Determinants of trophoblast lineage and cell subtype specification in the mouse placenta

Developmental Biology 284 (2005) 12 – 24 www.elsevier.com/locate/ydbio

Review

Determinants of trophoblast lineage and cell subtype specification in the mouse placenta David G. Simmons, James C. Cross* Genes and Development Research Group, Department of Biochemistry and Molecular Biology, University of Calgary, HSC Room 2279, 3330 Hospital Drive N.W., Calgary, AB, Canada T2N 4N1 Received for publication 21 January 2005, revised 4 May 2005, accepted 6 May 2005 Available online 16 June 2005

Abstract Cells of the trophoblast lineage make up the epithelial compartment of the placenta, and their rapid development is essential for the establishment and maintenance of pregnancy. A diverse array of specialized trophoblast subtypes form throughout gestation and are responsible for mediating implantation, as well as promotion of blood to the implantation site, changes in maternal physiology, and nutrient and gas exchange between the fetal and maternal blood supplies. Within the last decade, targeted mutations in mice and the study of trophoblast stem cells in vitro have contributed greatly to our understanding of trophoblast lineage development. Here, we review recent insights into the molecular pathways regulating trophoblast lineage segregation, stem cell maintenance, and subtype differentiation. D 2005 Elsevier Inc. All rights reserved. Keywords: Trophoblast lineage; Subtype specification; Mouse placenta; Stem cell maintenance

Introduction The placenta is the first organ to form in the mammalian embryo that promotes the survival and growth of the embryo in the uterus of the mother. The trophoblast cell lineage forms the epithelial part of the placenta and contains several specialized cell subtypes that play roles either in altering maternal physiology and blood flow to promote fetal growth or in nutrient uptake. Over the past decade, considerable insights have been gained into how the trophoblast lineage is segregated at the blastocyst stage and its subsequent differentiation, fueled largely by the generation of over 100 mutant mouse lines that manifest defects in placental development. In addition, the derivation of murine trophoblast stem cell lines has provided a powerful resource for understanding the molecular mechanisms governing trophoblast stem cell maintenance and differentiation. This review will focus on the current state of knowledge of trophoblast lineage formation, stem cell maintenance, and the subsequent * Corresponding author. Fax: +1 403 220 0737. E-mail address: [email protected] (J.C. Cross). 0012-1606/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.ydbio.2005.05.010

differentiation of the numerous trophoblast subtypes that make up the murine placenta.

Early segregation and specification of trophoblast lineage The first cell fate decision during murine embryonic development occurs at the 16-cell stage, where the polarized outer cells which later give rise to the trophectoderm are segregated from the apolar inner cells, the cells destined to become the inner cell mass (ICM). It is not until the blastocyst stage, however, that these cell fate decisions are considered irreversible as cells of the late morula can still adopt the opposite cell fate if their positions are reversed within the embryo (Ziomek et al., 1982). These early inner and outer cells can be distinguished not only by position but also by the expression of molecular markers. For example, the homeobox transcription factors Nanog (Chambers et al., 2003; Mitsui et al., 2003) and Cdx2 (Kunath et al., 2004) are differentially expressed within the inner apolar cells and outer polarized cells of the late morula, respectively.

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Cdx2 is a critical determinant of trophectoderm identity. Cdx2 null embryos form blastocysts but fail to maintain trophectoderm cell identity, instead forming a ball of Oct4 expressing cells incapable of hatching from the zona pellucida (Kunath et al., 2004). In addition, ectopic expression of Cdx2 in embryonic stem cell (ES) cells is sufficient to induce differentiation to the trophoblast cell lineage in vitro (Niwa, 2003). Cdx2 is the earliest known factor to have a role in trophoblast lineage development, although the molecular targets mediating its role in trophectoderm identity are still unknown. In contrast to specifying trophectoderm identity, Nanog is required to maintain ES cell self-renewal. Nanog-deficient ES cells lose their pluripotency and differentiate into endoderm (Mitsui et al., 2003), while overexpression of Nanog can override the dependency on LIF/STAT signaling for ES cell self-renewal, though pluripotency still requires Oct4 activity (Chambers et al., 2003). Oct4, a POU domain transcription factor, is also involved in ICM fate determination, although its expression does not become restricted until the late blastocyst stage (Okamoto et al., 1990; Rosner et al., 1990; Scholer et al., 1990), suggesting that it is not involved in initial lineage segregation. However, Oct4 is required to maintain ES cell fate and to suppress the differentiation of the ICM and cultured ES cells into trophectoderm derivatives (Nichols et al., 1998; Niwa et al., 2000). Regulatory sequences of the Oct4 gene are hypermethylated and associated with a closed chromatin structure in trophoblast stem cells, whereas these regions are hypomethylated with an open chromatin structure (acetylated histones) in ES cells, resulting in differential gene

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expression (Hattori et al., 2004). Another transcription factor, Sox2, has a similar function in repressing the trophoblast cell fate as that observed for Oct4 (Avilion et al., 2003) and works together with Oct4 to regulate downstream targets expressed in the ICM (Okuda et al., 1998).

