0 1992Harwood Academic Publishers GmbH
Growth Factors, 1992, Vol. 6, pp. 203-208 Reprints available directly from the publisher Photocopying permitted by license only
Printed in the United Kingdom
Distribution of Transforming Growth Factor Beta in a Two-Week-Old Human Embryo HALIT PINAR’, NANCY L. THOMPSON2,KATHLEEN C. FLANDERS3, MICHAEL B. SPORN3,JAMES SUNG’ and BEVERLY B. ROGERS’ ‘Program in Dmelopmental Pathology, Brown University and Women and Infants’ Hospital, Providence, RI, 2Department of Medical Oncology, Rhode Island Hospital, Prm’dence, RI and 3Laboratory of Chemopreventwn, National Cancer Institute, Bethesda, M D
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(Received August 6 1991, Accepted October 21 1991)
Using immunohistochemical methods we have investigated the presence of transforming growth factors f l , /32 and f l precursor in a two-week-old bilaminar human embryo. TGF-fl precursor was seen in both the epiblast and the hypoblast. In contrast to the widespread localization of TGF-fl precursor in the embryo proper, antibodies to the mature TGF-fl peptide localized preferentially to the hypoblast with only weak staining in the epiblast. Staining with antibodies to TGF-D was generally weak in both the epiblast and hypoblast layers of the embryo proper. These results show that TGF-fl and p2 peptides are detectable as early as the second week of human development. KEYWORDS: transforming growth factor-B, immunohistochemistry, early human embryo
INTRODUCTION
Transforming growth factor-beta (TGF-B) is a multifunctional regulatory peptide which occurs in several related forms (TGF-fl-p) and is evolutionarily highly conserved, emphasizing its critical roles in control of cell proliferation and function (Sporn, 1989; Moses, 1990). Although TGF-/3 was originally identified in an assay that measured its ability to enhance the growth of fibroblasts in soft agar, it has become clear that it has broad pleiotrophic actions on almost every type of cell (for reviews see Roberts, 1988; Sporn, 1989; Roberts, 1990). Peptide growth factors, hormones and hormone-like agents have been implicated as mediators of key steps in early development (dePablo, 1990). Several studies have linked TGF/3l and p2 with early embryogenesis, including the detection of this peptide and its messenger RNA in developing mouse and rat embryos Correspondence to: Halit Pinar, M.D., Women and Infants’ Hospital of Rhode Island, Department of Pathology and Laboratory Medicine, 101 Dudley Street, Providence, Rhode Island 02905-2499.
(Heine, 1987; Rapolee, 1988; Lehnert, 1988; Hill, 1986; Wilcox, 1988; Miller, 1989; Pelton, 1990; Gatherer, 1990). Additionally, TGF-@ alone or TGF-fl together with fibroblast growth factor have been shown to be potent inducers of mesoderm in amphibian embryos (Kimelman, 1987; Rosa, 1988). Mechanisms operative in the process of wound healing and carcinogenesis have long been thought to mimic mechanisms operative in embryonic development. The central role of TGF-/3 in wound healing and carcinogenesis, the almost universal distribution of the TGF-/3 receptor on cells (Wakefield, 1987), and the potent effects of the growth factor in control of cell migration, growth, differentiation, and regulation of extracellular matrix, strongly implicate it in embryonic development (Roberts, 1988). The present study was undertaken to obtain information about TGF-fl and TGF-@ at an early stage of human embryonic development. The embryo was in a bilaminar disc stage, consistent with a development age of two weeks. We have done immunohistochemical staining for TGF-/3 protein using avidin-biotin-peroxidase methodology and antibodies raised against syn-
203
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PINAR er al.
thetic peptides corresponding to the sequences of mature TGF-Pl, p2 .and TGF-/3l precursor. This study provides new insights into the patterns of expression of TGF-/? in a very early human embryo.
MATERIALS AND METHOD
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Tissue Preparation The specimen was received as a result of a hysterectomy on a patient with cervical neoplasia. The tissue was fixed in 10% buffered formalin, processed overnight in a Miles Scientific Tissue Processor@and embedded in paraffin. Serial sections 5 pm thick were cut and placed onto glass slides. Sections were de-paraffinized and either stained with hematoxylin-eosin or subjected to immunohistochemical staining (see below). Protocols for obtaining human pathological material for research have been approved by the hospitals' Institutional Review Board. Antibodies All polyclonal antibodies to TGF-fl precursor, TGF-fl and TGF-p2 were raised in New Zealand white rabbits using synthetic peptides as immunogens (Flanders, 1988). Anti-LC (1-30) antibody was generated against synthetic peptide corresponding to N-terminal amino acids 1-30 of mature TGF-fl sequence and affinity purified against TGF-fl (Flanders, 1989). Anti-m precursor antibody was generated against synthetic peptide corresponding to amino acids 266-278 of TGF-fl precursor, then affinity purified against peptide (Wakefield, 1988). Anti-P;! antibody was generated against synthetic peptide corresponding to amino acids 50-75 of mature TGF-P sequence and affinity purified against peptide (Flanders, 1990).
