Development 110, 435-443 (1990)Printed in Great Britain © The Company of Biologists Limited 1990

435

A mesoderm-inducing factor produced by WEHI-3 murine myelomonocytic

leukemia cells is activin A

R. M. ALBANO1, S. F. GODSAVE2*, D. HUYLEBROECK3, K. VAN NIMMEN3, H. V. ISAACS2,

J. M. W. SLACK2 and J. C. SMITH1

1 Laboratory of Embryogenesis, National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK^Imperial Cancer Research Fund, Developmental Biology Unit, Department of Zoology, South Parks Road, Oxford OX1 3PS, UK3Innogenetics, Industriepark Zwijnaarde 7, Box 4, B-9710 Ghent, Belgium

•Present Address: Furusawa Morphogenes Project, 5-9-6 Tohkohdai, Tsukuba 300-26, Japan

Summary

The first inductive interaction in amphibian develop-ment is mesoderm induction, during which a signal fromthe vegetal hemisphere of the blastula-staged embryoinduces mesoderm from overlying equatorial cells.Recently, a number of 'mesoderm-inducing factors'(MIFs), which may be responsible for this interaction,have been discovered. Examples of these MIFs includemembers of the fibroblast growth factor family as well asmembers of the TGF-/S superfamily such as TGF-/J2. Inaddition to these purified factors, several new sources ofmesoderm-inducing activity have been described. One ofthe most potent of these is the murine myelomonocyticleukemia cell line WEHI-3. Even at high dilutions,conditioned medium from WEHI-3 cells induces isolatedXenopus animal pole regions to form a variety of

mesodermal cell types. In this paper we show by severalcriteria, including iV-terminal amino acid sequencing,Northern blotting and various functional assays, that theWEHI-MIF is activin A. Activins are known to modulatethe release of follicle-stimulating hormone from culturedanterior pituitary cells and to cause the differentiation oftwo erythroleukemia cell lines. Our results, along withrecent data from other laboratories, indicate that thesemolecules may also act in early development in theformation of the mesoderm.

Key words: Xenopus laevis, mesoderm induction,mesoderm-inducing factors, morphogens, XTC-MIF, activinA, WEHI 3 cells

Introduction

In vertebrates, mesoderm is formed through a series ofinductive interactions that take place in the early stagesof development. In amphibians a great body ofexperimental evidence shows that cells in the vegetalhemisphere of the blastula-staged embryo releasesignals that cause cells in the equatorial region tobecome mesoderm. The cells in this region wouldotherwise give rise to ectodermal derivatives (Nieuw-koop, 1969, 1973; Sudarwati and Nieuwkoop, 1971;reviewed by Smith, 1989).

The molecular nature of these signals remainedunclear until recent findings revealed that polypeptidegrowth factors could mimic the mesoderm-inducingeffects of the vegetal cells (see Smith et al. 1989, forreview). The first of these mesoderm-inducing factors(MIFs) to be discovered was XTC-MIF, which isobtained from the conditioned medium of a Xenopuslaevis tadpole cell line called XTC (Smith, 1987). Thisfactor can induce notochord and segmented muscle,dorsal mesodermal derivatives, from isolated Xenopus

animal caps (Smith et al. 1988). Further characteriz-ation revealed that this factor is related to thetransforming growth factor type-/? (TGF-/?) polypeptidesuperfamily (Smith et al. 1988; Rosa et al. 1988), andmore recently we have shown the molecule to be aXenopus homologue of activin A (Smith et al. 1990).Other members of this family that may be involved inembryonic development and mesoderm induction areTGF-/31 and 2 (Lehnert and Akhurst, 1988; Pelton et al.1989; Rosa et al. 1988) and the localized mRNA Vgl(Weeks and Melton, 1987). Another class of growthfactors that is also capable of mesoderm induction is theheparin-binding growth factor family (Slack et al. 1987;Kimelman and Kirschner, 1987). mRNA and proteinfor one member of this family, basic fibroblast growthfactor (bFGF), has been detected in the early Xenopusembryo (Kimelman and Kirschner, 1987; Kimelman etal. 1988; Slack and Isaacs, 1989). The main difference inthe effects of the two families of MIFs is in the type ofmesodermal tissues they induce. XTC-MIF can inducethe dorsal mesoderm derivatives cited above, whilebFGF (or aFGF) can only induce unsegmented muscle

436 R. M. Albano and others

and mesenchyme, which are regarded as ventralmesodermal derivatives (Slack et al. 1988; Green et al.1990).