FGF4 signaling is required for maintenance of trophoblast stem cells The trophectoderm layer of the preimplantation embryo is the precursor to all trophoblast cell subtypes (Fig. 1). However, the entirety of the trophectoderm layer does not contribute equally to the various trophoblast subtypes. Upon implantation, the trophectoderm of the blastocyst not in contact with the ICM, the mural trophectoderm, differentiates to form post-mitotic primary trophoblast giant cells (TGC) that migrate into the antimesometrial portion of the implantation chamber and surround the future parietal yolk sac. In contrast, the trophectoderm directly overlying the ICM, known as the polar trophectoderm, retains its capacity to proliferate and expands to form the extraembryonic ectoderm and ectoplacental cone (Gardner et al., 1973). Maintenance of trophoblast proliferation and self-renewal is dependent on signals from the ICM. Indeed, ICM cells inserted into an empty sphere of trophectoderm can induce secondary sites of proliferation (Gardner et al., 1973). Pluripotent trophoblast stem cells reside within the extraembryonic ectoderm and later the chorionic ectoderm (Tanaka et al., 1998; Uy et al., 2002) and provide the ectoplacental cone with progenitors which give rise to the

Fig. 1. Overview of the trophoblast lineage and the expression patterns of trophoblast cell subtype specific genes.

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spongiotrophoblast and secondary TGC. Maintenance of trophoblast proliferation later in gestation is also dependant on close proximity with the embryonic-derived epiblast as isolated ectoplacental cone or extraembryonic ectoderm transplanted ectopically (Hunt and Avery, 1976) or cultured in vitro (Jenkinson and Billington, 1974; Rossant and Ofer, 1977) do not proliferate and in fact rapidly differentiate into TGCs. Molecular and genetic data indicate that FGF signaling plays a major role in regulating trophoblast proliferation and differentiation. Fgf4 is expressed in early embryos, becoming restricted to the ICM of the blastocyst and later to the epiblast of the early post-implantation embryo (Niswander and Martin, 1992; Rappolee et al., 1994). In contrast, Fgfr2, the receptor for FGF4, is reciprocally expressed in trophectoderm and extraembryonic ectoderm (HaffnerKrausz et al., 1999), strongly indicating that FGF4 may promote trophoblast proliferation via FGFR2 signaling. Genetic support for this came with the generation of mutant mice. Fgf4-deficient embryos die shortly after implantation (Feldman et al., 1995), likely resulting from a failure to maintain trophectoderm and primitive endoderm identity (Goldin and Papaioannou, 2003). In addition, the phenotype of mice with a deletion of exon 9 of the Fgfr2 gene, which deletes the IIIc isoform, resembles that of Fgf4 null mice (Arman et al., 1998). Interestingly, a second Fgfr2 mutant with a deletion of the exons 7 –9, the whole Ig loop, survives longer into gestation but dies of a defect in the labyrinth layer of the placenta by E10.5 (Xu et al., 1998). It is unclear whether the exon 9 deletion results in a dominantnegative form of Fgfr2 or whether the exons 7 –9 mutation results in a hypomorphic allele. Mutation of the Fgfr2 IIIb isoform results in perinatal death due to lung abnormalities (De Moerlooze et al., 2000), indicating that FGF4 likely signals through FGFR2 IIIc, and not IIIb, to promote trophoblast proliferation. In support of the importance of Fgf4, trophoblast stem (TS) cell lines can be derived from both blastocysts and E6.5 extraembryonic ectoderm by culturing in the presence of FGF4 and embryonic fibroblast conditioned medium (Tanaka et al., 1998). By removing FGF4 and embryonic fibroblast conditioned medium, TS cells differentiate into various trophoblast subtypes in culture (Hughes et al., 2004; Tanaka et al., 1998). Importantly, if injected back into blastocysts, TS cells retain the ability to differentiate into all trophoblast subtypes (Tanaka et al., 1998). The ERK1/2 MAP kinases have been shown to be downstream effectors of FGF receptor signaling (Ornitz and Itoh, 2001). Recently, two groups produced Erk2 mutant mice that die shortly after implantation due to a lack of ectoplacental cone and extraembryonic ectoderm formation (Saba-El-Leil et al., 2003; Yao et al., 2003). Intriguingly, a third group produced Erk2 mutant mice which survived until E11.5 after which they died due to defects in placental labyrinth development (Hatano et al., 2003). The prolonged survival in these latter mice may be due to the generation of

a hypomorphic allele rather than a null mutation as the mutation did not delete exon 2. Nevertheless, Erk2 is clearly required for both maintenance of trophoblast proliferation in the extraembryonic ectoderm and ectoplacental cone, as well as subsequent development of the labyrinth layer of the placenta.

