Expression of transforming growth factor β2 during the differentiation of murine embryonal carcinoma and embryonic stem cells

DEVELOPMENTAL

BIOLOGY

137,161-170(1990)

Expression of Transforming Growth Factor ,& during the Differentiation of Murine Embryonal Carcinoma and Embryonic Stem Cells CL. MUMMERY,* H. SLAGER, W. KR~IJER, A. FEIJEN, E. FREUND, I. KOORNNEEF, AND A.J.M. VANDEN EIJNDEN-VAN RAAIJ Hubrecht Laboratory, Netherlands Institute for Developmental Biology, Uppsalulaan 8, .%84 CT Utrecht, The Netherlands Accepted September 8, 1989 Transforming growth factor @a(TGF&) mRNA expression was studied by Northern blot analysis in a range of feeder-independent murine embryonal carcinoma (EC) cells and in feeder-dependent EC and embryonic stem (ES) cells. TGF& transcripts were not detected in any undifferentiated cells including P19, F9, PC13, C1003, PSA-1, PlO, and ES. Following induction of differentiation, however, TGF& became expressed, independently of the cell type formed. Retinoic acid (RA) addition and/or deprivation of the differentiation inhibiting activity of feeder cells resulted in the appearance of TGF@atranscripts within 2 days. These kinetics correlated entirely with the first appearance of the protein; an anti-peptide antibody specifically recognizing TGF& did not stain P19 EC cells by immunofluorescence but 2-3 days after RA addition, a significant proportion of the population was strongly labeled. In addition, primitive endoderm cells emerging from the inner cell mass of substrate attached blastocysts stained brightly with anti-TGF&, while the undifferentiated inner cell mass cells did not. Although all trophectoderm cells at the mid-blastocyst stage were stained, few had detectable levels of TGF& after plating on a substrate. Neither TGFfii nor TGF& affected the growth of EC cells, but a range of differentiated derivatives were all inhibited, with TGF& being marginally more effective than TGF& at the same concentration. D i99o Academic PWJ, IIK.

INTRODUCTION

Type /3 transforming growth factors (TGFPs) are a family of polypeptides that regulate cell growth and differentiation (Massaguk, 1987). This highly ubiquitous molecule has been identified in many nonneoplastic tissues, in transformed cells, and in media conditioned by several cell lines (Goustin et ak, 1986, Sporn et aL, 1987). TGF/3 was originally purified from human platelets as a homodimeric peptide with a molecular mass of 25,000 Da, based on its ability to induce anchorage-independent growth of normal fibroblast indicator cells (Assoian et a.& 1983). It is now apparent that at least five genetically distinct forms of TGF/3 exist, composed of closely related subunits that are found as homo- or heterodimers. The peptide first purified from human platelets has now been designated as TGF& and is the most predominant. A second form, TGF/3,, is a homodimer of subunits which share ‘71% amino acid homology with TGF/3, (Marquardt et ah, 1987). This form has been isolated from bovine bone (Seyedin et at, 1985,1987), from porcine platelets (Cheifetz et a& 1987), and from media conditioned by human glioblastoma (Wrann et al., 1987) and adenocarcinoma cells (Ikeda et ah, 1987). TGF& has been cloned (Derynck et aL, 1988; ten Dijke et aL, 1988; Jakowlew et aL, 198813)but as yet not biologically characterized. TGF& is closely related ’ To whom correspondence should be addressed.

to TGF& but the mRNA encodes a unique 11Gamino acid mature TGF/3 peptide and there is no signal peptide sequence in the precursor protein (Jakowlew et al., 1988a). More recently TGF&, has been identified (Sporn, personal communication) but few details on its characteristics are yet known. Many studies have shown that TGFB is secreted by virtually all cell types in a biologically inactive form (Keski-Oja et ah, 1987; Kryceve-Martinerie et aL, 1985; Lawrence et al., 1984, 1985). The biological latency appears to be due to an inability to bind to the TGFP receptor (Wakefield et aL, 1987). In purification procedures, this latent form has generally been activated by transient acidification (Lawrence et ah, 1984, 1985) which appears to disrupt a complex formed between mature, dimeric TGFP, a precursor remainder, and a third component which by analogy with epidermal growth factor might be a processing protease (Wakefield et aL, 1987). More recently, carbohydrate structures have been shown to be specifically involved with TGF/& latency (Miyazano and Heldin, 1989). Except perhaps in the vicinity of the osteoclast or in healing wounds, exposure to acidic (micro) environments is probably not a universal mechanism of activation of latent TGFP in viva. It is more likely that exogenous proteases, such as plasmin and cathepsin D (Keski-Oja et aL, 1987), would disrupt the quaternary structure of the complex and critically regulate TGFB action in viva.

161

0012-1606/90$3.00 Copyright All rights

0 1990 by Academic Press, Inc. of reproduction in any form reserved.

DEVELOPMENTALBIOLOGYV0~~~~137,1990

162

To date, few functional differences between TGF& and TGF& have been described, with one controversial exception, namely, the induction of mesoderm formation in explants of Xenopus Zaevis (Rosa et al, 1988). While TGFPl is only effective in the additional presence of fibroblast growth factor (FGF), TGF& induces striking mesoderm formation even when added alone. It is as yet unknown whether similar mechanisms might operate in murine development although TGFPl protein has been detected from Day 9 onward in postimplantation embryos (Heine et aZ., 1987); more recently the mRNA for TGF& has been detected as early as the 4- to &cell stage of preimplantation development (Rappolee et al., 1988). We now report that during the differentiation of embryonal carcinoma (EC) cells and embryonic stem (ES) cells, frequently used as a model system for early murine development because of their similarity with pluripotent cells of the inner cell mass in the blastocyst (Graham, 1977), striking changes occur in the expression of TGF&. Although undifferentiated EC and ES cells express TGF& mRNA (Mummery et al, 1989), TGFPz is only expressed after induction of differentiation. Using an antibody which recognizes TGF&, but not TGF&, the TGF& protein product was also only detectable after differentiation. Further, the inner cell mass of blastocyst stage embryo failed to stain but outgrowths of primitive endoderm formed after blastocysts were plated on a substrate stained strongly, confirming the results of two model systems. In addition, while TGFP2 was detectable in the trophectoderm of early- and mid-blastocyst stages, it was no longer detectable after blastocyst outgrowth. Two functionally similar growth factors are thus differentially regulated during the earliest differentiation steps of murine development. The results are discussed in the light of the effects of TGF& and TGF& on cell growth before and after differentiation and possible mechanisms of TGFP activation in the embryo. MATERIALS AND METHODS Cell Lines and Culture

