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Genes to Cells (2004) 9, 1083-1091. doi:10.1111/j.1365-2443.2004.00791.x
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Targeted disruption of the mouse ELYS gene results in embryonic death at peri-implantation development

Keisuke Okita1, Hiroshi Kiyonari2, Ikuo Nobuhisa1, Naoki Kimura3, Shinichi Aizawa2 and Tetsuya Taga1,*

1 Department of Cell Fate Modulation, Institute of Molecular Embryology and Genetics, Kumamoto University, Kumamoto 860-0811, Japan
2 Center for Developmental Biology, RIKEN, 2-2-3 Minatojima-minamimachi, Chuo-ku, Kobe 650-0047, Japan
3 Pharmaceutical Technology Laboratory I, Chugai Pharmaceutical Co. Ltd, Niihari, Ibaraki 300-4101, Japan


   Abstract
Top
 Abstract
Introduction
 Results
 Discussion
 Experimental procedures
 References
 
Early mouse development is a complicated process that is controlled by various proteins including transcription factors. Recently, we identified a putative transcription factor, ELYS (embryonic large molecule derived from yolk sac), using a subtraction strategy. To elucidate the role of ELYS in vivo, we generated ELYS-deficient mice by homologous recombination. Although heterozygous mice appeared to be healthy, fertile and normal, embryos homozygous for the ELYS mutation died between embryonic day (E) 3.5 and 5.5. Null mutant blastocysts collected from the uterus at E3.5 were viable and indistinguishable from wild-type littermates. However, when cultured in vitro, they showed impaired proliferation of the inner cells, because of apoptosis. The expression of ELYS mRNA was detected in both the inner cell mass (ICM) and the trophectoderm at the blastocyst stage, and persisted throughout the developing embryo during E4.5 to 6.5. These results indicate that ELYS is a critical factor for early mouse development and is essential for the survival of the inner cells.


   Introduction
Top
 Abstract
 Introduction
Results
 Discussion
 Experimental procedures
 References
 
Mouse development begins with a fertilized ovum. After an ovum is fertilized with a sperm in the ampullary region of the oviduct, the embryo continues cell division and goes down the oviduct. Around E3.5, it reaches the uterus and progresses from the morula to the blastocyst stage. The blastocyst is composed of two distinct kinds of cell lineage. The ICM, which is situated inside the blastocyst, will constitute the embryo proper during ontogeny, while the trophectoderm, which surrounds the ICM, constitutes the extra-embryonic tissues, including the foetal portion of the placenta. At the pre-implantation stage, mouse embryos change their gene expression pattern dramatically, where maternally stored RNAs are degraded and numerous genes for basic cellular activity and early developmental processes are activated (Hamatani et al. 2004; Wang et al. 2004). Studies of inactivation of these newly transcribed genes revealed, in some cases, early embryonic deaths. For instance, mice having mutation in the gene for cdc7, a cell cycle regulating factor, or SNF5, a component of the ATP-dependent chromatin remodelling enzyme SWI/SNF, die between E3.5 and E6.5 (Klochendler-Yeivin et al. 2000; Guidi et al. 2001; Kim et al. 2002). Glucose transporter 8, an insulin-responsive facilitative glucose transporter, is required for blastocyst survival (Pinto et al. 2002). Oct4 is a POU transcription factor which is thought to be a key regulator in maintaining pluripotency of inner cells and ES (embryonic stem) cells (Nichols et al. 1998; Niwa et al. 2000). The absence of Oct4 results in peri-implantation death before egg cylinder formation.

Recently, we performed a subtraction strategy using mRNAs from yolk sacs at two different developmental stages and isolated a putative transcription factor, ELYS (Kimura et al. 2002). Based on the cDNA sequence, ELYS protein is composed of 2243 amino acid residues, containing an AT-hook DNA-binding domain, nuclear localization signals and nuclear export signals. An in vitro transcription analysis using fusion proteins of the yeast GAL4 DNA-binding domain and various ELYS mutants revealed that ELYS has the ability to activate transcription. During mouse development, the expression of ELYS mRNA was mainly observed in haematopoietic organs. It was clearly present in the yolk sac, aorta-gonad-mesonephros, liver and spleen, where haematopoiesis occurs. However, in adult mice, the transcripts are broadly detectable in both haematopoietic and non-hematopoietic organs, such as the brain, lungs, heart, liver, thymus, spleen, kidneys and testes. The broad distribution of ELYS mRNA suggests that ELYS has a general function which is not restricted to haematopoiesis.