Nodal activity is essential for TS cell maintenance While it is now well established that ICM- or epiblastderived FGF4 promotes proliferation of the extraembryonic ectoderm through FGFR2 signaling, the initial attempts to generate TS cell lines required the presence of unknown factor(s) that could be provided by embryonic fibroblast conditioned medium (Tanaka et al., 1998). Recent studies have implicated several members of the TGFh superfamily in this role, in particular Nodal. Nodal plays an important role in trophoblast differentiation as conceptuses with a hypomorphic allele of Nodal have disrupted placental development characterized by the absence of a labyrinth layer and expanded spongiotrophoblast and TGC layers (Ma et al., 2001). A recent study has served to clarify the role of Nodal in the maintenance of trophoblast stem cells and to define in more detail the Fmicroenvironment_ in which they reside (Guzman-Ayala et al., 2004). The extraembryonic ectoderm expresses the transcription factor genes Err2/Errb (Luo et al., 1997; Pettersson et al., 1996; Tremblay et al., 2001), Eomesodermin (Eomes) (Ciruna and Rossant, 1999; Russ et al., 2000), and Cdx2 (Chawengsaksophak et al., 1997; Kunath et al., 2004) in response to FGF4 signaling and is required for the maintenance of proliferation. Nodal acts in an autocrine manner within the epiblast to maintain FGF4 expression but also acts upon the extraembryonic ectoderm, with FGF4, to sustain expression of Err2, Eomes, and Cdx2 and to suppress expression of Mash2 (Guzman-Ayala et al., 2004). In addition, extraembryonic ectoderm-derived Furin and PACE4, secreted proteases that cleave Nodal into a more active form (Beck et al., 2002), are also required for maintenance of the extraembryonic ectoderm (GuzmanAyala et al., 2004). Based on this work, it is clear that trophoblast stem cell maintenance is dependent upon crosstalk between the epiblast and extraembryonic ectoderm (Fig. 2). In addition to Nodal, other members of the TGFh superfamily can promote maintenance of extraembryonic ectoderm in culture. Activin can maintain expression of extraembryonic ectoderm markers and prevent ectopic Mash2 expression in cultured extraembryonic ectoderm (Guzman-Ayala et al., 2004). Activin can also replace embryonic fibroblast conditioned medium for the maintenance of TS cell proliferation in vitro (Erlebacher et al., 2004). In fact, TS cell lines can be established de novo in medium supplemented with FGF4 and Activin (Erlebacher et al., 2004). It may be that Activin simply mimics the

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effects of Nodal since they use the same receptors and signaling mechanisms (de Caestecker, 2004). Mutations in the Nodal/Activin receptors Alk2 (Gu et al., 1999) and Alk4 (Gu et al., 1998), double mutants for Type II receptors Act RII and Act RIIb (Song et al., 1999), and the downstream signaling molecules Smad2 (Waldrip et al., 1998) and Smad4 (Sirard et al., 1998) result in early defects similar to those seen with Nodal and Furin/Pace4 mutant mice. However, single mutant mice for Activin beta A (Matzuk et al., 1995), B (Matzuk et al., 1995; Vassalli et al., 1994), C (Lau et al., 2000), or double mutant mice for Activin beta A/B (Matzuk et al., 1995) do not have phenotypes resembling loss of FGF4 or Nodal. Based on these observations, it may be that, while Activin can compensate for Nodal through the use of shared receptors, it is not itself essential for maintenance of extraembryonic ectoderm in vivo. However, triple mutants have not yet been reported for the Activin ligands. TGFh can also replace embryonic fibroblast conditioned medium in maintaining TS cells in vitro (Erlebacher et al., 2004). TS cell lines passaged for more than 1.5 months in the presence of FGF4 and TGFh (without embryonic fibroblast conditioned medium) can still contribute to all trophoblast cell subtypes in chimeric embryos (Erlebacher et al., 2004). However, the physiological significance of TGFh in maintaining trophoblast stem cell proliferation in vivo remains to be clarified as neither TGFh ligand (Goumans et al., 1999; Kaartinen et al., 1995; Proetzel et

