Differentiation of the epithelial apical junctional complex during mouse preimplantation development: a role for rab13 in the early maturation of the tight junction

Mechanisms of Development 97 (2000) 93±104

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Differentiation of the epithelial apical junctional complex during mouse preimplantation development: a role for rab13 in the early maturation of the tight junction Bhavwanti Sheth a,1, Jean-Jacques Fontaine a,c,1, Elena Ponza a,1, Amanda McCallum a, Anton Page b, Sandra Citi d,e, Daniel Louvard f, Ahmed Zahraoui f, Tom P. Fleming a,* a

Division of Cell Sciences, School of Biological Sciences, University of Southampton, Bassett Crescent East, Southampton SO16 7PX, UK Biomedical Imaging Unit, School of Medicine, University of Southampton, Southampton General Hospital, Southampton SO16 6YD, UK c Departement des Sciences Biologiques et Pharmaceutiques, Ecole Nationale Veterinaire d'Alfort, 7 Avenue di General de Gaulle, 94704 Maisons Alfort Cedex, France d Department of Biology, University of Padova, 35121 Padova, Italy e Department of Molecular Biology, University of Geneva, 30 Quai Ernest Ansermet, 1211 Geneva 4, Switzerland f CNRS UMR 144, Institut Curie, 26 rue d'Ulm, 75248 Paris Cedex 05, France

b

Received 26 May 2000; received in revised form 13 July 2000; accepted 13 July 2000

Abstract We have investigated the mechanisms by which the epithelial apicolateral junctional complex (AJC) is generated during trophectoderm differentiation in the mouse blastocyst using molecular, structural and functional analyses. The mature AJC comprises an apical tight junction (TJ), responsible for intercellular sealing and blastocoel formation, and subjacent zonula adherens E-cadherin/catenin adhesion complex which also extends along lateral membrane contact sites. Dual labelling confocal microscopy revealed that the AJC derived from a single `intermediate' complex formed following embryo compaction at the 8-cell stage in which the TJ-associated peripheral membrane protein, ZO-1a2 isoform, was co-localized with both a- and b-catenin. However, following assembly of the TJ transmembrane protein, occludin, from the early 32-cell stage when blastocoel formation begins, ZO-1a2 and other TJ proteins (ZO-1a1 isoform, occludin, cingulin) colocalized in an apical TJ which was separate from a subjacent E-cadherin/catenin zonula adherens complex. Thin-section electron microscopy con®rmed that a single zonula adherens-like junctional complex present at the AJC site following compaction matured into a dual TJ and zonula adherens complex at the blastocyst stage. Embryo incubation in the tracer FITC-dextran 4 kDa showed that a functional TJ seal was established coincident with blastocoel formation. We also found that rab13, a small GTPase previously localized to the TJ, is expressed at all stages of preimplantation development and relocates from the cytoplasm to the site of AJC biogenesis from compaction onwards with rab13 and ZO-1a2 co-localizing precisely. Our data indicate that the segregation of the two elements of the AJC occurs late in trophectoderm differentiation and likely has functional importance in blastocyst formation. Moreover, we propose a role for rab13 in the speci®cation of the AJC site and the formation and segregation of the TJ. q 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Epithelial differentiation; Blastocyst; Trophectoderm; Mouse embryo; Tight junction; Zonula adherens; Apical junctional complex; ZO-1; rab13; Occludin; Cingulin; E-Cadherin; Catenin

1. Introduction Epithelial cells possess compositionally distinct apical and basolateral membranes necessary for polarized transport activity and tissue organization (reviewed in Yeaman et al., 1999). In simple epithelia, the basolateral surface is further divided into a lateral domain engaged in cell±cell * Corresponding author. Tel.: 144-23-8059-4398; fax: 144-23-80594319. E-mail address: [email protected] (T.P. Fleming). 1 These authors contributed equally to this study.

adhesion and communication and a basal domain which interacts with the extracellular matrix. At the apical extremity of the lateral membrane, the zonula occludens (tight junction; TJ) and zonular adherens junctions are located very close together in the belt-like `apical junctional complex' (AJC), further characterized by a circumferential ring of actin ®laments. The TJ acts both as a fence to the lateral diffusion of membrane constituents and as a regulated permeability seal to restrict paracellular transport across the epithelium (reviewed in Stevenson and Keon, 1998). The zonula adherens is a focus for the E-cadherin/

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B. Sheth et al. / Mechanisms of Development 97 (2000) 93±104

catenin-mediated cell±cell adhesion system which also extends along the entire lateral surface and contributes to intercellular signalling and cell polarity (Yeaman et al., 1999). In some epithelia, the zonula adherens is prominent (e.g. intestinal epithelium) while in others it is indistinct and represents the apical-most region of E-cadherin adhesion. Both junctions of the AJC comprise several interacting transmembrane and cytoplasmic proteins that mediate linkage to the actin cytoskeleton. Transmembrane proteins of the TJ include occludin (Furuse et al., 1993), claudin family members (Furuse et al., 1998; Morita et al., 1999) and junction adhesion molecule (JAM; Martin-Padura et al., 1998). Occludin comprises four membrane-spanning segments with two extracellular loops involved in intercellular sealing and a short N- and longer C-terminus, both located cytoplasmically (Furuse et al., 1993). Claudins are molecularly distinct from occludin but possess an equivalent structure with cytoplasmic C- and N-termini and two extracellular loops (Furuse et al., 1998; Morita et al., 1999). The occludin and claudin C-termini act as sites of interaction with a complex of peripheral membrane proteins, ZO-1, ZO-2 and ZO-3 (Stevenson et al., 1986; Itoh et al., 1993, 1999a; Willott et al., 1993; Furuse et al., 1994; Beatch et al., 1996; Haskins et al., 1998; Wittchen et al., 1999). ZO-1 interacts with actin (Fanning et al., 1998) and occurs in two alternately spliced isoforms, either with or without an 80 amino acid C-terminal a motif (ZO-1a1/a2; Balda and Anderson, 1993). Actin also binds directly in vitro with occludin and other ZO proteins (Wittchen et al., 1999). Similarly, the cytoplasmic plaque protein cingulin (Citi et al., 1988) interacts with ZO-1, ZO-2, ZO-3 and myosin (Cordenonsi et al., 1999a), and the C-terminus of occludin (Cordenonsi et al., 1999b). At the zonula adherens and lateral membrane, Ecadherin has a single transmembrane domain and interacts cytoplasmically with a complex containing b-catenin, plakoglobin and p120 CAS (Ozawa et al., 1989; McCrea et al., 1991; Staddon et al., 1995). This complex in turn associates with a-catenin which forms a direct link with the actin cytoskeleton (Hirano et al., 1987; Rimm et al., 1995; Aberle et al., 1996). The close positioning and fundamental roles of TJ and zonula adherens junctions is indicative of their coordinated activity in epithelial tissues. Thus, in mature epithelial cells induced to form a polarized epithelium in culture, Ecadherin adhesion has a primary and critical role in subsequent formation of the TJ (Gumbiner et al., 1988; Yap et al., 1995; Watabe-Uchida et al., 1998). Moreover, the two junction types contain constituents capable of cross-interaction; ZO-1 and ZO-2, in addition to binding to TJ proteins, can also associate with a-catenin and the E-cadherin membrane complex (Itoh et al., 1997, 1999b; Rajasekaran et al., 1996). Interestingly, during formation of an epithelium in vitro after replating of culture cells, ZO-1 transiently interacts with the E-cadherin complex before binding to occludin (Rajasekaran et al., 1996; Ando-Akatsuka et al., 1999). These studies suggest that associations between TJ and

