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Keywords:

  • lineage formation;
  • morula;
  • trophoblast;
  • trophectoderm;
  • primitive endoderm;
  • epiblast;
  • regulative;
  • inside-outside;
  • polarity;
  • polarization;
  • ICM;
  • Nanog;
  • Gata;
  • Cdx2;
  • Par;
  • PKC;
  • preimplantation;
  • embryo;
  • epithelialization;
  • asymmetric division

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. DEVELOPMENTAL STRATEGIES OF EARLY EMBRYOS
  5. DEVELOPMENT OF THE TE
  6. ESTABLISHMENT OF THE PE
  7. STEM CELLS FROM THE BLASTOCYST
  8. POLARITY AND AXIS FORMATION
  9. FUTURE PERSPECTIVES
  10. Acknowledgements
  11. REFERENCES

Animals use diverse strategies to specify tissue lineages during development. A common strategy is to partition maternally supplied and localized lineage determinants into progenitor cells. The mouse embryo appears to use a different, more regulative strategy to specify the first three lineages: the epiblast (EPI: future embryo), the trophectoderm (TE: future placenta), and the primitive endoderm (PE: future yolk sac). These lineages are specified during two successive differentiation steps leading to formation of the blastocyst. Here, we review classic and contemporary models of early lineage specification in the mouse, and describe recent efforts to understand the molecular regulation of these events. We describe evidence that trophectoderm differentiation bears resemblance to the process of epithelialization and describe the importance of apical/basal protein complexes in regulating this process. Next, we present a revised model of PE specification, and describe evidence that PE cells in the inner cell mass sort out to occupy their ultimate position on the surface of the EPI. Finally, we describe factors that reinforce these lineages and three distinct stem cell types that can be isolated from them. Together, these mechanisms guide the differentiation of the first lineages of the mouse and thereby set up tissues that will be important for the first steps of embryonic body patterning. Developmental Dynamics 235:2301–2314, 2006. © 2006 Wiley-Liss, Inc.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. DEVELOPMENTAL STRATEGIES OF EARLY EMBRYOS
  5. DEVELOPMENT OF THE TE
  6. ESTABLISHMENT OF THE PE
  7. STEM CELLS FROM THE BLASTOCYST
  8. POLARITY AND AXIS FORMATION
  9. FUTURE PERSPECTIVES
  10. Acknowledgements
  11. REFERENCES

The preimplantation mouse embryo is highly regulative. Cells of the early embryo maintain the ability to change fate after experimental manipulation or cell ablation for a considerable period of development. However, by the late blastocyst stage, three distinct tissue lineages are present: the epiblast (EPI), the primitive endoderm (PE), and the trophectoderm (TE). Only one of these, the EPI will give rise to the fetus, yet proper segregation and development of the other two extraembryonic lineages is crucial for the survival and even patterning of the embryo (reviewed in Beddington and Robertson,1999; Ang and Constam,2004; Rossant and Tam,2004). Given the importance of these tissues, understanding the molecular mechanisms establishing these three lineages is key to understanding early mouse development. This review will describe classic and recent studies of the mechanisms that allow establishment of the first three lineages of the mouse: the EPI, PE, and TE.

DEVELOPMENTAL STRATEGIES OF EARLY EMBRYOS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. DEVELOPMENTAL STRATEGIES OF EARLY EMBRYOS
  5. DEVELOPMENT OF THE TE
  6. ESTABLISHMENT OF THE PE
  7. STEM CELLS FROM THE BLASTOCYST
  8. POLARITY AND AXIS FORMATION
  9. FUTURE PERSPECTIVES
  10. Acknowledgements
  11. REFERENCES

Oocytes of many species have a clear polarity with distinct animal and vegetal (A/V) poles. Maternally supplied factors, generically termed determinants, localize to particular regions of the egg, often forming a gradient along the A/V axis. After fertilization, cell cleavage leads to segregation of determinants such that, after a couple of cleavages, individual blastomeres are not equivalent and have restricted developmental potential. Ablation of individual populations is not rescued by neighboring cell types. Often this process is accompanied by a stereotypical cleavage pattern, which is also important for reproducible generation of different kinds of blastomeres. Thus, intrinsic differences in blastomeres lead to the establishment of the first cell lineages in the embryo.

In mammals, early development and lineage specification has been best studied in the mouse, which appears to use a different strategy. The mouse oocyte has no clear polarity, and no molecules are yet known that are both important for lineage specification and exhibit a polarized distribution in the oocyte. Early cleavage patterns are not stereotypic, and remarkably, all blastomeres retain the potential to form all cell lineages until the eight-cell stage (reviewed in Johnson and McConnell,2004). Moreover, blastomere ablation at this stage does not affect the development of a normal fetus (Tarkowski,1959; Tsunoda and McLaren,1983; Zernicka-Goetz,1998; Ciemerych et al.,2000). These observations indicate that early mouse development is governed by a relatively flexible developmental program. Given this finding, elucidation of mechanisms driving early lineage specification was initially elusive.

To understand the cellular origins of the first three lineages, it is important to understand the morphological changes that lead to blastocyst formation. During the first two days of development, the embryo undergoes successive cleavage divisions to produce an eight-cell embryo (Fig. 1). At this stage, the morula stage, blastomeres increase cell–cell contact to produce a compacted morula. Subsequent divisions increase the topological complexity of the morula; cells can take up a position on the inside of the late morula, where they are enclosed entirely by other cells. Alternatively, cells can stay in contact with the external environment, and be outside cells. Lineage tracing experiments suggested that the TE is derived mainly from outside cells, whereas inside cells contribute to the inner cell mass (ICM; Fleming,1987). Later, the ICM will segregate into EPI and PE. Classical models of these processes recently have been complemented by molecular strategies, presenting a more complete view of the mechanisms by which these lineages are specified and maintained.

