The processes governing differential protein expression in preimplantation lineages were investigated using a monoclonal antibody recognising the tight junction polypeptide, ZO-1. ZO-1 localises to the maturing tight junction membrane domain in the polarised trophectoderm lineage from compaction (8-cell stage) onwards, ultimately forming a zonular belt around each trophectoderm cell of the blastocyst (32- to 64-cell stage). The protein is usually undetectable within the inner cell mass (ICM) although, in a minority of embryos, punctate ZO-1 sites are present on the surface of one or more ICM cells. Since ICM cells derive from the differentiative division of polarised 8- and 16-cell blastomeres, the distribution of ZO-1 following differentiative division in isolated, synchronised cell clusters of varying size, was examined. In contrast to the apical cytocortical pole, ZO-1 was found to be inherited by nonpolar (prospective ICM) as well as polar (prospective trophectoderm) daughter cells. Following division, polar cells adhere to and gradually envelop nonpolar cells. Prior to envelopment, ZO-1 localises to the boundary between the contact area and free membrane of daughter cells, irrespective of their phenotype. After envelopment, polar cells retain these ZO-1 contact sites whilst nonpolar cells lose them, in which case ZO-1 transiently appears as randomly-distributed punctate sites on the membrane before disappearing. Thus, symmetrical cell contact appears to initiate ZO-1 down-regulation in the ICM lineage. The biosynthetic level at which ZO-1 down-regulation occurs was investigated in immunosurgically isolated ICMs undergoing trophectoderm regeneration. By 6 h in culture, isolated ICMs generated a zonular network of ZO-1 at the contact area between outer cells, thereby demonstrating the reversibility of down-regulation. This assembly process was unaffected by alpha-amanitin treatment but was inhibited by cycloheximide. These results indicate that the ICM inherits and stabilises ZO-1 transcripts which can be utilised for rapid synthesis and assembly of the protein, a capacity that may have significance both in maintaining lineage integrity within the blastocyst and in the subsequent development of the ICM.

Two distinct cell populations emerge during early mammalian development. One is a polarised epithelial monolayer (trophectoderm, TE) located on the outer embryo surface, responsible for blastocoele fluid accumulation and the source of the postimplantation trophoblast lineages; the other is an internal non-epithelial cell cluster (inner cell mass, ICM) which will give rise to the future embryonic and remaining extraembryonic tissues. In the mouse, these two preimplantation phenotypes originate from differentiative events that begin at compaction in the 8-cell embryo (some 24 h and two cell cycles before blastocyst formation), when cells become adhesive and polarise along their apical-basal (outer-inner) axis (Lehtonen, 1980; Handyside, 1980; Reeve and Ziomek, 1981). Subsequent division of many polarised blastomeres is perpendicular to this axis such that the resulting outer and inner daughter cells (16-cell stage) inherit different cellular domains and exhibit distinct phenotypes. Outer daughter cells (prospective TE) display the polarised morphology of their parent cell while inner cells (prospective ICM) express a nonpolar cellular organisation (Johnson and Ziomek, 1981; Reeve, 1981; Ziomek and Johnson, 1981: Pickering et al. 1988; Houliston and Maro, 1989; Sutherland et al. 1990). The segregation of these two cell lineages during cleavage is coincident with their progressive structural divergence, with polar cells, from compaction onwards, gradually acquiring the TE epithelial characteristics necessary for blastocyst morphogenesis (Johnson and Ziomek, 1982; Fleming and Pickering, 1985; Maro et al. 1985; Fleming, 1986; Fleming and Goodall, 1986; Chisholm and Houliston, 1987; Watson and Kidder, 1988; Watson et al. 1990). These coordinated events of epithelial biogenesis and cell diversification in the early embryo have been the subject of several recent reviews (Johnson and Maro, 1986; Fleming and Johnson, 1988; Maro et al. 1988; Pratt, 1989; Fleming, 1990; Kimber, 1990; Wiley et al. 1990).

The divergence of preimplantation lineages has been shown to precede their commitment. For example, polar 16-cell blastomeres usually only give rise to TE cells, but can divide differentiatively, as at the 8-cell stage, to yield both TE and ICM progeny (Johnson and Ziomek, 1983), while nonpolar 16-cell blastomeres and early ICM cells can convert directly to a trophectodermal phenotype (Handyside, 1978; Johnson and Ziomek, 1983; Fleming et al. 1984). In both these examples of plasticity, the decision to maintain or alter the developmental pathway is governed by cellular interactions. In the case of polar cells, the orientation of their division plane appears to be influenced by cell shape, in turn modified by contact patterns, while nonpolar cells can eventually polarise and display an epithelial phenotype if they experience, for a prolonged period, an outside position where cell contacts are asymmetric (Johnson and Ziomek, 1983). These regulative characteristics can contribute to a balanced TE:ICM cell population ratio in the expanding blastocyst (Fleming, 1987).