zonula adherens/E-cadherin complex proteins may also occur as epithelia differentiate during development. The establishment and maintenance of the AJC is also dependent upon correct targeting of newly synthesized junctional proteins to this membrane domain (see Yeaman et al., 1999). Rab proteins are part of the Ras superfamily of small GTPases and are involved in the regulation and targeting of intracellular transport (reviewed in Novick and Zerial, 1997; Chavrier and Goud, 1999). To date, over 30 rab proteins have been identi®ed. They localize to the cytoplasmic face of vesicles and organelles involved in biosynthetic and endocytic pathways where they regulate discrete steps in vesicular transport. A number of rab proteins have been implicated in epithelial polarity including rab8 in basolateral membrane targeting (Huber et al., 1993), rab3B in the AJC region (Weber et al., 1994) and rab13 at the TJ (Zahraoui et al., 1994). In cells devoid of TJs, rab13 is dispersed in vesicles throughout the cytoplasm but in Caco-2 epithelial cells, rab13 co-localizes with ZO-1 at the TJ and not with E-cadherin at the zonula adherens and lateral membrane (Zahraoui et al., 1994). To date, it is unknown whether rab13 or related GTPases may have a role in the speci®cation of the TJ domain during epithelial differentiation. The trophectoderm is the ®rst epithelium to be generated after fertilization of the mammalian egg and forms the wall of the blastocyst (reviewed in Fleming et al., 1998). Ecadherin/catenin-mediated adhesion initiates at the 8-cell stage (compaction) when blastomeres polarize and begin to differentiate (Johnson et al., 1986; Vestweber et al., 1987; Ohsugi et al., 1996). E-cadherin expression and adhesion is essential for trophectoderm formation (Larue et al., 1994). By the 32-cell stage (,24 h after compaction), trophectoderm differentiation is complete, vectorial transport initiates and the blastocoel cavity forms (Fleming et al., 1998). Recently, we have investigated the biogenesis of the trophectoderm TJ during cleavage. We have found that the assembly of TJ proteins at the AJC begins after compaction, is dependent upon E-cadherin-mediated adhesion, and occurs in a step-wise manner due to a temporally regulated expression programme. Interestingly, the assembly sequence has revealed that the plaque proteins ZO-1a2 isoform and cingulin associate with the AJC at 8- and 16cell stages respectively (Fleming et al., 1989, 1993; Javed et al., 1993), and before the assembly, at the 32-cell stage, of their TJ transmembrane binding partner, occludin (Sheth et al., 2000). Here, we have investigated the mechanisms regulating emergence of the AJC during trophectoderm differentiation using a combination of dual labelling confocal microscopy, electron microscopy and a TJ permeability assay. We provide evidence that during the early phase of AJC formation at compaction, ZO-1a2 transiently co-localizes with the catenin proteins in a single, permeable, apicolateral `intermediate' junctional complex. Subsequently, after occludin assembly in the late morula, the AJC segregates

B. Sheth et al. / Mechanisms of Development 97 (2000) 93±104

into two distinct structural domains (apical, TJ; subjacent, zonula adherens), coincident with the apical separation of ZO-1, together with other TJ-associated proteins, from the E-cadherin complex, the establishment of a permeability seal and the formation of the blastocoel cavity. Moreover, we ®nd that the rab13 GTPase is expressed throughout cleavage but redistributes to the nascent AJC from compaction onwards, in precise co-localization with ZO-1a2. These data reveal a close coordination exists at the molecular level between the formation of TJ and zonula adherens during epithelial differentiation that contributes to the timing of blastocyst formation and indicate a role for rab13 in regulating de novo assembly and segregation of the TJ.

2. Results 2.1. Molecular maturation of the apicolateral junction complex Embryos at 2 h post-compaction (8-cell stage), 12 h postcompaction (mid 16-cell stage) and early blastocysts (approx. 32-cell stage, 24 h post-compaction) were singleor double-labelled for ZO-1 (using R40.76 recognizing both isoforms) and components of the E-cadherin adhesion system (either b-catenin or a-catenin). The R40.76 antibody will detect only ZO-1a2 isoform at 8- and 16-cell stages as the ZO-1a1 isoform is not expressed and assembled at cell contact sites until the 32-cell stage (Sheth et al., 1997). For each of these stages, catenins stained the cell contact sites within embryos, more intensely in the outer trophectoderm lineage than within the inner ICM lineage, and was absent from outer, apical membrane, as shown previously (Ohsugi et al., 1996). Compact 8-cell embryos labelled with ZO-1 and either b- or a-catenin, showed ZO-1 co-localized with catenins predominantly within the apicolateral cell contact region when embryo mid-plane optical sections were examined (Fig. 1A±D and Table 1). In 16-cell embryos, most contact sites between outer cells displayed the same colocalization pattern as observed at the 8-cell stage but in a minority (27%, n ˆ 102, for combined b- and a-catenin specimens; Table 1), ZO-1 and catenins were separately localized with ZO-1 present more apical to the catenin domain. This heterogeneous pattern was observed within individual embryos (Fig. 1E±I and Table 1). In contrast, in almost all trophectoderm contact sites within early blastocysts, ZO-1 was localized apical to and distinct from the catenin-positive lateral membrane (Fig. 1J±M and Table 1). To determine whether the ZO-1 binding site in early blastocysts represented the true TJ and a single complex of TJassociated proteins, double labelling between ZO-1 and either the TJ transmembrane protein, occludin, or the cytoplasmic plaque protein, cingulin, was performed. In both cases, most (,90%, Table 1) contact sites examined showed precise co-localization at the apicolateral region (Fig. 2A±