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Figure 1. Stages of preimplantation mouse development. Three rounds of cleavage division lead to the eight cell stage. Cells of the eight-cell embryo, or early morula, compact against each other so that cell outlines are no longer visible. Subsequent divisions lead to the late morula stage, where distinct inside and outside cell populations are established. At the cavitating late morula stage, the nascent blastocoele forms as an inside cavity. Blastocoele expansion continues through early blastocyst and late blastocyst stages. Two distinct lineages are apparent in the blastocyst stage: trophectoderm (TE) and inner cell mass (ICM). TE is also present in the late blastocyst, but the ICM has segregated into two lineages: epiblast (EPI) and primitive endoderm (PE).

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DEVELOPMENT OF THE TE

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. DEVELOPMENTAL STRATEGIES OF EARLY EMBRYOS
  5. DEVELOPMENT OF THE TE
  6. ESTABLISHMENT OF THE PE
  7. STEM CELLS FROM THE BLASTOCYST
  8. POLARITY AND AXIS FORMATION
  9. FUTURE PERSPECTIVES
  10. Acknowledgements
  11. REFERENCES

Classical Models of TE Formation

The TE is the first tissue type to be specified during mouse development and is morphologically distinct from the ICM by the early blastocyst stage (Fig. 1). The TE, which will form the fetal portions of the placenta, begins as an epithelial sheet enclosing the future embryo, the ICM. Molecularly, TE and ICM lineages can be distinguished by the expression patterns of several lineage-specific transcription factors. Cdx2, which is related to Drosophila Caudal, is expressed in the TE of the blastocyst where it is required for promoting TE fate (Beck et al.,1995; Strumpf et al.,2005). Two ICM-specific transcription factors are Oct4, a POU domain protein, and Nanog, a homeodomain protein (Palmieri et al.,1994; Chambers et al.,2003; Mitsui et al.,2003). Interactions among these factors during blastocyst formation are thought to reinforce TE and ICM fates. Although the molecular mechanisms regulating TE and ICM fates are beginning to be understood, the foundation of this field was provided by classic studies of early mouse development.

The Inside–Outside Model

The earliest models of mouse development suggested that, as in nonregulative embryos, inheritance of cytoplasmic determinants assigned cell fates (reviewed in Alexandre,2001). However, this proposal was rejected after the observation that cells of the early embryo can change fate if moved to a new position (Mintz,1965). These observations and others led to the development of a new model of early lineage determination. The Inside–Outside Model (Tarkowski and Wroblewska,1967) proposed that, instead of cytoplasmic determinants, cell fate is established by position in the late morula, the stage at which inside and outside cell populations are distinct (Fig. 2A). Position could be translated into cell fate in several ways. For example, the extent of cell–cell contact might inform a cell of its position and thereby determine cell fate: inside cells would symmetrically contact neighboring cells, whereas outside cells would asymmetrically contact other cells. Alternatively, inside and outside microenvironments could differ somehow chemically and thereby differentially affect cell fate.

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Figure 2. Classical models of lineage formation. A: The Inside–Outside Model proposes that, at the 16-cell stage, cell position on the outside or inside of the late morula determines its fate in the blastocyst. B: The Cell Polarity Model proposes that, at the 8-cell stage, cell polarity and cleavage patterns lead to establishment of outside and inside cells at the 16-cell stage and later. Symmetric cleavage produces two outside cells (Ba), whereas asymmetric cleavage produces one outside and one inside cell (Bb).

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The Cell Polarity Model

Efforts to understand how inside–outside positions are created led to the development of the Cell Polarity Model (Johnson et al.,1981). This model proposes that cell fate is established before the late morula stage (Fig. 2B). According to the Cell Polarity Model, cell fate is established at the eight-cell stage by the establishment of cell polarity along the radius of the early morula. Subsequent cell divisions lead to symmetric or asymmetric distribution of polarity information, based on the angle of cell division. Division parallel to the radial axis generates two identical, polarized cells that inherited the entire distribution of polarity information (Fig. 2Ba). In contrast, division perpendicular to the inside–outside axis creates two different cells: one that inherited all polarity information and stays outside, and one apolar cell that stays inside (Fig. 2Bb). Polar and apolar cells would then differ in their developmental potential, thereby setting up TE and ICM lineages.

The Inside–Outside and Cell Polarity models are not completely at odds with each other, because both rely on differences along the radial axis to break the symmetry of the early embryo rather than cytoplasmic determinants inherited from the oocyte. However, these models differ in an important way: the Inside–Outside Model predicts that cell position drives cell fate, whereas the Cell Polarity Model predicts that cell fate drives cell position. Extensive studies have lent support to the Cell Polarity Model and established a set of observations that have since guided molecular studies.

The Cell Polarity Model: The Evidence

Early experiments focused on determining whether cell polarity is affected by basic properties of the early embryo, such as mitosis, cell–cell contact, or protein synthesis. These experiments have provided a foundation for more recent efforts to identify the molecular mechanisms underlying the processes of polarization, compaction, and elaboration of cell fate. Cell polarity was first observed as the localization of a variety of subcellular components to the apical cell pole (outside-facing) around the time of compaction. These components included surface microvilli, cytoskeletal elements, endosomes, and microtubule organizing centers (reviewed in Johnson et al.,1986a; Johnson and McConnell,2004). Fluorescently labeled concanavalin A (Con A), a lectin that binds to certain proteoglycans, also becomes localized to the apical microvillus pole around the time of compaction (Handyside,1980; Ziomek and Johnson,1980; Johnson and Ziomek,1981a). Because inheritance of apical and basal regions is thought to lead to differences in cell fate, the stability of these domains is central to the Cell Polarity Model. It follows, therefore, that some aspect of cell polarity must be retained during mitosis. Although the cytoskeleton and endosomes are redistributed during cell division, mitosis does not disrupt Con A localization to the apical cell surface (Johnson and Ziomek,1981b).

