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Keywords:

  • Eomes;
  • Cdx2;
  • extraembryonic;
  • area opaca;
  • trophoectoderm;
  • primordial germ cells

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS AND DISCUSSION
  5. CONCLUSIONS
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

In the mouse blastocyst, Eomes and Cdx2 are critical for establishing the trophoectoderm, the precursor of the placenta. To better understand how the trophoectoderm lineage arose in mammals during evolution, we examined the expression of their orthologues in the pregastrulation chick embryo and found that, while both genes are expressed in extraembryonic tissues, their temporal pattern of expression differs from what occurs in mouse. Moreover, we failed to detect expression of other genes specific from the mouse trophoectoderm in extraembryonic regions of the chick. Also unlike the mouse, chick Eomes is expressed in primordial germ cells. Finally, conserved noncoding elements in the Eomes genomic region are unable to drive trophoectoderm restricted expression in the mouse blastocyst, but do so in conserved sites of expression such as the forebrain. These results suggest that critical changes in the gene regulatory networks controlling extraembryonic development accompanied the appearance of the trophoectoderm in mammals. Developmental Dynamics 239:620–629, 2010. © 2009 Wiley-Liss, Inc.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS AND DISCUSSION
  5. CONCLUSIONS
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

The first differentiation event in the mouse embryo is the segregation of embryonic and extraembryonic tissues, which occurs at 3.5 days after fertilization with the differentiation of the extraembryonic trophoectoderm (TE) from the inner cell mass (ICM). The TE is a cell population that gives rise to most of the lineages that form the invasive placenta, and is thought to be unique to mammals because no homologous structure has been described in nonmammalian vertebrates. The segregation of the TE from the ICM begins at the morula stage, with the segregation of outer from inner cells, and by the blastocyst stage these lineages are determined with the TE forming an outermost layer surrounding the ICM and the blastocyst cavity. The second lineage decision takes place in the late blastocyst, with some ICM cells segregating to form the primitive endoderm (PE), an extraembryonic endoderm cell layer; whereas the rest of the ICM will become the epiblast (Epi), which differentiates into all lineages of the embryo proper (reviewed in Rossant and Tam, 2009).

The first events during the development of the early chick embryo differ from the mouse in several respects. The most marked difference is the positioning of the embryo on top of the nutritive yolk, a structure absent from mammalian embryos, which are nourished by means of the TE-derived placenta. Nonetheless, very similar to the mouse, one of the earliest morphological differentiation events in the chick embryo is the separation of extraembryonic and embryonic territories (Bellairs and Osmond, 2005). This segregation occurs shortly before the time of laying (EGK-IX-X; Eyal-Giladi and Kochav, 1976), when two areas can be distinguished in the blastodisc—a thinner central layer, the area pellucida (AP), surrounded by a thicker layer, the area opaca (AO). The AO gives rise only to extraembryonic structures, while the embryo lineages are specified from the AP. Interestingly, one of the derivatives of the AO will be the chorion, that will later fuse with the allantois to form the chorioallantoic sac involved in gas exchange between the embryo and the environment (Bellairs and Osmond, 2005). This resembles closely the fusion of the trophoblast derived chorion in the mouse with the mesodermal allantois that produces the chorioallantoic placenta. By EGK-X some AP cells delaminate to generate the primary hypoblast beneath the epiblast of the AP, that will in turn originate the extraembryonic endoderm (Eyal-Giladi and Kochav, 1976). The appearance and formation of the different embryonic and extraembryonic populations in the chick embryo is clearly reminiscent of events in the mouse (O'Farrell et al., 2004).

Specification of the TE in the mouse embryo has been studied in detail, and this work has identified some of the main factors in the gene regulatory network that segregates extraembryonic and embryonic lineages at the blastocyst stage. Before formation of the blastocyst, the complementary expression patterns and reciprocal repression of the transcription factors Cdx2 (in the outermost cells) and Pou5f1 (in the innermost ones) defines the segregation of TE and ICM (Niwa et al., 2005). Cdx2 then activates Eomes expression in the TE, both transcription factors being essential for the specification and maintenance of the TE-derived trophoblast lineage. Conversely, the ICM differentially expresses the pluripotency gene Nanog in cells that will form the epiblast, and Gata4/Gata6 in future PE cells (Rossant and Tam, 2009).

