tissue-specific contro olf expression of the tight ... · but not all, te-specific polypeptides,...

Development 113, 295-304 (1991)Printed in Great Britain © The Company of Biologists Limited 1991

295

Tissue-specific control of expression of the tight junction polypeptide ZO-1

in the mouse early embryo

TOM P. FLEMING1-2* and MARK J. HAY1

^Department of Biology, School of Biological Sciences, University of Southampton, Bassett Crescent East, Southampton SO9 3TU, UK2Department of Anatomy, University of Cambridge, Downing Street, Cambridge CB2 3D Y, UK

* Author for correspondence

Summary

The processes governing differential protein expressionin preimplantation lineages were investigated using amonoclonal antibody recognising the tight junctionpolypeptide, ZO-1. ZO-1 localises to the maturing tightjunction membrane domain in the polarised trophecto-derm lineage from compaction (8-cell stage) onwards,ultimately forming a zonular belt around each troph-ectoderm cell of the blastocyst (32- to 64-cell stage). Theprotein 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 ICMcells. Since ICM cells derive from the differentiativedivision of polarised 8- and 16-cell blastomeres, thedistribution of ZO-1 following differentiative division inisolated, synchronised cell clusters of varying size, wasexamined. In contrast to the apical cytocortical pole,ZO-1 was found to be inherited by nonpolar (prospectiveICM) as well as polar (prospective trophectoderm)daughter cells. Following division, polar cells adhere toand gradually envelop nonpolar cells. Prior to envelop-ment, ZO-1 localises to the boundary between thecontact area and free membrane of daughter cells,irrespective of their phenotype. After envelopment,

polar cells retain these ZO-1 contact sites whilstnonpolar cells lose them, in which case ZO-1 transientlyappears as randomly-distributed punctate sites on themembrane before disappearing. Thus, symmetrical cellcontact appears to initiate ZO-1 down-regulation in theICM lineage. The biosynthetic level at which ZO-1down-regulation occurs was investigated in immunosur-gically isolated ICMs undergoing trophectoderm regen-eration. By 6 h in culture, isolated ICMs generated azonular network of ZO-1 at the contact area betweenouter cells, thereby demonstrating the reversibility ofdown-regulation. This assembly process was unaffectedby alpha-amanitin treatment but was inhibited bycycloheximide. These results indicate that the ICMinherits and stabilises ZO-1 transcripts which can beutilised for rapid synthesis and assembly of the protein, acapacity that may have significance both in maintaininglineage integrity within the blastocyst and in thesubsequent development of the ICM.

Key words: mouse embryo, tight junction, ZO-1,trophectoderm, inner cell mass, cell lineage, cell adhesion.

Introduction

Two distinct cell populations emerge during earlymammalian development. One is a polarised epithelialmonolayer (trophectoderm, TE) located on the outerembryo surface, responsible for blastocoele fluidaccumulation and the source of the postimplantationtrophoblast lineages; the other is an internal non-epithelial cell cluster (inner cell mass, ICM) which willgive rise to the future embryonic and remainingextraembryonic tissues. In the mouse, these twopreimplantation phenotypes originate from differenti-ative events that begin at compaction in the 8-cellembryo (some 24 h and two cell cycles before blastocystformation), when cells become adhesive and polarisealong their apical-basal (outer-inner) axis (Lehtonen,

1980; Handyside, 1980; Reeve and Ziomek, 1981).Subsequent division of many polarised blastomeres isperpendicular to this axis such that the resulting outerand inner daughter cells (16-cell stage) inherit differentcellular domains and exhibit distinct phenotypes. Outerdaughter cells (prospective TE) display the polarisedmorphology of their parent cell while inner cells(prospective ICM) express a nonpolar cellular organisa-tion (Johnson and Ziomek, 1981; Reeve, 1981; Ziomekand Johnson, 1981: Pickering et al. 1988; Houliston andMaro, 1989; Sutherland etal. 1990). The segregation ofthese two cell lineages during cleavage is coincidentwith their progressive structural divergence, with polarcells, from compaction onwards, gradually acquiringthe TE epithelial characteristics necessary for blastocystmorphogenesis (Johnson and Ziomek, 1982; Fleming

296 T. P. Fleming and M. J. Hay

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 andcell diversification in the early embryo have been thesubject 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 beenshown to precede their commitment. For example,polar 16-cell blastomeres usually only give rise to TEcells, but can divide differentiatively, as at the 8-cellstage, to yield both TE and ICM progeny (Johnson andZiomek, 1983), while nonpolar 16-cell blastomeres andearly ICM cells can convert directly to a trophectoder-mal phenotype (Handyside, 1978; Johnson and Zio-mek, 1983; Fleming et al. 1984). In both these examplesof plasticity, the decision to maintain or alter thedevelopmental pathway is governed by cellular interac-tions. In the case of polar cells, the orientation of theirdivision plane appears to be influenced by cell shape, inturn modified by contact patterns, while nonpolar cellscan eventually polarise and display an epithelialphenotype if they experience, for a prolonged period,an outside position where cell contacts are asymmetric(Johnson and Ziomek, 1983). These regulative charac-teristics can contribute to a balanced TE:ICM cellpopulation ratio in the expanding blastocyst (Fleming,1987).

