Cell Polarity and Mouse Early Development

CELL POLARITY AND MOUSE EARLY DEVELOPMENT

Tom P. Fleming, Elizabeth Butler, Jane Collins, Bhav Sheth, and Arthur E. Wild

I. Introduction

........................ A. Cell Polarity and Compaction B. Regulation of Cell Polarity . . . . . . . . . . . . . . . . . . . . . . . . ........

Stage in Epithelial Differentiation. . . . . . .

68

........... .73

. . . . . . . . . . ..75

........ . . . . . . . . . . . . 80 B. Desmosomes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 5 IV. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87

Advances in Molecular and Cell Biology Volume 26, pages 67-94. Copyright 0 1998 by JAI Press Inc. All right of reproductionin any form reserved. ISBN: 0-7623-0381-6

67

T.F! FLEMING, E. BUTLER, J. COLLINS, B. SHETH, and A.E WILD

68

I.

INTRODUCTION

Cell polarity occurs for the first time during mouse embryogenesis at the 8-cell stage, about 2.5 days after fertilization. This event is critical for the formation of the blastocyst and its subsequent development. First, cell polarity initiates the program of differentiation of the trophectoderm epithelium which forms the wall of the blastocyst. This tissue generates the blastocoel, regulates vectorial exchange of metabolites with the embryo interior, constitutes the embryonic surface that engages in uterine attachment, and, after implantation, gives rise to the chorio-allantoic placenta. Second, cell polarity underlies the concurrent program of cell diversification in the embryo in which differentiative cell divisions lead to the formation and segregation of the earliest embryonic tissues, the outer trophectoderm and the enclosed inner cell mass (ICM) from which the entire fetus is derived. Cell polarity is therefore of fundamental importance as a developmental mechanism in mammals. In this chapter, we first review the cell biological characteristics of cell polarity during mouse cleavage and the consequences for blastocyst differentiation and tissue diversification. Second, we consider the influence of polarity at a molecular level with respect to the differentiation of multimolecular adhesive junctions in trophectoderm and the origin of differential gene expression in the embryo. Different aspects of early mouse development have been reviewed elsewhere recently (Kimber, 1990; Wiley et al., 1990; Cruz, 1992; Fleming, 1992; Fleming et al., 1992, 1993a, 1994; Gueth-Hallonet and Maro, 1992; Watson, 1992; Collins and Fleming, 1995).

II.

CYTOLOGICAL ASPECTS OF CELL POLARITY AND TISSUE SEGREGATION A.

Cell Polarity and Compaction

Fertilization of the mouse egg is followed by three reduction cleavage divisions to produce an embryo composed of 8 spherical, loosely-associated and nonpolarized blastomeres (Figure 1A). By this stage, the embryo has activated its own genome (2-cell stage; Flach et al., 1982), degraded nearly all maternal transcripts (Paynton et al., 1988), and has transcribed and translated most of the proteins required to initiate cell polarity and differentiation (Levy et al., 1986). The switch in blastomere phenotype from nonpolar to polar at the 8-cell stage is comprehensive in nature and coincides with the onset of cell-cell adhesion, these combined events being referred to as “compaction” since blastomere outlines become indistinct as adhesive contacts form (Figures 1B-D). Blastomere polarity at compaction is detectable both within the deeper cytoskeletal and cytoplasmic zones and within the cell cortex and membrane. Thus, microfilaments and microtubules polymerize predominantly in the apical (outer-facing) cytoplasm (Johnson and Maro, 1984; Houliston et al., 1987) al-

Cell Polarity and Mouse Early Development

69

Figure 7. (A-C) Scanningelectron micrographs, of &cell embryos following removal of the zona pellucida (from Fleming et al., 1986a).(A) before compaction has occurred, each blastomere is non-adhesive and uniformly microvillous.(B) after compaction, blastomeres adhere closely together and display a pole of microvilli.(C) compact embryo following exposure to calcium-free medium, causingloss of adhesion and showing apical pole of microvilli. (D)Transmission electron micrograph of one blastomere from a compact 8-cell embryo (from Fleming and Pickering, 1985) showing apical pole of microvilli (M), and clustering of endocytic organelles (E) in the apical cytoplasm. Arrowheads indicate position of adhesive, non-microvillous, basolateral membrane. Bar = 10pm.

though a sub-population of stable acetylated microtubules polarizes basally (Houliston and Maro, 1989). Actin-associated proteins polarize mostly in the apico-lateral region (Sober, 1983; Lehtonen et al., 1988; Slager et al., 1992), nuclei relocate to the basal cytoplasm (Reeve and Kelly, 1983), endosomes and clathrincoated vesicles redistribute from a nonpolar distribution and become localized mostly in the apical cytoplasm (Reeve, 1981;Fleming and Pickering, 1985;Mar0 et dl., 1985; Figure lD), while mitochondria become cortically localized (Batten et al., 1987). Polarization of cytoplasmic components is dependent upon cytoskeletal organization and is consistently modified or inhibited by reagents affecting microfilament or microtubule integrity (Johnson and Maro, 1985; Fleming et al., 1986a,b). Polarization of the cytocortex (membrane and underlying cytoskeleton) is intimately associated with the initiation of cell-cell adhesion mediated by the calcium-dependent cadherin, uvomorulin (E-cadherin), which becomes localized at cell-cell contact sites at compaction (Hyafil et al., 1980; Peyrieras et al., 1983; Vestweber et al., 1987). This redistribution may be promoted by increased stabili-

70

T.P FLEMING, E. BUTLER, J. COLLINS, B. SHETH, and A.E WILD

zation (cytoskeletal anchorage?) of uvomorulin at contact sites and loss of stability at contact-free cell surfaces (Clayton et al., 1993). Uvomorulin appears to be the only mediator of cell adhesion at compaction although N-CAM (calciumindependent neural cell adhesion molecule) is also present in embryos at this time (Kimber et al., 1994). The newly-formed adhesive basolateral cell surfaces form functional gap junctions (Ducibella and Anderson, 1975; Magnuson et al., 1977; Lo and Gilula, 1979; McLachlin et al., 1983; Goodall and Johnson, 1984; Pratt, 1985) and, at their apicolateral border, focal tight junctions emerge (Ducibella and Anderson, 1975; Magnuson et al., 1977; Pratt, 1985) containing the marker protein ZO-1 (Heming et al.. 1989). Most significantly, a distinct apical cytocortex is formed at compaction, comprising a pole of microvilli (Ducibella et al., 1977;Handyside, 1980; Reeve and Ziomek, 1981; Figure 1). Unlike cytoplasmic polarity, the essential features of the apical microvillous pole can form and be maintained in the presence of cytoskeleton-disrupting agents (Johnson and Maro, 1984, 1985; Fleming et al., 1986a,b). Taken together, 8-cell blastomeres at compaction reorganize into a polarized proto-epithelial phenotype that marks the beginning of trophectoderm differentiation. We next consider the mechanisms by which the spatial patterning of cell polarity at compaction and the timing of its expression in the fourth cell cycle may be controlled. B.

Regulation of Cell Polarity

What is the role of cell adhesion in the establishment of blastomere polarity? In undisturbed 8-cell embryos, the apicobasal axis of polarity develops with respect to cell-cell contact sites (i.e., apical microvilli form opposite the contact points) and is not predetermined before the fourth cell cycle (Ziomek and Johnson, 1980; Johnson and Ziomek, 1981a; Figure 2). Culturing embryos in the absence of calcium or in the presence of antibodies against the ectodomain of uvomorulin, or in cytochalasin, inhibits or reverses the adhesive component of compaction (Pratt et al., 1982; Shirayoshi et al., 1983; Johnson et al., 1986; Figures lC, 2C) as do peptides containing the cadherin HAV recognition sequence (Blaschuk et al., 1990). Under conditions of uvomorulin neutralization, cell polarity can still occur for most components but does so over a more protracted time period and usually displays a random axis with respect to sites of cell-cell contact (Fleming et al., 1986a,b, 1989; Johnson et al., 1986; Figure 2C). The capacity of blastomeres to polarize is therefore “programmed” independently from their capacity to initiate adhesion, although the latter process would normally act to catalyze and synchronize cell polarity and to define in a permissive way the orientation of its axis (Johnson et aI., 1986). What is the relationship between the different features of cell polarity that are established at compaction following receipt of the “permissive” inductive signal mediated by uvomorulin adhesion? A number of experimental approaches indicate that the apical cytocortical domain of the cells (essentially where the apical pole of

71

Cell Polarity and Mouse Early Development

non-polar

polar

d

Figure2 (A-C) Disaggregated8-cell blastomeres labelled with FITC-concanavalinA to reveal distribution of microvilli (from Fleming et al., 1986a). (A) before compaction, staining is uniform.(B) after compaction, staining (microvillous pole) i s localised opposite the point of intercellular adhesion.(C) blastomeres exposed to cytochalasin fail to adhere together (see b) but can still generate microvillous polarity, along a random axis not related to the point of cell-cell contact (top blastomere). (D) Cell adhesion leads to conversion of 8-cell blastomeres from non-polar to polar phenotype; see text for consideration of mechanisms. Bar = IOpm.

microvilli forms) acts as a stable "memory" of the axis of polarity throughout the period of trophectoderm differentiation and is responsible for organizing polarization within the deeper cytoplasm (reviewed in Fleming, 1992; Figure 2D). Thus, in experimental conditions where microvillous polarity develops in the absence of cytoplasmic polarity (e.g., cytochalasin treatment, disrupting microfilaments, see earlier), returning embryos to normal medium permits cytoplasmic polarity to occur and, significantly, along the axis already defined by the pole of microvilli (Johnson and Maro, 1985). Similarly, cytoplasmic polarity, but not cortical polarity, is dissipated as 8-cell blastomeres enter mitosis, but is reestablished in the next interphase again along the axis defined by the stable microvillous pole (Fleming and Pickering, 1985; Maro et al., 1985; Johnson et al., 1988). Also, in heterokaryons formed by fusion of polarized 8- or 16-cell blastomeres with nonpolar 4-cell blastomeres, the apical cytocortex of the polarized cell induced polarization of cytoplasmic components derived from the 4-cell (Wiley and Obasaju, 1988). The question of how a contact-mediated signal might lead to a polarized cytocortical organization remains elusive. One possibility is that signal transduction

