Here we report that ski also induces QECs to grow rapidly in suboptimal medium. Surprisingly, ski also induces the capacity for muscle differentiation in QECs that are otherwise incapable of undergoing myogenesis. It is unexpected and paradoxical that an oncogene that induces rapid and anchorage-independent growth should simultaneously activate a program leading to the cessation of cell growth and terminal differentiation. We present data showing that a single cell type is the target of these contradictory activities, suggesting that the cellular controls leading to a choice between proliferation and differentiation are not necessarily exclusive in the muscle system. Several genes, including mpD1, mycf, myogenin, and my+5, have been shown to induce muscle differentiation in nonmyogenic cells (Davis et al., 1987; Pinney et al., 1988; Wright et al., 1989; Braun et al., 1989). It has been proposed that a set of regulatory genes exist that control determination and differentiation during muscle development (Pinney et al., 1988; Blau, 1988). ski, like mpD7 (Tapscott et al., 1988) encodes a nuclear protein (Barkas et al., 1988) whose myogenic activity may indicate that the c-ski proto-oncogene is a member of this regulatory gene set. Here we describe the process of muscle differentiation under the influence of ski at the cellular and molecular levels, and we discuss some of the roles ski might play in inducing this process. Results SKV Infection Induces Transformation and Muscle Differentiation in QECs infection of QECs by SKVs results in morphological transformation, the ability to clone in soft agar, and an enhanced growth rate (Table 1). Uninfected QECs are large, flat cells (Figure la) that grow slowly, with a doubling time of 48 hr, and require 2% chick serum in addition to 10% bovine serum for optimal growth. After SKV infection, QECs become smaller and spindle-shaped (Figure lc), resembling uninfected quail myoblasts (Konigsberg, 1979) and require only 5% bovine serum for optimal growth (data not shown). SKV-QECs grow to 2-3 times greater saturation densities than QECs and have a doubling time of 12-14 hr, about 4 times faster than the doubling rate for QECs in optimal growth medium (Table 1). QECs are completely anchorage dependent and fail to grow in soft agar,

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Table 1. Comparison

of Growth and Myogenesis

Cells

Doubling Time (hr)

QECS

40

SKV-QECs

14

in OECs and SKV-QECs

Population Doubling.9 18-20 >50

Saturation Densityb

Cloning in Agar (%)

l-l.5

x 105

0

3-3.5

x 105

3-7

Nuclei in Myotubes (%) O-5 70-90

B Number of doublings to reach senescence, starting at passage 1, and estimated from passage number and split ratios. Senescence fined as the point at which ceils required more than 4 times their normal doubling time to double. b Number of cells per cm*.

while SKV-QECs clone in soft agar at efficiencies (3%7%) comparable to those of both SKVinfected chick embryo fibroblasts (Stavnezer et al., 1981) and quail cells transformed by v-myc (Palmieri et al., 1983). In addition to the effects related to cell growth and transformation, skicontaining viruses induce QECs to undergo terminal muscle differentiation when placed under the appropriate conditions, as indicated by the formation of large networks of spontaneously contracting myotubes (Figure Id). Myogenic differentiation of SKV-QECs requires either depletion of growth factors from growth medium (GM), or a shift into serum-free differentiation medium (DM), where 700/o-90% of the cells fuse into myotubes. When 2% horse serum is added to DM, myotubes look thicker and can be maintained longer in culture, but differentiation is not enhanced and appears less synchronous than in serum-free DM (data not shown). Uninfected or helperinfected QECs shifted into DM have variable but low myogenie potential, with lo%-20% of the nuclei in myotubes at early passages, generally decreasing at later passages to 00/b-5%. In the experiments reported here, we have

DM

was de-

chosen to use only QECs with essentially 0% background of muscle differentiation (Figure lb). All ski-containing viruses that were tested, including natural isolates as well as viruses constructed using the cloned skigene, such as RAV-SKV (Stavnezer et al., 1986) and SRA-SKV (Teumer and Stavnezer, unpublished data; see Experimental Procedures for description), have indistinguishable activities on QECs. None of the helper viruses that were tested, including RAV-1, tdPrC, tdB77, or tdSR-A, influenced myogenesis in any way (data not shown). Expression of Mwcle-Specific Proteins and mRNAs in ski-Induced Myotubes We have determined whether QECs or SKV-QECs express the major components of the contractile apparatus, myosin and actin. Protein expression was assayed by indirect immunofluorescence with monoclonal antibodies against skeletal myosin (MF20; Bader et al., 1982), a-actin (84; Lessard, 1988) and the slow and fast isoforms of myosin heavy chain (MHC; Miller et al., 1985). None of these