In mammalian embryos, the function of growthfactors in mesoderm formation is not clear. Growthfactors are present in preimplantation mouse embryos(Rappolee et al. 1988) and the expression of the FGF-related proto-oncogene int-2 in this organism duringgastrulation (Wilkinson et al. 1988) implies a role inearly developmental decisions and possibly in meso-derm formation. To investigate this issue further theidentification of murine mesoderm-inducing factors andtheir isolation from embryos or from cultured cells inpure form is required. In a search for new MIFs,Godsave et al. (1988) found conditioned medium fromWEHI-3 cells, a murine myelomonocytic leukemia, tobe a source of a MIF that could induce dorsalmesodermal derivatives in the Xenopus animal capassay. They showed in a preliminary characterizationthat this factor, a protein, was very similar to XTC-MIFboth in chromatographic and biological properties. Inthis paper, we purify this mammalian-derived MIF fromthe conditioned medium of WEHI cells to homogeneityand show by N-terminal amino acid sequence analysis,specific functional assays and Northern hybridizationanalysis, that it is murine activin A, a homodimer of the)SA chains of inhibin. Our results support those recentlyobtained by Asashima et al. (1990), who showed thatrecombinant porcine activin A has mesoderm-inducingactivity.

Materials and methods

Embryos and mesoderm induction assayEmbryos of Xenopus laevis were obtained by artificialfertilization as described by Smith and Slack (1983). Fertilizedeggs were dejellied with 2% cysteine hydrochloride(pH7.8-8.1), washed and transferred to Petri dishes coatedwith 1 % Noble Agar and containing 10 % normal amphibianmedium (NAM; Slack, 1984). Embryos were staged accordingto Nieuwkoop and Faber (1967). Mesoderm-inducing activitywas assayed as described by Smith (1987) and Symes andSmith (1987). Briefly, animal pole regions were dissected fromstage-8 Xenopus embryos and transferred, blastocoel-facingsurface up, to the test solution. Fractions containing inducingactivity could be identified within 5-6 h; instead of remainingas spheres, induced explants begin to elongate. This change inshape is an early and reliable marker of mesoderm inductionand represents an attempt by the induced cells to undergogastrulation movements (Symes and Smith, 1987).

We define one unit of mesoderm-inducing activity as theminimum quantity that must be present in lml medium forinduction to occur. Thus if the highest active dilution is1/1000, the solution contains 1000 units ml"1 (Cooke et al.1987; Godsave et al. 1988).

Cells and conditioned mediumWEHI-3 cells (Warner et al. 1969) were grown in roller bottlescontaining 11 Iscove's medium with 5 % foetal calf serum,2.5/igml"1 fungizone, lOOumT1 penicillin and lOO^gml"streptomycin. When the cell density reached l^ml they

were resuspended in the same medium but without serum andwith 1/igmT1 endotoxin (Difco: E. coli 055-B 5) and40000 units ml"1 of aprotinin (Sigma). Incubation was con-tinued for 3-5 days and then the conditioned medium washarvested by centrifugation. It was concentrated tenfold usingan Amicon pumped ultrafiltration apparatus with YM10 spiralcartridge (nominal cut off IOXKPM,.).