72

T.P FLEMING, E. BUTLER, J. COLLINS, B. SHETH, and A.E WILD

mechanisms involving protein kinases may be involved in the propagation of a basal contact signal towards the apical domain in the plane of the membrane (reviewed in Fleming, 1992; Figure 2D; also see below). Alternatively, or in addition, transcellular ion currents, carried largely by Na+ ions and generated by the restricted membrane distribution of appropriate ion transporters, may be responsible for initiating a polarized state (Nuccitelli and Wiley, 1985; Wiley and Obasaju, 1988, 1989; reviewed in Wiley et al., 1990; Figure 2D). The identification of a Na+/glucose cotransporter at the apical microvillous domain of blastomeres from compaction onwards may be significant in initiating a Na+-based transcellular ion current (Wiley et al., 1991). For most events of compaction to proceed, neither proximate transcription nor translation are required (Kidder and McLachlin, 1985; Levy et al., 1986). suggesting that the timing of compaction at the 8-cell stage occurs through posttranslational modification of existing proteins. This is borne out in biogenetic studies on the uvomorulin adhesion system. Uvomorulin expression occurs both in unfertilized eggs and throughout cleavage, ruling out its biosynthesis as a mechanism initiating adhesion at compaction (Vestweber et al., 1987; Clayton et al., 1993). Prior to fertilization, however, uvomorulin is not transported to the cell surface, this is achieved from the zygote stage onwards (Clayton et al., 1993). Uvomorulin interacts with catenin proteins at its cytoplasmic tail which mediate the interaction with actin filaments, a requirement for adhesive function in tissue culture cells (reviewed in Takeichi, 1991; Geiger and Ayalon, 1992; Grunwald, 1993). Both a- and p-catenin are detectable during early development by immunoblotting and immunocytochemistry before compaction, indicating that their expression does not regulate uvomorulin adhesion at compaction (Ohsugi et al., 1996; J. Lewthwaite and T. Fleming, manuscript in preparation). It has been proposed that modifications which initiate compaction are prevented from occurring until the 8-cell stage by the synthesis of arestraining factor since, in the absence of protein synthesis, compaction takes place prematurely (Levy et al., 1986). Evidence suggests that phosphorylation events may be an important posttranslational mechanism to initiate compaction. The use of phorbol ester to activate protein kinase C (PKC) caused premature compaction of 4-cell embryos, coincident with redistribution of uvomorulin to regions of cell contact (Winkel et al., 1990; see also Bloom, 1989). Winkel et al. (1990) proposed that PKC activation may be an integral part of the cell-cell signaling mechanism that leads to the lifting of the putative compaction restraining factor. However, premature compaction induced by phorbol ester comprises uvomorulin-mediated cell adhesion but not polarization of blastomeres. A role for phosphorylation in the adhesive component of compaction is also supported by the fact that a number of cellular proteins become phosphorylated at the time of compaction or in response to agents that manipulate compaction (Bloom and McConnell, 1990; Bloom, 1991). Significantly, uvomorulin itself becomes phosphorylated for the first time at the beginning of the 8-cell stage (Sefton et al., 1992). However, recent studies indicate that the signaling path-

Cell Polarity and Mouse Early Development

73

way regulating compaction is more complex than first anticipated. First, the kinase inhibitor staurosporine, like the PKC activator phorbol ester, has been shown in embryos to induce premature adhesion mediated by uvomorulin but not premature polarization (O’Sullivan et al., 1993). Staurosporine is a potent inhibitor of PKC but is not entirely specific for this kinase group (Herbert et al., 1990). Second, treatment of embryos with 6-dimethyl-aminopurine (6-DMAP), a serine-threonine kinase inhibitor, also causes adhesion (but not polarization) to occur prematurely, again mediated by uvomorulin (Aghion et al., 1994). Collectively, these results suggest that both phosphorylation and dephosphorylation reactions contribute to the regulation of adhesion at compaction although the mechanism initiating cell polarity at compaction (involving transcellular ion current?) is yet to be identified. C.

Consequences of Cell Polarity

Cell polarity at compaction marks the initiation of trophectoderm differentiation and provides blastomeres with the essential spatial organization to give rise to divergent cell lineages. Division of polarized 8-cell blastomeres results in the formation of two distinct phenotypes in the 16-cell morula, a population of larger outer polar cells surrounding a group of smaller nonpolar cells (Handyside, 1980; Johnson and Ziomek, 1981b; Reeve and Ziomek, 1981; Figure 3). Various studies have demonstrated that the outer cell population tends to give rise to trophectoderm while the internal cells tend to form the ICM of the blastocyst (Tarkowski and Wroblewski. 1967: Hillman et al., 1972; Handyside and Johnson, 1978; Ziomek and Johnson, 1981; Balakier and Pedersen, 1982; Pedersen et al., 1986; Fleming, 1987a). These two cell types are distinct from the moment of their formation following differential inheritance of polarized cellular domains within parental 8-cell blastomeres (Johnson and Ziomek, 1981b). Thus, most, but not all, 8-cell blastomeres divide along an axis perpendicular to the axis of polarity, generating a polar cell which incorporates the apical pole of microvilli and a nonpolarcell incorporating the basal region (defined as a differenriarive division). A minority of blastomeres divide conservatively, parallel to the axis of polarity, such that the apical pole is bisected and inherited by both daughter cells (Johnson and Ziomek, 1981b). Thus, although cytoplasmic polarity is dissipated during mitosis, cytocortical polarity is maintained (Johnson et al., 1988) and provides the basis for establishment of separate trophectoderm and ICM cell lineages during late cleavage (Figure 3). The behavior of newly-formed polar and nonpolar 16-cell blastomeres ensure that their relative position within the embryo is maintained. Polar cells in culture tend to adhere to and envelop the nonpolar cells by virtue of the localization of uvomorulin, which is found on all cell surfaces except the apical membrane of polar cells (Ziomek and Johnson, 1981; Kimber et al., 1982; Surani and Handyside, 1983; Vestweber et al., 1987). During the fifth cell cycle, the outer polar cells continue and indeed extend their program of epithelial polarization (see later) while the internal cells remain nonpolar and gradually acquire the characteristics of ICM

T.F? FLEMING, E. BUTLER, J. COLLINS, B. SHETH, and A.E WILD

74 8-cell

16-Cell

co rnpaction

morula

32-cell early

blastocyst

Diagrammatic representation of the role of cell polarity in tissue formation and segregation during mouse cleavage (8- to 32-cell stage). Whole embryos shown above; the division plane options of individual polar cells from corresponding stage shown below. Trophectoderm lineage unshaded, ICM lineage shaded. Polar cells at the end of either 8- or 16-cell stages can divide along conservative or differentiative division planes to generate the blastocyst tissues. See text for details. Figure 3.

(Handyside and Johnson, 1978). At the end of the fifth cell cycle, polar cells can again divide either by differentiative or conservative divisions to yield either polar and nonpolar or two polar daughter cells, respectively. As in the previous cycle, nonpolar 32-cell blastomeres are located in the embryo interior, are surrounded by outer polar 32-cell blastomeres, and represent a second and final allocation of cells to the ICM lineage. Why should the mechanism of cell polarity and differentiative division be utilized twice to generate the ICM? Do the two rounds of differentiative cleavage contribute in distinct ways to early tissue segregation? Although it has been shown that the first allocation (8- to 16-cell transition) involves rnostjlastomeres dividing differentiatively, the numbers can vary considerably between embryos, from four to all eight (Johnson and Ziomek, 1981b; Balakier and Pedersen, 1982; Pedersen et al., 1986;Fleming, 1987a).This indicates that there is little control in situ on the orientation of cytokinesis in polarized 8-cell blastomeres. However, Pickering et al. (1988) have postulated from very convincing data that more advanced blastomeres (ie., those entering the fourth cell cycle earlier in typically asynchronous embryos) also engage in adhesive contacts at compaction earlier, thereby positioning deeper within the embryo and acquiring a smaller polarized apical cytocortical domain. This last attribute would in turn lead to an increased likelihood of such blastomeres dividing differentiatively rather than conservatively and would provide a cell bio-

Cell Polarity and Mouse Early Development

75

logical explanation of the long-standing observation that more advanced blastomeres tend to allocate disproportionately more progeny to the ICM than do later-dividing blastomeres (Surani and Barton, 1984;Garbutt et al., 1987; Pickering et al., 1988; Sutherland et al., 1990). Despite this effect of temporal order, the variable number of inner and outer 16cell blastomeres in embryos suggests that the primary role of the first round of differentiative division is to establish two populations of phenotypically distinct blastomeres in different locations within the embryo. Experimental evidence from isolated polar 16-cell blastomeres either cultured alone or in combination with other cells, has indicated that, in contrast to the first round, cell contact patterns could have a significant effect on the orientation of cleavage in the second round of differentiative division. Thus, polar 16-cell blastomeres were less likely to divide differentiatively when combined with other blastomeres, particularly nonpolar blastomeres, than when cultured alone (Johnson and Ziomek, 1983). The simplest interpretation of this phenomenon is that adhesive interactions with other blastomeres influence cell shape which in turn influence the orientation of the spindle. Moreover, support for a role for cell shape in the regulation of division plane orientation in situ has been forthcoming. It has been shown that the number of polar 16cell blastomeres dividing differentiatively in intact embryos is inversely related to the number of inner cells present within the morula (Fleming, 1987a). Thus, if the first allocation to the ICM is relatively small (fewer than normal 8-cell blastomeres dividing differentiatively) then the embryo can compensate by a relatively large allocation in the second round, and vice versa (Fleming, 1987a; Figure 4). This endogenous regulation mechanism can be best understood if the shape of polar 16-cell blastomeres is considered. A smaller than normal first allocation will lead to a larger population of polar cells enveloping this smaller core and displaying a more columnar disposition. Conversely, a large first allocation will lead to fewer polar cells of more flattened appearance enveloping a larger core (Figure 4). These shape changes, without further elaboration, could modify polar cell division orientations to ensure consistency in the cell population sizes forming the trophectoderm and ICM tissues of the blastocyst during the 32-cell stage. The use of cell polarity to establish tissue diversity by differentiative division can therefore be viewed as a two-phase event, the first concerned with establishing qualitative differences between cells (8- to 16-cell transition), the second with establishing quantitative differences between them (16- to 32-cell transition; Fleming, 1987a). D. Cell Polarity in 16-cell Blastomeres: An Intermediate Stage in Epithelial Differentiation

Maturation of the comprehensive features of cell polarity first observed at compaction continue during 16- and 32-cell cycles in outer blastomeres. At the 16-cell stage, the basolateral membrane of polar cells acquires a more complex molecular

76

T.P FLEMING, E. BUTLER, J. COLLINS, B. SHETH, and A.E WILD

16-cell morula

polar cell division plane

Dff

more

Con

less

Diff

less

Con

more

32-cell early blastocyst

Figure 4.

