Cell adhesion and gap junction formation in the early mouse embryo are induced prematurely by 6-DMAP in the absence of E-cadherin phosphorylation

During the preimplantation development of the mouse embryo, two phenotypically distinct cell populations appear at the 16-cell stage: non-polarized inner cells and polarized outer cells. These two populations give rise to the inner cell mass (mainly at the origin of the embryo proper) and the trophectoderm (the origin of extra-embryonic tissues), respectively (Fleming, 1987; Rossant, 1986). The first cellular event leading to the divergence between these two cell populations takes place at the 8-cell stage and is called compaction (for reviews see: Gueth-Hallonet and Maro, 1992; Johnson and Maro, 1986). During compaction, blastomeres flatten upon each other, due to the cell adhesion molecule E-cadherin, also known as uvomorulin (Hyafil et al., 1980; Vestweber and Kemler, 1985), and many cellular components are positionally reorganized. A cytoplasmic pole of endosomes (Maro et al., 1985; Reeve and Ziomek, 1981), microfilaments (Johnson and Maro, 1984) and microtubules (Houliston et al., 1987) forms in the apical domain of the blastomeres, and functional gap junctions appear in the basal domain (Goodall and Johnson, 1984; Lo and Gilula, 1979; McLachlin et al., 1983). The microvilli become restricted to the apical pole at the cell surface (Reeve and Ziomek, 1981) and tight junctions form (Ducibella et al., 1975; Fleming et al., 1989; Magnuson et al., 1977). Thus, at the end of the 8-cell stage, components of the blastomeres that were distributed homogeneously throughout the cytoplasm or on the cell surface are polarized with the axis of polarity being orthogonal to the zone of intercellular contacts (Johnson and Ziomek, 1981b; Ziomek and Johnson, 1980). During the fourth cleavage, asymmetric divisions take place and, due to processes of differential inheritance, give rise to the two phenotypically different cell populations (Houliston et al., 1989; Johnson and Ziomek, 1981a,b; Pickering et al., 1988). Natural compaction refers to both processes of intercellular flattening and polarization (cytoplasmic and surface).

The molecular mechanisms underlying compaction remain unknown. Since compaction can occur and can even be advanced in embryos cultured in the presence of protein synthesis inhibitors, it has been suggested that compaction may be controlled by releasing an inhibitory mechanism involving post-translational modification(s) of pre-existing protein(s) (Levy et al., 1986). Specific changes in the level of phosphorylation of certain proteins have been observed during compaction, whereas expression of the proteins themselves was not modified (Bloom, 1991; Bloom and McConnell, 1990). One such protein, the cell adhesion molecule E-cadherin, is phosphorylated just before compaction (Sefton et al., 1992).

One of the putative kinases involved in compaction is protein kinase C (PKC). However, the reported effects of PKC activation on compaction are contradictory. In some cases, the phorbol ester myristate acetate, which activates PKC, has been shown to induce the loss of cytoplasmic and surface polarity, and the loss of intercellular flattening, in compacted embryos. Myristate acetate was also shown to inhibit the appearance of these cellular changes during the 8-cell stage (Bloom, 1989). In other reports, the physiological activator of PKC, diacylglycerol (DAG), and phorbol esters at low concentrations, were shown to cause premature flattening at the 4-cell stage (Winkel et al., 1990).

In this study, we have investigated the effect of 6-dimethylaminopurine (6-DMAP), a Ser-Thr kinase inhibitor (Néant and Guerrier, 1988; Rebhun et al., 1973) on the process of compaction. We report that this drug accelerates compaction at the 8-cell stage and induces premature cell flattening and gap junction formation at the 4-cell stage. These latter processes are dependent upon E-cadherin function, but apparently not upon its phosphorylation.