To investigate the physiological role of ELYS, we generated mice deficient in ELYS by homologous recombination. The adult heterozygous mice showed no obvious phenotypes compared with wild-type mice. However, intercrosses of heterozygous mice did not produce offspring which had the double mutant allele. Surprisingly, the null embryos died between E3.5 and E5.5 before the onset of foetal haematopoiesis. Taken together with the results of in vitro blastocyst cultures and apoptotic cell staining, we propose that ELYS is an essential factor for inner cell proliferation and/or survival.


   Results
Top
 Abstract
 Introduction
 Results
Discussion
 Experimental procedures
 References
 
Targeted disruption of ELYS

To determine the requirement for ELYS in mice in vivo, we deleted the gene by homologous recombination. Previously, we showed that the mouse ELYS gene spans approximately 60 kbp in chromosome 1 and encodes 36 exons (Okita et al. 2003). According to this structural organization, a vector for targeted disruption of ELYS was designed that replaced an approximately 20-kbp portion of the ELYS gene, spanning exons 1–13 (containing most of the 5' untranslated region, the translation initiating methionine and all the three nuclear export signals), with the neomycin-resistance cassette (Fig. 1A). This construct was electroporated into the TT2 ES cell line. After G418 selection, the resistant clones were picked up and investigated for the correct recombination. Genomic DNA was extracted, digested with SacI and NheI and then analysed by Southern hybridization with a 3'-probe external to the vector (Fig. 1B). Analyses using probes for an external 5' flanking region were also performed for confirmation (Fig. 1C). Three independent clones among 192 resistant clones were verified to be homologous recombinants. Among these, two clones (nos 29 and 74) transmitted the ELYS mutation into the germ-line. The mutant mice from both clones showed the same phenotypes.



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  		Figure 1  Targeted disruption of the ELYS gene. (A) Schematic representation of the expected gene replacement at the ELYS locus. Restriction maps of the wild-type allele, targeting vector and targeted allele are shown. The filled boxes and the solid lines between them represent exons and introns, respectively. The wild-type ELYS gene shows exons 1 through 19, including the translation initiation codon (ATG). The restriction sites are BamHI (B), SacI (S) and NheI (N). The region for the probes used for Southern hybridization is also indicated. The targeting vector contains the neomycin-resistance gene (neo) under the control of the phosphoglycerate kinase gene promoter and the diphtheria toxin gene (DT) under the control of the MC1 promoter. (B) Southern blotting analysis of wild-type ES cells (wild) and three G418-resistant clones (nos 29, 74, and 111). DNA was digested with SacI and NheI, and a 3'-probe outside of the targeting vector was used for analysis. The positions of the wild-type (7.0 kbp) and recombined (8.2 kbp) alleles are indicated to the right. (C) Correct targeting was also confirmed by Southern blotting analysis using a probe for the 5' external region after BamHI digestion. The positions of the wild-type (7.8 kbp) and recombined (9.7 kbp) alleles are indicated to the right.

 
Embryonic lethality of ELYS homozygous mutant embryos

Animals heterozygous for the ELYS mutation were healthy and fertile. To generate homozygous mice, ELYS heterozygous mice were intercrossed. Genotyping of 89 newborn offspring by Southern blotting or PCR analysis revealed the absence of mice homozygous for the ELYS mutation (Table 1), indicating embryonic lethality. In order to determine when the ELYS mutation produced a lethal phenotype, embryos at several developmental stages were investigated. When blastocysts were flushed from the uterus at E3.5, ELYS null embryos were seen at the expected Mendelian ratio and were indistinguishable from the wild-type littermates. The TUNEL staining also showed no significant difference in the number of apoptotic cells in the wild-type, heterozygous and homozygous ELYS mutant blastocysts: 1.2 ± 1.3 (n = 11), 1.0 ± 1.2 (n = 9) and 1.0 ± 1.1 (n = 6), respectively. Furthermore, in situ hybridization with an Oct-3/4 probe revealed that all of the examined 31 blastocysts from the intercrosses of ELYS heterozygotes were comparably positive for the Oct-3/4 transcripts at their ICM (data not shown). Therefore, the ELYS null embryos appeared normal at E3.5 in terms of, at least, shape and Oct-3/4 expression. However, no homozygous mutants were observed in the implantation sites at E7.5, the neural plate stage. Wild-type or heterozygous embryos developed normally and only a few absorptions were observed. Therefore, ELYS null mutants died before E7.5. Next, we analysed the implantation sites at E5.5 histologically. Entire deciduas were isolated, fixed and stained with haematoxylin and eosin. Twenty implantation sites were examined and 14 embryos appeared to develop normally (Fig. 2A). The embryos in the other six sites showed abnormal development (Fig. 2B). Among these, two sites contained a few embryonic cells and four were almost empty. This high frequency (30%) implied that they were homozygous mutants. These results indicate that ELYS null mutants die between E3.5 and E5.5.