95

H). We have shown previously that ZO-1a1 isoform and occludin are precisely co-localized at this site (Sheth et al., 1997, 2000). Similarly, double labelling of early blastocysts with isoform speci®c antibodies revealed ZO-1a2 and ZO1a1 to be precisely co-localized at this apicolateral contact site in most (74%) junction sites (Fig. 2I±L and Table 1). Collectively, these experiments indicate that the molecular organization of the AJC changes during trophectoderm differentiation from a single complex present during 8and 16-cell morula stages composed of E-cadherin/catenins and ZO-1a2 to a double complex in the blastocyst where TJ (apical) and E-cadherin/catenins (subjacent and lateral) proteins are segregated. 2.2. Structural and functional maturation of the apicolateral junctional complex The AJC was examined by electron microscopy at different stages of trophectoderm differentiation. Prior to and during early stages of compaction at the 8-cell stage, the contact site between blastomeres showed no particular membrane specialization (Fig. 3A). As compaction proceeded, a small (,100 nm long), electron-dense, submembraneous plaque domain was evident within adjacent cells at the apicolateral contact site (Fig. 3B). During late 8and 16-cell stages, further electron-dense contact sites became apparent along lateral membranes (Fig. 3C). These structures resemble adherens junctions and an intercellular space was usually evident between their apposed membranes. In contrast, the AJC in early and late blastocysts sectioned in mid-plane was segregated into two domains. The most apical domain comprised one or more sites (,50 nm long) showing cytoplasmic electron density but where apposed membranes were in direct cell contact without an intercellular space (Fig. 3D±F) and resembling a TJ. Immediately subjacent to this domain, apposed membranes separated and formed one or more electrondense junctions with an intercellular space, resembling the adherens-like structures observed at 8- and 16-cell stages. Compact 8-cell, 16-cell, late morula and nascent and more expanding early blastocyst stages were incubated in FITC-dextran 4 kDa and viewed by confocal microscopy to determine the extent of paracellular permeability (Table 2). In all pre-blastocyst stages, ¯uorescent dextran was clearly visible between all the cells of the embryo (Fig. 4A). In a minority (23%) of nascent blastocysts where the cavity represented ,20% total volume, tracer permeability throughout the embryo was again evident (Fig. 4B). However, in remaining nascent and expanding blastocysts, tracer was excluded from the embryo, indicating a `global' permeability seal had been established (Fig. 4C). Collectively, these data provide evidence that the AJC segregates into two structural entities at the time of blastocyst formation, coinciding with the formation of a permeability seal at this site.

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Fig. 1. Dual labelling confocal microscopy of ZO-1 and a-catenin membrane localization in whole-mount embryos viewed in optical mid-plane at different stages of trophectoderm differentiation. (A±D) Compacted 8-cell stage embryo stained for ZO-1 (A, arrow) and a-catenin (B). In the merged image (C, arrow; D, magni®ed), co-localization of ZO-1 and a-catenin is evident at the apical cell contact site. (E±I) Sixteen-cell embryo stained for ZO-1 (E, arrow and arrowhead) and a-catenin (F). At most contact sites, ZO-1 and a-catenin are co-localized (G, arrow; H, magni®ed) but in some, ZO-1 is separate from and apical to a-catenin (G, arrowhead; I, magni®ed). (J±M) Early blastocyst stained for ZO-1 (J, arrows) and a-catenin (K). At this stage, ZO-1 is localized apical to a-catenin (L, arrows; M, magni®ed). Bar: 20 mm except (D,H,I,M) 5 mm.

Table 1 Immunolocalization of junctional proteins in double-labelled embryos at different stages of preimplantation development B positive only a

7 (16.7) 1 (2.7)

± ±

± ±

34 (82.9) 39 (63.9)

7 (17.1) 22 (36.1)

± ±

± ±

0 (0) 1 (0.8) 88 (88.9) 92 (90.2) 123 (74.1)

129 (100) 123 (99.2) 2 (2.0) 0 (0) 21 (12.7)

0 (0) 0 (0) 8 (8.1) 10 (9.8) 22 (13.3)

0 (0) 0 (0) 1 (1.0) 0 (0) 2 (1.2)

No. of embryos

Eight-cell stage A:B ZO-1:b-catenin ZO-1:a-catenin

15 15

37 42

36 (97.3) 35 (83.3)

Sixteen-cell stage A:B ZO-1:b-catenin ZO-1:a-catenin

16 18

41 61

Early blastocyst A:B ZO-1:b-catenin ZO-1:a-catenin ZO-1:cingulin ZO-1:occludin ZO-1a1:ZO-1a2

16 18 22 19 22

129 124 99 102 16

a

No. of junctions

A positive only a

Stage and antigen combination

Co-localized (%)

Not co-localized (%)

Only junctions positive for ZO-1 were analysed at 8- and 16-cell stages when all contact sites are positive for catenins.

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Fig. 2. Dual labelling confocal microscopy of TJ protein membrane localization in early blastocysts. (A±D) ZO-1 (A, arrow) and occludin (B, arrow) with merged image (C, arrow; D, magni®ed). (E±H) ZO-1 (E, arrow) and cingulin (F, arrow) with merged image (G, arrow; H magni®ed). (I±L) ZO-1a2 (I, arrow) and ZO-1a1 (J, arrow) isoforms with merged image (K, arrow; L, magni®ed). In all cases, proteins are co-localized (arrows) at the apical region of contact in the outer trophectoderm layer. Bar: 20 mm except (D,H,L) 5 mm.

2.3. Rab13 and the maturation of the apicolateral junctional complex To identify potential mechanisms responsible for the emergence and spatial segregation of the AJC, we examined the expression and localization pattern of the rab13 GTPase which has been implicated in the targeting of constituents to the TJ (Zahraoui et al., 1994). Rab13 mRNA expression was examined in single eggs and embryos at different stages of cleavage using two-stage RT±PCR. This analysis revealed the presence of rab13 transcripts at all preimplantation stages (Fig. 5A), con®rmed by cloning and sequencing of ampli®ed products. Rab13 mRNA was also detectable within ICMs isolated by immunosurgery from early blastocysts (Fig. 5B). Western blotting analysis of rab13 protein expression using a rab13-speci®c polyclonal antibody also showed a ubiquitous expression pro®le throughout cleavage. A major band migrating at ,26 kDa was observed in both embryos and control mouse lung and brain (Fig. 5C).

A further minor band migrating at ,24 kDa was also observed in the earlier stages (eggs, 2- and 8-cell) but not in blastocysts, lung or brain. Confocal microscopy revealed that membrane assembly of rab13 was coordinated with the onset of differentiation of the AJC. In 2-cell, 4-cell and precompact 8-cell embryos, rab13 was detectable as a weak, diffuse cytoplasmic reaction (Fig. 6A). In compact 8-cell embryos, however, punctate foci of rab13 were seen for the ®rst time at cell contact sites, either along the lateral membrane or more usually at the apicolateral margin (Fig. 6B). By comparing embryos that had compacted within 1±2 h with those compacted within 4±6 h, it was apparent that apicolateral concentration of rab13 occurred with time. In double-labelled compact 8cell embryos, a precise co-localization between the punctate sites of rab13 and those where ZO-1 ®rst assembles was evident (Fig. 6C±F). In 16-cell embryos, rab13 was restricted to the outer cell lineage and concentrated within the apicolateral contact site. By this stage, staining was

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linear or belt-like around each cell (Fig. 6G,H). In late morulae, early blastocysts (Fig. 6I,J) and more expanding blastocysts (Fig. 6K,L), rab13 was localized to the trophectoderm and labelled, in a linear way, the apicolateral region of cell contact. These results indicate that rab13 associates with the AJC from the earliest stage of its maturation and throughout the period of assembly of the TJ.

