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发表于 2009-3-5 10:47 |只看该作者 |倒序浏览 |打印

a ARC Centre of Excellence in Biotechnology and Development and Murdoch Children’s Research Institute, Royal Children’s Hospital, Melbourne, Victoria, Australia;
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b Wellcome Trust, Cancer Research UK Gurdon Institute and Department of Physiology, University of Cambridge, Cambridge, United Kingdom;

c Laboratory for Mammalian Germ Cell Biology, Center for Developmental Biology, RIKEN Kobe Institute, Kobe, Japan
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Key Words. Esg1 ? Oct4 ? Sox2 ? Germ cell ? Pluripotent ? Embryonic stem cell) |% M% i% |0 G) s' p
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Correspondence: M. Azim Surani, Ph.D, Wellcome Trust, Cancer Research UK Gurdon Institute and Department of Physiology, Tennis Court Road, University of Cambridge, Cambridge CB2 1QR, United Kingdom. Telephone: 44(0)1223-334136; Fax: 44(0)1223-334089; e-mail: as10021@mole.bio.cam.ac.uk

ABSTRACT

During mammalian preimplantation development, pluripotent cells are established within the inner cell mass (ICM). These pluripotent cells must not only contribute to all embryonic somatic cell lineages but also establish the germline, the only cell lineage to be transmitted to the next generation. It has also been possible to derive pluripotent stem cells from both epiblast and germ cells called embryonic stem (ES) and embryonic germ(EG) cells, respectively, which share similar phenotypic and molecular characteristics. Recently, human pluripotent ES (hES) and human EG cells, which are thought to be functionally equivalent to mouse ES (mES) and mouse EG (mEG) cells, have also been derived . Interestingly, differences exist between hES and mES cells with regard to their culture requirements . Although the molecular events leading to germ cell specification are not well understood, several genes that are critical for pluripotent cells and during preimplantation development are subsequently restricted to the germ lineage. Further understanding of the molecular basis of pluripotency would provide knowledge of the mechanisms that define stem cells and germ cells.

Several genes, including Oct4, nanog, and Sox2, have been strongly implicated in regulating pluripotency . In mice, OCT4 is essential for pluripotency in mES cells and preimplantation embryos and has been shown to act in conjunction with other proteins, such as SOX2. Despite data strongly indicating that OCT4 and SOX2 act together, their mode of action in maintaining a pluripotent state remains unclear. Since the molecular pathways through which these transcription factors operate are unknown, it is of value to identify further genes specifically expressed in pluripotent cells, including possible targets of OCT4, SOX2, and NANOG. By comparing genes expressed in ES cells with those in differentiated cells, we isolated the ES cell–specific gene Esg1, which has been previously shown to mark pluripotent cells and is a possible transcriptional target of OCT4 . Esg1 encodes a 13.7-KDa protein with a putative KH RNA binding domain. Interestingly, KH domains are also present in the Caenorhabditis elegans gld-1 and mex-3 proteins, which, as components of the germline P granules, have roles in the establishment of germ cells .

Although the expression of mouse Esg1 has been partially analyzed, it was thought to be expressed only in ES and embryonal carcinoma (EC) cells and during preimplantation development. Here we examine mouse Esg1, Sox2, and Oct4 expression, with particular reference to pluripotent cells and the developing primordial germ cells (PGCs). We show that Esg1 not only is expressed in pluripotent cells of the preimplantation embryo but is then specifically restricted to and maintained only in germ cells until after sex determination. Similarly, Sox2 expression in PGCs is maintained until after the PGCs enter the developing gonads and is then down-regulated in a manner comparable to Esg1 and Oct4. Esg1 exhibits a similar expression profile to Oct4 and Sox2, both of which have been shown to have critical roles in regulating pluripotency. Human ESG1, SOX2, and OCT4 are also coexpressed in hES cells, but in some human EC (hEC) cell lines, ESG1 expression is lost. These data suggest that Esg1 performs a conserved role in the regulation of pluripotency and PGC development.; {6 W* o  Z9 e