Diagrammatic representation of the relationship between polar cell division plane and blastocyst tissue sizes. 16-cell morulae vary substantially in the number of outer polar (unshaded) and inner non-polar (shaded) cells present, indicating inter-embryonic variation in the proportion of &cell blastomeres dividing differentiatively. The polar cells within morulae containing relatively few inner cells (top) tend to divide differentiatively (Diff) more frequently and conservatively (Con) less frequently than do the polar cells within morulae containing more inner cells (bottom). This distinction can be accounted for by polar cell shape in srtu and can regulate quantitatively trophectoderm and ICM tissue sizes in the blastocyst. See text for details.

organization. The apicolateral tight junction extends from a series of focal contacts to a discontinuous zonular configuration and becomes more complex in molecular composition (discussed later; Fleming et al., 1989, 1993b; Javed et al., 1993; Sheth et al., 1997). The adherens junction becomes distinct ultrastructurally (Reima, 1990) and may include increased assembly of myosin and actin at the cytoplasmic face (Slager et al., 1992). Calcium-independent adhesion systems have been identified to function in 16-cell morulae, particularly those based on highly branched lactosaminoglycans; their neutralization results in loss of adhesion between blastomeres (Bird and Kimber, 1984; Rastan et al., 1985; Bayna et al., 1988; Fenderson et al., 1990; Kimber, 1990). The extracellular matrix, particularly laminin, is expressed at this time yet its contribution to cell polarity and trophectoderm differentiation remains to be defined (Leivo et al., 1980; Cooper and MacQueen, 1983; Leivo and Wartiovaara, 1989; Thorsteinsdottir, 1992; Hierck et al., 1993; reviewed in Damsky et al., 1993). The laminin receptor component ahintegrin, is expressed on membranes throughout cleavage but does not begin to localize to basal surfaces until laminin A-chain is present, a feature indicative of the formation of the first basement membrane (Hierck et al., 1993).

Cell Polarity and Mouse Early Development

77

Polarity in the cytoplasm is extended such that preferential apical endocytic activity and apical endosome clustering is further enhanced relative to basolateral regions (Fleming and Pickering, 1985). The stability of this polarity is also increased, with the previously microtubular control of endosome distribution being supplemented by microfilament interactions (Fleming et al., 1986b). A secondary lysosome compartment, polarized to the basal cytoplasm, becomes detectable for the first time in this cell cycle (Fleming and Pickering, 1985; Figure 5). Polarized transcytosis via the endosomal compartment and membrane recycling pathways exist at this stage and may be involved in the stabilization of polarized membrane domains (Fleming and Goodall. 1986; Fleming, 1987b). In addition, Golgi bodies (Fleming and Pickering, 1985; Maro et al., 1985), mitochondria and lipid droplets (Wiley and Eglitis, 1981; Batten et a]., 1987) all polarize in the basal cytoplasm, and there is an increase in the assembly of cytokeratin filaments in cytocortical and perinuclear locations (Chisholm and Houliston, 1987; Emerson, 1988).

Figure 5. Maturation of endocytic system in polar cells during trophectoderm differentiation. (A) Secondary lysosomes (L) form during the 14-ceIl stage and polarise in the basal cytoplasm of each polar cell; endosomes (E) remain polarised in the apical cytoplasm. Bar = 5pm (B)Endocytic polarity is also present in blastocyst trophectoderm cells. (C)Trophectoderm cells demonstrate preferential endocytic activity from the apical membrane. Endocytic pathways across the epithelium, as well as recycling and lysosomal pathways (arrowed), all involve obligatory sorting at endosomes, suggesting these embryonic cells are capable of maintaining the distinct composition of apical and basolateral membrane domains. After Fleming and Goodall (1986) and Fleming (1987b).

78

E.

T.P FLEMING, E. BUTLER, J. COLLINS, B. SHETH, and A.E W I L D

Cell Polarity at the 32-cell Stage: Completion of Epithelial Biogenesis

Following division to the 32-cell stage, the polarization process culminates in the formation of the trophectoderm as a discrete epithelium as the blastocoel cavity is generated by vectorial fluid transport. However, cavity formation in the very early blastocyst is believed to involve the intercellular accumulation of water derived from the oxidation of cytoplasmic lipid before vectorial transport, mediated by the activity of Na+, K+-ATPase located on basolateral membranes, takes over (Wiley and Eglitis, 1981, Wiley, 1984). Na+ K+-ATPase activity has been detected along trophectoderm basolateral membranes from the 32-cell stage by ultrastructural cytochemistry (Vorbrodt et al., 1977) and by immunofluorescence microscopy (Watson and Kidder, 1988). In the latter study, thecatalytic nonglycosylated a subunit of Na+,K+-ATPase was first detectable as cytoplasmic foci in late morulae and redistributed to basolateral membranes just at the time cavitation began. However, the a subunit is transcribed from early cleavage (Watson et al., 1990b; Gardiner et al., 1990; MacPhee et al., 1994) and nonfluorescent techniques indicate that low levels of a subunit protein are present well in advance of cavitation (Gardiner et al., 1990; Van Winkle and Campione, 1991). Regulation of Na+, K+ATPase activity and basolateral localization may therefore be achieved by late expression of the glycosylated p subunit in the morula (Gardiner et al., 1990; Watson et al., 1990b; reviewed in Watson, 1992). The importance of basolateral localization of Na+, K+-ATPase in mediating cavitation has been demonstrated by the inhibitory effects of particular ionic conditions and ouabain, a specific inhibitor of the enzyme (DiZio and Tasco, 1977; Wiley, 1984; Manejwala et al., 1989). Culture media ion substitution experiments implicated Na+ (and not C l - ) ions as the major contributors to the osmotic gradient that drives water across the trophectoderm (Manejwala et al., 1989). Use of specific inhibitors suggests that transport of Na' into trophectoderm cells is carrier-mediated and may involve several apical routes of entry including Na+, K+ exchangers and Na+ channels (Manejwala et al., 1989). Na+-coupled amino acid and glucose transporters, though present, are not thought to play a significant role in blastocoel formation (DiZio and Tasco, 1977; Manejwala et al., 1989; Wiley et al., 1991). Physiological regulation of Na+,K+-ATPaseactivity at cavitation is mediated by CAMP since experimental elevation of this intracellular signaling pathway stimulates both Na+ uptake by trophectoderm and the rate of blastocoel accumulation (Manejwala et al., 1986; Manejwala and Schultz, 1989). Other important steps in the maturation of the polarized trophectoderm phenotype occur during the 32-cell stage and contribute to the functional capacity of the epithelium to engage in vectorial transport processes. Most significantly, tightjunctions become fully zonular in organization in freeze fracture replicas and functional as a permeability seal (Ducibella et al. 1975; Magnuson et al., 1977; Pratt, 1985; see later). The completion of tight junction formation in trophectoderm coincides with

Cell Polarity and Mouse Early Development

79

the polarized distribution of a number of membrane proteins involved in vectorial transport processes. In addition to Na+, K+-ATPase(see above), the Na+-independent GLUT2 glucose cotransporter is localized on trophectoderm basolateral membranes (Aghayan et al., 1992). This transporter may function in regulating glucose delivery to the blastocoel and ICM, demonstrating a further vectorial transport role for the polarized trophectoderm in controling the metabolic requirements of the ICM (Hewitson and Leese, 1993; Brison et al., 1993). This role is further served by an increase in the rate of endocytic activity in the trophectoderm compared with earlier stages, with endocytosis occurring preferentially at the apical surface (Fleming and Pickering, 1985; Fleming and Goodall, 1986; Pemble and Kaye, 1986; Figures 5B,C). Coordinated with endocytic polarization, the trophectoderm also engages in polarized secretion of polypeptides at apical (uterine) and basal (blastocoel) surfaces (Dardik and Schultz, 1991), an activity that is enhanced by transforming growth factor a (Dardik et al., 1993). The membrane distribution of growth factor receptors is also polarized in trophectoderm cells with the EGF receptor localized preferentially in the apical membrane (Wiley et al., 1992; Adamson, 1993) and the insulin receptor in the basolateral membrane (Heyner et al., 1989; Smith et al., 1993). These and other receptors interact with their ligands causing enhancement of trophectoderm metabolic, vectorial, and endocytic activity, including transcytosis of growth factors and their stimulation of ICM proliferation andmetabolism (Heyner et al., 1989; Harvey and Kaye, 1990, 1992; Kaye et al., 1992; Brice et al., 1993; Dunglison and Kaye, 1993; Smith et al., 1993; Shi et al., 1994). Desmosome formation, together with the assembly of major desmosomal cadherin glycoproteins and plaque proteins, occurs for the first time at punctate sites between apposed trophectoderm basolateral membranes at the 32-cell stage (Fleming et al., 1991; Collins et al., 1995; discussed later). These junctions associate with cytokeratin filaments synthesized within the cytoplasm and form i n particular when blastocoel accumulation is underway, indicating a role in stabilizing the new epithelium from stresses imposed by the expansion of the blastocoel (Fleming et al., 1991). Cavitation also coincides with increased laminin and type I11 collagen expression and deposition into basement membrane (Leivo et al., 1980; Sherman et al., 1980; Hierck et al., 1993). Recently, we have shown that a high molecular weight (330-380 kDa) membrane glycoProtein with certain characteristics of gp330 of the Heymann nephritis antigen complex (Orlando et al., 1992) and recognized by the monoclonal antibody 283D3 (Meads and Wild, 1993) is expressed along trophectoderm apical membranes from the 32-cell stage and relocates into the apical endosomal compartment from the time that blastocoel fluid accumulates; inhibition of cavitation by ouabain treatment disturbs 283D3 antigen redistribution (E. Butler, A. Wild, and T. Fleming, manuscript i n preparation). Although the precise function of this glycoprotein has yet to be defined, its activity provides a further example of cavitation coinciding with spatial reorganizaiion of blastomeres.

