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(1991)

Differential Localization of TGF-& in Mouse Preimplantation and Early Postimplantation Development H.G.

SLAGER,' K. A. LAWSON, A.J.M.

VANDENEIJNDEN-VANRAAIJ,S.

W. DELAAT,ANDC.

L. MUMMERY

The localization of transforming growth factor type /& (TGF-8,) has been followed during preimplantation and early postimplantation murine development using an anti-peptide antibody that specifically recognizes TGF-8,. The staining pattern showed that TGF-0, is expressed from the four-cell stage onward and is differentially regulated as cells diverge to various lineages. High levels of staining were found in the trophectoderm of the blastocyst but no staining was observed in the inner cell mass. During postimplantation development the primitive and embryonic ectoderm also lacked detectable staining while visceral endoderm stained well. Parietal endoderm cells also showed positive staining reaction although to a lesser extent than visceral endoderm cells. These findings were confirmed in model systems of the embryo, namely, embryonal carcinoma and embryonic stem cells differentiated to cells with either visceral or parietal endoderm characteristics. The possible regulatory role of this factor in early embryogenesis is discussed. I~#1991 Academic Press, Inc

INTRODUCTION

Transforming growth factor /3 (TGF-0) is secreted by a variety of neoplastic and nonneoplastic tissues and derives its name from its potency in phenotypically transforming fibroblasts i7~ vitro so that their growth becomes anchorage-independent (Roberts ef al., 1981). Depending on the nature of the target cells, TGF-P inhibits or stimulates cell proliferation (Moses et al, 1985) and induces a variety of other cellular effects in many nonneoplastic tissues (reviewed by Sporn et al., 1987). TGF-/3 appears to exert a complex physiological role in the process of bone formation (Robey et al., 1987), the formation of granulation tissue (Roberts et cd., 1986), and the formation of extracellular matrix (Laiho et al., 1986; Rizzino, 1988). At least five isoforms of TGF-/3 (TGF-&,) have now been described; these isoforms have extensive amino acid homology in the mature region, but differ in their precursor parts (Derynck et ul., 1986; de Martin ef al., 1987; ten Dijke et oh, 1988; Jakowlew et al., 1988; Kondaiah et ul., 1990). Interspecies sequence homology between mature human, porcine, and murine TGF-P, is 100% except for one amino acid in murine TGF-p,. Thus the TGF-Ps have been strongly conserved during evolution and may therefore have common functions in different species. TGF-/3s are secreted by many normal and transformed cell types in a latent form and can be activated by acidification. Mature TGF-P is then dissociated from

i To whom

correspondence

should

be addressed

the precursor and is the active form of TGF-P. Acidification, however, does not seem to be a physiological means of activation except perhaps in bone metabolism; proteolytic processing by specific enzymes has been suggested as being a more likely activation mechanism in Quito (Lyons et al., 1988; Miyazono and Heldin 1989). An important role for the TGF-P family of proteins has been implicated in early embryogenesis. When preimplantation mouse embryos are grown in coculture with normal rat kidney (NRK) fibroblasts in soft agar, the anchorage-dependent NRK cells are induced to form colonies (Rizzino, 1985), suggesting that (cultured) preimplantation embryos secrete transforming growth factor activity. Furthermore, TGF-/3, mRNA has been reported as early as the four to eight cell stage and up to at least the blastocyst stage. The TGF-P, protein product was also present in the blastocyst stage (Rappolee et al., 1988). In postimplantation development TGF-0, mRNA and protein have been detected in mesenchymal tissues such as bone and connective tissue (Heine et al., 198’7; Lehnert and Akhurst, 1988). TGF-& mRNA has been identified in Day 10.5 mouse embryos through postnatal stadia in the mesenchymal part of various tissues (Pelton et al., 1989). Recently, TGF-0, mRNA was demonstrated in Day 10.5-17.5 embryos and in adult tissues (Miller ef al., 1989). A role for TGF-&, has been suggested in murine palatogenesis since their mRNAs have been found to be restricted to specific areas in the palatal shelves (Fitzpatrick et al., 1990). For TGF-& at least, a paracrine function has been suggested as the mRNA was found in epithelia but the protein detected

205

0012-1606/91$3.00 Copyright All rights

‘c’ 1991 by Academic Press. Inc. of reproduction in any form reserved.