The T-box transcription factor Eomes was described as the first pan-mesodermal gene expressed in Xenopus (Ryan et al., 1996). Its expression in the primitive streak and role in mesoderm specification during gastrulation are widely conserved with other vertebrates, including zebrafish, chick, and mouse (Bulfone et al., 1999; Russ et al., 2000; Bruce et al., 2003; Arnold et al., 2008). A later expression pattern in the developing brain is also highly conserved in these species. Mouse Eomes is expressed specifically in the TE at blastocyst stages and later in the extraembryonic ectoderm, and mutant embryos for this gene die around implantation due to trophoblast defects. This places this transcription factor at a crucial position in the specification and maintenance of the trophoblast lineage, downstream of Cdx2 (Russ et al., 2000; Strumpf et al., 2005). Eomes thus appears to have been recruited during mammalian evolution to the regulatory network that determines the first lineage choice during development and trophoblast fate.

To gain insight into the evolutionary origin of the mammalian trophoblast and of early lineage determination, we analyzed the expression of Eomes and Cdx2 in prestreak and early gastrulating chick embryos. We find that Eomes and Cdx2 are sequentially expressed in extraembryonic regions of the chick embryos. However, these genes are expressed in the reverse order to that seen in the mouse TE, and conserved noncoding elements in the Eomes region were unable to drive TE-restricted expression in the mouse blastocyst. Analysis of Eomes expression in chick embryos also identified a novel and nonconserved expression domain in primordial germ cells (PGC). Our results point to fundamental differences in the regulation of extraembryonic fate in chick and mouse, despite morphological and gene expression similarities between them. Future studies will be needed to determine whether Eomes has a role in PGC development in chick.

RESULTS AND DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS AND DISCUSSION
  5. CONCLUSIONS
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

The First Lineage Decisions in Mouse and Chick Embryos Are Regulated Differently

In the mouse, Eomes is maternally expressed and thus can be detected in the oocyte and during the first cleavage stages (McConnell et al., 2005). At the late blastocyst stage Eomes expression, under the regulation of Cdx2 (Strumpf et al., 2005), is restricted to the TE and, after implantation, to its derivative, the extraembryonic ectoderm (Ciruna and Rossant, 1999; Russ et al., 2000). In the gastrulating embryo at E6.5, Eomes is also expressed on the posterior side of the embryo—a pattern similar to that seen in Xenopus, zebrafish, and chick—where it later plays a role in mesoderm specification and epithelium-to-mesenchyme transition (Arnold et al., 2008).

Analysis of the early chick embryo by in situ hybridization detected strong expression of Eomes at the earliest stage we could examine, corresponding to the freshly laid egg (EGK-X; Fig. 1A–A″; Eyal-Giladi and Kochav, 1976). At this stage one can distinguish the extraembryonic AO, and the epiblast and hypoblast of the AP. Eomes is expressed in the AO and the hypoblast but is excluded from the epiblast (Fig. 1A′–A″). We begin to detect expression in epiblast in the region of the Köller's sicke as it forms at the posterior side of the embryo at EGK-XI-XII stage (Fig. 1B–B″), as well as in the previous mentioned territories. At Hamburger and Hamilton stage 1 (HH1; Bellairs and Osmond, 2005), the expression in the AO and hypoblast persist (Fig. 1C–C′), but Eomes is also detected in the emerging primitive streak (Fig. 1C″). Expression in the AO and hypoblast decays by HH2, when we detected Eomes mRNA only in the primitive streak (Fig. 1D–D″).