Figure 1. Comparison of the Morphology of OECs and SW-OECs in Growth and Differentiation Media QECs at sixth passage (a and b) or sister cultures of sixth-passage QECs infected by SW SKV at second passags (SKV-OECs; c and d) were seeded at comparable densities, altowed to grow for 24 hr, and photographed in GM. Both cuftures were then shifted into DM and rephotographed 3 days later.

ski Induces Muscle Differentiation 295

Figure 2. lmmunofluorescence of QECs and SKV-GECs with Antibodies against Myosin and Skeletal aActin Cells were seeded in GM at comparable densities. Eighteen hours later, OECs (a and b) or RAV-SKV-infected QECs (e through h) were fed with DM; one plate of SKV-GECs (c and d) was refed GM. Cells were fixed in methanol 4 days after seeding and processed for immunofluorescence, as described in Experimental Procedures. Primary antibodies to myosin (a, b, e, and f) or skeletal a-actin (c, d, g, and h) yielded comparable results on QECs or SKVQECs in GM.

muscle-specific contractile proteins is expressed in QECs under any conditions (for example, Figures 2a and 2b). In SW-QECs kept in GM, an occasional cell expresses muscle-specific proteins, as shown in Figures 2c and 2d. After 3-5 days in DM, ski-induced myotubes express both myosin and skeletal a-actin (Figures 2e2h). Most of the ski-induced myotubes express the fast MHC isoform,

while only a few express the slow form (data not shown), as previously found in cultured quail myoblasts (Schafer et al., 1987). The timing and levels of muscle-specific gene transcription during in vitro terminal differentiation have been well described (Devlin and Emerson, 1979; Shani, et al., 1981; Caravatti et al., 1982). To compare those results with

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-SKV-QECs-

QECs MCK

,v-ski 0 24 48 72 72 hours in DM B SKV-QECs

M QECs

-MHC -cTNT 0 244872

0 72

hours in DM Figure 3. Transcription of MuscleSpecific into DM

Genes in SKV-OECs Shifted

(A) Identical Northern blots of RNA from QECs or SRA-WV-infected QECs (harvested at the indicated times after the cultures were shifted into DM) were probed with fragments of the indicated muscle-specific genes (described in Experimental Procedures). Each sample represents 10 ug of total RNA. glyoxalated and run on a 1% agarose gel. MCK, muscle-specific creatine phosphokinase; ACT, skeletal a-actin; TNI. troponin I. (B) Northern blot of the RNAs employed in (A), plus RNA from uninfected quail myoblasts (M), treated as in (A) above. MHC, myosin heavy chain; cTNT, cardiac troponin T.

muscle-specific gene expression in SKV-QECs during growth and differentiation, we have performed Northern analyses of RNAs before and after the shift into DM, using probes for MHC, skeletal a-actin, muscle-specific creatine kinase (M-CK), troponin I (TNI), and cardiac troponin T (cTNT). As shown in Figure 3, we detect increases in the steady-state levels of all of these mRNAs during skiinduced terminal differentiation. The kinetics of expression are somewhat different in each case: M-CK and TNI mRNAs steadily accumulate after the shift into DM (Figure 3A), while skeletal a-actin (Figure 3A) and MHC mRNAs (Figure 38) peak at 24 and 46 hr, respectively, after the shift into DM, then partially decrease. Similar patterns in the expression of skeletal a-actin mRNA have been observed by others in avian myogenic cultures (Hayward and Schwartz, 1966) and differing patterns of accumula-

