Expression of MyoD1 coincides with terminal differentiation in ...

The EMBO Journal vol.8 no.8 pp.2203 - 2207, 1989

Expression of MyoD 1 coincides with terminal differentiation in determined but inducible muscle cells

Didier Montarras, Christian Pinset, Jamel Chelly1, Axel Kahn1 and Fransois Gros Department of Molecular Biology, Pasteur Institute, 25, rue du Dr Roux, F-75724 Paris Cedex 15 and 'Unit6 129 INSERM, CHU Cochin, 25, rue du Faubourg St Jacques, 75014 Paris, France Communicated by D.Louvard

We have examined the expression of MyoDI, a potential determination factor of myogenic cells, in permissive and inducible C2 myoblasts. These two types of myoblasts exhibit distinct requirements to undergo terminal differentiation. Unlike permissive cells, inducible cells fail to differentiate in the presence of growth medium plus fetal calf serum and require insulin to undergo terminal differentiation. We show that while expression of MyoDI is constitutive in permissive cells, no trace of MyoDI transcripts is found in inducible cells at the myoblast stage. In these cells, however, expression of MyoDI accompanies differentiation. This indicates that MyoDl may not be required for the maintenance of the myoblast phenotype, and could act as an effector of terminal differentiation in already determined muscle cells. Our results provide new evidence that permissive and inducible cells represent two distinct stages of the progression of determined muscle cells toward terminal differentiation. Key words: cDNA-PCR/determination/differentiation/ MyoDI/myogenesis

Introduction The recent discovery of three genes, MyoDI (Davis et al., 1987) myogenin (Wright et al., 1989) and MyD (Pinney et al., 1988) that can convert cells of mesodermal origin to myoblasts represents a key step towards the understanding of the regulatory programme that controls myogenesis and ultimately leads to the coordinate activation of muscle specific genes. MyoD] was isolated as a cDNA clone that converts, upon transfection, IOTI/2 cells, 3T3 cells or adipoblasts into myoblasts, at a high frequency. Following the selection of myoblasts that have overcome the differentiation block caused by bromodesoxyuridine (BrdU), Wright et al. have identified a cDNA clone that corresponds to a transcript which is highly expressed in the BrdU-resistant myoblasts. Transfection of this cDNA clone, called myogenin, into lOT1/2 cells was shown to activate myogenesis (Wright et al., 1989). The existence of a third gene, MyD, that is capable of activating myogenesis in IOT1/2 cells was reported (Pinney et al., 1988). The fact that MyD-converted cells now express MyoD] suggests that MyD could act prior to MyoDl in the myogenic pathway (Pinney et al., 1988). The exact role(s) and position(s) of these three genes in the myogenic pathway remain to be

elucidated. MyoDl and myogenin cDNA sequences share a region of homology which is also found in the c-myc family. The MyoDi protein is localized in the nuclei of myoblasts and myotubes (Tapscott et al., 1988) and thus may interact with DNA. In the present study, we have analysed the patterns of expression of MyoD] and myogenin in inducible and permissive C2 myoblasts. We have tested the hypothesis that these two types of myoblasts, which display distinct requirements for terminal differentiation (Pinset et al., 1988), could differ by the expression of these two myogenic regulatory factors.

Results Permissive and inducible phenotypes We have recently defined two types of myoblasts, both derived from the original C2 myogenic cell line (Yaffe and Saxel, 1977), which we named permissive and inducible (Pinset et al., 1988). The properties of these cells are summarized in Table I. Unlike the progenitor cells, referred to as permissive, inducible myoblasts differentiate poorly in the presence of DME plus FCS and require the presence of insulin at high concentration of IGFI at a lower concentration, to undergo terminal differentiation. To further our study, we also analysed the phenotype of myoblasts from other sources, such as Sol8 myoblasts, derived from myosatellite cells of adult C3H mouse soleus muscle (Mulle et al., 1988). These cells, as shown in Table I, exhibit a permissive phenotype. Moreover, Sol8 myoblasts display a more pronounced ability to differentiate than C2 permissive cells in the presence of MCDB 202, a medium which was the most adverse for differentiation (Pinset et al., 1988). Since previous works have established that treatment of lOT1/2 cells with 5-azacytidine leads to myogenic conversion Table I. Properties of permTissive and inducible phenotypes % of fused colonies Sol8 Ind