Techniques

Feeder-independent EC cell lines (P19, F9, C1003, PC13) were cultured as described previously for PC13 EC cells (Mummery et aZ., 1984) on gelatinized flasks in growth medium consisting of a 1:l mixture of Dulbecco’s minimum essential medium (DMEM) and Ham’s F12 (DF) with 7.5% fetal calf serum (FCS) and buffered with 44 mM bicarbonate. Cells were passaged three times weekly. Feeder-dependent EC cell lines (PlO, PSA-1) and ES cells, derived basically as described by Evans and Kaufman (1981) from nondelayed blastocysts 129 strain mice, were maintained on primary embryonic fibroblasts from 13-day embryos,

treated with mitomycin C (10 pg/ml) for 3 hr. These cells were grown in MEM with 20% heat-inactivated FCS and 0.1 mM fi-mercaptoethanol. Prior to isolation of RNA, cells were transferred in the absence of feeder cells to growth medium containing 80% MEM conditioned by Buffalo rat liver (BRL) cells (kindly supplied by J. Pitts, Glasgow) with P-mercaptoethanol(O.1 mM) and heat-inactivated FCS, as described by Smith and Hooper (1987). RNA was isolated after 5-10 passages under these conditions. Characteristics of the ES cell line (ES5) have been described briefly previously (Mummery et al., 1989). It has a normal, male karyotype, will form tumors containing derivatives of all three germ layers when injected into syngeneic hosts, and forms chimaeric mice following aggregation with 8-cell stage embryos even after up to 15 passages on BRL-conditioned medium (BRL-CM) (not shown). The differentiated derivatives of P19 EC cells (END-2, EPI-7, MES-1) were cultured as described previously (Mummery et aZ., 1985, 1986). For immunofluorescence, cells were grown to 60-70% confluency on gelatinized glass coverslips in DF + 7.5% FCS in the presence or absence of RA (lop6 M) as required (see below). Induction

of Diferentiation

Feeder-independent EC cells were generally induced to differentiate by the addition of retinoic acid (10e6M from a stock solution of lo-’ M in DMSO stored at -70°C) for various periods to cultures in monolayer, as described previously (Mummery et al., 1984, 1985). Endoderm-like cells are then most frequently formed. Cl003 EC exceptionally were also induced to form neural derivatives by serum deprivation (Darmon et ah, 1982). Feeder-dependent cell lines were induced to differentiate by replacement of the BRL-conditioned medium by normal growth medium (Smith and Hooper, 1987). ES cells were also induced by RA (lo-? M) addition to monolayer cultures in BRL-conditioned medium to form parietal endoderm-like cells. RNA Isolation

and Northern

Blotting

RNA was extracted from nearly confluent cultures grown on 150-cm2tissue culture dishes. Following aspiration of the medium, the cells were lysed directly on the plates with 2.5 ml 4 M guanidine thiocyanate. RNA was isolated according to Chirgwin et al. (1977). Poly(A)+ RNA was isolated by oligo-dT chromatography and quantitated by measuring the optical density at 260 nm. Aliquots of 7.5 pg were denatured in 50% formamide containing 2.2 M formaldehyde and MEN buffer (20 mM 3-[N-morpholinolpropanesulfonic acid, 1.0 mM EDTA, 5.0 mM Na-acetate, pH 7.0) for 15 min at 65°C and electrophoresed in 0.8% agarose gels contain-

MUMMERYET AL.

TGFfiz mRNA

ing 2.2 M formaldehyde. After electrophoresis, RNA was stained with ethidium bromide and transferred to nitrocellulose filters in 20X SSC (1X SSC is 0.15 MNaCl and 0.015 MNa-citrate, pH 7.0). Following transfer, the filters were rinsed in 2~ SSC and baked at 80°C for 2 hr under vacuum. Filters were prehybridized for 4 hr in 5X SSC, 2X Denhardts, 50 mMNa-phosphate, pH 6.8,lO mMEDTA, 0.1% SDS, 0.1 mg/ml sheared salmon sperm DNA containing 50% formamide and hybridized in the same buffer with l-2 X lo6 cpm/ml of 32P-labeled probe for 16-24 hr. The plasmid (PBS, Stratagene) contains the 1.7-kb TGFP, fragment of clone sup 40/l cloned into the EcoRI site, as described (de Martin et ah, 1987). The 32P-labeled probe was obtained by multiprime labeling a DNA fragment with [32P]dCTP (>3000 mmole) and DNA polymerase I to a specific activity of 0.5-l X 10’ cpm/pg. Following hybridization, the filters were washed twice in 2~ SSC and once in 0.1X SSC for 30 min at room temperature followed by a single wash in 0.1X SSC at 55°C. Filters were autoradiographed at 70°C with Kodak X-Omat AR film and intensifying screens. Immunojluorescence