The role of cell–cell contact in setting up polarity is difficult to study in the intact embryo, because it is not possible to remove one cell without disrupting relationships between all the cells. Instead, disaggregated and reaggregated embryos have been used. These partial embryos compact essentially on schedule (Johnson and Ziomek,1981a) and have provided evidence that the development of polarity is initiated more effectively in the presence of cell–cell contact (Ziomek and Johnson,1980; Johnson and Ziomek,1981b). Also, the orientation of polarity may be influenced by regions of cell–cell contact. Couplets of cells isolated from the eight-cell embryo exhibit polarized Con A staining in a region opposite to their newly imposed region of cell contact (Ziomek and Johnson,1980; Johnson and Ziomek,1981b), suggesting that cell–cell contact gives orientation cues to the cell. This process has also been shown by disrupting cell adhesion in intact embryos by incubation in calcium-free medium (Pratt et al.,1982; Johnson et al.,1986b). The orientation of polarity is plastic for the first 5 hr of cell–cell contact, but becomes locked in thereafter (Johnson and Ziomek,1981b). Thus cell–cell contact is sufficient to define the angle of polarity, but is not necessary for the maintenance of polarity.

Remarkably, polarization and compaction not only proceed but occur prematurely after treatment with drugs that block protein synthesis (Levy et al.,1986) or activate protein kinase C (PKC; Winkel et al.,1990), offering three important clues about these processes: (a) that all components for polarization and compaction already exist in the early embryo, (b) that proteins made in the early embryo prevent precocious polarization and compaction, and (c) that posttranslational mechanisms, especially phosphorylation, oversee polarization and compaction. In more recent years, this last prediction has raised much interest in molecules such as PKC isoforms and their downstream targets.

Another important component of the Cell Polarity Model is that cell fate is influenced by the inheritance of polarity, as determined by the orientation of the cleavage plane (Fig. 2B). Cell division along the inside/outside axis produces two polar cells, both fated to occupy outside territory and become TE (Fig. 2Ba). On the other hand, division perpendicular to the inside–outside axis will produce one polar and one apolar cell, fated to occupy outside and inside space and to become TE and ICM, respectively (Fig. 2Bb). It follows, therefore, that every cell of the eight-cell stage will produce at least one TE cell during each ensuing division. In support of this proposal, isolated cells from the eight-cell stage give rise to polar–polar or polar–apolar couplets, but no apolar–apolar couplets (Johnson and Ziomek,1981a). Cell division planes occurring both parallel and perpendicular to the inside–outside axis have been observed using time-lapse microscopy (Sutherland et al.,1990), but evidence of a direct link between cleavage plane orientation and cell fate is still lacking.

Molecular Regulation of TE Vs. ICM Fate Choice

The link between cell polarity and cell fate suggests the exciting possibility that polarity-producing proteins may direct differentiation of the TE. The mature TE bears hallmarks of a polarized epithelium, with apical microvilli, and the polarized distribution of specialized protein complexes, such as tight and adherens junctions. As such, efforts in the field have focused on the examination of proteins known to be important for defining apical/basal polarity in other systems, such as Drosophila and cultured mammalian cells. Notably, because cells of the eight-cell embryo are initially unpolarized and nonepithelial, the preimplantion embryo offers a unique opportunity to study the process of mammalian epithelialization in vivo. Several proteins with compelling expression patterns as early as the eight-cell stage have been identified (Fig. 3A).

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Figure 3. Localization of apical/basal proteins during blastocyst formation. A: In the compacted early morula, apical proteins (yellow) localize to a pole opposite areas of cell–cell contact, while basolateral proteins (purple) localize to regions of cell–cell contact and comprise adherens junctions. B: In the blastocyst, the apical protein complex assembles as tight junctions apical to the adherens junctions.

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Based on timing of detection, adherens junction assembly appears to coincide with the assembly of an apical domain around the time of compaction in the eight-cell embryo. At this time E-cadherin, a cell adhesion molecule and a major component of adherens junctions, becomes greatly enriched at regions of cell–cell contact (Vestweber et al.,1987). In addition, the serine/threonine kinase EMK1 (Par1) is localized basolaterally (Vinot et al.,2005). Also at this time, several proteins localize to a small region of the apical cell cortex. These proteins include mouse homologues of Par3, Par6, and atypical PKCs (aPKCs; Pauken and Capco,2000; Thomas et al.,2004; Plusa et al.,2005; Vinot et al.,2005). Later, in the late morula, tight junctions begin to assemble just apical to the adherens junctions (Fleming et al.,1989; Sheth et al.,1997). Many apical proteins become localized to the tight junction around this time (Fig. 3B; Eckert et al.,2005; Vinot et al.,2005).

The distribution of these proteins is consistent with current models of apical/basal polarity formation in both Drosophila epithelium and mammalian cultured cells (reviewed in Nelson,2003; Margolis and Borg,2005). According to one current model, segregation of apical and basal domains is achieved through mutual antagonism between apical and basal protein complexes. Par3, Par6, and aPKC form an apical protein complex (reviewed in Etienne-Manneville and Hall,2003). Formation of this complex controls microtubule organization and excludes basolateral proteins, such as Lethal giant larvae (Lgl), from the apical membrane by direct aPKC-mediated phosphorylation (Plant et al.,2003; Yamanaka et al.,2003). Disruption of the apical protein complex or inhibition of aPKC function prevents formation of a functional apical domain and ultimately disrupts apical/basal polarity. Although it is unknown what initiates formation of the apical protein complex, these relationships can be adapted to the Cell Polarity Model to provide a molecular mechanism of cell fate segregation (Fig. 4). Assembly of the apical domain in the eight-cell embryo during compaction would exclude basolateral proteins from the apical membrane. Inheritance of the apical domain would lead to the continued polarization of daughter cells, whereas asymmetric division would result in the inheritance of the apical domain by only one daughter cell, whereas the other daughter cell would become apolar. Reassembly of the apical domain in the apolar daughter cell would be prevented by proteins associated with the basolateral domains.