Per

lOTAza T4

lOTl/2

MCDB 202a 17 ± 60 40 + 56 +3 0 + 20% FCS 0 DME + 2% FCSb94 4 3 8.1 ± 4 100 ± 0 45 i 15 85 DME + 10-6 Mb 0 98 ± 199 i 1 100 + 038 + 1094 Insulin Cells were plated at low density, 50 cells/dish, and allowed to form colonies in MCDB 202 plus 20% FCS and l0-7 M Dex, for 10 days in (a) and 7 days in (b). After 7 days, in (b), the medium was changed for DME plus 2% FCS or DME plus 10-6 M insulin to promote differentiation. Fused colonies were scored 3 days later after fixation and staining. Results, expressed as percentage of fused colonies SD, were determined after counting at least 400 colonies in three independent experiments: Per, permissive C2 cells; Ind, inducible C2 cells; Sol8, Sol8 cells; lOTaza, cell population obtained after treatment of lOT1/2 cells with 5-azacytidine; T4, myogenic clone isolated from the lOTaza population; lOTl/2, lOTl/2 cells.

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D.Montarras et al.

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Fig. 1. Northern blot analysis of MyoDI transcripts in inducible and permissive cells. Proliferating lOT1/2 cells (lane 1), inducible (lane 2) and permissive myoblasts (lane 3) were grown in MCDB 202 + 20% FCS and 10-7 M Dex. Differentiated inducible and permissive cells (lanes 4 and 5) were obtained after 3 days of growth in DME + 10-6 M insulin. Ten micrograms of total RNA from each type were hybridized with MyoDI and skeletal actin specific probes.

at high frequency (Konieczny and Emerson, 1984) we have also made use of azamyoblasts. Our results show (Table I) that azamyoblasts homogeneously behave as permissive cells according to the above criteria. As expected, untreated 10T1/2 cells fail to differentiate under any of the culture conditions tested (Table I).

MyoD 1 expression Results from Northern blot analysis (Figure 1) show that MyoDI transcripts are present in proliferating C2 permissive myoblasts. In contrast, no trace of these transcripts is found in inducible myoblasts or in 10TI/2 cells. Both types of myoblast, however, can undergo terminal differentiation (Pinset et al., 1988) as indicated (Figure 1) by the presence of skeletal actin transcripts. At the myotube stage, the level of MyoD] transcripts remains elevated in permissive cells, while these transcripts are still undetected in inducible cells. A detailed time-course analysis of inducible cell differentiation (Figure 2) shows that accumulation of MyoDI transcripts does not occur, even transiently, prior to skeletal actin transcript accumulation. No signal was detected between 0 and 24 h following addition of differentiation medium (data not shown). However, in some experiments, longer periods of exposure of the film elicited a weak signal in differentiated inducible cells, see times 50-72 h in Figure 2, suggesting that accumulation of MyoDI transcripts does occur at a low level in differentiated inducible cells. It is important to note that inducible cells exhibit a phenotype of determined myoblasts. Indeed these cells can undergo terminal differentiation even in the absence of DNA synthesis. Addition of differentiation medium containing 10-4 M AraC to quiescent cells, while totally preventing proliferation (data not shown), does allow the cells to differentiate, as probed by the time course of induction and accumulation of skeletal actin transcripts (Figure 2c). Since low amounts of transcripts would not be detected by Northern blot analysis we re-examined the expression of MyoDl in inducible and permissive cells, using the very sensitive procedure of amplification of single-stranded cDNA by the polymerase chain reaction (mRNA-PCR) (Chelly et al., 1988, and see Materials and methods). The specificity of the amplified products was established: (i) by the size of the fragments deduced from their electrophoretic mobility after staining with ethidium bromide; (ii) by hybridization,

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Fig. 2. Northern blot analysis of a time course of differentiation in inducible cells. Quiescent myoblasts (time 0 obtained by prolonged growth as described previously, Pinset et al., 1988) in MCDB 202 + 20% FCS and 10-7 M Dex were stimulated to differentiate by addition of DME + M 10-6 insulin in the absence (A), (B), (D), or presence (C), of l0-4 M cytosine arabinoside. Ten micrograms of RNA corresponding to the various times after addition of differentiation medium were hybridized with MyoDI, skeletal actin and myogenin specific probes. RNA from Sol8 myotubes were used as positive control in A, B and D.

