Cytotechnology 4: 227-242, 1990. 9 1990KluwerAcademic Publishers. Printed in the Netherlands.

Regulation and expression of transforming growth factor type-g during early mammalian development David Kelly, Wendy J. Campbell, Jay Tiesman and Angie Rizzino Eppley Institute for Cancer Research and Allied Diseases, University of Nebraska Medical Center, 42nd and Dewey Avenue, Omaha, NE 68198-6805, USA Received 13 March 1990; accepted in revisedform 9 May 1990

Key words: transforming growth factor type-beta, embryonal carcinoma cells, mammalian embryogenesis, polymerase chain reaction Abstract We have examined the effect of differentiation on the expression of different members of the transforming growth factor type-beta (TGF-6) family using embryonal carcinoma (EC) cells and early mammalian embryos. We determined that TGF-B activity increases approximately 25-100% when the mouse EC cell line, F9, is induced to differentiate with retinoic acid (RA). Interestingly, the increased TGF-B activity reflects the induction of TGF-B2 secretion following differentiation of both F9 EC cells and the human EC cell line, NT2/D1. Using the technique of reverse transcription-polymerase chain reaction (RT-PCR), we have verified that differentiation induces the expression of TGF-g2 as well as a distant member of the TGF-B family, Vgr-1. Transcripts for TGF-B2 and Vgr-1 were readily detected in the differentiated cells of F9 and PC- 13 but not in their undifferentiated counterparts. Moreover, TGF-B2 mRNA was readily detected in NT2/D1 cells following differentiation. In addition, transcripts for TGF-B2 were detected by RT-PCR in mouse morulae, preimplantation blastocysts and cultured blastocysts. Based on the data presented, it appears that the expression of both TGF-g2 and Vgr-1 is closely associated with the induction of differentiation during early development. Abbreviations: CS - Calf Serum, EC - Embryonal Carcinoma, FBS - Fetal Bovine Serum, RA Retinoic Acid, RT-PCR - Reverse Transcription-Polymerase Chain Reaction, TGF-B - Transforming Growth Factor type-beta

Introduction Transforming growth factor type-B (TGF-B) refers to a complex family of related polypeptides that are multifunctional regulators of cell growth and differentiation (reviewed in Rizzino, 1988). TGF-B was originally characterized by its ability to induce the anchorage-independent growth of

nontransformed cells (DeLarco and Todaro, 1978). Subsequent work established that TGF-B has potent effects on the proliferation of a wide variety of cells (reviewed in Sporn et al., 1986). In addition to the effects on cell proliferation, TGF-B has been shown to influence the differentiation of many cell types (reviewed in Rizzino, 1988; Kelly and Rizzino, 1989). Moreover, TGF-

228 B has also been shown to influence growth and differentiation in vivo (Silberstein and Daniel, 1987; Roberts et al., 1986). At present, five genetically distinct forms of TGF-B have been identified. The first three forms, TGF-B1, TGF-B2, and TGF-B3, have been isolated from a variety of mammalian sources and exhibit approximately 70-80% amino acid sequence similarity (Assoian et al., 1983; Derynck et al., 1985; Cheifetz et al., 1987; Derynck et al., 1988; ten Dijke et al., 1988; Jakowlew et al., 1988b). The other two forms, TGF-B4 and TGF-B5, have been cloned from cDNA libraries generated from chicken and amphibian embryos, respectively (Jakowlew et al., 1988a; Kondaiah et al., 1990). The different forms of TGF-B also share significant amino acid sequence similarity (greater than 30%) with the mammalian Vgr-1 gene product (Lyons et al., 1989), mammalian Mullerian inhibiting substance (MIS) (Care et al., 1986), the inhibins (Mason et al., 1985), the gene product of the Drosophila decapentaplegic complex (DPP-C) (Padgett et al., 1987), and the Xenopus Vg-1 gene product (Rebagliati et al., 1985). In most in vitro systems, TGF-B1 and TGF-B2 exert similar effects and appear to be functionally equivalent; however, several studies have shown that the two TGF-B species are not functionally identical in all systems (Ohta et al., 1987; Rosa et al., 1988). Similarly, recombinant human TGF/53 exerts similar activities as TGF-151 and TGFg2, but different dose response curves have been observed for the three forms of TGF-B (Graycar et al., 1989). These data, and the finding that TGF-B1, TGF-B2, and TGF-B3 are differentially expressed during mid- and late-gestation of mouse embryogenesis (Heine et al., 1987; Lehnert and Akhurst, 1988; Pelton et al., 1989; Miller et al., 1989a), suggest that different forms of TGF-B may have distinct physiological roles during mammalian development. Work with embryonal carcinoma (EC) cells also suggests that TGF-B may influence development prior to mid-gestation. EC cells, which can be induced by retinoic acid (RA) to differentiate into cells that exhibit the properties of early

embryonic cells (Strickland and Mahdavi, 1978; Strickland et al., 1980), have been shown to regulate the expression of TGF-B receptors when they differentiate (Rizzino, 1987). Whereas nearly all cells express TGF-15 receptors (Wakefield et al., 1987), F9 and PC-13 EC cells lack detectable TGF-B receptors. When these cells are induced to differentiate by RA, expression of TGF-B receptors is observed within 2 days and growth of the differentiated cells is inhibited by TGF-B (Rizzino, 1987). To characterize further the roles of TGF-B and the mechanisms by which TGF-B expression is regulated during development, we have examined TGF-B expression in EC cells and early mouse embryos. In this study, we report the differential expression of various members of the TGF-B family in undifferentiated and differentiated EC ceils as well as the detection of TGF-B2 transcripts in early mouse embryos.