Table 2 Development of a permeability seal in embryos analysed by FITC-dextran 4 kDa exclusion Stage

No. of embryos

Permeable

%

Late morulae 94 h post-hCG Nascent blastocysts (cavity ,20% volume) Blastocysts (cavity .20% volume)

35 22

35 5

100 23

18

0

0

3. Discussion In this study, we have investigated the maturation of the AJC during trophectoderm differentiation. From a combination of immunological, structural and functional analyses, we provide the ®rst evidence that the TJ and zonula adherens segregate from a common or intermediate apical junction at the terminal phase of epithelial differentiation (see Fig. 7 for summary diagram). We have also found that the TJ-associated rab GTPase, rab13 (Zahraoui et al., 1994), is intimately associated with the maturing AJC from the onset of assembly of TJ proteins at this site. Recently, we have shown that occludin, the transmembrane binding partner for ZO-1 and other TJ plaque proteins (see Section 1), ®rst assembles at the TJ as a late step in trophectoderm differentiation, during the early period of the 32-cell stage (Sheth et al., 2000). This assembly event is temporally regulated by the embryo since non-assembled forms of occludin protein are readily detectable by immunoblotting throughout cleavage. It is only after an apparent phosphorylation of one occludin form (designated band 2, 65±67 kDa) and its co-localization cytoplasmically with the ZO-1a1 isoform that band 2 occludin becomes competent to assemble at the TJ site and convert from a detergent soluble to insoluble form indicative of cytoskeletal anchorage (Sheth et al., 2000). The regulator of the timing of occludin assembly appears to reside in the expression pattern of ZO-1a1 which is not transcribed until the late morula stage. Newly expressed ZO-1a1 is immediately translated, associates intracellularly with occludin and the two proteins assemble together at the TJ site (Sheth et al., 1997). This is the ®nal step so far identi®ed in the trophectoderm model of TJ biogenesis and blastocoel cavity formation occurs rapidly afterwards, within the same cell cycle. Fig. 3. Ultrastructure of apical cell contact site between compacted 8-cell (A,B) and early 16-cell (C) blastomeres, and between trophectoderm cells of early blastocysts (D±F). At early compaction, no membrane specialization is evident (A), but subsequently an electron-dense plaque resembling an adherens junction is formed at the most apical contact site with a clear intercellular space present (B, arrowhead). Similar plaques then also form along the lateral membrane (C, arrowheads). In blastocysts, the most apical contact site comprises one or more electron-dense regions of direct membrane apposition without an intercellular space resembling a tight junction (D±F, arrows) with the adherens-like junctions situated below (D±F, arrowheads). Bar: 100 nm.

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Fig. 4. Living late morulae (A) and nascent early blastocysts (B,C) examined by confocal microscopy in the presence of FITC±dextran 4 kDa. In morulae and a minority of nascent blastocysts (cavity , 20% embryo volume), dextran tracer is evident between cells (A,B) but is excluded in the majority of blastocysts (C). Bar: 20 mm.

The FITC±dextran permeability data presented here showed that exclusion of ¯uorescent tracer from internal intercellular spaces was evident in the majority of embryos from when the nascent cavity was extremely small …,20% embryo volume). This is consistent with ultrastructural studies using lanthanum tracer for detection of trophectoderm sealing (Ducibella et al., 1975; Magnuson et al., 1978). Thus, occludin assembly in the late morula also leads to the establishment of a permeability seal to maintain the integrity of the cavitating blastocyst. Although the control of occludin membrane assembly provides a coherent mechanism to regulate the timing of blastocyst formation, we consider that earlier expression and membrane assembly events during cleavage are also contributory. We have found that the ZO-1a2 isoform

Fig. 5. Expression of rab13 during early development. (A) RT±PCR analysis of single eggs and single embryos showing ampli®ed 400 bp cDNA product (arrow). M, markers; E, unfertilized egg; 2, 2-cell embryo; 8, compact 8-cell embryo; LM, late morula; EB, early blastocyst; LB, late blastocyst; C, control, without template. (B) RT±PCR analysis of single early blastocyst (EB) and three isolated ICMs (I). (C) Western blot analysis of rab13 protein expression in equivalent numbers (n ˆ 300) of unfertilized eggs (E), late 2-cell embryos (2), compact 8-cell embryos (8), late blastocysts (LB) and in equivalent protein samples (7.5 mg) of mouse lung (L) and brain (Br). A major band at ,26 kDa is evident in each lane (arrow).

assembles at the apicolateral contact site from the time of compaction (8-cell stage: Fleming et al., 1989; Sheth et al., 1997) as does cingulin some 12 h later, during the 16-cell stage (Fleming et al., 1993). Recently, it has been shown that the N-terminus of cingulin binds to ZO-1 in vitro (Cordenonsi et al., 1999a) which may provide a molecular basis for cingulin and ZO-1a2 co-localization in the early embryo. Both of these TJ constituents have been shown to interact with the C-terminal domain of occludin (Furuse et al., 1994; Cordenonsi et al., 1999b). Since their assembly in the embryo precedes that of occludin, alternative binding partner(s) at the membrane must be involved. We speculate that this strategy of early assembly of TJ plaque proteins may contribute to the timing of blastocyst formation since it provides a local pool of constituent proteins to facilitate rapid TJ construction once membrane delivery of the occludin/ZO-1a1 complex has taken place. We used dual labelling confocal microscopy to investigate the mechanisms regulating early membrane assembly of ZO-1a2 in embryos prior to occludin assembly. In mature epithelial cells induced to form an epithelial monolayer in vitro after replating, ZO-1 has been shown to associate with catenins at contact sites in the absence of occludin (Rajasekaran et al., 1996; Ando-Akatsuka et al., 1999). We therefore analysed the distribution of ZO-1a2 and either bcatenin or a-catenin in midplane optical sections. Newlyassembled ZO-1a2 at the apicolateral contact site co-localized with both catenins at 8- and 16-cell stages but this association was not maintained in early blastocysts where ZO-1 and catenin positive sites became segregated (Table 1 and Fig. 1). This suggests that the early binding partner(s) for ZO-1a2 in the embryo may be catenins or associated Ecadherin complex proteins but con®rmation will require biochemical identi®cation of immunoprecipitated junctional protein complexes. Such studies are technically demanding in precisely staged preimplantation embryos due to the limited material available (,25 ng total protein per embryo). However, there is further supportive evidence for a role for the E-cadherin complex in early assembly of ZO-1a2. First, inhibition of E-cadherin adhesion at compaction (using either E-cadherin neutralizing antibody