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Figure 4. Molecular cell polarity model. In the early morula, asymmetric cleavage (dotted line) leads to asymmetric distribution of the apical protein complex, while symmetric cleavage (not shown) leads to symmetric distribution of the apical protein complex. Polar cells that have inherited the apical protein complex maintain polarity. The apical protein complex may influence expression of trophectoderm (TE) genes such as Cdx2. Apolar cells that have not inherited the apical protein complex fail to express Cdx2. The basolateral protein complex may suppress the formation of the apical protein complex in apolar cells.

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Functional analysis of many of these proteins has been provided using a variety of strategies (Table 1). Of interest, overexpression of either dominant-negative PKCλ, an aPKC, or to a lesser extent, RNA interference-mediated knockdown of ParD3 at the four-cell stage causes injected cells to preferentially occupy the ICM position at the blastocyst stage (Plusa et al.,2005b). Although it is not clear whether injected cells adopted an ICM fate, these observations support the notion that a functional apical domain is essential for polarity, outside cell formation, and probably TE fate (Fig. 4). However, it is less clear how the apical domain is established initially. One compelling model is that cell–cell contact and adherens junction assembly help establish or orient the apical domain. Of interest, ParD6b and EMK1 localization is disrupted in eight-cell embryos incubated in calcium-free medium (Vinot et al.,2005), suggesting cell–cell contact is important for the localization of these proteins. The absolute requirement for E-cadherin mediated cell–cell contact in cell polarization is not yet clear. Loss of zygotic E-cadherin expression does not disrupt the initiation of compaction, probably owing to perdurance of maternally supplied E-Cadherin (Larue et al.,1994; Riethmacher et al.,1995). Maternal germline knockout of E-cadherin postpones compaction, but embryos are soon rescued by zygotic expression of the paternal allele (De Vries et al.,2004). Examination of loss of both maternal and paternal alleles of E-cadherin will be important for establishing the role of cell–cell contact in establishment of cell polarity.

Table 1. Functional Studies on Epithelial Polarity Proteins in the Early Mouse Embryo
Gene/proteinType of proteinManipulationPhenotypeReference
ParD3PDZ domain proteinInjection of dsRNAInjected cells tend to occupy inside position in blastocystPlusa et al.,2005
PKCλAtypical protein kinase COverexpression of dominant/negativeInjected cells tend to occupy inside position in blastocyst 
PKCζAtypical protein kinase CInhibitory peptideDelayed cavitationEckert et al.,2004
PKCδNovel protein kinase CInhibitory peptideDelayed cavitation 
  Activating peptideAdvanced cavitation 
EzrinERM domain proteinOverexpression of non-phosphorylable formDisrupted adherens junctions (16-cell)Dard et al.,2004
  Overexpression of phosphomimetic formDisrupted adhesion, polarity (8-cell) 
Jam1Adhesion moleculeNeutralizing antibodyDelayed cavitationThomas et al.,2004
E-cadherinAdhesion moleculeNeutralizing antibodyRandomly oriented polarityFleming et al.,1989
  Zygotic nullCells dissociate after compaction, no cavitation, aberrant polarityLarue et al.,1994; Riethmacher et al.,1995; Oshugi et al.,1997
  Maternal germline nullBlastomeres do not associate until morula stagede Vries et al.,2004
β-cateninCadherin-assoicated proteinMaternal germline expression of truncated protein alleleBlastomeres do not adhere until four-cell stage 
VezatinTransmembrane adherens junction proteinMorpholino oligonucleotide-mediated knockdownArrestHyenne et al.,2005
EpithinMembrane serine proteaseInjection of dsRNACompaction inhibitedKhang et al.,2005
RhoASmall GTPaseOverexpression of constitutively active formPremature, disorganized polarizationClayton et al.,1999
Cdc42Small GTPase   

Acquisition of apical/basal polarity also precedes TE formation in experimentally manipulated embryos. First, forced localization of apolar cells to the outside eventually leads to polarization of these cells (Ziomek and Johnson,1982; Ziomek et al.,1982; Johnson and Ziomek,1983) and adoption of a TE fate. Second, TE regeneration from isolated ICMs follows the relocalization of polarity-associated proteins at the apical membrane in outer cells of ICMs (Eckert et al.,2005). These observations suggest that removal from symmetrical cell–cell contact allows spontaneous reassembly of the apical protein complex (Fig. 5). In other words, formation of the apical protein complex is repressed in apolar cells by cell–cell contact, probably by the basolateral protein complex (Figs. 4, 5).

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Figure 5. Model of trophectoderm/inner cell mass (TE/ICM) lineage determination. A: TE/ICM segregation during normal development. B: TE regeneration from ICM after immunosurgery.

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Maintenance of the ICM/TE Lineage Restriction

Several transcription factors are known to be important for ICM and TE fates. One gene important for ICM development is a POU domain transcription factor, Oct4. Oct4 is initially detectable in nuclei of all cells of the early embryo, but its expression becomes restricted to the ICM upon blastocyst formation (Palmieri et al.,1994). Oct4 null embryos implant, indicating formation of a functional TE, but they soon die and lack ICM derivatives such as epiblast and yolk sac (Nichols et al.,1998). Notably, Oct4 null embryos appear to form normal blastocysts with both TE and ICM-like cells present, suggesting that the initial generation of the ICM is not dependent upon Oct4. Furthermore, Oct4 protein is not detectable in nuclei of Oct4 null embryos at the eight-cell stage, arguing against rescue by maternally supplied Oct4. Of interest, the ICM of Oct4 mutants may be misspecified as TE, because half of the Oct4 mutant blastocysts expressed the TE marker, ENDO-A throughout the ICM.