Materials and methods Materials

Fetal bovine serum (FBS) and calf serum (CS) were obtained from Irvine Scientific (Santa Ana, CA). Dulbecco's modified Eagle's medium (DME), co-medium, Ham's F-12 nutrient mixture, and NCTC-109 were obtained from GIBCO (Grand Island, NY). Preparation and use of retinoic acid (RA) was as reported previously (Rizzino and Crowley, 1980). TGF-B1, TGF-B2, TGF-B neutralizing antibody and TGF-B2 specific neutralizing antibody were obtained from R & D Systems, Inc. (Minneapolis, MN). TGF-B1 specific neutralizing antibody was kindly provided by Dr. Anita Roberts. [3H]-thymidine was obtained from DuPont-New England Nuclear (Boston, MA). Restriction enzymes, reverse transcriptase and guanidine isothiocyanate were obtained from Bethesda Research Laboratories (Gaithersburg, MD). Hae III-digested CX174-RF DNA was obtained from New England Biolabs (Beverly, MA). NuSieve GTG agarose and SeaKem GTG agarose were obtained from FMC BioProducts (Rockland, ME). Taq DNA polymerase (Ce-

229 tus AmpliTaq) was obtained from United States Biochemical Corporation (Cleveland, OH). Polymerase chain reaction buffer was obtained from Stratagene, Inc. (La Jolla, CA).

medium were heat-treated at 70~ for 10 min and then acidified to pH 2 with 4 N HC1 at 4~ After 1 h, all aliquots were neutralized to pH 7.2 with 12 N NaOH and were concentrated further with a Centricon 10 membrane before being tested for the presence of TGF-B.

Cells and culture conditions Stock cultures of mouse F9 EC cells, mouse PC-13 EC cells, human NT2/D1 EC cells, and simian BSC-1 cells were maintained as previously described (Rizzino et al., 1983; 1988; 1990). CCL-64 cells were maintained in DME + 10% calf serum. F9 and PC-13 EC cells were induced to differentiate by treating the cells with 5 ~tM RA for 48 h. After this 2-day exposure, the medium was replaced with medium lacking RA. NT2/D1 EC cells were induced to differentiate by treating with 10 gM RA for 5 days. Mouse embryos were obtained from CF-1 females (Harlan Sprague-Dawley, Indianapolis, IN) that were mated with Eppley Swiss males. The embryos were collected in standard egg culture medium as described previously (Rizzino, 1985). Uncultured morulae and blastocysts were RNA extracted immediately, while cultured blastocysts were maintained in NCTC-109 medium supplemented with 10% FBS for 72 h before RNA extraction. All cell lines and embryos were cultured at 37~ in a humidified atmosphere of 95% air and 5% CO2.

Preparation of conditioned media Sub-confluent monolayers of F9 EC, F9-differentiated (day 4), NT2/D1 EC, and NT2/Dl-differentiated (day 5) cells were washed twice with serum-free medium and refed with a 1:1 mixture of DME and Ham's F-12 medium supplemented with 15 mM HEPES buffer and 25 nM selenous acid (Rizzino and Crowley, 1980). After 24 h, these conditioned media were collected, centrifuged for 15 min at 4~ in a Sorvall RC-2B centrifuge at a speed of 5000 rpm to remove cell debris, and concentrated with an Amicon (Danvers, MA) YM-5 membrane. Aliquots from each

Assay for TGF-fl activity TGF-B activity in the different conditioned media was determined by measuring the inhibition of [3H]-thymidine incorporation by CCL-64 cells (Tucker et al., 1984). In addition, by utilizing the TGF-B specific antibodies described above, this bioassay allowed for the characterization of the TGF-B activity produced by the EC cells and their differentiated cells. For each experiment, CCL-64 cells were seeded (8 x 103 cells/3.2 man tissue culture well) in DME + 10% CS. After 4 days, all cells were refed with DME + 5% FBS. On day 6, the cells were treated with the test factors. After 18 hrs, 0.2 gCi of [3H]-thymidine was added to each well, and the cells were incubated for another 2 h. Afterwards, the cells were washed twice with ice cold 5% trichloroacetic acid and solubilized with 0.25 N NaOH. The amount of [3H]-thymidine incorporated in the solubilized cells was determined with a scintillation counter. Relative [3H]-thymidine incorporation was calculated by dividing the average counts per minute (cpm) of each experimental condition by the average cpm of the 'untreated' control. Average cpm values were determined from triplicate samples that varied by less than 10% within each condition.

Synthetic oligonucleotides Oligonucleotide primers Were selected for PCR with consideration for the size of fragment amplified, internal restriction endonuclease sites, low GC content and the potential to reduce crosshybridization with non-specific sequences. Oligonucleotide primers were synthesized using an Applied Biosystems (Foster City, CA) Model

230 308B DNA synthesizer. The primers used to amplify transcripts for mouse and human TGF,131 were synthesized according to the human TGF131 sequence (Derynck et al., 1985) and encompass the base pair regions 1277-1296 and 15021521. These regions are identical to sequences reported for mouse TGF-131 (Derynck et al., 1986). The primers used to amplify the human, simian and mouse TGF-132 transcripts were synthesized according to the human TGF-B2 sequence reported by Madisen et al. (1988) and encompass the base pair regions of 1015-1034 and 1300-1319. These regions are identical to sequences for simian TGF-B2 (Hanks et al., 1988) and differ by only two base pairs in the mouse TGF-132 sequence (Miller et al., 1989b). Primers for Vgr-1 were synthesized according to the mouse Vgr-1 sequence (Lyons et al., 1989) and encompass the base pair regions 721-731 and 1373-1390. A 10 base pair (bp) sequence containing a NotI site was included on both Vgr-1 primers for future cloning purposes. The synthesized oligonucleotide primers were lyophilized and resuspended in 10 mM Tris-HC1 (pH 8). Concentrations were determined by measuring optical density at 260 nm.

Reverse transcription-polymerase chain reaction

Isolation procedures utilized to obtain total and polyadenylated RNA from EC cells and their differentiated cells have been described previously (Tiesman et al., 1988). RNA or polyadenylated RNA from these cells were reverse transcribed in a 50 gl reaction volume using oligo-dT priming and Moloney murine leukemia virus reverse transcriptase (M-MLVRT) (Gerard et al. 1987; Tiesman and Rizzino, 1989). Following reverse transcription, 1 gl of cDNA from EC cells or their differentiated cells was amplified in a 50 gl reaction mixture. Total cellular R N A was isolated from mouse embryos using the lysis and microultracentrifugation method of Iverson et al. (1987). Briefly, embryos were lysed in a solution containing guanidine isothiocyanate, sarkosyl, and 13-met-

captoethanol. RNA was isolated from this lysate by pelleting through a cesium chloride gradient using a Beckman TL-100 microultracentrifuge and a TLS-55 rotor. Total RNA from the embryos was reverse-transcribed using oligo-dT primers as described previously (Gerard et al., 1987; Tiesman and Rizzino, 1989) and 2 gl of the resulting cDNA were amplified according to the booster PCR technique of Ruano et al. (1989). The amplified DNA fragments from the EC cells, differentiated cells, and mouse embryos were electrophoresed through a 1% SeaKem/3% NuSieve agarose gel, stained with ethidium bromide, and viewed over an ultraviolet transilluminator.