Although the diversification process has been studied in some detail at the cellular level, the mechanisms leading to differential expression of individual molecules has received little attention. Clearly, the generation of distinct structural properties by prospective TE and ICM phenotypes will reflect differences in their underlying molecular composition which should be subject to modification (by, for example, cellular interactions) to accommodate the regulative capacity of the embryo. Indeed, several examples of lineage-specific expression of embryonic determinants have been reported (reviewed in Johnson, 1981; Kimber, 1990) and tissue-specific polypeptide synthesis has been identified at 16-cell (Handyside and Johnson, 1978) and blastocyst (Van Blerkom et al. 1976) stages. For some, but not all, TE-specific polypeptides, new gene expression is required for their synthesis to occur within ICM cells undergoing phenotypic transformation (Johnson, 1979).

In this paper, we examine the pattern of expression of a molecular marker for the tight junction, a structural component limited to the epithelial TE lineage (Ducibella et al. 1975; Magnuson et al. 1977) and necessary for vectorial fluid transport during blastocoele expansion. The marker, a 225×103Mr peripheral membrane phosphoprotein called ZO-1, was originally isolated from liver membrane fractions and has been shown to be present at the cytoplasmic membrane face of tight junctions in a variety of epithelia (Stevenson et al. 1986; Anderson et al. 1987). In a previous study we have shown that ZO-1 is present as a continuous belt circumscribing the apicolateral border of each TE cell in the blastocyst and gradually acquires this distribution pattern in the polar lineage from compaction onwards (Fleming et al. 1989). We consider the mechanisms regulating ZO-1 tissue-specificity in the early embryo by monitoring ZO-1 distribution during polar cell differentiative divisions and in isolated ICMs undergoing TE regeneration in the presence of biosynthetic inhibitors. We conclude that ZO-1, unlike certain structural features of cell polarity, is not differentially segregated at division but is subsequently down-regulated in nonpolar cells by cell interactions that cause nonpolar cells to become completely enclosed. Our results also suggest that down-regulation is limited to the protein level, with mRNA encoding ZO-1 being preserved within the ICM lineage, which we suggest may be significant for the viability and future development of the ICM.

Embryo collection, culture and manipulations

MFI female mice (3–4 week old, Olac-derived, Southampton University Animal House) were superovulated by intraperitoneal injections (5i.u.) of pregnant mares serum (PMS, Folligon, Intervet) and human chorionic gonadotrophin (hCG, Chorulon, Intervet), 46– 48 h apart. Mice were mated overnight with MFI males and checked for copulation plugs the following morning. Embryos were collected by flushing oviducts at 46– 48 h post-hCG (late 2-cell/early 4-cell stage) or at 67– 70 h post-hCG (8-cell stage) into warmed Hepes-buffered Medium 2 containing 4 mg ml-1 bovine serum albumin (M2+BSA, Fulton and Whittingham, 1978) and were cultured in Medium 16 containing 4 mg ml-1 BSA (M16+BSA, Whittingham and Wales, 1969) under paraffin oil at 37°C in 5% CO2 in air in sterile culture dishes. Blastocysts and isolated inner cell masses (ICMs) were cultured in Dulbecco’s Modification of Eagles Medium (DMEM, ICN Flow Labs) containing antibiotics and supplemented with 10% foetal calf serum (FCS, ICN Flow Labs). The zona pellucida of embryos at the appropriate stage was removed by brief incubation (15– 20 s) in acid Tyrode’s solution (Nicolson et al. 1975) and washing in M2+BSA.

For experiments with synchronised cell clusters, single 4-cell (1/4) or 8-cell (1/8) blastomeres were obtained near the end of their cell cycle by incubating zona-free late 4-cell (58– 60 h post-hCG) or compact 8-cell (72 h post-hCG) intact embryos in calcium-free M2 containing 6 mg ml”−1 BSA for 15 min and disaggregating embryos to single cells using a flame-polished micropipette. Isolated blastomeres were cultured in M16+BSA and examined hourly for evidence of division to 2/8 or 2/16 couplets respectively. All manipulations were carried out using a Wild stereomicroscope fitted with a 37°C heated stage. Both sets of newly-formed couplets were cultured individually for 18 h during which time they divided to 4/16 and 4/32 cell clusters respectively. In addition, 2/16 couplets were cultured for varying periods from 0– 8 h post division.

For experiments on isolated ICMs, zona-free early blastocysts (92–96 h post-hCG) were used in the immunosurgery procedure as described previously (Chisholm et al. 1985). ICMs were cultured for up to 6h in DMEM+FCS before fixation and immunocytochemistry. and Pickering, 1985; Maro et al. 1985; Fleming, 1986; Fleming and Goodall, 1986; Chisholm and Houliston, 1987; Watson and Kidder, 1988; Watson et al. 1990). These coordinated events of epithelial biogenesis and cell diversification in the early embryo have been the subject of several recent reviews (Johnson and Maro, 1986; Fleming and Johnson, 1988; Maro et al. 1988; Pratt, 1989; Fleming, 1990; Kimber, 1990; Wiley et al. 1990).

The divergence of preimplantation lineages has been shown to precede their commitment. For example, polar 16-cell blastomeres usually only give rise to TE cells, but can divide differentiatively, as at the 8-cell stage, to yield both TE and ICM progeny (Johnson and Ziomek, 1983), while nonpolar 16-cell blastomeres and early ICM cells can convert directly to a trophectodermal phenotype (Handyside, 1978; Johnson and Ziomek, 1983; Fleming et al. 1984). In both these examples of plasticity, the decision to maintain or alter the developmental pathway is governed by cellular interactions. In the case of polar cells, the orientation of their division plane appears to be influenced by cell shape, in turn modified by contact patterns, while nonpolar cells can eventually polarise and display an epithelial phenotype if they experience, for a prolonged period, an outside position where cell contacts are asymmetric (Johnson and Ziomek, 1983). These regulative characteristics can contribute to a balanced TE:ICM cell population ratio in the expanding blastocyst (Fleming, 1987).

