Mechanisms of Implantation in the Mouse: Differentiation and Functional Importance of Trophoblast Giant Cell Behavior

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Developmental Biology 258 (2003) 241–251 www.elsevier.com/locate/ydbio Review

Mechanisms of implantation in the mouse: differentiation and functional importance of trophoblast giant cell behavior

Ann Sutherland*

Department of Cell Biology, University of Virginia Health System, P.O. Box 800732, Charlottesville, VA 22908, USA Received for publication 17 December 2002, revised 21 February 2003, accepted 22 February 2003

Introduction in other developmental events: cell polarization, epithelial- ization, and epithelial–mesenchymal transition. The process According to Lewis Wolpert, “It is not birth, marriage or of transformation of trophectoderm into invasive tropho- death, but gastrulation which is truly the most important blast, and then into a vascular network, has many aspects in time in your life” (quoted in Slack, 1983). While it is common with other developmental events, and with gastru- certainly true that gastrulation is a vitally important mor- lation in particular. Understanding implantation is thus im- phogenetic event, the argument can be made for us, and for portant not only from a clinical standpoint, but unraveling all placental mammals, that implantation is an equally im- the complex mechanisms that establish and regulate the portant event in our lives. If implantation fails, so does behavior of this unique extra-embryonic lineage will com- gastrulation and all subsequent developmental events. Un- plement and extend our understanding of the cellular pro- like most vertebrate embryos, mammalian embryos require cesses that shape the embryo proper. an external source of nutrients to grow and develop. The Until recently, most of our knowledge of trophoblast mouse embryo and those of humans and other primates giant cell differentiation and function was morphological, accomplish this by forming contacts with the maternal and little was known of the underlying molecular or cellular blood supply, leading to the formation of a hemochorial events. The development of in vitro model systems, the placenta (Mossman, 1937; Perry, 1981). Two events are Rcho-1 and trophoblast stem (TS) cell lines (Faria and critical to the success of placentation: implantation and the Soares, 1991; Tanaka et al., 1998), and the ability to delete formation of vascular connections. Implantation begins with relevant genes in the mouse have facilitated the analysis of the apposition and attachment of the embryo to the uterine the molecular mechanisms regulating trophoblast giant cell epithelium, followed by displacement of this epithelium and differentiation (reviewed in Cross, 2000; Rinkenberger et invasion of the underlying stromal compartment. After im- al., 1997). However, exploration of the temporal and causal plantation has begun, an anastomotic network of blood relationships between molecular differentiation and func- spaces is formed surrounding the embryo that is continuous tional cell behavior is just beginning. This review will focus with maternal capillaries (Cross et al., 1994; Welsh and on the cellular aspects of trophoblast giant cell differentia- Enders, 1987). These blood-filled spaces provide nutrients tion and their correlation with known molecular aspects of and oxygen to the embryo during the period before the differentiation, as a means of promoting the synthesis of definitive chorioallantoic placenta is formed, allowing the these two approaches. embryo to grow and differentiate. Both implantation and early vasculogenesis are mediated by trophoblast giant cells, which arise from the epithelial The trophoblast lineage is established by changes in trophectoderm layer of the blastocyst. Trophoblast cell dif- cell polarity ferentiation represents the first divergence of embryonic lineage, and comprises many of the cellular processes seen Trophoblast differentiation in the mouse begins with the process of compaction and polarization at the eight-cell stage, which is essentially the organization of the blas- * Fax: ϩ1-434-982-3912. tomeres into a rudimentary epithelium (Fig. 1). It comprises E-mail address: [email protected]. the following processes: flattening of the blastomeres