Although the diversification process has been studiedin some detail at the cellular level, the mechanismsleading to differential expression of individual mol-ecules has received little attention. Clearly, thegeneration of distinct structural properties by prospec-tive TE and ICM phenotypes will reflect differences intheir underlying molecular composition which shouldbe subject to modification (by, for example, cellularinteractions) to accommodate the regulative capacity ofthe embryo. Indeed, several examples of lineage-specific expression of embryonic determinants havebeen reported (reviewed in Johnson, 1981; Kimber,1990) and tissue-specific polypeptide synthesis has beenidentified at 16-cell (Handyside and Johnson, 1978) andblastocyst (Van Blerkom et al. 1976) stages. For some,but not all, TE-specific polypeptides, new gene ex-pression is required for their synthesis to occur withinICM cells undergoing phenotypic transformation(Johnson, 1979).

In this paper, we examine the pattern of expression ofa molecular marker for the tight junction, a structuralcomponent limited to the epithelial TE lineage (Duci-bella et al. 1975; Magnuson et al. 1977) and necessaryfor vectorial fluid transport during blastocoele expan-sion. The marker, a 225xlO3Mr peripheral membranephosphoprotein called ZO-1, was originally isolatedfrom liver membrane fractions and has been shown tobe present at the cytoplasmic membrane face of tightjunctions in a variety of epithelia (Stevenson et al. 1986;Anderson et al. 1987). In a previous study we haveshown that ZO-1 is present as a continuous belt

circumscribing the apicolateral border of each TE cellin the blastocyst and gradually acquires this distributionpattern in the polar lineage from compaction onwards(Fleming et al. 1989). We consider the mechanismsregulating ZO-1 tissue-specificity in the early embryoby monitoring ZO-1 distribution during polar celldifferentiative divisions and in isolated ICMs undergo-ing TE regeneration in the presence of biosyntheticinhibitors. We conclude that ZO-1, unlike certainstructural features of cell polarity, is not differentiallysegregated at division but is subsequently down-regulated in nonpolar cells by cell interactions thatcause nonpolar cells to become completely enclosed.Our results also suggest that down-regulation is limitedto the protein level, with mRNA encoding ZO-1 beingpreserved within the ICM lineage, which we suggestmay be significant for the viability and future develop-ment of the ICM.

Materials and methods

Embryo collection, culture and manipulationsMF1 female mice (3-4 week old, Olac-derived, SouthamptonUniversity Animal House) were superovulated by intraperito-neal injections (5i.u.) of pregnant mares serum (PMS,Folligon, Intervet) and human chorionic gonadotrophin(hCG, Chorulon, Intervet), 46-48 h apart. Mice were matedovernight with MF1 males and checked for copulation plugsthe following morning. Embryos were collected by flushingoviducts at 46-48 h post-hCG (late 2-cell/early 4-cell stage) orat 67-70 h post-hCG (8-cell stage) into warmed Hepes-buffered Medium 2 containing 4mgml~1 bovine serumalbumin (M2+BSA, Fulton and Whittingham, 1978) andwere cultured in Medium 16 containing 4mgml~1 BSA(M16+BSA, Whittingham and Wales, 1969) under paraffinoil at 37°C in 5% CO2 in air in sterile culture dishes.Blastocysts and isolated inner cell masses (ICMs) werecultured in Dulbecco's Modification of Eagles Medium(DMEM, ICN Flow Labs) containing antibiotics and sup-plemented with 10% foetal calf serum (FCS, ICN FlowLabs). The zona pellucida of embryos at the appropriate stagewas removed by brief incubation (15-20 s) in acid Tyrode'ssolution (Nicolson et al. 1975) and washing in M2+BSA.

For experiments with synchronised cell clusters, single4-cell (1/4) or 8-cell (1/8) blastomeres were obtained near theend of their cell cycle by incubating zona-free late 4-cell(58-60 h post-hCG) or compact 8-cell (72 h post-hCG) intactembryos in calcium-free M2 containing 6mgml' ' BSA for15 min and disaggregating embryos to single cells using aflame-polished micropipette. Isolated blastomeres were cul-tured in M16+BSA and examined hourly for evidence ofdivision to 2/8 or 2/16 couplets respectively. All manipu-lations were carried out using a Wild stereomicroscope fittedwith a 37°C heated stage. Both sets of newly-formed coupletswere cultured individually for 18 h during which time theydivided to 4/16 and 4/32 cell clusters respectively. In addition,2/16 couplets were cultured for varying periods from 0-8 hpost division.