Protein purification proceduresThe WEHI-MIF purification scheme was based on thatrecently used for the purification of XTC-MIF by Smith et al.(1990). In a typical experiment, tenfold-concentrated WEHI-conditioned medium was adjusted to 1 M NaCl and pumped at40mlh~1 onto a 2.5x38cm column of Phenyl-Sepharose CL-4B equilibrated in 1M NaCl, 20mM Tris-HCl, pH8.0. Thecolumn was washed with one bed volume of this buffer andtwo bed volumes of 20 mM Tris-HCl, pH8.0 at 60 ml h"1. Thecolumn was then eluted at 60mlh~1 with a gradient ofethylene glycol in 20mM Tris-HCl, pH8.0 and 10ml fractionswere collected. Active fractions were pooled and pumped at40mlh~1 onto a 2.4x20cm column of DEAE-Sepharose CL-4B equilibrated in 20mM Tris-HCl, pH8.0. The column waswashed at 60 ml h"1 with one bed volume of the same bufferand eluted at 60mlh"1 with a gradient of NaCl in 20 mMTris-HCl, pH8.0. Active fractions were pooled and thismaterial was adjusted to pH2.2 with trifluoroacetic acid(TFA), degassed through a stream of helium, and pumped ata flow rate of 1.5 ml min"1 directly onto a 100x4.6mmRP-300column (Brownlee) attached to a Beckman System GoldHPLC system and equilibrated in buffer A (0.1% TFA).Absorbance was monitored at 210 run and when this fell tobase-line the column was developed with a gradient of bufferB (80 % acetonitrile in 0.1 % TFA, 0.2 % min"1) at a flow rateof O.Smlmin"1. Active fractions were pooled, diluted 4-foldwith buffer A, degassed and pumped onto a 100x2.1 mm RP-300 microbore column (Brownlee) at a flow rate ofO.Smlmin"1. Absorbance was monitored at 210 nm and,when it fell to base line, the column was developed with ashallow gradient (0.14% min"1) of buffer B at a flow rate ofO^mlmin"1. Active fractions were pooled, degassed, diluted4-fold with buffer A and applied to the same column.Conditions were as for the previous run except that thecolumn was developed with a shallower gradient of buffer B(0.056% min"1) at a flow rate of O^mlmin"1. Activefractions were pooled and subjected to polyacrylamide gelelectrophoresis, amino acid analysis and N-terminal aminoacid sequencing.

SDS-polyacrylamide gel electrophoresisMaterial from the final microbore column was analysed bySDS-PAGE under reducing and non-reducing conditions.Samples were mixed with an equal volume of 2xgel samplebuffer, with or without a reducing agent. They were applied tomini vertical slab gels containing 15 % acrylamide and usingthe buffer system of Laemmli (1970). Gels were silver stainedusing a modification of the technique of Wray et al. (1981),which can detect less than 1 ng of some protein species.

N-terminal amino acid sequencingThe protein concentration of purified WEHI-MIF wasdetermined by amino acid analysis and 50pmole wassubjected to Edman degradation on an Applied Biosystems477A pulsed liquid protein sequencer. The phenylthiohydan-toin (PTH) amino acid derivatives were identified by reverse-phase HPLC with an on-line 120 A PTH amino acid analyser.

Activin A is a mesoderm-inducing factor 431

Functional assaysCCL-64 assays

The effect of WEHI-MIF on the growth of CCL-64 mink lungepithelial cells was investigated using the method of Cone etal. (1988). This assay was used to test crude WEHI-conditioned medium, with and without acidification (1Macetic acid for 1 h), as well as purified WEHI-MIF.

Follicle-stimulating hormone (FSH) release assayThe release of FSH from cultured rat anterior pituitary cells inresponse to WEHI-MIF was assayed exactly as described byHuylebroeck et al. (1990). Vaccinia-virus-expressed recombi-nant activin A was used as a positive control (Huylebroeck etal. 1990). A reference standard preparation of bovinefollicular fluid, a source of inhibin activity, inhibited therelease of FSH in a dose-dependent fashion with a maximumof 45 % in the assay shown.

Erythroid differentiation assayDifferentiation of the K562 human erythroleukemia cell linein response to WEHI-MIF was assayed exactly as describedby Huylebroeck et al. (1990). Vaccinia virus-expressedrecombinant bovine activin A was used as a positive control(Huylebroeck et al. 1990).

Histological analysisAfter exposure to WEHI-MIF or a control solution, Xenopuslaevis animal cap explants were cultured at 20 °C for threedays until control embryos reached stage 40. They were thenfixed, sectioned and stained as described by Smith et al.(1988).

RNase protection assaysThese were performed exactly as described by Green et al.(1990).

Northern blot analysesTotal RNA was prepared from WEHI cells or adult mouseliver and ovary by the LiCl-urea method (Auffray andRougeon, 1979). Poly (A)+ RNA was selected by oligo (dT)-chromatography.