Although the diversification process has been studied in some detail at the cellular level, the mechanisms leading to differential expression of individual molecules has received little attention. Clearly, the generation of distinct structural properties by prospective TE and ICM phenotypes will reflect differences in their underlying molecular composition which should be subject to modification (by, for example, cellular interactions) to accommodate the regulative capacity of the embryo. Indeed, several examples of lineage-specific expression of embryonic determinants have been reported (reviewed in Johnson, 1981; Kimber, 1990) and tissue-specific polypeptide synthesis has been identified at 16-cell (Handyside and Johnson, 1978) and blastocyst (Van Blerkom et al. 1976) stages. For some, but not all, TE-specific polypeptides, new gene expression is required for their synthesis to occur within ICM cells undergoing phenotypic transformation (Johnson, 1979).

In this paper, we examine the pattern of expression of a molecular marker for the tight junction, a structural component limited to the epithelial TE lineage (Ducibella et al. 1975; Magnuson et al. 1977) and necessary for vectorial fluid transport during blastocoele expansion. The marker, a 225×103A7r peripheral membrane phosphoprotein called ZO-1, was originally isolated from liver membrane fractions and has been shown to be present at the cytoplasmic membrane face of tight junctions in a variety of epithelia (Stevenson et al. 1986; Anderson et al. 1987). In a previous study we have shown that ZO-1 is present as a continuous belt circumscribing the apicolateral border of each TE cell in the blastocyst and gradually acquires this distribution pattern in the polar lineage from compaction onwards (Fleming et al. 1989). We consider the mechanisms regulating ZO-1 tissue-specificity in the early embryo by monitoring ZO-1 distribution during polar cell differentiative divisions and in isolated ICMs undergoing TE regeneration in the presence of biosynthetic inhibitors. We conclude that ZO-1, unlike certain structural features of cell polarity, is not differentially segregated at division but is subsequently down-regulated in nonpolar cells by cell interactions that cause nonpolar cells to become completely enclosed. Our results also suggest that down-regulation is limited to the protein level, with mRNA encoding ZO-1 being preserved within the ICM lineage, which we suggest may be significant for the viability and future development of the ICM.

Embryo collection, culture and manipulations

MFI female mice (3-4 week old, Olac-derived, Southampton University Animal House) were superovulated by intraperitoneal injections (5i.u.) of pregnant mares serum (PMS, Folligon, Intervet) and human chorionic gonadotrophin (hCG, Chorulon, Intervet), 46-48 h apart. Mice were mated overnight with MFI males and checked for copulation plugs the following morning. Embryos were collected by flushing oviducts at 46– 48 h post-hCG (late 2-cell/early 4-cell stage) or at 67– 70 h post-hCG (8-cell stage) into warmed Hepes-buffered Medium 2 containing 4 mg ml-1 bovine serum albumin (M2+BSA, Fulton and Whittingham, 1978) and were cultured in Medium 16 containing 4 mg ml-1 BSA (M164-BSA, Whittingham and Wales, 1969) under paraffin oil at 37°C in 5% CO2 in air in sterile culture dishes. Blastocysts and isolated inner cell masses (ICMs) were cultured in Dulbecco’s Modification of Eagles Medium (DMEM, ICN Flow Labs) containing antibiotics and supplemented with 10% foetal calf serum (FCS, ICN Flow Labs). The zona pellucida of embryos at the appropriate stage was removed by brief incubation (15-20 s) in acid Tyrode’s solution (Nicolson et al. 1975) and washing in M2+BSA.

For experiments with synchronised cell clusters, single 4-cell (1/4) or 8-cell (1/8) blastomeres were obtained near the end of their cell cycle by incubating zona-free late 4-cell (58-60 h post-hCG) or compact 8-cell (72 h post-hCG) intact embryos in calcium-free M2 containing 6 mg ml-1 BSA for 15 min and disaggregating embryos to single cells using a flame-polished micropipette. Isolated blastomeres were cultured in M16+BSA and examined hourly for evidence of division to 2/8 or 2/16 couplets respectively. All manipulations were carried out using a Wild stereomicroscope fitted with a 37°C heated stage. Both sets of newly-formed couplets were cultured individually for 18 h during which time they divided to 4/16 and 4/32 cell clusters respectively. In addition, 2/16 couplets were cultured for varying periods from 0-8 h post division.

For experiments on isolated ICMs, zona-free early blastocysts (92–96 h post-hCG) were used in the immunosurgery procedure as described previously (Chisholm et al. 1985). ICMs were cultured for up to 6h in DMEM+FCS before fixation and immunocytochemistry.