For experiments on isolated ICMs, zona-free early blasto-cysts (92-96 h post-hCG) were used in the immunosurgeryprocedure as described previously (Chisholm et al. 1985).ICMs were cultured for up to 6 h in DMEM+FCS beforefixation 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 andcell diversification in the early embryo have been thesubject 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 beenshown to precede their commitment. For example,polar 16-cell blastomeres usually only give rise to TEcells, but can divide differentiatively, as at the 8-cellstage, to yield both TE and ICM progeny (Johnson andZiomek, 1983), while nonpolar 16-cell blastomeres andearly ICM cells can convert directly to a trophectoder-mal phenotype (Handyside, 1978; Johnson and Zio-mek, 1983; Fleming et al. 1984). In both these examplesof plasticity, the decision to maintain or alter thedevelopmental pathway is governed by cellular interac-tions. In the case of polar cells, the orientation of theirdivision plane appears to be influenced by cell shape, inturn modified by contact patterns, while nonpolar cellscan eventually polarise and display an epithelialphenotype if they experience, for a prolonged period,an outside position where cell contacts are asymmetric(Johnson and Ziomek, 1983). These regulative charac-teristics can contribute to a balanced TE:ICM cellpopulation ratio in the expanding blastocyst (Fleming,1987).

Although the diversification process has been studiedin some detail at the cellular level, the mechanismsleading to differential expression of individual mol-ecules has received little attention. Clearly, thegeneration of distinct structural properties by prospec-tive TE and ICM phenotypes will reflect differences intheir underlying molecular composition which shouldbe subject to modification (by, for example, cellularinteractions) to accommodate the regulative capacity ofthe embryo. Indeed, several examples of lineage-specific expression of embryonic determinants havebeen reported (reviewed in Johnson, 1981; Kimber,1990) and tissue-specific polypeptide synthesis has beenidentified at 16-cell (Handyside and Johnson, 1978) andblastocyst (Van Blerkom et al. 1976) stages. For some,but not all, TE-specific polypeptides, new gene ex-pression is required for their synthesis to occur withinICM cells undergoing phenotypic transformation(Johnson, 1979).

In this paper, we examine the pattern of expression ofa molecular marker for the tight junction, a structuralcomponent limited to the epithelial TE lineage (Duci-bella et al. 1975; Magnuson et al. 1977) and necessaryfor vectorial fluid transport during blastocoele expan-sion. The marker, a 225xlO3Mr peripheral membranephosphoprotein called ZO-1, was originally isolatedfrom liver membrane fractions and has been shown tobe present at the cytoplasmic membrane face of tightjunctions in a variety of epithelia (Stevenson et al. 1986;Anderson et al. 1987). In a previous study we haveshown that ZO-1 is present as a continuous belt

circumscribing the apicolateral border of each TE cellin the blastocyst and gradually acquires this distributionpattern in the polar lineage from compaction onwards(Fleming et al. 1989). We consider the mechanismsregulating ZO-1 tissue-specificity in the early embryoby monitoring ZO-1 distribution during polar celldifferentiative divisions and in isolated ICMs undergo-ing TE regeneration in the presence of biosyntheticinhibitors. We conclude that ZO-1, unlike certainstructural features of cell polarity, is not differentiallysegregated at division but is subsequently down-regulated in nonpolar cells by cell interactions thatcause nonpolar cells to become completely enclosed.Our results also suggest that down-regulation is limitedto the protein level, with mRNA encoding ZO-1 beingpreserved within the ICM lineage, which we suggestmay be significant for the viability and future develop-ment of the ICM.

Materials and methods

Embryo collection, culture and manipulationsMF1 female mice (3-4 week old, Olac-derived, SouthamptonUniversity Animal House) were superovulated by intraperito-neal injections (5i.u.) of pregnant mares serum (PMS,Folligon, Intervet) and human chorionic gonadotrophin(hCG, Chorulon, Intervet), 46-48 h apart. Mice were matedovernight with MF1 males and checked for copulation plugsthe following morning. Embryos were collected by flushingoviducts at 46-48 h post-hCG (late 2-cell/early 4-cell stage) orat 67-70 h post-hCG (8-cell stage) into warmed Hepes-buffered Medium 2 containing 4mgml~1 bovine serumalbumin (M2+BSA, Fulton and Whittingham, 1978) andwere cultured in Medium 16 containing 4mgml~1 BSA(M16+BSA, Whittingham and Wales, 1969) under paraffinoil at 37°C in 5% CO2 in air in sterile culture dishes.Blastocysts and isolated inner cell masses (ICMs) werecultured in Dulbecco's Modification of Eagles Medium(DMEM, ICN Flow Labs) containing antibiotics and sup-plemented with 10% foetal calf serum (FCS, ICN FlowLabs). The zona pellucida of embryos at the appropriate stagewas removed by brief incubation (15-20 s) in acid Tyrode'ssolution (Nicolson et al. 1975) and washing in M2+BSA.