domains into different daughter blastomeres, generating two different cell populations (Fleming, 1987; Johnson and Zi- omek, 1981a; Sutherland et al., 1990). The outer, polarized blastomeres form the epithelial trophectoderm layer of the blastocyst, which encloses the inner, highly cohesive non- polarized blastomeres of the inner cell mass (ICM). Thus, this segregation establishes the placental (trophectoderm) and embryonic (ICM) progenitors. The two cell populations immediately begin to diverge at the molecular level, expressing different repertoires of genes and proteins (John- son, 1979). In addition, the outer cells are already significantly biased in their developmental potential (Ziomek et al., 1982). In recombinations of blastomeres, an outer cell will always give rise to at least one outer descendant. This observation indicates that inheritance of all or part of the apical domains, first set up during polarization, leads to a trophectoderm fate (Ziomek et al., 1982). To understand how trophoblast differentiation is initiated, we need to understand how polarity is established at the eight-cell stage. The evidence indicates a central role for the cell–cell adhesion protein E-cadherin in both the induction and orientation of polarity (Johnson et al., 1986; Shirayoshi et al., 1983; Sutherland and Calarco-Gillam, 1983). The role of E-cadherin in establishing polarity may involve recruitment of the sec 6/8 complex to cell–cell contacts, thereby forming a target site for basolaterally oriented secretory vesicles and defining the basolateral domain (Grindstaff et al., 1998; Hsu et al., 1999). Consistent with this model, the location of the apical region of each blastomere is determined with respect to the position of the cell–cell contacts that it has with the surrounding blastomeres (Johnson and Ziomek, 1981b; Ziomek and Johnson, 1980) (Fig. 1). Deletion of the E-cadherin gene, as well as that of its intracellular binding partner ␣-catenin, results in Fig. 1. Polarization in the eight-cell mouse embryo. (A) Cellular events loss of trophectoderm epithelial organization (Larue et al., associated with polarization include a change in nuclear localization from 1994; Riethmacher et al., 1995; Torres et al., 1997). central to basal, a concentration of Con A binding sites and microvilli to the ␣ apical region, localization of alkaline phosphatase staining to basolateral The intracellular signals from E-cadherin and -catenin regions, and restriction of endocytic vesicles to the apical cytoplasm. (B) that orient cell polarity likely involve cytoskeletal reorga- Proposed mechanism for polarization of eight-cell blastomeres. Intercellu- nization by members of the Rho family of small GTPases. lar adhesion through E-cadherin leads to intracellular signals that orient the In cultured epithelial cells, both Rho and Rac are required forming axis of polarity. Subsequent directed transport of secretory vesi- for organization of cadherin-dependent cell–cell contacts cles to these points of contact (through recruitment of the Sec6/8 complex and microtubules?) stabilizes and reinforces the polarity by directing ba- and for recruitment of the actin cytoskeleton to these con- solateral and apical membrane proteins into different membrane compart- tacts (Braga et al., 1997; Lambert et al., 2002). In the early ments. The potential role of Sec6/8 is based on its demonstrated function eight-cell embryo, inhibition of Rho activity prevents po- in development of epithelial cell polarity in vitro (Grindstaff, 1998; Nelson, larization and compaction, while overexpression of a con- 2001). (C) Scanning electron micrographs of eight-cell embryos at various stitutively active form of Rho at the four-cell stage induces stages of compaction and polarization. premature polarization (Clayton et al., 1999). Another Rho family member, Cdc42, may contribute to the establishment against one another, formation of E-cadherin-dependent of polarity in the embryo. Cdc42 regulates cell polarity in a cell–cell adhesions, and apical–basal polarization of the number of other systems (Chant, 1999; Johnson, 1999), and blastomeres (Fig. 1, reviewed in Fleming et al., 2001). overexpression of a constitutively active form of Cdc42 at Compaction and polarization generate an asymmetry of the four-cell stage induces polarization (Clayton et al., adhesivity in each blastomere; the central, basolateral do- 1999). This may be related to sec 6/8 complex function, mains are highly adhesive while the outer, apical domains since Cdc42 controls secretory and endocytic vesicle target- are not. During the next cleavage, radially oriented divisions ing to the basolateral membrane (Cohen et al., 2001; Kros- of some of the blastomeres lead to segregation of these two chewski et al., 1999). However, Cdc42 is also linked to A. Sutherland / Developmental Biology 258 (2003) 241–251 243