MATERIALS AND METHODS
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Previous reports have shown that Esg1 is expressed in mES cells, mECs, and preimplantation embryos, although little Esg1 expression was detected in mature oocytes . In humans, expression has been observed in hES cells . We confirmed that Esg1 is expressed by mES cells and show that it is also expressed by mEG cells but not somatic cells of E13.5 developing embryos (Fig. 1A).
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Figure 1. (A): Northern blot containing total RNA isolated from mouse embryonic stem (mES) cells, E13.5 embryos (from which the gonads were removed), and mouse embryonic germ (mEG) cells probed with Esg1. The 28s ribosomal band is shown as a loading control. (B): Western blot containing total protein isolated from mES cells and E13.5 primary embryonic fibroblasts (PEFs) probed with the ESG1 antibody (ESG1) or the preimmune serum (PI) from the rabbit used to raise the antibody. (C): Western blot containing total protein isolated from mES cells transfected with an Esg1-HA expression plasmid or a control plasmid and probed with the ESG1 antibody (ESG1) or a mouse monoclonal anti-HA antibody (HA). The 15.9-KDa ESG1-HA fusion protein is detected by both the ESG1 antibody and the HA antibody (indicated by arrow). The endogenously expressed 13.7-KDa ESG1 protein is detected by the ESG1 antibody but not the HA antibody. (D): Western blot containing total protein isolated from embryonic stem cells, E13.5 PEFs, zygotes (Zyg), and blastocysts (Bl) probed with the ESG1 antibody.2 x% X5 u: |  R9 \% b7 j: u: m/ k
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To examine ESG1 protein expression, we raised and affinity-purified an ESG1 polyclonal antibody. On Western blots, this antibody recognized a band of the predicted size for ESG1 (13.7 KDa), which was present in protein extracts from ES cells but not primary embryonic fibroblasts. No bands were detected when an identical Western blot was exposed to preimmune serum (Fig. 1B). By overexpressing an Esg1-HA construct in mES cells and performing Western blotting, we confirmed that the ESG1 antibody specifically identifies the ESG1-HA fusion protein in addition to the endogenously expressed ESG1 (Fig. 1C). Using Western blotting and immunohistochemistry, we clearly detected expression of ESG1 protein in zygotes, strongly suggesting that it is maternally inherited, and later in cleavage-stage embryos and blastocysts (Figs. 1D, 2A). Immunofluorescence analysis showed that ESG1 is distributed in the cytoplasm and excluded from the nucleus in fertilized oocytes and in blastomeres of early embryos (Fig. 2A). No signal was obtained in embryos using preimmune serum. In E4.5 blastocysts, ESG1 was detected in both the trophectoderm and the ICM. As in ES cells, the protein was now localized both in the cytoplasm and the nucleus (Figs. 2A, 2B). Furthermore, during the derivation of mES cells from epiblast cells, we observed ESG1 in OCT4-positive cells, but this expression became weak or nonexistent in differentiating cells, where OCT4 expression was also downregulated (Fig. 2C). Because SOX2 is also required for pluripotent cells of the epiblast and has been shown to coregulate OCT4 target genes, we also examined SOX2 expression in outgrowing epiblast cells. SOX2 followed a similar expression pattern to that of OCT4 in the outgrowing epiblast (Fig. 2D), which is consistent with previous observations of SOX2 function ., M( `4 v+ C+ J, S$ M2 f
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Figure 2. (A): Zygote E4.5-stage blastocysts costained with ESG1 and OCT4 antibodies using indirect immunofluorescence. DNA has been stained using Toto3 (Molecular Probes, Inc., Eugene, OR, http://probes.invitrogen.com). ESG1 was observed only in the cytoplasm of zygotes, cleavage-stage embryos, and cavitating (E3.5) blastocysts. However, ESG1 staining in E4.5 blastocysts localized to both the cytoplasm and nucleus. Bars = 25 μm. (B): Mouse embryonic stem cells costained with ESG1 and OCT4 antibodies using indirect immunofluorescence. Bars = 10 μm. (C): Day-4 outgrowing blastocysts costained with ESG1 and OCT4 antibodies using indirect immunofluorescence. ESG1 staining becomes limited to the OCT4-positive cells. (D): Day-2 outgrowing blastocysts costained with SOX2 and OCT4 antibodies using indirect immunofluorescence. SOX2 staining is limited to the OCT4-positive cells. All immunofluorescence was visualized as optical sections using confocal microscopy. (E): RNA whole-mount in situ hybridization using an Esg1 probe and an Oct4 probe on E4.5 blastocysts and E5.5 and E6.5 embryos. Control samples hybridized with sense probes showed no staining. Bars = 25 μm (E4.5), 50 μm (E5.5), and 75 μm (E6.5).
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We then used RNA WISH to examine Esg1 expression in blastocysts and postimplantation embryos. Although the protein is more widespread in E4.5 blastocysts (possibly due to protein stability), Esg1 mRNA expression is limited to the ICM, whereas in E5.5 embryos, expression is confined to the epiblast but absent from the overlying visceral endoderm and the extraembryonic ectoderm. However, Esg1 was undetectable by E6.5, a time when Oct4 expression continues (Fig. 2E). This is noteworthy because in most respects, Esg1 expression is detected in Oct4-expressing cells.. t( x  O( G  a