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  		Table 1  Genotypes of offspring from heterozygous matings
 


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  		Figure 2  Histological analysis of embryos in the decidua at E5.5. Deciduas were isolated from intercrosses of ELYS heterozygous mice at E5.5, fixed, dehydrated through an ethanol gradient and xylene and then paraffin embedded. Series of sections (6 µm thickness) were stained with haematoxylin and eosin. A representative well-developed embryo (A) and severely underdeveloped embryo (B), which is a presumptive homozygous mutant, are shown. Bar, 100 µm.

 
Expression of ELYS in early mouse embryos

ELYS mRNA was previously shown to be expressed in the liver, thymus, lungs, spinal cord and eyes in E14.5 mouse embryos. However, the expression in the early stages of embryogenesis was not determined. We investigated the spatial expression profiles of ELYS by whole mount RNA in situ hybridization in E3.5 to E6.5 mouse embryos (Fig. 3). In the blastocyst at E3.5, dark blue signals were detected in both the ICM and the trophectoderm layers, indicating that ELYS mRNA was expressed in every part of the embryo. At E4.5, immediately after implantation, the expression persisted throughout the embryo. The signals were seen in the epiblast, parietal endoderm, visceral endoderm and mural trophectoderm layers. The ELYS anti-sense probe also revealed broad expression of ELYS mRNA in both embryonic and extra-embryonic portions of the embryo at E5.5 and E6.5. The transcripts were present in the epiblast, visceral endoderm, extra-embryonic ectoderm and ectoplacental cone. The two anti-sense probes, which recognized the 5' or 3' part of the ELYS mRNA, showed the same expression patterns, while no staining was observed with the sense probes. For confirmation of the technique, the hybridization was also performed using an anti-sense probe for Oct-3/4. This probe specifically stained the epiblast of the embryo at E6.5 as previously described (Rosner et al. 1990; Scholer et al. 1990). Hence, the transcripts of ELYS are expressed throughout the embryo at E3.5 to E6.5.



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  		Figure 3  Expression of ELYS mRNA in wild-type embryos at stages E3.5 through E6.5. Whole mount in situ hybridization was performed with ELYS anti-sense (A, B, C and D), ELYS sense (E) and Oct-3/4 anti-sense (F) probes. The dark blue signals for ELYS mRNA are present in both the ICM and the trophoblast portion of the blastocyst at E3.5 (A). The signals are also detected throughout the embryos at E4.5 (B), E5.5 (C) and E6.5 (D), whereas Oct-3/4 mRNA was only detected in the epiblast at E6.5 (F). E indicates background staining. ep, epiblast; exe, extra-embryonic ectoderm. Bar, 50 µm.

 
ELYS-deficient blastocysts cultured in vitro show impaired ICM outgrowth

Because of the early lethal phenotype of ELYS null mutants, it is difficult to further study the function of ELYS in vivo. Hence, we employed an in vitro blastocyst culture system. Embryos at E3.5 were isolated by flushing from the uterus and cultured in ES derivation medium containing leukaemia inhibitory factor (LIF). Under these conditions, inner cells should expand, outgrow and frequently differentiate into extra-embryonic endoderm, while the trophectoderm layer should become adherent and differentiate into giant cells. At the beginning of the culture, ELYS null blastocysts were morphologically normal and indistinguishable from wild-type or heterozygous embryos. They hatched from their zona pellucida and attached normally to the gelatin-coated dishes similarly to their littermates. During a 5-day culture, the wild-type or heterozygous blastocysts showed continuous growth and their ICMs formed the typical structure of dense cell masses (Fig. 4A). In contrast, none of ELYS null mutants showed a distinguishable ICM-like structure, although the trophectoderm layer differentiated into giant cells (Fig. 4B). Among the 53 blastocysts tested, one heterozygous and two homozygous embryos revealed delayed attachment, although they exhibited similar outgrowth to other embryos of the same genotype over a prolonged culture period. As summarized in Fig. 4(C), the null mutants were never associated with robust outgrowth of the inner cells. These results demonstrate that ELYS is important for development of the ICM.