For experiments with synchronised cell clusters, single4-cell (1/4) or 8-cell (1/8) blastomeres were obtained near theend of their cell cycle by incubating zona-free late 4-cell(58-60 h post-hCG) or compact 8-cell (72 h post-hCG) intactembryos in calcium-free M2 containing 6mgml~1 BSA for15min and disaggregating embryos to single cells using aflame-polished micropipette. Isolated blastomeres were cul-tured in M16+BSA and examined hourly for evidence ofdivision to 2/8 or 2/16 couplets respectively. All manipu-lations were carried out using a Wild stereomicroscope fittedwith a 37°C heated stage. Both sets of newly-formed coupletswere cultured individually for 18 h during which time theydivided to 4/16 and 4/32 cell clusters respectively. In addition,2/16 couplets were cultured for varying periods from 0-8 hpost division.

For experiments on isolated ICMs, zona-free early blasto-cysts (92-96 h post-hCG) were used in the immunosurgeryprocedure as described previously (Chisholm et al. 1985).ICMs were cultured for up to 6 h in DMEM+FCS beforefixation and immunocytochemistry.

DrugsEarly blastocysts were cultured in medium containing alpha-amanitin (Sigma) at 100 f.ig ml"1 to inhibit RNA polymerase IIactivity (Braude, 1979; Kidder and McLachlin, 1985; Fleminget al. 1989) or cycloheximide (Sigma) at 400 ,UM to inhibitprotein synthesis (Fleming et al. 1989; TCA-precipitableincorporation of [35S]methionine was reduced to 6 % normallevel at this concentration) for lh prior to immunosurgery.Drug treatment was maintained during immunosurgery andduring subsequent culture of isolated ICMs until fixation.

Immunocytochemistry.Embryos, cell clusters and cultured ICMs were processed forZO-1 immunofluorescence as described previously (Flemingetal. 1989). Cell clusters at the 16-cell stage were first labelledwith rhodamine-conjugated Concanavalin A (Sigma) todetect microvillous polarity as described (Fleming etal. 1989).Specimens were viewed on a Leitz Diaplan microscope usingappropriate filters and photographs were taken on KodakT-max film.

Results

In the early blastocyst (32- t9 64-cell stage), ZO-1protein is detectable as a continuous zonular belt at theapicolateral borders of each cell in the trophectoderm(TE) (Fig. 1A; see also Fleming et al. 1989). In mostwholemount blastocysts (52/83, 62%) viewed in mid-sectional plane, ZO-1 sites were confined to the TEjunctional zone (Fig. IB), but in a minority (38%),punctate ZO-1 sites were also evident on the surface ofone or two ICM cells (Fig. 1C). Previously (Fleming etal. 1989), we have shown that ZO-1 is first detectableimmunocytochemically in embryos at the compacting8-cell stage where it localises as a series of dots to theapicolateral contact region between blastomeres, coin-cident with the onset of tight junction formation(Ducibella and Anderson, 1975; Ducibella et al. 1975;Magnuson et al. 1977). Since initial ZO-1 expression inthe 8-cell embryo precedes the diversification ofprospective TE and ICM cell lineages (beginning at the8- to 16-cell division), the origin of ZO-1 expression inthe ICM (where at this stage of development, tight

ZO-1 expression in mouse embryos 297

junctions are absent) might result from inheritancerather than local synthesis. We therefore evaluatedwhether or not ZO-1 protein differentially segregatedinto the polar (prospective TE) lineage during differen-tiative division of isolated blastomeres.