A gene important for early TE development also has been identified. Cdx2 encodes a Caudal-like transcription factor and is expressed in the TE of the blastocyst (Beck et al.,1995; Strumpf et al.,2005). Cdx2 protein is first detectable in the nuclei of select cells at the eight-cell stage (Niwa et al.,2005) and becomes restricted to outer cells of the late morula (Niwa et al.,2005; Strumpf et al.,2005). Cdx2 mutant embryos form a normal-appearing blastocyst, although ICM markers such as Oct4 and Nanog fail to be repressed in the TE (Strumpf et al.,2005), indicating a defect in restriction of the ICM/TE lineage. Cdx2 mutant TE eventually collapses and undergoes apoptosis, leading to blastocyst lethality before implantation. The Cdx2 mutant phenotype thus parallels the Oct4 mutant phenotype: both mutants form morphologically normal blastocysts, but exhibit defects in segregation of the ICM and TE lineages. This finding and other data have led to a model in which mutual antagonism between Oct4 and Cdx2 reinforce the segregation of ICM and TE fates in the blastocyst (Fig. 8A). This model has been supported by studies using stem cells isolated from the blastocyst lineages and will be discussed more later.

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Figure 8. Stem cells from the blastocyst. A: Trophoblast stem (TS) cells are derived from the trophectoderm (TE) lineage, embryonic stem (ES) cells from the epiblast (EPI) lineage, and extraembryonic endoderm (XEN) cells from the primitive endoderm (PE) lineage of the blastocyst. B: Stem cell fates are reinforced by lineage-specific transcription factors: Cdx2 in TS cells, Oct4 and Nanog in ES cells, and Gata4/6 in XEN cells. Many of these factors positively regulate their own expression (circular arrow), whereas negative interactions (bars) maintain lineage restrictions.

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Recently, it has been suggested that localized maternal Cdx2 mRNA and protein can be detected before the eight-cell stage and that maternal Cdx2 is involved in TE formation (Deb et al.,2006). However, knockdown experiments using RNA interference strategies have led to conflicting results, with one group finding preblastocyst lethality (Deb et al.,2006) and another finding phenocopy of zygotic null Cdx2 mutants (Meissner and Jaenisch,2006). Ultimate proof of the functional role of maternal Cdx2 will be provided by analysis of germline null mutants.

ESTABLISHMENT OF THE PE

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. DEVELOPMENTAL STRATEGIES OF EARLY EMBRYOS
  5. DEVELOPMENT OF THE TE
  6. ESTABLISHMENT OF THE PE
  7. STEM CELLS FROM THE BLASTOCYST
  8. POLARITY AND AXIS FORMATION
  9. FUTURE PERSPECTIVES
  10. Acknowledgements
  11. REFERENCES

Segregation of Two Lineages Within the ICM: the EPI and the PE

Twenty-four hours after blastocyst formation, the second lineage decision leads to the establishment of two morphologically distinct populations from the ICM: the EPI, which is the embryonic lineage, and the PE, which will give rise to another extraembryonic tissue, the yolk sac. The PE forms as a monolayer on the surface of the ICM directly facing the blastocoele, while the EPI lies between the PE and the TE (Fig. 1). The PE plays important roles not only in supporting fetal development but also in establishing the anterior/posterior (A/P) axis of the embryo (reviewed in Beddington and Robertson,1999; Ang and Constam,2004; Rossant and Tam,2004). EPI and PE cells from the late blastocyst appear to be lineage-restricted, because they contribute only to their respective lineages in chimeric embryos (Gardner and Rossant,1979; Gardner,1982,1984). However, the mechanisms behind the establishment and segregation of these two lineages within the ICM have been unclear until recently.

Models of EPI/PE Lineage Formation

The original model of establishment of the EPI and PE lineages proposed that ICM cells of the early blastocyst are a homogeneous population of bipotential cells, each with the ability to become either EPI or PE (Fig. 6A). Similar to the Inside–Outside Model described above, cell fate within the ICM would be determined by position within the ICM. Surface cells would differentiate as PE, while enclosed cells would differentiate as EPI. This model is consistent with the ultimate arrangement of EPI and PE cells within the late blastocyst. Supporting data for this model were provided mainly by in vitro studies using embryonic stem (ES) and embryonic carcinoma (EC) cells, which recapitulate many features of the EPI lineage. When grown as embryoid bodies, a layer of PE-derived tissue is found on the outside surface (Martin and Evans,1975; Becker et al.,1992; Murray and Edgar,2001). However, there is little experimental evidence that the PE lineage is derived from cells occupying outside positions in vivo and in vitro.

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Figure 6. Models of epiblast/primitive endoderm (EPI/PE) lineage formation. A: The classic model assumes that the inner cell mass (ICM) is a homogenous population of cells with equal potential (gray) to form either EPI (red) or PE (blue) in the late blastocyst. These lineages are specified on the basis of their position within the ICM, as cells facing the blastocoele become PE, while internal cells become EPI. B: The new model proposes that the ICM is a heterogeneous population of cells with distinct developmental potentials to become either EPI or PE. Cell sorting leads to the segregation of these two lineages in the late blastocyst.

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The above model presumes the ICM is a homogeneous population. ES and EC cells are relatively homogeneous populations, but the ICM may not be. Indeed, several pieces of evidence suggest that the ICM is a heterogeneous population. First, a subset of ICM cells express the extraembryonic lineage-specific cytokeratin ENDO-A in the early blastocyst (Chisholm and Houliston,1987). Second, lineage-specific markers for the EPI and the PE are expressed in nonoverlapping domains in the ICM of the early blastocyst, before morphological segregation of these two lineages (Chazaud et al.,2006). Third, individual ICM cells from the early blastocyst have distinct Epi-like or PE-like gene expression profiles by single cell microarray analysis (Kurimoto et al.2006). Finally, individual labeled ICM cells from the early blastocyst can contribute to either EPI or PE lineages but rarely to both (Chazaud et al.,2006). These results suggest that the early ICM is not a homogeneous population but a mosaic of EPI and PE progenitors. An alternative model of EPI and PE formation suggests that cells of the ICM already have different developmental potential and that the ICM is a heterogeneous mixture of EPI and PE cells that will sort out into the appropriate layers (Fig. 6B).