tion of several human muscle-specific mRNAs during in vitro differentiation have been reported (Gunning et al., 1967). cTNT is transcriptionally activated during early stages of cardiac and skeletal muscle development, but becomes repressed in skeletal muscle after day 14 of gestation (Cooper and Ordahl, 1964; Long and Ordahl, 1966). In culture, cTNT is constitutively expressed in skeletal muscle cells (Toyota and Shimada, 1963). We find low levels of cTNT mRNA in proliferating SKV-QECs, which increase approximately lo-fold during terminal differentiation (Figure 38). In uninfected QECs, cTNT transcripts are undetectable. All of the results described above show that muscle-specific mRNA accumulation during ski-induced differentiation is comparable to that observed in other myogenic cultures. Commitment to Terminal Differentiation during ski-Induced Myogenesis Because of ski’s effects on the growth rate of QECs, we were particularly interested in the timing of commitment to terminal differentiation, i.e., of irreversible withdrawal from the cell cycle during ski-induced myogenesis. Identical plates of SKVQECs were seeded at low density, fed DM 12 hr later, then switched back to complete GM at the indicated times. The experiment was terminated by fixing the cultures 72 hr after the shift into DM. The results show that cultures that were refed with GM after only 6 hr in DM continued to proliferate and formed no myotubes (Figure 4a). Cells refed after 12 hr formed some myotubes, but unfused cells were still able to grow (Figure 4b). By 16 hr in DM, most cells had irreversibly withdrawn from the cell cycle, because cultures refed GM after 16 hr in DM contained no more cells than cultures maintained in DM for 24 hr or longer (Figures 4c and 4d, and data not shown). In addition, the cultures maintained in DM for 16 hr formed myotubes with the same efficiency as those maintained in DM throughout the experiment. Therefore, commitment to fusion and terminal differentiation in SKV-QECs occurs between 12 and 16 hr after removal of growth factors. This timing is consistent with experiments performed on fusion-blocked quail myoblasts, which show a loss of proliferative capacity after comparable intervals in differentiation-promoting medium (Devlin et al., 1962; Devlin and Konigsberg, 1963). inhibition of s&i-induced Myogenesis by TGF-p, and by the myc and src Oncogenes Another hallmark of in vitro myogenic differentiation is the fact that it can be inhibited by certain growth factors, such as TGF-6 (Massague et al., 1966; Olson et al., 1966) and by many oncogenes, such as src and myc (Falcone et al., 1965). We tested whether ski-induced myogenic differentiation was still subject to the action of these inhibitors. TGF61 (300 PM) was added to SKVQECs upon addition of DM, and cells were stained to examine skeletal a-actin expression 46 hr later. The results in Figure 5 show that TGF61 inhibits myogenic differentiation of SKV-QECs, although it does not abolish it completely. (Myosin staining yielded comparable results.) Complete inhibition was

ski Induces Muscle Differentiation 297

Figure 4. Timing of Commitment Differentiation in SW-GECs

to Terminal

Sets of six identical plates of SRASW-infected QECs were seeded at low density and shifted into DM the following day. After the indicated times in DM, cells were refed with complete GM. then fixed 2 days later. Control cultures, which remained in DM for the whole 3 days, contained approximately the same number of cells. and the same proportion of myotubes, as the 16 and 24 hr cultures (data not shown).

16hr

24hr

found when we superinfected SKVQECs with viruses containing myc or sm, then tested the doubly-infected cells by shifting them into DM (data not shown). Therefore, the induction of myogenesis by ski does not sidestep the control of those factors that can influence uninfected myoblast differentiation in vitro.

CONTROL

ski induces Terminal Muscle Differentiation in ski-Wansformed Ceils One possible explanation for skr’s paradoxical activitythe simultaneous induction of transformation and terminal differentiation-is that these effects are exerted on two different cell types. Because QECs are most likely a mixture of cell types, it is possible that one type of cell might be transformed by ski while another would be induced to differentiate. To test this possibility we used soft-agar cloning, which would allow us to analyze ski’s myogenic activity on single clones of transformed SKV-QECs. Uninfected and WV-infected QECs were cultured in soft agar, and clones of SKV-GECs were picked after 2 weeks. Uninfected cells produced no clones (see Table 1). Of the SKVQEC clones picked from soft agar, 70% contained fused myotubes after being cultured in monolayer for 5 days in GM and 2 days in DM, as shown in the example in Figure 6. Therefore, the ski oncogene can have seemingly opposite effects simultaneously and on a single cell type.