ZO-1 localisation in cell clustersSingle 8-cell blastomeres (1/8 cells) were allowed todivide in culture to 2/16 couplets and cultured forvarying periods up to 8h before analysis of cellphenotypes present and ZO-1 distribution (Table 1).Using Concanavalin A staining to identify polar cells,on average 76% couplets were polannonpolar (P:NP)pairs, while 24% were polanpolar (P:P) in phenotype;this ratio is consistent with previous data (Pickering etal. 1988). In most P:NP couplets at Oh and lh post-division, ZO-1 was distributed as dots apparentlyrandomly-placed on the membrane of both cells thatwere usually rounded and not adhering together(Fig. 2A,B; Table 1). At later time points (3-5h post-division), polar cells flattened against and beganenveloping nonpolar cells in P:NP couplets, and ZO-1became localised to the periphery of the contact zone,firstly in a punctate pattern and then as a continuousline (Fig. 2C-F). In some couplets, a transitional statewas evident with both random and contact-associatedZO-1 membrane sites being present (scored as contact-localised in Table 1). After 8h post-division, most(68 %) P:NP couplets showed complete envelopment ofthe nonpolar cell by the polar cell such that a peripheralcontact zone was no longer present; in these couplets,ZO-1 staining again appeared as randomly-distributeddots mainly at the contact site but also on the polar cellouter membrane (Fig. 2G,H; Table 1). In P:P couplets,cells flattened against each other but envelopment didnot take place; here, ZO-1 membrane staining changedfrom a random to a contact-associated pattern as seen inP:NP couplets, but this was maintained up to 8 h post-division (Table 1). These results indicate that, unlikethe apical microvillous pole, ZO-1 is inherited by bothcells during differentiative division of polar 8-cells.Subsequently, ZO-1 distribution is modified according

Fig. 1. Wholemount early blastocysts immunolabelled for ZO-1 detection. (A) Blastocyst viewed en face, showing belt-likeZO-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 mid-sectional plane showing punctate ZO-1 sites on the surface of certain ICM cells (arrow). Bar=10^m.


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

Time post 2/16formation (h)

Control medium01358

Cycloheximide135

3*

* Cycloheximide treatmentdivision onwards.

n

861041007589

548163

48

spanned

%P:NPpairs

6775867181

787886

81

1 h prior to division

Surface ZO-1

P:NP pairs

Random dotsn (%)

53 (91)58 (74)8(9)8(15)

49 (68)

42 (100)42 (67)27 (50)

33 (85)

to 3h postdivision.

Periphery ofcontact zone

5(9)20 (26)78 (91)45 (85)23 (32)

0(0)21 (33)27 (50)

6(15)

distribution

P:P

Random dotsn

28 (100)22 (85)0(0)1(4)0(0)

11 (92)3(50)3(33)

6(67)

For remaining groups, exposure to

pairs

Periphery ofcontact zone

(%)

0(0)4(15)

14 (100)21 (96)17 (100)

1(8)3(50)6(67)

3(33)

cycloheximide was from

to cell contact patterns. In experiments where 2/16couplets were cultured in the presence of cycloheximidefor up to 5 h from the time of division of 1/8blastomeres (or from prior to division), the ZO-1distribution pattern in P:NP couplets was similar to thatdescribed above, although a delay was evident in theacquisition of contact-localised sites (Table 1). Thisfurther indicates that ZO-1 in nonpolar cells resultsfrom inheritance rather than de novo synthesis.

To evaluate whether ZO-1 inheritance by nonpolarcells was influenced by the breakdown of putative tightjunctions (and hence dispersion of ZO-1) duringdisaggregation of compact 8-cell embryos into 1/8blastomeres, the distribution of ZO-1 was also analysedin newly-formed 4/16 cell clusters derived from thedivision of 2/8 couplets where ZO-1 is known to belocalised to the periphery of the contact zone (Fleminget al. 1989). In these experiments, 2/8 couplets weregenerated from 1/4 blastomeres and cultured for 18 h toallow for division to the 16-cell stage to occur beforeanalysis (Table 2). Three types of 4/16 clusters were

Table 2. ZO-1 distribution in 4/16 and 4/32 cellclusters derived from division in vitro of 2/8 and 2/16

couplets respectively. Data from six experiments

Phenotype(polar:nonpolarcell ratio)

4/164:03:1

. 2:2

4/324:03:12:2

n (%)

99 (45)75 (34)45 (20)

22 (12)31 (17)

132 (71)

ZO-1 staininjsites

; present at contactinvolving

Both polar andPolar cells only nonpolar cells

(%)

1009273

1009091

(%)

08

27

0109

observed, depending upon the number of blastomeresdividing differentiatively. The ratio of outer polaninnernonpolar cells in these clusters was 4:0 (45%), 3:1(34 %) or 2:2 (20 %), frequencies consistent with earlierdata using this mouse strain (Pickering et al. 1988). Inall clusters, ZO-1 was present along the cell bordersbetween outer polar cells. In most (85%) clusterscontaining inner nonpolar cells, ZO-1 was only locatedbetween polar cells, while the negative nonpolar cellswere completely internalised (Fig. 3A-E). However, ina minority (15 %) of heterogeneous 4/16 clusters, ZO-1was also present at contact sites involving nonpolar cells(Table 2). In these latter examples, ZO-1 eitherappeared as randomly-placed dots where the nonpolarcells were fully internalised (as seen previously in 2/16couplets, Fig. 2H) or was localised to the periphery ofthe contact zone where the nonpolar cells had not beencompletely enclosed (Fig. 3F-I). These experimentstherefore confirm that ZO-1 can be inherited by bothpolar and nonpolar 16-cell daughters, irrespective ofwhether natural cell contacts are maintained in parental8-cell blastomeres prior to division. They also indicatethat the internalisation of nonpolar cells leads to thedispersion and ultimate loss of ZO-1 membranestaining.