Molecular Regulation of EPI/PE Lineage Formation

The identification of genes required for PE formation has given insight into mechanisms regulating the initiation and maintenance of EPI/PE lineage restriction. In the late blastocyst, the homeobox transcription factor Nanog is expressed in the EPI (Chambers et al.,2003; Mitsui et al.,2003), and the zinc finger transcription factor Gata6 is expressed in the PE (Morrisey et al.,1998; Koutsourakis et al.,1999). Mice lacking both alleles of Nanog form a morphologically normal blastocyst (Mitsui et al.,2003), indicating that the ICM and TE form normally. These embryos die after implantation and lack recognizable EPI tissues. In contrast, Gata6 null embryos exhibit defects in the PE lineage, although not as early as the Gata6 expression pattern might suggest, perhaps because of functional redundancy between Gata6 and the related protein Gata4 (Morrisey et al.,1998; Koutsourakis et al.,1999). Gata6 mutants die nearly 4 days after blastocyst formation and exhibit defects in PE-derivatives, the visceral and parietal endoderm.

Several lines of evidence indicate that the fibroblast growth factor (FGF) family of receptor tyrosine kinases (RTK) is required for Gata6 expression and PE formation. Mice lacking either Fgf4 or Fgf receptor2 (Fgfr2) do not form PE in vivo or in vitro (Feldman et al.,1995; Wilder et al.,1997; Arman et al.,1998). Overexpression of a dominant-negative form of the Fgf receptor prevents formation of PE in vitro in embryoid bodies (Li et al.,2001). Importantly, this phenotype can be rescued by overexpression of Gata6 and Gata4 (Li et al.,2004). PE also fails to develop in Grb2 mutant mice (Cheng et al.,1998). Grb2 encodes an SH2/SH3 containing adaptor protein important for RTK-mediated activation of the Ras-MAPK cascade. Late blastocysts lacking Grb2 do not form a morphologically distinct layer of PE cells, and express only markers of EPI (Chazaud et al.,2006). Furthermore, Gata6 is not expressed in the ICM of Grb2 mutants at the early blastocyst stage, and Nanog is expressed in all ICM cells at this stage (Chazaud et al.,2006).

Taken together, these results suggest a new model in which Grb2/RTK signaling within the ICM of the early blastocyst selects certain cells for PE fate, PE fate is reinforced by Gata factors, and PE and EPI cell types sort out by the late blastocyst stage (Fig. 6B). However, it is not clear how Grb2/RTK activity initially is restricted to a subset of cells within the ICM. One possibility is that the differences in the developmental origins of the ICM may set up different cellular responses to Grb2/RTK signaling. Cells in the ICM derive from the inner cells of the late morula. These inner cells are produced by two successive rounds of asymmetric division (Fig. 7A): first, during the 8- to 16-cell transition and then during the 16- to 32-cell transition. These two populations, the first and second inner cells, are set aside at distinct developmental time points and therefore may contribute heterogeneity to the nascent ICM. Moreover, cells that express the extraembryonic marker ENDO-A were found to derive almost exclusively from second inner cells (Chisholm and Houliston,1987). From this result, it was suggested that the second inner cells form PE and that induction of the PE did not depend on location within the ICM. Another possibility is that cell fates are specified after blastocyst formation, through some kind of cell–cell interaction, such as lateral inhibition (Fig. 7B).

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Figure 7. Two models of epiblast/primitive endoderm (EPI/PE) determination. A: Cleavage-driven model. Aa: First inner cells produced in the 8- to 16-cell division become EPI progenitors (red). Ab: Second inner cells produced in the 16- to 32-cell division become PE progenitors (blue). Trophectoderm (TE; green). B: Stochastic model. Inner cells are randomly determined to become either EPI or PE progenitors.

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Several pieces of evidence suggest that segregation of EPI and PE cells into two separate layers by the late blastocyst stage is directed by the Gata factors. Evidence from in vitro studies suggests that EPI and PE cells acquire different adhesive characteristics that allow them to sort out from each other. In ES cells, Gata4/6 overexpression leads to a strong up-regulation of LamininB1, which changes the adhesion properties of Gata-expressing cells (Fujikura et al.,2002). Mutation of Dab2, a direct target of Gata6 (Morrisey et al.,2000) seems to cause a defect in the sorting out process in vivo. Cells labeled with PE markers are found scattered in the middle of the ICM (Yang et al.,2002). Dab2 is a PTB domain containing adaptor protein that modulates cellular adhesiveness during megakaryocytic differentiation of the human chronic myeloid leukemic cell line K562 (Tseng et al.,2003) and transiently interacts with Integrin β1 to regulate its activation during induced epithelial to mesenchymal transition in normal murine mammary gland cells (Prunier and Howe,2005). Moreover, mutation of LamC1 and Integrinβ1, genes known to modulate cell adhesiveness, causes similar phenotypes (Stephens et al.,1995; Smyth et al.,1999). These genes thus represent excellent candidates for regulating cell sorting since PE lineage progenitors form but do not segregate to the surface of the ICM in these mutants.

STEM CELLS FROM THE BLASTOCYST

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. DEVELOPMENTAL STRATEGIES OF EARLY EMBRYOS
  5. DEVELOPMENT OF THE TE
  6. ESTABLISHMENT OF THE PE
  7. STEM CELLS FROM THE BLASTOCYST
  8. POLARITY AND AXIS FORMATION
  9. FUTURE PERSPECTIVES
  10. Acknowledgements
  11. REFERENCES

An interesting feature of the first three lineages of the mouse is that stem cells can be derived from each (Fig. 8A). ES cells have been derived from the epiblast lineage (Evans and Kaufman,1981; Martin,1981), trophoblast stem cells (TS cells) from the trophectoderm lineage (Tanaka et al.,1998), and extraembryonic endoderm (XEN) cells from the primitive endoderm lineage (Kunath et al.,2005). Like other stem cells, these cells can either self-renew or differentiate into lineage-specific cell types. Each stem cell type also exhibits morphology, gene expression, and growth factor requirements characteristic of the lineage from which it derives. Importantly, all have been shown to contribute mainly to their lineage of origin in chimeras (Beddington and Robertson,1989; Tanaka et al.,1998; Kunath et al.,2005). Stem cells from the blastocyst have provided a valuable tool for the genetic analysis of lineage-promoting factors and have confirmed the central importance of transcription factors in maintaining cell fates. Of interest, because many of these factors regulate each other's expression, manipulation of levels of these factors can cause existing stem cell lines to adopt properties of other lineages.