Swiss female mice (10- to 12-week-old; Animalerie Spécialisée de Villejuif, Centre National de la Recherche Scientifique, France) were superovulated by intraperitoneal injection of 6.5 i.u. of pregnant mare’s serum gonadotrophin (PMSG, Intervet) and human chorionic gonadotrophin (hCG, Intervet) 48 hours apart. Females were paired overnight with Swiss males and checked for vaginal plugs 10 to 15 hours later. In these conditions, fertilization is considered to occur 12 hours post-hCG. Two-cell stage embryos were recovered by flushing from the oviduct 48 hours post-hCG. Embryos were recovered and manipulated in medium 2 (Fulton and Whittingham, 1978) containing 4 mg/ml bovine serum albumin (M2+BSA), at 37°C. Embryos were cultured in medium T6 containing BSA (T6+BSA; Howlett et al., 1987), under paraffin oil at 37°C with 5% CO₂in air in plastic dishes (Falcon).

Late 2-cell or 4-cell stage embryos were examined every hour for cell division and their passage to the following stage (i.e. 4 or 8, respectively). They were then cultured either in control T6 medium, or in T6 medium containing 6-DMAP (Sigma) at various concentrations. The intensity of flattening between the blastomeres within each embryo was scored hourly during the next 9 hours using a binocular microscope (Leica). Three different categories have been considered: (i) if all blastomeres were rounded and clearly distinguishable, the embryo was non-flattened and was given a score of zero; (ii) if individual blastomeres were still visible but difficult to distinguish, the embryo was semi-flattened and was given a score of one; (iii) if individual blastomeres were indistinguishable, the embryo was completely flattened and was given a score of two.

The flattening index was obtained by dividing the total score by twice the number of embryos, multiplied by 100.

Zonae pellucidae were removed by brief incubation in acid Tyrode’s solution (Nicolson et al., 1975). Newly formed 4-cell embryos were placed in Ca²⁺-free M2 containing 6 mg/ml BSA for 15-25 minutes, during which time they were disaggregated to single blastomeres using a flame-polished micropipette. Isolated cells were cultured further in individual microdrops of T6+BSA. Half of the single cells were then labeled during a 3 minute incubation in M2 containing 4 mg/ml polyvinylpyrrolidone (PVP) and 4 μg/ml 6-carboxyfluorescein diacetate (CFDA; stock solution ×250 in acetone; Sigma) as described previously (Goodall and Johnson, 1982). Aggregation was performed by bringing two isolated blastomeres adjacent to one another (one labeled and one non-labeled) in M2+BSA containing 50 μg/ml phytohaemagglutinin (Sigma) on a layer of 1% gelose in a Petri dish. To prevent the embryos from sticking to the dishes during both the staining and the pairing steps, we used Falcon dishes coated with 4 mg/ml BSA and dried. After a 10 minute incubation in the presence of phyto- haemagglutinin, the pairs were washed and incubated further for 5 hours either in T6 control medium, or in T6 containing 0.6 mM 6-DMAP. They were then placed on a glass slide in a drop of medium under oil and examined using a Leitz Diaplan epifluorescence microscope.

Zona-free embryos were rinsed in T6 containing 4 mg/ml PVP (T6+ PVP) and placed in specially designed chambers containing T6+PVP, as described by Maro et al. (1984). The chambers were coated with 0.1 mg/ml concanavalin A in phosphate-buffered saline (PBS) before use. The chambers containing samples were centrifuged at 450 gfor 10 minutes at 37°C.

To assess cell surface polarity, the cells were decompacted by incu- bating the embryos in Ca²⁺-depleted medium for 10 minutes so as to localize better the surface microvilli. They were then fixed rapidly with 1% paraformaldehyde in PBS for 2 minutes followed by a 2 minute incubation in 0.7 mg/ml FITC-labeled concanavalin A (ConA- FITC) at room temperature and three washes in M2+BSA. They were then further fixed with 3.7% formaldehyde (BDH) in PBS for 30 minutes at 37°C and post-permeabilized with 0.25% Triton X-100 in PBS for 10 minutes.

For E-cadherin staining, embryos were either fixed in 3.7% formaldehyde for 30 minutes at room temperature and then perme- abilized in 0.25% Triton X-100 for 10 minutes or first permeabilized for 5 minutes with 0.25% Triton X-100 in PHEM buffer (60 mM PIPES, 25 mM HEPES, 10 mM EGTA, 2 mM MgCl₂, 0.6 μM taxol) and then fixed as described above. All samples were then quenched with 50 mM NH₄Cl in PBS for 10 minutes. Immunological staining was performed using anti-E-cadherin antibody (Peyrieras, 1984) at a dilution of 1/150, followed by anti-rabbit antibody labeled with fluo- rescein (KPL, 1/50).