and Antibodies

The presence of TGFP2 in undifferentiated and differentiating P19 EC cells was determined by indirect immunofluorescence using an anti-peptide antiserum. This antibody raised in rabbits against a polypeptide corresponding to the N-terminal amino acid sequences l-29 of porcine platelet TGFP2 specifically recognized the original peptide and porcine TGFP2 in several immunological assays, including ELISA and immunoblotting. The anti-TGFP, antiserum recognized both active and latent TGFP2 as shown by immunoprecipitation analysis. Furthermore, this antiserum completely neutralized the growth inhibitory effect of TGFP2 on mink lung carcinoma (ML-CCL64) cells and the transforming capacity of this factor on quiescent monolayers of NRK cells in the presence of epidermal growth factor (van den Eijnden-van Raaij, submitted). The antiserum was affinity-purified on a Reactigel MW-65F (agarose gel beads; Pierce) column that had been coupled to the TGFP2 peptide. After elution the specifically bound antibodies were used for all experiments, with the nonbound fraction as control. Prior to labeling, cells on coverslips were incubated in serum-free medium for 2 hr, fixed with 2% glutaraldehyde in PBS for 20 min, incubated with sodium borohydride (0.5 mg/ml) for 5 min, and permeabilized with 0.1% Triton X-100 for 4 min., at room temperature. The cells were then exposed to the affinity-purified anti-TGFP, anti-serum or control serum (18 pg/ml in PBS + 2 mg/ml ovalbumin) for 1 hr at room temperature, rinsed with PBS, incubated

163

Expessicm

with fluorescein isothiocyanate-conjugated goat antirabbit IgG (diluted 1:40 in PBS/ovalbumin) for 1 hr at room temperature, rinsed with PBS, and mounted in Moviol. Embryo Collection and Manipulation

For immunofluorescence at the mid-blastocyst stage, embryos were either freshly isolated by flushing from the uterus of superovulated Swiss 3T3 mice 4 days postcoitum (PC),with the plug day as Day 1, or isolated from the oviduct on Day 2 pc and cultured to the blastocyst stage in Ml6 medium (without FCS). This was to establish whether any TGFP2 detected was of embryonic origin or taken up from exogenous (maternal) sources. For further differentiation, blastocysts were plated on to mitomycin C-treated embryonic feeder cells in MEM with 20% heat-inactivated FCS and 0.1 mM ,&mercaptoethanol; under these conditions they attached rapidly, and following trophoblast outgrowth, egg cylinder development occurred and a layer of primitive endoderm cells formed over the remaining embryo. Embryos were fixed and stained as above, except that procedures were carried out in microdrops with micropipets for embryos in suspension. Growth Inhibition

Assay

Cells were plated at 5000 cells per 1.5cm-diameter well in 1 ml DF + 7.5% FCS to attach. TGFP was then added for 64 hr with [3H]TdR (0.5 j&i/ml; 55 mCi/ mmole; Amersham) present for the last 16 hr. Cultures were then aspirated, washed four times with phosphate-buffered saline (PBS), treated with methanol for 15 min, dried in air, and dissolved in 0.1 N NaOH for liquid scintillation counting. Inhibition was expressed as a percentage relative to control cultures in the absence of TGFP. RESULTS Diferentiation and the Appearance TGFP, Expression

of

P19 cells. The expression of TGFP, in EC cells and their differentiated derivatives was initially examined in P19 EC cells. In this model system RA addition to monolayer cultures irreversibly induces differentiation to a mixed population of fibroblast-like cells with endodermal (Mummery et al., 1986) and mesodermal (Roguska and Gudas 1985) characteristics. Figure la shows that TGFP2 expression is not detectable in undifferentiated P19 EC cells, but between 1 and 2 days of RA addition, transcripts of 5.8, 4.8, 4.2, and 3.5 kb were visible on Northern blots hybridized with a cDNA probe for TGFP2. This is approximately the same time at which transcripts for Endo-A, which codes for an in-

164

DEVELOPMENTAL BIOLOGY b

VOLUME 137,199O

Whereas the EC cell lines described remain undifferentiated under standard culture conditions and require chemical induction, for example by RA, to form other cell types, PlO and PSA-1 EC cells are dependent for maintainance of their undifferentiated state on coculture with fibroblast feeder cells; medium conditioned by 5.8 kb BRL cells contains a factor(s) (differentiation inhibiti:; kk ing activity or DIA) also able to maintain these cells in 3.5 kb an undifferentiated state (Smith and Hooper, 1987). Culture in BRL-conditioned medium facilitates isolation of mRNA from feeder-dependent EC cell lines in the absence of contaminating feeder cells. In neither PlO or PSA-1 EC cells are TGF& transcripts detectable prior to differentiation (Fig. 2). FIG. 1. Northern blot analysis of TGFPz expression in P19 EC cells. Embryonic stem cells. ES cells isolated directly from (a) Effect of RA on P19 EC cells in monolayer. RA (1O-6M) was added preimplantation embryos are also feeder dependent; at Time 0 and poly(A)+ RNA was isolated at the time indicated, as described under Materials and Methods. (b) TGF& expression in dif- upon removal of DIA, ES cells acquire a flattened morferentiated clonal derivatives of P19 cells; MES-1 is a mesodermal phology (Smith et al., 1988), while addition of RA in the derivative (Mummery et aL, 1986), END-2, an endoderm-like deriva- presence of DIA/LIF induces differentiation to parietal tive and EPI-7 has neuroepitheleal characteristics (Mummery et al., endoderm-like cells (Smith and Hooper, 1987; Heath et 1985). al., 1989). Undifferentiated ES cells do not express TGFPB, but following differentiation, induced by either RA addition or DIA/LIF deprivation, all four TGFPB termediate filament protein expressed in endoderm transcripts are expressed (Fig. 3). In view of the close cells (Duprey et aZ.,1985), first becomes evident (Mum- relationship between ES cells and preimplantation emmery et al, 1989). TGFPz transcripts are not observed bryos, evidenced by the high efficiency with which they exclusively in RA-treated cells; MES-1 cells, mesoderma1 derivatives of P19EC isolated from DMSO-treated embryoid bodies (Mummery et ah, 1986), also express TGFPB as shown in Fig. lb. Expressions in END-2 and EPI-7, endodermal and neuroepithelial derivatives of P19 (Mummery et al, 1985), are shown for comparison. Feeder-dependent versus-independent EC cells. Expression of TGFPz was also determined in three other EC cell lines, F9, PC13, and C1003. Transcripts were not detectable on Northern blots of any cell type prior to differentiation (Fig. 2). RA addition to F9 cells in monolayer in the presence of dbcAMP induces differentiation to a homogeneous population of parietal endoderm-like cells (Hogan et al, 1981) expressing laminin. TGF& expression is also induced during differentiation with approximately the same kinetics as in P19 cells, appearing primarily as transcripts of 4.2 and 3.5 kb 2 days after RA addition (Fig. 2). The 5.8- and 4.8-kb transcripts observed in differentiated P19 are much reduced in differentiated F9 cells. In the human EC cell line Tera-2 clone 13 (Engstrom et ah, 1984) RA also strongly inFIG. 2. Northern blot analysis of TGF& expression in feeder-indeduces TGFPz when added to monolayer cultures (Fig. 2); pendent versus feeder-dependent EC cells. Poly(A)+ RNA was isothree of the four transcripts seen in differentiated P19 lated from F9 EC cells, F9 EC cells treated for 5 days with RA (10e6 cells (5.8, 4.8, and 4.2 kb) are also visible in Tera-2, as M) and dbCAMP (2 mM), Cl003 EC cells either deprived of serum or well as a 7.5-kb mRNA. TGF& is only weakly induced in treated with RA (10m6M) for 5 days, and human Tera-2 clone 13 EC cells (T2) treated with or without RA (5 X 10m5M) for 5 days, as Cl003 cells although four transcripts are evident on described under Materials and Methods. PSA-1 and PlO EC cells were Northern blots whether differentiation is induced by transferred from feeder layers and cultured for five passages in BRL-conditioned medium prior to RNA isolation. RA or by serum deprivation. a