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in mesenchymal cells underlying these epithelia (Lehnert and Akhurst, 1988). Interactions of epithelia and mesenchyme may be mediated through the extracellular matrix in which TGF-0, also has been found (Thompson et al., 1989; Flanders et al., 1989). In vitro model systems for embryonic development have also provided evidence for a role for TGF-Ps in development. Embryonal carcinoma (EC) cells (Graham, 1977) and embryo-derived stem (ES) cells (Evans and Kaufman, 1981; Martin, 1981) express TGF-& mRNA (Mummery et al., 1989) but TGF-/I, is only expressed upon differentiation (Mummery et al., 1990). TGF-/3, protein was not detected in either undifferentiated EC cells or the inner cell mass (ICM) of the blastocyst, but trophectoderm in the blastocyst as well as endodermal cell outgrowths of plated blastocysts grown in vitro were positive for TGF-0, staining. Furthermore, differentiated EC cells stained positively for TGF-0,; in both P19 EC cells treated with retinoic acid (RA) and P19 END-2 (Mummery et al., 1985), a cloned derivative of P19 EC cells, resembling endoderm, high levels of TGF-& staining were detected (Mummery et al., 1990). Using a confocal laser scanning microscope (CLSM), we have now followed in detail the localization of TGF& protein throughout the preimplantation and early postimplantation period. Blastocysts plated on tissue culture substrates and grown in vitro, to mimic the early postimplantation period, were also investigated. MATERIALS

AND

METHODS

Embryos Preimplantation embryos were obtained from outbred Swiss 3T3 mice. Embryos were collected from both normal pregnant and superovulated mice in M2 or Dulbecco’s minimal essential medium (DMEM) with 0.5% w/v bovine albumin (BSA). Superovulation was induced by intraperitoneal injection of 5 IU pregnant mare serum (Sigma) followed by 5 IU of human chorionic gonadotrophin (Sigma) 46 hr later. Postimplantation embryos were obtained from Fl crosses between C57BL6 females and CBA males. Taking the midpoint of the dark cycle as t = 0, the conceptuses were dissected from the decidua at 6.4,6.7, and 7.4 days post coitum (PC) in PBS. They were then transferred to the fixative; Reichert’s membrane (RM) and the ectoplacental cone were removed after fixation. Embryo

Culture

Blastocysts collected at Day 3.5 were cultured on feeder layers of primary mouse embryo fibroblasts (MEF), treated with mitomycin C (10 pg/ml, 3 hr), in MEM with 20% heat-inactivated (56°C 30 min) fetal

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calf serum (Integro) immunofluorescence

and 0.1 mMP-mercaptoethanol studies.