The segregation pattern of ZO-1 during the nextcleavage division (16- to 32-cell stage) was analysed in asimilar manner to that described above. Single 8-cellblastomeres were disaggregated from compact embryosand allowed to divide in culture to 2/16 couplets. Thesewere cultured for 18 h during which time division to4/32 cell clusters occurred. The ratio of outer polar:in-ner nonpolar cells in these clusters was 4:0 (12 %), 3:1(17%) and 2:2 (71%), figures consistent with earlierdata (Johnson and Ziomek, 1983). In these moreadvanced specimens, nonpolar cells were always fullyenclosed 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 involvinginner nonpolar cells (Fig. 4G,H; Table 2). These resultssuggest that ZO-1 can also be inherited (and sub-sequently down-regulated) by nonpolar cells at the 16-

Fig. 2. Polar:nonpolar 2/16 couplets at different timespostdivision from 1/8 blastomeres;(A,C,E,G) Concanavalin A staining to identify the polarcell (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 bothpolar and nonpolar cells (arrows), at positions bearing norelationship with the contact area. (C,D) At 3hpostdivision, ZO-1 is localised to the periphery of thecontact area between cells where a linear series of punctatesites are present (arrow). (E,F) At 5h postdivision, ZO-1at the contact site is now continuous rather than punctatein appearance. (G,H) By 8h postdivision, the polar cell hascompletely enveloped the nonpolar cell; punctate ZO-1sites are found throughout the contact area between cells.Bar=10f<m.

to 32-cell transition, presumably by polar cells dividingdifferentiatively.

ZO-1 expression in isolated ICMsIn order to investigate in more detail the process of ZO-1 down-regulation within the nonpolar ICM lineage, wemade use of the developmental potential of ICMs toregenerate trophectoderm during culture followingimmunosurgical isolation from early blastocysts (Flem-ing et al. 1984). In ICMs processed immediatelyfollowing isolation (0 h time-point), over 50 % showedno evidence of ZO-1 immunolabelling, the remainderpossessed faint punctate ZO-1 sites at the contactborder between two or more adjacent outer cells(Fig. 5A,B; Table 3). After culture for 1-3h, all ICMsshowed this discontinuous ZO-1 staining pattern, whichwas brighter than previously and involved progressivelymore of the outer cell population (Fig. 5C). From 6hculture, 85 % ICMs had acquired linear ZO-1 sitesbetween outer cells (Fig. 5D; Table 3). The temporalaccumulation of ZO-1 specifically at junctional sitesbetween outer ICM cells during trophectoderm regen-eration was also analysed in the presence of biosyn-thetic inhibitors. Treatment with the transcriptionalinhibitor alpha-amanitin, administered from 1 h prior toimmunosurgery and during culture (0-6 h) of isolatedICMs up until the time of fixation, had no observableeffect on the acquisition of linear ZO-1 junctional sitesbetween outer ICM cells (Table 3). However, a similartreatment with the protein synthesis inhibitor cyclohexi-mide blocked the formation of linear ZO-1 sites,although faint discontinuous sites between outer cellswere evident in a minority of ICMs (Table 3).

Discussion

In this study, we have investigated the mechanismsregulating lineage-specific expression of the tightjunction protein, ZO-1, in the preimplantation embryo.ZO-1 is a prominent component of the junctionalcomplex circumscribing the apicolateral border be-tween trophectoderm cells (Fleming et al. 1989), but isusually undetectable in the ICM, except for occasionalcells that show punctate membrane sites in a minority of


embryos. One possible explanation for differentialexpression might be that the protein, first evident atcompaction in all cells of the 8-cell embryo prior totissue diversification, is segregated by inheritance intothe polar (trophectoderm) lineage where ZO-1 syn-

thesis continues, in order to facilitate tight junctionmaturation and turnover. Segregation of molecularcomponents is known to contribute to Drosophilapattern formation (Ingham, 1988) and, in the presentstudy, segregation might also include ZO-1 mRNA in

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 sitebetween outer polar cells (arrows), as shown in tangential (B) and mid-sectional (C) planes. (D,E) Cluster containing threeouter 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 localisedto 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 contactbetween nonpolar cells (arrow in I). Bar=10,um.