TS Cells Vs. ES Cells and the TE/ICM Lineage Restriction

Several lines of evidence suggest that ES and TS cells recapitulate the lineages from which they derive. Like the ICM, ES cells express Oct4, and ES cells cannot be derived from Oct4 null embryos (Nichols et al.,1998), suggesting Oct4 is essential for ES cell fate. Like the TE, TS cells express Cdx2 (Tanaka et al.,1998), and TS cells cannot be derived from Cdx2 null embryos, underscoring its importance for TS cell fate (Strumpf et al.,2005). Reduction of Oct4 in existing ES cells leads to derepression of Cdx2 expression (Niwa et al.,2000), consistent with proposals that Oct4 reinforces segregation of ICM and TE fates. Similarly, overexpression of Cdx2 in ES cells causes repression of Oct4 and other ES cell genes (Niwa et al.,2005). Notably, both Oct4 mutant and Cdx2-overexpressing ES cells can grow as TS-like cells in the presence of TS growth medium (Niwa et al.,2000,2005). These two cell lines exhibit similar, although not identical, morphology and gene expression profiles, but only Cdx2-overexpressing cells have been shown to contribute to the placental lineage in chimeras (Niwa et al.,2005).

These cell lines have provided a level of detail not easily obtained in the intact embryo. For example, genetic and biochemical evidence from ES cells recently has suggested that Cdx2 and Oct4 also regulate each other at the protein level and that this interaction allows Cdx2 to block Oct4 from reinforcing its own expression (Niwa et al.,2005). This observation has extended our understanding of how Cdx2 and Oct4, initially coexpressed in outer cells of the late morula (Niwa et al.,2005; Strumpf et al.,2005) become restricted to distinct lineages of the blastocyst. Of interest, in the absence of Oct4, Cdx2 is not necessary for driving ES cells toward TE-like fate, although it does appear to be required for their self-renewal (Niwa et al.,2005). Thus, Cdx2 promotes TE fate primarily by repressing Oct4 activity but may promote stem cell self-renewal through a separate pathway.

XEN Cells Vs. ES Cells and the PE/EPI Lineage Restriction

ES cell lines, which are established from outgrowth of blastocysts, are often compared with the ICM. However, given the mosaic makeup of the early ICM (Chazaud et al.,2006), ES cells are likely to derive from a select population of ICM cells in the blastocyst. Although cell types derived from other blastocyst lineages are initially present in outgrown blastocysts, these are not propagated during ES cell derivation, suggesting that other ICM-derived cell types are selected against. In fact, if outgrowths are cultured in non-ES cell culture conditions, stem cells from the PE lineage can be recovered. These stem cells, called XEN for extraembryonic endoderm, proliferate in culture and contribute only to the PE lineage when injected into blastocysts (Kunath et al.,2005).

Transcription factors important for the PE/EPI lineage restriction are also important for ES and XEN cell fates. The EPI-expressed gene Nanog encodes a protein required for maintenance of ES cell pluripotency (Chambers et al.,2003; Mitsui et al.,2003). Knockout of Nanog in ES cells leads to differentiation of PE-like cells and expression of the PE marker Gata6 (Mitsui et al.,2003). These studies have been complemented by studies on Gata6 in ES cells. Overexpression of Gata4/6 in ES cells also leads to differentiation into a PE-like cells and down-regulation of the ICM/EPI marker Oct4 (Fujikura et al.,2002). Thus, antagonism between Nanog and Gata factors may reinforce XEN and ES cell fates to promote self-renewal and prevent switching among these lineages, analogous to the activity of Oct4 and Cdx2 in ES and TS cell fates (Fig. 8B).

POLARITY AND AXIS FORMATION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. DEVELOPMENTAL STRATEGIES OF EARLY EMBRYOS
  5. DEVELOPMENT OF THE TE
  6. ESTABLISHMENT OF THE PE
  7. STEM CELLS FROM THE BLASTOCYST
  8. POLARITY AND AXIS FORMATION
  9. FUTURE PERSPECTIVES
  10. Acknowledgements
  11. REFERENCES

Polarity in the Oocyte and Blastocyst

The blastocyst has a natural morphological polarity, with ICM and polar trophectoderm at the embryonic pole and the blastocoele and mural TE at the abembryonic pole. How the embryonic/abembryonic (em/ab) axis is established is a controversial topic in the field of early mouse development. Two independent groups have reported that this axis can be traced back to the first cleavage of the embryo (Gardner,2001; Piotrowska et al.,2001; Plusa et al.,2005; reviewed in Zernicka-Goetz,2005). According to these studies, the first cleavage plane coincides with the animal/vegetal axis of the oocyte and will ultimately coincide with an axis perpendicular to the em/ab axis of the blastocyst. The descendants of one of the two-cell blastomeres tended to occupy the polar trophectoderm and nearby ICM cells, whereas the other's descendents occupied the mural trophectoderm and surface cells of ICM. However, other groups have reported that there is no correlation between the first cleavage plane and the em/ab axis (Alarcon and Marikawa,2003,2005; Chroscicka et al.,2004; Hiiragi and Solter,2004). An alternative model suggested that the first cleavage plane is determined by the angle of pronuclear aposition and does not always cross the animal pole (Hiiragi and Solter,2004; reviewed in Rossant and Tam,2004). It is not clear whether this discrepancy is due to technical differences or differences in the mouse strains used for each study. Because these observations have been based only on morphology, it will be important to analyze early cleavage and lineage formation at the molecular level. Knowledge of molecular mechanisms behind the blastocyst formation will help settle the debate.