TGF-B

Figure 5. Inhibition

of ski-lnduceo

Myogenesis

by TGF6

Duplicate cultures of RAV-SW-infected QECs were shifted into DM with or without l’GF-51 (300 PM). The cells were fixed 3 days after the medium shift, and reacted with monoclonal anti-skeletal a-actin (84) followed by FITC-labeled rabbit anti-mouse lg. Anti-myosin antibodies gave indistinguishable results. SKV-GECs without TGF61: a and b; SKV-GECs plus lGF61: c and d.

Uninfected QECs Cannot Differentiate into Muscle skh myogenic activity could be due simply to an enhancement of differentiative potential in cells already within the myogenic lineage. For example, given its effects on cell growth, ski could act by replacing a missing factor possi-

Cell 296

Figure 6. Muscle Differentiation

in a s/d-Transformed

Agar Clone

Clones of RAV-SW-infected QECs which grew after 2 weeks in soft agar were picked from agar plugs and allowed to grow. When the cells appeared healthy, the clones were shifted into DM. Seventy percent of all surviving clones showed some evidence of myotubes.

bly required for differentiation, or ski might selectively expand a subpopulation of myoblasts. To test the first possibility, we attempted to “replace” ski during terminal differentiation by plating QECs on gelatin-coated dishes and shifting them into DM, or DM plus varying concentrations of horse serum and chick embryo extract, in an attempt to duplicate conditions previously described as supporting myoblast growth and differentiation (Konigsberg, 1979; Schafer et al., 1997). We observed no myotubes in any of these QEC cultures. To ensure that these negative results were not due to inadequate reagents, we simultaneously cloned uninfected quail myoblasts, then scored clones for their ability to differentiate in DM or in low-growth medium (5% horse serum, 1% embryo extract). After 2 weeks, the

vast majority of cells in more than 90% of the clones had fused into myotubes (as in Figure 7a), a few clones had small numbers of myotubes, and approximately 5% showed no evidence of differentiation (as in Figure 7b). Some cells appear to lose their differentiated phenotype when placed in tissue culture; thus QECs, which were established and passaged under conditions optimized for fibroblasts, might really be myoblasts that have lost the capacity to differentiate. We therefore decided to test ski’s ability to induce differentiation in clones established and grown under conditions designed for myoblast culture, which nevertheless failed to form myotubes when shifted intooptimal differentiation medium. Each nonmuscle clone from the previous experiment was split into two wells, one of which was kept uninfected, while one was infected with SKV. After two passages, each uninfected clone and its SKV-infected sister culture were shifted into DM, and scored 4 days later for the presence of myotubes. Eight of 11 clones retained their nonmyogenic phenotype in the absence of SKV infection (as in Figure 7c), and in five of these eight clones, myotubes developed only in the SKV-infected plates (as in Figure 7d). Therefore, QECs and selected clones of cells from muscle tissue, both of which lack the capacity to differentiate in vitro, can be induced to do so by the ski oncogene. These results show that ski does not simply substitute for appropriate culture conditions to stimulate differentiation of a myogenic component in QECs. Three out of 11 surviving clones were myogenic with or without SKV infection (data not shown). These may represent the type of clone seen initially in which only a few of the cells formed myotubes, and were mistakenly scored as nondifferentiating clones. In any event, this result formally demonstrates that ski does not inhibit quail myoblast differentiation.

Figure 7. Induction of Myotubes in Nondifferentiating, Muscle-Derived Clones after SKV Infection Uninfected quail myoblasts were cloned as described, and plates were shifted into DM when the majority of clones reached a diameter of 0.5 mm. Two weeks later, most of the clones had differentiated (a), while some showed no evidence of differentiation (b). The latter were picked, divided in half, and either kept uninfected (c) or infected with SRASKV (d). Cells were photographed in DM, either 2 weeks (a and b) or 3 days (c and d) after medium shift.

ski Induces Muscle Differentiation 299

Figure8 lmmunofiuorescence of QECs or SKV-OECs with L4 Antibody Uninfected or SRASW-infected QECs maintained in GM were fixed in cold methanol and rehydrated in PBS. Indirect immunofluorescence with monoclonal L4 antibody was performed as described in Experimental Procedures.