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  		Figure 4  In vitro outgrowth of cultured blastocysts. Blastocysts were flushed from the uterus of heterozygous females 3.5 days after intercrosses and grown in vitro. Representative heterozygous (A) and homozygous (B) blastocysts are shown. At the beginning of the culture (0 d), blastocysts of both genotypes appear normal. Their ICMs are indicated with black arrowheads. The blastocysts hatched and attached to the gelatin-coated dishes at around day 1. The inner cells of the heterozygous embryo continues to proliferate and forms a dense core structure (arrow) surrounded by primitive endoderm cells. However, no distinct ICM is seen in the homozygous culture. Outgrowth of polyploid trophectoderm cells is observed in all the cultures (white arrowhead). Bars: 0 d in A and B, 100 µm; 1 d–5 d in A and B are at the same magnification (bar in 1 d represents 200 µm). (C) Summary of the blastocyst outgrowth. The null genotype is never associated with robust outgrowth of the inner cells and is the only genotype associated with undetectable inner cells outgrowth. Although one heterozygous and two homozygous blastocysts only adhered after 6 days of the culture, they show similar phenotypes to other embryos of the same genotype over a prolonged culture period.

 
Cultured ELYS null blastocysts only showed spreading of cells of the trophectoderm lineage, implying two possibilities. First, the inner cells of the blastocyst may die during the culture period. Second, the inner cells may become trophectoderm-like cells and show an extended cell shape, thereby rendering the ICM-like cell mass deficient. In order to analyse the ICM abnormalities in detail, ICMs were isolated from the blastocysts using an immunosurgery method and cultured in vitro. The inner cells from wild-type or heterozygous embryos continued to grow vigorously during the 5-day culture and produced primitive endoderm cells in many cases (Fig. 5A). Although similar inner cells were obtained from the homozygous embryos, none of them showed proliferation (Fig. 5B). Instead, they floated in the medium and never adhered to the dishes. The cultured inner cells were recovered on day 2 and investigated for apoptotic cell death using the TUNEL assay. Fluorescence indicative of DNA fragmentation was only observed in the ELYS null mutants (Fig. 5C), thus revealing that they undergo apoptosis.



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  		Figure 5  Impaired proliferation of the inner cells isolated from ELYS null embryos in culture. (A) Representative inner cells outgrowth derived from a heterozygous blastocyst. ICMs were obtained from blastocysts using an immunosurgery method and cultured in ES cell derivation medium containing LIF. Cultures from wild-type and heterozygous embryos show typical formation of a compacted core structure and the appearance of primitive endoderm cells. Although the ICMs of null embryos were also obtained and appeared similar to those of the other genotypes, they neither attached to the dishes nor expanded. Bars: 0 d, 50 µm; 2 d and 5 d, 200 µm (B) Summary of the inner cells outgrowth. (C) TUNEL assays of the cultured ICM. After 2 days of culture, the cell clumps of heterozygous and homozygous mutants were recovered and TUNEL staining was performed. Phase contrast (left) and fluorescence (right) photographs are shown. No staining is evident in the heterozygous ICM, whereas fluorescence is observed throughout the ELYS-deficient embryo. Bar, 50 µm.

 

   Discussion
Top
 Abstract
 Introduction
 Results
 Discussion
Experimental procedures
 References
 
In this study, we created ELYS mutant mice that lacked a functional ELYS gene. The homozygous mutants died at an early stage of development. When blastocysts were recovered from the uterus of heterozygous mice at E3.5, embryos of each genotype existed at the expected rate. All the embryos were morphologically normal and identical to their littermates. Outgrowth cultures revealed that the ELYS null blastocysts escaped from their zona pellucida and attached to the dishes in vitro. These results indicate that the homozygous blastocysts are viable and have the ability to implant. Usually, mouse implantation occurs around E4.5. We investigated embryos in the decidua at E5.5, just after implantation, and found a high frequency of impaired development. At E7.5, almost all the embryos developed normally, but ELYS null mutants were absent. Taking these observations together, it is most likely that, although ELYS-deficient blastocysts undergo implantation, they die shortly afterwards and are immediately absorbed.