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al., 1995; Sanford et al., 1997) nor TGFh type II receptor (Oshima et al., 1996) mutant mice have trophoblast phenotypes. The details of how FGF4 and TGFh superfamily ligands (Nodal, Activin, or TGFh) cooperate to maintain trophoblast stem cell identity are now being elucidated. In the absence of any growth factor, isolated extraembryonic ectoderm explants undergo differentiation into ectoplacental cone derivatives, upregulating Mash2 expression while dowregulating Cdx2, Eomes, and Err2 (Guzman-Ayala et al., 2004). The addition of FGF4 alone can inhibit the induction of Mash2 but cannot maintain expression of Cdx2, Eomes, and Err2. Conversely, addition of Nodal or Activin alone cannot inhibit Mash2 expression in extraembryonic ectoderm but, in combination with FGF4, can maintain Cdx2, Eomes, and Err2 expression (GuzmanAyala et al., 2004). These data indicate that FGF4 and Nodal (or Activin or TGFh) have distinct but cooperative functions in TS cell maintenance.

Developmental loss of trophoblast stem cell potential While TS cell lines can be derived from blastocysts and early post-implantation extraembryonic ectoderm (Tanaka et al., 1998), this potential disappears completely by the 11 somite pair stage (Uy et al., 2002), which corresponds to approximately E8.5– E8.75 (Kaufman and

Fig. 2. The trophoblast stem cell microenvironment. Diagrams depict mouse conceptuses at E6.5 and E7.5 indicating the molecular interactions required for the maintenance of trophoblast stem cell proliferation. At E6.5, Fgf4 from the embryonic ectoderm signals through Fgfr2 to sustain Cdx2, Eomes, and Err2 expression and suppress Mash2 expression in the extraembryonic ectoderm. Nodal from the epiblast maintains Fgf4 expression and cooperates with Fgf4 to maintain trophoblast stem cell marker expression in extraembryonic ectoderm. Pace4 and Furin from extraembryonic ectoderm process Nodal into a mature more active form. At E7.5, Fgf4 and Nodal are expressed in distal embryonic ectoderm which, at this stage, is separated from the chorion and Fgfr2 by the exocoelomic cavity. It is unclear how Fgf4/Nodal dependent trophoblast stem cells are maintained at this and later stages. It is hypothesized that trophoblast stem cell maintenance at this stage is dependent upon factors within the ectoplacental cavity. Blue—extraembryonic ectoderm (trophoblast), Gray—embryonic ectoderm, Light green—parietal endoderm, Dark green—visceral endoderm, Red—mesoderm.

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Bard, 1999). The loss of TS cell potential is not dependent upon contact of the allantois with the chorion, although this event (approximately 5 somite pair stage) corresponds to an initial drop in TS cell potential (Uy et al., 2002). Instead, disappearance of TS cell potential appears to be associated with the occlusion of the ectoplacental cavity (Uy et al., 2002), the space formed after the anterior and posterior amniotic folds meet to produce the chorion and spatially separate it from the ectoplacental cone (Kaufman and Bard, 1999). In cultures of whole mouse conceptuses, removal of the ectoplacental cone prior to chorion– ectoplacental fusion increases the TS cell potential of extraembryonic ectoderm over that of intact conceptuses (Uy et al., 2002). Based on this, factors within the ectoplacental cavity fluid may contribute to trophoblast stem cell maintenance at the later egg cylinder stage rather than contact with the epiblast which has ceased by this stage of development (Uy et al., 2002) (Fig. 2). The finding that TS cell potential is lost by E8.5 has important developmental implications since the placenta continues to grow almost until the end of term. It may be that as yet uncharacterized progenitor cells are formed that account for the development of the subsequent placental layers and various trophoblast subtypes which comprise them.

Trophoblast giant cell differentiation Trophoblast giant cells are terminally differentiated, polyploid cells that arise through the process of endoreduplication, an unusual cell cycle with successive rounds of DNA synthesis in the absence of intervening mitoses (Gardner and Davies, 1993; MacAuley et al., 1998). Since they initially form at the periphery of the conceptus, they are in contact with maternal tissue and blood at virtually every stage of development and serve to mediate invasion into the decidua (Cross, 2000) (Fig. 3). In addition, TGCs assist in adapting maternal physiology during pregnancy through luteotrophic and lactogenic hormones (Cross et al., 2002; Linzer and Fisher, 1999), as well as synthesize angiogenic factors (Carney et al., 1993; Lee et al., 1988; Voss et al., 2000), vasodilators (Gagioti et al., 2000; Yotsumoto et al., 1998), and anticoagulants (Weiler-Guettler et al., 1996) which likely help to promote local blood flow to the implantation site. Of the various trophoblast subtypes formed during development, the molecular mechanisms regulating TGC formation are by far the best characterized. Factors known to maintain the extraembryonic ectoderm (FGF4 and Nodal/ Activin/TGFh) actively suppress TGC formation, and withdrawal of these factors results in rapid differentiation into TGCs (Erlebacher et al., 2004; Hughes et al., 2004; Rossant and Ofer, 1977; Tanaka et al., 1998). As a result, TGC formation is often considered the Fdefault_ differentiation pathway in the absence of TS cell maintenance.