Fig. 2. Transition of the trophectoderm to an invasive phenotype. The hatched blastocyst has a polarized, transporting, epithelial trophectoderm layer (TE), which exhibits no motility. The trophectoderm can be divided into two regions based on proximity to the inner cell mass (ICM): the polar TE (in green) and the mural TE (in yellow). As the blastocyst becomes competent to implant, the TE cells alter their polarity and begin to extend apical protrusions. During implantation in vivo, these protrusions aid in displacement and phagocytosis of the apoptotic uterine epithelial cells, and give rise to the laminar extensions that ultimately form the blood sinuses of the yolk sac placenta. formation of adherens junctions through activation of its the ICM, and all blastomeres differentiate as trophectoderm effector protein IQGAP (Fukata et al., 2001), and is re- (Nichols et al., 1998). cruited to the membrane in the Par3/6 multiprotein complex associated with polarization and formation of tight junctions (Joberty et al., 2000; Qiu et al., 2000). Implantation occurs by an invasive transformation of A major unanswered question is how cellular polariza- the trophectoderm tion results in differential gene expression and onset of trophectoderm differentiation. An important aspect of this At the time of implantation, the epithelial trophectoderm question is whether trophectoderm differentiation is specif- cells transform into an invasive population, the trophoblast ically induced, or whether it is a default pathway in the giant cells. These are the cells that mediate penetration of preimplantation embryo. The ﬁndings of Ziomek et al. the embryo into the uterus and formation of connections (1982) that cells inheriting any part of the original oocyte with the maternal blood supply. The transition from tro- cortical region are biased to form trophectoderm certainly phectoderm to trophoblast involves morphologic and behav- suggest the existence of localized factors that direct trophec- ioral transformations that resemble an epithelial–mesenchy- toderm differentiation. However, this notion is refuted by mal transition. There are three major elements: a change in the observation that ICM cells isolated from the early blas- cell polarity, a change in cell motility, and a change in tocyst, which have little if any of either the original oocyte intercellular adhesion (Figs. 2 and 3). The trophectoderm is cortex or the polarized surface of the eight-cell blastomeres, a transporting epithelium with functional tight junctions are fully capable of regenerating a trophectoderm layer (Fleming et al., 2000; McLaren and Smith, 1977; Sheth et (Handyside, 1978; Hogan and Tilly, 1978; Spindle, 1978; al., 1997) which can be divided into two regions, mural and Wiley, 1978). In addition, when compaction is prevented, polar, by the position of the cells relative to the ICM (Fig. when blastomeres are cultured individually, or when there 2). This distinction is not trivial, as the mural and polar are only a small number of them, they all differentiate into trophectoderm have different fates in the postimplantation trophectoderm (Johnson et al., 1986; Shirayoshi et al., 1983; embryo. The mural trophectoderm cells are the ﬁrst to Tarkowski and Wroblewska, 1967). Recent work demon- differentiate, and they give rise to the primary trophoblast strates that the POU transcription factor Oct-4 is required to giant cells, which establish a network of anastomotic chan- maintain the totipotent phenotype of ICM cells. Oct-4 is nels surrounding the embryo, known as the yolk sac pla- expressed in pluripotent or totipotent cells, and either loss of centa (Bevilacqua and Abrahamsohn, 1988; Copp, 1978; expression or overexpression leads to differentiation (Pesce Dickson, 1963; Dickson and Araujo, 1966). The cells of the and Scholer, 2001). In embryos, Oct-4 is expressed in all polar trophectoderm, in contrast, are maintained in a prolif- blastomeres until the morula stage, when it becomes re- erative, undifferentiated state by signals derived from prox- stricted to the inner cells. Deletion of Oct-4 leads to loss of imity with the ICM (Ansell and Snow, 1975; Chai et al., 244 A. Sutherland / Developmental Biology 258 (2003) 241–251

Fig. 3. Correlation of cellular and molecular aspects of trophoblast giant cell differentiation in the preimplantation trophectoderm. The trophectoderm cells of the early blastocyst (96–120 h phCG) are polarized, with a differential distribution of intracellular organelles and surface proteins. At this stage, eomesodermin is expressed, and is required for later onset of protrusive activity. At 120 h, the trophectoderm require a signal to activate mTOR, a serine–threonine kinase that regulates translation. Amino acids can provide this signal, as well as other potential signals that affect later changes in cell–cell adhesion. By 144–168 h, the trophectoderm cells have begun to demonstrate the outcome of these earlier signals. Changes in apical membrane integrin distribution and in the organization of the cytoskeleton due to Rac 1, LIMK, and palladin are evident in changes in apical adhesivity and protrusive activity. The activity of mSna and MASH2 transcription factors restrain terminal differentiation to trophoblast giant cells, while mSna may also affect the expression of E-cadherin, altering the nature of the adherens junctions (and potentially tight junctions). The function of mSna and MASH2 in the trophectoderm are hypothetical, and are included here based on their demonstrated functions in the ectoplacental cone (Cross, 2000).