RA (days)

+T I.ocn

7

MUMMERYET AL.

TGFB, mRNA Expression

a a u u?

5.8 4.8

kb kb

“3:: kbb

FIG. 3. Effect of differentiation on TGF& expression in ES cells. Poly(A)+ RNA was isolated for Northern blot analysis from ES cells grown for five passages in BRL-conditioned medium (BRL-CM) after transfer from feeder layers. Differentiated derivatives were obtained by the addition of RA (5 X lo-’ M) to BRL-conditioned medium (3 days, 5 days) or the transfer of cells to identical, nonconditioned medium (MEM) for 5 days.

form germ line chimaeras when injected in blastocysts (Bradley et ah, 1984), similar changes in the pattern of TGF& expression might be expected during endoderm formation in preimplantation and early postimplantation embryos. Detection of TGFP, by Immunojuwescence during Werentiation of EC Cells To establish when the TGF& protein first became detectable after the induction of differentiation, P19 EC cells were plated in DF + 7.5% FCS in the presence of RA (10e6 M) for 1 to 5 days, incubated in serum-free medium for 2 hr prior to collection to remove any traces of exogenous TGFB from FCS, and then fixed as described under Materials and Methods. An antibody to a partial peptide sequence corresponding to the N-terminal residues l-29 of porcine platelet TGF& has recently been described in detail (van den Eijnden-van Raaij et aZ.,submitted for publication); this antibody recognizes both the TGF& peptide and native porcine TGFBz, without cross-reactivity with TGF&, and was used in the studies described below. Figures 4a and 4b show that no staining above background is observed in undifferentiated P19 EC cells; incidentally, rounded cells in mitosis are stained but this is apparently artifactual since similar cells labeled with control serum are also stained (not shown). The first positively labeled cells appear 2 days after RA addition with a substantial proportion of cells stained by Day 3 (Figs. 4c and 4d).

165

The cell population is then of mixed morphology with both fibroblast- and endoderm-like cells evident. Clearly not all cells were positively stained, even on Day 5 (Figs. 4e and 4f), when labeling is very intense. Whether labeling was restricted to one particular cell phenotype was not clear. Background labeling of differentiated cells with control serum was slightly lower than that of undifferentiated EC cells, probably because of their flattened morphology (not shown). Detection of TGFP, bp Immunofluorescence in Embryos In a series of blastoeysts at the same developmental stage, no staining above background was detectable in the ICM of embryos. By contrast, all cells of the trophectoderm were brightly stained (Figs. 5a and 5b) independently of whether embryos were freshly isolated or maintained for 2 days in culture (not shown). This would suggest that the TGF& was of embryonic and not of maternal origin. After replating embryos to feeder layers to encourage trophectoderm outgrowth and endoderm differentiation, however, the majority of trophectoderm cells completely lacked anti-TGF& staining, while the emerging primitive endoderm clearly labeled above background, the apparently undifferentiated, underlying ICM cells remaining negative (Figs. 5c and 5d). Eflect of TGF@ on the Growth of EC Cells The effects of TGF& on the growth of F9 and PC13 EC cells and their differentiated progeny have been described previously by Rizzino (1987). Prior to differentiation, the growth of neither EC cell line was affected by TGF& addition under serum-free conditions, but following 48 hr induction by RA, cell numbers were significantly lower than those in control cultures when TGF& had been present over a 72-hr period. We have now investigated the effect of TGF& on several EC cell lines and some of their differentiated derivatives. The results are shown in Table 1. Addition of 1 rig/ml of TGF/3, to exponentially growing P19, PC13, C1003, and F9 EC cells had no significant effect on [3HJTdR incorporated during the last 16 hr of the assay; by contrast all differentiated derivatives tested under identical conditions were significantly inhibited in growth. The EPI-‘7 cell line, an epithelial cell type derived from P19 EC, was particularly strongly inhibited, with levels of [3H]TdR lower than even those in ML-CCL 64 cells, a cell line frequently used in bioassays to detect TGFB-like activity (van Zoelen et aL, 1986). The effects of 1 rig/ml of TGFPi in a parallel assay are shown for comparison. The lack of effect of TGF& on undifferentiated EC cells was confirmed and in all differentiated cells TGF& inhibited growth to a somewhat lesser extent than TGFP2.

