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- EXPERIMENTAL PROCEDURES
Mammalian development begins following sperm and egg fusion in the ampulla of the oviduct. In the mouse, the first cleavage division occurs within 24 hr, with additional asynchronous cell divisions producing the ∼64-cell embryo approximately 100 hr post-human chorionic gonadotropin (hCG; Barlow et al., 1972). As the blastocyst forms several morphologically distinct features appear. The blastocoel cavity expands and two distinct cell populations emerge that have restricted fates: the inner cell mass (ICM) and trophectoderm (TE). The expanded blastocyst then undergoes a morphological change in shape from round to elongated, concurrent with or before implantation (Finn and McLaren, 1967). The embryo hatches from the zona pellucida, and extensive interactions between the embryo and maternal uterine tissue (involving epidermal growth factor, prostaglandins, cytokines, and other signaling pathways) lead to blastocyst activation, uterine receptivity, uterine epithelium extracellular matrix degradation, and implantation (Schlafke and Enders, 1975; Strickland et al., 1976; Copp and Rossant, 1978; Parr et al., 1987; Brenner et al., 1989; Yamazaki and Kato, 1989; Rohde and Carson, 1993; Kamijo et al., 1998; Galan et al., 2000; Paria et al., 2001; Zhang et al., 2013). In humans, it is estimated that as many as 75% of early embryo losses are thought to result from implantation failure (Wilcox et al., 1988; Norwitz et al., 2001; Cockburn and Rossant, 2010). Many important questions remain about mechanisms that control implantation, as well as the morphogenetic changes that occur during peri-implantation embryo development.
Beginning at the morula stage and throughout blastocyst formation, there are well-documented molecular events that influence and distinguish early cell fates. For example, OCT4, SOX2, fibroblast growth factor-4 (FGF4), and NANOG are restricted to the inner cell mass (ICM), while GATA3, CDX2, and EOMES specifically mark the cells of the TE (Feldman et al., 1995; Nichols et al., 1998; Ciruna and Rossant, 1999; Hancock et al., 1999; Avilion et al., 2003; Mitsui et al., 2003; Home et al., 2009). In the late blastocyst, a sheet of primitive endoderm (PrE) covers the ICM and can be identified by GATA4, GATA6, SOX7, and SOX17 expression (Cai et al., 2008; Plusa et al., 2008; Artus et al., 2011). Progress defining the mechanisms responsible for initial lineage specific gene expression and cell fate has been made largely through analysis of knockout (KO) embryos that have revealed genetic interactions and hierarchies of these critical proteins (reviewed in Rossant and Tam, 2009; Stephenson et al., 2012).
Specification of the ICM and TE are closely intertwined. Oct4 null embryos fail to form pluripotent ICM and although trophoblast cells are observed, neither ICM nor PrE proliferate in outgrowth assays (Nichols et al., 1998). Sox2 KO embryo outgrowths express reduced levels of Oct4 and Fgf4, and an abundance of the TE marker Pl1 (Avilion et al., 2003). In contrast, Nanog knock out embryos make blastocysts, but outgrowths differentiate specifically into PrE (Chambers et al., 2003; Mitsui et al., 2003). More recent work has shown that SOX2 activates Fgf4 and positively regulates Oct4 expression in the ICM (Avilion et al., 2003), while the TE marker CDX2 acts to repress Oct4 and Nanog and promote Eomes expression in the TE (Strumpf et al., 2005). Other studies show that GATA3 activates Cdx2 specifically in the TE through interaction with an intronic GATA binding site (Home et al., 2009), but control of Gata3 expression remains unclear.
Development of the PrE is also closely connected to ICM and TE formation. PrE precursors identified by expression of GATA-6 and platelet-derived growth factor receptor-α (PDGFRα) first arise within the ICM in the 64-cell embryo in a “salt and pepper” heterogenic pattern (Chazaud et al., 2006; Plusa et al., 2008; Yamanaka et al., 2010; Grabarek et al., 2012). The primitive endoderm marker GATA6 is not required for formation of OCT4-positive ICM in vivo, but Gata6 KO embryos fail to develop GATA4-positive PrE, DAB2-positive visceral endoderm (VE), or parietal endoderm (PE; Cai et al., 2008). Sox7 and Sox17 (both PrE markers) are also expressed in a mosaic pattern in the 64-cell embryo, in the PrE of the late blastocyst and in the extraembryonic VE of the gastrulating mouse embryo (Artus et al., 2011). It has been suggested that the two proteins are functionally redundant; however, the absence of Sox17 in implantation-delayed blastocysts results in a loss of PrE epithelial integrity, including premature delamination and migration of PE (Artus et al., 2011). Although the initial expression of Sox17 and Gata6 are not reliant upon FGF4, the level and heterogeneity of Fgf4 expression is crucial for differentiation of both the ICM and PrE lineages (Nichols and Smith, 2009; Yamanaka et al., 2010; Kang et al., 2013). Shortly after the “salt and pepper” GATA6/NANOG gene expression pattern is observed in the ICM, a single sheet of PrE is observed overlaying the ICM of the ∼80-cell embryo and then clearly covering the surface of the ICM of the 100-cell embryo (Plusa et al., 2008; Rossant and Tam, 2009; Morris and Zernicka-Goetz, 2012). However, the dynamics of the morphogenetic sorting events that occur during these stages (64–80 cell embryo) are not well documented in vivo.