QECs Do Not Express a Muscle Lineage Marker Although QECs might be incapable of differentiation, they might represent a precursor cell already determined to the muscle lineage. if so, ski’s myogenic activity identifies a myoblast precursor that has not been previously described. We used an immunochemical approach to test this possibility. The antigen detected by the L4 monocional antibody is present very early during embryogenesis on undifferentiated cells of the myogenic or chondrogenic lineages, but not on fibroblasts (George-Weinstein et al., 1988). This antibody has previously been used to identify myoblasts in culture (Menko and Boettiger, 1987). As seen in Figure 8, indirect immunofluorescence showed that the vast majority of QECs (>99%) did not express L4, which was detected on all quail myotubes (data not shown) and in virtually all SKV-QECs, even in GM and before terminal differentiation. These data are consistent with our failure to observe differentiation in QECs, and indicate that before infection the majority of these cells did not belong to the muscle lineage. The occasional SKV-QECs that do not express L4 may represent a subset of cells that cannot be converted into myogenic cells-possibly the same subset observed in our cloning experiments (i.e., 30% of agar clones and 37% of nonmyogenic clones). The Target for s&i-induced Myogenesis is a Major Component of QEC Populations The target(s) for ski’s myogenic activity could be a small fraction of QECs that might be selectively expanded because of ski’s effects on their growth rate. Such a minor population could consist of determined muscle cells not detected in our previous experiments. We found this possibility unlikely, based on the following observations. Multiple passage is widely believed to result in the overgrowth of fibroblasts and the loss of other cell types from the initially heterogeneous cultures prepared from embryos. As mentioned earlier, myogenic cells do rapidly disappear af-

ter a few passages of our QEC populations. In spite of this, in QECs that were passed up to ten times (15-20 doublings) before SKV infection, the proportion of nuclei in myotubes after SKV infection and shift into DM (84%87%) did not differ significantly from the value obtained with cells infected at second passage (72%-92%). Admittedly, selection and expansion of a minor target population could still have occurred, because one or two additional passages are required for virus spread in the siowergrowing, late-passage ceils. However, such selective expansion of a minor muscle component would also require that ski not transform or enhance the growth rate of other cell types, such as fibroblasts. We have observed ski’s transformation and growth-rate effects on primary cells derived from many quail tissues, including brain, liver, kidney, and lung, as well as on melanocytes (data not shown) and on chicken fibrobiasts (Stavnezer et al., 1981) and neuroretina ceils (S. Saule, D. Steheiin, and E. Stavnezer, unpublished data). Nevertheless, we devised an experiment based on the fluctuation test (Luria and Delbriick, 1943) to examine the possibility of selective expansion by ski of a minor myogenie component, and to obtain an estimate of the size of the target population for ski-induced myogenesis. We plated uninfected QECs at clonal density in microtiter wells, as described in Experimental Procedures. One day later, SKV was added to all microtiter plates except controls. After 2 weeks, all plates were shifted into DM and scored 2 days later for myogeniclnonmyogenic colonies. The results of this experiment (Table 2) show that 83% of the wells contained myogenic colonies. With an average of about two colonies per well, the data indicate that the target population is at least 40% of all QECs, and that number is likely to be an underestimate since we could not expect all of the cells to become infected under this protocol. Furthermore, the proportion of QECs susceptible to myogenic conversion does not decrease with increasing

Cdl 300

Table 2. Proportion

Number Number Percent Percent Percent

of Cloned, Uninfected

QECs Susceptible

to Myogenic

of Colonies per Well (range) of Myogenic Colonies per Well (range) Myogenic Colonies Wells with Myogenic Colonies Wells with >l Myogenic Colony

Conversion

by SKV

QECs Uninfected

QECs Plus SKV

1.3 (O-5) 0 0 0 0

1.9 (O-5) 1.2 (O-5) 63 + 3 63 f 3 25 f 2

QECs at both early (passage 2) and late (passage 10) passages were plated at clonal density in microtiter plates. Some were infected 1 day later with SRA-SKV, and all were tested for myogenesis after 2 weeks. Number of wells scored: OECs uninfected, 192; QECs plus SKV, 266. No differences were observed as a result of passage in culture.