DEVELOPMENTALBIOLOGY

166

VOLUME137,199O

FIG. 4. Immunofluorescent staining of P19 EC cells with anti-TGF& antiserum during differentiation. RA (10m6M) was added at Time 0 to P19 EC cells in monolayer culture. Cells were fixed and stained with affinity-purified anti-TGF& peptide anti-serum at Time 0 (a, b), after 3 days (c, d) and after 5 days (e, f). a, e, and c show the fluorescence staining and b, d, and f the corresponding phase-contrast field.

DISCUSSION

In the present study we have shown that none of a variety of murine and human EC cells express TGF&

mRNA prior to differentiation, but that after induction of differentiation to endoderm- or mesoderm-like cells, TGF& becomes expressed. Apart from the usual 5%, 4%, and 4.2-kb transcripts, differentiated murine EC

MUMMERY ET AL.

TGF@, rnRNA Expression

167

FIG. 5. Immunofluorescent staining of embryos with anti-TGF@, antiserum. Embryos were collected at the hlastocyst stage, fixed immediately, and stained with anti-TGF& (a, b) or were plated onto flbroblast feeder layers for 2-3 days, to facilitate endoderm outgrowth, prior to fixation and staining, as described under Materials and Methods (c, d). b and d show fluorescence while a and c show the corresponding phase-contrast field.

cells, but not human Tera-2, also express a 3.5kb transcript. The high degree of homology between members of the TGF/3 family increases the possibility of crosshybridization so that this 3.5-kb mRNA could represent TGFP3. Studies in progress comparing human and murine EC cells with a specific TGF& probe (Jakowlew et ak, 1988b) should establish whether this is indeed the case. On the other hand, the murine TGFPz cDNA has recently been cloned (Miller et al, 1989) and five different transcripts have been observed at all stages, from

10.5 to 17.5 days, of murine development. The mRNA species were however differentially regulated, the 3.5kb transcript being specifically expressed late in development, the 5.0 (4.8)-kb transcript being transiently expressed, and the other mRNA species continually increasing. We observe similar differences in the abundance of different transcripts in the differentiated cells in the present study, for example RA-treated P19 EC cells compared with P19 MES-1 and ES + RA with ES + MEM, but it will require characterization of their struc-

168

DEVELOPMENTAL BIOLOGY

TABLE 1

VOLUME 13’7.1990

receptor. Scatchard analysis has already shown that TGFPi receptors at least are detectable on the surface of the differentiated derivatives of P19 used in the present study (Mummery et al, 1989). Similar studies are % Growth inhibition now in progress with TGF&. The mechanism of signal Cell type TWA TGWz transduction of these receptors is as yet unknown (Cheifetz et aL, 1987). 6 -8 P19 EC All members of the TGFP gene family studied to date -5 -5 PC13 EC are encoded as larger precursor peptides (330-575) and -5 0 Cl003 EC generally contain signal peptide sequences near their 0 0 F9 EC N-termini; the mature peptide is derived by proteolytic 50 P19 MES-1” 38 cleavage from the precursor protein. Either or both the 75 95 P19 EPI-7b precursor (or latent) and mature form of TGFP may be 40 65 P19 END-2b 40 45 Cl003 Fib-g” produced by cells and secreted into their medium. Only 56 60 ML-CCL 64d the mature TGF@ is able to bind to specific receptors and is biologically active, although the latent precursor Note. Data are expressed as the percentage of [3H]TdR incorporated in cells exposed to TGFP relative to that of cells in the absence of can be activated by acidification. By determining activity before and after exposure to pH 2, the relative TGFP. Standard errors were <5% in all cases. u Mesodermal derivative of P19; Mummery et aL, 1986. amounts of active and latent TGFP produced by differbNeuroectodermaVendodermal derivatives of P19; Mummery et aL, ent cell types can be determined. Most cells produce 1985. latent TGFP and preliminary results (van den EijndencMesodermal derivative of C1003; Mummery and Piersma, unpubvan Raaij et al., in preparation) have shown this is also lished observations. the case for differentiated EC cells; by contrast, howd Mink lung cells. ever, extracts of undifferentiated EC cells appear to contain active TGF/3. Since we know that all EC cells tural differences before questions regarding their dif- investigated so far and ES cells express TGF& (Mumferential transcriptional regulation can be addressed. mery et al, 1989) but not TGF& it is probable that the Using anti-peptide antibodies which detect the TGFflz growth inhibitory activity the undifferentiated cells but not TGFPl protein, it was also shown that in P19 EC express is TGF&, assuming that no other members of cells, studied so far in most detail, TGF& became de- TGFP family are involved. Since EC and ES cells have low levels of surface receptors for TGF/3 (Rizzino, 1987), tectable at approximately the same time after induction of differentiation as the mRNA. On the basis of the TGFP receptors are not down-regulated upon ligand failure of both TGFPi and TGF/?, to affect the growth of binding (Massague and Kelly, 1986) and differentiated the undifferentiated EC cells and published data that EC cells only secrete latent TGFP, it would appear unhave shown that EC cells lack or have low levels of likely that TGF/3 operates via a simple autocrine loop stages. However, receptors for TGFPl (Rizzino, 1987), it would be reason- during these early differentiation able to assume that these cells also have low levels of noncomplex interactions between cells via TGFP in early development are quite feasible. Pluripotent stem receptors for TGF&. On the other hand, three distinct TGFP receptor types have been identified on the surface cells may secrete active TGFPi, which binds to recepof almost all cell types examined (Cheifetz et ah, 1986); tors on more differentiated cell types. In turn, this may these have been operationally defined as TGF-P recep- induce secondary gene activation resulting in, for examtor types I (53 kDa), II (73-95 kDa), and III (300 kDa). ple, increased production of extracellular matrix facTypes I and II bind TGFPl with higher affinity than tors (Ignotz and Massague, 1986; Ignotz et aZ., 1987; TGF&, while type III binds with equal affinity. It has Roberts et uZ.,1986; reviewed Rizzino, 1988). In addition, more recently been shown (Boyd and Massague, 1989) it has recently been shown that proteolytic enzymes are that cell surface expression of the type I receptor is able to activate latent TGFP in vitro (Lyons et al, 1989). correlated with growth inhibition. EC cells may lack the Parietal endoderm cells in preimplantation embryos type I receptor but express the other receptor types, secrete high levels of plasminogen activator (PA) (Strickland and Mahdavi, 1978); this proteolytic enzyme Studies are currently in progress to establish whether low levels of binding are correlated with other bioef- may be able to activate latent TGF/3 produced either by fects. By contrast, all differentiated EC cells tested the cells themselves or by other cells also present at were strikingly inhibited in exponential growth by that stage of development. At present there are few TGFPl and TGFPz, suggesting that they do have surface other clues to the exact role of TGFP in development. receptors for both factors probably including the type I Data on cells in vitro have shown that TGFP can act as a TGF&INDUCED GROWTH INHIBITION OF EC CELLS AND THEIR DIFFERENTIATED DERIVATIVES