Although previous reports have suggested that Esg1 is not expressed after the preimplantation and peri-implantation stages , we examined Esg1 expression during germ cell development. A unique quality of segregating PGCs is the expression of many pluripotency-associated genes, which are downregulated in somatic cells. We therefore used WISH to examine Esg1, Sox2, and Oct4 expression in gonads from E10.5–E15.5 embryos. At these stages, Esg1 and Oct4 expression was detected only in the developing gonads, whereas Sox2 was expressed in neural tissues as well (Fig. 3A). In our experiments, we focused on the gonadal expression of Sox2 as its expression elsewhere has been previously reported . Oct4 is expressed only in germ cells after E9.0 and is therefore an excellent marker of developing PGCs. Comparison of Esg1, Sox2, and Oct4 expression in developing gonads using WISH indicated that all three genes were coexpressed at similar levels in E10.5-E13 male and female gonads. Then, in females, all three genes were downregulated in an anterior-posterior wave during E13.5-E15.5, with the downregulation of Esg1 appearing to lag behind that of Oct4 and Sox2 (Fig. 3B). Recent observations also show an anterior-posterior wave of Oct4 down-regulation in female gonads marking entry into meiosis . By E15.5, Esg1, Sox2, and Oct4 expression was undetectable in the developing ovary. In males, the expression of these markers was detected without significant decrease until and including E14.5. By E15.5, Oct4 RNA expression was detected in male gonads, although its level seemed to be moderately decreased, whereas Esg1 and Sox2 expression had been abolished (Fig. 3C).1 [! I4 _% J; b3 d

Figure 3. (A–C): RNA whole-mount in situ hybridization using riboprobes specific for Oct4, Esg1, and Sox2. (A): E12.5 partially dissected embryos. (B): Female E12.5-E15.5 gonad/mesonephros tissues. All three genes are downregulated in an anterior-posterior wave in female gonads. (C): Male E12.5-E15.5 gonad/mesonephros tissues. (A–C): For consistency, all samples were treated together in the same experiment. Control samples hybridized with sense probes showed no staining. Arrows indicate the gonad. Bars = 500 μm. (D): Expression analysis in single gonadal cells. Southern blot analysis of single-cell cDNAs isolated from E11.5 gonads hybridized with Esg1 and Sox2 probes are indicated together with probes for the germ cell–specific markers Oct4 and stella and the ubiquitously expressed control ribosomal S12. G and S indicate lanes containing germ and somatic cell cDNA, respectively. (E): Immunohistochemical analysis of ESG1 and OCT4 expression in primordial germ cells (PGCs) and mouse embryonic germ cells (mEGs). E11.5 gonadal and mEG cells costained with ESG1 and OCT4 antibodies using indirect immunofluorescence. ESG1 locates to the nucleus and cytoplasm of E11.5 PGCs and mEG cells. Bars = 10 μm. All immunofluorescence was visualized as optical sections using confocal microscopy. (F): Western blot containing total protein isolated from two male or two female gonads from E12.5-E15.5 embryos probed with OCT4 and ESG1 antibodies. To ensure equal loading between samples, the protein from two gonads was run in each lane.
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To assess whether the expression of Esg1 and Sox2 was limited to PGCs, we isolated single cells and prepared cDNA pools representative of genes expressed in each randomly sampled single-gonadal cell (either a somatic or germ cell) . To identify which cDNA pools were PGC derived, each pool was examined for expression of the PGC-specific markers Oct4 and stella. All samples were also examined for expression of Esg1, Sox2, and the ribosomal gene S12, which was included as a loading control. The Esg1 and Sox2 probes hybridized only to cDNAs that were also positive for stella and Oct4, strongly indicating that Esg1 and Sox2 are expressed only in PGCs during E10.5-E13.5 and not in somatic cells of the gonad (Fig. 3D, E11.5 only shown). In support of this result, we used immunohistochemistry to examine ESG1 and OCT4 protein expression in gonadal PGCs. Consistent with its localization in mES cells, ESG1 protein was usually detected in the nucleus and cytoplasm of mEG cells and E10.5-E13.5 PGCs that were also positive for OCT4 (Fig. 3E, E11.5 PGCs only shown). It also appeared that OCT4 and ESG1 protein expression were substantially decreased in most female germ cells by E13.5 and that there are many OCT4/ESG1-positive cells at E12.5 but few at E15.5 in male gonads. This observation was supported by Western analysis of OCT4 and ESG1 expression in E12.5-E15.5 gonads. OCT4 and ESG1 bands of approximately 40 and 13.7 KDa, respectively, were detected in E12.5 and E13.5 samples from both sexes. However, very low or no expression of ESG1 and OCT4 was detected in female gonads by E14.5, and ESG1 was substantially decreased and OCT4 moderately decreased in male gonads by E15.5 (Fig. 3F). One interesting but perhaps unsurprising observation from these data is that the expression level of Oct4, Sox2, and Esg1 is greater in proliferating PGCs (before meiotic or mitotic arrest). Thus, it appears that expression of pluripotency-associated markers is associated with proliferating germ cells as well as pluripotent cells (ES, EG, and EC)." V. F% Y' C! _' e+ O