1998; Copp, 1979; Gardner and Beddington, 1988; Gardner types in both rat and mouse (Poelmann, 1975; Potts, 1968; et al., 1973). These include a short-range signal by FGF4, Schlafke and Enders, 1975; Tachi et al., 1970). Several although FGF4 alone is not sufficient to fully inhibit the observations suggest that this is one aspect of a general onset of giant cell differentiation (Chai et al., 1998; Ras- functional change in trophectoderm cell polarity. As they soulzadegan et al., 2000; Tanaka et al., 1998). Prior to initiate cell contacts through their apical domain, the implantation, the proliferating polar trophectoderm cells trophectoderm cells change their response to contact with feed asymmetrically into and expand the mural population extracellular matrix proteins (Schultz and Armant, 1995; (Copp, 1979; Gardner, 2000). After implantation, the polar Schultz et al., 1997). At the early blastocyst stage, trophec- trophectoderm gives rise to the extraembryonic ectoderm toderm cells neither adhere to nor respond to contact with and the ectoplacental cone (EPC) (Copp, 1979), which con- fibronectin. However, by the late blastocyst stage, when the tribute to the labyrinth and spongiotrophoblast of the defin- embryo is implanting, contact with fibronectin induces in- itive chorioallantoic placenta (reviewed in Cross, 2000; tegrin-dependent adhesivity of the apical domain, which is Rossant and Croy, 1985). brefeldin-A-sensitive and involves a new localization of The process of giant cell differentiation, which began integrin ␣5␤1 on the apical domain of the trophectoderm with the mural trophectoderm cells, continues after implan- cells (Schultz et al., 1997; Sutherland et al., 1993; Wang et tation in the EPC. The EPC contains proliferating cells that al., 2002) (Fig. 3). Previous to this, ␣5␤1 is only detectable provide a continuing supply of differentiating trophoblast at the basal surface of the trophectoderm (Sutherland et al., giant cells. These trophoblast cells are known as secondary 1993). Together, these findings suggest that, with differen- trophoblast giant cells, and they serve to expand the area of tiation, the restricted trafficking of ␣5␤1 integrin to strictly the yolk sac placenta during the period of rapid growth basolateral regions seen in the early blastocyst is altered to between implantation and formation of the chorioallantoic include the apical regions of the trophectoderm. The inte- placenta (Cross, 2000; Rossant, 1986). grin ␣7␤1 is also relocalized from basal to apical domains, likely as a result of contact with its ligands in the uterine Changes in polarity epithelial basement membrane (Klaffky et al., 2001). Polar- ized epithelial cells maintain their specialized plasma mem- One of the first events associated with trophoblast inva- brane domains through distinct protein trafficking mecha- sive differentiation is a change in cell polarity. At implan- nisms (reviewed in Brown and Breton, 2000; Nelson and tation, the apical domain of the trophectoderm becomes Yeaman, 2001), suggesting that changes in protein traffick- adhesive, and the trophectoderm and uterine epithelium ing in the trophectoderm of the late blastocyst allows pro- attach to one another through their apical domains (Reinius, teins of the basal and lateral domains to be directed to the 1967; Schlafke and Enders, 1975). In fact, specialized ad- apical domain as well. Once there, contact with other cells herens junctions are even observed between the two cell or with extracellular matrix would stabilize the location of A. Sutherland / Developmental Biology 258 (2003) 241–251 245

Fig. 4. Correlation of cellular and molecular aspects of trophoblast giant cell differentiation in the postimplantation embryo. The least differentiated cells, the extraembryonic ectoderm, are a polarized epithelium that exhibits no motility and expresses eomesodermin. More differentiated cells are found in the core of the ectoplacental cone, which are cohesive (but not epithelial), nonmotile, and express Mash-2 and mSna. Further differentiation leads to the onset of motility and phagocytosis, expression of Hand-1, and loss of Mash-2 and mSna expression. Finally, the fully differentiated trophoblast giant cells of the yolk sac placenta are not motile but have reepithelialized, and retain their phagocytic ability and Hand-1 expression. these proteins in the apical membrane, allowing the change after zona release, and likely functions in the displacement in adhesivity. One intriguing possibility is that the new and phagocytosis of the apoptotic uterine epithelial and cell–cell and cell–extracellular matrix interactions estab- decidual cells (Bevilacqua and Abrahamsohn, 1988, 1989; lished in this way lead to changes in gene expression, Rassoulzadegan et al., 2000). This interpretation is sup- promoting further differentiation of the trophectoderm to ported by the data of Rassoulzadegan and colleagues, who trophoblast giant cells. showed that the ability of trophoblast cells to phagocytose large