80

111.

T.f? FLEMING, E. BUTLER, J. COLLINS, B. SHETH, and A.E WILD

MOLECULAR ASPECTS OF CELL POLARITY AND TISSUE SEGREGATION

Cell biological studies of cell polarity and its contribution to the differentiation and diversification of tissues in the preimplantation embryo have demonstrated the following: (a) cell polarity, although programmed independently of cell interactions, is dependent upon asymmetric adhesive cell contacts to define the orientation of its axis; (b) cell polarity is a stable state and represents the proto-epithelial phenotype that is extended and elaborated during late cleavage, culminating in the formation of the polarized trophectoderm epithelium; and (c) cell polarity is utilized by the embryo as a mechanism to generate phenotypically distinct cell subpopulations by differentiative division. These cell populations express many identical proteins but two-dimensional gel electrophoresis or immunolocalization studies have demonstrated that some polypeptides are specific to either trophectoderm or ICM, or their polar and nonpolar progenitors in 16-cell morulae (Van Blerkom et al., 1976; Handyside and Johnson, 1978; Johnson, 1979; Slager et al., 1991). The glycogen content of the two tissues is also distinct (Edirisinghe et al., 1984). In order to identify how these basic cellular mechanisms might regulate at the molecular level the emergence and differentiation of the two tissues in the blastocyst, in recent years we have focused our studies on the expression and distribution of cell adhesions systems during cleavage. Many elegant studies have demonstrated that the pattern of cell adhesion expression, particularly that of cadherins, has a pivotal role in the formation and segregation of tissues during development (reviewed in Takeichi, 1991). As discussed earlier, cell adhesion mediated by uvomorulinlcatenin is initiated at compaction and continues to operate throughout preimplantation development, causing adhesion between all blastomeres of the blastocyst (Vestweber et al., 1987). Uvomorulinkatenin adhesion is therefore not expressed tissue specifically in the early embryo. A.

Tight Junction

Although analysis of the uvomorulin adhesion system has not contributed to our understanding of tissue divergence, biosynthetic studies on the tight junction adhesion system have been more fruitful in this respect. Recently, it has become apparent that the apicolateral tight junction is a multimolecular complex. It is composed of at least one transmembrane protein, occludin, and several cytoplasmic “plaque” proteins including ZO- 1, localized very close to the membrane domain, and cingulin, located more internally and possibly also interacting with actin filaments (reviewed in Anderson et al., 1993; Citi, 1993;Furuse et al.. 1993). As discussed above, tightjunction formation begins at compaction but is not complete until about the 32-cell stage, approximately 24 hours later, when blastocoel fluid accumulation occurs (reviewed in Fleming et al., 1993a, 1994; Collins and Fleming, 1995). Thus, the intramembraneous component (although not yet identified in molecular terms) assembles at the

Cell Polarity and Mouse Early Development

81

apicolateral region of cellkell contact between %cell blastomeres at compaction (Ducibella and Anderson, 1975; Pratt, 1985), ZO-1 protein assembles at this site about 1-2 hours later (Fleming et al., 1989), and cingulin assembles some 10 hours later again and usually during the 16-cell stage (Fleming et al. 1993b; Figure 6). Fi-

Figure 6.

Tight junction formation and tissue segregation during cleavage. (A) ZO-1 protein (arrows) assembles for the first time at discontinuous sites along the apicolateral margin between &cell blastomeres after compaction has occurred; here shown in two isolated cells. (B) Cingulin protein (arrows) assembles apicolaterally for the first time usually during the 16-cell stage; here shown in cluster of four 16-cell blastomeres (from Fleming et al., 1993b). (C) At the blastocyst stage, ZO-1 (and cingulin, not shown) is distributed as a continuous belt around the apicolateral contact site between trophectoderm cells, here shown en face. (D,E) Blastocyst viewed by confocal brightfield and fluorescent imaging showing ZO-1 staining essentially restricted to the trophectoderm layer (arrowheads) and absent from ICM (I). Bar = 20pm (A,B same mag).

82

T.P FLEMING, E. BUTLER, J. COLLINS, B. SHETH, and A.E WILD

nally, a new splice variant of ZO- 1 is transcribed for the first time at approximately the 32-cell stage and may have important implications for the functional activity of the tight junction. This isoform, ZO-la+, assembles at the tight junction at the early 32cell stage after intracellular association with occludin; the two proteins assembly at the membrane as a complex (Sheth et al., 1997). This event appears to complete tight junction formation such that blastocoel fluid accumulation occurs almost immediately afterward. The sequential nature of the molecular assembly of the junction appears to be coordinated by sequential expression of the proteins involved. Thus, synthesis of ZO-1 (now known to the ZO1 a-isoform) is detectable before synthesis of cingulin from the embryonic genome (Fleming et al., 1989; Javed et al., 1993), and then finally Z O - l a + (Sheth et al., 1997). Significantly, tight junction assembly is specific to the polar cell lineage generating the trophectoderm and does not occur in the ICM. Thus, both ZO- 1 isoforms and cingulin are detectable immunocytochemically in trophectoderm and are essentially absent from the ICM (Figures 6C-E, but see below). However, since the expression of tightjunction constituents spans the period during which differentiative divisions give rise to these blastocyst tissues, there is the opportunity to investigate, in biogenetic terms, the basis of tight junction tissue specificity. We have utilized synchronized clusters of isolatTd blastomeres to determine whether ZO- 1 is inherited by one or both daughter cells following differentiative division (Fleming and Hay, 1991). We have shown that at 8- to 16-cell and at 16- to 32-cell cycles, ZO-I a-isoform is inherited by both polar and nonpolar daughter cells following division, ruling out a mechanism based upon differential inheritance to explain trophectoderm-specificity of tight junction formation (Figure 7). In such polar: nonpolar cell clusters, putative apicolateral tight junction contacts containing ZO-1 are transient and remain intact only as long as the nonpolar cell retains a contact-free membrane face. Once this is lost, as occurs in intact embryos as nonpolar cells become internalized and surrounded by polar cells, then the tight junction link between polar: nonpolar sister cells is rapidly degraded with ZO-1 fragmenting into a series of randomly distributed membrane-associated foci before disappearing altogether (Fleming and Hay, 1991; Figure 7). Conversely, po1ar:polar daughter cells of conservative divisions (from 8- to 16-cell cycles onwards) establish stable apicolateral tight junctions containing ZO- 1 .This can be explained by their retention of a contact-free membrane face, the nonadhesive apical membrane domain, which ensures that they remain in an outer position in the intact embryo. The capacity to assemble a stable multimolecular tightjunction only in the outer epithelial lineage therefore appears to be regulated by cell position, (interpreted by cell contact asymmetry) rather than by a mechanism involving differential inheritance. However, it should be noted that cell positionperse is in fact regulated by differential inheritance of the apical polar domain (see earlier). A positional model to explain tight junction tissue specificity is supported by experiments in which ICMs are immunosurgically isolated from early blastocysts and cultured in vitro. Here, the outer ICM cells, now in an outer position and experiencing a contact-free sur-

Cell Polarity and Mouse Early Development

83

figure 7. ZO-I localization following differentiative division of polar &cell biastomeres (after Fleming and Hay, 1991). (A,B) 2/16 couplet 5 hours after differentiative division stained with TRITC-concanavalin A (A) to identify polar cell (left, with microvillous pole) and with anti ZO-1 antibody (B) showing membrane assembly at the contact site (arrow). Here, the non-polar cell still retains a contact-free membrane face. (C,D) 2/16 couplet 8 hours after differentiative division; TRITC-concanavalin A staining (C) identifies the surface of the polar cell which has now entirely enveloped the non-polar cell seen in (D)which shows ZO-1 at the contact site between cells (arrows). Here, ZO-1 appears fragmented and soon disappears since the nonpolar cell no longer possesses a contact-free membrane face. (E,F) Early 4/16 cell cluster comprising three outer polar cells and one central nonpolar cell (derived from two &cell sister blastomeres, one dividing differentiatively, the other conservatively). The nonpolar cell isstill not fully enveloped and retains a contact-free membrane face and displays ZO-1 at contact sites with polar cells (arrows). ( G I ) Later 4/16 cell cluster comprising two outer polar cells and two inner nonpolar cells (derived from two &cell blastomeres, both having divided differentiatively). Here, both nonpolar cells are now fully enclosed and are negative for ZO-1 which is found only at contact sites between polar cells (arrows) shown en face (H) and in midsection (I) of cluster. Bar = 1 Oprn.

face, rapidly reassemble an apicolateral tight junction belt containing ZO-1 (Fleming and Hay, 1991). We next investigated the effect that cell position might have on the synthesis of tight junction-associated proteins. We have shown by immunoprecipitation that