for

Immunojluorescence Preimplantation embryos were fixed immediately after collection or after a 3- to 6-day culture period on MEF cells. The zona pellucida was not removed. Fixation was in 2% paraformaldehyde/O.l% glutaraldehyde (Merck) in 0.1 M phosphate buffer, pH 7.4, for 30 min at room temperature (RT). For embryos cultured on MEF cells, fixation was preceded by a 1-hr culture period at 37°C in medium without serum. After washing, fixed embryos were treated for 5 min at RT with a 0.05% solution of sodium borohydride (Merck) in phosphatebuffered saline (PBS) without Ca2+ and Mg2+ and washed again. Cells were permeabilized by incubation for 10 min at RT in 0.1% Triton X-100 in PBS. After washing, specimens were preincubated for 60 min at RT in a 0.5% w/v solution of BSA in DMEM (Hepes buffered, 25 mM). This solution was also used to dilute the first antibody and the second fluorescein isothiocyanate (FITC)-coupled goat anti-rabbit IgG (H + L) antibody 1:50 (Tago). Incubation times were 1 hr at RT; washing steps between each incubation were 5 times for 10 min. Finally the specimens were embedded in a 90/10 mixture of glycerol and PBS containing paraphenylenediamine (Johnson and Nogueira Araujo, 1981). Postimplantation embryos were fixed in the Reichert’s membrane and treated in the same way as preimplantation embryos except as follows: fixation was for 1 hr, sodium borohydride was for 20 min, permeabilization was for 30 min, preincubation and antibody dilutions were in DMEM containing 1% BSA, first antibody incubation was for 7 hr, washing was for 12 hr at 4°C second FITC-coupled antibody incubation was for another 7 hr, and washing was for 12-18 hr at 4°C. Smaller embryos were embedded as described above; larger embryos were dehydrated in a graded ethanol series and mounted in benzyl alcohol:benzyl benzoate 1:2 (Dent et al., 1989). During dehydration and mounting a ZO-25% reduction in size was observed compared to embryos measured shortly after fixation (results not shown). The embryos in Fig. ZD-F and Fig. 8 were mounted following this treatment; they therefore do not represent their size at dissection. Specimens were viewed in a Bio-Rad Lasersharp MRC-500 CLSM and photographed from a photographic line screen (Lucius and Baer) on Kodak PX 125 ASA film. Microscope settings were kept identical when comparing test and control samples. Antibodies We have recently prepared and characterized an antipeptide antiserum specifically recognizing TGF-/3, (van

SLAGERETAL.

TGF-fl, in Mouse

den Eijnden-van Raaij et ah, 1990). In short, this antiserum was raised in rabbits against a synthetic peptide of the first 29 amino acid N-terminal portion of human TGF-0,. The antiserum was then affinity-purified on a column of Reactigel HW65F agarose gel beads (Pierce) to which l-2 mg of the immunizing peptide was coupled by means of an 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (Pierce) bridge according to manufacturer’s instructions. In immunofluorescence experiments the nonbound fraction of the affinity-purified antiserum was used as a control for nonspecific fluorescence. Affinity-purified antibodies were used at a concentration of 20-25 pg/ml, and controls were diluted to the same IgG concentration, determined by an enzyme linked immunosorbent assay (ELISA). Rabbit anti-uvomorulin antibodies were used at 1:60 dilution in the same manner as described above as a control for antibody penetration. Troma-1 antibodies were used at a dilution of 1:300; the secondary FITC conjugate used was goat anti-rat IgG, for cu-uvomorulin (CY-UM) the same FITC conjugate was used as for o(-TGF-&. Both ol-UM and Troma-1 antibodies were kindly provided by Dr. R. Kemler.

RESULTS

ImmunoJluorescent Detection of TGF-/3, in Differentiated ES/EC Cells It was recently shown that TGF-P protein could be localized in preimplantation mouse embryos by immunofluorescence using antibodies raised against a synthetic peptide corresponding to the first 29 amino acids of TGF-0, (Rappolee et al., 1988). Unstained cells were also found in these embryos but no further identification of cell type and developmental stage was made. We were therefore interested to look more systematically at the possible expression of TGF-/3 in early embryos and in EC and ES cells. In view of our previous results on TGF& mRNA expression by EC and ES cells and the availability of antibodies specifically recognizing TGF-fi type 2, we selected this isoform for detailed investigation. EC and ES cells were induced to differentiate by RA either in monolayer or as aggregates as well as cloned EC-derived differentiated cell lines were investigated for the presence of TGF-0,. The cells were divided into two groups: cells with visceral endoderm characteristics such as F9 EC and P19 EC aggregates treated with RA or cloned cells like PSA-5E and P19 END-2 (Mummery et al., 1985) and cells with parietal endoderm characteristics such as F9 EC and ES-5 cells treated in monolayer with either RA and dibutyryl-CAMP or RA alone. In Fig. 1 these cells are shown labeled with the anti-TGF-& antibodies. In general, cells with visceral endoderm