MUMMERY ET AL.

TGFfle mRNA E~p~s~ion

169

W. (1977). Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistrg l&5294-5299. DARMON, M., STALLCUP,W., and PITTMAN, Q. (1982). Induction of neural differentiation by serum-deprivation in cultures of the embryonal carcinoma cell line 1003. Exp. Cell. Res. 138, 73-78. DE MARTIN, R., HAENDLER, B., HOFERWARBINEK,R., GAUGITSCH,H., WRANN,M., SCHL~SENER,H., SEIFERT,S. M., BODMER, S., FONTANA, A., and HOFER, E. (1987). Complementary DNA for human glioblastoma-derived T cell suppressor factor, a novel member of the transforming growth factor-p gene family. EMBO .J. 6,3673-3677. DERYNCK,R., LINDQUIST,P. B., LEE, A., WEN, D., TAMM, J., GRAYCAR, J. L., RHEE, L., MASON,A. J., MILLER, D. A., COFFEY,R. J., MOSES, H. L., and CHEN, E. Y. (1988). A new type of transforming growth factor, TGF-PB. EMBO J. 7.3737-3743. DUPREY,P., MORELLO,D., VASSEUR,M., BABINET, C., LONDAMINE,H., BR~LET, and JACOB,F. (1985). Expression of the cytokeratin endo A gene during early mouse embryogenesis. Proc NatL Acad Sci. USA 82,8535-8539. ENGSTRBM,W., REES,A. R., and HEATH, J. K. (1985). Proliferation of a human embryonal carcinoma-derived cell line in serum-free medium: Inter-relationship between growth factor requirements and membrane receptor expression. J. CeUSci. 73,361-373. EVANS, M. J., and KAUFMAN, M. H. (1981). Establishment in culture of pluripotential cells from mouse embryos. Nature (London) 292, 154-156. GOUSTIN,A. S., LEOF, E. B., SHIPLEY, G. D., and MOSES,H. L. (1986). Cancer Res. 46,1015-1029. GRAHAM, C. F. (1977). Teratocarcinoma cells and normal mouse embryogenesis. In “Concepts in Mammalian Embryogenesis” (M. I. Sherman and C. F. Graham, Eds.), pp. 315-394. MIT Press, Cambridge, MA. HEATH, J. K., SMITH, A. G., WILLS, A. J., and EDWARDS,D. R. (1989). Growth and differentiation in factors of embryonic stem cells. in “Cell to Cell Signals in Mammalian Development” (S. W. de Laat, J. G. Bluemink, and C. L. Mummery, Eds.), NATO AS1 Series, We thank T. van Achterberg, S. van Genesen, W. van Rotterdam, Springer Verlag, Heidelberg. Vol. H26, pp. 219-229. and J. Schoorlemmer for their contributions to cell culture and pro- HEINE, V. I., FLANDERS,K., ROBERTS,A. B., MUNOZ,E. F., and SPORN, ducing blots. The ES cell line used was isolated by Dr. H. C. Tsung of M. B. (1987). Immunocytochemical localization of TGF/3 during emthe Academia Sinica, Shanghai, during a working visit to the Hubryonal development in mice. Proc. Amer. Assoc. Cancer Res. 28,53. brecht Laboratory in 1987. We are also indebted to Dr. M. Wrann for HOGAN, B. L. M., TAYLOR, A., and ADAMSON,E. (1981). Cell interacthe TGF& probe and to Drs. E. J. J. van Zoelen and S. W. de Laat for tions modulaste embryonal carcinoma cell differentiation into parimany discussions and their continued support of this project. The eta1 or visceral endoderm. Nature (London) 291,235. work was in part financed by the Koningin Wilhelmina Fonds, the IGNOTZ,R. A., ENDO, T., and MASSAGUI?,J. (1987). Regulation of fibroDutch Cancer Society (A.J.v.d.E.; I.K.). nectin and type I collagen mRNA levels by transforming growth factor+?. J. BioL Chem. 262,6443-6446. IGNOTZ,R. A., and MASSAGUE,J. (1986). Transforming growth factorREFERENCES p stimulates the expression of fibronectin and collagen and their incorporation into the extracellular matrix. J. Biol. Chem. 261, ASSOIAN, R. K., KOMORIYA, A., MEYERS, C. A., MILLER, D. M., and 4337-4345. SPORN,M. B. (1983). Transforming growth factor-p in human plateIKEDA, T., LIOUBIN, M. N., and MARQUARDT,H. (1987). Human translets. J. BioL Chem. 258,7155-7X0. forming growth factor type @a:Production by prostatic adenocarciBOYD, F. T., and MASSAGU~,J. (1989). Transforming growth factor-8 noma cell line, purification and initial characterization. Biochemisinhibition of epithelia cell proliferation linked to the expression of a trg 26,2406-2410. 53 kD membrane receptor. J. BioL Chem 264,22’72-2278. JAKOWLEW,S. B., DILLARD, P. J., SPORN,M. B., and ROBERTS,A. B. BRADLEY,A., EVANS, M., KAUFMAN, M. H., and ROBERTSON, E. (1984). (1988a). Complementary deoxyribonucleic acid cloning of a messenFormation of germ-line chimaeras from embryo-derived teratocarger ribonucleic acid encoding transforming growth factor P4 from cinema cell lines. Nature (London) 309,255-256. chicken embryo chondrocytes. MoL EndocrimL 2,1186-1195. CHEIFETZ,S., LIKE, B., and MASSAGU~,J. (1986). Cellular distribution JAKOWLEW,S. B., KONDAIAH, P., DILLARD, P. S., SPORN,M. B., and of type I and type II receptors for transforming growth factor /3.J. ROBERTS,A. B. (198813).Complementary deoxyribonucleic acid BioL Chem. 261,9972-9978. cloning of a novel transforming growth factor-p messenger ribonuCHEIFETZ,S., WEATHERBEE,J. A., TSANG, M. L. S., ANDERSON,J. K., cleic acid from chicken embryo chondrocytes. Mol. Endocrinol 2, MOLE, J. E., LUCAS,R., and MASSAGU~,J. (1987). The transforming 747-755. KESKI-OJA, J., LYONS, R. M., and MOSES,H. L. (1987). Inactive segrowth factor-p system, a complex pattern of cross-reactive ligands creted form(s) of transforming growth factor-p: Activation by proand receptors. Cell 48.409-415. teolysis. J. Cell B&hem. (Suppl.) IlA, 60. CHIRGWIN,J. M., PRYZBALA,A. E., MACDONALD,R. Y., and RUTTER,