However, TGC differentiation depends upon the coordinated activity of a number of factors, most notably basic helix –loop –helix transcription factors (bHLH) and their interacting proteins (Fig. 4). The bHLH factor Mash2 is expressed within the ectoplacental cone and is thought to suppress TGC formation (Cross et al., 1995; Kraut et al., 1998; Scott et al., 2000), potentially maintaining TGC precursors. Indeed, overexpression of Mash2 inhibits TGC formation in vitro, albeit transiently (Cross et al., 1995; Hughes et al., 2004; Kraut et al., 1998; Scott et al., 2000). Mash2 expression is downregulated as differentiation into TGCs proceeds (Hughes et al., 2004; Scott et al., 2000) and a targeted deletion of Mash2 results in a reduction of ectoplacental cone cells, loss of the subsequent spongiotrophoblast layer, and a concurrent expansion in TGC numbers (Guillemot et al., 1995; Guillemot et al., 1994; Tanaka et al., 1997). The E proteins Alf1 and Itf2, bHLH factors which interact with Mash2, and the bHLH binding proteins Id1 and Id2 are also downregulated upon TGC differentiation (Jen et al., 1997; Scott et al., 2000). Eed, a polycomb group protein, may be required for repression of Mash2 during TGC formation as Eed mutants show ectopic and persistent Mash2 expression and a significant decrease in secondary TGC differentiation (Wang et al., 2002). In contrast to suppressing TGC formation, several factors are known to directly promote TGC differentiation. The bHLH transcription factors Hand1 (Cross et al., 1995) and Stra13 (Hughes et al., 2004) can overcome FGF4/embryonic fibroblast conditioned medium to promote TGC differentiation (Cross et al., 1995), while deletion of Hand1 results in a block to TGC formation (Riley et al., 1998; Scott et al., 2000). The bHLH antagonist Imfa promotes TGC differentiation (Kraut et al., 1998), possibly by inhibiting the function of Mash2. Thus, an opposing network of bHLH transcription factors and bHLH interacting proteins regulate TGC differentiation. In addition to cell intrinsic factors, extrinsic factors also influence TGC formation. Retinoic acid, for example, can promote TGC formation both in vitro and in vivo (Yan et al., 2001), similar to the effects of overexpression of the retinoic acid responsive gene Stra13 (Hughes et al., 2004).

Trophoblast giant cells—origins and subtypes The mural trophectoderm gives rise to primary TGCs immediately following implantation, while secondary TGCs originate from precursors within the ectoplacental cone and later the spongiotrophoblast (Carney et al., 1993; Johnson and Rossant, 1981; Rossant et al., 1978) (Fig. 4). Isolated extraembryonic ectoderm or cultures of TS cells can also give rise to TGCs in vitro, but not before transiently expressing markers of ectoplacental cone and spongiotrophoblast (Carney et al., 1993; Hughes et al., 2004; Tanaka et

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Fig. 3. Histological sections of the mouse placenta and trophoblast subtypes at embryonic days (E) 8.0, 10.5, and 14.5. (A – D) Paraffin sections at E8.0 stained with H&E. At this stage, the ectoplacental cavity has collapsed, and the chorion, a tightly packed layer of trophoblast stem cells, is in contact with the base of the ectoplacental cone. The edge of the chorion is covered by mesothelium, a single cell layer derived from embryonic mesoderm. The dotted line demarcates the outer border of the trophoblast giant cell layer. (E – H) Embryonic day 10.5. Paraffin (E) and semi-thin plastic (F – H) sections stained with H&E and toluidine blue, respectively. By E10.5, the structure of the mature placenta has begun to form, and the three distinct placental layers can be readily identified. (I – L) Embryonic day 14.5. Paraffin (I) and semi-thin plastic (J – L) sections stained with H&E and toluidine blue, respectively. By E14.5, numerous glycogen trophoblast cell clusters can be seen within the spongiotrophoblast layer as well as beyond the trophoblast giant cell layer. The labyrinth layer contains a considerably higher density of both maternal blood spaces and fetal capillaries. Ch, chorion trophoblast; EPC, ectoplacental cone; f, fetal capillary space; GlyT, glycogen trophoblast cell; Lab, labyrinth; m, maternal blood sinusoid space; Mes, chorionic mesothelium; SpT, spongiotrophoblast; TGC, trophoblast giant cell.

al., 1998), suggesting a three-step model for TGC formation in which trophoblast stem cells first pass through a progenitor phase characterized by Mash2 and Tpbpa expression (Carney et al., 1993). While this model is almost certainly true, this model is based on expression studies of whole populations of cells and does not preclude the possibility that some extraembryonic ectoderm cells could give rise to TGCs directly or through a previously uncharacterized precursor. In support of this possibility, TS cells cultured with exogenous retinoic acid readily form TGCs and can do so without transiently expressing the ectoplacental/spongiotrophoblast marker Tpbpa/4311 (Yan et al., 2001). Cell lineage-tracing studies have confirmed the hypothesis that not all Fsecondary_ trophoblast giant cells are derived from Tpbpa-positive precursors (A. Fortier and J.C. Cross, unpublished data). TGCs display a previously underappreciated level of sub-specialization (Fig. 4). For example, endovascular TGCs which invade great distances into the maternal spiral arteries to replace endothelial cells express Plf but not Pl1, whereas endovascular TGCs more proximal to the placenta express both genes (Hemberger et al., 2003).

The morphology of endovascular TGCs is also clearly different to that of interstitial TGCs as they are much smaller and more spindle-like (Hemberger et al., 2003). In addition, several genes characteristically expressed in TGCs are not uniformly expressed in all trophoblast giant cells, including Mrj (Hunter et al., 1999), Thrombomodulin (Weiler-Guettler et al., 1996), and members of the Cathepsin family (Hemberger et al., 2000). This indicates the existence of several functionally distinct subsets of TGCs.

Spongiotrophoblast cell differentiation Spongiotrophoblast cells comprise the middle layer of the placenta sandwiched between the outer secondary TGCs and the inner labyrinth layer (Fig. 3). The function of the spongiotrophoblast layer (or junctional zone) is poorly understood. However, it could act as structural support for the developing villous structures of the labyrinth and is also known to express several unique genes (Cross et al., 2002; Iwatsuki et al., 2000).

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Fig. 4. Trophoblast giant cell origins, differentiation, and subtypes. The gene names in blue text are trophoblast cell subtype specific in their expression. The gene names in red text indicate genes that promote the differentiation step indicated. The open arrow indicates that retinoic acid (RA) is a cell extrinsic factor that promotes differentiation.

Precursors for spongiotrophoblast cells reside within the ectoplacental cone. However, observations from several mouse mutants suggest that spongiotrophoblasts and TGCs can arise from a common ectoplacental cone precursor (Fig. 5). For example, Mash2 has opposing effects on spongiotrophoblast and TGC development; loss of Mash2 expression results in loss of the spongiotrophoblast layer and an increase in secondary TGCs (Guillemot et al., 1994). Mutations in the Choroideremia (Shi et al., 2004), Cx31 (Plum et al., 2001), and Keratin8/19 (Tamai et al., 2000) genes also have opposing effects on these two cell types, indicating that these genes may influence cell fate decisions in common ectoplacental cone precursors. Interestingly, formation and/or maintenance of a common ectoplacental cone precursor population is affected by mutations in p185/ Cul7 or Nodal. Deletion of the p185/Cul7 gene results in a decrease in both TGCs and spongiotrophoblast cells (Arai et al., 2003), whereas mice with a hypomorphic allele of Nodal (Ma et al., 2001) have increased cell numbers in both these layers. In contrast, mutations in Rap250 (Antonson et al., 2003) or Hsf1 (Xiao et al., 1999) result in a decrease in spongiotrophoblast cells without any apparent effect on TGC number, indicating a role in spongiotrophoblast differentiation downstream of precursor commitment (Fig. 5). Interestingly, genes expressed exclusively in labyrinth trophoblast can also affect spongiotrophoblast formation. For example, Ipl, a cytoplasmic adaptor protein, and Esx1, a homeobox gene, are both expressed in labyrinth cells and not spongiotrophoblast, but their deletion results in an expanded spongiotrophoblast layer (Frank et al., 2002; Li

and Behringer, 1998). It is unclear how these genes exert their effects on spongiotrophoblast cells, although it is certain that a cross-talk exists between these layers within the placenta. As with the TGC population, the spongiotrophoblast layer is not homogenous at the cellular level, and several subtypes are known to exist. In rats, large numbers of spongiotrophoblast cells become polyploid by undergoing endoreduplication during the second half of gestation (Zybina et al., 2000). It is unknown if any, or at what extent, polyploid spongiotrophoblast cells exist in mice or if there is any functional significance of polyploidy. In addition, a unique trophoblast subtype arises from the spongiotrophoblast layer later in gestation, the glycogen trophoblast cell, which migrates great distances into the interstium of the decidua basalis (Adamson et al., 2002).