Lkwloptno~f

207

characteristics stained well but cells resembling parieta1 endoderm stained weakly. Immunofluorescent Detection of TGF-/3, in Preimplantation Embryos Antibody penetration. A principal advantage of confocal laser microscopy is that large fluorescently labeled specimens can be examined by optical sectioning (White et ab, 1987; Shotton, 1989). Serial Z-axis sections are digitally stored and than used for reconstruction (alternatively termed “projection”) and stereo imaging. However, in large specimens antibody penetration may be a limiting factor. In order to rule out penetration artifacts with anti-TGF-P, antibodies, anti-uvomorulin (UM) antibodies were used to label the embryos. Uvomorulin is a cell adhesion molecule (Vestweber and Kemler, 1984) expressed in preimplantation embryos and in all cells of the embryonic ectoderm. In the protocol used if permeabilization with Triton X-100 was insufficient, imperfect penetration by cr-UM antibodies (also rabbit IgGs) would be expected. However, staining of UM could be detected both in the blastocyst and in the postimplantation embryos (Fig. 2). Z-sectioning revealed the presence of UM in endoderm and ectoderm up to at least the mid streak/late streak stage before the amniotic cavity is closed. TGF/3, localization. Embryos from the two-cell to blastocyst stages were tested with the affinity-purified antibodies for the presence of TGF-P,. As shown in Figs. 3A and 3B, two-cell embryos did not stain above controls, while four-cell embryos clearly did. At this point, varying degrees of fluorescent intensity between individual blastomeres and individual embryos were observed, suggesting possible regulation in the cell cycle. The exact time at which the protein first became detectable at the four-cell stage was not determined. It seems likely that staining is the result of the onset of major embryonic genome activation during the G2 phase of the second cell cycle. As development progressed, staining continued through to morula stage embryos as shown in Fig. 3C. The cells on the outside of the embryo tended to stain more intensively than cells positioned inside. This became more apparent when blastocysts were investigated. Trophectoderm cells stained intensively but the ICM cells were devoid of staining during the period of blastocoel formation (Figs. 3D, 3E). Trophoblast formation is the first differentiation step during development. Nonstaining trophoblast cells were also observed. In all the cells the nucleus remained unstained, whereas the cytoplasm generally stained uniformly. Also, brightly fluorescing areas adjacent to the nucleus reminiscent of the Golgi apparatus could be observed (data not shown). Fluorescence localized in the ICM cells facing the blas-

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FIG, 1. Immunofluorescent detection of TGF-/3, in differentiated EC/ES cells. (A, B) P19 END-2. (C, D) PSA-5E. (E, F) F9 EC aggregate treated with 10e6 M RA for 7 days. (G, H) P19 EC aggregate treated with 10m7 M RA for 6 days. (I, J) F9 EC monolayer treated with lo-? M and 1 mM dibutyryl-CAMP for 6 days. (K, L) ES-5 cells in monolayer treated with 10m7 M RA for 6 days. Culture medium was a 1:l mixture of DMEM and Ham’s F12 medium with 7.5% FCS for A-H. For ES cells, Buffalo rat liver cell-conditioned medium with 20% FCS and 10e4 M 2-mercaptoethanol was used. (A-H) Cells with visceral endoderm morphology. (I-L) Cells with parietal endoderm morphology; parietal endoderm cells stain more weakly than visceral endoderm cells with anti-TGF-8,. Photographs represent one optical section by CLSM. Scale bar in H, 100 pm (for E-H). Scale bar in L = 50 pm (for A-D and I-L). The first member of a pair shown is taken with Nomarski interference optics, the second is viewed with fluorescence.

tocoelic cavity was observed in some embryos (data not shown). Primitive endoderm cells are formed in this area between Day 4.5 and Day 5.5 (Gardner, 1981,1985). Therefore it would be reasonable to assume that this

TGF-& staining indicates the presence of primitive endoderm cells. However, in the majority of blastocysts examined we could not detect TGF-&-positive primitive endoderm.