Table 3. ZO-1 localisation in ICMs isolated immunosurgically from early blastocysts and cultured for variousperiods in the presence or absence of biosynthetic inhibitors. Data from three experiments

Time post- ZO-1 staining pattern (%)

Treatment1

Control

a-Amanitin(100^g/ml)

Cycloheximide(400 fm)

ISUldUUll

(h)

01361224036036

n

626062674836604174407454

Negative

5608iOr0

630

t258980

Discontinuous2

441001001500

3710019751120

Linear3

000

85100100

00

81

000

'Treatment with inhibitors commenced lh prior to immunosurgery and continued up until fixation of ICMs.2Discontinuous pattern: discontinuous dot-like ZO-1 sites present at lateral contact sites between two or more outer cells (e.g. Fig. 5C).3Linear pattern: Linear, belt-like, ZO-1 staining at the contact site between outer cells (e.g. Fig. 5D).


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

order to maintain phenotypic specificity in ZO-1synthesis. Alternatively, differential expression mightnot be coupled directly with phenotypic divergence, butmay be achieved as a consequence of positionaldifferences between lineages that lead to up- or down-regulation of specific proteins. Our findings indicatestrong support for the latter model.

The analysis of ZO-1 distribution following differen-tiative divisions at 8- to 16-, and 16- to 32-cell stages insynchronised cell clusters revealed that ZO-1 wasinherited by both polar and nonpolar daughter cells.The presence of ZO-1 in nonpolar cells is alsosupported by its localisation to the contact site betweenpolar and nonpolar cells prior to envelopment, since wehave shown previously that the formation of suchcontacts between earlier-staged blastomeres is depen-dent upon both cells being competant to express ZO-1

Fig. 5. ZO-1 immunolabelling of ICMs, isolated from earlyblastocysts and fixed immediately (A,B) or cultured for 3h(C) or 6h (D) before fixation. (A,B) Punctate ZO-1 sitesare 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,um.

(Fleming et al. 1989). The inheritance of ZO-1 by bothcells following differentiative division therefore con-trasts with the segregation of the apical microvillouspole into the trophectoderm lineage exclusively (John-son and Ziomek, 1981). The ZO-1 pattern more closelyresembles features of the cytoplasm of polarisedblastomeres (cytoskeletal elements, membraneous or-ganelles) that lose their polarised distribution duringmitosis and, following differentiative division, areinherited by both cells (Reeve, 1981; Johnson andMaro, 1984; Fleming and Pickering, 1985; Maro et al.1985; Chisholm and Houliston, 1987; Houliston andMaro, 1989). The basolateral epithelial surface com-ponents uvomorulin (Vestweber et al. 1987), vinculin(Lehtonen and Reima, 1986), spectrin (Sobel andAlliegro, 1985), gap junctions (Lo and Gilula, 1979) andplakoglobin (Fleming et al. 1991) are similarly allpartitioned into both TE and ICM lineages duringblastocyst development. We have shown previouslythat ZO-1 contact sites between polarised 8-cellblastomeres in different conformations also representspecialisations of the basolateral epithelial membranerather than of the apical-basolateral membrane bound-ary (Fleming et al. 1989). Thus, it appears that thenonpolar ICM lineage inherits all the cellular features


that are epithelial in character except those associatedwith the apical cytocortex. It is these apical componentsthat have been shown to be instructive in the globalreorganisation of cells into an overtly polarised pheno-type (Johnson and Maro, 1985; Johnson et al. 1988;Wiley and Obasaju, 1988, 1989).

The contact-associated membrane assembly of ZO-1by nonpolar cells was found to persist until they becameinternalised by enveloping polar cells, at which timeZO-1 membrane sites became disorganised into ran-dom punctate foci and then disappeared. Thus,position, or more specifically the loss of cell contactasymmetry, appeared to initiate ZO-1 down-regulation.Earlier studies on cultured epithelial cell lines haveshown that deprivation of normal cell-cell contacts,either by extracellular calcium depletion or treatmentwith anti-uvomorulin antibody, inhibited or reversedthe association of ZO-1 with the tight junctionmembrane (Gumbiner et al. 1988; Siliciano andGoodenough, 1988; Anderson et al. 1989). • Similartreatments on mouse embryo 8-cell blastomeres alsoinhibited or reversed the normal contact-localisedpattern of ZO-1 assembly (Fleming et al. 1989). Ourpresent result therefore demonstrates that uvomorulin-mediated cell-cell adhesion is not the sole regulator ofZO-1 assembly at the membrane. Rather, this assemblyis dependent upon both adhesion and the presence ofcontact-free membrane. The relevance of the latterfactor may be attributable to the molecular character ofthe, as yet, unidentified ZO-1 membrane binding sitewhich may require the presence of both membranefaces for its stabilisation.