particles or whole cells emerges in the mural TE at the Changes in motility blastocyst stage, coincident with the development of protrusive activity (Rassoulzadegan et al., 2000). Trophoblast The motility of the trophectoderm cells changes as they protrusive activity is also critical for morphogenesis of the are undergoing the changes in polarity. At the early blasto- blood sinuses of the yolk sac placenta. To form these si- cyst stage, the trophectoderm cells exhibit no protrusive nuses, the trophoblast cells extend long laminar protrusions, activity. As they progress to the late blastocyst stage, when which meet and adhere to form the cavities through which implantation is initiated, they extend protrusions from their maternal blood will flow (Bevilacqua and Abrahamsohn, apical domains, and by the late blastocyst stage, they exhibit 1989) (Fig. 2). a high frequency of protrusion formation (Martin and Suth- In blastocysts or ectoplacental cone explants cultured in erland, 2001; McRae and Church, 1990) (Figs. 2 and 3). The vitro, the onset of protrusive activity leads to spreading and onset of protrusive activity in the trophectoderm at the time migration of the trophoblast cells on the substrate (Armant of implantation has been noted in embryos of several other et al., 1986; Chavez, 1984; Jenkinson, 1973; Martin and mammalian species (Gonzales et al., 1996a,b), where it Sutherland, 2001). Mouse trophoblast cells do not migrate appears to facilitate release of the embryo from the zona significantly in vivo but invade primarily by directed phago- pellucida and to position the embryo within the uterus. In cytosis of apoptotic decidual cells (Bevilacqua and Abraha- the mouse, trophectoderm protrusive activity is initiated msohn, 1989; Ilgren, 1983; Welsh and Enders, 1987). How- 246 A. Sutherland / Developmental Biology 258 (2003) 241–251 ever, phagocytosis of apoptotic cells also requires tight junctions (Bevilacqua and Abrahamsohn, 1989; Bev- protrusive activity and depends on the activity of the small ilacqua et al., 1985). In addition, gaps in the intercellular GTPase Rac for formation of the enveloping lamellipodia apposition of trophoblast cells develop as the blood sinuses (Caron and Hall, 1998; Tosello-Trampont et al., 2001). form (Bevilacqua and Abrahamsohn, 1988; Welsh and End- Consistent with their phagocytic behavior, the peripheral ers, 1987). It is likely that in vivo, the many adhesive cells of the ectoplacental cone exhibit large numbers of contacts between the trophoblast and the surrounding de- podosomes, cytoskeletal structures associated with endocy- cidual cells maintain the structure of the blastocoele cavity tosis and proteolytic activity in phagocytic cells such as when the permeability seal between the trophoblast cells macrophages and osteoclasts (Linder et al., 2000; Parast et disappears (Bevilacqua and Abrahamsohn, 1989; Bevilac- al., 2001; Zambonin-Zallone et al., 1988). The onset of qua et al., 1985). During this period of development, main- motility in the differentiating trophoblast cells also corre- taining the form of the blastocoele cavity allows the thick lates with the onset of expression of other proteins involved basement membrane known as Reichert’s membrane to in actin filament assembly; for example, LIM kinase, a key form under the trophoblast cell layer. Reichert’s membrane regulator of cofilin function (Cheng and Robertson, 1995), allows efficient diffusion of gases and nutrients between the and palladin, a scaffolding molecule (Parast and Otey, maternal blood in the yolk sac placenta and the embryo, but 2000) (Fig. 3). protects the embryo from contact with the maternal immune system. Interestingly, Reichert’s membrane and the tropho- Changes in cell–cell adhesion blast blood spaces seen in the mature yolk sac placenta have never been generated by in vitro cultured blastocysts under Attachment and the onset of motility are followed by any culture conditions. It is likely that the morphogenesis of changes in cell–cell contacts and in the types of cadherins these structures requires the stabilizing interactions between expressed by the trophoblast cells (Fig. 3). In the blastocyst, the trophoblast cells and the surrounding decidual cells. the trophectoderm cells are linked by E-cadherin-containing adherens junctions (Damjanov et al., 1986; Fleming et al., 2001). At the time of implantation, trophoblast cells cease to express E-cadherin (Damjanov et al., 1986), express P- Trophoblast cell behavior may be regulated at many cadherin transiently, and then downregulate the expression levels of that cadherin as well (Nose and Takeichi, 1986). In the yolk sac placenta, there are adherens junctions both between Investigations at the molecular level have identified a trophoblast giant cells and between giant cells and the number of transcription factors that progressively regulate uterine decidual cells (Bevilacqua et al., 1985; Bevilacqua trophoblast differentiation (reviewed in Cross, 2000). The and Abrahamsohn, 1988). When