By embryonic day (E) 5.5, the embryo adopts a radially symmetric egg cylinder morphology that has been well described (Wiley and Pedersen, 1977; Gonda and Hsu, 1980; Copp, 1981; Skreb et al., 1991; Hishinuma et al., 1995; Perea-Gomez et al., 2007; Rossant and Tam, 2009). Although the initial dynamics of egg cylinder formation are not completely defined, the directional growth of the egg cylinder has been detailed; egg cylinder expansion first occurs antimesometrially into the blastocoel cavity and then mesometrially toward the primary uterine lumen (Copp, 1979; Skreb et al., 1991). The enlarged primitive endoderm cells differentiate into visceral (classically named proximal) endoderm, the proamniotic cavity forms within the epiblast (EPI), and the polar trophectoderm differentiates into the extraembryonic ectoderm (Snell and Stevens, 1966). Additionally, many details regarding morphogenetic movements and distinct tissue formation have been defined during gastrulation in the postimplantation embryo (Downs and Davies, 1993). Although there is a large body of preimplantation (E0.5–E3.5) and postimplantation (E5.5–E8.5) literature, little is known about peri-implantation development in utero. This critical developmental window encompasses the embryo's first interaction with and subsequent implantation into the uterine luminal epithelium (ULE) as well as the transition from the blastocyst to the egg cylinder. An informative implantation staging description was produced by Finn and McLaren in 1967, which includes informative data collected through electron microscopy techniques (Finn and McLaren, 1967). More recently, a study of ex vivo E5.0–E6.5 embryos has revealed a new model for formation of both the extraembryonic ectoderm and visceral endoderm that suggests that the anterior visceral endoderm arises from polyclonal origins during peri-implantation (Perea-Gomez et al., 2007).
While both preimplantation and postimplantation embryos can be readily dissected and cultured, analysis of peri-implantation stages presents unique challenges as development of the embryo is reliant on interactions with maternal tissue. Although current protocols allow for relatively successful ex vivo embryo culture during the blastocyst to early egg cylinder transition (Morris et al., 2012), the molecular dynamics within blastomeres have not been thoroughly detailed in utero. Adding to the difficulty of isolating E4.0–E5.0 embryos, the uterus displays no obvious external clues to guide embryo collection until egg cylinder stages when decidual swellings permit embryo localization (Snell and Stevens, 1966). As a result, detailed molecular knowledge of peri-implantation development remains a gap in the understanding of mammalian development. Study of these stages in vivo may provide distinct insight regarding mechanisms that control the initial formation of a primitive endoderm sheet, the ICM to EPI transition, and embryo/uterus interactions or signaling.
Here, we present morphological and gene expression data collected in a systematic manner by sequential immunofluorescence to examine peri-implantation development within the context of the uterus in vivo. We document expression of key lineage-specific transcription factors and markers of epithelial biogenesis. Our data support current models but also suggest that in vivo: (1) the first GATA6-positive primitive endoderm cells are sorted to the periphery of the ICM and still express OCT4; (2) the GATA6-positive primitive endoderm cells that form a sheet over the ICM are OCT4 and Laminin negative, but E-Cadherin positive; (3) parietal endoderm cells exhibit strikingly higher GATA6 expression when compared with visceral endoderm cells; (4) maternal SOX17 in the uterine epithelium may guide or mark implantation sites. Our results are largely consistent with current models of PrE formation/specification, and add insight to the sorting events occurring as the embryo transitions from the blastocyst to early egg cylinder stage (reviewed in Stephenson et al., 2012).
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- EXPERIMENTAL PROCEDURES
Although there is a significant body of literature on peri-implantation anatomy, there is relatively little in vivo molecular analysis available. We have examined peri-implantation developmental dynamics and integrated our results with known key morphogenetic events and models (Fig. 4). This molecular and morphogenetic map of peri-implantation will be useful for analysis of embryonic phenotypes that occur between blastocyst formation and gastrulation. Through analysis of gene expression at precise time points during peri-implantation development in utero, we have documented subtle changes in primitive endoderm morphogenetic differentiation.