209

FIG. 2. Immunofluorescent detection of uvomorulin in embryos. (A) blastocyst, Nomarski interference optics. (B) One optical section taken approximately through the middle of the embryo. ((21 Projection of serial sections. (D) Day 7.5 pc embryo, Nomarski interference optics. (E) One optical sections taken approximately through the middle of the embryo. (F) projection of serial sections. Scale bar for A-C, 20 em for D-F. 75 pm.

Iwmuw.&orescent Blastocysts

Detection

of’ TGF-P,

in Pluted

Soon after blastocoel formation and hatching the embryo implants in the uterine wall. When collected before

implantation and grown in vitro blastocysts continue to grow in a fashion similar to normal development (Sherman, 1975, review; Robertson and Bradley, 1986; Robertson, 198’7). A prominent feature of these plated blastocysts is the formation of endodermal cells. They can be

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FIG. 3. Immunofuorescent detection of TGF-P2 in preimplantation midline of the embryo, is shown. On the left side: anti-TGF-@, antibodies, H) morula; (D, I) early blastocyst; (E, J) late hlastocyst. Phase-contrast bar is 10 pm.

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embryos by CLSM. One optical section, taken approximately at the on the right: controls. (A, F) Two-cell stage; (B, G) four-cell stage; (C, images are shown on the right-hand side of fluorescence images. Scale

recognized by their initially small round appearance derm could be identified. In Fig. 5 three of these plated and by the endoderm marker Troma-1 (Kemler et aZ., blastocysts are shown, stained with anti-TGF-/3, anti1981; Fig. 4). In general, 4-6 days after plating, endo- bodies. We observed similar staining in cultured blasto-

211

FIG. 4. Immunofluorescent labeling of Troma-l-positive cells on plated of focus taken at the top of the embryo. Scale bar = 50 pm.

blastocyst.

cysts previously (Mummery et ab, 1990). Several days after plating, staining can still be seen in endodermal cells but only weak staining is observed in outgrowing trophectoderm cells. Trophectoderm cells apparently lose their TGF-& during culture in vitro. The underlying ICM cells, analogous to the embryonic ectoderm, again do not stain (Figs. 5A, 5B). Before extensive endoderm outgrowth is apparent, high levels of staining on and between the ICM-derived cells can be observed that may be associated with the extracellular matrix (Fig. 5C); a similar staining has been described recently for TGF-0, in mouse neonatal and adult tissues (Flanders et al., 1989; Thompson et ah, 1989).

p) axis of the embryo, the posterior side being the side of the streak. The position of the initiating primitive streak and early mesoderm is extremely difficult to identify (-6.5 days), consequently we could not recognize the a-p axis at this and earlier stages. By the time the streak has extended halfway and further toward the distal tip of the egg cylinder (-6.7 days and later) the orientation of the a-p axis is readily identifiable. The Fl litters collected showed normal variations in size and, as far as could be judged, in developmental stage. In total, 74 embryos from 6.4, 6.7, and 7.5 days pc were labeled with the anti-TGF-/3, antibodies, divided over nine experiments. Most of these embryos were collected at Day 6.4 and Day 6.7 pc. The immunofluorescence pattern of TGF-8, in embryos without obvious mesoderm formation was heterogeneous but reproducible (Figs. 6,7). Shared phenomena were lack of localization in the embryonic ectoderm and extraembryonic ectoderm in all embryos investigated, but visceral embryonic endoderm and visceral extraembryonic endoderm stained strongly. In general visceral embryonic endoderm staining was more prominent than visceral extraembryonic endoderm staining. At higher magnification fluorescence could be seen around the cell nucleus, in agreement with the fluorescence pattern in preimplantation embryos. In Figs. 6A and 6B one of the smallest embryos recovered is shown. The proamniotic cavity is barely visible and extraembryonic ectoderm is only slightly extended distally. This embryo is equivalent to 5.5 days of development (Theiler, 1972). The endoderm stains moderately but significantly with the anti-TGF-& antibodies. The embryo in Figs. 6C and 6D