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Figure 1. Expression of trophoblast markers in prestreak chick embryos. A–D: Eomes expression is detected from stage EGK-X to the beginning of primitive streak formation. A: At EGK-X (A) Eomes is expressed in the area opaca (A′) and the hypoblast of the area pellucida, whereas it is excluded from the epiblast (A″). B,C: From EGK-XII to HH1 (B,C) the expression is maintained in the area opaca (B′,C′) and hypoblast of the area pellucida (C′), and it also appears in the region of the Köller's sickle (B,B″) and the emerging primitive streak (C,C″). D: With the extension of the primitive streak (HH2, D) Eomes expression disappears from the area opaca (D′) and becomes restricted to the primitive streak (D″). E–H: Cdx2 expression is not detected until EGK-XII (F), when it begins to be weakly expressed in the area opaca (F′; F″ section corresponds to Köller's sickle region). Cdx2 only becomes strong in the area opaca by HH1–HH2 (G–G′,H–H′); at these stages there is no expression in embryonic regions. The darker region in the primitive streak observed in whole-mount at Hamburger and Hamilton stage (HH) 2 (H) does not correspond to true hybridization signal, as is confirmed by the complete lack of signal seen in sections of stage-matched embryos (H″). I–L: Bmp4 and Fgfr2 are not expressed in extraembryonic structures at prestreak stages (I,K) and they are first detected at HH4 (J,L). AO, area opaca; AP, area pellucida; Epi, epiblast; Hypo, hypoblast; KS, Köller's sickle; PS, primitive streak. Horizontal lines indicate the plane of the sections shown in panels below of the same embryos (A–F) or in stage-matched specimens (G,H).

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Expression of Eomes in the chick hypoblast might be related to the role of this gene in endoderm specification in both zebrafish and mouse. However, mouse Eomes is not expressed in the PE, the equivalent of the chick hypoblast (O'Farrell et al., 2004), but instead in the anterior primitive streak, from where the definitive endoderm will form (Arnold et al., 2008). Nevertheless, there is a transient expression of Eomes in the visceral endoderm (Ciruna and Rossant, 1999; Kwon and Hadjantonakis, 2007), a PE derivative, that could relate to the expression we observe in the chick hypoblast. In zebrafish, it is the maternal Eomes product that is localized to the marginal blastomeres, where it induces formation of the endoderm (Bjornson et al., 2005). Maternal expression of Eomes has also been reported in mouse (McConnell et al., 2005), although with no spatial restriction. We cannot rule out that the high amounts of Eomes mRNA detected at EGK-X might represent maternal transcripts, because this stage coincides with the transition from maternal to zygotic gene expression in the chick embryo (Zagris et al., 1998; Elis et al., 2008). Further studies will be required to determine whether a role for Eomes in endoderm specification is conserved in the chick embryo and if this is related to its expression in the hypoblast. In this regard, it is interesting to note that recent results show that, contrary to established views, the extraembryonic endoderm contributes to the definitive endoderm in the mouse (Kwon et al., 2008).

The early expression of chick Eomes in the AO suggests that the similarities between extraembryonic tissues in mouse and chick may extend beyond mere morphological features. We, therefore, examined the expression of orthologues of other genes that are TE-specific in mouse. Cdx2 is the first gene known to be specifically expressed in the mouse blastocyst and has been shown to be upstream of Eomes during TE specification. Cdx2-deficient embryos manifest an earlier TE phenotype than Eomes-deficient embryos, and while Eomes expression is lost in Cdx2 knockouts, Cdx2 expression is intact in Eomes knockouts (Strumpf et al., 2005; Ralston and Rossant, 2008). However, in the early chick embryo, and contrary to what we see for Eomes, we did not detect Cdx2 expression at stage EGK-X (Fig. 1E–E″), and only by EGK-XII we began to detect weak expression in the AO (Fig. 1F–F″). By HH1 Cdx2 expression was found only in the AO with no expression in the nascent primitive streak (Fig. 1G–G′). Finally, strong expression of Cdx2 restricted to the AO was seen at HH2, when Eomes expression is clearly down-regulated in this domain (Fig. 1H–H″). Apparent expression in the primitive streak at this stage is due to background, as no staining is observed in sections. At later stages Cdx2 expression is restricted to the caudal part of the primitive streak (data not shown), in a pattern identical to that previously reported (Marom et al., 1997). According to these results, Eomes and Cdx2 expression domains only overlap during a restricted period of time in the extraembryonic region of the chick embryo at pregastrulation stages. By HH1, when Cdx2 begins to be expressed in the AO, Eomes expression is about to decay in this territory. This differs markedly from what happens in the mouse blastocyst, and makes it difficult to position Cdx2 upstream of Eomes in the chick embryo. These results thus argue against an overall conservation in amniotes of the gene regulatory networks that control extraembryonic fate.