Drugs

Early blastocysts were cultured in medium containing alpha-amanitin (Sigma) at 100 μgml-1 to inhibit RNA polymerase II activity (Braude, 1979; Kidder and McLachlin, 1985; Fleming et al. 1989) or cycloheximide (Sigma) at 400μM inhibit protein synthesis (Fleming et al. 1989; TCA-precipitable incorporation of [35S]methionine was reduced to 6 % normal level at this concentration) for 1 h prior to immunosurgery. Drug treatment was maintained during immunosurgery and during subsequent culture of isolated ICMs until fixation.

Immunocytochemistry

Embryos, cell clusters and cultured ICMs were processed for ZO-1 immunofluorescence as described previously (Fleming et al. 1989). Cell clusters at the 16-cell stage were first labelled with rhodamine-conjugated Concanavalin A (Sigma) to detect microvillous polarity as described (Fleming et al. 1989). Specimens were viewed on a Leitz Diaplan microscope using appropriate filters and photographs were taken on Kodak T-max film.

In the early blastocyst (32- to 64-cell stage), ZO-1 protein is detectable as a continuous zonular belt at the apicolateral borders of each cell in the trophectoderm (TE) (Fig. 1A; see also Fleming et al. 1989). In most wholemount blastocysts (52/83, 62%) viewed in mid-sectional plane, ZO-1 sites were confined to the TE junctional zone (Fig. IB), but in a minority (38%), punctate ZO-1 sites were also evident on the surface of one or two ICM cells (Fig. 1C). Previously (Fleming et al. 1989), we have shown that ZO-1 is first detectable immunocytochemically in embryos at the compacting 8-cell stage where it localises as a series of dots to the apicolateral contact region between blastomeres, coincident with the onset of tight junction formation (Ducibella and Anderson, 1975; Ducibella et al. 1975; Magnuson et al. 1977). Since initial ZO-1 expression in the 8-cell embryo precedes the diversification of prospective TE and ICM cell lineages (beginning at the 8- to 16-cell division), the origin of ZO-1 expression in the ICM (where at this stage of development, tight junctions are absent) might result from inheritance rather than local synthesis. We therefore evaluated whether or not ZO-1 protein differentially segregated into the polar (prospective TE) lineage during differentiative division of isolated blastomeres.

Fig. 1.

Wholemount early blastocysts immunolabelled for ZO-1 detection. (A) Blastocyst viewed en face, showing belt-like ZO-1 sites encircling each trophectoderm cell. (B) Blastocyst viewed in mid-sectional plane, showing foci of ZO-1 (arrows) between trophectoderm cells (t) but no staining in the ICM (i) lying above the blastocoele (b). (C) Blastocyst in midsectional plane showing punctate ZO-1 sites on the surface of certain ICM cells (arrow). Bar=10 μm.

Fig. 1.

Wholemount early blastocysts immunolabelled for ZO-1 detection. (A) Blastocyst viewed en face, showing belt-like ZO-1 sites encircling each trophectoderm cell. (B) Blastocyst viewed in mid-sectional plane, showing foci of ZO-1 (arrows) between trophectoderm cells (t) but no staining in the ICM (i) lying above the blastocoele (b). (C) Blastocyst in midsectional plane showing punctate ZO-1 sites on the surface of certain ICM cells (arrow). Bar=10 μm.

ZO-1 localisation in cell clusters

Single 8-cell blastomeres (1/8 cells) were allowed to divide in culture to 2/16 couplets and cultured for varying periods up to 8h before analysis of cell phenotypes present and ZO-1 distribution (Table 1). Using Concanavalin A staining to identify polar cells, on average 76% couplets were polarmonpolar (P:NP) pairs, while 24% were polar:polar (P:P) in phenotype; this ratio is consistent with previous data (Pickering et al. 1988). In most P:NP couplets at Oh and Ih postdivision, ZO-1 was distributed as dots apparently randomly-placed on the membrane of both cells that were usually rounded and not adhering together (Fig. 2A,B; Table 1). At later time points (3–5h postdivision), polar cells flattened against and began enveloping nonpolar cells in P:NP couplets, and ZO-1 became localised to the periphery of the contact zone, firstly in a punctate pattern and then as a continuous line (Fig. 2C-F). In some couplets, a transitional state was evident with both random and contact-associated ZO-1 membrane sites being present (scored as contactlocalised in Table 1). After 8h post-division, most (68 %) P:NP couplets showed complete envelopment of the nonpolar cell by the polar cell such that a peripheral contact zone was no longer present; in these couplets, ZO-1 staining again appeared as randomly-distributed dots mainly at the contact site but also on the polar cell outer membrane (Fig. 2G,H; Table 1). In P:P couplets, cells flattened against each other but envelopment did not take place; here, ZO-1 membrane staining changed from a random to a contact-associated pattern as seen in P:NP couplets, but this was maintained up to 8 h postdivision (Table 1). These results indicate that, unlike the apical microvillous pole, ZO-1 is inherited by both cells during differentiative division of polar 8-cells. Subsequently, ZO.-l distribution is modified according to cell contact patterns. In experiments where 2/16 couplets were cultured in the presence of cycloheximide for up to 5h from the time of division of 1/8 blastomeres (or from prior to division), the ZO-1 distribution pattern in P:NP couplets was similar to that described above, although a delay was evident in the acquisition of contact-localised sites (Table 1). This further indicates that ZO-1 in nonpolar cells results from inheritance rather than de novo synthesis.