passage. Two of the microtiter plates (one infected, one uninfected) contained tenth-passage QECs, and we observed no differences when these were compared with second-passage QECs. We would have expected only a few of the wells to contain myogenic colonies if the target cells comprised only a small fraction of the population. For example, if there were as many as 10% potentially myogenie cells, then only 20% of the wells, at most, would contain myogenic colonies. This experiment shows that ski does not selectively amplify a minor proportion of QECs into a myogenic population. Therefore, either ski induces determination of nonmuscle cells to the myogenic lineage, or the target population in QECs represents a novel myogenic precursor that is unable to differentiate in vitro and does not express the early muscle lineage marker detected by the L4 antibody. Discussion We have described terminal muscle differentiation of primary QECs under the influence of the v-skioncogene. Because terminal muscle differentiation in vitro has been extensively studied, we are able to compare the ski-induced process to that observed with cultured myoblasts. Our data show that by several criteria, including the timing of irreversible commitment, the induction of muscle-specific gene expression, and the inhibition of differentiation by myc, src, and TGF-81, myogenesis stimulated by ski is remarkably similar to that of normal myoblasts. There is, however, a significant difference. Unlike primary myoblasts and some myoblast cell lines, SKV-QECs do not require special medium supplements (embryo extracts) or protein-coated culture dishes for extended proliferation. Thus ski-induced muscle differentiation is an excellent in vitro model because SKV-QECs are easier to culture and clone at higher efficiencies than uninfected myoblasts. Furthermore, SKV-QECs can be passaged for long periods without the overgrowth of fibroblasts. We have shown that QECs transformed by the ski oncogene proliferate 3-4 times faster than uninfected cells, and have decreased growth factor requirements. This rapid growth rate is similar to that observed in myc-transformed QECs (Palmieri et al., 1983). In view of this growthenhancing activity, it is surprising that ski also confers on the transformed cells the capacity for terminal differentiation, which involves withdrawal from the cell cycle. By demonstrating that clones of transformed cells grown in

soft agar can differentiate into contracting myotubes, we have shown that these seemingly “opposite” effects are not due to ski’s activity on two different cell types: one which could differentiate but not become transformed, and a second which would transform but not differentiate. The stimulation of both processes in a single cell type by a single gene, ski, is paradoxical because it has generally been accepted that the choice between proliferation and differentiation in muscle cells reflects the activation of mutually exclusive pathways (Nadal-Ginard, 1978). A precedent for reconciling ski’s dual activities may be found in the effects of growth hormone (GH) on adipocyte differentiation. G H appears to stimulate directly the differentiation of preadipocytes when these cells enter a resting state (Nixon and Green, 1984). In addition, although G H is not a mitogen, it indirectly stimulates the proliferation of adipocytes by inducing the production of insulin-like growth factor 1 (IGF-l), which is a more potent mitogen for adipocytes than for their precursors (Zezulak and Green, 1988). Although this model cannot be applied directly to myogenesis, because myoblasts stop proliferating after differentiation, it does suggest some testable mechanisms by which ski could induce both growth factor and “differentiation factor” effects. Preliminary studies indicate that SKV-QECs can produce a PDGF-like growth factor activity (C. Colmenares, E. Stavnezer, and M. A. Lieberman, unpublished data). We have found that v-ski induces muscle differentiation in fibroblast-like QECs and in otherwise nonmyogenic clones derived from muscle tissue. Prior to SKV infection, QECs are nonmyogenic and do not express the L4 antigen, which identifies cells in the myogenic and chondrogenie lineages (George-Weinstein et al., 1988). We have also shown that at least 40% of QECs are targets for ski’s myogenic activity. These results suggest that ski can either induce determination of nonmuscle cell types to the myogenic lineage, or induce the capacity for differentiation in a precursor that cannot be identified as a muscle cell. v-ski has also induced muscle differentiation in cells isolated from brain, liver, and skin. In some of these cases (i.e., brain), multiple passages after infection are required to observe this effect, suggesting that the targets are likely to be minor components of these cell populations (such as fibroblasts or uncommitted mesodermal cells). In contrast, primary liver cultures develop myogenic ability quite rapidly, within 2-3 passages after infection. We do not know which cell type in these tissues can be converted