Given that Cdx2 is absent from the early extraembryonic lineages of the chick embryo, we examined whether the same was also the case for other mouse TE-specific genes. Bmp4 and Fgfr2 are expressed in the mouse TE and its derivative the extraembryonic ectoderm (Haffner-Krausz et al., 1999; Lawson et al., 1999; Rossant and Cross, 2001). These factors form part of the trophoblast-epiblast crosstalk network: Fgfr2 expressed on trophoblast cells binds Fgf4 from the epiblast, activating signals that maintain the trophoblast (reviewed in Rossant and Cross, 2001), while trophoblast-expressed Bmp4 maintains the patterning of the epiblast (Fujiwara et al., 2002; Di-Gregorio et al., 2007). Whole-mount in situ hybridization in early chick embryos revealed that neither Bmp4 (Fig. 1I,J) nor Fgfr2 (Fig. 1K,L) are expressed until stage HH4, confirming previous reports (Chapman et al., 2002; Lunn et al., 2007): these factors show no expression whatsoever in chick extraembryonic domains. Similarly, we found no expression in pregastrulation stages of Tead4 (data not shown), a gene that in mouse has been found to be upstream of Cdx2 in specification of the trophoectoderm (Yagi et al., 2007; Nishioka et al., 2008), in response to the Hippo signaling pathway (Nishioka et al., 2009).

In both chick and mouse, expression of Eomes is initially restricted to extraembryonic lineages, what could indicate a similar regulation of the first lineage decisions in avians and mammals. However, other factors involved in the specification and maintenance of mammalian TE are not expressed in a comparable manner in the early chick extraembryonic domains, and the later onset of Cdx2 expression in chick appears to indicate that Eomes expression in extraembryonic lineages is not regulated in the same manner as in mouse. Moreover, the absence of Bmp4 and Fgfr2 suggests that the extraembryonic–epiblast communication network is a novel acquisition in mammals.

Eomes Is Expressed in the Primordial Germ Cells of the Chick but Not the Mouse Embryo

Bulfone et al. (1999) have previously described the expression pattern of chick Eomes from stages HH3 to HH28. According to this report, at HH3 Eomes is expressed in the anterior-most part of the AP, in the hypoblast, and ectoderm anterior and lateral to the PS. This expression decays by HH5 with the regression of the PS, and disappears by HH6. Later in development, at HH25, Eomes is expressed in the telencephalic pallium of the developing brain. This pattern, with expression in the gastrulating embryo and later in the central nervous system, is widely conserved in Xenopus, zebrafish, and mouse (Bulfone et al., 1999; Ciruna and Rossant, 1999; Kimura et al., 1999; Mione et al., 2001; Bachy et al., 2002). In embryos from HH19 to HH22, we detected Eomes expression in the previously described territories, such as the telencephalon (Fig. 2D,F), but also detected Eomes mRNA in a novel and nonconserved domain: the PGC (Fig. 2E,G).

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Figure 2. Eomes is expressed in primordial germ cells in the chick. A–C: At HH4, Eomes expression is detected in the anterior half of the embryo (A) and in scattered cells (primordial germ cells [PGC]) in the boundary between the anterior area opaca and area pellucida (A′: magnification of the square in A). By Hamburger and Hamilton stage (HH) 4+ (B) the expression in the epiblast is restricted to the PGC and the node (B′: magnification of the square in B; arrowhead indicates PGC). With the regression of the primitive streak (HH5, C) epiblast expression is seen only around the node and in the head fold, and the signal in the PGC along the germinal crescent is very apparent (C′: magnification of the square in C). D–F: At later stages Eomes is expressed in the forebrain of the chick (black arrow in D; F) and along the genital ridges (red arrow in D; E: magnification of the chick in D). G: The PGC expressing Eomes along the genital ridge of a HH22 chick are shown in this section. H–J: In sections of mouse embryos, Eomes is detected in a conserved pattern in the forebrain of the E10.5 embryo (black arrow in H) but not in the genital ridges (red arrow in H; I: magnification of the embryo in H) or in the gonads at E13.5 (J). K,L: The PGC marker Pou5f1 is clearly detected in these territories (K, expression in PGC along the genital ridge of the E10.5 embryo; L, expression in E13.5 gonads). Ovaries are shown on the left and testis on the right (J, L).