explanted in vitro, tropho- patterns of expression of the transcription factors eomeso- blast giant cells form intercellular contacts that contain dermin, Mash-2, mSna, and Hand-1 (among others) support ␤-catenin (Parast et al., 2001) and VE-cadherin (AES; un- a three-step model for trophoblast giant cell differentiation published observations). The progressive switching of cad- (Cross, 2000). This pathway begins with stem cells in the herin isoforms is accompanied by functional changes in trophectoderm and extraembryonic ectoderm expressing cell–cell interactions. The intercellular contacts between the eomesodermin; followed by cells of an intermediate pheno- cells that express E-cadherin (trophectoderm cells in the type in the EPC, which express Mash-2 and mSna; and ends blastocyst, extraembryonic ectoderm and the core cells of with trophoblast giant cells in the periphery of the EPC and the EPC) are strong and stable. In contrast, the cell–cell in the yolk sac placenta expressing Hand-1 (Figs. 3 and 4). contacts between the motile trophoblast cells (which ex- This model correlates very well with the progression of press P-cadherin) are patchy and dynamic, allowing cells to changes in cell behavior observed during differentiation slide past one another (Parast et al., 2001). Finally, the (Parast et al., 2001); however, the correlation between mo- intercellular contacts between trophoblast giant cells in the lecular and behavioral changes cannot be direct, as molec- yolk sac placenta are strong and stable, consistent with their ular differentiation data are not strictly predictive of the endothelial function (Parast et al., 2001). cellular phenotypes in each region (Parast et al., 2001). For Another change in cell–cell adhesion that occurs during example, the peripheral ectoplacental cone cells and the trophoblast giant cell differentiation is in the integrity of the cells in the yolk sac placenta exhibit substantially different tight junctions between trophoblast cells (Fig. 3). In em- morphology and motile behavior, yet both express Hand-1. bryos cultured in vitro, changes in tight junction integrity Likewise, Rcho-1 and TS stem cells both express eomeso- lead to collapse of the blastocoele cavity, followed by re- dermin, but TS cells are epithelial while Rcho-1 cells are traction of the mural trophoblast cells to the embryonic end, highly motile. However, peripheral ectoplacental cone and leaving the ICM exposed to the external milieu (Martin and undifferentiated Rcho-1 cells are much different by molec- Sutherland, 2001; Olovsson and Nilsson, 1993). Micro- ular criteria, but exhibit similar motile behavior. Thus, scopic analysis of trophoblast giant cells in vivo shows that while the molecular model mirrors the behavioral model for they have many adherens junctions and desmosomes, but no trophoblast differentiation, there are clearly other factors A. Sutherland / Developmental Biology 258 (2003) 241–251 247 influencing the translation of changes in gene expression for the invasive transformation of the trophectoderm. The into changes in cell behavior. need for this signal is evidenced by the phenomenon of Some of the cellular changes observed during trophoblast diapause, or delayed implantation. Diapause is a naturally differentiation may not be regulated at the transcriptional level. occurring event in many species, in which development One potential influence is cell–extracellular matrix interac- beyond the blastocyst stage is delayed for weeks to months, tions. Trophoblast proliferation and motile behavior are regu- until a hormonal signal triggers a surge of estrogen which lated in unique ways on different isoforms of laminin, through induces the uterus to become receptive, and the embryo to different combinations of receptors (Klaffky et al., 2001). In implant (Mead, 1993; Renfree and Shaw, 2000; Van the implanted embryo, the cells in the extraembryonic ecto- Blerkom et al., 1978). Diapause can be induced in vivo derm, the EPC, and the yolk sac placenta encounter different either by ovariectomy or by a lack of leukemia inhibitory combinations of laminin isoforms in their microenvironment factor (LIF) (Stewart et al., 1992), and in vitro by lack of (Klaffky et al., 2001). These interactions may act at the post- leucine or arginine in the medium (Gwatkin, 1966; Martin translational level to alter cell motility and proliferation, lead- and Sutherland, 2001; Naeslund, 1979; Van Blerkom et al., ing to the observed differences in their behavior. 