We have investigated the events leading to formation of the primitive endoderm in the preimplantation mouse embryo. Our results indicate that, during early stages of development, cells co-express markers for different lineages. Subsequently PrE fate results from a progressive restriction of expression of PrE-specific markers followed by cell sorting.
Pdgfrα is a novel marker for PrE. A mouse line expressing histone H2B-EGFP reporter from the Pdgfra
locus served as a live imaging reporter of PrE. Pdgfrα H2B-GFP
expression is initially distributed in an apparently random, heterogeneous manner until approximately the 64-cell stage, or after six rounds of divisions. The early heterogeneous expression of Pdgfrα H2B-GFP
is reminiscent of the recently reported salt-and-pepper expression of Nanog and Gata6 in the ICM of the E3.5 blastocyst (Chazaud et al., 2006
; Rossant et al., 2003
). This, along with the lineage tracing studies, has led to a model for lineage specification in which EPI and PrE precursors are specified in a possibly random manner within the ICM and later segregate to their respective layers. This is in contrast to the previously assumed model that cell position with respect to the blastocyst cavity is the primary determinant of PrE fate (reviewed in Yamanaka et al., 2006
Our results revealed a multi-step process of PrE formation that shares features of both of these models. As also shown by Dietrich and Hiiragi (Dietrich and Hiiragi, 2007
), lineage-specific factors initially exhibit widespread and overlapping expression in early blastocysts. This is followed by progression towards the mutually exclusive expression of Cdx2 in the TE (Dietrich and Hiiragi, 2007
) (this study) and Nanog, Gata6 and Pdgfrα in the ICM (this study, see ) and, subsequently, by the mutually exclusive expression of EPI- and PrE-specific markers in the ICM during the 32- to 64-cell transition.
Multi-step model of EPI/PrE lineage formation
We also noticed that at early stages (morula and early blastocyst), the levels of Gata6 and Nanog vary among cells throughout the embryo and generally appear to be mutually independent. A similar observation was described by Dietrich and Hiiragi for TE and ICM markers (Dietrich and Hiiragi, 2007
). There are two possible explanations for this type of expression. First, the relative levels of different factors, while varying among cells, may remain fairly constant within individual cells. Differences would then become amplified by the maturation of mutually inhibitory pathways at later stages. Alternatively, it is possible that the relative levels of factors fluctuate with time. Interestingly, it was reported recently that Nanog
expression fluctuates in ES cells (Chambers et al., 2007
). Low levels of Nanog predisposed cells towards differentiation, but did not mark commitment. In another study, cells were flow-sorted according to their level of Nanog expression and, when subsequently cultured, they reverted to their original heterogeneous state. An association between stochastic gene expression and differentiation has been reported in other systems (Hu et al., 1997
), but evidence during embryo development, especially in the mouse, has previously been lacking (reviewed by Laforge et al., 2005
; Martinez-Arias and Hayward, 2006
It seems plausible that such a mechanism may operate within the ICM. We propose that before blastocyst formation, a large number of genes exhibit fluctuating and mutually independent expression at low levels. As transcription factor levels increase, mutually inhibitory regulatory pathways begin to take effect. The latter is supported by our observation that mutually exclusive expression is typically observed only in the subset of cells with the highest expression levels. Heterogeneous expression of Nanog that correlated negatively with Gata6 expression has recently been reported in mouse ES cells (Singh et al., 2007
). Nanog was shown to repress Gata6 directly, through binding of its promoter. Conversely, the Grb/Mek pathway, which regulates Gata6 expression, has been shown to repress Nanog expression (Hamazaki et al., 2006
Our data suggest that downregulation of Nanog and Gata6 occurs in two phases. The first reflects downregulation in TE cells, which was often evident immediately after blastocyst formation but had invariably commenced in all blastocysts of at least 37 cells. The second phase initiates around the 64-cell stage, when the number of Nanog-positive cells drops dramatically and thereafter remains relatively constant. This phase reflects the maturation of the presumed inhibitory mechanisms between Nanog- and Gata4/Gata6-expressing pathways, analogous to the state of ES cells.