To determine the cause of the ELYS mutant death, we investigated the homozygous embryos in vitro. When whole blastocysts were cultured, they hatched, attached and expanded comparably with those of the other genotypes for the first 2 days. However, the mutant ICM did not expand thereafter, while the trophectoderm cells differentiated into giant cells. After 5 days in culture, ELYS null mutants showed only flattened cells of the trophectoderm lineage. Similar phenotypes have been reported for blastocysts which have null mutations for genes important for proliferation, survival or differentiation of inner cells. The Chk1 and ATR genes play essential roles at cell cycle checkpoints. ATR regulates Chk1 phosphorylation in a DNA damage-dependent manner (Liu et al. 2000). In culture, ATR- or Chk1-deficient blastocysts exhibit impaired outgrowth of the inner cells and die by apoptosis (Brown & Baltimore 2000; Takai et al. 2000). Similarly, ES cells deficient in Chk1 also show a severe defect in proliferation, and undergo apoptotic death (Liu et al. 2000). The transcription factors Oct-3/4, Sox-2 and Nanog are known to strictly control the differentiation of inner cells and ES cells (Nichols et al. 1998; Avilion et al. 2003; Chambers et al. 2003; Mitsui et al. 2003). Their null blastocysts also fail to form the ICM-like structure in vitro, probably because their inner cells promptly differentiates into another cell lineage. For example, inner cells isolated from Oct-3/4 null blastocysts proliferate and enter the trophectoderm cell lineage, even in ES cell derivation medium containing LIF (Nichols et al. 1998). According to detailed analyses using ES cells, the expression level of Oct-3/4 is a striking factor for the fate decision of inner cells and ES cells. A 1.5-fold increase in Oct-3/4 expression in ES cells results in determination of the primitive endoderm and mesoderm, while a decrease causes determination of the trophectoderm lineage (Niwa et al. 2000). Hence, cultured Oct-3/4 null blastocysts only show trophectoderm cells. Based on these reports, we examined the nature of ELYS-deficient inner cells in detail. ICMs were isolated from the blastocysts with immunosurgery and cultured in vitro. Although inner cells from ELYS heterozygous grew robustly, inner cells from the null mutants did not show any proliferation and were stained with the TUNEL assay, indicative of apoptosis. These results suggest that ELYS is an essential factor for the proliferation or survival of inner cells. As the expression of ELYS mRNA was observed throughout the blastocysts, including the ICM and the trophectoderm, we could not rule out the possibility that ELYS deficiency might also cause trophectoderm failure. This is because fibroblast growth factor-4, which is produced from the inner cells, is known to be an essential factor for trophectoderm proliferation (Niswander & Martin 1992; Feldman et al. 1995). The loss of the ICM might lead to delayed depletion of trophectoderm as a result of exhaustion of such growth factor(s). The trophectoderm appeared to be more resistant to ELYS depletion than inner cells, as shown by the in vitro outgrowth experiments of the blastocysts. However, over a prolonged culture period, presumptive ELYS null trophectoderm cells decreased gradually. Although ATR and Chk1 are thought to be important for all cell proliferation, cultured blastocysts of their mutants also reveal a sustained trophectoderm (Brown & Baltimore 2000; Takai et al. 2000). Because of the rapid proliferation of the inner cells, inner cells would be much more sensitive to metabolic inhibitors or disruption of growth control genes than trophectoderm cells and would die promptly. The ICM abnormality observed in the ELYS null culture experiment is most likely as a result of the cell-autonomous requirements for ELYS, rather than the secondary effect of the trophectoderm failure. This is because ELYS null blastocysts at E3.5 were viable and their ICMs showed normal Oct-3/4 expression. In addition, inner cells isolated from the wild-type but not the ELYS-deficient embryos by immunosurgery grew vigorously in vitro at least for 5 days.

ELYS, Chk1 and ATR mutant blastocysts showed similar phenotypes in culture, i.e. they all produced trophoblast cells, but not inner cells. However, they differed in detail. Chk1 null blastocytes at E3.5 showed aberrant nuclei and gross apoptotic cell death as revealed by the TUNEL staining, whereas ELYS and ATR null blastocysts at the same stage appeared normal, but exhibited apoptosis only in the culture period. The timing of the occurrence of apoptosis in these mutants may reflect the stage or environment where each gene is necessary. In such a stage or environment, null mutation may not be compensated by other proteins or the maternally produced gene product may be depleted. Although most maternal gene products are destroyed at the 2- or 8-cell stage of development (Nothias et al. 1995; Schultz 2002), some maternal proteins, such as Sox-2, are known to persist until the blastocyst stage (Avilion et al. 2003). Detection of maternal ELYS gene transcripts or products in ELYS-deficient mutants by in situ hybridization or immunocytochemistry needs to be performed to clarify this point.

