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EMBRYONIC STEM CELLS

Tex19, a Mammalian-Specific Protein with a Restricted Expression in Pluripotent Stem Cells and Germ Line

^aDepartment of Developmental Biology and Bioinformatics Platform of Strasbourg, Institut de Génétique et de Biologie Moléculaire et Cellulaire (Institut National de la Santé et de la Recherche Médicale, Centre National de la Recherche Scientifique, Université Louis Pasteur), Illkirch, France;
^bFaculté de Médecine, Centre Hospitalier Universitaire, Strasbourg, France;
^cLaboratoire de Biologie Expérimentale, Aspects Cellulaires et Moléculaires de la Reproduction et du Développement, Faculté des Sciences, Vandoeuvre-les-Nancy, France;
^dDépartement de Pharmacologie et Physicochimie, UMR 7175 (Centre National de la Recherche Scientifique, Université Louis Pasteur), Illkirch, France

Key Words. Tex19 • Oct4 • Embryonic stem cells • Germ cells • Preimplantation embryo • Pluripotency

Correspondence: Correspondence: Stéphane Viville, Pharm.D., Ph.D., IGBMC, Department of Developmental Biology, 1 Rue Laurent Fries, Illkirch, F-67400 France. Telephone: 33-3-88-65-33-22; Fax: 33-3-88-65-32-01; e-mail: viville{at}igbmc.u-strasbg.fr

Received on September 14, 2007; accepted for publication on December 10, 2007.

First published online in STEM CELLS EXPRESS  December 20, 2007.

	ABSTRACT

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Although the properties of embryonic stem (ES) cells make these cells very attractive in the field of replacement therapy, the molecular mechanisms involved in the maintenance of their pluripotency are not fully characterized. Starting from the observation that most pluripotent markers are also expressed by spermatogonia stem cells, we identified Tex19 as a new potential pluripotency marker. We show that Tex19 is a mammalian-specific protein duplicated in mouse and rat, renamed Tex19.1 and Tex19.2, whereas only one form is found in human. In mouse, both forms are localized on chromosome 11 and transcribed in opposite directions. Tex19 proteins are well conserved, showing two highly conserved domains that do not present any similarity with any other known domains. We show that Tex19.2 is specifically detected in the male somatic gonad lineage, whereas Tex19.1 expression is very similar to that of Oct4. Transcripts are maternally inherited, and expression starts as soon as the early embryo and later is limited to the germ line. Tex19.1 transcripts were also detected in mouse pluripotent stem cells, and expression of Tex19.1, like that of Oct4, decreases after murine embryonic stem and germ cell differentiation. Human TEX19 was more closely related to murine Tex19.1 and was also detected in adult testis and in undifferentiated ES cells. By immunofluorescence, we found that Tex19.1 protein localizes to the nucleus of mouse ES and inner cell mass cells. All these results suggest that Tex19.1, as well as human TEX19, could be a new factor involved in the maintenance of self-renewal or pluripotency of stem cells.

Disclosure of potential conflicts of interest is found at the end of this article.

	INTRODUCTION

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Since the establishment of human embryonic stem (ES) cells and embryonic germ (EG) cells [1, 2], the study of pluripotency has become one of the major research areas in medicine. Indeed, these cells, because of their self-renewal and pluripotency properties, are promising tools in cell replacement therapy. The strategy of such therapeutic treatment is based on the production, in vitro, of specific cell types that, after transplantation, are able to restore physiological functions in diseases such as diabetes, Huntington disease, Parkinson disease, and heart failure. However, these approaches are confronted, as for organ transplants, with the major problem of tissue rejection. A potential solution is the production of patient-histocompatible ES cells or ES-like cells. For this purpose, three methods allowing the reprogramming of differentiated cells have been proposed: (a) nuclear transfer using enucleated oocyte reprogramming capabilities; (b) fusion of somatic cells with pluripotent cells; and (c) transfection with four transcription factors (Oct4, Sox2, c-Myc, and Klf4), allowing the reprogramming of mouse fibroblasts [3–7].