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Fig. 3. PCR amplification of MyoDJ, cDNA, in myoblasts. Fifteen cycles of amplification were performed as described in Materials and methods, with RNA prepared from proliferating cells. Lane 1, Sol8 myoblasts; lane 2, inducible myoblast; lane 3, lOTl/2 cells, lane 4, T4 azamyoblasts. MyoDI, 149 bp mouse MyoDI specific amplified fragment; PK, 67 bp rat pyruvate kinase specific amplified fragment, used as a standard (see Materials and methods).

under stringent conditions, with the appropriate probes; (iii) by the presence of restriction sites at the expected positions, based on the cDNA sequence (data not shown); and (iv) by the fact that mouse DNA did not direct amplification of these fragments (data not shown). The results are presented in Figure 3. As expected, the MyoDJ-specific cDNA fragment was strongly amplified in Sol8 and T4 myoblasts, which both exhibit a permissive phenotype. In contrast, no signal was detected even after 15 cycles of amplification in inducible myoblasts or in 1OT1/2 cells. Results of a time-course analysis of inducible cell

Expression of MyoDl

inducible C2 lOT'

0

12

24

and accumulation of myogenin mRNA coincided with skeletal actin accumulation (see Figure 2D) and terminal differentiation (Pinset et al., 1988).

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Fig. 4. PCR amplification of MvoDI cDNA during a time course of differentiation of inducible cells. See Figure 2 for culture conditions and Figure 3 and Materials and methods for PCR technique.

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Fig. 5. Northern blot analysis of myogenin transcripts in myoblasts (B) and myotubes (A). Lane 1, IOTI/2 cells; lane 2, inducible cells; lane 3, permissive cells; lane 4, Sol8 cells. See legend of Figure 1 for culture conditions. Ten micrograms of total RNA from each cell type were hybridized with myogenin specific probe.

Discussion MyoD] has been shown to be sufficient for activation of myogenesis in several cell types of mesodermal origin (lOT1/2 cells, 3T3 cells and adipoblasts), and expression of MyoDI appears to be restricted to determined muscle cells and skeletal muscle tissue. These data have led to the proposition that MyoD 1 should play a key role in myogenic determination (Davis et al., 1987). In the present study, we show that MyoD] is not expressed in determined but inducible cells at the myoblast stage, its expression being inducible and accompanying terminal differentiation. MyoD 1 is not obligatory for the maintenance of the myoblast phenotype Inducible C2 myoblasts were derived and cloned from permissive C2 cells after prolonged repeated passaging (Pinset et al., 1988), an approach that we used to study the stability of the myogenic characteristics of permissive cells. Unlike the progenitor cells, inducible cells fail to differentiate in the presence of DME plus FCS, and require insulin at high concentration (10-6 M) or IGFI at a lower concentration (2.5 x 10-8 M) for terminal differentiation. In all the cells that exhibit a permissive phenotype (C2.7, Sol8 and Aza myoblasts), expression of MyoDi is constitutive. By contrast no trace of these transcripts is found in inducible myoblasts, even after 15 cycles of amplification by PCR. It must be noted, however, that after 30 cycles of amplification it is possible to detect MyoDI transcripts, not only in inducible cells but also in lOT1/2 cells and cardiac tissue (data not shown). This reflects that transcription of MyoDI may occur at a very low level in these cells. A level of transcripts corresponding to less than one copy per 100 cells would be detected under these conditions (Chelly et al.,