used at a final concentration of approximately 1-5 pg IgG/ml. Sections were counterstained lightly with 1%methyl green attempting not to obscure weak positive peroxidase staining. As a control for staining specificity (a) the anti-TGF-P rabbit IgG was replaced with normal rabbit IgG or (b) the antibody was blocked by preincubation with a 20-fold molar excess of the appropriate synthetic peptide prior to use on sections. Controls were compared with unblocked antibody for staining patterns. Sections of mouse embryo were included in each experiment as a positive control (Heine et al., 1987). The staining was graded subjectively (0-4+) on the basis of intensity where (0) was absence of staining and (4+) was the presence of intense brown pigment seen at 400x magnification. RESULTS
Hematoxylin and eosin stained sections showed a two-week-old bilaminar embryo with the epiblast and hypoblast comprising the embryo proper (Fig. 1). The embryonal sac consisted of the amniotic cavity and the primary yolk sac. This, in turn, was surrounded by a larger sac composed of the cytotrophoblast and the extraembryonic coelom/mesoderm. At the site of implantation the syncytiotrophoblast and lacunae of blood
Immunohistochemical Staining Pre-TGF-PI, TGF-P1 and TGF-P were localized in 5 pm sections of paraffin-embedded tissue using avidin-biotin-peroxidase immunohistochemistry and a Vectastain ABC Elite Kit@ (Vector Laboratories, Burlingame,' CA) essentially as described by Flanders et (1988) and Thompson et al. (1988). Primary antibodies were
FIGURE 1. The two week old bilaminar embryo. Hypoblast (H) and epiblast (El layers comprise the embryo proper. Yolk sac (Y) is located adjacent to the hypoblast layer. Amniotic cavity (A) is adjacent to the epiblast layer. Extraembryonic mesoderm (EM) is present in the larger extraembryoniccoelom (EC). Surrounding this sx is the cytotrophoblastic layer (cy). The syncytiotrophoblastic layer (Sy)showing large, syncytial cells located in the endometrium (EN). ~ 1 0 0 .Hematoxylineosin. See Color Plate at back of issue.
TGF-B IN EARLY HUMAN EMBRYO
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vessels formed a primitive placenta. The endometrial glands and stroma showed changes consistent with pregnancy. TGF-/3 is synthesized as a latent form (TGF-/3 precursor) incapable of binding to receptors. The mature TGF-/3 peptide is formed by cleavage of precursor sequences to release the active, receptor binding peptide (Roberts, 1988). Presumably, the localization of the precursor sequences reflects sites of synthesis (Flanders, 1989). An antibody to the amino acid sequence corresponding to the TGF-fl precursor revealed light to moderate staining of both the epiblast and hypoblast layers of the embryo proper, the yolk sac and the extra-embryonic mesoderm (Fig. 2A)
205
(Table I). Marked staining was seen in the cytotrophoblast, the syncytiotrophoblast, the endometrial glands, stroma and myometrium (Fig. 3A). Specificity of staining was demonstrated by lack of reactivity on a contiguous section incubated with an aliquot of the primary antibody that had been preadsorbed with TGF-fl prercursor peptide (Fig. 2B). In contrast to the widespread localization of TGF-fl precursor in the embryo proper, antibodies to the mature TGF-fl peptide showed light staining of the hypoblast. The staining of the epiblast was barely detectable (Fig. 2C). There was weak staining of the extraembryonic mesoderm however. Some cells in the cytotrophoblast were also positive, but the cells in the syncytiotrophoblast did not stain (Fig. 3B). Antibodies to TGF-fl mature peptide on the
FIGURE 2A. Staining of the embryo with TGF-/3 precursor. The epiblast (E) and hypoblast (H) are light to moderately stained. x400. Avidin-biotin-peroxidase, with 1% methyl green counterstain. See Color Plate at back of issue. FIGURE 2C. Staining of the embryo with TGF-/3 shows light staining of the hypoblast (H). The staining of the epiblast (E)is barely detectable. x400. See Color Plate at back of issue.
FIGURE 2B. Specificityof staining is demonstrated by lack of reactivity on a contiguous section incubated with an aliquot of the primary antibody that had been preadsorbed with TGF-/3l precursor peptide. x400. See Color Plate at back of issue.
FIGURE 2D. Staining of the embryo with TGF-/32 shows weak staining of both the hypoblast (HI and the epiblast (E). ~ 4 0 0See . Color Plate at back of issue.
PINAR et al.