Several genes, such as Oct4, Sox2, Nanog, and FoxD3 or, more recently, Zic3, Zfx, and Zfp206, have been shown to be implicated in regulating self-renewal and/or pluripotency [8–17]. The complexity of the process allowing self-renewal and pluripotency of a cell line has been underlined by showing the importance of the fine-tuning of the Oct4 expression level [18]. This complexity was further highlighted by recent studies showing that Oct4, Sox2, and Nanog are the key regulators of the transcriptional network that cooperatively controls self-renewal and pluripotency in ES cells [19]. Furthermore, genome-wide studies on mouse and human ES cells have underlined many downstream target genes that are activated or repressed by Oct4 and/or Nanog [19–21]. In addition, ES cell pluripotency is associated with unusual chromatin architecture. Indeed, ES cell chromatin is in a relatively decondensed state [22], complemented by a unique epigenetic status with the coexistence of activating and repressive histone modifications [23, 24].

Despite results strongly suggesting that Oct4, Sox2, Nanog, and FoxD3 act together rather than individually, the hierarchy of renewal and pluripotency pathways has not yet been established. Therefore, there is still a need to characterize new actors involved in self-renewal and pluripotency properties of stem cell lines and of totipotent and pluripotent cells of the early embryo.

Starting from the observation that for most markers of pluripotency, such as Oct4, Sox2, and Nanog, there is a continuum in their expression from the epiblast to the primordial germ cells (PGC) up to the spermatogonia stem cells, we checked spermatogonia stem cell-expressed genes for their expression in pluripotent stem cells. We began our study by analyzing the 25 spermatogonia-specific genes described by Wang et al., [25] among which Tex20, a murine homolog of Sall4, has been recently shown to be involved in the proliferation of embryonic stem cells [26]. We focused our attention on Tex19, because of its specific 12.5-day post coitum (dpc) genital ridge expression. Previously, a unique Tex19 gene has been described in mouse, and no ortholog was found in human [25]. Here, we show that Tex19 is a mammalian-specific protein. Moreover, sequence analysis revealed that the Tex19 gene, which is duplicated in mouse and rat species (Tex19.1 and Tex19.2) is unique on the human genome. TEX19 proteins are well conserved among mammals; in particular, two highly conserved domains could be delineated. These domains do not share sequence similarity with any known protein domains. Tex19.1 is expressed in mouse pluripotent stem cells (ES, EG, F9, and P19) and localizes to the nucleus. Tex19.1 expression, like that of Oct4, disappears upon mouse embryonic stem (mES) or mouse EG cell differentiation by leukemia inhibitory factor (LIF) retrieval. During mouse development, Tex19.1 presents an expression profile very similar to that of Oct4, restricted to the inner cell mass (ICM), PGC, and testis; no other organs, with the exception of the placenta, express Tex19.1. Tex19.2 expression starts later during testicular development and is specific to somatic lineages in male gonads. In human, we found that TEX19 is more closely related to murine Tex19.1 and is expressed in adult testis and undifferentiated human ES (hES) cells.

	MATERIALS AND METHODS

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Bioinformatic Tools
Protein sequence homology searches were performed with the q99mv2_mouse protein (Tex19) [25] as a query in the UniProt [27] database using the PipeAlign tool [28]. Additional Tex19 protein homologs were found using tblastn searches [29] on ongoing sequencing eukaryotic genomes. We used the DbClustal [30] program to generate a multiple alignment of complete sequences [31]. The multiple alignment was manually refined in the SeqLab (Wisconsin Package; Accelrys, San Diego, http://www.accelrys.com) editor. Protein secondary structure predictions were generated by the PHDsec program [32]. Synteny analysis was carried out on the National Center for Biotechnology Information Map Viewer (http://www.ncbi.nlm.nih.gov/mapview) [33]. Using blastn homology searches with the 25 cDNAs specifically expressed by spermatogonia A0 cells [25] as queries in the mouse Expressed Sequence Tags (EST) public database, PGC-expressed cDNAs were selected.

Isolation of Germ Cells from Embryonic Gonads and Germ Cell Depletion Experiments
Oct4-GOF-18 PE:green fluorescent protein (GFP) transgenic males [34] were bred with CD1 females to collect gonad-mesonephros complexes at 12.5, 16.5, and 18.5 dpc. Samples were dissociated with 0.05% trypsin and filtered through 3-inch filcons (BD Bioscience, San Diego, http://www.bdbiosciences.com). GFP-positive and -negative cells were sorted by fluorescence-activated cell sorting (FACS). Then, cells were pelleted and resuspended in 350 µl of RLT buffer for RNA extraction as described below.