1989). Despite their characteristics, inducible cells display differentiation using the PCR technique are presented in Figure 4. The presence of the MyoDI-specific cDNA fragment was not detected in quiescent myoblasts (time 0). This fragment became detectable 12 h after addition of differentiation medium and its level increased during the next 2 days as differentiation occurred. As expected, the internal standard was amplified to the same level in each sample. Quantification of these results indicated that the level of the MyoDI cDNA fragment increased 100-fold between 12 and 72 h. These data clearly illustrated that induction and accumulation of MyoDi transcript occurred during terminal differentiation of inducible cells. Quantification of Northern blot analyses indicated, however, that the level of MyoDl transcript in differentiated inducible cells remained -75 times lower than in permissive cells. Detection of myogenin transcripts No Myogenin transcript was detected by Northern blot analysis (Figure SB) at the myoblast stage, in any of the cells that we tested (C2 permissive, Sol8, T4 and C2 inducible). The presence of these transcripts is readily detected in all these cells at the myotube stage. A time-course analysis performed with inducible cells revealed that the induction

a phenotype of determined myoblasts (Turner, 1978; Pinset and Whalen, 1985) since they can undergo terminal differentiation in absence of DNA synthesis. The mechanism that controls MyoD] expression in inducible cells remains to be elucidated. Unlike what is observed with lOT1/2 cells, 5-azacytidine treatment does not lead to any phenotypic conversion and MyoDI expression in inducible cells (D.Montarras and C.Pinset, unpublished observation). This suggests that MyoDI expression is controlled by different mechanisms in inducible and lOTI/2 cells. It has also been observed that lOT1/2 cells treated with 5-azacytidine or transfected with MyoD] lose their ability to differentiate when they cease expressing MyoDI (Davis et al., 1987). In contrast, despite the lack of MyoDi expression, inducible cells still retain the ability to differentiate terminally. These results indicate that MyoDI may not be required for the maintenance of the myoblast phenotype and suggest that other determination factors must be operating in inducible

cells. MyoD 1 expression accompanies differentiation of inducible cells By using the PCR amplification procedure we have observed that induction and accumulation of MyoDI transcripts 2205

D.Montarras et al.

coincide with terminal differentiation of inducible cells. These transcripts become detectable 12 h after addition of differentiation medium and their level subsequently undergoes a 100-fold increase as differentiation proceeds. The relative amounts of MyoD] transcripts in differentiated inducible and permissive cells could not be determined from PCR analysis since in permissive cells (because of their abundance) amplification of MyoD] transcripts is no longer exponential at 15 cycles. However, it appears from quantification of Northern blot analysis that, in differentiated inducible cells, the level of MyoD] transcripts remains much lower (75 times lower) than in differentiated permissive cells. Despite this fact, both cell types finally display the same differentiated phenotype as evaluated by myotube formation and expression of muscle-specific genes. It must be noted, however, that differentiation occurs more slowly in inducible cells (Pinset et al., 1988). This may be a consequence of the pattern of MyoD] expression. Expression of MyoD] was not detected in L6 myoblasts, which exhibit an even slower kinetics of differentiation than inducible cells (Wright et al., 1989). Our results suggest that MyoDl could act as an effector of differentiation. This notion is further sustained by the finding that MyoD 1 could interact with 5' enhancer of the muscle-specific creatin kinase gene (Buskin et al., 1988). In this line, it is interesting to note that, in vivo, appearance of MyoD] transcripts represents a relatively late event in the formation of myotomal muscles (D.Sassoon et al., submitted). It appears also from our study that the level of MyoD] transcripts does not have to be as elevated as it is in permissive cells to promote myogenesis. Unlike what has been observed with MyoD], permissive and inducible cells exhibit similar patterns of myogenin expression. Myogenin transcripts become detectable only when these cells undergo differentiation. The myogenin cDNA was cloned from L6 myoblasts that have overcome the differentiation block induced by BUdR (Wright et al., 1989), a strategy designed to identify factors involved in the decision to differentiate in already determined myoblasts (Wright, 1985). Nevertheless, it was shown recently that myogenin can also activate myogenesis in 10T1/2 cells (Wright et al., 1989). It is conceivable that events of determination and differentiation, the latter being in the sense of the decision to differentiate, may not be so easy to distinguish; indeed from the differences exhibited by permissive and inducible cells one might consider the MyoDl could act by increasing the probability for a myoblast to differentiate. Inducible myoblasts differ strikingly from permissive myoblasts by the media they require to undergo