We propose that upregulation of Gata4 and downregulation of Nanog in a subset of ICM cells at around the 64-cell stage reflects a crucial stage of stabilisation, or fixing, of a previously fluctuating expression state. Lineage tracing data (Perea-Gomez et al., 2007
) suggests that 30% of ICM cells from early blastocysts have a dual fate, with daughter cells occupying both EPI and PrE layers. This is in contrast to the restricted fate of ICM cells found in what were likely to be later stage blastocysts (Chazaud et al., 2006
). This would suggest that fate becomes restricted around the mid-blastocyst (~64-cell) stage, and is coincident with changes in marker expression.
Our data support the restriction of ICM cell fate preceding cell sorting. Despite this, our live imaging data revealed an upregulation of Pdgfrα H2B-GFP
expression in cells lining the blastocyst cavity and downregulation in deeper-lying cells, supporting a role for positional signals. The variety of behaviours of GFP-positive cells we observed in our live imaging studies suggests a more complex model of cell sorting than a simple segregation of precursors to the respective EPI and PrE layers. The failure of some GFP-positive cells to contribute to the PrE suggests that, although the mutually exclusive expression of Nanog and Gata6 biases cells towards a particular fate, positional signals are required to complete or reinforce specification. Cell polarisation is likely to be involved, consistent with the observation that, prior to PrE formation, the sub-cellular localisation of the PrE markers Lrp2 (megalin) and Dab2 becomes polarised only in cells lining the cavity (Gerbe et al., 2008
We observed that GFP-positive cells lining the cavity rarely changed their position. By contrast, deeper-lying cells tended to be more migratory, but lost this property upon reaching the cavity. Thus, even a randomly directed migration of deeper-lying cells would suffice to drive the majority of cell sorting because PrE precursors, once in contact with the cavity, will remain there. The probability of PrE precursors reaching the cavity would also be enhanced by adhesive contacts with any neighbouring PrE precursors that already line the cavity. The adhesive properties of PrE precursors have previously been implicated in PrE formation (reviewed by Yamanaka et al., 2006
Once a PrE precursor reaches the cavity, its position may be maintained by establishing cell polarity and epithelial properties, to the competitive exclusion of non-polarised cells. In a study of mouse blastocyst formation, it was shown that differences in epithelial properties between subsets of cells were sufficient to influence their propensity to occupy an outer position and to contribute to the TE (Plusa et al., 2005
). Thus, PrE precursors occupying the cavity surface may have a propensity to flatten, due to cell polarisation. Increased flattening would cause cells to occupy a greater surface area of the cavity, to the competitive exclusion of non-polarised EPI precursors. Deeper-lying PrE precursors that maintain contact with such cells would thus be able to maintain proximity to the cavity.
An additional selective mechanism, using apoptosis, appears to account for the minority of PrE precursors that fail to come into contact with the cavity. The prevalence of apoptosis in deeper-lying GFP-positive cells after the 64-cell stage suggests that the stabilisation of PrE-specific transcription factor expression in strongly GFP-positive cells excludes EPI potential, and leaves the cell with either of two possible outcomes: contributing to the PrE or defaulting to apoptosis. More weakly GFP-expressing cells showed a tendency to downregulate expression, suggesting that they may retain the potential to form EPI. In agreement with our observations, a surge in the frequency of cell death in the ICM of embryos of 60–110 cells was previously reported (Copp, 1978
). We suggest that one of the primary functions of apoptosis within the ICM is the elimination of inappropriately positioned PrE precursors. This is supported by our observation that the probability of GFP-positive cells undergoing apoptosis is much greater for inner cells than for those in the PrE layer.
In summary, our results reconcile the two apparently disparate models of PrE lineage specification within the ICM of the mouse blastocyst. Our observations lead us to propose a three-step model in which stochastic expression of lineage-specific transcription factors, at the 16- to 32-cell stage, precedes the maturation of mutually inhibitory regulatory pathways, leading to a salt-and-pepper distribution of EPI and PrE precursors in the ICM after the 64-cell stage. This is followed by cell sorting, which may be largely, if not completely, explained by a passive, selective mechanism involving cell movement, cell adhesion and apoptosis.