Glycogen trophoblast cells Sometime after E12.5, glycogen trophoblast cells begin to appear in the spongiotrophoblast layer and subsequently migrate interstitially into the decidua, in some cases well beyond the boundaries of endovascular TGC invasion (Adamson et al., 2002) (Fig. 3). In the second half of gestation, they become the main source of IGF II within the placenta (Redline et al., 1993). Very little is known about the molecular regulators of glycogen trophoblast cell formation or function. Several mouse mutants have shown a disruption in glycogen trophoblast cell numbers, but almost always in conjunction with similar defects in the

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Fig. 5. Spongiotrophoblast differentiation. Red text denotes genes that affect spongiotrophoblast/TGC common precursor maintenance/formation, cell fate decisions, or spongiotrophoblast differentiation downstream of precursor cell fate decision. Blue text denotes genes that are expressed within specific placental compartments or differentiated trophoblast cell subtypes.

spongiotrophoblast layer itself, including Esk1 (Li and Behringer, 1998), Ipl (Frank et al., 2002), and Pkba (Yang et al., 2003). By contrast, Igf2 null mice have a disproportionate loss in glycogen trophoblast cells (Takahashi et al., 2000), whereas p57kip2 mutant mice have placentomegaly, with greatly increased numbers of trophoblast cells in the spongiotrophoblast and labyrinth layers, but, intriguingly, glycogen trophoblast cells and TGCs are not affected. Glycogen trophoblast cells therefore share several features with both TGCs and spongiotrophoblast cells, but they are clearly unique.

Labyrinth trophoblast subtypes The innermost layer of the mouse placenta, termed the labyrinth, consists of branched villi that provide a large surface area for nutrient, waste, and gas exchange and is composed of trophoblast cells with underlying blood vessels derived from the allantois (Fig. 3). It is important to acknowledge, however, that the labyrinth layer does not contain a homogeneous group of trophoblast cells (Fig. 6). In fact, the labyrinth contains several distinct trophoblast subtypes that reside in fixed locations with respect to maternal and fetal blood spaces. The cells lining the maternal blood sinusoids are mononuclear trophoblast cells

that express placental lactogen II, considered a marker of TGCs (Fig. 7). Adjacent to this cell layer are two layers of syncytiotrophoblast cells followed by fetal endothelial cells which are in contact with the fetal blood (Cross, 2000). All of these cell types can be readily identified based on position and morphology (Cross, 2000). In addition to the cells comprising the villi, clusters of Eomes positive cells can be detected scattered throughout the labyrinth (Wu et al., 2003). Ehox, another marker of extraembryonic ectoderm at E6.5, can also be detected in islands of cells within the labyrinth layer (Jackson et al., 2003). It would be interesting to know whether these markers co-localize as it is tempting to speculate that these cells may represent labyrinth trophoblast precursors.

Syncytiotrophoblast differentiation Precursors for syncytiotrophoblast most likely reside within the chorionic plate, based on the fact that many genes expressed exclusively in the labyrinth are expressed initially in chorionic trophoblast cells including Gcm1 (Basyuk et al., 1999), Esx1 (Li and Behringer, 1998), Dlx3 (Morasso et al., 1999), Tef5 (Jacquemin et al., 1998), and Err2 (Luo et al., 1997). However, there is no direct evidence of this. Gcm1 is the only gene known thus far to be specifically

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Syncytiotrophoblast formation is a two-step process involving first cell cycle exit and then subsequent fusion of these cells to form a syncytium. Gcm1 regulates the initiation of syncytiotrophoblast formation as ectopic expression of Gcm1 in TS cells in vitro promotes cell cycle exit (Hughes et al., 2004) and Gcm1-deficient mice have a block in syncytiotrophoblast formation (AnsonCartwright et al., 2000). In addition, Rb-deficient mice have excessive proliferation in the labyrinth layer and show a relative decrease in syncytiotrophoblast formation (Wu et al., 2003), indicating that cell cycle exit is critical for syncytiotrophoblast formation. Very little is known

Fig. 6. Differentiation of trophoblast cell subtypes within the labyrinth. Red text denotes genes that regulate the differentiation step indicated. Blue text denotes genes that are expressed within specific placental compartments or differentiated trophoblast cell subtypes.

required for syncytiotrophoblast differentiation in mice (Anson-Cartwright et al., 2000). Gcm1 expression first appears in equally spaced clusters of cells within the chorion beginning at E7.5 (Basyuk et al., 1999). By E9.0, after the allantois has attached to the basal surface of the chorion (Cross et al., 2003), Gcm1 expressing cell clusters define the initiation sites for primary branching morphogenesis (Anson-Cartwright et al., 2000). As branches elongate and protrude into the chorion, Gcm1 expression is restricted to the tips of the branches (Anson-Cartwright et al., 2000), and syncytiotrophoblast cells begin to form (Cross, 2000). In Gcm1 mutant mice, syncytiotrophoblast formation is blocked, villous branching morphogenesis is not initiated, and the chorioallantoic interface remains flat (Anson-Cartwright et al., 2000). Factors directly regulating Gcm1 expression in the early chorion have not yet been identified. However, maintenance and further expansion of Gcm1 expression require contact and signaling with the allantois. Gcm1 expression is dramatically reduced in Mrj mutant mice which have a failure of chorioallantoic attachment (Hunter et al., 1999). Furthermore, Hsp90b-deficient mice have a phenotype that is similar to that of Gcm1 mutants with failure of branching morphogenesis and labyrinth formation (Voss et al., 2000). Maintenance and expansion of Gcm1 expression are lost in these mice (Stecca et al., 2002), even though Hsp90b is expressed exclusively in allantoic mesoderm. Thus, initial expression of Gcm1 appears to be independent of chorioallantoic contact, but subsequent maintenance and expansion of Gcm1 expression require contact with and signaling from the allantois (Stecca et al., 2002).

Fig. 7. Trophoblast cell subtypes in the labyrinth layer. (A) Electron microscopy of E14.5 labyrinth layer. The cells lining the maternal blood sinusoids (m) are large mononuclear trophoblast cells (Mono). Adjacent to the mononuclear trophoblast cells are two layers of syncytiotrophoblast (SynT). Endothelial cells (Endo) line the fetal capillary space (f). Scale bar = 5 Am. (B) Placental lactogen II immunostaining in E12.5 and E16.5 placenta. PL II immunoreactivity can be detected in the labyrinth layer by E12.5 in cells lining the maternal blood sinusoids. Cells lining the fetal blood vessels do not stain for PL II. Note that, by E16.5, all mononuclear trophoblast cells in the sinusoids express PL II. Brown color is DAB staining of PL II (1:500 dilution of PL II antibody from Chemicon). Sections were counterstained with nuclear fast red (Dako). Black arrowhead = maternal red blood cell; open arrowhead = fetal red blood cell. Scale bar = 50 Am.

D.G. Simmons, J.C. Cross / Developmental Biology 284 (2005) 12 – 24

about the process of trophoblast cell – cell fusion in mice. In humans, however, trophoblast fusion is thought to be mediated by Syncytin, a transmembrane protein encoded by an endogenous retrovirus (Mi et al., 2000). When Syncytin is ectopically expressed in cells, it can mediate fusion even if it is expressed in only one of the two cells, inferring that fusion is asymmetric and involves unequal partners (Mi et al., 2000). Importantly, GCM1 can regulate the Syncytin promoter (Yu et al., 2002). Recently, homologues of human Syncytin and Syncytin2 (Blaise et al., 2003) have been identified in mice (Dupressoir et al., 2005). They are expressed in the labyrinth layer of the murine placenta from E9.5 and have been shown to trigger cell fusion in cultured cells (Dupressoir et al., 2005). Whether they are regulated by Gcm1 is currently unknown.

Concluding remarks Much progress has been made in recent years towards understanding the molecular mechanisms governing trophoblast development and the subsequent differentiation of the various trophoblast subtypes comprising the placenta. This comes mainly through the generation of a large number of mutant mice, as well as the identification of trophoblast subtype specific markers. It is now clear that the trophoblast lineage comprises a previously underappreciated diversity of subtypes. In the future, a more detailed analysis of mouse mutants with defects in placental development and how they influence the various trophoblast subtype populations would greatly enhance our knowledge of the mechanisms of trophoblast differentiation. The derivation of trophoblast stem cells in vitro has proved invaluable to our understanding of trophoblast stem cell maintenance and subtype specialization and will continue to be a powerful tool to provide further insights into the regulation of trophoblast proliferation and differentiation.

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