Table 1.

ZO-1 distribution in polar.polar (P:P) and polar:non-polar (P:NP) 2/16 couplets at different times postdivision in vitro of 1 /8 blastomeres derived from compact 8-cell embryos. Data from four experiments

ZO-1 distribution in polar.polar (P:P) and polar:non-polar (P:NP) 2/16 couplets at different times postdivision in vitro of 1 /8 blastomeres derived from compact 8-cell embryos. Data from four experiments
ZO-1 distribution in polar.polar (P:P) and polar:non-polar (P:NP) 2/16 couplets at different times postdivision in vitro of 1 /8 blastomeres derived from compact 8-cell embryos. Data from four experiments
Fig. 2.

Polarmonpolar 2/16 couplets at different times postdivision from 1/8 blastomeres; (A,C,E,G) Concanavalin A staining to identify the polar cell (top cell in A,C and E, outer cell in G), (B,D,F,H) ZO-1 staining. (A.B) At lh postdivision, punctate ZO-1 sites are evident at the surface of both polar and nonpolar cells (arrows), at positions bearing no relationship with the contact area. (C,D) At 3h postdivision, ZO-1 is localised to the periphery of the contact area between cells where a linear series of punctate sites are present (arrow). (E,F) At 5h postdivision, ZO-1 at the contact site is now continuous rather than punctate in appearance. (G,H) By 8h postdivision, the polar cell has completely enveloped the nonpolar cell; punctate ZO-1 sites are found throughout the contact area between cells. Bar=10 μm.

Fig. 2.

Polarmonpolar 2/16 couplets at different times postdivision from 1/8 blastomeres; (A,C,E,G) Concanavalin A staining to identify the polar cell (top cell in A,C and E, outer cell in G), (B,D,F,H) ZO-1 staining. (A.B) At lh postdivision, punctate ZO-1 sites are evident at the surface of both polar and nonpolar cells (arrows), at positions bearing no relationship with the contact area. (C,D) At 3h postdivision, ZO-1 is localised to the periphery of the contact area between cells where a linear series of punctate sites are present (arrow). (E,F) At 5h postdivision, ZO-1 at the contact site is now continuous rather than punctate in appearance. (G,H) By 8h postdivision, the polar cell has completely enveloped the nonpolar cell; punctate ZO-1 sites are found throughout the contact area between cells. Bar=10 μm.

To evaluate whether ZO-1 inheritance by nonpolar cells was influenced by the breakdown of putative tight junctions (and hence dispersion of ZO-1) during disaggregation of compact 8-cell embryos into 1/8 blastomeres, the distribution of ZO-1 was also analysed in newly-formed 4/16 cell clusters derived from the division of 2/8 couplets where ZO-1 is known to be localised to the periphery of the contact zone (Fleming et al. 1989). In these experiments, 2/8 couplets were generated from 1/4 blastomeres and cultured for 18 h to allow for division to the 16-cell stage to occur before analysis (Table 2). Three types of 4/16 clusters were observed, depending upon the number of blastomeres dividing differentiatively. The ratio of outer polar:inner nonpolar cells in these clusters was 4:0 (45%), 3:1 (34 %) or 2:2 (20 %), frequencies consistent with earlier data using this mouse strain (Pickering et al. 1988). In all clusters, ZO-1 was present along the cell borders between outer polar cells. In most (85%) clusters containing inner nonpolar cells, ZO-1 was only located between polar cells, while the negative nonpolar cells were completely internalised (Fig. 3A-E). However, in a minority (15 %) of heterogeneous 4/16 clusters, ZO-1 was also present at contact sites involving nonpolar cells (Table 2). In these latter examples, ZO-1 either appeared as randomly-placed dots where the nonpolar cells were fully internalised (as seen previously in 2/16 couplets, Fig. 2H) or was localised to the periphery of the contact zone where the nonpolar cells had not been completely enclosed (Fig. 3F-I). These experiments therefore confirm that ZO-1 can be inherited by both polar and nonpolar 16-cell daughters, irrespective of whether natural cell contacts are maintained in parental 8-cell blastomeres prior to division. They also indicate that the internalisation of nonpolar cells leads to the dispersion and ultimate loss of ZO-1 membrane staining.

Table 2.

ZO-1 distribution in 4/16 and 4/32 cell clusters derived from division in vitro of 2/8 and 2/16 couplets respectively. Data from six experiments

ZO-1 distribution in 4/16 and 4/32 cell clusters derived from division in vitro of 2/8 and 2/16 couplets respectively. Data from six experiments
ZO-1 distribution in 4/16 and 4/32 cell clusters derived from division in vitro of 2/8 and 2/16 couplets respectively. Data from six experiments
Fig. 3.

Heterogeneous 4/16 clusters containing outer polar and inner nonpolar cells; (A,D,F,H) Phase-contrast, (B,C,E,G,I) ZO-1 staining. (A–C) Cluster containing two outer and two inner cells; ZO-1 is restricted to the contact site between outer polar cells (arrows), as shown in tangential (B) and mid-sectional (C) planes. (D,E) Cluster containing three outer cells and one inner cell; ZO-1 is present only at the contact site between outer polar cells, seen in mid-sectional view (E). (F,G) Cluster with three outer cells and one inner nonpolar cell which has not been fully enclosed; ZO-1 is localised to the contact site between polar and nonpolar cells (arrows in G). (H,I) Cluster with two polar and two nonpolar cells, the latter being central in the cluster and not fully enclosed; ZO-1 is present at all contact sites, including the contact between nonpolar cells (arrow in I) Bar=10 μ m.