Recently, Smith et al. (2004) isolated a novel gene, Mtb, whose gene product interacts with SCL. SCL is an important transcription factor for primitive haematopoiesis. Mtb is expressed widely in mouse embryos before gastrulation, but subsequently its expression is restricted to the sites of neurogenesis and haematopoiesis. Mtb null mutants show the apoptotic cell death in the ICM of E3.5 blastocysts and die immediately following implantation. Like the blastocysts deficient for ELYS, the Mtb null blastocysts fail to exhibit outgrowth of inner cells (Smith et al. 2004). Because of the similar expression pattern and early embryonic lethality, ELYS might in part function in cooperation with Mtb in early embryonic development and haematopoiesis.

ELYS was broadly expressed at early stages of development (E3.5 to E6.5) in both embryonic and extra-embryonic regions. Expression is also observed in several tissues of adult mice (Kimura et al. 2002). Considering our present results, ELYS might be involved in the survival or proliferation of various cell types. The process of the cell cycle is strictly controlled. Cyclin and cyclin-dependent kinase are thought to be the key regulators of the system. These factors regulate proteins, which function specifically in the G1, S, G2 and M phases. For instance, cyclin E signals activate the transcription factor E2F1 which regulates essential genes during G1/S (DeGregori et al. 1995). Previously, we revealed the promoter region important for ELYS expression in an aorta-gonad-mesonephros culture system (Okita et al. 2003). It is noteworthy that this region contains the E2F1 cis-element, indicating the possibility of cell cycle-dependent regulation of the ELYS gene. In addition to this possibility, there is another possibility for ELYS function which could be imagined. At the pre-implantation stage, mouse embryos degrade most of the maternally stored RNAs and activate numerous genes for energy metabolism, protein synthesis, morphogenesis and cell signalling to prepare for further development (Hamatani et al. 2004; Wang et al. 2004). As ELYS has transcriptional activity and is broadly expressed in early embryos, it might control the gene expressions essential for cell survival or peri-implantation development. Additional experiments are required to clarify the precise function of ELYS in development.


   Experimental procedures
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
References
 
Gene targeting

The mouse ELYS gene was isolated by screening a phage library derived from the C57BL/6 strain of mice as described before (Okita et al. 2003). A 6.1-kbp PvuII–BamHI-digested genomic DNA fragment containing a part of exon 1 and the 5' flanking region was used as the 5' arm of the targeting vector and ligated to a neomycin-resistance cassette. For the 3' arm, a 3.0-kbp BamHI–EcoRI fragment containing exons 14–16 was placed downstream of the neomycin-resistance cassette. The neomycin-resistance gene was driven by the phosphoglycerate kinase gene promoter and responsible for positive selection. A diphtheria toxin A cassette flanking the 3' arm provided negative selection against random integration of the construct. The targeting vector was linearized with NotI and electroporated into TT2 ES cells (Yagi et al. 1993). Selection (300 µg/mL G418) was applied 24 h after electroporation and resistant clones were picked up 8 days later. These clones were screened by PCR and Southern blotting using a 3' external probe after SacI/NheI digestion. Correct targeting was also confirmed using a probe for the 5' external region after BamHI digestion. Chimeras were produced by microinjection into ICR blastocysts and transferred to the uterus of pseudopregnant ICR females. Germ-line chimeras were mated to C57BL/6 females to generate mice heterozygous for the disrupted ELYS allele. Two independent ES cell clones (nos 29 and 74) produced germ-line chimeras. Mutant mice were backcrossed at least one time before the analysis. All embryos were generated by natural matings. The genotypes of the offspring were determined by Southern blotting analysis. Embryos at various developmental stages were genotyped by PCR using the following primers: 5'-TGCACCTATGGCACTGCTCC-3' and 5'-GCTTATCCTGAATCCAGACTTC-3' to amplify a 294-bp fragment from the wild-type allele, and 5'-GCTCATTCCTCCCACTCATG-3' and 5'-ACCACTTTATTCCATGTCCACTC-3' to amplify a 238-bp fragment from the recombined allele. The products were resolved by electrophoresis and confirmed by Southern blotting analysis.