1978). In each case, embryos remain as expanded blasto- In at least two cases, a correlation between transcription cysts, and cannot attach and spread when tested in vitro. factor expression and cellular differentiation has been charac- The requirement for LIF expression to make the uterus terized (Figs. 3 and 4). First, the T-box transcription factor receptive was demonstrated in LIF knock-out mice. Uterine eomesodermin is expressed in trophectoderm and in undiffer- LIF is normally produced in response to the estrogen surge entiated cells in the extraembryonic ectoderm, where it regu- that induces receptivity. Strikingly, LIF null uteri display lates the transition from epithelial trophectoderm to invasive none of the usual signs of receptivity. In addition, LIF null trophoblast. Embryos lacking eomesodermin are unable to embryos can implant into wild type uteri, and injection of implant, and do not attach and spread in vitro but rather remain LIF directly into LIF-deficient mice allows implantation and as expanded blastocysts (Russ et al., 2000). Eomesodermin- normal development to occur (Chen et al., 2000). However, null embryos do exhibit the normal changes in integrin local- LIF may also act directly on the embryo as well. For ization, suggesting that the primary function of eomesodermin example, treatment of human trophoblast cells with LIF target genes may be to regulate protrusive activity, rather than leads to an increase in the expression of VASP, an intracel- cell polarity. Eomesodermin may not be involved in inducing lular protein involved in re-modelling the actin cytoskeleton the changes in cell–cell adhesion seen during giant cell differ- (Kayisli et al., 2002). entiation, as its expression normally disappears in the EPC, Exposure to amino acids is required at the early blasto- where the changes in cadherin expression and the onset of cyst stage (108 h postfertilization) for successful develop- changes in cell–cell contacts occur. Consistent with a general ment (Fig. 3). This appears to be a developmental check- role in regulating the onset of protrusive activity, eomesoder- point, as transient exposure to medium containing all amino min is also required for the epithelial–mesenchymal transition acids prior to this point does not allow the trophectoderm to of the embryonic ectoderm to mesoderm during gastrulation undergo their normal transformation (Blake et al., 1982; (Russ et al., 2000). Martin et al., 2001). However, once the blastocyst stage of Second, the transcription factor Snail (mSna) is ex- development is reached, a 4- to 8-h exposure to the normal pressed in the intermediate population of trophoblast cells in complement of amino acids will fully induce the embryo to the EPC, where it acts to inhibit giant cell differentiation proceed (Blake et al., 1982; Martin et al., 2001) Exposure to (Nakayama et al., 1998). This expression pattern correlates amino acids leads to activation of the serine–threonine ki- with the onset of cadherin switching (Nose and Takeichi, nase mTOR, which is specifically required for initiating 1986). Several studies have shown that mSna promotes changes in protrusive activity (Martin and Sutherland, epithelial–mesenchymal transition by repressing E-cadherin 2001) (Fig. 3). However, lack of amino acids does not transcription (Batlle et al., 2000; Cano et al., 2000), and inhibit changes in functional cell polarity, and blastocoele during gastrulation, mSna functions in this manner to pro- collapse is not affected by direct inhibition of mTOR with mote the transition from embryonic ectoderm to mesoderm rapamycin (Martin and Sutherland, 2001). These results (Ciruna and Rossant, 2001). In the EPC, mSna may be suggest that the changes in polarity, motility, and cell–cell acting to promote the switch from E-cadherin to P-cadherin- adhesion associated with trophoblast giant cell differentia- dependent adhesion, in addition to repressing the onset of tion are not necessarily regulated coordinately, and that giant cell differentiation (Figs. 3 and 4). amino acids may be activating more than one signalling pathway in the embryo. Much about this system remains to be characterized, including the proximal downstream ef- Environmental regulation of trophoblast fects of mTOR activity and the nature of the amino acid- differentiation dependent signalling pathways regulating mTOR and blastocoele collapse. In addition to the activity of genetic pathways within the The manner in which this amino acid-mediated signal is trophectoderm, an external signal from the uterus is required presented to the embryo in vivo is not clear. Experimental 248 A. Sutherland / Developmental Biology 258 (2003) 241–251 manipulations suggest that the signal is provided by the phoblast differentiation involves cellular and molecular uterine environment, as mouse embryos prevented from mechanisms that have been identified in other embryonic entering the uterus due to ligation of the oviduct exhibit the differentiative events. For example, eomesodermin expres- same phenotype as those cultured in the absence of amino sion is common between mesoderm and trophoblast, and acids (Lee et al., 2000). One appealing possibility is that a Hand-1 expression is critical for cardiac differentiation as change in sodium levels in the uterine fluid affects the well as trophoblast giant cell differentiation. Likewise, the sodium-dependent transport of leucine that is initiated at the cellular events that these molecules regulate are changes in blastocyst stage (Van Winkle, 1977; Van Winkle and Cam- cell polarity, epithelial–mesenchymal transitions, and pione, 1987; Van Winkle et al., 1985a,b). In fact, the so- changes in cytoskeletal organization and motility, all of dium level in fluid from uteri in diapause is lower than that which are fundamental elements of many other develop- in fluid from receptive uteri (Van Winkle, 1977; Van Win- mental events. Far from being an isolated system with kle et al., 1983). However, a study showing that maternal limited applicability, the process of trophoblast differentia- malnutrition during the preimplantation period affects de- tion has many general aspects that are highly relevant to velopment both pre- and postnatally suggests that, under other differentiative and morphogenetic events. This means certain circumstances, changes in amino acid concentration that understanding the underlying cellular and molecular can be involved directly (Kwong et al., 2000). events can provide paradigms that are applicable to both implantation and embryonic development. Accordingly, the kinds of questions to be answered are General relevance of the mouse as a model system fundamental and have broad applicability. First of all, to uncover the elements that are critical to establishment of the Trophoblast function during implantation and placenta- trophoblast lineage, we must investigate the mechanisms tion is critical for developmental and reproductive success regulating the establishment and maintenance of cell polar- in mammals. Many problems of fertility and pregnancy ity. This is a very general cell biological question, which is encountered in humans are due to failure or abnormalities in important not only to developing systems, but also to ho- implantation and placentation, and defects in early events meostasis in the adult. From the developmental perspective, can have drastic effects later in gestation. A better under- we need to determine how the process of polarization leads standing of trophoblast differentiation and function will thus to the molecular events regulating trophectoderm commit- improve reproductive success, as well as maternal and fetal ment. Changes in polarity are also a key aspect of the health. Toward this end, the mouse can be an effective transition to an invasive phenotype. However, we under- model system. The fundamental steps of trophoblast differ- stand neither how changes in polarity are temporally regu- entiation in the mouse are very similar to that of other lated nor what molecular mechanisms are involved. The embryos exhibiting interstitial implantation, in particular, humans and other primates (Enders, 1989; Enders et al., developmental regulation of motility and phagocytic behav- 1989). At the time of implantation, the trophectoderm of the ior also needs to be better characterized, and their function human/primate blastocyst also undergoes an epithelial–mes- in implantation and formation of the yolk sac placenta enchymal transition, requiring changes in polarity, cell–cell defined mechanistically. The factors regulating onset of adhesions, and motility (Enders, 1989; Enders et al., 1989). motility in epithelial cells are of profound importance not Similarly to behavior of mouse trophoblast, human tropho- only for other developmental events such as gastrulation, blast cells become invasive, mediating entry of the embryo but also for initiation of metastasis of cancerous cells. into the endometrial stroma, and then forming a network of Furthermore, the role of intrinsic factors and environ- lacunae that surrounds the embryo and connections to ma- mental cues from the uterus in regulating both polarity and ternal vessels. These lacunae are quite homologous in struc- motility need to be defined. The importance of amino acid ture and function to the trophoblast blood sinuses of the signalling to trophoblast motility suggests that changes in mouse, although the mechanism of their formation is dif- either amino acid concentration or sodium levels in the ferent. Thus, defining the mechanisms underlying the uterine fluid may affect fertility and embryonic develop- changes in polarity, motility, and cell adhesion in the mouse ment. This is supported by evidence that there are both embryo can provide paradigms that can be further tested in immediate and long-term postnatal effects on the embryo of primate models (Enders, 2000). maternal malnutrition during the preimplantation period in the rat (Kwong et al., 2000). These findings have important implications for clinical management of pregnancy as well Future directions as to our basic understanding of the mechanisms regulating implantation. Analysis of trophoblast giant cell differentiation is Finally, and most importantly, the molecular and cellular clearly at an early stage, and much more work both at the elements of trophoblast differentiation need to be linked molecular and cellular level remains to be done. The excit- through a vertical analysis. 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