Immunojluorescent PostCmplantation

Detection Embryos

of TGF-p,

in

At Day 6.5 pc, the embryo consists of embryonic and extraembryonic ectoderm enveloped by visceral embryonic and visceral extraembryonic endoderm, respectively. The proximal part of the embryo forms the ectoplacental cone, originating from polar trophectoderm. At the ectoplacental cone, the primitive endoderm cells have migrated onto the mural trophoblast to become parietal endoderm (PE) which forms Reichert’s membrane. At Day 6.5 pc, the primitive streak arises locally at the junction of embryonic and extraembryonic ectoderm and extends distally (Hashimoto and Nakatsuji, 1989). The mesoderm is formed at the streak and migrates anteriorly and laterally between the embryonic ectoderm and endoderm (Nakatsuji et al, 1986). The formation of the streak defines the anterior-posterior (a-

(A) Phase-contrast.

(B) One CLSM

section

with

high depth

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VOLUME 145.1991

is a little larger and shows a moderately labeled endoderm with a few strongly positive endoderm cells. Such strong positive staining is also shown in Fig. 7 but instead of just a few cells, staining is localized over one whole region. A similar pattern was observed in a minority of the embryos recorded. When older (7.5 days PC.) embryos were examined, the whole endoderm was stained as a continuous layer with equal staining intensity in all cells (Fig. 8). An embryo stained with control IgGs is shown as a control. Isolated RM (from 7.5 day pc embryos) was also examined as shown in Fig. 9. Strongly fluorescing cells resembling trophoblast giant cells could be identified. PE cells could also be identified (arrow in Fig. 9A) but they stained only weakly. At the proximal part of the embryos in Fig. 6 some strongly fluorescing cells are observed which are most likely remnants of the RM and/ or the ectoplacental cone. Trophectoderm stained directly after collection in vivo therefore does not give the same staining pattern as trophectoderm cultured in vitro (Fig. 5). DISCUSSION

detection of TGF-/& in plated blastoFIG. 5. Immunofluorescent cysts. Three CLSM optical sections. (A) Low magnification image (with high depth of focus) of strongly staining embryo, trophectoderm loosing expression, 5 days after plating. (B) Positive staining in endoderm outgrowth, remaining inner cell mass cells not staining, 6 days after plating. (C) Extracellular matrix-like fluorescence around inner cell mass-derived cells, 3 days after plating. Arrowheads indicate

Following previous experiments on expression and localization of TGF-& in ES/EC cells and in early embryos (Mummery et al., 1990) we now report the localization pattern of TGF-0, throughout the preimplantation period and in early postimplantation embryos. As a model for early postimplantation embryogenesis, plated blastocysts grown in vitro until the formation of endoderm were also studied. The anti-peptide antibody used recognized TGF-& but not TGF-0, protein (van den Eijnden-van Raaij et al, 1990). Control experiments using anti-uvomorulin antibodies excluded the possibility of penetration artifacts in multiple cell layers (Fig. 1). The diagram in Fig. 10 represents a schematic overview of TGF-& staining during early development and is used as a guideline for the discussion below. TGF-& protein becomes expressed after embryonic genome activation in the G2 phase of the second cell cycle, with the first detectable protein in the four-cell embryo. Expression could be cell cycle-dependent since variations in staining intensity between individual cells in an embryo are observed. In two-cell embryos, however, staining above background is never observed. Earlier, TGF-0, mRNA and protein have been shown to become detectable first at the four- to eight-cell stage (Rappolee et al, 1988). Although detected at the same time, TGF-0, and TGF-/3, are not necessarily regulated in the same way. As development progresses expression trophectoderm growth. Scale

cells; dashed line indicates the border bar in A is 100 Km; in B, C 50 Wm.

of ICM

out-

213

FIG. 6. Immunofluorescent detection sections. Staining is only in endoderm photographs. Scale bar = 50 urn.