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Eomes expression in PGC becomes apparent by HH4 in the germinal crescent, located at the anterior boundary of the AO and AP (Fig. 2A,A′). PGC are large cells that can be found dispersed in the germinal crescent and have a very characteristic rounded shape, making them easily distinguishable from other cell populations (Bellairs and Osmond, 2005). At HH4+, Eomes expression in the primitive streak decreases and becomes restricted to the node (Fig. 2B), and by HH5 expression is detected anterior to the node along the regressing primitive streak and in the head process and forming head folds (Fig. 2C). At these stages, the PGC in the germinal crescent remain positive for Eomes (Fig. 2B′,C′). Starting at HH10, PGC migrate from the germinal crescent by means of the vascular system toward the gonads that they reach by HH16 (Bellairs and Osmond, 2005). We detected Eomes expression along the genital ridges in whole-mount embryos from HH19 to HH24 (Fig. 2D,E; and data not shown), and restriction of this expression to PGC was confirmed in tissue sections of HH22 embryos (Fig. 2G).

Expression of Eomes in PGC has not been reported in any other vertebrate. We, therefore, re-examined the expression pattern of Eomes in the mouse embryo, focusing on the genital ridges and gonads. PGC are specified in mouse at around E7.0, in the region where the mesoderm is being determined (reviewed in Matsui and Okamura, 2005). At this time, Eomes is highly expressed in mesoderm, making it difficult to ascertain if it is expressed in the PGC. At E10.5, we detected Eomes in the previously described domain in the developing brain, but found no expression in the genital ridges (Fig. 2H,I). Conversely, at this stage, we found clear expression of Pou5f1, a well characterized PGC marker (Fig. 2K; Scholer, 1991). Similarly, no expression of Eomes was detected in the gonads of E13.5 embryos (Fig. 2J), contrasting with the gonadal expression of Pouf51 at this stage (Fig. 2L). The lack of expression of mouse Eomes in gonads was confirmed by reverse transcriptase-polymerase chain reaction (RT-PCR; data not shown). PGC, therefore, represent a novel and nonconserved expression domain of Eomes in chick.

Several genes are expressed in developing PGC in chick, such as Vasa, Dead end, Nanog, and the Pou5f1-related gene PouV (Tsunekawa et al., 2000; Stebler et al., 2004; Canon et al., 2006; Lavial et al., 2007). Of special interest are Nanog and Pou5f1, because of their pivotal role in specifying the early lineages of the mouse blastocyst and in maintaining embryonic stem cell pluripotency within the epiblast at this stage (Boiani and Scholer, 2005; Niwa, 2007). This places them as repressors of extraembryonic fate in mouse, contrary to Eomes, which contributes to TE formation and maintains trophoblast stem cells. Furthermore, mouse Oct4 and Nanog bind to genomic regions in the vicinity of Eomes, and are thought to directly repress its transcription (Loh et al., 2006). Accordingly, Nanog and Pou5f1, but not Eomes, are expressed in PGC in mouse (Scholer, 1991; Hatano et al., 2005; Yamaguchi et al., 2005). In contrast, in chick, we find Eomes expressed together with Nanog and PouV in this stem cell population. The role of Eomes in PGC and its relation to Nanog and PouV in the chick remain to be elucidated.

Conserved Elements in the Genomic Region of the Mouse and Chick Eomes Genes Are Unable to Drive TE-Specific Reporter Expression in Blastocysts

Given the common expression of Eomes in extraembryonic domains of the mouse blastocyst and the prestreak chick embryo, we reasoned that these domains might be controlled by conserved regulatory mechanisms. We, therefore, examined whether noncoding regions surrounding the mouse Eomes gene and conserved with chick were able to direct restricted reporter expression in the TE of transgenic mouse blastocysts.