25 fig of total or 5 /ig of poly (A)+ RNA were electrophor-esed on a 1 % agarose gel containing formaldehyde. This wasblotted onto nitrocellulose or nylon membranes which werebaked for 2h at 80 °C. Membranes were probed with thefollowing:

1. A Pstl/EcoRl fragment of clone £A30 (Esch et al. 1987)encoding the mature portion of rat activin A together with 16amino acids of the non-mature portion.

2. An EcoRl/Styl fragment of clone ^ u (Esch etal. 1987)encoding 163 amino acids of the non-mature portion of ratactivin B.

3. An 0.6kb fragment encoding the JV-terminal precursorportion of human TGF-/31.

4. A 1.6 kb fragment containing the entire coding regionfor human TGF-^2.

5. A lkb fragment encoding the N-terminal precursorportion of TGF-/33.

The probes were labelled with 32P by random priming(Feinberg and Vogelstein, 1984) and filters were hybridized in50% formamide, 5xSSPE (TGF-/3 probes) or 5xSSC with50mM Tris-Cl pH7.6 (activin probes), 5xDenhardt's sol-ution and 100-250 u% salmon sperm DNA, at 42°C (TGF-/3)or 50°C (activin). Washes for TGF-/3 probes were 5xSSPE atroom temperature followed by O.lxSSPE, 0.1% SDS for30min. Washes for activin probes were lxSSC, 0.2% SDS at

room temperature followed by 3x20min 0.2xSSC, 0.2%SDS, at 64°C. Membranes were autoradiographed with anintensifying screen at —70°C. They were stripped of probesand rehybridized under the same conditions with a mouseactin cDNA to verify the loading (Minty et al. 1981).

Results

Cell culture and MIF productionA preliminary screen of mammalian cell lines showedthat about one in ten produced mesoderm-inducingactivity. WEHI cells were selected because of theirability to grow in suspension. It was found that a densityof 10 ml"1 was the highest that could safely be reachedwithout overacidification of the medium and so thisdensity was used for preparing conditioned medium.Iscove's medium was used in place of RPMI because ofits superior buffering capacity. Cultures of up to 11would grow satisfactorily in stirred cells or rollerbottles, but above this volume aeration becamelimiting.

WEHI cells are extremely sensitive to endotoxin andlow endotoxin serum (<lngml~1) was used for thegrowing cells. But we found that endotoxin alsoincreased production of the MTF by a factor of about 10and so it was included at 1 ng ml"1 during preparation ofconditioned medium. Exposure to serum-free mediumalso seemed to increase the MIF yield by a factor of 10.We included aprotinin during the conditioning step toreduce proteolysis, but its effect on yield was probablyless than that of serum deprivation or endotoxin.Normal WEHI growth medium contained about16-32 u ml"1 of MIF, and following the conditioningprocedure described in Materials and methods thiscould be boosted to about 4000 u ml"1. In fact the 101batches of medium used for purification were grown atthe ICRF cell culture facility and for these the yield wasless good, about 320 u ml"1 before concentration. Afterconcentration, the specific activity of the conditionedmedia increased, but it fell during storage at — 70°C toabout 500 u ml"1.

Purification of WEHI-MIFIn a preliminary characterization of WEHI-MIF,Godsave et al. (1988) showed that this factor hasproperties in common with the TGF-/3 family. WhenWEHI-conditioned medium was assayed for the abilityto inhibit the growth of CCL-64 cells, however, itproved to have no detectable TGF-/3-like activity (datanot shown). This indicates that WEHI-conditionedmedium contains less than O.Sngml"1 TGF-/3 (seeFig. 4), despite the presence in WEHI cells oftranscripts for TGF-zfFs 1 and 3 (see Fig. 6).