mitogen, stimulating anchorage independent growth of nontransformed cells, as a growth inhibitor, or as an inducer of differentiation, depending on the nature of the target cell and the circumstances under which it is added (reviewed Sporn et ah, 1987). Immunofluorescence with the anti-TGF& antibody in the present study has clearly demonstrated that the cellular localization of the protein is developmentally regulated during the pre- and peri-implantation stages. If the protein is also activated, by PA for example, and secreted in the blastocyst, it could be involved in directly regulating growth or the very specific pattern of extracellular matrix production and secretion in the embryo which in particular results in the formation of Reichert’s membrane. One approach to investigating the function of TGFP in development is to use specific antibodies to neutralize secreted TGFP-like activity. Since the antipeptide antibody recognizing native TGF& used in the present study is also able to neutralize specifically TGFPz activity (van den Eijnden-van Raaij et al, submitted for publication), studies are currently in progress to follow the effect of its presence on preimplantation development both in terms of the efficiency with which embryos develop to blastocysts in vitro and the levels at which TGFfl-regulated protein products are expressed. Microinjection of the antibody into cells at various developmental stages will be used to establish whether TGFP, has to be secreted to exert its effect.

170

DEVELOPMENTAL BIOLOGY

KRY&VE-MARTINERIE, C., LAWRENCE, D. A., CROCHET, J., JULLIEN, P., and VIGIER, P. (1985). Further study of P-TGFs released by virally transformed and non-transformed cells. Int. J. Cancer 35, 553-558. LAWRENCE, D. A., PIRCHER, R., and JULLIEN, P. (1985). Convension of a high molecular weight latent @-TGF from chicken embryo fibroblasts into low molecular weight active P-TGF under acidic conditions. Biochem. Biophys. Res. Ccnnmun. 133,1026-1034. LAWRENCE, D. A., PIRCHER, R., KRYC$VE-MARTINERIE, C., and JULLIEN, P. (1984). Normal embryo fibroblasts release transforming growth factors in a latent form. J CelL Physiol. 121,184188. LYONS, R. M., KESKI-OJA, J., and MOSES, H. L. (1989). Proteolytic activation of latent transforming growth factor fl from fibroblastconditioned medium. J. Cell BioL 106,1659-1665. MARQUARDT, H., LIOUBIN, M. N., and IKEDA, T. (1987). J. Biol. Chem. 262, l2,127-12,131. MASSACU$, J. (1987). The TGF-fi family of growth and differentiation factors. Cell 49,437-438. MASSAGUI?, J., and KELLY, B. (1986). Internalization of transforming growth factor p and its receptor in Balb/C 3T3 fibroblasts. J. Cell. PhysioL 125,216-222. MILLER, D. A., LEE, A., PELTON, R. W., CHEN, E. Y., MOSES, H. L., and DERYNCK, R. (1989). Murine transforming growth factor & cDNA sequence and expression in adult tissues and embryos. MoL End+ crinol. 3,1108-1114. MIYAZONO, K., and HELDIN, C. H. (1989). Role for carbohydrate structures in TGF@, latency. Nature (London) 338,158-160. MUMMERY, C. L., VAN DEN BRINK, C. E., VAN DER SAAG, P. T., and DE LAAT, S. W. (1984). The cell cycle, cell death and cell morphology during retinoic acid induced differentiation of embryonal carcinoma cells. Dev. BioL 104,297-307. MUMMERY, C. L., FEIJEN, A., VAN DER SAAG, P. T., VAN DEN BRINK, C. E., and DE LAAT, S. W. (1985). Clonal variants of differentiated P19 EC cells exhibit EGF receptor kinase activity. Dev. BioL 109, 402-410. MUMMERY, C. L., FEIJEN, A., VAN DEN BRINK, C. E., MOOLENAAR, W. H., and DE LAAT, S. W. (1986). Establishment of a differentiated mesodermal line P19 EC cells expressing functional PDGF and EGF receptors. Exp. Cell. Res. 165,229-242. MUMMERY, C. L., VAN DEN EIJNDEN-VAN RAAIJ, J., FEIJEN, A., TSUNG, H. C., and KRUIJER, W. (1989). Regulation of growth factors and their receptors in early murine embryogenesis. In “Cell to Cell Signals in Mammalian Development” (S. W. de Laat, J. G. Bluemink, and C. L. Mummery, Eds.), NATO AS1 Series, Springer Verlag, Heidelberg. Vol H26. RAPPOLEE, D., BRENNER, C. A., SCHULTZ, R., MARK, D., and WERB, Z. (1988). Developmental expression of PDGF, TGFol, and TGFP genes in preimplantation mouse embryos. Science 241,1823-1825. RIZZINO, A. (1987). Appearance of high affinity receptors for type fl transforming growth factor during differentiation of murine embryonal carcinoma cells. Cancer Rex 47,4386-4390. RIZZINO, A. (1988). Transforming growth factor @ Multiple effects on cell differentiation and extracellular matrices. Dev. BioL 130, 411-422. ROBERTS, A. B., SPORN, M. B., ASSOIAN, R. K., SMITH, J. M., ROCHE, N. S., WAKEFIELD, L. M., HEINE, V. I., LIOTTA, L. A., FALANG, A. V.,