differentiation, by the pattern of expression of MyoDl and, as recently shown (Crowder and Merlie, 1988), by the presence of distinct DNase I hypersensitive sites in the acetylcholine receptor 6 and -y subunit genes. These differences strongly argue in favour of the fact that these two determined cell lines represent two distinct stages of the progression of myoblasts toward terminal differentiation. It will be informative to determine whether inducible myoblasts express the MyD gene, which has been hypothesized recently to act prior to MyoDI in myogenic conversion of lOT1/2 (Pinney et al., 1988). The use of antisense RNA to MyoDl and the transfection of MyoDI cDNA in inducible cells should prove very useful to clarify the role of MyoD1 as an effector to terminal differentiation.

Materials and methods Cell culture Permissive C2 myoblasts (clone C2.7) and inducible C2 myoblasts (clone C2.7 40.4) have been described previously (Pinset et al., 1988). Sol8 myoblasts were isolated from the soleus muscle of adult C3H mice (Mulle et al., 1988). The embryonic cell line, C3H lOT1/2 (Taylor and Jones, 1979) was obtained from H.Arnold (Medical School, University of Hamburg, FRG). The myogenic clone T4 was derived from 5-azacytidine-treated C3H lOTI/2 cells as previously described (Konieczny and Emerson, 1984). Tissue culture products MCDB 202 and DME media came from Biochrome (Angouleme) and were prepared as described (Pinset et al., 1988). Fetal calf serum (FCS) was from Gibco. Bovine insulin and dexamethasone were from Sigma. RNA RNA preparation, Northern blot analysis and densitometry were performed as previously described (Pinset et al., 1988).

DNA probes The mouse a skeletal actin mRNA specific probe used as the 185 bp PstI-PstI fragment from pAM91.1 which is uniquely homologous to the 3' non-coding portion of the mouse a skeletal actin mRNA (Minty et al., 1981). The mouse MvoDJ mRNA-specific probes used were the 1200 and 585 bp EcoRI-HindHI fragments from PVZ CII (Davis et al., 1987), kindly provided by A.Lassar. Myogenin mRNA specific probe, a gift from W.Wright, was the 1500 bp cDNA clone, which is homologous to the rat myogenin mRNA (Wright et al., 1989). All probes were labelled by random priming (Feinberg and Vogelstein, 1984). Synthesis and amplification of specific cDNAs These are shown in Table II. Specific first strand cDNA synthesis Ten micrograms of total RNA were incubated at 42°C for 1 h in 10 /d of 50 mM Tris-HCI buffer, pH 8.3, containing 75 mM KCI, 3 mM MgCI,, 10 mM dithiothreitol with 5 pmol of the oligonucleotide primer complementary to the transcript and corresponding to the 3' end of the fragment to be amplified. The cDNA was synthesized by primer extension,

Table II. Oligonucleotide primers Location in nucleotide sequence

Predicted size of

amplified fragment

MvoDl complementary primer

5'-TGGTGCGCCCTCTGCTGCTGCAGTC-3'

1147-1122

MyoDI identical primer 5'-TCCGCCAGAGGGGGCATCCCTAAGC-3' Pyruvate kinase complementary primer 5'-GGGTCAGTTGAGCCACACTCG-3'

998-1023

149 bp

617-594

Pyruvate kinase identical primer

5'-AAGCAACGTAGCAGCATGGAA-3'

2206

67 bp

0-21

Expression of MyoD1 using 100 U/itg RNA of MMLV reverse transcriptase (BRL) at 42°C for 2 h in 100 p] of the same buffer containing 1 mM of each dNTP, 50 U placental ribonuclease inhibitor (RNasin, Promega Biotech), I mM sodium pyrophosphate and 50 itg/ml bovine serum albumin.