In the 64-cell embryo, a “salt and pepper” pattern is observed which reflects a differentiation of primitive endoderm cells that lack both SOX2 and NANOG and express GATA6 and PDGFRα (Chazaud et al., 2006; Plusa et al., 2008; Yamanaka et al., 2010; Grabarek et al., 2012). In the 80-cell embryo, the primitive endoderm cells largely cover the ICM and the primitive endoderm cells clearly cover the surface of the ICM in the 100-cell embryo (Plusa et al., 2008). Our data revealed a consistent localization of primitive endoderm cells at the periphery of the ICM in vivo between these stages (between the 64-cell “salt and pepper” stage and the 80-cell stage “PrE covered ICM” stage). At 115 hp-hCG, these peripheral primitive endoderm cells express high levels of OCT4, GATA6, and Laminin, but are SOX2 and E-Cadherin negative. Slightly later at 121 hp-hCG, the GATA6-expressing PrE cells that cover the ICM are E-Cadherin positive and Laminin/OCT4 negative (Fig. 2). The PPrE are likely the descendants of the GATA6-positive primitive endoderm first observed in the “salt and pepper” ICM (Rossant et al., 2003; Chazaud et al., 2006). However, our data suggest that morphogenetic events result in precise localization of these cells at the periphery before covering the ICM, and that migration across the ICM may be the mechanism by which a single sheet of PrE is formed. Alternatively, a Cadherin-mediated sorting may be responsible for localization of the CDH1-negative/GATA6-positive PPrE; Cadherins are known to be involved in several developmentally regulated sorting events (reviewed in Nelson et al., 2013), and appropriate regulation of cell adhesion is essential for TE formation and implantation (Larue et al., 1994; Fleming et al., 2001; Jha et al., 2006). Additionally, several reports have suggested a sorting model of “directionally biased relocation and apoptosis,” which results in survival of those PrE precursors that are at the ICM surface and apoptosis of precursors within deeper the ICM (Rossant et al., 2003; Chazaud et al., 2006; Plusa et al., 2008). Our descriptive observations are not inconsistent with this model, and suggest that this sorting may direct cells precisely toward the intersection of the ICM, TE, and blastocoel cavity.
Recently, a model of sequential transcription factor activation in the primitive endoderm was suggested in which Gata6 is first expressed, followed by Sox17, Gata4, and finally Sox7 (Artus et al., 2011). It has also been suggested that Nanog expression in the ICM prevents ectopic expression of Gata6, which in turn silences Nanog in the differentiating PrE (Chambers et al., 2003; Mitsui et al., 2003). At the time of lineage allocation, this mechanism is also controlled by specific levels of heterogeneous FGF4, which plays an important role in acquisition of cell lineage bias through positively regulating Gata6 expression and negatively regulating Nanog (Kang et al., 2013). The mutually exclusive expression patterns of Gata6 and Nanog observed in utero are consistent with this model. Functional investigation of signaling between the ICM, TE and PPrE cells at this time may shed light on these important developmental events.
In vitro preimplantation analyses have revealed that Laminins (1, 2/4 and 10/11) and their receptor integrin α7β1 may be key players in trophoblast adhesion and invasion (Klaffky et al., 2001). Importantly, deletion of either Lamb1 or Lamc1 (genes that encode two of the three chains that comprise Laminin 1) results in peri-implantation lethality (Smith and Wilson, 1971; Miner et al., 2004). Our analyses of in utero gene expression confirm that, as development progresses, Laminin is produced by the primitive endoderm (Chazaud et al., 2006; Niakan et al., 2010), but also show that Laminin production is highest in the PPrE and parietal endoderm cells at 115 hp-hCG (∼E4.25), and down-regulated in the cells of the PrE when they cover the ICM at 121 hp-hCG (Figs. 23). These results raise the possibility that the Laminin knockout phenotypes may be due to defects in PPrE cell function.
The mouse uterus is bifurcated with two fluid filled uterine horns. Sites of embryo implantation have been shown to be random in both mouse and rat models (Krehbiel and Plagge, 1962). However, we show that the great majority of embryos implant specifically at SOX17-expressing regions of the ULE. Consistent with the assertion of random implantation sites (Krehbiel and Plagge, 1962), these areas of SOX17 expression are not uniform, appearing in patches throughout the ULE (Fig. 6). Recently, Sox17 was identified as a target of progesterone in uterine tissue (Rubel et al., 2012), and progesterone is known to control implantation in all mammals examined (Zhang et al., 2013). Although functional experiments are needed to test this hypothesis, our data raise the possibility that progesterone is required to elevate SOX17 in regions of the ULE, permitting implantation at these sites. The initiation of the ULE degradation has been suggested to be independent of embryonic signaling, as decidualization can be induced by the presence of glass beads (Blandau, 1949a, 1949b). However, molecular crosstalk between the embryo and maternal tissue has been demonstrated for several signaling pathways (reviewed in Zhang et al., 2013). Our data indicate that uterine SOX17 may play a functional role during implantation and should be added to the growing list of genes and pathways known to be involved.