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TABLE 1 Staining Intensity of Different Parts of the Specimen
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TGF-/?I PRE TGF-fl TGF-@
Epiblast
Hypoblast
Extraembryonic
Cytotrophoblast
Syncytiotrophoblast
++
ct
++
+ti+
ttt+
+/-
++
+/+/-
+
FIGURE 3A. The cytotrophoblast (Cy) and the syncytiotrophoblast (Sy) layers are markedly stained with TGF-P precursor. x400. Avidin-biotin-peroxidase, with 1% methyl green counterstain. See Color Plate at back of issue.
+
* *
tct.
FIGURE 3C. The cytotrophoblast (Cy) and syncytiotrophoblast (Sy) both stained positively with TGF-@, with greater staining seen in the syncytiotrophoblast. x400. See Color Plate at back of issue.
after preincubation of antibody with the appropriate TGF-P peptides.
DISCUSSION
FIGURE 3B. Some cells in the cytotrophoblast (Cy) layer stained positively for TGF-fl but the cells in the syncytiotrophoblast (Sy) layer are negative. x400. See Color Plate at back of issue.
other hand, weakly stained both the epiblast and the hypoblast of the embryo proper (Fig. 2D). The extraembryonic mesoderm was moderately stained. The cytotrophoblast and syncytiotrophoblast also stained positively, with greater intensity seen in the latter (Fig. 3'2). Specificity of the reaction was again demonstrated by absence of peroxidase reaction product on control sections
We were able to show the presence of T.GF-Pl and TGF-@ in the hypoblast and epiblast cells of a two-week-old human embryo. This is the earliest demonstration of this important growth factor in a human embryo. Immunohistochemical and in situ hybridization studies have implicated TGF-P as an important morphogenetic substance in the mammalian embryo (Heine, 1987; Lehnert, 1988; Wilcox, 1988). TGF-P appears to be especially important in the remodeling that occurs during formation of vertebrae, limb buds, teeth, facial bones and the valves of the heart (Heine, 1987; Lehnert, 1988; Sandberg, 1988a and 1988b; Gatherer, 1990; Pelton, 1990). High levels of TGFP normally are found in embryonic structures that are often sites of congenital malformation, such as the palate or the interventricular septum of the heart (Heine, 1987). The formation and
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TGF-B IN EARLY HUMAN EMBRYO
remodeling of connective tissue that occurs during normal embryogenesis involves biochemical mechanisms similar to those used during repair of injury in the adult, and it seems that TGF-fi is an important mediator of both processes. Heine et al. (1987) demonstrated the expression of TGF-P in a unique pattern, both spatially and temporally, in the developing mouse embryo. The highly specific pattern of histochemical staining of TGF-/?in the mouse embryo described in that study appeared to correlate with specific morphogenetic and histogenetic events, particularly those involving cells and tissues of mesenchymal or mesodermal origin. It has been shown that TGF-B can exert a potent inductive effect on the formation of mesoderm in amphibian embryos, prior to gastrulation (Kimelman, 1987; Rosa, 1988). Because the bulk of the vertebrate organism is composed of mesodermal cells and tissues, it is apparent that TGF-P participates in some fundamental way in the basic architecture and organization of almost the entire developing embryo. The two-week-old human embryo is bilaminar with only two layers of cells in the embryo proper. The epiblast lines the amniotic cavity and the hypoblast lines the primitive yolk sac. The epiblast gives rise to ectoderm and the hypoblast gives rise to endoderm. During the third week the embryo becomes trilaminar with newly formed mesoderm between the original epiblast and hypoblast. In our study TGF-fl and p2 were seen in the relatively undifferentiated epiblast and hypoblast layers characteristic of the bilaminar stage. Since TGF-P- is a known inducer of mesenchyme/mesoderm (Rosa, 1988), it is possible that this action occurs before the visible appearance of the mesenchyme. Alternatively, TGF-P may have another function in the early embryo in addition to mesoderm induction. It is possible that TGF-P modifies growth in all primitive cells. This is supported by the fact that TGF-P has been detected in pre-implantation blastocysts of the mouse (Rappolee, 1988). Angiogenesis and wound healing are two of the primary functions of TGF-/? (reviewed in Roberts 1988 and 1990). Its presence in the trophoblast may be related to the future development of blood vessels in chorionic villi and/or to invasion of maternal tissue by the trophoblast. TGF-P is the most potent known endogenous
201
suppressant of lymphocyte proliferation and function (Kehrl, 1989). In our study there was marked presence of TGF-P in the cyto- and syncytiotrophoblast. High levels of TGF-p2 have also been demonstrated in pregnant uteri of Swiss mice (Altman, 1990). Clark et al. (1990) have demonstrated that TGF-B;! is produced in decidua and that it is immunosuppressive. This could possibly play a role in maternal immunity, in the retention of the fetal allograft, as well as in regulating fetal and neonatal immunological competence. We cannot conclude that the immunohistochemically demonstrable TGF-P is active in these sites. It may be preFent in a latent form, waiting to be activated at a later time. It is also not clear which cells actually synthesize the protein. We do not know the temporal or spatial pattern of TGF-P receptor expression in the embryo which might identify sensitive cell populations. TGF-P has been implicated in both autocrine as well as paracrine mechanisms of action in different tissues and presence of the protein may indicate either synthesis, uptake or both in a specific cell. In developing mouse embryos, it is expressed by epithelial cells as demonstrated by in situ hybridization (Lehnert, 1988) yet it is present in mesenchymal tissue as demonstrated by antibody localization (Heine, 1987). We used antibodies to the precursor form of TGF-fl as a means of addressing this issue, since the mature peptide is cleaved from precursor sequences presumably only after secretion. However, in situ hybridization for mRNA is the only definitive method of ascertaining sites of synthesis in vivo. Additional studies with increased numbers of early human embryos are needed to further define the role of TGF-P in early human development.
ACKNOWLEDGMENTS The authors acknowledge t h e continued support of Dr.
D. B. Singer.
REFERENCES Altman, D. J., Schneider, S. L., Thompson, D. A., Hwei-Ling, Cheng and Tomasi, T. B. (1990) A transforming growth factor /32 (TGF-mblike immunosuppressive factor in amniotic
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fluid and localization of TGF-m mRNA in the pregnant uterus. J, E x p . Med. 172; 1391-1401. Clark, D. A., Flanders, K. C., Banwatt, D., Millar-Book, W., Manual, J., Stedronska-Clark and Rowley, B. (1990)Murine pregnancy decidua produces a unique immunosuppressive molecule related to transforming growth factor 8 2 . J. Immunol. 144,3008-3014. defablo, F. and Roth, J. (1990)Endocrinization of the early embryo: an emerging role for hormones and hormone-like factors. TIBS 15,3-6. Flanders, K. C., Roberts, A. B., Ling, N., Fleurdelys, B. E. and Sporn, M. B. (1988)Antibodies to peptide determinants of transforming growth factor /3 and their applications. Biochemistry 27,739-746. Flanders, K. C., Thompson, N. L., Cissell, D. S., Van Obberghen-Schilling, E., Blaker, C. C., Kass, M. S., Ellingsworth, L. R., Roberts, A. B. and Sporn, M. B. (1989)Transforming growth factor-/3l: histochemical localization with antibodies to different epitopes. 1. Cell. Biol. 108,653-660. Flanders, K. C., Cissell, D. S., Mullen, L. T., Danielpour, D., Sporn, M. B. and Roberts, A. B. (1990)Antibodies to transforming growth factor-/32 peptides: ,specific detection of TGF-fl in immunoassays. Growth Factors 3,45-52. Gatherer, D., Ten Dijke, P., Baird, D. T. and Akhurst, R. J. (1990)Expression of TGF-/3 isoforms during first trimester human embryogenesis. Development 110,445-460. Heine, U. I., Munoz, E. F., Flanders, K., Ellingsworth, L. R., Lam, P. H.-Y., Thompson, N. L., Roberts, A. B. and Sporn, M. B. (1987)Role of transforming growth factor-/3 in the development of the mouse embryo. J. Cell. Biol. 105, 2861-2876. Hill, D. J., Strain, A. J. and Milner, R. D. G. (1986)Present of transforming growth factor-beta-like activity in multiple fetal rat tissues. Cell B i d . Int. Rep. 10,915-922. Kehrl, J. H., Taylor, A. S., Delsing, G. A., Roberts, A. B., Sporn, M. B. and Fauci, A. S. (1989)Further studies of the role of TGF-/3 in human B cell function. 1. Immunol. 143, 1868-1874. Kimelman, D. and Kirschner, M. (1987)Synergistic induction of mesoderm by FGF and TGF-P and the identification of an mRNA coding for FGF in the early Xenopus embryo. Cell 51, 869-877. Lehnert, S.A. and Akhurst, R. J. (1988)Embryonic expression pattern of TGF-/3 type 1 FWA suggests both paracrine and autocrine mechanisms of action. DeveZopment 104,263-273. Mason, I. J.,Murphy, D., Munke, M., Franke, U., Elliott, R. W. and Hogan, B. L. (1986)Developmental and transformation-sensitive expression of the SPARC gene on mouse chromosome 11. Eur. Mol. Biol. Organ. 5,1831-1837. Miller, D. A., Lee, A., Pelton, R. W., Chen, E. Y., Moses, H. L. and Derynck, R. (1989)Murine transforming growth factorp2 cDNA sequence and expression in adult tissues and embryos. Molec. Endocrinol. 3,1108-1114. Moses, H. L., Yang, E. Y. and Pietenpol, J. A. (1990)TGF-P
stimulation and inhibition of proliferation: new mechanistic insights. Cell 63,245-247. Pelton, R. W., Dickinson, M. E., Moses, H. L. and Hogan, L. M. (1990) In situ hybridization analysis of TGF-B3 RNA expression during mouse development: comparative studies with TGF-/3l and TGF-m. Development 110,609-620. Rappolee, D. A., Brenner, C. A., Schultz, R., Mark, D. and Werb, 2. (1988)Developmental expression of PDGF, TGFalpha, and TGF-/3 genes in preimplantation mouse embryos. Science 242,1823-1825. Roberts, A. B., Flanders, K. C., Kondaiah, P., Thompson, N. L., Van Obberghan-Schilling, E., Wakefield, L., Rossi, P., de Crombrugghe, B., Heine, U. L. and Sporn, M. B. (1988) Transforming growth factor fi biochemistry and roles in embryogenesis, tissue repair and remodeling, and carcinogenesis. Recent Prog. Horn. Res. 44,157-197. Roberts, A. B. and Sporn, M. B. (1990)Transforming growth factor-@ In Handbook of Experimental Pharmacoloy, Val. 95/I, Peptide Growth Factors and Their Receptors I , M. B. Sporn and A. B. Roberts, eds, Springer Verlag, New York, pp. 419-472. Rosa, F., Roberts, A. B., Danielpour, D., Dart, L. L., Sporn, M. B. and Dawid, I. B. (1988) Mesoderm induction in amphibians: the role of TGF-m-like factors. Science 239, 783-786. Sandberg, M., Vuorio, T., Hivonen, H., Alitalo, K. and Vuorio, E. (1988a)Enhanced expression of TGF-/3 and c-fos mRNAs in the growth plates of developing human long bones. Development 102,461-470. Sandberg, M., Autio-Harmainen, H. and Vuorio, E. (1988b) Localization and expression of types I, I11 and IV collagen, TGF-fl and c-fos genes in developing human calvarial bones. Dev. Biol. 130,324-334. Seyedin, S . M., Thomas, T. C., Thompson, A. Y., Rosen, D. M. and Piez, K. A. (1985)Purification and characterization of two cartilage-inducing factors from bovine demineralized bone. Proc. Natl. Acad. Sci. U S A 82,2267-2271. Sporn, M. B. and Roberts, A. B. (1989)Transforming growth factor+ Multiple actions and potential clinical applications. J.A.M.A.262,938-941. Thompson, N.L., Flanders, K. C., Smith, M., Ellingsworth, L. R., Roberts, A. B. and Sporn, M. B. (1989)Expression of transforming growth-/TI in specific cells and tissues of adult and neonatal mice. J. Cell. B i d . 108,661-669. Wakefield, L. M., Smith, D. M., Flanders, K. C. and Sporn, M. B. (1988) Latent transforming growth factor-/3 from human platelets: a high molecular weight complex containing precursor sequences. ]. Biol. Chem. 263,7646-7654. Wakefield, L. M., Smith, D. M., Masui, T., Harris, C. C. and Sporn, M. 8 . (1987)Distribution and modulation of the cellular receptor for transforming growth factor-beta. J. Cell. Biol. 105,965-975. Wilcox, J. N. and Derynck, R. (1988) Developmental expression of transforming growth factors alpha and beta in mouse fetus. Mol. Cell. Biol. 8,3415-3422.
Growth Factors, 1992, Vol. 6, pp. 203-208 Reprints available directly from the publisher Photocopying permitted by license only
Printed in the United Kingdom
Distribution of Transforming Growth Factor Beta in a Two-Week-Old Human Embryo HALIT PINAR’, NANCY L. THOMPSON2,KATHLEEN C. FLANDERS3, MICHAEL B. SPORN3,JAMES SUNG’ and BEVERLY B. ROGERS’ ‘Program in Dmelopmental Pathology, Brown University and Women and Infants’ Hospital, Providence, RI, 2Department of Medical Oncology, Rhode Island Hospital, Prm’dence, RI and 3Laboratory of Chemopreventwn, National Cancer Institute, Bethesda, M D
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(Received August 6 1991, Accepted October 21 1991)
Using immunohistochemical methods we have investigated the presence of transforming growth factors f l , /32 and f l precursor in a two-week-old bilaminar human embryo. TGF-fl precursor was seen in both the epiblast and the hypoblast. In contrast to the widespread localization of TGF-fl precursor in the embryo proper, antibodies to the mature TGF-fl peptide localized preferentially to the hypoblast with only weak staining in the epiblast. Staining with antibodies to TGF-D was generally weak in both the epiblast and hypoblast layers of the embryo proper. These results show that TGF-fl and p2 peptides are detectable as early as the second week of human development. KEYWORDS: transforming growth factor-B, immunohistochemistry, early human embryo
INTRODUCTION
Transforming growth factor-beta (TGF-B) is a multifunctional regulatory peptide which occurs in several related forms (TGF-fl-p) and is evolutionarily highly conserved, emphasizing its critical roles in control of cell proliferation and function (Sporn, 1989; Moses, 1990). Although TGF-/3 was originally identified in an assay that measured its ability to enhance the growth of fibroblasts in soft agar, it has become clear that it has broad pleiotrophic actions on almost every type of cell (for reviews see Roberts, 1988; Sporn, 1989; Roberts, 1990). Peptide growth factors, hormones and hormone-like agents have been implicated as mediators of key steps in early development (dePablo, 1990). Several studies have linked TGF/3l and p2 with early embryogenesis, including the detection of this peptide and its messenger RNA in developing mouse and rat embryos Correspondence to: Halit Pinar, M.D., Women and Infants’ Hospital of Rhode Island, Department of Pathology and Laboratory Medicine, 101 Dudley Street, Providence, Rhode Island 02905-2499.
(Heine, 1987; Rapolee, 1988; Lehnert, 1988; Hill, 1986; Wilcox, 1988; Miller, 1989; Pelton, 1990; Gatherer, 1990). Additionally, TGF-@ alone or TGF-fl together with fibroblast growth factor have been shown to be potent inducers of mesoderm in amphibian embryos (Kimelman, 1987; Rosa, 1988). Mechanisms operative in the process of wound healing and carcinogenesis have long been thought to mimic mechanisms operative in embryonic development. The central role of TGF-/3 in wound healing and carcinogenesis, the almost universal distribution of the TGF-/3 receptor on cells (Wakefield, 1987), and the potent effects of the growth factor in control of cell migration, growth, differentiation, and regulation of extracellular matrix, strongly implicate it in embryonic development (Roberts, 1988). The present study was undertaken to obtain information about TGF-fl and TGF-@ at an early stage of human embryonic development. The embryo was in a bilaminar disc stage, consistent with a development age of two weeks. We have done immunohistochemical staining for TGF-/3 protein using avidin-biotin-peroxidase methodology and antibodies raised against syn-
203
204
PINAR er al.
thetic peptides corresponding to the sequences of mature TGF-Pl, p2 .and TGF-/3l precursor. This study provides new insights into the patterns of expression of TGF-/? in a very early human embryo.
MATERIALS AND METHOD
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Tissue Preparation The specimen was received as a result of a hysterectomy on a patient with cervical neoplasia. The tissue was fixed in 10% buffered formalin, processed overnight in a Miles Scientific Tissue Processor@and embedded in paraffin. Serial sections 5 pm thick were cut and placed onto glass slides. Sections were de-paraffinized and either stained with hematoxylin-eosin or subjected to immunohistochemical staining (see below). Protocols for obtaining human pathological material for research have been approved by the hospitals' Institutional Review Board. Antibodies All polyclonal antibodies to TGF-fl precursor, TGF-fl and TGF-p2 were raised in New Zealand white rabbits using synthetic peptides as immunogens (Flanders, 1988). Anti-LC (1-30) antibody was generated against synthetic peptide corresponding to N-terminal amino acids 1-30 of mature TGF-fl sequence and affinity purified against TGF-fl (Flanders, 1989). Anti-m precursor antibody was generated against synthetic peptide corresponding to amino acids 266-278 of TGF-fl precursor, then affinity purified against peptide (Wakefield, 1988). Anti-P;! antibody was generated against synthetic peptide corresponding to amino acids 50-75 of mature TGF-P sequence and affinity purified against peptide (Flanders, 1990).