of TGF-0, in early postimplantation embryos, Day 6.4 pc. (B, D) Linear projection of a Z-series of optical and remnants of Reichert’s membrane. On the left-hand side: (A, C) Nomarski interference optics

continues into the morula stage. Again, variability in staining intensity between individual cells is observed. In some embryos it is confined to the outer cells leaving inside cells completely unstained. In the blastocyst differential expression is more obvious; ICM cells do not stain at all while trophectoderm clearly does. The inner cells of the morula contribute substantially to the ICM (Balakier and Pedersen, 1982 and references therein). Our results indicate that these cells lose TGF-P, staining when they become ICM cells, the pluripotent stem cells that will form the embryo proper. This lack of staining continues until endoderm is formed. Endoderm formation by plated blastocysts in vitro was accompanied by strong anti-TGF-0, staining 4-6 days after plat-

ing. Endodermal outgrowths in this stage in vitro have not been very well characterized, so we could not establish whether primitive, visceral, or parietal endoderm was stained. However, with marker studies we have identified Troma-l-positive cells on these cultured blastocysts, indicating that fully differentiated endoderm is present (Fig. 4). In intact postimplantation embryos, however, more information on each endoderm population could be obtained since the different endoderm types can be unequivocally identified on the basis of their position. Prestreak embryos showed TGF-&-positive visceral endoderm cells with a broad spectrum of staining patterns; single, strongly positive endoderm cells could be identified, as well as localized groups or

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FIG. ‘7. Immunofluorescent detection of TGF-0, in early postimplantation embryo, Day 6.7 pc. (A) Nomarski interference optics. (B) Linear projection of a Z-series of optical sections. (C) Serial Z-series of optical sections through the embryo. Scale bar is 50 km.

regions of cells (Figs. 6 and 7). Staining intensities, as in preimplantation embryos, also varied between individual embryos but were always significantly above the controls. Despite the different staining patterns, in none of these embryos were there morphological signs of primitive streak or mesoderm formation. Based on this and on the size of the embryos they were designated as prestreak embryos. Larger embryos, with mesoderm,

clearly showed a uniformly stained TGF-&-positive endoderm layer, both in the embryonal and in the extraembryonal regions (Fig. 8). In all postimplantation embryos examined, the embryonic and extraembryonic ectoderm were negative for TGF-0, staining. Isolated Reichert’s membrane had both TGF-&-positive and TGF-&negative cells. Based on morphology and size, the positive cells resembled trophectoderm cells and

FIG.

through optical

8. Immunofluorescent detection of TGF-6, in Day 7.5 pc embryo. (A) Nomarski the embryo. (C) Linear projection of optical sections. (D) Control embryo, sections made at the same instrument settings as C. Scale bar 100 pm.

negative cells resembled parietal endoderm cells. TGF& staining in EC/ES cell systems after differentiation into cells with PE-like characteristics revealed low levels of TGF-0, protein. In the same studies, cells with visceral endoderm characteristics stained well (Fig. 1). Thus results from in vitro model systems are in agreement with TGF-0, localization in different cell layers of the embryo itself, with the exception of the trophectoderm of plated blastocysts. These trophectoderm cells do not stain, while at the equivalent stage in the embryo trophectoderm cells contain TGF-& protein. This may represent a difference in the balance between uptake, synthesis, and secretion between in viva and ilz vitro. During culture of blastocysts, extracellular fluorescence is observed in the ICM in some cases even before extensive endoderm formation is apparent (Fig. 5C).

interference Normarski

optics. (B) Serial Z-series of optical sections interference optics. (E) Linear projection of

The fluorescence observed might represent either latent and/or active TGF-& as only neutral fixatives were used but as yet we have no means of discriminating between the two. The observed perinuclear fluorescence might be antibody reactivity with precursor protein in the Golgi apparatus. It is interesting therefore that TGF-P has been described as being associated with extracellular matrix proteins in a number of cases. TGF-/3 can bind to fibronectin (Fava and McClure, 1987, Mooradian et ul., 1989); the type III receptor for TGF-P is a membrane bound heparan/chondroitin sulfate proteoglycan (Segarini and Seyedin, 1988; Cheifetz and Massague, 1989) and the TGF-8 1,3, and 4 precursors contain an RGD sequence, known for its specific binding to receptors of the integrin family. Regulation of epithelialmesenchymal cell interactions through the extracellu-

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FIG. 9. Immunofluorescent denotes PE cells. (B) Single cells. Scale bar = 100 km.