ski Induces Muscle Differentiation 301

into muscle; the identification of ski’s potential target(s) will await its expression in characterized cell lines. Several genes have been shown to induce myogenic determination in nonmuscle cells; these include myd, rnyoD7, and myogenin and myM, which are related to myoD7 (Davis et al., 1987; Pinney et al., 1988; Wright et al., 1989; Braun et al., 1989). It has been suggested that these are members of a set of regulatory genes that control determination and differentiation during muscle development (Pinney et al., 1988; Blau, 1988). ski is not related to the my007 family at the sequence level (Tapscott et al., 1986; Stavnezer et al., 1989) and does not hybridize to the myd gene in Southern blots of myd-myoblasts (E. Stavnezer, S. Pearson-White, D. Pinney, and C. Emerson, unpublished data). However ski, like myoD7, encodes a nuclear protein (Barkas et al., 1986) whose myogenic activity may indicate a role for the c-ski proto-oncogene in the cascade that controls muscle development. Consistent with this hypothesis, recent data show that c-ski cDNAs, expressed at high levels from a retroviral vector, are also capable of inducing myogenesis in QECs (C. Colmenares, P Sutrave, S. Hughes, and E. Stavnezer, unpublished data). Transfection of myd into nonmuscle cells induces m)aDl expression, leading to the suggestion that these genes may be members of a cascade of genes that regulate muscle differentiation (Pinney et al., 1988). Since expression of myuD7 seems to antagonize cell growth (Davis et al., 1967), it is likely to act late within the muscle differentiation pathway. Because ski stimulates proliferation and induces differentiation, it is tempting to speculate that it might act early within this regulatory pathway to achieve both myogenie determination and expansion of the resulting myoblast population. A major difference between other muscle determination genes and ski is that, at least at the mRNA level, ski expression is not muscle specific. c-ski mRNA is expressed in chicken fibroblasts (Li et al., 1986) and we have detected ski mRNA in multiple avian tissues, although at low but varying levels (Stavnezer, 1988; A. Barkas and E. Stavnezer, unpublished data). This finding does not rule out a role for ski in muscle differentiation. ski is now known to be alternatively spliced (Sutrave and Hughes, 1969) and it could be that one spliced form is muscle specific. Alternatively, ski could act like some transcription factors (or cofactors), that may contribute to tissue-specific expression, yet may be present in “inappropriate” tissues (for reviews see fvluller et al., 1988; Wasylyk, 1988). In contrast to ski, many activated oncogenes have been reported to inhibit muscle differentiation when expressed in quail myoblasts; these include v-m& v-src, v-enbB, and vJps (Falcone et al., 1985). The same effect is observed when expression vectors encoding N- and H-ras (Olson et al., 1987) ElA (Webster et al., 1988) and, to a lesser extent, c-m)nc (Schneider et al., 1987) are introduced into mammalian myoblast cell lines. It has been suggested that growth stimulation and altered responses to growth factors may be the mechanisms by which genes like myc affect muscle differentiation. Our results indicate that the situation is likely to be more complex, since ski does not

inhibit myogenesis although, like myc, it does stimulate proliferation. ExPewlmental