Results Effect of differentiation on the release of TGF-fi

To examine the effects of differentiation on the release of TGF-B by EC cells, we have utilized the ability of TGF-13 to inhibit the growth of the mink lung epithelial cell line, CCL-64 (Tucker et al., 1984). Homogenous preparations of TGF-B1 generated a steep growth inhibition curve of CCL-64 cells with an EDs0 of approximately 0.1 ng/ml (Fig. 1). Homogenous preparations of TGFB2 also generated a steep growth inhibition curve (data not shown) and, as others have reported (Graycar et al., 1989), TGF-B2 appears to be a more potent growth inhibitor of CCL-64 cells than TGF-B1. In several experiments using this bioassay, we determined that the release of TGF13by F9 EC cells ranged from an increase of 25% to an increase of 100% when these cells were induced to differentiate with RA (Fig. 1). The majority (greater than 80%) of the TGF-13 activity released by F9 EC and their differentiated cells was in a non-active or latent form (data not shown), since acidification of the conditioned medium from both cell types was required to obtain an accurate determination of the amount of TGF-13 activity that was present. To confirm that the observed TGF-13 activity was due to a member(s) of the TGF-13 family, rather than to another

231 Table 1. Antibody neutralization of the TGF-g-like activity in conditioned media from F9 EC and F9-differentiated cells a Factors addedb

Relative incorporation of 3H-thymidineC

No addition (control) F9 EC F9 EC + TGF-13 neutralizing antibody F9 EC + TGF-gl antibody F9 EC + TGF-B2 antibody F9 EC + TGF-B1 antibody + TGF-B2 antibody

1.00 0.31 0.86 0.79 0.37 0.85

F9-differentiated F9-diff + TGF-B neutralizing antibody F9-diff + TGF-B1 antibody F9-diff + TGF-B2 antibody F9-diff + TGF-131 antibody + TGF-B2 antibody

0.26 0.79 0.42 0.59 0.96

TGF-B 1 TGF-B1 + TGF'B neutralizing antibody TGF-BI + TGF-B1 antibody TGF-B1 + TGF-B2 antibody

0.18 0.76 0.88 0.22

TGF-B2 TGF-B2 + TGF-B neutralizing antibody TGF-B2 + TGF-B1 antibody TGF-B2 + TGF-B2 antibody

0.10 0.84 0.13 0.92

aCCL-64 bioassay was performed as described in the Materials and methods. bConditioned media from F9 EC and F9-differentiated cells were heat-treated (70~ for 10 min) followed by acid-treatment (pH 2 for 1 h at 4~ and added at a concentration equivalent to the amount of TGF-g activity secreted by 6 x 105 ceils in a 24 h period. All other factors were added at the following optimal concentrations: TGF-B1 (250 pg/ml), TGF-B2 (250 pg/ml), TGF-B neutralizing antibody (7.5 gg/ml), TGF-B1 specific antibody (equivalent to the amount that will neutralize 250 pg/ml of TGF-B1), and TGF-132 specific antibody (7.5 gg/ml). CRelative [3H]-thymidine incorporation was determined as described in the Materials and methods. The control was designated as 1.00 and represented 5,235 cpm. This experiment was repeated twice and similar results were obtained.

lOO g

factor(s) that could interfere with the incorporation of [3H]-thymidine by CCL-64 cells, a TGF-B neutralizing antibody was used. When an optimal concentration of the antibody was incubated with the conditioned medium from both F9 EC and F9-differentiated cells, virtually all of the TGF-B

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1'5 2'5

go

r'516o I~o

TGF-pl

Added (Pg/ml)

2

2'so

4

Cell Number ( 1 0 -5 )

5bo 20

Fig. 1. Release of TGF-B-Iike activity by F9 EC and F9-differentiated cells as determined by measuring the inhibition of [3H]-thymidine incorporation by CCL-64 cells. Conditioned media from F9 EC (-A-) and F9-differentiated (--~-) cells were processed as described in the Materials and Methods. After concentration, the conditioned media were added to CCL64 cells at a concentration equivalent to that amount conditioned by the cell numbers indicated. TGF-gl ( - O - ) was added at the concentrations indicated.

232 Table 2. Antibodyneutralization of the TGF-B-likeactivityfrom the conditionedmedia of NT2/D1 EC and NT2/D1 differentiated cellsa

Factors addedb

Relative incorporation of 3H-thymidinec

No addition (control) NT2/D1 EC NT2/D1 EC + TGF-B neutralizing antibody NT2/D1 EC + TGF-gl antibody NT2/D1 EC + TGF-B2 antibody NT2/D1 EC + TGF-BI antibody + TGF-B2 antibody

1.00 0.24 0.84 0.81 0.23 0.82

NT2/D1-differentiated NT2/Dl-diff + TGF-B neutralizing antibody NT2/Dl-diff + TGF-BI antibody NT2/Dl-diff + TGF-B2 antibody NT2/Dl-diff + TGF-B1 antibody+ TGF-B2 antibody

0.43 0.96 0.86 0.64 0.99

TGF-B1 TGF-B1 + TGF-B neutralizing antibody TGF-B1 + TGF-B1 antibody TGF-B1 + TGF-B2 antibody

0.29 0.95 0.94 0.33

TGF-132 TGF-B2 + TGF-B neutralizing antibody TGF-B2 + TGF-B1 antibody TGF-B2 + TGF-B2 antibody