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or calcium-free culture medium) delays the timing of ZO1a2 membrane assembly and severely disrupts its spatial organization (Fleming et al., 1989). Second, the membrane localization of ZO-1 as well as catenin proteins is abnormal in E-cadherin null mutant preimplantation embryos (Ohsugi et al., 1997). Third, an alternative candidate for an early membrane binding partner for ZO-1a2 is claudin which, like occludin, interacts with ZO-1 at the C-terminal domain (Itoh et al., 1999a). However, this possibility is unlikely since, in a recent analysis, we have found claudin membrane assembly in the mouse embryo model to initiate only from the late morula stage (F. Thomas et al., manuscript in preparation). Fourth, ZO-1a2 might associate indirectly with the membrane by binding to other pre-assembled TJassociated plaque proteins that it is known to interact with. Candidates here would include cingulin (Cordinonsi et al., 1999a), ZO-2 (Wittchen et al., 1999) or ZO-3 (Haskins et al., 1998). Again, this is unlikely since both cingulin (Fleming et al., 1993) and ZO-2 (R. Nowak et al., in preparation) assemble later than ZO-1a2. The expression pattern of ZO3 is currently under investigation. The switch from co-localized to segregated distribution patterns for ZO-1 and catenins at the blastocyst stage provides evidence of a compositional nature that the AJC derives from a single, intermediate junction during differentiation, separating as a late event upon membrane assembly of occludin (Fig. 7). From this time, occludin, ZO-1 (both isoforms) and cingulin are all co-localized in an apparent TJ complex, apical to the E-cadherin complex. The behaviour of mature cultured cells during early stages of forming an epithelium in vitro where transient interaction between ZO-1 and catenins has been identi®ed (Rajasekaran et al., 1996; Ando-Akatsuka et al., 1999) therefore mimics that of the differentiating trophectoderm. Thin-section examination of embryo stages provided further structural evidence in support of this model for AJC biogenesis. Thus, a single zonula adherens-type junction with a clear intercellular space at 8- and 16-cell stages differentiated further into an apical TJ with no intercellular space and a subjacent zonula adherens equivalent to earlier stages. Although the ultrastructure of preimplantation embryo cell Fig. 6. Confocal microscopy of rab13 localization during early development. (A) Precompact 8-cell embryo with a diffuse cytoplasmic reaction. (B) Compact 8-cell embryo, viewed in tangential section showing some cytoplasmic reaction and discontinuous membrane localization of rab13 at the apical region of contact between blastomeres (arrows). (C±F) Compact 8-cell embryo viewed tangentially shown in bright-®eld (C) with speci®c blastomeres identi®ed, 1±4, and double-labelled for rab13 (D, arrows) and ZO-1 (E, arrows) and with images merged (F, arrows), showing precise colocalization of both proteins at contact sites between cells. (G,H) Sixteencell embryo viewed in midplane (G) and tangentially (H) showing rab13 restricted to apicolateral contact regions between outer blastomeres (arrows). (I,J) Early blastocyst viewed in midplane (I) and tangentially (J) showing continuous linear staining of rab13 between the outer trophectoderm cells. (K,L) Late blastocyst viewed in midplane (K) and in z-series projection (L) showing trophectoderm staining of rab13. b, blastocoel; i, ICM. Bar: 20 mm.

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Fig. 7. Schematic diagram of preimplantation stages and proposed pattern of membrane assembly of junctional proteins during trophectoderm differentiation. Below each stage is shown the composition of apicolateral contact sites between outer cells. Following the assembly of occludin at the 32-cell stage, separation of apical TJ and subjacent zonula adherens/E-cadherin domains is apparent, coinciding with blastocyst cavitation and the establishment of a permeability seal.

contacts has been investigated previously (e.g. Ducibella and Anderson, 1975; Ducibella et al., 1975; Magnuson et al., 1977), the timing and pattern of trophectoderm AJC formation has not been reported. From our studies on the mouse embryo, ZO-1a2 is therefore the ®rst TJ-associated protein known to undergo membrane assembly during trophectoderm differentiation. In addition to identi®cation of its binding partner(s), a mechanism to permit targeting of ZO-1a2 to the apicolateral region of cell contact must be considered. In this context, we have found that the small rab GTPase, rab13, is expressed throughout preimplantation development at mRNA and protein levels. Interestingly, Western blotting analysis of rab13 protein expression showed a major band migrating at ,26 kDa in both embryos and control tissues and a further minor band migrating at ,24 kDa detected speci®cally in the earlier stages (eggs, 2- and 8-cell) but not in blastocysts, lung or brain. The protein sequence of human rab13 has several putative sites for post-translational modi®cation, for example two N-glycosylation and ®ve phosphorylation sites are present. Phosphorylation of other rab proteins has also been reported (Ayad et al., 1997; Fitzgerald and Reed, 1999). Thus, the 26 kDa band in embryos may represent a developmentally regulated, post-translational modi®cation of rab13. Rab proteins are a diverse group of proteins that regulate vesicle traf®cking particularly in exocytic and endocytic pathways and are involved in the targeting of components in polarized cells (Novick and Zerial, 1997; Chavrier and Goud, 1999). Rab13 contains a C-terminal CaaX motif (C, cys; a, mostly aliphatic; X, any amino acid) similar to those of Ras/Rho proteins and is isoprenylated in vivo and gera-

nylgeranylated in vitro which may facilitate its anchorage to the lipid bilayer during membrane attachment (Joberty et al., 1993). Regulation of membrane association and disassociation is achieved by rab13 speci®c binding to rod cGMP phosphodiesterase d subunit (d-PDE; Marzesco et al., 1998). Signi®cantly, rab13 has been shown to associate with the tight junction in mature epithelial cells, indicative of a speci®c role in targeting and docking of TJ proteins (Zahraoui et al., 1994). We have found that rab13 assembles at the apicolateral region of contact between blastomeres from compaction onwards and does so in precise co-localization with ZO-1a2. The apicolateral polarized distribution of rab13 from the earliest stage of assembly of TJ-associated proteins indicates, therefore, that it may be involved not only in maintaining the integrity of the TJ in mature cells but also the speci®cation of this domain during differentiation. Rab13 regulatory protein, d-PDE, exhibits two putative C-terminal sequences necessary for interaction with PDZ motifs (PSD95, Dlg, ZO-1) (Marzesco et al., 1998) which, signi®cantly, are present on ZO-1 and other ZO family TJassociated proteins (Anderson, 1996). Rab13/d-PDE is therefore suitably equipped to initiate ZO-1 targeting to the putative TJ site. In summary, we provide molecular and structural evidence that during trophectoderm epithelial differentiation, a close interaction occurs between components of the TJ and zonula adherens (E-cadherin) complex. This association contributes to the mechanism of blastocyst formation since it permits premature membrane assembly of TJ-associated plaque proteins prior to the late assembly of occludin (or claudin) which can then act as a limiting factor to activate blastocoel formation. In addition, we have shown that