Fig. 3.

Heterogeneous 4/16 clusters containing outer polar and inner nonpolar cells; (A,D,F,H) Phase-contrast, (B,C,E,G,I) ZO-1 staining. (A–C) Cluster containing two outer and two inner cells; ZO-1 is restricted to the contact site between outer polar cells (arrows), as shown in tangential (B) and mid-sectional (C) planes. (D,E) Cluster containing three outer cells and one inner cell; ZO-1 is present only at the contact site between outer polar cells, seen in mid-sectional view (E). (F,G) Cluster with three outer cells and one inner nonpolar cell which has not been fully enclosed; ZO-1 is localised to the contact site between polar and nonpolar cells (arrows in G). (H,I) Cluster with two polar and two nonpolar cells, the latter being central in the cluster and not fully enclosed; ZO-1 is present at all contact sites, including the contact between nonpolar cells (arrow in I) Bar=10 μ m.

The segregation pattern of ZO-1 during the next cleavage division (16- to 32-cell stage) was analysed in a similar manner to that described above. Single 8-cell blastomeres were disaggregated from compact embryos and allowed to divide in culture to 2/16 couplets. These were cultured for 18 h during which time division to 4/32 cell clusters occurred. The ratio of outer polar:inner nonpolar cells in these clusters was 4:0 (12 %), 3:1 (17%) and 2:2 (71%), figures consistent with earlier data (Johnson and Ziomek, 1983). In these more advanced specimens, nonpolar cells were always fully enclosed within the cluster. In most (approx 90%) heterogeneous clusters ZO-1 staining was restricted to the contact sites linking outer polar cells (Fig. 4A-F), although in remaining specimens randomly placed ZO-1 dots also occurred along membrane sites involving inner nonpolar cells (Fig. 4G,H; Table 2). These results suggest that ZO-1 can also be inherited (and subsequently down-regulated) by nonpolar cells at the 16- to 32-cell transition, presumably by polar cells dividing differentiatively.

Fig. 4.

Heterogeneous 4/32 clusters containing outer polar and inner nonpolar cells; (A,D,G) Phase contrast, (B,C,E,F,H) ZO-1 staining. (A-C) Cluster with two outer and two inner cells; ZO-1 sites are present only at the contact site between outer polar cells (arrows), seen in tangential (B) and mid-sectional (C) planes. (D-F) Cluster with three outer and one inner cell; ZO-1 sites are restricted to outer polar cell contacts (E, tangential, and F, mid-sectional, planes). (G,H) Cavitated cluster in which punctate ZO-1 sites are evident on the surface of an inner nonpolar cell (arrow, H). Bar=10lum.

Fig. 4.

Heterogeneous 4/32 clusters containing outer polar and inner nonpolar cells; (A,D,G) Phase contrast, (B,C,E,F,H) ZO-1 staining. (A-C) Cluster with two outer and two inner cells; ZO-1 sites are present only at the contact site between outer polar cells (arrows), seen in tangential (B) and mid-sectional (C) planes. (D-F) Cluster with three outer and one inner cell; ZO-1 sites are restricted to outer polar cell contacts (E, tangential, and F, mid-sectional, planes). (G,H) Cavitated cluster in which punctate ZO-1 sites are evident on the surface of an inner nonpolar cell (arrow, H). Bar=10lum.

ZO-1 expression in isolated ICMs

In order to investigate in more detail the process of ZO-1 down-regulation within the nonpolar ICM lineage, we made use of the developmental potential of ICMs to regenerate trophectoderm during culture following immunosurgical isolation from early blastocysts (Fleming et al. 1984). In ICMs processed immediately following isolation (Oh time-point), over 50% showed no evidence of ZO-1 immunolabelling, the remainder possessed faint punctate ZO-1 sites at the contact border between two or more adjacent outer cells (Fig. 5A,B; Table 3). After culture for 1-3h, all ICMs showed this discontinuous ZO-1 staining pattern, which was brighter than previously and involved progressively more of the outer cell population (Fig. 5C). From 6h culture, 85 % ICMs had acquired linear ZO-1 sites between outer cells (Fig. 5D; Table 3). The temporal accumulation of ZO-1 specifically at junctional sites between outer ICM cells during trophectoderm regeneration was also analysed in the presence of biosynthetic inhibitors. Treatment with the transcriptional inhibitor alpha-amanitin, administered from 1 h prior to immunosurgery and during culture (0-6 h) of isolated ICMs up until the time of fixation, had no observable effect on the acquisition of linear ZO-1 junctional sites between outer ICM cells (Table 3). However, a similar treatment with the protein synthesis inhibitor cycloheximide blocked the formation of linear ZO-1 sites, although faint discontinuous sites between outer cells were evident in a minority of ICMs (Table 3).

Table 3.