We first compared the Eomes genomic regions in human, mouse, opossum, and chick, using the gene annotations provided by the Ensembl Genome Browser server (www.ensembl.org) (Fig. 3A). Human EOMES is located on chromosome 3, flanked by two intergenic regions of 519 kb (upstream) and 260 kb (downstream). Murine Eomes is located on chromosome 9, opossum Eomes on chromosome 8, and the chick Eomes orthologue on chromosome 2. There is conserved synteny among the upstream regions of all species. However, the downstream region found in human, opossum, and chick is absent in mouse. This is likely due to an intra-chromosomal rearrangement, resulting in the region downstream of mouse Eomes being syntenic with a region located 10 Mb upstream of human EOMES. The fact that the downstream region of mouse Eomes has suffered a rearrangement, suggesting a major reduction in the intergenic noncoding region, strongly indicates that most Eomes regulatory elements are likely to be located in the upstream region. Recently, a BAC transgenic mouse strain has been described (Tg(Eomes::GFP)) that recapitulates most aspects of Eomes expression in the mouse. Green fluorescent protein (GFP) expression is detected in the TE of the late blastocyst and in the extraembryonic ectoderm at early streak stages, but is not maintained in the chorion at late streak stages (Kwon and Hadjantonakis, 2007). This bacterial artificial chromosome (BAC) construct covers ∼186 kb upstream and ∼18 kb downstream of Eomes, confirming that most if not all regulatory elements are located upstream of Eomes, and for that reason we restricted our analysis to that region (Fig. 3A,B). Furthermore, a homozygous translocation involving a breakpoint upstream of EOMES has been found in humans (Baala et al., 2007), which maps inside the region covered by the BAC construct described above (Fig. 3B). This translocation results in microcephaly and other neuroanatomical defects, but obviously does not affect the earlier role of EOMES in TE development, what would argue that all the regulatory information required for proper expression of EOMES at preimplantation stages has not been disconnected from the gene by this translocation.

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Figure 3. Analysis of Eomes regulation in the mouse blastocyst. A: The genomic region containing Eomes is conserved between mammals and chick with the exception of the region downstream of mouse Eomes, which has undergone rearrangement and lost synteny with respect to other vertebrates. The region covered by the bacterial artificial chromosome (BAC) used to create the mouse transgenic line Tg(Eomes::GFP), which recapitulates Eomes expression (Kwon and Hadjantonakis, 2007), is indicated. B: This region was used in a global multiple alignment analysis, with the mouse as base organism, to identify elements conserved in all species examined (highly conserved elements [HCE]). The five HCE identified (Eo1 to Eo5) are highlighted blue. The position of the breakpoint involved in a homozygous translocation described in humans that results in late neurodevelopmental defects (Baala et al., 2007) is shown by a vertical green line. C: Eo1 to Eo5 were tested for their enhancer activity in blastocysts, and none was able to drive expression of lacZ to a level comparable to that of the previously described Pou5f1 enhancer (Pou5f1DE). Scattered dots were detected inside cells of HCE transgenic and control blastocysts, in a pattern like that shown for Eo1. The table shows the percentage of lacZ positive blastocysts obtained for each construct. D: Conversely we could detect enhancer activity for Eo5, that drives expression of the transgene to the forebrain (arrowhead), an evolutionarily conserved domain of Eomes expression among vertebrates.

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Multiple-alignment of sequences from diverse organisms corresponding to the same genomic region is a powerful tool for identifying cis-regulatory elements (Boffelli et al., 2004; de la Calle-Mustienes et al., 2005). This approach is based on the assumption that a high level of conservation in a noncoding region must be due to a functional constraint. We performed multiple-alignment analysis of the noncoding regions flanking Eomes in mouse, human, opossum, and chick, and selected highly conserved elements (HCE) present in all four species. Five HCE (Eo1 to Eo5) were selected for testing of their enhancer activity in a mouse transient transgenic assay (Fig. 3B). None of the HCE tested was able to drive expression of the reporter gene lacZ in blastocysts to the level induced by a previously described Pou5f1 enhancer element, used as positive control (Fig. 3C; Yeom et al., 1996). Instead, all the HCE drove low-level punctuated expression indistinguishable from a negative control (Fig. 3C; see Experimental Procedures). It is interesting that HCE Eo5 lies within one of the regions detected in a genome-wide chromatin immunoprecipitation assay with p300 designed to identify forebrain enhancers with extremely high accuracy (Visel et al., 2009). When we tested this HCE for its enhancer activity in transgenic embryos at E10.5, we could detect specific expression of the reporter gene lacZ in the telencephalon (Fig. 3D). This element also drove expression of the reporter consistently in the midbrain, although this is not a site of endogenous Eomes expression in mouse or chick. This suggests that elements conserved between mouse and chick do indeed control conserved aspects of Eomes expression, such as in the forebrain.