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the rab13 GTPase localizes at the AJC site throughout the time that TJ-associated proteins assemble there. This may indicate an important role for rab13 in specifying the site of TJ maturation during development. 4. Experimental procedures 4.1. Embryo collection, culture and manipulations MF1 4±5-week-old female mice (University of Southampton Biomedical Facility) were superovulated by intraperitoneal injection of 5 IU pregnant mare serum (PMS; Folligon, Intervet) followed 48 h later by 5 IU human chorionic gonadotrophin (hCG; Chorulon, Intervet) and mated overnight with MF1 males. Embryos were collected at the late 2-cell stage (,48 h post-hCG) or 8-cell stage (,68 h post-hCG) by ¯ushing dissected oviducts with H6 medium (containing 4 mg/ml bovine serum albumin, BSA, Sigma) and cultured in T6 medium (containing 4 mg/ml BSA) in 5% CO2 at 378C as described previously (Sheth et al., 1997). Unfertilized eggs within cumulus masses were collected at 12 h post-hCG and treated with hyaluronidase to remove cumulus cells; eggs and embryos were treated with acid Tyrode's solution to remove the zona pellucida; and ICMs from early blastocysts were isolated by immunosurgery exactly as described (Fleming et al., 1991; Chisholm et al., 1985; Sheth et al., 1997). Embryos were staged as follows from the time of hCG injection: 2-cell, 48 h; 4cell, 56 h; pre-compact 8-cell, 68 h; compact 8-cell, 70 h; 16-cell morula, 78 h; late morula, 90±96 h; early blastocysts, 94±98 h; late (expanded) blastocysts, 115 h. In certain experiments, embryos were staged from compaction during the 8-cell stage by hourly inspection of cultures; newly compacted embryos were removed and cultured separately until required. 4.2. Antibodies Rabbit polyclonal antibodies to human rab13 (Zahraoui et al., 1994), human occludin (Fallon et al., 1995; Van Itallie and Anderson, 1997), chicken cingulin (Citi et al., 1988), mouse a-catenin (Herrenknecht et al., 1991), mouse b-catenin (Braga et al., 1995), rabbit and guinea-pig polyclonals to mouse ZO-1a1 and ZO-1a2 isoforms (Sheth et al., 1997) and rat monoclonal antibody R.40.76 to ZO-1 (Anderson et al., 1988) were used in this study. 4.3. Immunocytochemistry and confocal microscopy For all antibodies except rab13, zona-free embryos were ®xed in 1% formaldehyde in PBS, attached onto coverslips coated with 1 mg/ml poly-l-lysine hydrobromide (Sigma) and processed for immunocytochemistry as described previously (Fleming et al., 1991; Sheth et al., 1997). For rab13 staining (either single- or double-labelled), zonafree embryos were placed on coverslips within processing

chambers pre-coated by incubation with 1 mg/ml concanavalin A in PBS (10 min), before ®xation in methanol at 2208C. Embryos were single- or double-labelled using appropriate dilutions of primary antibodies for 1 h in PBS/ 0.1% Tween-20 (PBS-T), washed in PBS-T and incubated in appropriate secondary antibodies diluted in PBS-T. In double-labelling experiments, FITC-, TRITC-/Texas red(Amersham), Alexa 488- or Alexa 546- (Cambridge Biosciences) conjugated secondary antibodies were used and in all staining combinations, secondary antibodies were checked to ensure that they did not react with the inappropriate primary antibody. Specimens were visualized using a £63 oil-immersion objective on a Nikon inverted microscope linked to a Bio-Rad MRC-600 series confocal imaging system equipped with a krypton±argon laser. 4.4. Electron microscopy Specimens were ®xed in 3% glutaraldehyde, post-®xed in 1% osmium tetroxide (0.1 M cacodylate buffer, pH 7.3), alcohol dehydrated and embedded in Spurr's resin. Thin sections were stained and examined on a Hitachi H7000 transmission electron microscope. 4.5. Paracellular permeability Compact 8-cell, 16-cell, late morulae and early blastocysts were incubated in H6 plus BSA containing 1 mg/ml FITC±dextran of size ,4 kDa (Sigma) for 10 min and viewed immediately by confocal microscopy. Each embryo was scored for the presence or absence of ¯uorescence within intercellular spaces or in the blastocoel. 4.6. Reverse transcriptase±polymerase chain reaction (RT± PCR) Single, cumulus-free, unfertilized eggs, single staged embryos or isolated ICMs were lysed onto messenger af®nity paper (mAP, Amersham) and processed for RT±PCR as described by Collins and Fleming (1995) with the modi®cations reported in Sheth et al. (2000). Four percent (2 ml) of the ®rst stage product was subsequently added to the second stage reaction and ampli®ed using nested primers. Mouse rab13 5 0 -outer and inner sense primers were GCCTACGACCACCTCTTC and TCTTCAAGTTGCTGC, the 3 0 antisense outer and inner primers were CGCCTGCTCTCTCTGCA and TCTGCACCTGCCGCT, respectively. Both ®rst- and second-stage ampli®cation was achieved using 25 cycles at 958C (30 s), 558C (30 s) and 728C (30 s). cDNA ampli®ed from early 4-cell, compact 8-cell and late blastocyst stages was cloned into pBluescript (Stratagene) and sequenced in both directions using a Thermosequenase kit (Amersham). 4.7. Electrophoresis and immunoblotting Mouse lung and brain extract for electrophoresis was generated using frozen tissue powder boiled for 5 min in

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PBS/1% sodium dodecyl sulphate (SDS) and centrifuged at 10 000 £ g for 3 min. Mouse eggs and embryos were brie¯y washed in H6 plus PVP (Sigma) before solubilisation in boiling SDS-sample buffer for 5 min. Samples were run on 12% polyacrylamide gradient gels (Novex), blotted onto Hybond-C nitrocellulose (Amersham) and the blots processed for detection of rab13 (antibody diluted 1:100) using methods as described previously (Sheth et al., 2000). Acknowledgements We thank The Wellcome Trust, Medical Research Council and European Union (Biomedical Programme, BMH4CT95-0090) for ®nancial support to T.P.F., and James M. Anderson (Yale University), Vania Braga (University College London), and Rolf Kemler (Freiburg) for the supply of antibodies. J.-J.F. was in receipt of an EMBO Fellowship and E.P. in receipt of a SOCRATES studentship to work in T.P.F.'s laboratory. References Aberle, H., Schwartz, H., Kemler, R., 1996. Cadherin±catenin complex: protein interactions and their implications for cadherin function. J. Cell Biochem. 61, 514±523. Anderson, J.M., 1996. Cell signalling: MAGUK magic. Curr. Biol. 6, 382± 384. Anderson, J.M., Stevenson, B.R., Jesaitis, L.A., Goodenough, D.A., Mooseker, M.S., 1988. Characterization of ZO-1, a protein component of the tight junction from mouse liver and Madin-Darby canine kidney cells. J. Cell Biol. 106, 1141±1149. Ando-Akatsuka, Y., Yonemura, S., Itoh, M., Furuse, M., Tsukita, S., 1999. Differential behavior of E-cadherin and occludin in their colocalization with ZO-1 during the establishment of epithelial cell polarity. J. Cell. Physiol. 179, 115±125. Ayad, N., Hull, M., Mellman, I., 1997. Mitotic phosphorylation of Rab4 prevents binding to a speci®c receptor on endosome membranes. EMBO J. 16/15, 4497±4507. Balda, M.S., Anderson, J.M., 1993. Two classes of tight junctions are revealed by ZO-1 isoforms. Am. J. Physiol. 264, C918±C924. Beatch, M., Jesaitis, L.A., Gallin, W.J., Goodenough, D.A., Stevenson, B.R., 1996. The tight junction protein ZO-2 contains three PDZ (PSD-95/Discs-Large/ZO-1) domains and an alternatively spliced region. J. Biol. Chem. 271, 25723±25726. Braga, V.M.M., Hodivala, K.J., Watt, F.W., 1995. Calcium-induced changes in distribution and solubility of cadherins and their associated cytoplasmic proteins in human ketaniocytes. Cell Adhes. Commun. 3, 201±215. Chavrier, P., Goud, B., 1999. The role of ARF and rab GTPases in membrane transport. Curr. Opin. Cell Biol. 11, 466±475. Chisholm, J.C., Johnson, M.H., Warren, P.D., Fleming, T.P., Pickering, S.J., 1985. Developmental variability within and between mouse expanding blastocysts and their ICMs. J. Embryol. Exp. Morph. 86, 311±336. Citi, S., Sabanay, H., Jakes, R., Geiger, B., Kendrick-Jones, J., 1988. Cingulin: a new peripheral component of tight junctions. Nature 333, 272± 276. Collins, J.E., Fleming, T.P., 1995. Speci®c mRNA detection in single lineage-marked blastomeres from preimplantation embryos. Trends Genet. 11, 5±7. Cordenonsi, M., D'Atri, F., Hammar, E., Parry, D.A., Kendrick-Jones, J.,

103

Shore, D., Citi, S., 1999a. Cingulin contains globular and coiled-coil domains and interacts with ZO-1, ZO-2, ZO-3, and myosin. J. Cell Biol. 147, 1569±1582. Cordenonsi, M., Turco, F., D'atri, F., Hammar, E., Martinucci, G., Meggio, F., Citi, S., 1999b. Xenopus laevis occludin: identi®cation of in vitro phosphorylation sites by protein kinase CK2 and association with cingulin. Eur. J. Biochem. 264, 374±384. Ducibella, T., Anderson, E., 1975. Cell shape and membrane changes in the eight-cell mouse embryo: prerequisites for morphogenesis of the blastocyst. Dev. Biol. 47, 45±58. Ducibella, T., Albertini, D.F., Anderson, E., Biggers, J.D., 1975. The preimplantation mammalian embryo: characterization of intercellular junctions and their appearance during development. Dev. Biol. 45, 231± 250. Fallon, M.B., Brecher, A.R., Balda, M.S., Matter, K., Anderson, J.M., 1995. Altered hepatic localisation and expression of occludin after common bile duct ligation. Am. J. Physiol. 269, C1057±C1062. Fanning, A.S., Jameson, B.J., Jesaitis, L.A., Anderson, J.M., 1998. The tight junction protein ZO-1 establishes a link between the transmembrane protein occludin and the actin cytoskeleton. J. Biol. Chem. 273, 29745±29753. Fitzgerald, M.L., Reed, G.L., 1999. Rab6 is phosphorylated in thrombinactivated platelets by a protein kinase C-dependent mechanism: Effects on GTP/GDP binding and cellular distribution. Biochem. J. 342, 353± 360. Fleming, T.P., McConnell, J., Johnson, M.H., Stevenson, B.R., 1989. Development of tight junctions de novo in the mouse early embryo: control of assembly of the tight junction-speci®c protein, ZO-1. J. Cell Biol. 108, 1407±1418. Fleming, T.P., Garrod, D.R., Elsmore, A.J., 1991. Desmosome biogenesis in the mouse preimplantation embryo. Development 112, 527±539. Fleming, T.P., Hay, M., Javed, Q., Citi, S., 1993. Localisation of tight junction protein cingulin is temporally and spatially regulated during early mouse development. Development 117, 1135±1144. Fleming, T.P., Butler, E., Collins, J., Sheth, B., Wild, A.E., 1998. Cell polarity and mouse early development. Adv. Mol. Cell Biol. 26, 67±94. Furuse, M., Hirase, T., Itoh, M., Nagafuchi, A., Yonemura, S., Tsukita, S., Tsukita, S., 1993. Occludin: A novel integral membrane protein localising at tight junctions. J. Cell Biol. 123, 1777±1788. Furuse, M., Itoh, M., Hirase, T., Nagafuchi, A., Yonemura, S., Tsukita, S., Tsukita, S., 1994. Direct association of occludin with ZO-1 and its possible involvement in the localisation of occludin at tight junctions. J. Cell Biol. 127, 1617±1626. Furuse, M., Fujita, K., Hiiragi, T., Fujimoto, K., Tsukita, S., 1998. Claudin1 and -2: novel integral membrane proteins localizing at tight junctions with no sequence similarity to occludin. J. Cell Biol. 141, 1539±1550. Gumbiner, B., Stevenson, B., Grimaldi, A., 1988. The role of the cell adhesion molecule uvomorulin in the formation and maintenance of the epithelial junctional complex. J. Cell Biol. 107, 1575±1587. Haskins, J., Gu, L., Wittchen, E.S., Hibbard, J., Stevenson, B.R., 1998. ZO3, a novel member of the MAGUK protein family found at the tight junction, interacts with ZO-1 and occludin. J. Cell Biol. 141, 199±208. Herrenknecht, K., Ozawa, M., Eckerskorn, C., Lottspeich, F., Lenter, M., Kemler, R., 1991. The uvomorulin-anchorage protein, a-catenin is a vinculin homologue. Proc. Natl. Acad. Sci. USA 88, 9156±9160. Hirano, S., Nose, A., Hatta, K., Kawakami, A., Takeichi, M., 1987. Calcium-dependent cell±cell adhesion molecules (cadherins): subclass speci®cities and possible involvement of actin bundles. J. Cell Biol. 105, 2501±2510. Huber, L.A., Pimplikar, S., Parton, R.G., Virta, H., Zerial, M., Simons, K., 1993. Rab8, a small GTPase involved in vesicular traf®c between the TGN and the basolateral plasma membrane. J. Cell Biol. 123, 35±45. Itoh, M., Nagafuchi, A., Yonemura, S., Kitani-Yasuda, T., Tsukita, S., Tsukita, S., 1993. The 220-kD protein colocalising with cadherins in non-epithelial cells is identical to ZO-1, a tight junction-associated protein in epithelial cells: cDNA cloning and immunoelectron microscopy. J. Cell Biol. 121, 491±502.