Blastocyst and outgrowth cultures

Blastocysts were flushed from the uterus at E3.5 using M2 medium and cultured on gelatinized 24-well culture plates in ES cell derivation medium (Glasgow modification of Eagle's medium supplemented with 10% foetal calf serum, 1 mM sodium pyruvate, 100 µM ß-mercaptoethanol, 100 µM non-essential amino acids and 1000 U/mL leukaemia inhibitory factor) at 37 °C in a 5% CO2 incubator. The morphology was observed every day. After 5 days, the cells were lysed on the bottom of the well and their genotypes were determined by PCR as described above.

ICM isolation was performed by immunosurgery as previously described (Solter & Knowles 1975). Briefly, the zona pellucida was removed from blastocysts with acid Tyrode's solution (Sigma, St Louis, MO) and embryos were incubated with anti-mouse antiserum for 30 min at 37 °C. After washing with M16 medium, they were incubated with guinea pig serum (Sigma) containing complement until lysis of the trophectoderm occurred. The lysates were recovered for determination of their genotype by PCR. ICMs were cultured in ES cell derivation medium as described above.

For TUNEL assays, cells were picked up and fixed in 2% paraformaldehyde for 30 min. Permeabilization and TUNEL assays were performed using an In Situ Cell Death Detection Kit, Fluorescein (Roche Diagnostics, Basel, Switzerland), according to the manufacturer's instructions.

Histology and whole mount in situ hybridization

Embryos and surrounding tissues were removed from timed pregnant mice and fixed overnight by immersion in 4% paraformaldehyde. They were dehydrated in an ethanol series, and then paraffin-embedded, sectioned and stained with haematoxylin and eosin. Whole mount in situ hybridization was performed following the method of Rosen and Beddington (Rosen & Beddington 1994). Digoxigenin-11-UTP-labelled riboprobes were prepared using a DIG (digoxigenin) RNA Labeling Kit (Roche Diagnostics). For the detection of ELYS mRNA, two independent probes were designed: ELP12 corresponded to amino acid residues 12–164 of ELYS and ELP1674 corresponded to amino acids 1674–1842. An Oct-3/4 anti-sense probe was prepared using the AGS122 plasmid (kindly provided by Dr J. Nichols) which contains the POU homeodomain of the Oct-3/4 gene.


   Acknowledgements
 
We are very grateful to Drs Jennifer Nichols, Ian Chambers, Mia Buehr, Frances Stenhouse, Austin Smith and Kimi Araki for their encouraging suggestions and generous help in analysing the early mutant embryos. We thank Ms Yuko Saiki, Ms Kaori Kaneko and Ms Sayomi Iwaki for their technical assistance. We also thank Ms Yuki Noguchi and Ms Michiko Ohta for their excellent secretarial assistance. This work was supported in part by Grants-in-Aid for 21st Century COE Research from the Ministry of Education, Culture, Sports, Science and Technology ‘Cell Fate Regulation Research and Education Unit’, the Human Frontier Science Program, and the Virtual Research Institute of Ageing of Nippon Boehringer Ingelheim. KO is affiliated to the 21st Century COE program ‘Cell Fate Regulation Research and Education Unit’, Kumamoto University.


   Footnotes
 
Communicated by: Noriko Osumi

* Correspondence: E-mail: taga{at}kaiju.medic.kumamoto-u.ac.jp


   References
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
 References
 
Avilion, A.A., Nicolis, S.K., Pevny, L.H., Perez, L., Vivian, N. & Lovell-Badge, R. (2003) Multipotent cell lineages in early mouse development depend on SOX2 function. Genes Dev. 17, 126–140.[Abstract/Free Full Text]

Brown, E.J. & Baltimore, D. (2000) ATR disruption leads to chromosomal fragmentation and early embryonic lethality. Genes Dev. 14, 397–402.[Abstract/Free Full Text]

Chambers, I., Colby, D., Robertson, M., et al. (2003) Functional expression cloning of Nanog, a pluripotency sustaining factor in embryonic stem cells. Cell 113, 643–655.[CrossRef][Medline]

DeGregori, J., Kowalik, T. & Nevins, J.R. (1995) Cellular targets for activation by the E2F1 transcription factor include DNA synthesis- and G1/S-regulatory genes. Mol. Cell. Biol. 15, 4215–4224.

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Received: 29 April 2004
Accepted: 12 August 2004




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