For connexin43 staining, embryos were fixed in 3% paraformalde- hyde in PBS for 10 minutes at room temperature and then permeabi- lized in 0.1% Triton X-100 for 30 minutes. Immunological staining was performed using anti-connexin43 antibody (El Aoumari et al., 1990) at a dilution of 1/20 followed by fluorescein-labeled anti-rabbit antibody (KPL, 1/50).

Coverslips were removed from the chambers and the specimens were mounted in Citifluor (City University, London). They were examined with a BioRad MRC 600 Confocal Laser Microscope, mounted on an Optiphot II Nikon microscope (60× objective Nikon Plan Apo; NA, 1.4), using an argon ion laser adjusted at 488 nm wave- length to visualize the fluorescein. The signal was averaged using a Kalman filter on eight images. Micrographs were recorded using Kodak T-Max film on a VM 1710 Lucius and Baer, black and white high-resolution monitor.

For transmission electron microscopy, the embryos were placed in chambers (see above) and fixed in 2% glutaraldehyde in 100 mM cacodylate buffer for 30 minutes and postfixed on ice with 0.5% OsO₄in cacodylate buffer for 10 minutes. They were then stained en bloc with 2% aqueous uranyl acetate before dehydration. Extracted embryos were first dipped quickly in 0.5% Triton X-100 in PHEM buffer before a pre- fixation step with 0.3% Triton X-100 in the same buffer for 10 minutes. After a further extraction with 0.3% Triton X-100 for 30 minutes, the embryos were fixed with 2% glutaraldehyde and 0.2% tannic acid in a phosphate buffer, postfixed on ice with 0.5% OsO₄for 10 minutes and stained en bloc with uranyl acetate. Both kinds of samples were then embedded in Epon in capsule beam and sectioned on a Reichert ultra- microtome before observation under a Philips EM410 at 80 kV.

The procedure used was modified from that of Houliston et al. (1989). Briefly, clean glass coverslips were coated with poly-L-lysine (Sigma type 1B, 1 mg/ml fresh solution) for 15 minutes and washed two or three times in cacodylate buffer (0.1 M, pH 7.4) before being placed in wells of a Limbro 24-well tissue culture dish containing cacody- late buffer. Cells were fixed in 3% glutaraldehyde (Sigma) in cacody- late buffer containing 2% sucrose for 1 hour at room temperature and then transferred to the center of freshly prepared coverslips. Samples were dehydrated through a graded series of ethanol (30 minutes each in 20%, 40%, 60%, overnight in 70%, 30 minutes each in 80%, 90%, 95% and 100%) and then dried from 100% ethanol via CO₂in a Polaron E3000 critical-point drying apparatus. Coverslips were mounted on stubs with silver glue and coated with a 50 nm layer of gold in a Polaron E5000 Diode sputtering system. Finally, embryos were examined in a ISM-35CF Jeol microscope under 20 kV.

Embryos were washed three times in a small drop of M2 with 4 mg/ml polyvinylpyrrolidone, mixed with the same volume of double-strength loading buffer (83 mM Tris-HCl, pH 6.8, 2.7% SDS, 6% 2-β-mer- captoethanol, 13% glycerol and 0.001% Bromophenol Blue) and boiled for 2 minutes (Laemmli, 1970). Proteins were separated using a 4% polyacrylamide gel of 0.75 mm (run at 200 V for 1 hour) on a Microgel apparatus (Bio-Rad). Proteins were transferred elec- trophoretically (Bio-Rad) onto nitrocellulose. E-cadherin was detected using the anti-E-cadherin serum (1/200; Peyrieras, 1984) by overnight incubation at 4°C, followed by a second layer of anti-rabbit antibody linked to peroxidase (Amersham, 1/300). PBS buffers con- taining 3% milk powder and 0.1% Tween-20 were used for both incu- bations and washes. The peroxidase activity was detected using the ECL Western Blotting Detection System (Amersham) according to the manufacturer’s instructions.