Polymerase chain reaction (PCR) After NaOH hydrolysis and neutralization the cDNA was co-precipitated with 100 pmol of the oligonucleotide primers complementary and identical to the transcript (see below). cDNA and primers were resuspended in 50 /II of Taq DNA polymerase buffer: 16.6 mM ammonium sulphate, 67 mM Tris-HCI, pH 8.8, 6.7 mM MgCI2, 10 mM ,-mercaptoethanol, 6.7 yM EDTA, 1 mM of each dNTP, and 10% (v/v) of dimethyl sulphoxide. After 10 min at 94°C and 2 min at room temperature, 2 units of Taq DNA polymerase (New England Biolabs) were added and the second strand of cDNA was synthesized for 5 min at 70°C. This step was followed by 15-30 cycles of amplification (denaturation: 1 min at 92°C; annealing: 1 min at 42°C; extension: 2 min at 70°C). To prevent evaporation solutions were covered with mineral oil. As an internal control of amplification to compare relative amounts of MyoDI transcripts, 200 ng of total RNA from rat liver was added to each sample to perform co-reverse transcription and coampliciation of MyoDI cDNA with the rat liver specific pyruvate kinase cDNA (Cognet et al., 1987). No cross-reactivity was detected between mouse myogenic cell RNA and rat pyruvate kinase oligonucleotides and between rat liver RNA and mouse MvoDI oligonucleotides. We also determined that the MAyoDI fragment could not be amplified from mouse DNA. Therefore it is likely that the two MyoDJ oligonucleotides belong to two distinct exons.

Mulle,C., Benoit,P., Pinset,C., Roa,M. and Changeux,J.-P. (1988) Proc. Natl. Acad. Sci. USA, 85, 5928-5932. Pinney,D., Pearson-White,S., Konieczny,F., Latham,K. and Emerson,C,Jr (1988) Cell, 53, 781-793. Pinset,C. and Whalen,R. (1985) Dev'. Biol., 108, 284-289. Pinset,C., Montarras,D., Chenevert,J., Minty,A., Barton,P., Laurent,C. and Gros,F. (1988) Differentiation, 38, 28-34. Tapscott,S.J., Davis,R.L., Thayer,M.J., Cheng,P.-F., Weintraub,H. and Lassar,A.B. (1988) Science, 242, 405-411. Taylor,S.M. and Jones,P.A. (1979) Cell, 17, 771-779. Turner,D.C. (1978) Differentiation, 10, 81-93. Wright,W. (1985) J. Cell. Biol., 100, 311-316. Wright,W., Sassoon,D.A. and Lin,V.L. (1989) Cell, 56, 607-617. Yaffe,D. and Saxel,O. (1977) Nature, 270, 725-727.

Received on March 13, 1989; revised on May 2, 1989

Note added in proof A human muscle factor related to, but distinct from MyoDI and myogenin, was recently described (Braun et al., 1989).

Analysis of the cDNA amplified fragments The amplified products were separated by electrophoresis on 8% (w/v) polyacrylamide gels (8% monoacrylamide, 0.4 % bisacrylamide). After alkaline denaturation for 30 min by 0.2 M NaOH, 0.6 M NaCI solution and washing for 30 min with a 7% v/v formaldehyde solution, gels were blotted overnight onto a nylon filter and then hybridized with specific probes. The rat liver pyruvate kinase (PK) probe used, corresponded to the first common exon of the PK gene (Cognet et al., 1987). The MvoDJ probe used was the 1200 bp EcoRI-HindIII fragment (see above). Final wash of filters was performed at 65°C in 0. 1 x SSC, 0. 1% sodium dodecylsulphate (1 x SSC = 0.15 mM NaCl, 0.015 M sodium citrate, pH 7). Filters were applied to X-Omat Kodak X-ray film with an intensifying screen for different periods of time at -80°C. Levels of amplification of the MyoDI cDNA fragment were determined by densitometry and expressed relative to the level of amplification of the pyruvate kinase cDNA fragment that was used as a standard.

Acknowledgements We thank Drs M.Ontell, M.Buckingham and D.Louvard for critical reading of this manuscript and Dr D.Sassoon for sharing results prior to publication; G.Antolini for preparing this manuscript and the photo service of the Pasteur Institute for the illustrations. This work was supported by grants from the Centre National de la Recherche Scientifique, the Institut National de la Sante et de la Recherche Medicale, the Pasteur Institute, the Association Fran4aise contre les Myopathies, the Association Fan(aise contre le Cancer, the Fondation pour la Recherche Medicale Franfaise and the Ligue FranVaise contre le Cancer.

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