0.24 1.00 0.27 0.92

aCCL-64 bioassay was performed as described in the Materials and methods. bConditioned media from NT2/D1 EC and NT2/D1 differentiated cells were heat-treated (70~ for 10 min) followed by acid-treatment (pH 2 for 1 h at 4~ and added at a concentrationequivalent to the amountof TGF-B activity secreted by 2 x 106 cells in a 24 h period~ All other factors were added at the concentrations indicated in Table 1. eRelative [3H]-thymidineincorporationwas determined as described in the Materials and methods. The control was designated as 1.00 and represented 12,943 cpm. This experiment was repeated twice and similar results were obtained.

activity released from both cell types was neutralized (Table 1). To characterize further the TGF-B activity that was expressed by F9 EC and their differentiated cells, we incubated the conditioned m e d i u m from these cells with optimal concentrations of specific neutralizing antibodies that distinguish between TGF-B1 and TGF-B2. Using the CCL-64 growth inhibition bioassay, we determined that while TGF-B1 specific antibody neutralized virtually all of the TGF-B activity released by F9 EC cells, the antibody was only capable of neutralizing about one-third of the TGF-B activity released by F9-differentiated cells (Table 1). In addition, TGF-B2 specific antibody was unable to neutralize a significant amount o f the TGF-B activity released by F9 EC cells but was able to

neutralize up to two-thirds of the activity released by the differentiated cells (Table 1). Finally, TGF-B1 and TGF-B2 specific antibodies when added together, neutralized the TGF-B activity released by F9 E C and their differentiated cells in a similar fashion to that seen with the TGF-B neutralizing antibody (Table 1). Therefore, the greater TGF-B activity observed in F9-differentiated cells appears to be due to the induction o f TGF-B2 and not to increases in the amount o f TGF-B1 secreted. To determine if differentiation causes the induction of TGF-B2 in other EC cells, we examined the h u m a n EC cell line, NT2/D1. As was observed in F9 cells, the conditioned medium from both NT2/D1 EC and their differentiated ceils contained TGF-B activity (Table 2). The

233 Table 3. Oligonucleotideprimers used to examineexpression of transcripts for the TGF-Bgrowth factor familya

Amplified transcript

Predicted size (bp)

Restriction site (bp of fragments)

Sequence reference

TGF-B1, mouse human

244 244

BstNI (174,70) BstNI (153,70,21)

Derynck et al. 1986 Derynck et al. 1985

TGF-B2, human simian mouse mouse

303 303 303 303

AvalI (155,148) AvaII (155,148) HindIII (189,114) DdeI (161,142)

Madisen et aI. 1988 Hanks et al. 1988 Miller et al. 1989 Miller et al. 1989

Vgr-1, mouse

690

BglI (570,120)

Lyons et al. 1989

aOligonucleotideprimers for each amplifiedfragment were selected and synthesizedas described in the Materials and methods. To confirm the identity of the amplifiedfragments, restriction endonucleasedigestions were performed. Representativerestriction sites along with the predicted sizes of restriction fragments are described above. majority of the TGF-B activity released by NT2/ D 1 EC and NT2/D 1-differentiated cells was in a latent form (data not shown) and, when activated, this activity was able to be neutralized with the TGF-B neutralizing antibody (Table 2). In the conditioned medium from NT2/D1 EC cells, the TGF-B 1 specific antibody neutralized virtually all of the TGF-B activity that was present, while the TGF-B2 specific antibody was unable to neutralize any of the TGF-B activity released by NT2/D1 EC cells (Table 2). In the case of NT2/Dl-differentiated cells, the TGF-B1 specific antibody could only neutralize approximately two-thirds of the TGF-B activity released into the medium, while the TGF-B2 specific antibody was able to neutralize almost one-third of the TGF-B activity that was released (Table 2). The above findings indicate that in both human and mouse EC cell lines, differentiation induces the secretion of TGF-f32.

E x p r e s s i o n o f transcripts f o r different TGF-fis by E C cells a n d their differentiated cells

To verify the production of TGF-131 and TGF-B2 by EC cells and their differentiated cells, we used a combination of reverse transcription and polymerase chain reaction (RT-PCR) (Rappolee et al., 1988). In RT-PCR, polyadenylated RNA is reverse-transcribed into a cDNA strand that is

subsequently used as a template to exponentially amplify a specific DNA fragment that is flanked by two oligonucleotide primers. The technique of RT-PCR allows for the detection of low levels of gene expression that are undetectable by northern blot analysis. Moreover, due to its specificity, RT-PCR allows the examination of the expression of members of gene families that share high degrees of sequence similarity, as is true for the TGF-B gene superfamily. Finally, RT-PCR allows for the more rigorous identification of transcripts by restriction analysis of the amplified fragments. Using RT-PCR and primers specific for TGF131 (Table 3), we were able to detect transcripts for TGF-131 in F9, PC-13, and NT2/D1 EC cells, as well as in the differentiated cells of F9 and PC-13. RT-PCR of R N A isolated from both the EC cells and differentiated cells resulted in the amplification of a single 244 bp fragment (Figs. 2a and 2b), which is the size of the predicted nucleotide sequence (Table 3). In addition, digestion of the amplified fragment with BstNI restriction endonuclease generated the patterns predicted from the sequence for both mouse and human TGF-131 (Fig. 2, Table 3). Expression of TGF-B1 transcripts in both the EC cells and their differentiated cells supports the results observed in bioassay. When primers specific for TGF-B2 (Table 3) were used in RT-PCR, we readily detected tran-

234

235 Fig. 2 (facing page). Expression of transcripts for TGF-131 by F9, PC-13, and NT2/D1 EC cells (Panel A) and the differentiated counterparts of F9 and PC-13 EC cells (Panel B). RT-PCR was performed for each sample as described in the Materials and Methods with the following thermal cycler conditions: For 30 cycles of RT-PCR, samples were heated to 94~ for 3 min to denature DNA, cooled to 65~ for 2 min to allow primer annealing, and heated to 72~ for 1 min to extend primers. Primers for TGF-131 that are conserved between human and mouse sequences are described in Materials and Methods. Expected sizes of amplification products and restriction endonuclease (BstNI) fragments are indicated in Table 3. The undigested (lane U) and restriction endonuclease-digested (lane C) DNA fragments can be compared for each cell line. Note that the restriction pattern of the human EC cell line, NT2/DI, differs from the restriction pattern of the mouse F9 and PC-13 EC cell lines and their differentiated counterparts. Sizes of HaeIII-digested CX174-RF DNA size marker (lane M) are indicated at the left in kilobases. This experiment was repeated twice and similar results were obtained.

scripts for TGF-B2 in F9-, PC-13- and NT2/D1differentiated cells but not in the undifferentiated parental EC cells. RT-PCR of RNA isolated from the differentiated cells of F9, PC- 13, and NT2/D 1 resulted in the amplification of a single 303 bp fragment (Fig. 3a), which is the size of the predicted nucleotide sequence (Table 3). In addition, a single fragment of the same size was amplified in the simian cell line, BSC-1, which is known to produce TGF-g2 (Tucker et al., 1984; Hanks et al., 1988) (Fig. 3). Digestion of the amplified fragment with restriction endonucleases generated the patterns predicted from the sequence for TGF-132 (Figs. 3b and 3c, Table 3). The large contrast in the expression of TGF-132 transcripts between the undifferentiated cells and the differentiated cells of three different EC cell lines argues that the expression of TGF-g2 during differentiation is due to the increase in the steady state levels of transcripts for TGF-B2 rather than to an enhancement in its release. Differentiation also appears to be required for the expression of a distant member of the TGF-g family, Vgr-1. Previous studies utilizing northern blot analysis have detected the expression of Vgr-1 mRNA upon the differentiation of F9 EC cells (Lyons et al., 1989). In this study, using the highly sensitive and specific technique of RTPCR, transcripts for Vgr-1 were readily detected in the differentiated cells of F9 and PC-13, but not in their undifferentiated counterparts (Fig. 4). When primers specific for Vgr-1 (Table 3) were used, RT-PCR of RNA isolated from F9- and PC-13-differentiated cells resulted in the amplification of a single 690 bp fragment (Fig. 4). The amplified fragment is the size of the predicted

nucleotide sequence and when digested with BglI restriction endonuclease, the pattern predicted from the sequence for Vgr-1 is generated (Fig. 4 and Table 3). The above data suggest that the ap9pearance of Vgr-1 transcript is closely associated with the induction of differentiation. Whether this is due to increased transcription or decreased turnover of TGF-g2 mRNA remains to be determined. However, since the appearance of transcripts for both Vgr-1 and TGF-f32 occur after EC cells are induced to differentiate, their expression m a y be regulated by similar mechanisms.

Presence of TGF-fl transcripts during early development With the finding that TGF-B2 expression is induced by differentiation in three EC cell lines, we examined mouse embryos during early development for the expression of TGF-B2 transcripts. Other investigators have detected transcripts for TGF-B1 as early as the eight-cell stage during mouse embryogenesis using RT-PCR (Rappolee et al., 1988) and we have confirmed these results (data not shown). Using RT-PCR and primers specific for TGF-B2 (Table 3), we also detected transcripts for TGF-B2 in morulae, blastocysts, and blastocysts cultured for 3 days in serumcontaining medium. RT-PCR of RNA isolated from morulae and uncultured blastocysts resulted in the amplification of a single 303 bp fragment (Fig. 5), which is the size predicted by the nucleotide sequence for mouse TGF-g2 (Table 3). RT-PCR of RNA isolated from cultured blastocysts resulted in the amplification of the 303 bp

236

237

Fig. 3. Expression of transcripts for TGF-B2 in the differentiated cells of F9, PC-13, and NT2/D1. RT-PCR was performed for each sample as described in the Materials and Methods with the following thermal cycler conditions. For 30 cycles of RT-PCR, samples were heated to 94~ for 3 rain to denature DNA, ramped for 1 min to 56~ and maintained at 56~ for 2 rain for primer annealing, and heated to 72~ for 1 min to extend primers. Primers for TGF-B2 are described in the Materials and methods. Expected sizes of amplification products and restriction endonuclease fragments are indicated in Table 3. a) (facing page) Detection of transcripts for TGF-B2 when mouse and human EC cells (Fg, NT2/D1) are induced to differentiate with RA (F9-diff, NT2/Dl-diff). TGF-B2 transcripts were also detected in PC-13-differentiated cells and the simian cell line, BSC-1. b) (facing page) Restriction enzyme (AvaII) analyses of the NT2/I)l-differentiated and BSC-1 TGF-g2 DNA fragments generated by RT-PCR. c) Restriction enzyme (HindIII) analyses of the F9and PC-13-differentiated TGF-B2 DNA fragments generated by RT-PCR. In Figs. 3b and 3c, undigested (U) and restriction endonuclease-digested (C) DNA fragments are shown. Sizes of HaeIII-digested CX174-RF DNA size marker (lane M) are indicated at the left in kilobases. This experiment was repeated twice and similar results were obtained.

f r a g m e n t as well as a f r a g m e n t o f a p p r o x i m a t e l y 160 bp (Fig. 5). D i g e s t i o n o f the 303 bp a m p l i f i e d f r a g m e n t with D d e I restriction e n d o n u c l e a s e generated the pattern predicted f r o m the s e q u e n c e for T G F - B 2 (Fig. 5 a n d T a b l e 3). A t present, the identity o f the 160 bp f r a g m e n t is unclear. W h e t h e r it is a p r o d u c t o f an alternative splice site within T G F - B 2 p r i m a r y transcript or another f o r m o f T G F - B that shares s e q u e n c e similarity with T G F B2 is currently u n d e r investigation.

Discussion T h e data presented in this report s t r o n g l y s u g g e s t that the e x p r e s s i o n o f T G F - B 2 is closely associated with the induction o f differentiation in e m b r y o n a l c a r c i n o m a (EC) cells and during early m a m m a l i a n e m b r y o g e n e s i s . Transcripts for T G F B2 w e r e detected in the differentiated cells o f both m u r i n e and h u m a n E C cell lines but w e r e n o t detected in their undifferentiated counterparts. T h e s e data are consistent with r e c e n t findings that utilize n o r t h e r n blot analysis ( M u m m e r y

238

Fig. 4. Detection of transcripts for Vgr-1 when mouse EC cells (F9, PC-13) are induced to differentiate with RA (F9-diff, PC-13-diff). RT-PCR was performedfor each sample as described in the Materials and Methods with the followingthermal cycler conditions. For 30 cycles of RT-PCR, samples were heated to 94~ for 2 min to denature DNA, ramped for 1 min to 65~ and maintained at 65~ for 2 min to allow primer annealing, and heated to 72~C for 1 rain to extend primers. Primers for Vgr-1 are described in the Materials and Methods. Expected sizes of amplification products and restriction endonuclease (BglI) fragments are indicated in Table 3. undigested (lane U) and restriction endonuclease-digested (lane C) DNA fragments are represented in the two lanes on the far right. Sizes of HaelII-digested CX174-RFDNA size marker (lane M) are indicated at the left in kilobases. This experiment was repeated twice and similar results were obtained.

et al., 1990); however, our data extend this report in two important aspects. First, given the large differential in the expression o f TGF-B2 transcripts by E C cells and their differentiated cells, our data argue that the expression of TGF-B2 after differentiation is due to the increase in the steady state levels of transcripts for TGF-B2 rather than to an enhancement in its release. Second, we report that TGF-B-like activity increases with differentiation and this increase appears to be due to the secretion o f a latent form o f TGF-B2

that can be activated to a biologically mature form. Upon acid-treatment o f conditioned medium, TGF-B2 specific antibody partially neutralized the TGF-f3-1ike activity released from mouse and human differentiated EC cells, but was unable to neutralize any of the TGF-B-like activity released from their undifferentiated counterparts. The TGF-B-like activity released by the undifferentiated EC cells is primarily, if not exclusively, due to the release of TGF-f31. W e also report the detection o f TGF-f32 transcripts during early mouse

239

Fig. 5. Detection of transcripts for TGF-B2 during early mouse embryogenesis. RT-PCR was performed as described in the Materials and Methods with the following thermal cycler conditions described below. For 20 cycles of RT-PCR, reaction volumes containing a 20 nM primer mix were heated to 94~ for 1 min to denature DNA, cooled to 56~ for 4 rain to allow primer annealing, and heated to 72~ for 3 min to extend primers. Subsequently, primer concentrations were increased to 2 gM and an additional 50 cycles of RT-PCR were performed at identical temperature shifts but with denaturing, annealing, and extension times of 1, 2, and 3 min, respectively. Primers for TGF-B2 are described in the Materials and Methods. Expected sizes of amplification products and restriction endonuclease (DdeI) fragments are indicated in Table 3. TGF-B2DNA fragments were detected in mouse morulae (Mor.), blastocysts (BI.), and blastocysts cultured for 3 days in serum-containing medium (C. B1.). Undigested (lane U) and restriction endonuclease-digested (lane C) DNA fragments are compared for each embryo stage, Sizes of HaeIII-digestedOX174-RF DNA size marker (lane M) are indicated at the left in kilobases. This experiment was repeated twice and similar results were obtained. embryogenesis. TGF-B2 transcripts were detected with R T - P C R as early as the morula stage of d e v e l o p m e n t and continued to be expressed in the preimplantation blastocysts and the cultured blastocysts. While recent findings of M u m m e r y et al. (1990) indicate the ability of a TGF-132 specific antibody to bind to the cells o f the trophectoderm in the blastocyst, this is the first report of the detection o f transcripts for TGF-B2 during the earliest events of m a m m a l i a n embryogenesis. Although w o r k with the EC m o d e l s y s t e m suggests that differentiation induces the expression o f T G F B2, it remains to be determined whether transcripts for TGF-B2 are differentially expressed by the cells of the early embryo. It has been established that the expression of

T G F - g l , TGF-132 and TGF-B3 is both spatially and temporally regulated in mid- and late-stage m o u s e e m b r y o s ( 1 1 - 1 8 days gestation) suggesting that TGF-B m a y exert effects on cell differentiation (Heine et al., 1987; Lehnert and Akhurst, 1988; Pelton et al., 1989; Miller et al., 1989). In addition, the differential effects of TGF-fS1 and TGF-132 on m e s o d e r m formation in Xenopus e m b r y o s is consistent with TGF-B having an important regulatory role(s) during development (Rosa et al., 1988). Evidence that the TGF-Bs can exert differential effects in various systems m a y lie in the fact that T G F - g appears to bind to at least two different receptors (reviewed in Segarini, 1989). In the case of EC cells, differentiation leads to the formation of cells that

240 exhibit receptors for TGF-B1 and respond to TGF-B1 by growth inhibition (Rizzino, 1987). Similarly, TGF-f52 inhibits the growth of differentiated EC cells, but does not appear to affect the proliferation of the undifferentiated cells (Mummery et al., 1990; Kelly and Rizzino, unpublished data). Interestingly, although EC cells lack detectable receptors for TGF-B and fail to respond to TGF-B, they release significant amounts of TGF-B1. At present, the roles played by different TGF13s during early development are unknown. The induction of TGF-B2 production in the EC differentiated cells and the expression of TGF-B1 and TGF-B2 during the early stages of embryogenesis suggest that TGF-B may act to control specific regulatory events of the differentiating cells. One possible mechanism by which TGF-B2 may exert its effects on the differentiated cells is by regulating the deposition of extracellular matrices. TGFB has been shown to influence the extracellular matrices produced by many different cell types (reviewed in Rizzino, 1988) and extracellular matrices have been shown to affect cell shape, which in turn affects RNA and protein synthesis. While TGF-B acts to increase the production of extracellular matrix components in most systems, at least in the case of F9-differentiated and PYS-2 cells, TGF-B has been shown to inhibit the production of the extracellular matrix component, laminin (Kelly and Rizzino, 1989). Whether TGF-B exerts effects on other components of the extracellular matrix in the differentiated EC cells or affects extracellular matrix production in the early embryo is currently unknown. Clearly, additional studies will be needed to understand the mechanisms by which TGF-13 affects EC-derived endoderm-like differentiated cells as well as the possible role(s) that TGF-B may exert during early mammalian development.