Groups of embryos at selected stages were washed twice in phosphate-free T6+BSA medium and then incubated in 25 μl drops of phosphate-free T6+BSA containing 1 mCi/ml of [³²P]orthophos- phate (PBS13, Amersham) for 2 hours and washed three times in phosphate-free T6+PVP. They were then harvested in 10 μl of loading buffer. For immunoprecipitations, the labeled embryos were recovered in 10 μl of the same medium and lysed by adding 20 μl RIPA (20 mM Tris, pH 8, 1 mM EDTA, 0.15 M NaCl, 1% NP40, 1% deoxycholate, 0.1% SDS) containing protease inhibitors (1 mM phenylmethanesulfonyl fluoride, 0.1 μg/ml aprotinin, 0.1 μg/ml leupeptin and 0.1 μg/ml pepstatin). The lysate was spun to pellet the insoluble material and the recovered supernatant was incubated with 100 μl StaphylococcusA cells (Immuno-Precipitin, Gibco-BRL) that had been boiled previously for two periods of 30 minutes each in PBS containing 10% β-mercaptoethanol and 3% SDS, washed twice with RIPA after spinning and recovered in RIPA (v/v). Non-specific immunoprecipitation was reduced by an incubation for 1 hour at 0°C without antibody, followed by centrifugation. The supernatant was then incubated with the anti-E-cadherin serum (1/75; Peyrieras, 1984) overnight at 0°C before the addition of 40 μl of the prepared 50% sus- pension StaphylococcusA cells for a further 30 minute incubation at 0°C. The pellet was washed seven times with 1 ml RIPA and the immunoprecipitated proteins were removed from the last pellet by boiling it in 20 μl Laemmli sample buffer for 10 minutes. The super- natants were then stored at −20°C before SDS-PAGE electrophoresis performed according to Laemmli (1970). Acrylamide gels (7.5%) were fixed, treated for 20 minutes with Amplify (Amersham), dried and exposed to β-Max film (Amersham) in film cassettes containing intensifying screens for 10 days at −80°C.

The monoclonal antibody ECCD-1 (ascites fluid; Yoshida-Noro et al., 1984) directed against E-type cadherin was diluted 1/50 in T6+BSA. The antibody is active in preventing flattening at dilutions in excess of 1/200 (Johnson et al., 1986). A stock solution of 1 mg/ml cyto- chalasin D (CCD; Sigma) in dimethylsulphoxide, stored at −20°C, was diluted in T6+BSA to a final concentration of 0.5 μg/ml. A stock solution of 12 mM 6-dimethylaminopurine (6-DMAP; Sigma) in T6+PVP was prepared before each experiment.

At compaction, flattening normally took 6-7 hours after entry to the 8-cell stage and the flattening index reached 90%, since almost all embryos had become completely flattened (Fig. 1). In the presence of 6-DMAP, flattening was accelerated and took about 3-4 hours at the lower dose used (0.3 mM). At higher doses (0.6 and 1.2 mM), corresponding to the doses used in various systems for kinase inhibition, complete cell flattening occurred after only 2 hours of culture.

6-DMAP was also able to induce premature flattening of 4- cell stage embryos (Fig. 2); however, a clear effect was observed only with concentrations between 0.6 and 1.2 mM (Fig. 3). At these higher doses, flattening proceeded over a 5 hour period and gave rise mainly to semi-flattened embryos (80% semi-flattened and 20% fully flattened).

For further investigations, 4-cell stage embryos were cultured for 5 hours, immediately after the 2- to 4-cell stage transition, with 0.6 mM 6-DMAP, the lowest concentration at which the maximum effect on flattening was observed. This effect took place during a single interphase, since maximal flat- tening had occurred in less than 5 hours after entry to the 4- or 8-cell stage, whereas the third and fourth cell cycles last at least 10 hours (Smith and Johnson, 1985). Under these conditions, the drug-induced flattening effect was reversed completely within 30 minutes after transfer into normal medium. Further- more, embryos left in this dose of 6-DMAP showed only a slight delay in the rate of development, suggesting that this treatment did not interfere with subsequent cytokinesis and karyokinesis (data not shown).