ZO-1 localisation in ICMs isolated immunosurgically from early blastocysts and cultured for various periods in the presence or absence of biosynthetic inhibitors. Data from three experiments

ZO-1 localisation in ICMs isolated immunosurgically from early blastocysts and cultured for various periods in the presence or absence of biosynthetic inhibitors. Data from three experiments
ZO-1 localisation in ICMs isolated immunosurgically from early blastocysts and cultured for various periods in the presence or absence of biosynthetic inhibitors. Data from three experiments
Fig. 5.

ZO-1 immunolabelling of ICMs, isolated from early blastocysts and fixed immediately (A,B) or cultured for 3h (C) or 6h (D) before fixation. (A,B) Punctate ZO-1 sites are present between outer cells in certain regions only (arrows). (C) Punctate (discontinuous) ZO-1 staining (arrow) is present between all outer cells. (D) A belt-like (linear) network of ZO-1 is present between outer cells. Bar=10 μ m.

Fig. 5.

ZO-1 immunolabelling of ICMs, isolated from early blastocysts and fixed immediately (A,B) or cultured for 3h (C) or 6h (D) before fixation. (A,B) Punctate ZO-1 sites are present between outer cells in certain regions only (arrows). (C) Punctate (discontinuous) ZO-1 staining (arrow) is present between all outer cells. (D) A belt-like (linear) network of ZO-1 is present between outer cells. Bar=10 μ m.

In this study, we have investigated the mechanisms regulating lineage-specific expression of the tight junction protein, ZO-1, in the preimplantation embryo. ZO-1 is a prominent component of the junctional complex circumscribing the apicolateral border between trophectoderm cells (Fleming et al. 1989), but is usually undetectable in the ICM, except for occasional cells that show punctate membrane sites in a minority of embryos. One possible explanation for differential expression might be that the protein, first evident at compaction in all cells of the 8-cell embryo prior to tissue diversification, is segregated by inheritance into the polar (trophectoderm) lineage where ZO-1 syn thesis continues, in order to facilitate tight junction maturation and turnover. Segregation of molecular components is known to contribute to Drosophila pattern formation (Ingham, 1988) and, in the present study, segregation might also include ZO-1 mRNA in order to maintain phenotypic specificity in ZO-1 synthesis. Alternatively, differential expression might not be coupled directly with phenotypic divergence, but may be achieved as a consequence of positional differences between lineages that lead to up- or downregulation of specific proteins. Our findings indicate strong support for the latter model.

The analysis of ZO-1 distribution following differentiative divisions at 8- to 16-, and 16- to 32-cell stages in synchronised cell clusters revealed that ZO-1 was inherited by both polar and nonpolar daughter cells. The presence of ZO-1 in nonpolar cells is also supported by its localisation to the contact site between polar and nonpolar cells prior to envelopment, since we have shown previously that the formation of such contacts between earlier-staged blastomeres is dependent upon both cells being competant to express ZO-1 (Fleming et al. 1989). The inheritance of ZO-1 by both cells following differentiative division therefore contrasts with the segregation of the apical microvillous pole into the trophectoderm lineage exclusively (Johnson and Ziomek, 1981). The ZO-1 pattern more closely resembles features of the cytoplasm of polarised blastomeres (cytoskeletal elements, membraneous organelles) that lose their polarised distribution during mitosis and, following differentiative division, are inherited by both cells (Reeve, 1981; Johnson and Maro, 1984; Fleming and Pickering, 1985; Maro et al. 1985; Chisholm and Houliston, 1987; Houliston and Maro, 1989). The basolateral epithelial surface components uvomorulin (Vestweber et al. 1987), vinculin (Lehtonen and Reima, 1986), spectrin (Sobel and Alliegro, 1985), gap junctions (Loand Gilula, 1979) and plakoglobin (Fleming et al. 1991) are similarly all partitioned into both TE and ICM lineages during blastocyst development. We have shown previously that ZO-1 contact sites between polarised 8-cell blastomeres in different conformations also represent specialisations of the basolateral epithelial membrane rather than of the apical-basolateral membrane boundary (Fleming et al. 1989). Thus, it appears that the nonpolar ICM lineage inherits all the cellular features that are epithelial in character except those associated with the apical cytocortex. It is these apical components that have been shown to be instructive in the global reorganisation of cells into an overtly polarised phenotype (Johnson and Maro, 1985; Johnson et al. 1988; Wiley and Obasaju, 1988, 1989).

The contact-associated membrane assembly of ZO-1 by nonpolar cells was found to persist until they became internalised by enveloping polar cells, at which time ZO-1 membrane sites became disorganised into random punctate foci and then disappeared. Thus, position, or more specifically the loss of cell contact asymmetry, appeared to initiate ZO-1 down-regulation. Earlier studies on cultured epithelial cell lines have shown that deprivation of normal cell-cell contacts, either by extracellular calcium depletion or treatment with anti-uvomorulin antibody, inhibited or reversed the association of ZO-1 with the tight junction membrane (Gumbiner et al. 1988; Siliciano and Goodenough, 1988; Anderson et al. 1989). Similar treatments on mouse embryo 8-cell blastomeres also inhibited or reversed the normal contact-localised pattern of ZO-1 assembly (Fleming et al. 1989). Our present result therefore demonstrates that uvomorulin-mediated cell-cell adhesion is not the sole regulator of ZO-1 assembly at the membrane. Rather, this assembly is dependent upon both adhesion and the presence of contact-free membrane. The relevance of the latter factor may be attributable to the molecular character of the, as yet, unidentified ZO-1 membrane binding site which may require the presence of both membrane faces for its stabilisation.