used at a final concentration of approximately 1-5 pg IgG/ml. Sections were counterstained lightly with 1%methyl green attempting not to obscure weak positive peroxidase staining. As a control for staining specificity (a) the anti-TGF-P rabbit IgG was replaced with normal rabbit IgG or (b) the antibody was blocked by preincubation with a 20-fold molar excess of the appropriate synthetic peptide prior to use on sections. Controls were compared with unblocked antibody for staining patterns. Sections of mouse embryo were included in each experiment as a positive control (Heine et al., 1987). The staining was graded subjectively (0-4+) on the basis of intensity where (0) was absence of staining and (4+) was the presence of intense brown pigment seen at 400x magnification. RESULTS
Hematoxylin and eosin stained sections showed a two-week-old bilaminar embryo with the epiblast and hypoblast comprising the embryo proper (Fig. 1). The embryonal sac consisted of the amniotic cavity and the primary yolk sac. This, in turn, was surrounded by a larger sac composed of the cytotrophoblast and the extraembryonic coelom/mesoderm. At the site of implantation the syncytiotrophoblast and lacunae of blood
Immunohistochemical Staining Pre-TGF-PI, TGF-P1 and TGF-P were localized in 5 pm sections of paraffin-embedded tissue using avidin-biotin-peroxidase immunohistochemistry and a Vectastain ABC Elite Kit@ (Vector Laboratories, Burlingame,' CA) essentially as described by Flanders et (1988) and Thompson et al. (1988). Primary antibodies were
FIGURE 1. The two week old bilaminar embryo. Hypoblast (H) and epiblast (El layers comprise the embryo proper. Yolk sac (Y) is located adjacent to the hypoblast layer. Amniotic cavity (A) is adjacent to the epiblast layer. Extraembryonic mesoderm (EM) is present in the larger extraembryoniccoelom (EC). Surrounding this sx is the cytotrophoblastic layer (cy). The syncytiotrophoblastic layer (Sy)showing large, syncytial cells located in the endometrium (EN). ~ 1 0 0 .Hematoxylineosin. See Color Plate at back of issue.
TGF-B IN EARLY HUMAN EMBRYO
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vessels formed a primitive placenta. The endometrial glands and stroma showed changes consistent with pregnancy. TGF-/3 is synthesized as a latent form (TGF-/3 precursor) incapable of binding to receptors. The mature TGF-/3 peptide is formed by cleavage of precursor sequences to release the active, receptor binding peptide (Roberts, 1988). Presumably, the localization of the precursor sequences reflects sites of synthesis (Flanders, 1989). An antibody to the amino acid sequence corresponding to the TGF-fl precursor revealed light to moderate staining of both the epiblast and hypoblast layers of the embryo proper, the yolk sac and the extra-embryonic mesoderm (Fig. 2A)
205
(Table I). Marked staining was seen in the cytotrophoblast, the syncytiotrophoblast, the endometrial glands, stroma and myometrium (Fig. 3A). Specificity of staining was demonstrated by lack of reactivity on a contiguous section incubated with an aliquot of the primary antibody that had been preadsorbed with TGF-fl prercursor peptide (Fig. 2B). In contrast to the widespread localization of TGF-fl precursor in the embryo proper, antibodies to the mature TGF-fl peptide showed light staining of the hypoblast. The staining of the epiblast was barely detectable (Fig. 2C). There was weak staining of the extraembryonic mesoderm however. Some cells in the cytotrophoblast were also positive, but the cells in the syncytiotrophoblast did not stain (Fig. 3B). Antibodies to TGF-fl mature peptide on the
FIGURE 2A. Staining of the embryo with TGF-/3 precursor. The epiblast (E) and hypoblast (H) are light to moderately stained. x400. Avidin-biotin-peroxidase, with 1% methyl green counterstain. See Color Plate at back of issue. FIGURE 2C. Staining of the embryo with TGF-/3 shows light staining of the hypoblast (H). The staining of the epiblast (E)is barely detectable. x400. See Color Plate at back of issue.
FIGURE 2B. Specificityof staining is demonstrated by lack of reactivity on a contiguous section incubated with an aliquot of the primary antibody that had been preadsorbed with TGF-/3l precursor peptide. x400. See Color Plate at back of issue.
FIGURE 2D. Staining of the embryo with TGF-/32 shows weak staining of both the hypoblast (HI and the epiblast (E). ~ 4 0 0See . Color Plate at back of issue.
PINAR et al.
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TABLE 1 Staining Intensity of Different Parts of the Specimen
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TGF-/?I PRE TGF-fl TGF-@
Epiblast
Hypoblast
Extraembryonic
Cytotrophoblast
Syncytiotrophoblast
++
ct
++
+ti+
ttt+
+/-
++
+/+/-
+
FIGURE 3A. The cytotrophoblast (Cy) and the syncytiotrophoblast (Sy) layers are markedly stained with TGF-P precursor. x400. Avidin-biotin-peroxidase, with 1% methyl green counterstain. See Color Plate at back of issue.
+
* *
tct.
FIGURE 3C. The cytotrophoblast (Cy) and syncytiotrophoblast (Sy) both stained positively with TGF-@, with greater staining seen in the syncytiotrophoblast. x400. See Color Plate at back of issue.
after preincubation of antibody with the appropriate TGF-P peptides.