104

B. Sheth et al. / Mechanisms of Development 97 (2000) 93±104

Itoh, M., Nagafuchi, A., Moroi, S., Tsukita, S., 1997. Involvement of ZO-1 in cadherin-based adhesion through its direct binding to a-catenin and actin ®laments. J. Cell Biol. 138, 181±192. Itoh, M., Furuse, M., Morita, K., Kubota, K., Saitou, M., Tsukita, S., 1999a. Direct binding of three tight junction-associated MAGUKs, ZO-1, ZO2 and ZO-3, with the COOH termini of claudins. J. Cell Biol. 147, 1351±1363. Itoh, M., Morita, K., Tsukita, S., 1999b. Characterization of ZO-2 as a MAGuK family member associated with tight as well as adherens junctions with a binding af®nity to occludin and a-catenin. J. Biol. Chem. 274, 5981±5986. Javed, Q., Fleming, T.P., Hay, M., Citi, S., 1993. Tight junction protein cingulin is expressed by maternal and embryonic genomes during early mouse development. Development 117, 1145±1151. Joberty, G., Tavitian, A., Zahraoui, A., 1993. Isoprenylation of rab proteins possessing a C-terminal CaaX motif. FEBS Lett. 330, 323±328. Johnson, M.H., Maro, B., Takeichi, M., 1986. The role of cell adhesion in the synchronisation and orientation of polarization in 8-cell mouse blastomeres. J. Embryol. Exp. Morphol. 93, 239±255. Larue, L., Ohsugi, M., Hirchenhain, J., Kemler, R., 1994. E-cadherin null mutant embryos fail to form a trophectoderm epithelium. Proc. Natl. Acad. Sci. USA 91, 8263±8267. Magnuson, T., Demsey, A., Stackpole, C.W., 1977. Characterization of intercellular junctions in the preimplantation mouse embryo by freeze-fracture and thin-section electron microscopy. Dev. Biol. 61, 252±261. Magnuson, T., Jacobson, J.B., Stackpole, C.W., 1978. Relationship between intercellular permeability and junction organization in the preimplantation mouse embryo. Dev. Biol. 67, 214±224. Martin-Padura, I., Lostaglio, S., Schneemann, M., Williams, L., Romano, M., Fruscella, P., Panzeri, C., Stoppacciaro, A., Ruco, L., Villa, A., Simmons, D., Dejana, E., 1998. Junctional adhesion molecule, a novel member of the immunoglobulin superfamily that distributes at intercellular junctions and modulates monocyte transmigration. J. Cell Biol. 142, 117±127. Marzesco, A.-M., Galli, T., Louvard, D., Zahraoui, A., 1998. The rod cGMP phosphodiesterase d subunit dissociates the small GTPase rab13 from membranes. J. Biol. Chem. 273, 22340±22345. McCrea, P.D., Turck, C.W., Gumbiner, B., 1991. A homolog of the armadillo protein in Drosophila (plakoglobin) associated with E-cadherin. Science 254, 1359±1361. Morita, K., Furuse, M., Fujimoto, K., Tsukita, S., 1999. Claudin multigene family encoding four-transmembrane domain protein components of tight junction strands. Proc. Natl. Acad. Sci. USA 96, 511±516. Novick, P., Zerial, M., 1997. The diversity of rab proteins in vesicle transport. Curr. Opin. Cell Biol. 9, 496±504. Ohsugi, M., Hwang, S., Butz, S., Knowles, B.B., Solter, D., Kemler, R., 1996. Expression and cell membrane localisation of catenins during mouse preimplantation development. Dev. Dyn. 206, 391±402. Ohsugi, M., Larue, L., Schwarz, H., Kemler, R., 1997. Cell-junctional and cytoskeletal organization in mouse blastocysts lacking E-cadherin. Dev. Biol. 185, 261±271. Ozawa, M., Baribault, H., Kemler, R., 1989. The cytoplasmic domain of the cell adhesion molecule uvomorulin associates with three independent proteins structurally related in different species. EMBO J. 8, 1711± 1717.

Rajasekaran, A.K., Hojo, M., Huima, T., Rodgiguez-Boulan, E., 1996. Catenins and zonula occludens-1 form a complex during early stages in the assembly of tight junctions. J. Cell Biol. 132, 451±463. Rimm, D.L., Koslov, E.R., Kebriaei, P., Cianci, C.D., Morrow, J.S., 1995. Alpha 1(E)-catenin in an actin-binding and ±bundling protein mediating the attachment of F-actin to the membrane adhesion complex. Proc. Natl. Acad. Sci. USA 92, 8813±8817. Sheth, B., Fesenko, I., Collins, J.E., Moran, B., Wild, A.E., Anderson, J.M., Fleming, T.P., 1997. Tight junction assembly during mouse blastocyst formation is regulated by late expression of ZO-1a1 isoform. Development 124, 2027±2037. Sheth, B., Moran, B., Anderson, J.M., Fleming, T.P., 2000. Post-translational control of occludin membrane assembly in mouse trophectoderm: a mechanism to regulate timing of tight junction biogenesis and blastocyst formation. Development 127, 831±840. Staddon, J.M., Smales, C., Schulze, C., Esch, F.S., Ribin, L.L., 1995. p120, a p120-related protein (p100), and the cadherin/catenin complex. J. Cell Biol. 130, 369±381. Stevenson, B.R., Keon, B.H., 1998. The tight junction: morphology to molecules. Annu. Rev. Cell Dev. Biol. 14, 89±109. Stevenson, B.R., Siliciano, J.D., Mooseker, M.S., Goodenough, D.A., 1986. Identi®cation of ZO-1: A high molecular weight polypeptide associated with the tight junction (zonula occludens) in a variety of epithelia. J. Cell Biol. 103, 755±766. Van Itallie, C.M., Anderson, J.M., 1997. Occludin confers adhesiveness when expressed in ®broblasts. J. Cell Sci. 110, 1113±1121. Vestweber, D., Gossler, A., Boller, K., Kemler, R., 1987. Expression and distribution of cell adhesion molecule uvomorulin in mouse preimplantation embryos. Dev. Biol. 124, 451±456. Watabe-Uchida, M., Uchida, N., Imamura, Y., Nagafuchi, A., Fujimoto, K., Uemura, T., Vermeulen, S., van Roy, F., Adamson, E.D., Takeichi, M., 1998. a-Catenin±vinculin interaction functions to organize the apical junctional complex in epithelial cells. J. Cell Biol. 142, 847±857. Weber, E., Berta, G., Tousson, A., St. John, P., Gree, M.W., Gopalokrishnam, U., Jilling, E.J., Sorscher, T.S., Abrahamson, D.R., Kirk, K.L., 1994. Expression and polarisation of a Rab3 isoform in epithelial cells. J. Cell Biol. 125, 583±594. Willott, E., Balda, M.S., Fanning, A.S., Jameson, B., Van Itallie, C., Anderson, J.M., 1993. The tight junction protein ZO-1 is homologous to the Drosophila discs-large tumour suppressor protein of septata junctions. Proc. Natl. Acad. Sci. USA 90, 7834±7838. Wittchen, E.S., Haskins, J., Stevenson, B.R., 1999. Protein interactions at the tight junction. Actin has multiple binding partners, and ZO-1 forms independent complexes with ZO-2 and ZO-3. J. Biol. Chem. 274, 35179±35185. Yap, A.S., Stevenson, B.R., Keast, J.R., Manley, S.W., 1995. Cadherinmediated adhesion and apical membrane assembly de®ne distinct steps during thyroid epithelial polarization and lumen formation. Endocrinology 136, 4672±4680. Yeaman, C., Grindstaff, K.K., Nelson, W.J., 1999. New perspectives on mechanisms involved in generating epithelial cell polarity. Physiol. Rev. 79, 73±98. Zahraoui, A., Joberty, G., Arpin, M., Fontaine, J.J., Hellio, R., Tavitian, A., Louvard, D., 1994. A small rab GTPase is distributed in cytoplasmic vesicles in non-polarised cells but colocalised with the tight junction marker ZO-1 in polarised epithelial cells. J. Cell Biol. 124, 101±115.