84

T.P FLEMING, E. BUTLER, J. COLLINS, B. SHETH, and A.E WILD

cingulin synthesis in metabolically-labeled blastocysts is significantly greater, up to a 15-fold difference, in the trophectoderm than the ICM (Javed et al., 1993). Thus, it appears that loss of cell contact asymmetry in the ICM lineage not only leads to loss of assembly competence but also to down-regulation of expression of tightjunction components. Recently, we have analyzed whether the reduction in expression of tight junction proteins in the ICM was regulated by transcriptional or translational mechanisms. Utilizing the RT-PCR technique, transcripts for both isoforms of ZO-1 were detected in both trophectoderm and ICM of early blastocysts, suggesting that the reduction in ICM expression is controlled by reduction in mRNA translation (Sheth et al., 1997). Moreover, the reformation of a ZO-1containing tightjunction network in outer cells of isolated ICMs in culture is insensitive to a-amanitin treatment, confirming that new transcription is not required for this up-regulation event (Fleming and Hay, 1991). Why might transcripts for tight junction proteins be retained within the ICM? Presumably such transcripts (as well as protein, see above) will be inherited by differentiative divisions during the morula stage, but we have not yet established whether ICM nuclei in situ engage in transcription of mRNA for tight junction constituents. We suspect that these transcripts may serve a role in the developmental program undertaken by the ICM. In the late blastocyst, a new epithelium, the primary endoderm (progenitor of extra-embryonic parietal and visceral endoderm tissues), is delaminated at the blastocoelic face of the ICM and typically contains tight junctions (Nadijcka and Hillman, 1975; reviewed in Gardner and Beddington, 1988). We have proposed that the same pool of transcripts may be utilized for the expression of tight junctions in both trophectoderm and primary endoderm epithelia (Fleming and Hay, 1991; Fleming et al., 1993a). Thus, during blastocyst expansion, this pool would be maintained in a state of low translation by the presence of cellular processes, derived from nearby trophectoderm cells, that cover the blastocoelic face of the ICM and prohibit the formation of contact-free membrane surfaces at this site (Fleming et al., 1984; shown in Figures 3 and 4). These processes withdraw in the late blastocyst, concommitant with the differentiation of primary endoderm and up-regulation of tight junction expression and assembly. Taken together, our analysis of the tight junction adhesion system during blastocyst formation has demonstrated that cell polarity plays a significant role in its maturation and tissue specificity. The sequential pattern of tight junction protein expression and membrane assembly is dependent upon the continued presence of a contact-free cell surface provided by the nonadhesive apical membrane domain. The loss of such a domain, as occurs in cells entering the ICM lineage, leads to reduction or cessation in synthesis of tight junction proteins and their disassembly at membrane contact sites. This down-regulation is reversible if a contact-free surface is reestablished. However, these dramatic changes in biosynthetic events do not appear to be coordinatined with, or dependent upon, changes in transcriptional activity of tight junction constituents.

Cell Polarity and Mouse Early Development

B.

85

Desmosomes

The formation of disc-shaped desmosome adhesive junctions represents a late maturation step in the cell polarization program underlying trophectoderm differentiation and occurs during the 32-cell stage Just as tight junction formation is completed and blastocoel fluid accumulation has initiated (see above). The principal components of desmosome junctions are two adhesive transmembrane glycoproteins belonging to the cadherin superfamily (desmoglein and desmocollins) and three associated cytoplasmic proteins (plakoglobin, desmoplakin I and 11) which together form a plaque into which cytokeratin filaments insert (reviewed in Schwarz et al., 1991; Buxton and Magee, 1992; Garrod, 1993). Like tight junctions, desmosomes are trophectoderm-specific in the blastocyst (Ducibella et al., 1975; Magnuson et a]., 1977; Fleming et al., 1991) but appear to have a different mechanism for regulating their initial construction. Metabolic labeling and immunoprecipitation analysis of carefully-staged preimplantation embryos has revealed that the desmosomal plaque constituents are first synthesized during cleavage (8- and 16-cell stages) and in advance of the desmosomal cadherins (32-cell stage). However, all major constituents first assemble along apposed trophectoderm membrane contact sites at approximately the same time (32-cell stage) when desmosomes first form (Fleming et al., 199 1). These biogenetic characteristics indicate that the initiation of desmosoma1 cadherin expression may regulate the timing of desmosome formation which may utilize preexisting nonassembled plaque components. Desmosome formation therefore occurs rapidly, within a single cell cycle, and is mediated by the delay in availability of the membrane-spanning components whereas the tight junction is formedprogressively with cytoplasmic plaque constituents assembling in a sequential manner during cleavage. These differing strategies may reflect the difference in shape, size, and morphology of these junction types (discussed in Fleming et al., 1993a) but ensure that they become functional at about the same time, as the trophectoderm vectorial transport activity gets underway. The biogenetic control of the timing of desmosome formation i n the early embryo has been studied further by analyzing the timing of transcription of desmocollin, the desmosomal cadherin that has been implicated a significant role in desmosome adhesion. The two alternatively-spliced variants (a and b; Collins et al., 1991) of the mouse desmocollin gene 2 (DSC2) are transcribed coordinately from the embryonic genome beginning at the late 16-cell stage or the early 32-cell stage (Collins et al., 1995; Figure 8A). This transcriptional event correlates well with the onset of desmocollin translation (Fleming et al., 199 1 ; see above). Taken together, our data strongly suggests that desmosome formation in trophectoderm is controled by desmocollin transcriptional activation.

86

T.f? FLEMING, E. BUTLER, J. COLLINS, B. SHETH, and A.E WILD

Reverse transcriptase PCR amplification of DSC2 (desmocollin)transcripts in embryos and blastomeres. (A) Lanes labeled 1 are DNA amplified from single 16-cell morulae showing either negative or weak positive signal indicative of the initiation of DSC2 transcription from the embryonic genome (confirmed by sequencing of product). 3 , three 16-cell morulae, B, single early blastocyst, where more product has been amplified; C, control samples minus template but with complete reaction. Arrowheads adjacent to marker lanes indicate 564bp. (B) DSC2 transcript detection in single trophectoderm (T) or ICM ( I ) cells derived from early blastocysts. Trophectoderm cells consistently demonstrate the presence of DSC2 mRNA whereas only a minority of ICM cells do. B, single intact blastocyst; C, controls minus template as above. Arrowheads adjacent to marker lanes indicate 600 bp. After Collins et al. (1 994).

Figure 8.

The mechanism regulating desmosome tissue specificity has also been investigated. The presence of DSC2 mRNA is detectable by a sensitive modification of the RT-PCR technique within single blastomeres of known phenotype isolated from early blastocysts. This analysis has demonstrated that all trophectoderm cells contain DSC2 mRNA but in most ICM cells (approximately 75%) the transcript is not detectable despite the reliable identification of uvomorulin mRNA in all cells irrespective of their phenotype (Collins et al., 1995; Figure 8B). Desmosome tissue specificity in the early embryo can best be explained therefore by differential transcription of the desmocollin gene in the early blastocyst. Moreover, the detection of DSC2 mRNA in a minority of ICM cells need not represent “leakiness” in regulation of transcriptional activity. This proportion of ICM cells (25%) is exactly the average proportion known to be allocated from the polar lineage following the second round of differentiative divisions at the 16- to 32-cell stage (Fleming, 1987a). Since DSC2 transcription may just precede this division cycle, the ICM DSC2 mRNA pool is presumably generated by inheritance from outer polar cells rather than by inherent transcription. Significantly, isolation and culture of ICMs from early blastocysts leads to a substantial increase in the level of DSC2 mRNA detected and to the expression and assembly of desmocollin protein at putative desmosomes present between outer cells (Collins et al., 1995).Thus, the positional signal of the presence or absence of a contact-free membrane face identified to regulate tight junction protein expression also appears to operate to regulate desmocollin transcription and translation.

Cell Polarity and Mouse Early Development

IV.

87

CONCLUSIONS

Cell polarity established at compaction in the mouse embryo can be viewed as the foundation of several interrelated processes governing blastocyst formation. Blastomere polarization is likely to be of widespread importance in the early development of eutherian mammals since it occurs in several species other than the mouse (e.g., Koyama et al., 1994). In the mouse, it is a stable state in that polarized cells have not been shown to lose their polarity even in isolation. In the intact embryo, this stability is conducive with the polar lineage maintaining cell position and orientation, and in acquiring further structural and molecular features of polarity with time, such as occurs in intercellular junction formation. The molecular nature of this stability is unknown, but given the relative importance of the cytocortex over cytoplasmic domains of the cell in establishing and maintaining polarity, the apical membrane and cortex may be regarded as the most likely center where stability is controlled. The microvilli of the apical pole, unlike others on blastomere membranes, are not disrupted by prolonged cytochalasin treatment (Pratt et al., 1982; Fleming et al., 1986a), which suggest the actin bundles that structure them are biogenetically stable, turning over very slowly. The composition and molecular organization of actin-associated proteins in the apical cortex may therefore be important in conferring stability to cell polarity (see Johnson et al., 1988). In addition to providing a stable "framework" upon which the polarized cellular organization of the trophectoderm can be manifest, cell polarity also regulates tissue divergence by differentiative division. Our studies on cell adhesion maturation have identified that the presence or absence of a contact-free membrane surface on blastomeres controls up- or down-regulation of gene and protein expression which in turn underlies tissue divergence. It appears, therefore, that the pattern of blastomere biogenesis is controlled by whether or not it inherits part or all of the apicaI cortical domain which will ensure the continuance of a contact-free cell surface. Our next task will be to unravel the molecular signaling pathway leading from cellular interaction pattern to gene expression pattern.

ACKNOWLEDGMENTS We are grateful to the Wellcome Trust, the Medical Research Council, the Science and EngineeringResearch Council, and the Wessex Medical Trust for funding of research in our laboratory and for provision of studentships. We thank many collaborators for gifts of precious antibodies, Sue Pickeringfor scanningEM micrographs,and Mark Hay for his skill in generating the computerized diagrams.