Acknowledgements Dr. Anita Roberts (National Cancer Institute, Bethesda, MD) is thanked for her gift of TGF-B1 specific neutralizing antibody. This work was

supported by grants from the Council of Tobacco Research (2520), the National Institute of Child Health and Human Development (ND 21568), the National Cancer Institute (Laboratory Cancer Research Center Support Grant, CA 36727), and the American Cancer Society (ACS SIG-16). Dr. Wendy J. Campbell was supported by a National Institutes of Health training grant (T32 CA09476). David Kelly and Jay Tiesman were supported by fellowships from the Nebraska Governor's Research Initiative in Biotechnology.

References 1. Assoian RK, Komoriya A, Meyers CA, Miller DM and Sporn MB (1983) Transforming growth factor-f3 in human platelets. J. Biol. Chem. 258: 7155-7160. 2. Cate RL, Mattaliano RJ, Hession C, Tizard R, Farber NM, Cheung A, Ninfa EG, Frey AZ, Gash DJ, Chow EP, Fisher RA, Bertonis JM, Tones G, Wallner BP, Ramachandran KL, Ragin RC, Manganaro TF, Maclaughlin DT and Donahoe PK (1986) Isolation of the bovine and human genes for Mullerian inhibiting substance and expression of the human gene in animal ceils. Cell 45: 685-698. 3. Cheifetz S, Weatherbee JA, Tsang MLS, Anderson JK, Mole JE, Lucas R and Massague J (1987) The transforming growth factor-f3 system, a complex pattem of cross-reactive ligands and receptors. Cell 48: 409-415. 4. De Larco JE and Todaro GJ (1978) Growth factors from murine sarcoma virus-transformed cells. Proc. Natl. Acad. Sci. USA 75: 4001=4005. 5. Derynck R, Jarrett JA, Chen EY and Goeddel DV (1986) The murine transforming growth factor-f3precursor. J. Biol. Chem. 261: 4377-4379. 6. Derynck R, Jarrett JA, Chen EY, Eaton DH, Bell JR, Assoian RK, Roberts AB, Spom MB and Goeddel DV (1985) Human transforming growth factor-f3 complementary DNA sequence and expression in normal and transformed ceils. Nature 316: 701-705. 7. Derynck R, Lindquist PB, Lee A, Wen D, Tamm J, Graycar JL, Rhee L, Mason AJ, Miller DA, Coffey RJ, Moses HL and Chert EY (1988) A new type of transforming growth factor, TGF-f33.EMBO J. 7: 3737-3743. 8. Gerard GF (1987) Making effective use of M-MLV reverse transcriptase. Focus (Bethesda Research Laboratories, Gaithersburg, MD) 9: 5-6. 9. Graycar JL, Miller DA, Arrick BA, Lyons RM, Moses HL and Derynck R (1989) Human transforming growth factor133: Recombinant expression, purification, and biological activities in comparison with transforming growth factorsBi and -f32. Mol. Endocrinol. 3: 1977-1986. 10. Hanks SK, Armour R, Baldwin JH, Maldonado F, Spiess J and Holley RW (1988) Amino acid sequence of the BSC-I

241 cell growth inhibitor (polyergin) deduced from the nucleotide sequence of the cDNA. Proc. Natl. Acad. Sci. USA 85: 79-82. 11. Heine UI, Munoz EF, Flanders KC, Ellingsworth LR, Lain H-YP, Thompson NL, Roberts AB and Spore MB (1987) Role of transforming growth factor in the development of the mouse embryo. J. Cell Biol. 105: 2861-2876. 12. Iverson PL, Mata JE and Hines RN (1987) Rapid isolation of both RNA and DNA from cultured cells or whole tissues with a benchtop ultracentrifuge. BioTechniques 5: 521523. 13. Jakowlew SB, Dillard PJ, Spore MB and Roberts AB (1988a) Complementary deoxyribonucleic acid cloning of a messenger ribonucleic acid encoding transforming growth factor-B4 from chicken embryo chondrocytes. Mol. Endocrinol. 2: 1186-1195. 14. Jakowlew SB, Kondaiah P, Dillard PS, Spore MB and Roberts AS (1988b) Complementary deoxyribonucleic acid cloning of a novel transforming growth factor-B messenger ribonucleic acid from chicken embryo chondrocytes. Mol. Endocrinol. 2: 747-755. 15. Kelly D and Rizzino A (1989) Inhibitory effects of transforming growth factor-B on laminin production and growth exhibited by endoderm-like cells derived from embryonal carcinoma cells. Differentiation 41: 34--41. 16. Kondaiah P, Sands MJ, Smith JM, Fields A, Roberts AB, Spore MB and Melton DA (1990) Identification of a novel transforming growth factor-g (TGF-B5)mRNA in Xenopus laevis. J. Biol. Chem. 265: 1089-1093. 17. Lehnert SA and Akhurst RJ (1988) Embryonic expression pattern of TGF beta type-1 RNA suggests both paracrine and autocrine mechanisms of action. Development 104: 263-273. 18. Lyons K, Graycar JL, Lee A, Hashmi S, Lindquist PB, Chen EY, Hogan BLM and Derynck R (1989) Vgr-1, a mammalian gene related to Xenopus Vg-1, is a member of the transforming growth factor-B gene superfamily. Proc. Natl. Acad. Sci. USA 86: 4554-4558. 19. Madisen L, Webb NR, Rose TM, Marquardt H, Ikeda T, Twardzik D, Seyedin S and Purchio AF (1988) Transforming growth factor-l]2: cDNA cloning and sequence analysis. DNA 7: 1-8. 20. Mason AJ, Hayrick JS, Ling NL, Esch F, Ueno N, Ying S-Y, Guillemin R, Niall H and Seeburg PH (1985) Complementary DNA sequences of ovarian follicular fluid inhibin show precursor structure and homology with transforming growth factor-I]. Nature 318: 659-663. 21. Miller DA, Lee A, Matsui Y, Chen EY, Moses HL and Derynck R (1989a) Complementary DNA cloning of the murine transforming growth factor-l]3 (TGF-l]3) precursor and the comparative expression of TGF-l]3 and TGF-I]I messenger RNA in murine embryos and adult tissues. Mol. Endocrinol, 3: 1926-1934. 22. Miller DA, Lee A, Pelton RW, Chen EY, Moses HL and Derynck R (1989b) Murine transforming growth factor-l]2 cDNA sequence and expression in adult tissues and embryos. Mol. Endocrinol. 3:1108-1114.