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detection of TGF-8, in isolated Reichert’s membrane optical section with high depth of focus. Some maternal

lar matrix might be a mechanism by which TGF-P exerts its effect(s) (Flanders et ah, 1989; Thompson et al., 1989). In the postimplantation embryo, however, we were not able to identify possible extracellular TGF-& due to limitations in optical resolution. The function of TGF-P, in early embryogenesis can at this moment only be speculated upon. The EC/ES cells do not have, or have in low numbers, receptors for TGF/3 and their growth rate is not affected by TGF-0s. Differentiated EC cells, however, display high affinity receptors and are growth inhibited by TGF-/3s (Mummery et al., 1990; Mummery and van den Eijnden-van Raaij, 1990; Rizzino, 1987). Besides a lack of effect of TGF-0s on

of 7.5-day embryo. (A) Nomarski interference tissue may also be present. Arrow denotes

optics, arrow trophectoderm

the growth rate of undifferentiated EC/ES cells no other information is available on possible alterations of gene expression under the influence of TGF-fls in the undifferentiated cells. However, in differentiated F9 EC, resembling PE cells, it is known that TGF-0, influences proliferation, morphology, and laminin production, indicating regulation of processes in early development (Kelly and Rizzino, 1989). Also, in differentiated clones of P19 EC, P19 END-2, P19 MES-1, and P19 EPI-7 the mRNA levels of plasminogen activator inhibitor are transiently increased after 4 hr of TGF-/3, treatment. Leukemia inhibitory factor mRNA expression was also affected by TGF-& treatment of these cells (Mummery

FIG. 10. Schematic diagram of TGF-& staining during early development. Shading intensities correspond to levels of TGF-& staining observed in embryos and EC/ES cell model systems. Trophectodermderived giant cells and ectoplacental cone are not represented explicitly in the bottom tier of the diagram as they were not examined in detail. Extraembryonic ectoderm will form chorionic ectoderm. The time scale of the diagram is up to 7.5 days pc of development.

and van den Eijnden-van Raaij, 1990). Furthermore, TGF-P, has been shown to enhance embryonic development of preimplantation embryos in culture (Paria and Dey, 1990). Only recently, TGF-& transcripts have been found in preimplantation embryos by reverse transcription polymerase chain reaction techniques as well as in differentiated F9 EC cells (Kelly et al, 1990). Considering the specific localization patterns of TGF-0, in this study, a regulatory role for TGF-0, in early development would seem likely. Possibly, and in analogy with the Xenopus ectoderm explant model system, TGF-/I, may have a role in mesoderm formation in mammalian development (Rosa et al., 1988). Current experiments with TGF-P, on in vitro model systems of embryogenesis should supply us with more information on the role of TGF-/3, in early development. This work was initiated in collaboration with Drs. A. J. Verkleij and J. Boonstra, Department of Molecular Cell Biology, University of Utrccht, The Netherlands. We thank W. Hage for instruction on the CLSM and Dr. A. Rizzino for sharing information prior to publication. REFERENCES BALAKIER, H., and PEDERSEN, R. A. (1982). Allocation of cells to inner cell mass and trophectoderm lineages in preimplantation mouse embryos. Dep. Bid. 90, 352-362. CHEIFETZ, S., and MASSAGUE, J. (1989). Transforming growth factor /j (TGFB) receptor proteoglycan. J. Bid. Chem. 264, 12,025-12,028. DENT, J. A., POLSON, A. G., and KLYMKOWSKY, M. W. (1989). A whole mount immunocytochemical analysis of the expression of the intermediate filament protein vimentin in Xerq~,~ Lkoc~lopn/cllf 105, 61-74. DERYNCK, R., JARRET, J. A., CHEN, E. Y., and GOEDDEL, D. V. (1986). The murine transforming growth factor @precursor. J. Biol. (‘hem. 261,4377-4379. DIJKE TEN, P., HANSEN, P., IWATA, K. K.. PIELER, C., and FOULKES,

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