Proceduree

Cell Cultum and Vlrusee QECs were prepared by trypsinization of day 9 embryos of Japanese quails, as described (Stavnezer et al., 1991). In brief, minced embryos were trypsinized after removal of the head, limbs, and viscera, and the released cells were immediately frozen in medium containing 10% DMSO. Quail myoblasts were prepared by mechanical disruption of day 9 quail embryo leg or breast muscle, as described (Konigsberg, 1979). cultured for one or two passages, and frozen. QECs were grown in GM consisting of medium 199 (Ear& salts) buffered with HEPES (25 mhl), and supplemented with 10% tryptose phosphate broth, 10% calf serum, and 2% chick serum; for SKV-QECs, chick serum was reduced to 1%. Quail myoblasts were grown on gelatincoated plates in Ham’s F-IO medium, supplemented with 15% horse serum and 10% embryo extract (Konigsberg. 1979; Schafer, et al., 1997). DM was Ham’s F-12, supplemented with CaCls and 05% polyvinylpyrrolidone, as de scribed (Konigsberg, 1979). Uninfected QECs were plated at clonal density in microtiter plates in medium 199 plus 10% tryptose phosphate broth, 15% fetal calf serum, and 3% chick serum. Fifty percent of this medium was preconditioned by QECs for at least 24 hr. Microtiter plates were fed every other day. Differentiation was tested by shifting cells from GM into DM and scoring for myotube formation and expression of muscle-specific markers after 3-5 days in DM. As a quantitative measure of differentiation, cells were fixed with methanol after 3-5 days in DM, stained with Giemsa, and the fraction of nuclei in myotubes was determined. The extent of muscle differentiation observed by this method was variable, depending greatly on both total and local cell concentrations. To test for anchorage independence, cells were cloned in agar as described (Stavnezer et al., 1981) at three different cell densities. After 2 weeks, colonies were counted; cloning efficiencies did not increase with increasing cell density, and the percentages were averaged. Clones were then picked from agar and placed in individual wells; not all of the clones picked emerged from the agar plugs. Cells were infected and passaged two to three times to ensure infection of all cells, as described previously (Stavnezer et al., 1991). The viruses used were: transformation-defective B77, RAV-1, MC29, RSV SRA, four natural isolates of SW (Li et al., 1985), and the constructs RAV-SKV (Stavnezer et al., 1986) and SRASKV fleumer and Stavnezer, unpublished data). RAV-SKV is a replicationdefective virus for which RAV-1 is used as the helper virus; SRA-SKV is a nondefective virus in which the cloned v-ski has been inserted in place of v-src in a RSV SR-A provirus. Viral stocks had titers ranging from lo5 to 10s infectious units per ml. Immunofluoreecence Cells plated on 35mm, Wmm, or 100 m m plates, in GM or DM, were rinsed with phosphate-buffered saline (PBS; calcium and magnesium free), fixed in cold methanol for 6 min and immediately rehydrated with PBS Cultures were stored for up to 1 week or used immediately. Plates were allowed to dry, and “wells” were created by circles drawn with Sharpie markers. Appropriate dilutions of different antibodies in PBS were added to the wells. The plates were then incubated at 3pc for 30 min. extensively rinsed with PBS, and incubated again for 30 min at 3pc with FITC-labeled rabbit anti-mouse antibody (Cappel). After washing, samples were examined and photographed on a Zeiss IM35 microscope. Northern Blots RNA was isolated as described by Chomczynski and Sacchi (1997). with minor modifications. In brief, after the first isopropanol precipitation, the second addition of guanidine isothiocyanate was omitted; instead, the RNAs were resuspended in water and extracted with phenol-chloroform (1:1) and chloroform until the interface was clean, then precipitated in ethanol. RNAs were glyoxalated, electmphoreeed on 1% agarose gels in 10 m M phosphate, and transferred to nitrocelluloee or Nytran (Schleicher and Schuell). Probes were labeled by the random priming method (Feinberg and Vogelstein, 19&3), and blots were processed as described (Stavnezer et al.. 1991). The probes for

Cell 302

muscle-specific transcripts were: for skeletal a-actin, 261 bp from the 3’untranslated region of chick skeletal a-actin; for M-CK, 250 bp of the coding sequence, from Hpall to Pstl, of chick muscle crealine phosphokinase; for cTNT, 132 bp from the 3’ untranslated sequence, plus 10 bp of the coding sequence, of chick cTNT; these three probes were obtained from Charles Ordahl, UCSF (see Coleman and Ordahl, 1986). Other probes used were: for TNI, 680 bp of quail TNI cDNA, obtained from Charles Emerson, University of Virginia (Hastings and Emerson, 1982); for MHC, 3.1 kb of chick MHC cDNA. obtained from Jeffrey Robbins, University of Cincinnati (Molina et al., 1987); for glyceraldehyde 3-phosphate dehydrogenase (GAPDH), used as a control for the amount of RNA loaded on the Northern blots, 1.26 kb of chick GAPDH cDNA, obtained from Robert Schwartz, Baylor College of Medicine (Dugaiczyk et al., 1983). Acknowledgments We would like to acknowledge Alex Barkas’ contribution of the initial observation of ski-induced myogenesis. We thank C. Ordahl, C. Emerson, J. Robbins, and R. Schwartz, who supplied probes; D. Fischman, J. Lessard, and F. Stockdale. who provided us with monoclonal antibodies; and W. Bacon, who kindly supplied us with quail embryos. We are grateful to C. Emerson for his helpful suggestions, and to M. Lieberman and D. Luse for their critical comments on the manuscript. This work was supported by Public Health Service grants CA32187 and CA43660, and by a National Institutes of Health postdoctoral fellowship to c. c. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 USC. Section 1734 solely to indicate this fact. Received

February

21, 1989; revised July 24, 1989.

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