DISCUSSION
FIGURE 3B. Some cells in the cytotrophoblast (Cy) layer stained positively for TGF-fl but the cells in the syncytiotrophoblast (Sy) layer are negative. x400. See Color Plate at back of issue.
other hand, weakly stained both the epiblast and the hypoblast of the embryo proper (Fig. 2D). The extraembryonic mesoderm was moderately stained. The cytotrophoblast and syncytiotrophoblast also stained positively, with greater intensity seen in the latter (Fig. 3'2). Specificity of the reaction was again demonstrated by absence of peroxidase reaction product on control sections
We were able to show the presence of T.GF-Pl and TGF-@ in the hypoblast and epiblast cells of a two-week-old human embryo. This is the earliest demonstration of this important growth factor in a human embryo. Immunohistochemical and in situ hybridization studies have implicated TGF-P as an important morphogenetic substance in the mammalian embryo (Heine, 1987; Lehnert, 1988; Wilcox, 1988). TGF-P appears to be especially important in the remodeling that occurs during formation of vertebrae, limb buds, teeth, facial bones and the valves of the heart (Heine, 1987; Lehnert, 1988; Sandberg, 1988a and 1988b; Gatherer, 1990; Pelton, 1990). High levels of TGFP normally are found in embryonic structures that are often sites of congenital malformation, such as the palate or the interventricular septum of the heart (Heine, 1987). The formation and
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TGF-B IN EARLY HUMAN EMBRYO
remodeling of connective tissue that occurs during normal embryogenesis involves biochemical mechanisms similar to those used during repair of injury in the adult, and it seems that TGF-fi is an important mediator of both processes. Heine et al. (1987) demonstrated the expression of TGF-P in a unique pattern, both spatially and temporally, in the developing mouse embryo. The highly specific pattern of histochemical staining of TGF-/?in the mouse embryo described in that study appeared to correlate with specific morphogenetic and histogenetic events, particularly those involving cells and tissues of mesenchymal or mesodermal origin. It has been shown that TGF-B can exert a potent inductive effect on the formation of mesoderm in amphibian embryos, prior to gastrulation (Kimelman, 1987; Rosa, 1988). Because the bulk of the vertebrate organism is composed of mesodermal cells and tissues, it is apparent that TGF-P participates in some fundamental way in the basic architecture and organization of almost the entire developing embryo. The two-week-old human embryo is bilaminar with only two layers of cells in the embryo proper. The epiblast lines the amniotic cavity and the hypoblast lines the primitive yolk sac. The epiblast gives rise to ectoderm and the hypoblast gives rise to endoderm. During the third week the embryo becomes trilaminar with newly formed mesoderm between the original epiblast and hypoblast. In our study TGF-fl and p2 were seen in the relatively undifferentiated epiblast and hypoblast layers characteristic of the bilaminar stage. Since TGF-P- is a known inducer of mesenchyme/mesoderm (Rosa, 1988), it is possible that this action occurs before the visible appearance of the mesenchyme. Alternatively, TGF-P may have another function in the early embryo in addition to mesoderm induction. It is possible that TGF-P modifies growth in all primitive cells. This is supported by the fact that TGF-P has been detected in pre-implantation blastocysts of the mouse (Rappolee, 1988). Angiogenesis and wound healing are two of the primary functions of TGF-/? (reviewed in Roberts 1988 and 1990). Its presence in the trophoblast may be related to the future development of blood vessels in chorionic villi and/or to invasion of maternal tissue by the trophoblast. TGF-P is the most potent known endogenous
201
suppressant of lymphocyte proliferation and function (Kehrl, 1989). In our study there was marked presence of TGF-P in the cyto- and syncytiotrophoblast. High levels of TGF-p2 have also been demonstrated in pregnant uteri of Swiss mice (Altman, 1990). Clark et al. (1990) have demonstrated that TGF-B;! is produced in decidua and that it is immunosuppressive. This could possibly play a role in maternal immunity, in the retention of the fetal allograft, as well as in regulating fetal and neonatal immunological competence. We cannot conclude that the immunohistochemically demonstrable TGF-P is active in these sites. It may be preFent in a latent form, waiting to be activated at a later time. It is also not clear which cells actually synthesize the protein. We do not know the temporal or spatial pattern of TGF-P receptor expression in the embryo which might identify sensitive cell populations. TGF-P has been implicated in both autocrine as well as paracrine mechanisms of action in different tissues and presence of the protein may indicate either synthesis, uptake or both in a specific cell. In developing mouse embryos, it is expressed by epithelial cells as demonstrated by in situ hybridization (Lehnert, 1988) yet it is present in mesenchymal tissue as demonstrated by antibody localization (Heine, 1987). We used antibodies to the precursor form of TGF-fl as a means of addressing this issue, since the mature peptide is cleaved from precursor sequences presumably only after secretion. However, in situ hybridization for mRNA is the only definitive method of ascertaining sites of synthesis in vivo. Additional studies with increased numbers of early human embryos are needed to further define the role of TGF-P in early human development.
ACKNOWLEDGMENTS The authors acknowledge t h e continued support of Dr.
D. B. Singer.
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