REFERENCES Adamson, E.D. (1993).Activities of growth factors in preimplantation embryos. J. Cell, Biochem. 53, 280-287.

88

T.P FLEMING, E. BUTLER, J. COLLINS, B. SHETH, and A.E WILD

Aghayan, M., Rao, L.V., Smith, R.M., Jarett, L., Charron, M.J., Thorens, B. & Heyner, S. (1992). Developmental expression and cellular localization of glucose transporter molecules during mouse preimplantation development. Development 115,305-312. Aghion, J., Gueth-Hallonet, C., Anthony, C., Gros, D. & Maro, B. (1994). Cell adhesion and gap junction formation in the early mouse embryo are inducedprematurelyby 6-DMAP in the absence of E-cadherin phosphorylation. J. Cell Sci. 107, 1369-1379. Anderson, J.M., Balda, M.S. & Fanning, A S . (1993). The structure and regulation of tight junctions. Curr. Opin. Cell Biol. 5, 772-778. Balakier, H. & Pedersen, R.A. (1982). Allocation ofcells to inner cell mass and trophectoderm lineages in preimplantation mouse embryos. Dev. Biol. 90,352-362. Batten, B.E., Albertini, D.F. & Ducibella, T. (1987). Patterns of organelle distribution in mouse embryos during preimplantation development. Amer. J. Anat. 178,204213. Bayna, E.M., Sharper, J.H. & Shur, B. (1988). Temporally specific involvement of cell surface 8-1,4 galactosyltransferase during mouse embryo morula compaction, Cell 53, 145-157. Bird, J.M. & Kimber, S.J. (1984). Oligosaccharides containing fucose linked a(1-3) and a(I-4) to N-acetylglucosamine cause decompaction of mouse morulae. Dev. Biol. 104,449460. Blaschuk, O.W., Sullivan, R., David, S. & Pouliot Y. (1990). Identification of a cadherin cell adhesion recognition sequence. Dev. Biol. 139,227-229. Bloom, T.L. (1989). The effects of phorbol ester on mouse blastomeres: a role for protein kinase C in compaction? Development 106, 159-171. Bloom, T.L. (1991). Experimental manipulation of compaction of the mouse embryo alters patterns of protein phosphorylation. Molec. Reprod. Devel. 28, 23g244. Bloom, T.L. & McConnell, J. (1990). Changes in protein phosphorylation associated with compaction of the mouse preimplantation embryo. Molec. Reprod. Devel. 26, 199-210. Brice, E.C., Wu, J.X., Muraro, R., Adamson, E.D. & Wiley, L.M. (1993). Modulation of mouse preimplantation development by epidermal growth factor receptor antibodies, antisense RNA, and deoxyoligonucleotides. Devel. Genet. 14, 174-1 84. Brison, D.R., Hewitson, L.C. & Leese, H.J. (1993). Glucose, pyravate, and lactate concentrations in the blastocoel cavity of rat and mouse embryos. Molec. Reprod. Devel. 35,227-232. Buxton, R.S. & Magee, A.I. (1992). Structure and interactionsofdesmosomal and other cadherins. Sem. Cell Biol. 3, 157-167. Chisholm, J.C. & Houliston, E. (1987). Cytokeratin filament assembly in the preimplantation mouse embryo. Development 101,565-582. Citi, S. (1993). The molecular organization of tightjunctions. J. Cell Biol. 121,485489. Clayton, L., Stinchcombe, S.V. & Johnson, M.H. (1993). Cell surface localisation and stability of uvomorulin during early mouse development. Zygote 1,333-344. Collins, J.E. & Fleming, T.P. (1995). Epithelial differentiation in the mouse preimplantation embryo: Making adhesive cell contacts for the first time. Trends. Biochem. Sci. 20, 307-312. Collins, J.E., Legan, P.K., Kenny, T.P., Macgarvie, J., Holton, J.L. & Garrod, D.R. (1991). Cloning and sequencing of desmosomal glycoproteins 2 and 3 (desmocollins): cadherin-like desmosomal adhesion molecules with heterogeneous cytoplasmic domains. J. Cell Biol. 113,381-391. Collins, J,E., Lorimer, J.E., Garrod, D.R., Pidsley, S., Buxton, R. & Fleming, T.P. (1995). Regulation of desmocollin transcription in mouse preimplantation embryos. Development l 2 t , 743-753. Cooper, A.R. & MacQueen, H.A. (1983). Subunits of laminin are differentially synthesised in mouse eggs and early embryos. Dev. Biol. 96,467471. Cruz, Y.P. (1992). Role of ultrastructural studies in the analysis of cell lineage in the mammalian pre-implantation embryo. Microsc. Res. Tech. 22, 103-125. Damsky, C., Sutherland, A. & Fisher, S. (1993). Extracellular matrix 5: adhesive interactions in early mammalian embryogenesis, implantation, and placentation. FASEB J. 7, 1320-1329.

Cell Polarity and Mouse Early Development

89

Dardik, A. & Schultz, R.M. (1991). Protein secretion by the mouse blastocyst: differences in the polypeptide composition secreted into the blastocoel and medium. Biol. Reprod. 45,328-333. Dardik, A,, Doherty, A.S. & Schultz, R.M. (1993). Protein secretion by the mouse blastocyst: stimulatory effect on secretion into the blastocoel by transforming growth factor-a. Molec. Reprod. Devel. 34,396401. DiZio, S.M. & Tasca, R.J. (1977). Sodium-dependentaminoacid transport in preimplantationembryos. 111. Na+, K+-ATPase linked mechanism in blastocysts. Dev. Biol. 59, 198-205. Ducibella, T. & Anderson, E. (1975). Cell shape and membrane changes in the 8-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. Ducibella, T., Ukena, T., Karnovsky, M. & Anderson, E. (1977). Changes in cell surface and cortical cytoplasmic organization during early embryogenesis in the preimplantation mouse embryo. J. Cell Biol. 74, 153-167. Dunglison, G.F. & Kaye, P.L. (1993). Insulin regulates protein metabolism in mouse blastocysts. Molec. Reprod. Devel. 36, 4 2 4 8 . Edirisinghe, W.R., Wales, R.G. &Pike, I.L. (1984). Studies ofthe distributionofglycogen between the inner cell mass and trophoblast cells of mouse embryos. J. Reprod. Fert. 71, 533-538. Emerson, J.A. (1988). Disruption of the cytokeratin filament network in the preimplantation mouse embryo. Development 104,219-234. Fenderson, B.A., Eddy, E.M. & Hakomori, S. (1990). Glycoconjugate expression during embryogenesis and its biological significance. BioEssays 12, 173-179. Flach,G., Johnson,M.H.,Braude,P.R. ,Taylor,R. &Bolton,V.N. (1982).Thetransitionfrommaternal to embryonic control in the 2-cell mouse embryo. EMBO J. 1,681-686. Fleming, T.P. (1987a). A quantitative analysis ofcell allocation to trophectoderm and inner cell mass in the mouse blastocyst. Dev. Biol. 119, 520-531. Fleming, T.P. (1987b). Endocytosis and epithetial biogenesis in the mouse early embryo. BioEssays 4, 105-109. Fleming, T.P. (1992). Trophectoderm biogenesis in the preimplantation mouse embryo. In: Epithelial Organization and Development (T.P. Fleming, Ed.), pp. 1 1 1-136. Chapman and Hall, London. Fleming, T.P. & Goodall, H. (1986). Endocytic traffic in trophectoderm and polarised blastomeres of the mouse preimplantation embryo. Anat. Rec. 216,490-503. Fleming, T.P. & Hay, M.J. (1991). Tissue-specific control of expression of the tight junction polypeptide ZO-1 in the mouse early embryo. Development 113,295-304. Fleming, T.P. & Pickering, S.J. (1985). Maturationand polarizationofthe endocytoticsystem in outside blastomeres duringmouse preimplantation development. J. Embryol.exp. Morph. 89,175-208. Fleming, T.P., Warren, P.D., Chisholm, J.C. & Johnson, M.H. (1984). Trophectodermal processes regulate the expression of totipotency within the inner cell mass of the mouse expending blastocyst. J. Embryol. exp. Morph. 84, 63-90. Fleming, T.P., Pickering, S.J., Qasim, F. & Maro, B. (1986a). The generation ofcell surface polarity in mouse 8-cell blastomeres: the role of cortical microfilaments analysed using cytochalasin D. J. Embryol. exp. Morph. 95, 169-191. Fleming, T.P ,Cannon, P M. & Pickering, S.J. (1986b). The cytoskeleton, endocytosis and cell polarity in the mouse preimplantation embryo. Dev. Biol. I 13,406419. Fleming, T.P., McConnell, J., Johnson, M.H. &Stevenson, B.R. (1989). Development oftightjunctions de novo in the mouse early embryo: control of assembly of the tight junction-specific 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 1 12,527-539.