VOLUME 137,199O

KEHRL, J. H., and FAUCI, A. S. (1986). Transforming growth factor type p: Rapid induction of fibrosis and angiogenesis in vivo and stimulation of collagen formation in vitro. Proc. NatL Acad. Sci. USA 83,4167-4171. ROGUSKA, M. A., and GUDAS, L. S. (1985). An increase in prolyl-4-hydroxylase activity occurs during the retinoic acid-induced differentiation of mouse teratocarcinoma stem cell lines F9 and P19. J. Biol. Chem. 260, 13,893-13,896. ROSA, F., ROBERTS, A. B., DANIELPOUR, D., DART, L. L., SPORN, M. B., and DAVID, I. B. (1988). Mesoderm induction in amphibians: The role of TGF&-like factors. Science 239,783-785. SEYEDIN, S. M., SEGARINI, P. R., ROSEN, D., THOMPSON, A. Y., BENTZ, H., and GRAYCAR, J. (1987). Cartilage inducing factor-p is a unique protein structurally and functionally related to transforming growth factor 0. J. BioL Chem 262,1946-1949. SEYEDIN, S. M., THOMAS, T. C., THOMPSON, A. Y., ROSEN, D. M., and DIEZ, K. A. (1985). Purification and characterization of two cartilage-inducing factors from bovine demineralized bone. Proc. NutL Acad. Sci. USA 82,2267-2271. SMITH, A. G., HEATH, J. K., DONALDSON, D. D., WONG, G. G., MOREAU, J., STAHL, M., and ROGERS, D. (1988). Inhibition of pluripotential embryonic stem cell differentiation by purified polypeptides. Nature (London) 336,688-690. SMITH, A. G., and HOOPER, M. L. (1987). Buffalo rat liver cells produce a diffusible activity which inhibits the differentiation of murine embryonal carcinoma and embryonic stem cells. Dev. BioL 121,1-9. SPORN, M. B., ROBERTS, A. B., WAKEFIELD, L. M., and DE CROMBRUGGHE, B. (1987). Some recent advances in the chemistry and biology of transforming growth factor-&J. Cell Biol. 105,1039-1045. STRICKLAND, S., and MAHDAVI, V. (1978). The induction of differentiation in teratocarcinoma stem cells by retinoic acid. Cell 15, 393-403. TEN DIJKE, P., HANSEN, P., IWATA, K. K., PIELER, C., and FOULKES, J. G. (1988). Identification of another member of the transforming growth factor type 0 gene family. Proc. NatL Acud. Sci. USA 85, 4715-4719. VAN ZOELEN, E. J. J., VAN OOSTWAARD, T. M. J., and DE LAAT, S. W. (1986). PDGF-like growth factor induces EGF-potentiated phenotypic transformation of normal rat kidney cells in the absence of TGFP. B&hem. Biwphys. Res. Commun. 141,1229-1935. WAKEFIELD, L. M., SMITH, D. M., MASUI, T., HARRIS, C. C., and SPORN, M. B. (1987). Distribution and modulation of the cellular receptor for transforming growth factor /3. J. Cell BioL 105,965-975. WILLIAMS, R. L., HILTON, D. J., PEASE, J., WILLSON, T. A., STEWART, C. L., GEARING, D. P., WAGNER, E. F., METCALF, D., NICOLA, N. A., and GOUGH, N. M. (1988). Myeloid leukaemia inhibitory factor maintains the developmental potential of embryonic stem cells. Nature (London) 336,684-687. WOZNEY, J. M., v. ROSEN, A. J., CELESTE, L. M., MITSOCK, M. J., WHITTERS, R. W., KRIZ, R. M., HEWICK, and WANG, E. A. (1989). Novel regulators of bone formation: Molecular clones and activities. Science 242,1528-1534. WRANN, M., BODMER, S., DE MARTIN, R., SIEPL, C., HOFER-WARBINEK, R., FREI, K., HOFER, E., and FONTANA, A. (1987). T Cell suppressor factor from human glioblastoma cells is a 12.5 kd protein closely related to transforming growth factor-p. EMBO J. 6,1633-1636.