23. Mummery CL, Slager H, Kruijer W, Feijen A, Freund E, Koornneef I and van den Eijnden-van Raaij AJM (1990) Expression of transforming growth factor-l]2 during the differentiation of routine embryonal carcinoma and embry0nic stem cells. Dev. Biol. 137: 161-170. 24. Ohta M, Greenberger JS, Anklesaria P, Bassols A and Massaque J (1987) Two forms of transforming growth factor-l] distinguished by multipotential haematopoietic progenitor cells. Nature 329: 539-541. 25. Padgett RW, St Johnston RD and Gelbart WM (1987) A transcript from a Drosophila pattern gene predicts a protein homologous to the transforming growth factor-B-family. Nature 325: 81-84. 26. Pelton RW, Nomura S, Moses HL and Hogan BLM (1989) Expression of transforming growth factor-f32 RNA during murine embryogenesis. Development 106: 759-767. 27. Rappolee D, Brenner CA, Schultz R, Mark D and Werb Z (1988) Developmental expression of PDGF, TGF~x, and TGFB genes in preimplantation mouse embryos. Science 241: 1823-1825. 28. Rebagliati MR, Weeks DL, Harvey RP and Melton DA (1985) Identification and cloning of localized maternal RNAs from Xenopus eggs. Cell 42: 769-777. 29. Rizzino A (1985) Early mouse embryos produce and release factors with transforming growth factor activity. In Vitro Cell. Dev. Biol. 21: 531-536. 30. Rizzino A (1987) Appearance of high affinity receptors for type l] transforming growth factor during differentiation of murine embryonal carcinoma cells. Cancer Res. 47: 43864390. 31. Rizzino A (1988) Transforming growth factor-B: Multiple effects on cell differentiation and extracellular matrices. Dev. Biol. 130: 411-422. 32. Rizzino A and Crowley C (1980) Growth and differentiation of embryonal carcinoma cell line F9 in defined media. Proc. Natl. Acad. Sci. USA 77: 457-461. 33. Rizzino A, Kazakoff P and Nebelsick J (1990) Densityinduced down-regulation of epidermal growth factor receptors. In Vitro Cell. Dev. Biol. 26: 537-542. 34. Rizzino A, Kuszynski C, Ruff E and Tiesman J (1988) Production and utilization of growth factors related to fibroblast growth factor by embryonal carcinoma cells and their differentiated cells. Dev. Biol. 129: 61-71. 35. Rizzino A, Orme L and De Larco JE (1983) Embryonal carcinoma cell growth and differentiation: Production of and response to molecules with transforming growth factor activity. Exp. Cell Res. 143: 143-152. 36. Roberts AB, Sporn MB, Assoian RK, Smith JM, Roche NS, Wakefield LM, Heine UI, Liotta LA, Falanga V, Kehrl JH and Fauci AS (1986) Transforming growth factor type 6: Rapid induction of fibrosis and angiogenesis in vivo and stimulation of collagen formation in vitro. Proc. Natl. Acad. Sci. USA 83: 4167-4171. 37. Rosa F, Roberts AB, Danielpour D, Dart LL, Sporn MB and Dawid IB (1988) Mesoderm induction in amphibians: The role of TGF-l]2-1ikefactors. Science 239: 783-785.

242 38. Ruano G, Fenton W, Kidd KK (1989) Biphasic amplification of very dilute DNA samples via 'booster' PCR. Nucl. Acids Res. 17: 5407. 39. Segarini PR (1989) Transforming growth factor-f5 receptors. Cytotechnology 2: 307-316. 40. Seyedin SM, Segarini PR, Rosen D, Thompson AY, Bentz H and Graycar J (1987)' Cartilage inducing factor-B is a unique protein structurally and functionally related tO transforming growth factor-fs. J. Biol. Chem. 262: 1946-1949. 41. Silberstein GB and Daniel CW (1987) Reversible inhibition of mammary gland growth by transforming growth factor-B. Science 237: 291-293. 42. Sporn MB, Roberts AB, Wakefield LM and Assoian RK (1986) Transforming growth factor-fs: Biological function and chemical structure. Science 233: 532-534. 43. Strickland S and Mahdavi V (1978) The induction of differentiation in teratocarcinoma stem cells by retinoic acid. Cell 15: 393-403. 44. Strickland S, Smith KK, Marotti KR (1980) Hormonal induction of differentiation in teratocarcinoma stem cells: Generation of parietal endoderm by retinoic acid and dibutyryl cAMP. Cell 21: 347-355. 45. ten Dijke P, Hansen P, Iwata KK, Pieler C and Foulkes JG (1988) Identification of another member of the transforming factor type f5 gene family. Proc. Natl. Acad. Sci. USA 85: 4715-4719.

46. Tiesman J, Meyer A, Hines RN and Rizzino A (1988) Production of growth factors related to fibroblast growth factor and platelet-derived growth factor by human embryonal carcinoma ceils. In Vitro Cell. Dev. Biol. 24: 1209-1216. 47. Tiesman J and Rizzino A (1989) Expression and developmental regulation of the k-FGF oncogene in human and murine embryonal carcinoma cells. In Vitro Cell. Dev. Biol. 25: 1193-1198. 48. Tucker RF, Shipley GD, Moses HL and Holley RW (1984) Growth inhibitor from BSC-1 cells closely related to platelet type B transforming growth factor. Science 226: 705707. 49. Wakefield LM, Smith DM, Masui T, Harris CC, Sporn MB (1987) Distribution and modulation of the cellular receptor for transforming growth factor-B. J. Cell Biol. 105: 965975.

Address for offprints: A. Rizzino, Eppley Institute for Cancer Research and Allied Diseases, University of Nebraska Medical Center, 42nd and Dewey Avenue, Omaha, NE 68198-6805, USA