90

T.P FLEMING, E. BUTLER, J. COLLINS, B. SHETH, and A.E WILD

Fleming, T.P., Javed, Q. & Hay, M. (1992). Epithelial differentiation and intercellular junction formation in the mouse early embryo. Development supplement 105-1 12. Fleming, T.P., Javed, Q., Collins, J. & Hay M. (l993a). Biogenesis of structural intercellularjunctions during cleavage in the mouse embryo. J. Cell Science supplement 17, 119-125. Fleming, T.P., Hay, M.J., Javed, Q. & Citi, S. (1993b). Localization oftightjunction protein cingulin is temporally and spatially regulated during early mouse development. Development 117, 1135-1 144. Fleming, T.P., Butler E., Lei, X., Collins, J., Javed, Q., Sheth, B., Stoddart, N., Wild, A. & Hay, M. (1994). Molecular maturation of cell adhesion systems during mouse early development. Histochemistry 101, 1-7. Furuse, M., Hirase, T., Itoh, M., Nagahchi, A., Yonemura, S., Tsukita, S. & Tsukita, S. (1993). Occludin: a novel integral membrane protein localizing at tight junctions. J. Cell Biol. 123, 1777-1788. Garbutt, L.C., Johnson, M.H. &George, M.A. (1987). When and how does cell division order influence cell allocation to the inner cell mass of the mouse blastocyst? Development 100,325-332. Gardiner, C.S., Williams, J.S. & Menino, A.R. (1990). Sodium/potassium adenosine triphosphatase aand p-subunit and a-subunit mRNA levels during mouse embryo development in vifro. Biol. Reprod. 43,788-794. Gardner, R.L. & Beddington, R.S.P. (1988). Multi-lineage “stem” cells in the mammalian embryo. J. Cell Sci. supplement 10, 11-27. Garrod, D.R. (1993). Desmosomes and hemidesmosames. Curr. Opin. Cell Biol. 5 , 3 W O . Geiger, B. & Ayalon, 0. (1992). Cadherins. Ann. Rev. Cell Biol. 8,307-332. Goodall, H. & Johnson, M.H. (1984). The nature of intercellular coupling within the preimplantation mouse embryo. J. Embryol. exp. Morph. 79, 53-76. Grunwald, G.B. (1993). The structural and functional analysis of cadherin calcium-dependent cell adhesion molecules, Curr. Opin. Cell Biol. 5 , 797-805. Gueth-Hallonet, C. & Maro, B. (1992). Cell polarity and cell diversification during early mouse embryogenesis. Trends Genet. 8,274279. Handyside, A.H. (1980). Distribution of antibody- and lectin-binding sites on dissociated blastomeres of mouse morulae: evidence for polarization at compaction. J . Embryol. exp. Morph. 60,99-116. Handyside, A.H. & Johnson, M.H. (1978). Temporal and spatial patterns of the synthesis of tissue-specific polypeptides in the preimplantation mouse embryo. J. Embryol. exp. Morph. 44, 191-199. Harvey, M.C. & Kaye, P.L. (1990). Insulin increases the number ofthe inner cell mass and stimulates morphological development of mouse blastocysts in vifro. Development 110,963-967. Harvey, M.C. & Kaye, P.L. (1992). IGF-2 stimulates growth and metabolism ofearly mouse embryos. Mechanisms Devel. 38, 169-174. Herbert, J.M., Augereau, J.M., Gleye, J. & Maffrand, J.P. (1990). Chelerythrine is apotentand specific inhibitor of protein kinase C. Biochem. Biophys. Res. Commun. 172,993-999. Hewitson, L.C. & Leese, H.J. (1993). Energy metabolism of the trophectoderm and inner cell mass of the mouse blastocyst. J. Exp. Zool. 267,337-343. Heyner, S., Rao, L.V., Jarett, L. & Smith, R.M. (1989). Preimplantation mouse embryos internalize maternal insulin via receptor-mediated endocytosis: pattern of uptake and functional correlations. Dev. Biol. 134,48-58. Hierak, B.P., Thorsteinsdottir, S., Niessen, C.M., Freund, E., Iperen, L.V., Feyen, A,, Hogervorst, F., Poelmann, R.E., Mummery, C.L. & Sonnenberg, A. (1 993). Variants ofthe a6pIlaminin receptor in early murine development: distribution, molecular cloning and chromosomal localization of the mouse integrin a6subunit. Cell Adhesion and Regulation 1,33-53. Hillman, N., Sherman, M.1. & Graham, C. (1972). The effect of spatial arrangement on cell determination during mouse development. J. Embryol. exp. Morph. 28, 263-278.

Cell Polarity and Mouse Early Development

91

Houliston, E. & Maro, B. (I 989). Posttranslational modification of distinct microtubule subpopulations during cell polarization and differentiation in the mouse preimplantation embryo. J. Cell Biol. 108,543-551. Houliston, E., Pickering, S.J. & Maro, B. (1987). Redistribution of microtubules and pericentriolar material during the developmentof polarity inmouse blastomeres. J. Cell Biol. 104,1299-1 308. Hyafil, F., Morello, D., Babinet, C. & Jacob, F. (1980). A cell surface glycoprotein involved in the compaction of embryonal carcinoma cells and cleavage stage embryos. Cell 21,927-934. 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 I 17,1145-1 I5 I. Johnson, M.H. (1979). Molecular differentiation of inside cells and inner cell masses isolated from the preimplantation mouse embryo. J. Embryol. exp. Morph. 53,335-344. Johnson, M.H. & Maro, B. (1 984). The distribution of cytoplasmic actin in mouse 8-cell blastomeres. J. Embryol. exp. Morph. 82,97-I 17. Johnson, M.H. & Maro, B. (1985). A dissection ofthe mechanisms generating and stabilisingpolarity in mouse 8- and 16-cell blastomeres: the role ofcytoskeletal elements. J. Embryol. exp. Morph. 90, 3 11-334. Johnson, M.H. & Ziomek, C.A. (1981a). Induction of polarity in mouse 8-cell blastomeres: specificity, geometry and stability. J. Cell. Biol. 91, 303-308. Johnson, M.H. & Ziomek, C.A. (198 1b). The foundation of two distinct cell lineages within the mouse morula. Cell 24, 71-80. Johnson, M.H. & Zionek, C.A. (1983). Cell interactions influence the fate of mouse blastomeres undergoing the transition from the 16- to the 32-cell stage. Dev. Biol. 95,211-218. Johnson, M.H., Maro, B. & Takeichi, M. (1986). The role of cell adhesion in the synchronization and orientation of polarization in 8-cell mouse blastomeres. J. Embryol. exp. Morph. 93,239-255. Johnson, M.H., Pickering, S.J., Dhiman, A,, Radcliffe, G.S. & Maro, B. (1988). Cytocortical organisation during natural and prolonged mitosis in mouse 8-cell blastomeres Development 102, 143-158. Kaye, P.L.,Bell, K.L., Beebe, L.F.S., Dunglison, G.F., Gardner,H.G. & Harvey, M.B. (1992). Insulin and IGFs in preimplantation development. Reprod. Fertil. Devel. 65,367-375. Kidder, G.M. & McLachlin, J.R. (1985). Timing of transcription and protein synthesis underlying inorphogenesis in preimplantation mouse embryos. Dev. Biol. 1 12,265-275. Kimber, S.J. (1990). Glycoconjugates and cell surface interactions in pre- and peri-implantation mammalian embryonic development. Int. Rev. Cytol. 120, 53-167. Kimber, S.J., Surani, M.A.H. & Barton, S.C. (1982). Interactions ofblastomeres suggest changes in cell surface adhesiveness during the formation of inner cell mass and trophectoderm in the preimplantation mouse embryo. J. Embryol. exp. Morph. 70, 133-152. Kimber, S.J., Bentley, J., Ciemerych, M., Moller, C.J. & Bock, E. (1994). Expression of N-CAM in fertilized pre- and peri-implantation and parthenogenetically activated mouse embryos. Eur. J. Cell Biol. 63, 102-1 13. Lehtonen, E., Ordonez, G. & Reima, I. (1988). Cytoskeleton in preimplantation mouse development. Cell Differentiation 24, 165-1 78. Leivo, 1. & Wartiovaara, J. (1989). Basement membrane matrices in mouse embryogenesis, teratocarcinoma differentiation and in neuromuscular maturation. Int. J. Dev. Biol. 33, 81-89. Leivo, I., Vaheri, A., Timpl, R. & Wartiovaara, J. (1980). Appearance and distribution ofcollagens and laminin in the early mouse embryo. Dev. Biol. 76, 1OC-114. &Maro,B. (1986). Control ofthetimingofcompaction: amajor Levy, J.B.,Johnson,M.H.,Goodall,H. developmental transition in mouse early development. J. Embryol. exp. Morph. 95,213-237. LO,C.W. & Gilula,N.B. (1979). Gap junctional communication in the preimplantationmouse embryo. Cell 18, 399409. MacPhee, D.J., Barr, K.J., De Sousa, P.A., Todd, S.D.L. &Kidder, G.M. (1994). RegulationofNa+, K+-ATPase alpha subunit gene expression during mouse preimplantation development. Dev. Biol. 162,259-266.