To examine whether the 6-DMAP-induced intercellular flat- tening was dependent upon E-cadherin, the calcium-dependent adhesion molecule involved in compaction (Hyafil et al., 1980; Vestweber and Kemler, 1985), we performed the following experiments.

First, we tested the dependence of the 6-DMAP-induced cell flattening upon the presence of Ca²⁺in the medium. We found that 4-cell stage embryos cultured in the presence of 6-DMAP did not flatten in the absence of extracellular Ca²⁺(Table 1). Furthermore, at the 8-cell stage, 6-DMAP-treated and control embryos did not compact in the absence of Ca²⁺(data not shown).

Second, we cultured 4-cell stage embryos in the presence of both 6-DMAP and ECCD-1, an anti-E-cadherin antibody able to inhibit compaction in culture (Johnson et al., 1986; Yoshida- Noro et al., 1984). This led to a complete inhibition of the 6- DMAP-induced intercellular flattening (Table 1).

Third, we observed that like the normal process of com- paction (Pratt et al., 1982), 6-DMAP-induced intercellular flat- tening was also dependent upon an intact microfilament network, since it was inhibited by cytochalasin D (Table 1).

Finally, 6-DMAP induced a redistribution of E-cadherin at the 4-cell stage, as observed with confocal microscopy after immunostaining with an anti-E-cadherin antibody (Peyrieras, 1984). In control embryos, E-cadherin was distributed evenly on all the surface membranes (Fig. 4a). In 6-DMAP treated embryos, E-cadherin staining was more intense and restricted to the membranes involved in intercellular contacts (Fig. 4b), as it is in compacted 8-cell embryos (Johnson et al., 1986). When the embryos were extracted with detergent before fixation, the staining pattern observed differed, since the staining was restricted to the intercellular membranes in both control and 6-DMAP-treated embryos. However, under these conditions, the staining after the 6-DMAP treatment was more intense than in controls (Fig. 4c, d). This suggests that the state of E-cadherin present on the apical and baso-lateral membranes may be different from that at the surface, even before redistribution of that latter induced by 6-DMAP (see Discussion).

In control compacted 8-cell embryos, differentiated junctions can be observed, including gap junctions and intermediate junctions (Fig. 5d). An ultrastructural study of the morpho- logical changes taking place in 6-DMAP-induced flattened 4- cell embryos was performed. In 6-DMAP-treated embryos, a set of intercellular junctions was observed along apposed membranes between the blastomeres (Fig. 5b, c), whereas such complexes were not found in control 4-cell stage embryos (Fig. 5a). These differentiating junctions were het- erogeneous, sometimes similar to gap junctions (Fig. 5c) and sometimes more like intermediate junctions (maculaadherens, Fig. 5b). Frequently, they were underlined by cytoplasmic densities.

The characterization of the proteins in these junctions was carried out by immunofluorescence using an antibody directed against connexin43, a subunit of gap junctions (anti-CX43). Optical sections of compacted 8-cell embryos showed multiple discrete reactive foci along the baso-lateral membranes of the blastomeres (Fig. 6a), whereas in control 4-cell stage embryos, no specific membrane labeling could be detected (Fig. 6b). By contrast, in 6-DMAP-treated embryos we observed the appear- ance of some large foci in areas of cell-cell adhesion (Fig. 6c). Thus, 6-DMAP seems able to induce the premature assembly of connexons in baso-lateral membranes of 4-cell stage embryos.

The existence of intercellular coupling in 6-DMAP-treated embryos was tested by studying the transfer of carboxyfluo- rescein-diacetate (CFDA) in reconstituted pairs of blastomeres, where one of the cells was prelabeled with CFDA (Goodall and Johnson, 1982). In reconstituted pairs of 8-cell blas- tomeres, dye transfer was observed in 81% of the pairs (Table 2), whereas non-treated 4-cell blastomeres showed virtually no dye transfer (1%; Table 2and Fig. 7a, a′). In contrast, 12% of the 6-DMAP-treated 4-cell blastomeres did transfer dye (Table 2and Fig. 7b, b′, c, c′). Thus, it can be concluded that treatment with 6-DMAP induces the precocious assembly of a small number of functional gap junctions between blastomeres of 4- cell stage embryos.

Both cell flattening and cell polarization take place during natural compaction. They are not only concomitant, but also dependent upon the same cellular mechanisms, involving the cytoskeleton and intercellular contacts (for review, see Gueth- Hallonet and Maro, 1992). We tested whether 6-DMAP was able to induce premature surface polarization, as judged by concanavalin A staining and scanning electron microscopy. In both 4-cell control and 6-DMAP-treated embryos, the staining was weak and homogeneous, whereas it was intense and restricted to the apical surface of control compacted 8-cell embryos (Fig. 8a-c). This was confirmed at the scanning electron microscope level, where an even distribution of microvilli was observed (Fig. 8d, e) in both control and 6- DMAP-treated embryos. The microvilli in 6-DMAP-treated embryos were clumped, suggesting that they were very adhesive (Fig. 8e, g). Thus, treatment with 6-DMAP has no effect on the blastomere surface polarization.

As described previously (Rime et al., 1989), we confirmed that 6-DMAP did not inhibit protein synthesis in early mouse embryos (data not shown). Moreover, [³²P]orthophosphate incorporations into total proteins of treated 4-cell stage embryos labeled for 2 hours at either the end (Fig. 9a) or the beginning (Fig. 9b) of the 6-DMAP treatment showed that, at concentrations up to 1.2 mM, this drug had no effect on global phosphorylation. The same result was obtained with pulses of 30 minute labeling (data not shown)

It has been shown recently that E-cadherin is phosphorylated at the entry into the 8-cell stage (Sefton et al., 1992). This temporal correlation with the onset of cellular flattening suggested that this phosphorylation event may be essential for E-cadherin function. Since we have shown that the effect of 6- DMAP is mediated by E-cadherin, we analyzed the state of phosphorylation of E-cadherin in premature flattened 4-cell embryos treated with 6-DMAP. The fact that 6-DMAP is a kinase inhibitor does not exclude the possibility of the induction of some phosphorylation events, through indirect phosphoryla- tion/dephosphorylation cascades. To perform this study, we used a polyclonal anti-E-cadherin antibody (Peyrieras, 1984) that specifically recognizes E-cadherin as a 120 kDa band after immunoblotting in normal 8-cell stage embryos (Fig. 10a, lane 1) as well as in 4-cell stage embryos (lane 3). Thus, this antibody recognizes both the phosphorylated and the non-phos- phorylated states of the E-cadherin. The presence of a band of the same intensity with 8-cell control and 4-cell stage treated with 6-DMAP (Fig. 10a, lane 2) shows that: (i) 6-DMAP does not interfere with the recognition of the antigen by the antibody; and (ii) the total amount of E-cadherin is not affected by the treatment with this drug. Therefore, we performed immunopre- cipitations of E-cadherin with this antibody after [³²P]orthophosphate labeling of embryos. First, we confirmed that E-cadherin is phosphorylated in control embryos at the 8- cell stage (Fig. 10band c, lanes 3) and that it is not phospho- rylated in control embryo at the 4-cell stage (Fig. 10band c, lanes 1). The presence of several other bands that co-precipi- tate with E-cadherin has been observed already with the same antibody (Sefton et al., 1992). Second, at the 4-cell stage, after labeling during the 2 last hours (Fig. 10b, lane 2) or during the first 2 hours (Fig. 10c, lane 2) of the 5 hour 6-DMAP treatment period, no phosphorylation of E-cadherin could be detected within the [³²P]proteins immunoprecipitated. The same result was obtained even if the [³²P]orthophosphate was present 1 hour before the 2 hour 6-DMAP treatment (data not shown).

We report that 6-DMAP at low concentration (0.6 mM) accel- erates normal flattening at the 8-cell stage and can induce premature flattening of 4-cell mouse embryos. 6-DMAP- induced flattening mimics natural compaction to some extent, since treated embryos have the appearance of normally compacted embryos, with obvious flattening of blastomeres and disappearance of cell outlines. Furthermore, differentiated and functional junctions are set up in the areas of cell-cell adhesion, similar to those observed during normal compaction (Lo and Gilula, 1979; Reima, 1990). The junctions are thus most likely involved in the intense intercellular adhesion taking place following 6-DMAP treatment.

Flattening and polarization occur concomitantly during the 8-cell stage and seem to be controlled by both intercellular contacts and reorganization of the cytoskeleton (Gueth- Hallonet and Maro, 1992). However, 6-DMAP treatment does not induce cell surface polarization, as judged by the persis- tence of a uniform spread of microvilli over the apical surface. Previous observations have shown that the surface polarization can be accomplished by two alternative routes, one of which is independent of cellular flattening (Houliston et al., 1989). Our result confirm that flattening and polarization events can occur independently.

It may be suggested that 6-DMAP induces flattening of embryos through its effects upon the Ca²⁺-dependent cell adhesion molecule E-cadherin, since we observed that the effect of 6-DMAP could be inhibited either by an anti-E- cadherin antibody or by the absence of extracellular Ca²⁺. Fur- thermore, treatment of 4-cell stage embryos with the drug induces a redistribution of E-cadherin in the contact areas. At the 4-cell stage, E-cadherin is detected over all surface membranes of directly fixed embryos. In contrast, E-cadherin is present only in the cell-cell contact areas when equivalent embryos are extracted with a non-ionic detergent before fixation. This suggests that E-cadherin in the plasma membrane exists in a less extractable form when it is involved in inter- cellular contacts. It has been observed in different epithelial cell lines that E-cadherin is linked to catenins, cytoplasmic proteins that are also anchored to the cytoplasmic actin network (Kemler and Ozawa, 1990; Ozawa et al., 1989, 1990). In addition, E-cadherin belongs to a membrane multi-protein complex that also contains fodrin, ankyrin and the Na⁺,K⁺- ATPase (Nelson and Veshnock, 1987). The formation of this latter complex seems to be induced by cell contacts (Nelson et al., 1990). These data support the hypothesis that in mouse embryos at the 4-cell stage, only E-cadherin present in the surface membranes involved in intercellular contacts, is anchored to the membrane and cytoplasmic cytoskeleton, and is thus resistant to detergent extraction. In this model, 6-DMAP would be responsible for the recruitment of E-cadherin from the apical membranes to the baso-lateral membranes and its stabilization by anchorage. However, E-cadherin staining observed in the baso-lateral membranes of 6-DMAP-treated 4- cell stage embryos is homogeneous, and does not therefore mimic completely the redistribution observed during normal compaction when, at the end of the 8-cell stage, E-cadherin staining becomes more intense in the membranes close to the surface of the embryo (Reima, 1990; Vestweber et al., 1987).

We have observed that 6-DMAP (0.6 mM) is able to induce the early formation of functional gap junctions in some coupled pairs of 4-cell stage blastomeres. The incidence of such coupled pairs is low but significant. This can be explained in three ways: (i) connexin43, the only connexin yet demon- strated to be involved in junctional communication at com- paction, only begins to accumulate in the embryo at the 4-cell stage (De Sousa et al., 1993; Valdimarsson et al., 1991); (ii) the isolated blastomeres are paired by simple apposition and, even 5 hours later, intercellular contacts are not as extensive as in a whole embryo; (iii) both blastomeres must be ‘competent’ to establish functional gap junctions. Statistically, this suggests that 6-DMAP induces this competence in 35% of individual 4-cell blastomeres (the probability of getting a pair of competent cells is equal to the square of the probability of getting one competent blastomere: 0.12 = 0.35×0.35).

It has been shown in various systems that gap junction formation is dependent upon cadherin-dependent cell adhesion (Bryant et al., 1988; Jongen et al., 1991; Mahoney et al., 1991; Mege et al., 1988; Musil et al., 1990). Since connexin43 is present in the mouse embryo before the 8-cell stage (De Sousa et al., 1993; Valdimarsson et al., 1991), an E-cadherin- dependent cell interaction can trigger the formation of func- tional gap junctions. It is also possible that junctional commu- nication within the embryo is controlled through direct phosphorylation of connexin43 on serine residues, as observed in 3T3 cells (Lau et al., 1992). A causal link between the setting-up of gap junctions and the polarization of blastomeres during normal compaction has been suggested, because of their temporal association (Lo, 1982; Lo and Gilula, 1979). Indeed, certain studies have established on essential role for gap junctions in the transmission of information between blas- tomeres during development (Warner, 1985). However, it has also been shown that polarity can appear even if the intercel- lular coupling is blocked and that polarity can be perturbed without communication through gap junctions (Goodall, 1986; Goodall and Maro, 1986). Our results confirm that the premature appearance of gap junctions does not induce premature polarization.

Induction of premature compaction in 4-cell stage embryos has been obtained by PKC activation (Winkel et al., 1990). Changes in phosphoprotein profiles during compaction have also been observed (Bloom and McConnell, 1990). This suggests that phosphorylations play a key role in the regula- tion of compaction. The fact that inhibition of phosphorylation by 6-DMAP provokes effects that are similar to PKC-directed phosphorylation could appear paradoxical. However, it can be explained by an indirect effect through phosphorylation/ dephosphorylation cascades. Firstly, E-cadherin is phosphory- lated at the beginning of the 8-cell stage (Sefton et al., 1992) and it was suggested that this phosphorylation is involved in the general process of compaction. Our data show that E- cadherin is not phosphorylated prematurely by 6-DMAP at the 4-cell stage, suggesting that phosphorylation is not required for its specific function in cell flattening and junction formation during compaction. It is still possible that E-cadherin phos- phorylation is involved in some other features of compaction, such as cell polarization. Secondly, treatment of 4-cell stage embryos by 6-DMAP at doses up to 1.2 mM does not signifi- cantly affect the global cellular phosphorylation events, and the specific target(s) of 6-DMAP action are not detectable by one- dimensional gel electrophoresis. Thus, they most likely corre- spond to minor phosphoprotein(s) turning over rapidly.

Although other mechanisms have not been reported, we cannot completely exclude the possibility of an action of 6- DMAP that is independent of its effect on phosphorylation. However: (1) 6-DMAP is a well known inhibitor of the mitotic kinase, p34^cdc2(Jessus et al., 1991). Experiments performed in our laboratory showed that, in vitro, 6-DMAP inhibits histone kinase activity (p34^cdc2) in metaphase-II-arrested oocytes (Szöllösi et al., 1993) and MAP kinase activity in metaphase- II-arrested and activated oocytes (Verlhac et al., 1993). (2) In compacted mouse embryos, during mitosis, blastomeres round up and gap junctions are switched off (Goodall and Maro, 1986). The observed effects of 6-DMAP at the 4-cell stage, induction of cell flattening and setting-up of gap junctions, suggests that a kinase with a specificity similar to the mitotic kinase may be controlling compaction negatively. In addition, okadaic acid, a potent phosphatase inhibitor, inhibits flattening of 8-cell stage embryos and induces their rapid decompaction when they are already flattened, confirming the implication of phosphorylation events in this phenomenon. If 4-cell stage embryos treated by 6-DMAP are transferred to medium con- taining okadaic acid, we observe rapid reversibility of the flat- tening (data not shown). Similarly, 4-cell embryos transferred to control medium round up, return to normal behavior and go on to form a normal blastocyst without delay. These results confirm that the steady state of phosphorylation of the putative target of 6-DMAP is less stable than during the natural phe- nomenon of compaction. We are currently performing analysis by two-dimensional gel electrophoresis in order to identify this target.

We thank Angelica Santa-Maria for her expert technical assistance with the SEM; Jean-Claude Courvalin, Marie-Anne Félix and Nicola Winston for critical reading of the manuscript; Richard Schwartzmann and Gérard Géraud for photographic work. This work was supported by grants from the Institut National pour la Santé et la Recherche Médicale, the Ligue Nationale contre le Cancer, the Association pour la Recherche contre le Cancer and the Fondation pour la Recherche Médicale to B.M. C.G.-H. was the recipient of a fellowship from the Association pour la Recherche contre le Cancer.