Our experiments on isolated ICMs were designed toidentify the biosynthetic level at which down-regulationof ZO-1 within the ICM lineage is accomplished. Theformation of a zonular pattern of ZO-1 contact sitesbetween outer cells of ICMs occurred rapidly (by 6 h)during trophectoderm regeneration, well in advance offluid accumulation (by approx 24 h, Handyside, 1978;Johnson, 1979; Fleming et al. 1984) and the expressionof all trophectoderm-specific polypeptides (24 h, John-son, 1979). Indeed, contact-associated ZO-1 wasevident in certain surface regions of some ICMs fixedimmediately following isolation, and may reflect eitherthe redistribution of inherited protein or new synthesisof ZO-1 during the period between trophectoderm lysisand ICM isolation (approx 30min). The assembly of azonular ZO-1 network in isolated ICMs was unaffectedby alpha-amanitin treatment, but was inhibited bycycloheximide treatment (Table 3). The simplest in-terpretation of this result is that ZO-1 expression andassembly takes place in the absence of transcriptionalactivity but does require protein synthesis. Thus,sufficient mRNA transcripts for ZO-1 assembly wouldappear to be present within the ICM lineage prior toimmunosurgery, indicating that ZO-1 RNA, in additionto ZO-1 protein, is inherited by nonpolar cells duringdifferentiative division. The preservation of ZO-1mRNA in the ICM is further supported by data fromCaco-2 cells showing stable, elevated levels of ZO-1mRNA, (analysed directly) in conditions where cell-

cell contacts are inhibited and ZO-1 translation isminimal (Anderson et al. 1989). However, full confir-mation of the presence of functional ZO-1 mRNA inthe ICM awaits a direct analysis, which we intend topursue. The dependence upon protein synthesis forZO-1 assembly in ICMs suggests that the posttranscrip-tional regulation of ZO-1 expression is mediated at thetranslational level. We, therefore, hypothesise thatalthough nonpolar cells preserve inherited ZO-1mRNA, they do not preserve ZO-1 protein which isdown-regulated by natural turnover in the absence ofmembrane assembly sites (when cell contacts aresymmetrical) but which needs to be resynthesised denovo once assembly sites are reestablished (cell contactasymmetry). This model is consistent with the obser-vation that the ZO-1 protein level in Caco-2 cellsrapidly declines when cell contacts are inhibited(Anderson et al. 1989). However, we cannot as yetexclude the possibility that ZO-1 protein in a diffuseform, not readily detectable by our immunocytochemi-cal technique, is retained by nonpolar cells and isavailable for membrane assembly. In this case, toexplain our cycloheximide data, the synthesis ofundefined protein(s) would be required for ZO-1assembly to occur. We are currently investigating byquantitative means the level of ZO-1 total protein andsynthesis in ICMs compared with trophectoderm, toresolve this issue.

In conclusion, our results indicate that ZO-1 proteinrequired for tight junction assembly in the trophecto-derm lineage is, nevertheless, inherited, along with itsmRNA, by the ICM lineage. The loss of cell contactasymmetry in the ICM leads to the down-regulation ofZO-1 protein, but the mRNA appears to be stabilised.What might be the significance of message stability? Ithas been shown previously that totipotent ICM cellsmust undergo transcription to express the full comp-lement of trophectoderm-specific polypeptides duringtrophectoderm regeneration (Johnson, 1979). How-ever, in the absence of transcription, certain trophecto-derm marker polypeptides can be expressed in the earlyphase of the regeneration process (Johnson, 1979). Thisis entirely consistent with our findings, since ZO-1assembly occurs rapidly following immunosurgery. Thecapacity of ICMs to engage in rapid and transcription-ally-independent expression of certain key epithelialcomponents might be responsible for the observedflattening and envelopment of outer ICM cells over theICM core that occurs as an initial event duringtrophectoderm regeneration (Fleming et al. 1984). Thisprocess is necessary to retain contact symmetrybetween core cells and hence stabilise them fromphenotypic transformation. Thus, in the intact embryo,ZO-1 message stability within the ICM might contrib-ute to the continued viability of this lineage indepen-dently of protection provided by the trophectoderm. Itis noteworthy that in blastocysts in which the polartrophectoderm has been injured deliberately, few if anyICM cells enter the trophectoderm lineage (Dyce et al.1987). An additional role for ZO-1 message stabilitywithin the ICM concerns the next phase in the ICM


developmental programme, that of delamination ofprimary endoderm at the interface with the blastocoele.It will be of interest to establish whether epithelialbiogenesis of trophectoderm and primary endodermtissues are linked to promote kinetic efficiency by theconservation of transcripts encoding common epithelialproteins.

We thank The Wellcome Trust for financial support for thiswork. Certain preliminary experiments were undertaken atthe Department of Anatomy, Cambridge, for which weacknowledge grants from the Medical Research Council andthe 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 CharlieMcFadden and Barry Lockyer for technical assistance.

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{Accepted 30 May 1991)

tissue-specific contro olf expression of the tight ... · but not all, te-specific polypeptides,...

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