Of interest, blastomeres from the four-cell mouse embryo may not be equivalent in terms of fate and developmental potential. First, not all blastomeres from the four-cell embryo gave rise to all tissue lineages (Fujimori et al.,2003). Second, one four-cell blastomere, identified on the basis of its unique cleavage pattern, was unable to form an entire embryo on its own, despite being able to contribute to all cell lineages in chimeras (Piotrowska-Nitsche and Zernicka-Goetz,2005). Together these data suggest that as-yet unidentified patterning information may be differentially distributed among early blastomeres, although this information is modifiable because cell fates are yet plastic.

Recently, Deb et al. (2006) reported localization of maternal Cdx2 mRNA to one half of the oocyte and to only one of the two-cell blastomeres (Deb et al.,2006). Using a combination of a time course of Cdx2 expression and lineage tracing, the Cdx2-expressing blastomere and its descendents were proposed to divide later than the nonexpressing blastomere and, surprisingly, to give rise to the entire trophectoderm (both mural and polar), occupying both embryonic and abembryonic regions of the blastocyst. The earlier-dividing two-cell blastomere did not give rise to any trophectoderm. However, this proposal contradicts all previously published observations regarding developmental potential and fate of cells from the two-cell embryo, because all other groups have reported contribution of both two-cell blastomeres to both ICM and TE (Gardner,2001; Piotrowska et al.,2001; Alarcon and Marikawa,2003,2005; Chroscicka et al.,2004; Hiiragi and Solter,2004; Plusa et al.,2005a; Motosugi et al.,2005; Piotrowska-Nitsche and Zernicka-Goetz,2005). Before these observations can be considered as changing the paradigms of early mouse development, it is essential to explore whether this lineage relationship holds true in other mouse strains and if it can be reproduced by other groups using alternate lineage tracing methods.

Formation of the Anterior/Posterior Axis

Formation of the lineages of the blastocyst is driven by cell polarity and cell segregation mechanisms. However, these processes do not in themselves tell us how the next important event of development, the formation of the A/P body axis, is brought about. The earliest sign of a definitive axis in mouse embryos is observed before gastrulation, when a subpopulation of PE derivatives, known as the distal visceral endoderm (DVE), moves anteriorly to become the anterior visceral endoderm (AVE; reviewed in Beddington and Robertson,1998; Beddington and Robertson,1999; Ang and Constam,2004; Rossant and Tam,2004). This extraembryonic cell population sends signals and initiates the A/P axis in the embryo. However, it is not yet understood how and when this population is specified, nor is it clear whether there is a direct relationship between these cells and any prior asymmetries—at the blastocyst stage or earlier (reviewed in Tam and Rossant,2003; Rossant and Tam,2004; Zernicka-Goetz,2004).

Insight into this issue has been provided recently by careful examination of the Nodal antagonist and DVE/AVE marker encoded by Lefty. Surprisingly, Lefty expression is restricted to a tiny subset of cells in the ICM of the early blastocyst (Takaoka et al.,2006). Twenty-four hours later, in the late blastocyst, Lefty expression is localized to a small region of the PE. These observations support the notion that the ICM is initially a mosaic of EPI and PE cells and suggest that an embryonic axis prepattern may exist as early as the blastocyst stage. Another interesting finding recently obtained in zebrafish suggests that a homologue of Nodal, squint (sqt), mRNA localizes to the future dorsal side of the embryo as early as the four-cell stage (Gore et al.,2005). Initially, maternal sqt mRNA is distributed evenly throughout the cytoplasm of the egg but, after fertilization, maternal sqt mRNA gradually becomes enriched in future dorsal cells. A localization signal identified in the 3′-untranslated region of the mRNA is conserved between fish and humans, although mouse Nodal mRNA lacks this sequence. It will be interesting nonetheless to analyze whether this localization is conserved in mouse embryos.

FUTURE PERSPECTIVES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. DEVELOPMENTAL STRATEGIES OF EARLY EMBRYOS
  5. DEVELOPMENT OF THE TE
  6. ESTABLISHMENT OF THE PE
  7. STEM CELLS FROM THE BLASTOCYST
  8. POLARITY AND AXIS FORMATION
  9. FUTURE PERSPECTIVES
  10. Acknowledgements
  11. REFERENCES

After more than 25 years since the first models of lineage restriction were proposed, we have begun to understand the molecular underpinnings of lineage restriction and fate commitment in the early mouse embryo. Technological advancements have contributed and will continue to contribute to the success of this field. The development of novel techniques for lineage tracing and live imaging, and novel methods for the identification of new genes are a few examples. Investigation of early patterning mechanisms in non-mouse mammals has and will continue to illuminate the similarities and differences among mammalian developmental strategies (reviewed in Selwood and Johnson,2006). In addition, stem cell lines from the first three lineages of the mouse and from the ICM of human embryos have not only provided an invaluable tool for developmental biologists, but they have emerged as a research model in their own right. It is already clear that lessons learned through these strategies extend beyond blastocyst formation, and have contributed to advancement in our understanding of other processes, such as body patterning and stem cell therapeutics.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. DEVELOPMENTAL STRATEGIES OF EARLY EMBRYOS
  5. DEVELOPMENT OF THE TE
  6. ESTABLISHMENT OF THE PE
  7. STEM CELLS FROM THE BLASTOCYST
  8. POLARITY AND AXIS FORMATION
  9. FUTURE PERSPECTIVES
  10. Acknowledgements
  11. REFERENCES

We thank Dr. Cheryle Séguin for careful reading of this manuscript and other members of the Rossant Lab for input. We also thank the pioneers of this field for providing careful analyses, insightful interpretations, and prescient models.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. DEVELOPMENTAL STRATEGIES OF EARLY EMBRYOS
  5. DEVELOPMENT OF THE TE
  6. ESTABLISHMENT OF THE PE
  7. STEM CELLS FROM THE BLASTOCYST
  8. POLARITY AND AXIS FORMATION
  9. FUTURE PERSPECTIVES
  10. Acknowledgements
  11. REFERENCES