In view of these results, which indicated similaritiesbetween WEHI-MIF and XTC-MIF, the WEHI-MIFpurification scheme was based on that recently used forXTC-MIF by Smith et al. (1990). The first stepemployed hydrophobic interaction chromatography.1.71 of tenfold-concentrated WEHI-conditioned me-

438 R. M. Albano and others

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Fig. 1. Initial steps in the purification of WEHI-MIF.(A) 1.51 of concentrated WEHI conditioned medium wasadjusted to 1 M NaCl and applied to a column ofPhenyl-Sepharose. The column was washed with 1 M NaCland with buffer containing no NaCl before mesoderm-inducing activity was eluted with a gradient of 0-80 %ethylene glycol. The solid horizontal bar represents thefractions containing inducing activity, which were pooledfor the next purification step. (B) Pooled fractions from thePhenyl-Sepharose column were applied to aDEAE-Sepharose column. The column was washed untilthe absorbance of the eluate fell to base line prior toelution with a gradient of 0-0.6M NaCl. The solidhorizontal bar represents the fractions containing MIFactivity, which were pooled for the next purification step.

dium containing 1530 mg protein and 850000 units ofmesoderm-inducing activity was adjusted to 1 M NaCland loaded onto a Phenyl-Sepharose CL-4B column.All the mesoderm-inducing activity bound to the geland when the column was developed with a gradient of0-60% ethylene glycol the activity eluted at approxi-mately 38% (Fig. 1A). In this step the bulk ofcontaminating proteins came out during the washes andat the beginning of the gradient. The pooled activefractions (2.8 mg in 87 ml; 174000 units) were thenloaded directly onto a DEAE-Sepharose CL-4Bcolumn and developed with a gradient of 0-0.6 M NaCl.A major protein peak was separated from the active

fractions which eluted at a concentration of approxi-mately 0.5 M NaCl (Fig. IB). The column was sub-sequently washed with 1 M NaCl but no inducing activityeluted. At this stage the protein concentration of thepooled active fractions was very low (<1 /zgml"1), witha total volume of 117 ml containing 117000 units ofmesoderm-inducing activity. Preliminary experimentshad shown that concentration by ultrafiltration resultedin significant loss of activity. Therefore the material wasadjusted to pH2.2 with TFA and pumped directly ontoa C8 reverse-phase column. All of the inducing activitybound to the column and after development it eluted atapproximately 30% acetonitrile (Fig. 2A). The pooledactive fractions were then diluted 4-fold with 0.1%TFA and loaded directly onto a C8 microbore reverse-phase column. This column has the advantage ofincreasing resolution while reducing fraction size. Thecolumn was eluted with a shallower gradient ofacetonitrile and all activity eluted as two peaks atapproximately 30 % acetonitrile. The active peak couldbe resolved from the other by assaying the columnfractions at a dilution of 1/40000. This was verified after4-fold dilution of the pooled active fractions andloading onto the same column. This time the columnwas developed with an even shallower gradient ofacetonitrile. As shown in Fig. 2C all mesoderm-inducing activity co-eluted with a single UV-absorbingpeak at approximately 30 % acetonitrile. The homogen-eity of this peak was checked by SDS-PAGE underreducing and non-reducing conditions. One proteinspecies is visible following silver staining of the gel(Fig. 3A). This migrates as a 23.5 xlO3 MR band undernon-reducing conditions and as a 14.5xlO3 MR bandafter reduction. This indicates that this protein is adimer of two subunits which display the same apparentmolecular weight. This is common for members of theTGF-£ family such as XTC-MIF (Smith et al. 1990),TGF-iSl and -pi (Ikeda et al. 1987) and activin A (Valeet al. 1986). The final yield of purified WEHI-MIF wasdetermined by amino acid analysis and, from a batch of1.7 litres of 10xconcentrated conditioned medium, weobtained 1.7 fig of highly purified factor, representing8500 units. This represents a purification of 9000-fold,and a recovery of 1 %.

Approximately SOpmole of highly purified WEHI-MIF was subjected to amino acid sequencing. Seven-teen N-terminal amino acids were identified and, after acomparison with sequences for other members of theTGF fi family, we found it to be identical to theN-terminus of activin A, a protein that is active inreleasing FSH by anterior pituitary cells and in inducingerythroid differentiation. In Fig. 3B we align theA'-terminus amino acid sequence of WEHI-MIF withthat of rat activin A and, for comparison, activin B. Allof the identified amino acids are identical with activin Aand the three unknowns are very likely to be cysteineresidues (see Fig. 3 legend). In fact, the sequence is thesame for all mammalian activin A chains purified so far(pig, human, bovine and porcine: Ling et al. 1988) andalso for the recently identified frog activin A (Smith etal. 1990).

Activin A is a mesoderm-inducing factor 439

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Fig. 2. Purification of WEHI-MIF using reverse-phaseHPLC. (A) Pooled fractions from the DEAE-Sepharosecolumn were adjusted to pH2.2, pumped directly onto aRP-300 C& column and eluted with an acetonitrile gradientas described in Materials and methods. The solid horizontalbar represents the fractions containing MIF activity, whichwere pooled for the next step. (B) Pooled fractions fromthe previous step were pumped directly onto a RP-300microbore column and eluted with a shallow gradient ofacetonitrile as described in Materials and methods. Thesolid horizontal bar represents the fractions containingactivity, which were pooled for next step. (C) The finalpurification step consisted of pumping the pooled fractionsfrom the previous step onto the same microbore columnand eluting with a much shallower gradient of acetonitrileas described in Materials and methods. The solid horizontalbar represents the two fractions containing activity whichcorresponded to the single UV-absorbing peak.

WEHI-MIF behaves as an activin in specific biologicalassaysTo confirm that WEHI-MIF is an activin and not arelated member of the TGF-/3 family, three specificbiological assays were performed. In the first assay, wetested whether purified WEHI-MIF could inhibit thegrowth of CCL-64mink lung epithelial cells. Thegrowth of this cell line is strongly inhibited by TGF-/J1

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Fig. 3. Analysis of purified WEHI-MIF by polyacrylamidegel electrophoresis. TV-terminal amino acid sequencingshows that the molecule is mouse activin A. (A) Aliquotsfrom the pooled fractions from the final purification stepwere electrophoresed on a 15 % polyacrylamide gel and thegel was silver stained. The samples were run under non-reducing (NR) or reducing (R) conditions. (B) 50pmole ofthe single protein species observed on the polyacrylamidegel was subjected to TV-terminal amino acid sequencing asdescribed in Materials and methods. The sequence obtainedis compared with that of rat activin A and also activin B. Xin the sequence corresponds to a lack of signal in a positioncorresponding to cysteine in rat activin A. Amino acids arelisted using the one letter code.

and TGF-/32 (Tucker et al. 1984). Since these proteinshave been shown to possess moderate mesoderm-inducing activity alone or acting synergistically to otherfactors (Kimelman and Kirschner, 1987; Rosa et al.1988), this assay could indicate whether WEHI-MIF ismore related to these polypeptides or to the activins,which have no significant anti-proliferative effect onthese cells (KVN and DH, unpublished results; Smith etal. 1990). As shown in Fig. 4, porcine TGF-/32 has astrong growth-inhibiting activity, while WEHI-MIF hasno activity, even at lOngmT1. This indicates that thisfactor is not functionally homologous to TGF-^1 orTGF-02.

The two other assays employed are specific foractivins. In the first assay we tested the ability ofWEHI-MIF to stimulate the release of FSH fromanterior pituitary cells. Fig. 5 shows that WEHI-MIFstimulates the release of FSH from these cells in a dose-dependent fashion (but not luteinizing hormone; datanot shown). The ED50 of WEHI-MIF in this assay isapproximately 2.2ngml~1. This is similar to thereported ED50 for porcine activin A (Vale et al. 1986;Ling et al. 1986), human recombinant activins A and B(Mason et al. 1989), frog activin A (Smith et al. 1990)and recombinant bovine activin A (KVN and DH,unpublished results). The other assay relies on therecently described activity of activins on the differen-

440 R. M. Albano and others

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Fig. 4. Purified WEHI-MIF does not inhibit the growth ofCCL-64 cells. WEHI-MIF was assayed in the CCL-64growth inhibition assay and compared with a porcine TGF-Pl standard. Solid circles: TGF-/S2; open circles: WEHI-MIF.

0 1 2 3 4 5 6 7 8 9 10 11 12WEHI-MIF (ngmP1)

Fig. 5. Purified WEHI-MIF stimulates the release of FSHfrom rat anterior pituitary cells. WEHI-MIF was assayedfor FSH-release activity as described in Materials andmethods. Assays were done in triplicate and mean valuesare expressed as % change over basal levels obtained withBSA/HC1 or PBS. Statistical analysis was performed usingthe Mann-Whitney Latest. Results were consideredsignificant when P<0.05. In this experiment vaccinia virus-expressed bovine activin A (approx 10-20ngmP1)stimulated FSH release by 117%.

tiation of erythroleukemia cell lines. Activins A and Bcause these cells to differentiate terminally intohaemoglobin-synthesizing cells (Murata et al. 1988;Mason et al. 1989). WEHI-MIF induced haemoglobinsynthesis in K562 cells with a specific activity similar tothat of recombinant bovine activin A, while TGF-/31and TGF-/S2 showed no effect (data not shown). Theseresults show, therefore, that WEHI-MIF behaves as anactivin in specific biological assays.

Activin f5A chain mRNA is expressed in WEHI cellsTo confirm further that WEHI-MIF is mouse activin A,we took advantage of the high degree of conservation ofthe mature region of activin A, and used a rat cDNA

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Fig. 6. (A) mRNA encoding the activin /3A chain isexpressed in WEHI cells. Approximately 25 jig of totalRNA from WEHI cells (W) and ovary (O) were subject todenaturing agarose gel electrophoresis, blotted ontonitrocellulose and hybridized to probes encoding parts ofthe y3A or /3B chains of the rat activins as described inMaterials and methods. Hybridization and washes weredone under high stringency. (B) Presence of mRNAs forTGF-^s 1 and 3 but not TGF-/32 in WEHI cells. Each lanecontains 5/ig poly (A)+ RNA. Lane A was hybridized witha probe to human TGF-/3 1, lane B with a probe to TGF-/32and lane C with a probe to TGF-/S3.

probe corresponding to this region to hybridize aNorthern blot of total RNA from WEHI cells andmouse ovary and liver. The latter was used as a negativecontrol since a recent study by Meunier et al. (1988),using Sl-nuclease analysis, showed that although theycould detect activin A and B chain expression in avariety of rat tissues, no expression was seen in liver.Fig. 6A shows that the probe hybridizes to a major

transcript of 6.9 kb present in both WEHI cells andovary, with three other minor transcripts of 4.2, 3.5 and1.8 kb. Similar minor transcripts were also observed inovary by Mason et al. (1985). The same blot washybridized with a rat activin B probe and no expressionin WEHI cells was observed although a major transcriptwas present in ovary (Fig. 6A). This rules out thepossibility that the activin A probe could be hybridizingto an activin B mRNA (activin A and activin B areabout 70% homologous at the amino acid level: Masonet al. 1989). The membrane was also probed with alabelled fragment of the rat inhibin a chain (Esch et al.1987) but again only the ovary contained an a transcript(Albano and Smith, unpublished results). The integrityof all RNAs was monitored by hybridizing filters withan or actin probe (data not shown). The rat activin Aprobe has recently been used to clone mouse activin Afrom a WEHI cDNA library. This cDNA was used toprobe the above filters and the same transcripts wereobserved (Albano and Smith, unpublished results).

WEHI poly (A)+ RNA was also probed using clonesfor human TGF-/J1, 2 and 3 (Fig. 6B). This showedtranscripts of the expected size for TGF-)3s 1 (2.5 kb and1.8 kb) and 3 (3.5 kb) but TGF-/32 transcripts were notobserved.

Mesoderm-inducing activity of WEHI-MIF (mouseactivin A)The mesoderm-inducing activity of WEHI-MIF wastested on groups of ten stage-8 Xenopus animal poleexplants using histological analysis and groups of threeexplants for RNase protection. Concentrations of 0.01to lOngml"1 were used. Both techniques showed thatconcentrations in excess of O.SngmP1 WEHI-MIFwere sufficient to induce abundant muscle and neuraltissue (data not shown for histological analysis; seeFig. 7 for RNase protection analysis using muscle-specific actin probe). Notochord formation was notobserved in this experimental series, probably becauseconcentrations of approximately 25ngml~1 arerequired to induce this tissue type (Green et al. 1990).However, notochord has been seen to be induced byconditioned medium from the WEHI cell line (Godsaveet al. 1988) and by recombinant porcine activin A(Asashima et al. 1990). We are currently undertaking adetailed examination of the inductive effects of murineactivin A.

Discussion

WEHI cells are thought to be myelomonocytic cellsproducing both granulocytes and macrophages, and thenumber of potential growth-regulatory secretory prod-ucts is thus very large. One product of these cells, forexample, is known to be interleukin 3 (Lee et al. 1982).A preliminary characterization of the MIF secreted bythe cells suggested that it was TGF-y3-like (Godsave etal. 1988) but in the present work it is shown that thecells do not secrete detectable TGF-/3 protein althoughat least two forms, TGF-^ 1 and 3, appear to be

Activin A is a mesoderm-inducing factor 441WEHI-MIF

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i

CYTO—ACTIN

Fig. 7. RNase protection assay shows that highly purifiedWEHI-MIF induces expression of cardiac actin in Xenopusanimal caps at concentrations of 0.3ngml~' and above.Explants were analyzed at stage 40. Numbers giveconcentrations of WEHI-MIF in ngml"1; WE, wholeembryo at stage 40; cyto actin, cytoskeletal actin; Y, yeasttRNA control.

transcribed. We showed that production of the MIFcould be boosted by endotoxin, and developed a large-scale culture method appropriate to its purification.

The purification protocol used here is based on thatdeveloped for XTC-MIF (Smith et al. 1990). N-terminalamino acid sequence analysis reveals that the firstseventeen amino acids of WEHI-MIF are identical tothose of activin A from other mammalian species and tothe first ten amino acids of the activin A homologuefrom Xenopus laevis. WEHI-MIF shares biologicalproperties with activin A in that it stimulates the releaseof FSH from rat anterior pituitary cells and inducesdifferentiation of erythroleukemia cells. Transcripts foractivin A but not activin B are present in RNAextracted from the WEHI cells. These data lead us toconclude that the MIF purified from WEHI cells ismouse activin A.

The mesoderm-inducing activity of mouse activin Ais similar to that of its frog homologue as judged byRNase protection and histological analysis of treatedanimal cap explants. Activin A from both species caninduce differentiation of dorsal mesodermal derivativesas well as neural epithelium in isolated animal caps atconcentrations around 0.3 ngml"1. It has been reportedthat related molecules like TGF-/32 induce muscle in

442 R. M. Albano and others

isolated caps but only at concentrations of 3-12 ng ml 1

(Rosa et al. 1988). In accordance with our results,Asashima et al. (1990) recently reported that porcinerecombinant activin A has mesoderm-inducing ability.In their report, however, the lowest concentration ofactivin A that was used was 10 ng ml"1; our results showthat the protein is active at much lower concentrations.

The recent findings that activins are erythroiddifferentiation factors and are expressed in a variety ofextragonadal tissues in the adult animal also predictsdiverse functions for these proteins (Murata et al. 1988;Meunier etal. 1988). It is intriguing that a protein that isthought to be involved in oocyte maturation may alsoplay important roles in later developmental stages. Ourresults showing that mouse activin A has the samemesoderm-inducing activity as frog activin A, which upto now has been known as the most powerful mesoderminducer, opens up the possibility of studying the role ofgrowth factors in the formation of mesoderm in themouse. In this organism, the work of Wilkinson et al.(1988) shows that transcripts for a member of the FGFfamily, the gene int-2, are expressed during gastrulationin migrating cells of the mesoderm. Since variousmembers of this family, including the int-2 protein, areknown mesoderm inducers (Paterno et al. 1989), it ispossible that growth factors are playing a role inmesoderm formation and specification in mouse em-bryos. The identification of a powerful murine meso-derm-inducing protein has given us a valuable tool withwhich to investigate this hypothesis.

We thank Ruth Martin of the ICRF cell culture facility forpreparing the 101 batches of conditioned medium, DrShunichi Shimasaki for generously providing rat /3A, /SB and *chain cDNA clones, Nancy Papalopolu for the mouse a-actincDNA clone, Geoff Howes and Gary Paterno for help withNorthern blots, Jeremy Green for help with RNase protec-tions, Alastair Aitken and Alan Harris for amino acidsequencing, and Jean-Marie Jaspar and Paul Franchimont forthe FSH assays. RMA is supported by a CAPES, Brazil PhDstudentship. This work was supported by the MRC and theICRF.

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{Accepted 19 June 1990)