The results of our transgenic analysis suggest that the enhancer elements responsible for Eomes expression in the TE of the mouse blastocyst are not located in regions highly conserved in chick. This supports the idea that Eomes is regulated differently in the extraembryonic tissues of mouse and chick embryos. Nevertheless, we cannot rule out the possibility that conserved binding sites might exist in less conserved regions that fall below the threshold used in our analysis, or that other conserved regions located outside of the 200-kb region analyzed would drive expression in extraembryonic tissues.

CONCLUSIONS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS AND DISCUSSION
  5. CONCLUSIONS
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

By examining the early expression pattern of Eomes in early chick embryos we have found surprising similarities as well as differences with its mouse orthologue. Although Eomes expression in the chick AO at pregastrulation stages seems comparable to that in the TE of the mouse blastocyst, it is not driven by conserved noncoding regions acting as cis-regulatory elements. We cannot exclude the possibility that a common mechanism of Eomes expression is mediated by conserved upstream regulatory elements that were missed by our analysis; however, the analysis of Cdx2 expression in the chick supports the conclusion that the restricted expression of Eomes in extraembryonic regions of avians and mammals is established by different mechanisms. In this regards, it is interesting to note that, in the mouse morula, cell polarity of outer cells seems to be a critical factor in the establishment of the trophoectoderm and the initial expression of Cdx2, while inner cells showing no polarity will give raise to the ICM (Rossant and Tam, 2009). In the case of the pregastrulation chick embryo, cells from the area pellucida show clear apical–basal polarity, and form a continuous epithelial layer with cells from the area opaca (Bellairs and Osmond, 2005), precluding differences in cell polarity as a conserved mechanism for activation of Cdx2 or Eomes.

We have also found that, unexpectedly, chick Eomes is expressed in PGC, which does not occur in mouse. Because chick PGC also express Nanog and the Oct4-related PouV genes, and in mouse Eomes is repressed by these factors as part of the embryonic pluripotency network, it is tempting to speculate that a change might have occurred in the interrelationship of these genes in the avian and mammalian lineages. This would lend further support to the idea that significant changes in the regulation of Eomes expression in the early embryo occurred concomitantly with the appearance of the blastocyst and the early lineage segregation observed in mammals.

EXPERIMENTAL PROCEDURES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS AND DISCUSSION
  5. CONCLUSIONS
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

Gene Expression Analysis

Whole-mount in situ hybridization in chick and mouse embryos was carried out following previously described procedures (Nieto et al., 1996; Ariza-McNaughton and Krumlauf, 2002). In all cases, embryos of different stages where processed in parallel and for the same amount of time. This included embryos at later stages of development for which the expression pattern of the genes analyzed has already been described in detail. Negative controls using sense probes were included. The chick Eomes probe was generated by amplification of cDNA obtained from whole HH6 embryos with the primers cEoF (5′-TGACT GAAGATGGCGTTGAG- 3′) and cEoR (5′-CTTCAGAGAAGCCTGGAGGA-3′). The amplification product was then cloned into pGEM-T Easy Vector for use as the template for reverse transcription. The probes for chick Cdx2 and Fgfr2 were obtained from the BBSRC chick expressed sequence tag (EST) collection (Boardman et al., 2002). The ESTs selected were ChEST626o23 (Cdx2) and ChEST420f6 (Fgfr2). The probe for chick Bmp4 was a gift from Dr. J.J. Sanz-Ezquerro, and mouse Eomes and Pou5f1 probes were gifts from Dr. T. Rodriguez. After whole-mount in situ hybridization, embryos were cryosectioned for microscopy analysis.