Our experiments on isolated ICMs were designed to identify the biosynthetic level at which down-regulation of ZO-1 within the ICM lineage is accomplished. The formation of a zonular pattern of ZO-1 contact sites between outer cells of ICMs occurred rapidly (by 6 h) during trophectoderm regeneration, well in advance of fluid accumulation (by approx 24 h, Handyside, 1978; Johnson, 1979; Fleming et al. 1984) and the expression of all trophectoderm-specific polypeptides (24 h, Johnson, 1979). Indeed, contact-associated ZO-1 was evident in certain surface regions of some ICMs fixed immediately following isolation, and may reflect either the redistribution of inherited protein or new synthesis of ZO-1 during the period between trophectoderm lysis and ICM isolation (approx 30min). The assembly of a zonular ZO-1 network in isolated ICMs was unaffected by alpha-amanitin treatment, but was inhibited by cycloheximide treatment (Table 3). The simplest interpretation of this result is that ZO-1 expression and assembly takes place in the absence of transcriptional activity but does require protein synthesis. Thus, sufficient mRNA transcripts for ZO-1 assembly would appear to be present within the ICM lineage prior to immunosurgery, indicating that ZO-1 RNA, in addition to ZO-1 protein, is inherited by nonpolar cells during differentiative division. The preservation of ZO-1 mRNA in the ICM is further supported by data from Caco-2 cells showing stable, elevated levels of ZO-1 mRNA, (analysed directly) in conditions where cell-cell contacts are inhibited and ZO-1 translation is minimal (Anderson et al. 1989). However, full confirmation of the presence of functional ZO-1 mRNA in the ICM awaits a direct analysis, which we intend to pursue. The dependence upon protein synthesis for ZO-1 assembly in ICMs suggests that the posttranscriptional regulation of ZO-1 expression is mediated at the translational level. We, therefore, hypothesise that although nonpolar cells preserve inherited ZO-1 mRNA, they do not preserve ZO-1 protein which is down-regulated by natural turnover in the absence of membrane assembly sites (when cell contacts are symmetrical) but which needs to be resynthesised de novo once assembly sites are reestablished (cell contact asymmetry). This model is consistent with the observation that the ZO-1 protein level in Caco-2 cells rapidly declines when cell contacts are inhibited (Anderson et al. 1989). However, we cannot as yet exclude the possibility that ZO-1 protein in a diffuse form, not readily detectable by our immunocytochemical technique, is retained by nonpolar cells and is available for membrane assembly. In this case, to explain our cycloheximide data, the synthesis of undefined protein(s) would be required for ZO-1 assembly to occur. We are currently investigating by quantitative means the level of ZO-1 total protein and synthesis in ICMs compared with trophectoderm, to resolve this issue.

In conclusion, our results indicate that ZO-1 protein required for tight junction assembly in the trophectoderm lineage is, nevertheless, inherited, along with its mRNA, by the ICM lineage. The loss of cell contact asymmetry in the ICM leads to the down-regulation of ZO-1 protein, but the mRNA appears to be stabilised. What might be the significance of message stability? It has been shown previously that totipotent ICM cells must undergo transcription to express the full complement of trophectoderm-specific polypeptides during trophectoderm regeneration (Johnson, 1979). However, in the absence of transcription, certain trophectoderm marker polypeptides can be expressed in the early phase of the regeneration process (Johnson, 1979). This is entirely consistent with our findings, since ZO-1 assembly occurs rapidly following immunosurgery. The capacity of ICMs to engage in rapid and transcriptionally-independent expression of certain key epithelial components might be responsible for the observed flattening and envelopment of outer ICM cells over the ICM core that occurs as an initial event during trophectoderm regeneration (Fleming et al. 1984). This process is necessary to retain contact symmetry between core cells and hence stabilise them from phenotypic transformation. Thus, in the intact embryo, ZO-1 message stability within the ICM might contribute to the continued viability of this lineage independently of protection provided by the trophectoderm. It is noteworthy that in blastocysts in which the polar trophectoderm has been injured deliberately, few if any ICM cells enter the trophectoderm lineage (Dyce et al. 1981). An additional role for ZO-1 message stability within the ICM concerns the next phase in the ICM developmental programme, that of delamination of primary endoderm at the interface with the blastocoele. It will be of interest to establish whether epithelial biogenesis of trophectoderm and primary endoderm tissues are linked to promote kinetic efficiency by the conservation of transcripts encoding common epithelial proteins.

We thank The Wellcome Trust for financial support for this work. Certain preliminary experiments were undertaken at the Department of Anatomy, Cambridge, for which we acknowledge grants from the Medical Research Council and the Cancer Research Campaign to Drs M. H. Johnson and P. R. Braude. The ZO-1 antibody was kindly provided by Dr B. Stevenson, University of Alberta. Our thanks to Charlie McFadden and Barry Lockyer for technical assistance.

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