Examination of Oct4, Sox2, and Esg1 expression using single-cell cDNA analysis also indicates that, although Oct4 and Sox2 are both expressed in nascent PGCs (E7), high levels of Esg1 expression in the developing germline occurs later, from approximately E8.5 (data not shown). This indicates that although Sox2 and Oct4 may both be required for PGC specification, Esg1 may not function at this early stage of PGC development.

Significantly, ESG1, SOX2, and OCT4 have been conserved in humans. We therefore used RT-PCR to examine ESG1, SOX2, OCT4, and ribosomal protein RPL32 (positive control) expression in two hES lines, two hEC lines, and a germ cell carcinoma sample (testis). ESG1, SOX2, and OCT4 were strongly expressed in both hES cell lines and the germ cell carcinoma sample, but only one of the two hEC lines expressed ESG1 despite expression of OCT4 and SOX2 (Fig. 4). This is partly in contrast with a very recent report that indicates that hESG1 is not expressed by hEC cells . The expression of hESG1 in some but not all hEC lines may reflect different levels of pluripotency in these cells and may provide a novel and important way to monitor different lines. Generally, however, the data presented here and by Kim et al.  are consistent with a conserved role for these three genes in human pluripotent cells and the germline.; J" Q& e1 }, k7 [. S& n

Figure 4. Human ESG1, SOX2, and OCT4 are coexpressed in human embryonic stem (hES) and in germline carcinoma but only in some human embryonal carcinoma (hEC) cells. Reverse transcription (RT)–polymerase chain reaction analysis of ESG1, SOX2, and OCT4 expression is shown in NTERA2 (hEC line), germ cell carcinoma (GCC), HES2 (hES line), HES4 (hES line), and GCT27X1 (hEC line) samples. Ribosomal protein 32 (RPL32) was used as a positive control. For each sample, 50 ng of cDNA was subjected to 30 cycles of polymerase chain reaction.( B8 Q/ t7 ^3 P  G
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Several genes, including Oct4, Sox2, and nanog , are critical for maintaining pluripotency in mouse cells. Although all three of these genes are detected in early mouse embryos and germ cells, their expression patterns are not identical. For example, nanog is only initiated in the inner cells of the developing morula , whereas Esg1 is more broadly expressed throughout pre-implantation development, suggesting that nanog does not have a role in Esg1 activation. Oct4 expression also differs from that of Esg1, since the latter is downregulated in the epiblast after E5.5 (like nanog), a time that precedes the initiation of lineage specification from the pluripotent epiblast cells. Also, both Oct4 and Sox2 are highly expressed, whereas Esg1 seems to be expressed at significantly lower levels in early germ cells. Similarly, some hEC cells express SOX2 and OCT4 but not ESG1. Despite these differences in Oct4, Sox2, and Esg1 expression, cells that express Esg1 also express Oct4 and Sox2. Taken together, it seems plausible that a functional relationship may exist between OCT4, SOX2, and ESG1, a possibility supported by the presence of OCT4 binding sites in the Esg1 promoter .& s5 {  {. Z% H1 z

From cumulative studies, it is evident that key genes associated with pluripotency are used in a context-dependent manner. For example, although the zygote and early blastomeres are totipotent, they are unlike pluripotent stem cells in that they lack the capacity for sustained self-renewal. In a different context, differentiation of the epiblast cells into somatic cells occurs in the presence of tightly controlled levels of Oct4, when Esg1 and nanog are downregulated. Finally, in another context, the retention or reexpression of many pluripotency-associated genes such as Oct4, Sox2, nanog, and Esg1 may be important during the specification, maintenance, or function of PGCs. It is worth noting that some germline-specific control of Oct4 expression is evident, because two distinct enhancer elements (distal and proximal) are needed to drive expression in pluripotent stem cells and the germline . Similar situations may occur for other genes that are expressed in ES cells and the developing germline and may influence properties of cells exhibiting different aspects of the pluripotent state. Intriguingly, the expression of hESG1 by some but not all hEC lines may also indicate important differences in pluripotency in hEC cells, which may also occur in some hES lines. It is therefore important to evaluate the molecular basis of pluripotency more precisely to facilitate more informed application of technologies involving pluripotent cell types.

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