Sequence Analysis

Study of the synteny of the Eomes genomic context was based on annotations obtained from the Ensembl Genome Browser (www.ensembl.org), from which we also downloaded the genomic sequences used in the comparative analysis. Global multiple alignments were performed using the Vista package (Frazer et al., 2004). Only sequences with at least 70% identity over 100 bp and conserved from human to chicken were categorized as HCE.

Transgenic Analysis

HCE selected according to the global multiple alignment analysis were amplified using four selected BACs as templates: RP23-478D17, RP24-77J5, RP23-272A7, RP23-235G22. These BACs cover the whole intergenic region flanking mouse Eomes and were obtained from the BACPAC Resources Center (http://bacpac.chori.org/). Primers used for PCR and the lengths of corresponding amplified fragments were as follows. Eo1F: 5′-GCAGGGAGTGCTTCTGTTTATT-3′, Eo1R: 5′-ACCCCTTGAGGTTGAGT CATAA-3′ (2,072 bp); Eo2F: 5′-TGGG GTGTATATAAGCAGTCCTC-3′, Eo2R: 5′-TCTGTCTGTCTGTCTATCATCAAC C-3′ (1,402 bp); Eo3F: 5′-AGAGGACTT GGGTTTAGTTTCCA-3′, Eo3R: 5′-GAC CCAGGGAACTACCTTTCTTA-3′ (6,653 bp); Eo4F: 5′-GCAGTACACACACGCA TGAA-3′, Eo4R: 5′-TCATTTCCCAGGA TTCTTCG-3′ (1,402 bp); Eo5F: 5′-AT GCCCACAACACACACAGA-3′, Eo5R: 5′-TGTTACTGGGGCTTGGATGT-3′ (3,658 bp). As a positive control, we amplified the previously described Pou5f1 distal enhancer element (Yeom et al., 1996), using BAC RP23-152G18 as template (http://bacpac.chori.org/); the primers for PCR were Pou5f1DE-F: 5′-AGCGGCCGCCTCTG CTACATGTAAATTTGTCT-3′ and Pou 5f1DE-R: 5′-AGCGGCCGCCTAAACA AGTACTCAACCCTTGAA-3′ (3,368 bp). Each fragment was subcloned in pGEM-T Easy Vector and then excised and cloned into a modified pBluescript vector (Yee and Rigby, 1993) containing the lacZ reporter gene under the control of the human beta-globin minimal promoter and with an SV40 polyadenylation signal. Constructs were linearized and plasmid sequences removed before micro-injection.

For generation of transient transgenics, females from a F1 C57/CBA background were superovulated to obtain fertilized oocytes (Nagy et al., 2003). Each construct was micro-injected into the pronucleus of fertilized oocytes at 0.5 dpc and a concentration of 4 ng/μl. Micro-injected oocytes were cultured in microdrops of M16 medium (SIGMA) covered with mineral oil (SIGMA) at 37°C, 5% CO2 until blastocyst stage. For lacZ staining, blastocysts were fixed in buffer containing 1% formaldehyde, 0.2% glutaraldehyde, 2 mM MgCl2, 5 mM ethyleneglycoltetraacetic acid (EGTA), and 0.02% Igepal for 5 min at room temperature. After washes in phosphate buffered saline (PBS), blastocysts were transferred to X-Gal staining solution for 24 hr at room temperature in the dark. A minimum of 50 blastocysts were used per construct to calculate the percentage of lacZ positive embryos. When using the empty vector containing only the minimal promoter and the lacZ reporter as a negative control, we routinely obtain low-level punctuated lacZ expression in approximately 4% of blastocysts. The embryos at embryonic day 10.5 were fixed for 30 min at 4°C and then washed several times in PBS-0.02% Igepal at room temperature before staining.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS AND DISCUSSION
  5. CONCLUSIONS
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

We thank Tristan Rodriguez and Juan Jose Sanz-Ezquerro for in situ probes, Robb Krumlauf for reporter constructs, the Transgenic Unit at CNIC for generating embryos, Eva Alonso and Cristina Arias for fruitful discussions, and Simon Bartlett for critical reading and editing of the manuscript.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS AND DISCUSSION
  5. CONCLUSIONS
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES