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Proc. Natl. Acad. Sci. USAVol. 88, pp. 6511-6514, August 1991Physiology/Pharmacology
Presence of activin (erythroid differentiation factor) in unfertilizedeggs and blastulae ofXenopus laevis
(mesoderm induction/folfistatin)
MAKOTO ASASHIMA*t, HIROSHI NAKANOt, HIDEHO UCHIYAMAt, HIROMU SUGINOf, TAKANORI NAKAMURAt,YUZURU ETO§, DAISUKE EJIMA§, SHIN-ICHIRO NISHIMATSU¶, NAOTO UENOI, AND KEI KINOSHITAIItDepartment of Biology, Yokohama City University, Kanazawa, Yokohama 236, Japan; tFrontier Research Program, Institute of Physical and ChemicalResearch (RIKEN), Wako, Saitama 351-01, Japan; §Central Research Laboratories, Ajinomoto, Inc., Kawasaki, Kawasaki 210, Japan; Institute of AppliedBiochemistry, Tsukuba University, Tennodai, Tsukuba 305, Japan; and I'Biological Laboratory, Nippon Medical School, Nakahara, Kawasaki 211, Japan
Communicated by Howard A. Bern, April 3, 1991 (received for review February 1, 1991)
ABSTRACT Activin A, a member of the transforminggrowth factor if superfamily, has recently been found to havepotent mesoderm-inducing activity on isolated early Xenopusanimal-cap cells. We measured the activin activity of theXenopus egg extract by using an erythroid-differentiating testwith Friend leukemia cells. The results showed that an activinhomologue is, indeed, contained in unfertilized eggs and blas-tulae ofXenopus laevis in a considerable amount. This activitywas eluted at the same retention time as human activin A whenfractionated by reversed-phase HPLC. Furthermore, the frac-tion containing erythroid-differentiating factor activity hadmesoderm-inducing activity on Xenopus animal-cap cells. Themesoderm-inducing activity of this fraction was suppressedwhen coincubated with follistatin, an activin-binding protein.These results suggest that an endogenous activin may be anatural mesoderm-inducing factor acting in Xenopus embryo-genesis.
Mesoderm induction is the earliest interaction that occursduring the morula and blastula stage of amphibian develop-ment (1-4). In this process, the signal(s) from the vegetalhemisphere is postulated to induce cells of the animal hemi-sphere to form mesodermal tissues, such as muscle andnotochord. Under experimental conditions, the animal-capexplants from blastula stage embryos form only atypicalepidermis when cultured in isolation but form mesodermaltissues when cultured in contact with vegetal cells or treatedwith some mesoderm-inducing factors (MIFs). Several poly-peptide growth factors, such as acidic and basic fibroblastgrowth factors (FGFs) (5-8) and transforming growth factorp (TGF-p) (9, 10), have been identified as MIFs that canmimic the mesoderm induction of vegetal cells.
Activin A, a member ofthe TGF-13 superfamily, is the thirdmolecule that has potent mesoderm-inducing activity (11, 12)and whose chemical structure is known (13, 14). It wasoriginally isolated from the mammalian gonadal fluid as astimulator of the release of pituitary follicle-stimulating hor-mone (14). Activins A and B are now attracting interest fromdevelopmental biologists because they possess high meso-derm-inducing activity (12, 15) and because the MIFs purifiedfrom many sources have been identified as being homologuesof or related to activin. These MIFs include factors obtainedfrom Xenopus XTC cells (16, 17), chicken embryos, calfkidney (18), and mouse macrophage cells (15). Activin Ainduces mesodermal tissues in a concentration-dependentmanner; a high concentration induces dorsal tissues, a me-dium concentration induces intermediate tissues, and a lowconcentration induces ventral tissues (19-21). Therefore,activins are candidates for molecules active in the formation
of the embryonic dorso-ventral axis. The genes encodingXenopus activin A and B (inhibin PA and OB chains, respec-tively) have been cloned (15), and the transcripts of bothchains were detected after blastula stage in embryos. How-ever, the existence of activin protein has been suggested onlyby a preliminary measurement in the blastula stage (17).Although these reports suggested a possible relationship ofactivins and a natural MIF in Xenopus development, to ourknowledge, the existence of active activin protein in the egghas not yet been shown.
Erythroid differentiation factor (EDF) obtained from theconditioned medium of THP-1 and K-562 myelomonocyteswas found to be identical to activin A (22). The factor causesF5-5 Friend leukemia cells to synthesize hemoglobin, whichis readily detectable by dianisidine staining. This EDF test ishighly quantitative and sensitive. TGF-p, inhibin, and FGFdo not have EDF activity (22). Therefore, the EDF test is anexcellent method to measure activin activity. We used thisassay to determine the activin content of Xenopus eggs.
MATERIALS AND METHODSMaterials. Recombinant human activin A (EDF) was pre-
pared as described (23). Porcine follistatin (activin-bindingprotein) was purified from porcine ovaries as described (24).Embryos ofXenopus laevis were staged according to Nieuw-koop and Faber (25).
Reversed-Phase HPLC and the EDF Test. Unfertilized eggs(1782 eggs) were homogenized with a glass homogenizer at00C in 3.6 ml of 2 M guanidine hydrochloride containing 0.1%trifluoroacetic acid (TFA). Blastula embryos (3354 embryosat stages 8 and 9) were homogenized likewise in 10 ml of theguanidine/TFA solution. The homogenate was centrifuged at12,000 rpm in a 6-cm rotor for 5 min. After centrifugation, 2M guanidine hydrochloride containing 0.1% TFA was addedto the supernatant to adjust the volume to 5 ml for the eggs(or to 10 ml for the blastulas, of which 5 ml was used for theEDF test and the rest was used for the mesoderm-inductionassay). An equal volume of0.1%TFA was then added and themixture was centrifuged to remove insoluble materials. Thesupernatant was desalted by using a PD-10 column (Pharma-cia LKB) equilibrated with 0.1% TFA. The desalted samplewas injected into a STMS-300 column (Cosmosil PackedColumn, 4.6 x 250 mm, Nacalai Tesque, Kyoto, Japan),equilibrated with 0.1% TFA, and eluted with a linear gradientof 20-40o (vol/vol) acetonitrile in 0.1% TFA at a flow rateof 1 ml/min. To each fraction of 1 ml, fresh Friend cellmedium [Ham's F12 medium supplemented with 10%o (vol/
Abbreviations: TGF-/3, transforming growth factor A; EDF, eryth-roid differentiation factor; MIF, mesoderm-inducing factor; FGF,fibroblast growth factor; TFA, trifluoroacetic acid.*To whom reprint requests should be addressed.
6511
The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.
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6512 Physiology/Pharmacology: Asashima et al.
I01
5 10 15 20 25 30 0 5 10 15 20 2530Fraction Fraction
FIG. 1. Reversed-phase HPLC chart ofXenopus egg materials (Aand B) and their EDF activities (C and D). The estimated amount ofactivin in each fraction is shown above the bars in ng. Here, the EDFactivities of fractions obtained from 1782 unfertilized eggs and 1677stage 9 blastula embryos are presented. AN, acetonitrile.
vol) fetal calf serum] was added and lyophilized. Afterreconstitution with distilled water, the sample was neutral-ized, filtered, and assayed by an EDF test as described (22).Mesoderm-Induction Assay. The presumptive ectoderm of
a stage 9 blastula was cut at an angle of =30° around theanimal pole with fine tungsten needles. These explants of=0.3 mm x 0.3 mm were exposed to Steinberg's solution (pH7.4) containing material from HPLC fractions and 0.2%bovine serum albumin (Sigma, no. A7888) for 3 days at 20°Cin a 24-well polystylene dish in suspension culture (SumilonMS-8024R, Sumitomo Bakelite, Tokyo). To prepare a testsolution of the egg extract, two HPLC fractions (1 ml perfraction) were combined, mixed with 2 mg of bovine serumalbumin, lyophilized, reconstituted with 100 ,Al of distilledwater, neutralized, and added to 900 ,ul of Steinberg's solu-tion. Histological analyses were presented elsewhere (12).Induced tissues could be identified clearly by the histologicalfeatures. In the coincubation experiment, material from theHPLC fractions and follistatin were incubated together for=1 hr at 20°C prior to the addition of the explant.Northern Blot Analysis of Activin mRNA from Early Em-
bryos. Early embryos were stored at -80°C until RNA
extraction. The embryos and an ovary were homogenizedand total RNA was extracted as described by Chirgwin et al.(26). Poly(A) RNA was purified on an oligo(dT) column(Pharmacia, Uppsala, Sweden). Each of the poly(A) RNAs(10 ,ug) was denatured with 5 M glyoxal, electrophoresed ina 1% agarose gel with 10mM sodium phosphate (pH 7.0), andtransferred in 20x standard saline citrate (SSC) to a nitro-cellulose membrane (Schleicher & Schuell).
Activin Probe. Activin-related genes were isolated from aX. laevis liver genomic DNA library using rat inhibin PAcDNA (27) as a probe. Among isolated Xar (Xenopus activinrelated) genes, Xar9 was found to encode a protein thatshows 87% amino acid homology with human inhibin PAsubunit (28) in the predicted mature region. A Sau3AI-digested Xar9 DNA fragment (341 base pairs) that corre-sponds to the mature region (29) was labeled with [32P]dCTP(specific activity, 1 x 109 cpm/pg) and used as a hybridiza-tion probe. The hybridization was done in 1 M NaCl/0.2%bovine serum albumin/0.2% Ficoll/0.2% polyvinylpyrroli-done/50 mM Tris-HCl, pH 7.4/20 mM EDTA/0.1% SDScontaining yeast tRNA (0.2 mg/ml) at 60°C for 15 hr. Filterswere washed at 50°C for 20 min with lx SSC/0.1% SDS.
RESULTSXenopus Egg Extract Has Activin Activity. Unfertilized eggs
and blastulae (stages 8 and 9) ofX. laevis were homogenizedin 2 M guanidine hydrochloride containing 0.1% TFA. Theextract was desalted and fractionated by reversed-phaseHPLC, and the activin activity in each fraction was assayedby an EDF test (Fig. 1). The activin activities of the extractsfrom unfertilized eggs and blastulae were detected in frac-tions 17 and 18. The retention times of recombinant humanactivin A and activin activities from these extracts wereidentical. Other growth factors belonging to the TGF-p familydid not possess EDF activity and their retention times weredifferent from that of activin A (Y.E., unpublished data).These results indicate that an activin homologue(s) is presentin Xenopus unfertilized eggs and blastulae. As shown in Fig.1, the content of activin homologue in each fraction wasestimated from its EDF activity. Total content of extractedactivin homologue was =w1.6 ng from 1782 unfertilized eggsand =2.0 ng from 1677 blastulae (stages 8 and 9) embryos. Byassuming that recovery of the activin homologue is 100%, theamount of activin homologue is roughly 1 pg per unfertilizedegg or blastula embryo.
Table 1. Results of the mesoderm-inducing test
Human activin ABlastula extract (EDF)
9/10 13/14 17/18 21/22 25/26 17/18/follistatin* Alone + follistatinExplants, total no. 12 10 16 12 13 10 11 10Mesoderm-induced explants,
total no. 1 (8) 1 (10) 12 (75) 1 (8) 2 (15) 2 (20) 10 (91) 1 (10)Ectodermal tissue
Atypical epidermis, no. 12 (100) 10 (100) 9 (56) 11 (92) 13 (100) 10 (100) 1 (9) 10 (100)Epidermis, no. 0 (0) 1 (10) 8 (50) 1 (8) 2 (15) 1 (10) 10 (91) 1 (10)Neural tissues, no. 0 (0) 0 (0) 1 (6) 0 (0) 1 (8) 0 (0) 3 (27) 1 (10)
Mesodermal tissueNotochord, no. 0 (0) 0 (0) 3 (19) 0 (0) 0 (0) 0 (0) 3 (27) 0 (0)Muscle, no. 0 (0) 0 (0) 6 (38) 0 (0) 0 (0) 1 (10) 7 (64) 1 (10)Blood cells, no. 0 (0) 0 (0) 2 (13) 0 (0) 1 (8) 1 (10) 4 (37) 0 (0)Coelomic epithelium, no. 0 (0) 1 (10) 4 (25) 1 (8) 2 (15) 1 (10) 5 (45) 1 (10)Mesenchyme, no. 1 (8) 1 (10) 5 (31) 1 (8) 1 (8) 2 (20) 5 (45) 1 (10)The blastula extract was prepared from 1677 blastulas (stages 8 and 9) and various HPLC fractions, as indicated, were tested. Human activin
A was added at 10 ng/ml alone or with follistatin at 100 ng/ml. Numbers in parentheses are the differentiation frequency (%).*Fractions from another batch of blastula embryos (stages 8 and 9); material from fractions 17 and 18 was added at 2 ng/ml of estimated activinhomologue and follistatin was at 100 ng/ml.
Proc. Natl. Acad. Sci. USA 88 (1991)
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Proc. Natl. Acad. Sci. USA 88 (1991) 6513
A B
/ not
C DFIG. 2. Histological section of the induced explants with fractionated egg extracts. (A) Explants cultured in Steinberg's solution differen-
tiated to atypical epidermis (ep). (B and C) Explants treated with material in fractions 17 and 18 differentiated to notochord (not), muscle (mus),mesenchyme (mes), and epidermis (epi). (D) Explants cultured with material in fractions 17 and 18 and follistatin (100 ng/ml) showed nomesoderm differentiation.
Xenopus Egg Extract Contains Mesoderm-Inducing Activ-ity. The mesoderm-inducing activity (MIF-like activity) ofthe activin-homologue-containing fractions was examined bythe mesoderm-inducing test. As shown in Table 1 and Fig. 2,only the animal-cap explants treated with the material infractions 17 and 18 formed highly developed mesodermaltissues, such as muscle, coelomic epithelium, blood cells,mesenchyme, and notochord. Material in fractions 17 and 18obtained from unfertilized eggs gave similar results (data notshown). On the other hand, treatment of the explants withmaterial in fractions 1-15 and 18-22 resulted in no significantinduction of mesodermal tissues. Fractions 17 and 18 fromblastulae were estimated by the EDF test to contain anactivin homologue concentration equivalent to human activinA at 2 ng/ml. The mesoderm-inducing activity of this fractionwas roughly comparable to that of human activin A, whichwas used at 10 ng/ml.Mesoderm-Inducing Activity of the Egg Extract Is Sup-
pressed by Foflistatin. The mesoderm-inducing activity of thematerial in fractions 17 and 18 of the blastula extract wasstrongly inhibited by the addition of porcine follistatin, anactivin-binding protein, at 100 ng/ml (Table 1 and Fig. 2).Follistatin binds activin to form an inactive equimolar com-plex, resulting in inhibition of various activin actions (24, 30).Follistatin does not bind TGF-,B and inhibin A (31). Theresults clearly indicate that there are many biological andprotein-chemical properties common to the activin homo-logue from egg extract and human activin A: induction ofmesodermal tissues, inhibition of the mesoderm-inducingactivity by follistatin, and behavior in reversed-phase HPLC.Therefore, it is highly probable that the mesoderm-inducingactivity in the egg extract is due to the activin homologue.
Activin A Gene Is Not Tralscibed in Unfertilized Eggs andEarly Embryos of Xenopus. We searched a genomic library
obtained from Xenopus liver for aXenopus activin A gene usingrat activin A cDNA as a probe and found a positive clone, Xar9.The amino acid sequence of Xenopus activin A, which waspredicted from Xar9 cDNA encoding mature activin A, wasfound to have 87% homology with that of rat activin A (27) andwas identical to that of Thomsen et al. (15). Northern blotanalyses with the Xar9 cDNA probe showed the presence ofactivinAmRNA in stage 32 larva andXenopusXTC cells but notin unfertilized mature egg and stage 7 embryo. Activin A expres-sion was also observed in heart, lung, kidney, and testis of theadult Xenopus (data not shown).
DISCUSSIONThe results suggest that an activin homologue is alreadypresent in an unfertilized egg at a concentration comparableto that in a blastula embryo. This is in part compatible withthe findings that the mesoderm-inducing activity of the pre-sumptive endoderm appears quite early in amphibian devel-opment (32). However, the mesoderm-inducing activity ofthe endoderm increases markedly during cleavage. One ex-planation for this contradiction is that activin may be presentin unfertilized eggs in an inactive form (for instance, as aprecursor) that was artificially activated during the purifica-tion procedures. Another explanation, which is even moreplausible, is that the inducing activity of activin is blocked inearly embryos by some inhibitor or binding protein, such asfollistatin, and then activin is released from the inhibitor toact as a MIF during the morula and blastula stages. It is alsopossible that activin is stored in membrane-bound vesicles inthe early blastomeres and secreted in later stages.The EDF test estimated that -1 pg of the activin homo-
logue is present in a Xenopus egg. From this result theconcentration in the egg is calculated to be f500 pg/ml. Since
Physiology/Pharmacology: Asashima et aL
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6514 Physiology/Pharmacology: Asashima et al.
recombinant human activin A can effectively induce meso-dermal tissues from 300 pg/ml (20), Xenopus egg appears tocontain enough activin homologue, even assuming that it isuniformly distributed in the egg. These results suggest thatthe activin homologue is a natural inducer in Xenopus em-bryogenesis.Thomsen et al. (15) have reported that the gene for activin
B is transcribed in the blastula stage, the gene for activin Ais not transcribed before the end ofgastrula stage ofXenopusdevelopment, and no maternal activinmRNA was found. Ourpresent result using Xar9 gene probe also supports theabsence of activin A mRNA in very early embryos. Thesedata suggest that newly synthesized activin induces meso-derm after the late blastula stage. However, the mesoderm-inducing activity of the presumptive endoderm appears ear-lier than this and disappears during gastrula stage in variousamphibians (1, 2, 32). The present activin homologue did notincrease much from the unfertilized egg to the blastula stage,suggesting that this homologue is likely to be of maternalorigin, being incorporated into the oocyte by some mecha-nism. It is not yet known whether this activin homologuecorresponds to activin A, activin B, activin AB, or a closelyrelated factor.As for FGF, the presence ofmRNA and peptide in oocytes
and embryos has been reported (33, 34). FGF, however,hardly induces dorsal mesodermal tissues, such as notochord(5, 19). Although it is possible that FGF plays important rolesin the developmental processes (for instance, to act syner-gistically with activin), activin is the key substance for anatural MIF, in that it exists in the egg in a sufficient amountand that it can induce almost all mesodermal tissues in aconcentration-dependent manner beginning from very lowconcentrations (12, 19-21).
This work was supported in part by a Grant-in-Aid from theMinistry of Education, Science, and Culture of Japan.
1. Nieuwkoop, P. D. (1969) Roux's Arch. Dev. Biol. 162, 341-373.2. Nakamura, O., Takasaki, H. & Ishihara, M. (1971) Proc. Jpn.
Acad. 47, 313-318.3. Gurdon, J. B., Fairman, S., Mohun, T. J. & Brennan, S. (1985)
Cell 41, 913-922.4. Smith, J. C. (1990) Development 105, 665-677.5. Slack, J. M. W., Darlington, B. G., Heath, J. K. & Godsave,
S. F. (1987) Nature (London) 326, 197-200.6. Kimelman, D. & Kirschner, M. (1987) Cell 51, 869-877.7. Paterno, G. D., Gillespie, L. L., Dixon, M. S., Slack,
J. M. W. & Heath, J. K. (1989) Development 106, 79-83.8. Grunz, H., McKeehan, W. L., Knochel, W., Born, J., Tiede-
mann, H. & Tiedemann, H. (1988) Cell Differ. 22, 183-190.9. Rosa, F., Roberts, A. B., Danielpour, D., Dart, L. L., Sporn,
M. B. & Dawid, I. B. (1988) Science 239, 783-785.10. Knochel, W., Born, J., Hoppe, P., Loppnow-Blinde, B., Tiede-
mann, H., Tiedemann, H., McKeehan, W. L. & Grunz, H.(1987) Naturwissenschaften 74, 604-606.
11. Nakano, H., Kinoshita, K., Ishii, K., Shibai, H. & Asashima,M. (1990) Dev. Growth Differ. 32, 165-170.
12. Asashima, M., Nakano, H., Shimada, K., Kinoshita, K., Ishii,K., Shibai, H. & Ueno, N. (1990) Roux's Arch. Dev. Biol. 198,330-335.
13. Mason, A. J., Hayflick, J. S., Ling, N., Esch, F., Ueno, N.,Ying, S.-Y., Guillemin, R., Niall, H. & Seeburg, P. (1985)Nature (London) 318, 659-663.
14. Vale, W., Rivier, J., Vaughan, J., McClintock, R., Corrigan,A., Woo, W., Karr, D. & Spiess, J. (1986) Nature (London) 321,776-779.
15. Thomsen, G., Woolf, T., Whitman, M., Sokol, S., Vaughan, J.,Vale, W. & Melton, D. A. (1990) Cell 63, 485-493.
16. Smith, J. C., Price, B. M. J., Van Nimmen, K. & Huylebro-eck, D. (1990) Nature (London) 345, 729-731.
17. van den Eijinden-Van Raaij, A. J. M., van Zoelent, E. J. J.,van Nimmen, K., Koster, C. H., Snoek, G. T., Durston, A. J.& Huylebroeck, D. (1990) Nature (London) 345, 732-734.
18. Asashima, M., Nakano, H., Uchiyama, H., David, M., Ples-sow, S., Loppnow-Blinde, B., Hoppe, P., Dau, H. & Tiede-mann, H. (1990) Naturwissenschaften 77, 389-391.
19. Green, J. B. A., Howes, G., Symes, K., Cooke, J. & Smith,J. C. (1990) Development 108, 173-183.
20. Ariizumi, T., Moriya, N., Uchiyama, H. & Asashima, M.(1991) Roux's Arch. Dev. Biol., in press.
21. Green, J. B. A. & Smith, J. C. (1990) Nature (London) 347,391-394.
22. Eto, Y., Tsuji, T., Takezawa, M., Takano, S., Yokogawa, Y.& Shibai, H. (1987) Biochem. Biophys. Res. Commun. 142,1095-1103.
23. Murata, M., Eto, Y., Shibai, H., Sakai, M. & Muramatsu, M.(1988) Proc. Natl. Acad. Sci. USA 85, 2434-2438.
24. Kogawa, K., Nakamura, T., Sugino, K., Takio, K., Titani, K.& Sugino, H. (1991) Endocrinology 128, 1434-1440.
25. Nieuwkoop, P. D. & Faber, J. (1956) Normal Table ofXenopusLaevis (Daudin) (North-Holland, Amsterdam).
26. Chirgwin, J. M., Przybyla, A., MacDonald, R. J. & Rutter, W.(1979) J. Biochem. 18, 5294-5299.
27. Esch, F. S., Shimasaki, S., Mercado, M., Cooksey, K., Ling,N., Ying, S.-Y., Ueno, N. & Guillemin, R. (1987) Mol. Endo-crinol. 1, 849-855.
28. Mason, A. J., Hayflick, J., Ling, N., Esch, F., Ueno, N., Ying,S.-Y., Guillemin, R., Niall, H. & Seeburg, P. (1985) Nature(London) 318, 659-663.
29. Ueno, N., Asashima, M., Nishimatsu, S., Suzuki, A. & Mu-rakami, K. (1991) in Frontiers in Muscle Research, eds. Ozawa,E., Masaki, T. & Nabeshima, Y. (Elsevier, Amsterdam), pp.17-27.
30. Asashima, M., Nakano, H., Uchiyama, H., Sugino, H., Na-kamura, T., Eto, Y., Ejima, D., Davids, M., Plessow, S.,Cichocka, I. & Kinoshita, K. (1991) Roux's Arch. Dev. Biol.,in press.
31. Nakamura, T., Takio, K., Eto, Y., Shibai, H., Titani, K. &Sugino, H. (1990) Science 247, 836-838.
32. Asashima, M. (1975) Roux's Arch. Dev. Biol. 177, 301-308.33. Kimelman, D., Abraham, J. A., Haaparanta, T., Palisi, T. M.
& Kirschner, M. W. (1988) Science 242, 1053-1056.34. Slack, J. M. W. & Isaacs, H. V. (1989) Development 105,
147-153.
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