92

T.P FLEMING, E. BUTLER, J. COLLINS, 6.SHETH, and A.E WILD

Magnuson, T., Demsey, A. & Stackpole, C.W. (1977). Characterization of intercellularjunctions in the preimplantation mouse embryo by freeze-fracture and thin-section electron microscopy. Dev. Biol. 61, 252-261. Manejwala, F.M. & Schultz, R.M. (1989). Blastocoel expansion in the preimplantation mouse embryo: stimulation of sodium uptake by CAMPand possible involvement of CAMP-dependent protein kinase. Dev. Biol. 136, 560-563. Manejwala, F.M., Cragoe, E.J. & Schultz, R.M. (1989). Blastocoel expansion in the preimplantation mouse embryo: role of extracellular sodium and chloride and possible apical routes oftheir entry. Dev. Biol. 133,210-220. Manejwala, F.M., Kali, E. & Schultz, R.M. (1986). Developmentofactivatable adenylate cyclase in the preimplantation mouse embryo and a role for cyclic AMP in blastocoel formation. Cell 46, 95-1 03, Maro, B., Johnson, M.H., Pickering, S.J. & Louvard, D. (1985). Changes in the distribution of mernbraneous organelles during mouse early development. J. Embryol. exp. Morph. 90, 287-309. McLachlin, J.R., Caveney, S. & Kidder, G.M. (1983). Control ofgapjunction formation in early mouse embryos. Dev. Biol. 98, 155-164. Meads, T.J. & Wild, A.E. (1993). Apical expression of an antigen common to rabbit yolk sac endoderm and kidney proximal tubule epithelium. J. Reprod. Immunol. 23,247-264. Nadijcka, M. & Hillman, N. (1974). Ultrastructural studies of the mouse blastocyst substages. J. Embryol. exp. Morph. 32,675495. Nuccitelli, R. & Wiley, L. (1985). Polarity of isolated blastomeres from mouse morulae: detection of transcellular ion currents. Dev Biol. 109,452463. Ohsugi, M., Hwang, S.Y., Butz, S., Knowles, B.B., Solter, D. & Kemler, R. (1996). Expression and cell-membrane localization of catenins during mouse preimplantation development. Devel. Dynam. 206,391-402. Orlando, R.A., Kerjaschki, D., Kurihara, H., Biemesderfer, D. & Farquhar, M.G. (1992). gp330 associates with a 44-kDa protein in the rat kidney to form the Heymann nephritis antigenic complex. Proc. Natl. Acad. Sci. USA 89,6698-6702. O’Sullivan, D.M., Johnson, M.H. & McConnell, J.M.L. (1993). Staurosporine advances interblastomeric flattening of the mouse embryo. Zygote 1, 103-1 12. Paynton, B.V., Rempel, R. & Bachvarova, R. (1988). Changes in state of adenylation and time course of degradation of maternal mRNAs during oocyte maturation and early embryonic development in the mouse. Dev. Biol. 129,304-314. Pedersen, R.A., Wu, K. & Balakier, H. (1986). Origin of the inner cell mass in mouse embryos: cell lineage analysis by microinjection. Dev. Biol. 117, 581-595. Pemble, L.B. & Kaye, P.L. (1986). Whole protein uptake and metabolism by mouse blastocysts. J. Reprod. Fert. 78, 149-157. Peyrieras, N., Hyafil, F., Louvard, D., Ploegh, H. L. & Jacob, F. (1983). Uvomorulin: a non-integral membrane protein of early mouse embryo. Proc. Natl. Acad. Sci USA 80,6274-6277. Pickering, S.J., Maro, B., Johnson, M.H. & Skepper, J.N. (1988). The influence of cell contact on the division of mouse 8-cell blastomeres. Development 103,353-363. Pratt, H.P.M. (1985). Membrane organization in the preimplantation mouse embryo. J. Embryol. exp. Morph. 90, 101-121. Pratt, H.P.M., Ziomek, C.A., Reeve, W.J.D. & Johnson, M.H. (1982). Compaction of the mouse embryo: an analysis of its components. J. Embryol. exp. Morph. 70, 1 13-132. Rastan, S., Thorpe, S.J., Scudder, P., Brown, S., Gooi, H.C. & Feizi, T. (1985). Cell interactions in preimplantation embryos: evidence for involvement of saccharides of the poly-N-acetyllactosamineseries. J. Embryol. exp. Morph. 87, 115-128. Reeve, W.J.D. (1981). Cytoplasmic polarity develops at compaction in rat and mouse embryos. J. Embryol exp. Morph. 62,351-367.

Cell Polarity and Mouse Early Development

93

Reeve, W.J.D. & Kelly, F. (1983). Nuclear position in cells ofthe mouse early embryo. J. Embryol exp. Morph. 75, 117-139. Reeve, W.J.D. & Ziomek, C.A. (I98 I). Distribution ofmicrovilli on dissociated blastomeres from mouse embryos: evidence for surface polarization at compaction. J. Embryol. Exp. Morph. 62,339-350. Reima, I. (1990). Maintenance of compaction and adherent-type junctions in mouse morula-stage embryos. Cell. Differen. 29, 143-153. Schwarz, M., Owaribe, K., Kartenkeck, J, & Franke, W.W. (1991). Desmosomes and hemidesmosomes: constitutive molecular components. Ann. Rev. Cell Biol. 6,461491. Sefton, M., Johnson, M.H. & Clayton, L. (1992). Synthesis and phosphorylation of uvomorulin during mouse early development, Development I 15,313-318. Sherman, M.I., Gay, R., Gay, S. & Miller, E.J. (1980). Association ofcollagen with preimplantationand periimplantation mouse embryos. Dev. Biol. 74,470-478. 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-la+ isoform. Development 124,2027-2037. Shi, C.Z., Collins, H.W., Buettger, C.W., Garside, W.T., Matschinsky, F.M. & Heyner, S. (1994). Insulin family growth factors have specific effects on protein synthesis in preimplantation mouse embryos. Molec. Reprod. Devel. 37,398-406. Shirayoshi, Y., Okada, T.S. & Takeichi, M. (1983). The calcium-dependent cell-cell adhesion system regulates inner cell mass formation and cell surface polarization in early mouse development. Cell 35,631438. Slager, H.G., Lawson, K.A., Van Den Eijnden-Van Raaij, A.J.M., DeLaat, S.W. & Mummery, C. ( 1991). Differential localization of TGF-P2 in mouse preimplantation and early post-implantation development. Dev. Biol. 145,205-218. Slager, H.G., Good, M.J., Schaart, G., Groenewoud, J.S. & Mummery, C.L. (1992). Organization of non-muscle myosin during early murine embryonic differentiation. Differentiation 50,47-56. Smith, R.M., Garside, W.T., Aghayan, M., Shi, C.Z., Shah, N., Jarett, L. & Heyner, S. (1993). Mouse preimplantation embryos exhibit receptor-mediated binding and transcytosis of maternal insulin-like growth factor I . Biol. Reprod. 49, 1-12. Sobel, J.S. (1983). Localization of myosin in the preimplantation mouse embryo. Dev. Biol. 95, 227-23 1. Surani, M.A.H. &Barton, S.C. (1984). Spatial distributionofblastomeres is dependent on cell division order and interactions in mouse morulae. Dev. Biol. 102,335-343. Surani, M.A.H. & Handyside, A.H. (1983). Reassortment of cells according to position in mouse morulae. J. Embryol. exp. Morph. 225,505-51 1. Sutherland,A.E., Speed, T.P. & Calarco, P.G. (1990). Inner cell allocation in the mouse morula: the role of orientated division during fourth cleavage. Dev. Biol. 137, 13-25. Takeichi, M. (1991). Cadherin cell adhesion receptors as a morphogenetic regulator. Science 251, 1451-1455. Tarkowski, A.K. & Wroblewska, J. (1967). Development of blastomeres of mouse eggs isolated at the 4- and 8-cell stage. J. Embryol. exp. Morph. 18, 155-180. Thorsteinsdottir, S. (1992). Basement membrane and fibronectin matrix are distinct entities in the developing mouse blastocyst. Anat. Rec. 232, 141-149. Van Blerkom, J., Barton, S.C. &Johnson, M.H. (1976). Molecular differentiation in the preimplantation mouse embryo. Nature 259,3 19-32 I . Van Winkle, L.J. & Campione, A.L. (1991). Ouabain-sensitive Rb+ uptake in mouse eggs and preimplantation conceptuses. Dev. Biol. 146, 158-166. Vestweber, D.,Gossler, A,, Boller, K. & Kemler, R. (1987). Expressionanddistributionofcell adhesion molecule uvomorulin in mouse preimplantation embryos. Dev. Biol. 124,45 1 4 5 6 . Vorbrodt, A,, Konwinski, M., Solter, D. & Koprowski, H. (1977). Ultrastructural cytochemistry of membrane-bound phosphatases in preimplantation mouse embryos. Dev. Biol. 55, 1 17-1 34.

94

T.P FLEMING, E. BUTLER, J. COLLINS, B. SHETH, and A.E WILD

Watson, A.J. (1992). The cell biologyofblastocyst development. Molec. Reprod. Devel. 33,492-504. Watson, A.J. & Kidder, G.M. (1988). Immunofluorescence assessment ofthe timing of appearance and cellular distribution of NdK-ATPase during mouse embryogenesis. Dev. Biol. 126, 80-90. Watson, A.J., Damsky, C.H. & Kidder, G.M. (1990a). Differentiation of an epithelium: factors affecting the polarized distribution of Na+, K+-ATPase in mouse trophectoderm. Dev. Biol. 141, 104-1 14. Watson. A.J., Pape, C.. Emanuel, J.R., Levenson, R. & Kidder, G.M. (1990b). Expression of Na,K-ATPase a and p subunit genes during preimplantation development of the mouse. Devel. Genet. 1 1 , 4 4 8 . Wiley, L.M. (1984). Cavitation in the mouse preimplantation embryo: NdK-ATPase and the origin of nascent blastocoele fluid. Dev. Biol. 105,33&342. Wiley, L.M. & Eglitis, M.A. (1981). Cell surface and cytoskeletal elements: cavitation in the mouse preimplantation embryo. Dev. Biol. 86,493-501. Wiley, L.M. & Obasaju, M F. (1988). Induction of cytoplasmic polarity in heterokaryons of mouse 4-cell-stage blastomeres fused with 8-cell- and 16-cell-stage blastomeres. Dev. Biol. 130, 276-284. Wiley, L.M. & Obasaju, M.F. (1989). Effects of phlorizin and ouabain on the polarity of mouse 4-ce11/16-celI stage blastomere Ireterokaryons. Dev. Biol. 133,375-384. Wiley, L.M., Kidder, G.M. & Watson, A.J. (1990) Cell polarity and development of the first epithelium. BioEssays 12,67-73. Wiley, L.M., Lever, J.E., Pape, C. & Kidder, G.M. (1991). Antibodies to a renal Na+/glucose cotransport system localize to the apical plasma membrane domain of polar mouse embryo blastomeres. Dev. Biol. 143, 149-161. Wiley, L.M., Wu, J.X., Harari, I. &.Adamson, E.D. (1992). Epidermal growth factor receptor mRNA and protein increase after the four-cell preimplantation stage in murine development. Dev. Biol. 149,247-260. Winkel, G.K., Ferguson, J.E., Takeichi, M. & Nuccitelli, R. (1990). Activation of protein kinase C triggers premature compaction in the 4-cell stage mouse embryo. Dev. Biol. 138, 1-15. Ziomek, C.A. & Johnson, M.H. (1980). Cell surface interactions induce polarization of mouse 8-cell blastomeres at compaction. Cell 21, 935-942. Ziomek, C.A. & Johnson, M.H. (1981). Properties of polar and apolar cells from the 16-cell mouse morula Roux’s Arch. Dev. Biol, 190,287-296.