CD1 pregnant females received a single intraperitoneal injection of 200 µl of busulfan solution (8 mg/ml in dimethyl sulfoxide [DMSO]) or 200 µl of DMSO (control) at 10.5 dpc. Gonad-mesonephros complexes of embryos were collected at 14.5 dpc and analyzed by whole-mount in situ hybridization (WISH) and reverse transcription (RT)-polymerase chain reaction (PCR).

Collection of Human Samples
Total RNA of adult human testis and undifferentiated and differentiated hES cells (VUB01 cell line [35]) were obtained from the Research Centre for Reproduction and Genetics of the Vrije Universiteit Brussels. The human placenta sample was provided by Syndicat Inter-Hospitalier de la Communaté Urbane de Strasbourg-Centre Médico-Chirurgical et Obstétrical (SIHCUS-CMCO).

Cell Culture and Cell Collections
P1, D3, and CK35 ES cell lines were routinely cultured as described [36]. EG1 and EG3 cell lines were derived from GFP-positive PGC sorted from Oct4-GOF-18 PE:GFP transgenic CD1 embryos at 12.5 and 8.5 dpc, respectively, and cultured as described [37]. For differentiation experiments, embryoid bodies were prepared as described [38]. After 10 days, EB were collected in RLT buffer for RNA extraction as described below. Different mammalian cell lines were provided by the Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC) cell culture facility (supplemental online Table 1).

RNA Isolation and RT-PCR Analyses
Total RNA was extracted using the RNeasy mini or micro kit from Qiagen (Hilden, Germany, http://www1.qiagen.com) following the manufacturer's instructions. One microgram of total RNA was treated with RNase-free DNase I (Roche Diagnostics, Basel, Switzerland, http://www.roche-applied-science.com) and then was retrotranscribed using SuperScript II (Invitrogen, Carlsbad, CA, http://www.invitrogen.com) with hexanucleotides as primers, following the manufacturer's instructions. Then, cDNAs were diluted in 80 µl, and 2 µl of cDNA was used for PCR amplification.

For mouse β-actin, Gapdh, Oct4, Vasa, and P450ssc and human β-actin and Oct4 amplification, PCR was carried out with 0.25 µl of Taq polymerase (5 U/µl) (Roche Diagnostics), 2.5 µl of 10x Taq buffer, 25 mM each dNTP, 0.4 pM each primer, and 2 mM MgCl₂ in a total volume of 25 µl. A unique pair of primers was used to amplify both forms of mouse Tex19. Amplifications were performed with the Qiagen Multiplex system following the manufacturer's instructions. For each gene, PCR conditions and fragment size are summarized in supplemental online Table 2.

In Situ Hybridization on Histological Section and WISH Analyses
In situ hybridization (ISH) and WISH were carried out using mouse Oct4 and Tex19 probes of 312 and 487 base pairs (bp), respectively. RNA sense and antisense digoxigenin-labeled probes were synthesized using the Dig RNA Labeling Mix kit (Roche Diagnostics) following the manufacturer's instructions. Embryos or isolated gonad-mesonephros complexes were collected and fixed overnight in 4% paraformaldehyde (PFA; wt/vol) in phosphate-buffered saline (PBS), pH 7.2, at 4°C.

For ISH, sections were prepared as described [39]. Then, they were postfixed for 10 minutes in 4% PFA in PBS at 4°C before the prehybridization step, which was performed for 2 hours at 70°C in the following mixture: 1 x salt (1.95 M NaCl, 0.05 M Tris, pH 7.2, 0.5 M EDTA, pH 8.0, 7.8 g/l Na₂H₂PO₄, 7.1 g/l Na₂HPO₄), 10% dextran sulfate, 1 mg/ml tRNA, 1 x Denhardt's, and 50% deionized formamide. Hybridization was carried out overnight at 70°C with appropriate denatured probes in a humid chamber. Then, sections were washed at 70°C in a solution of 1x standard saline citrate (SSC), 50% formamide, and 0.1% Tween. Detection of digoxigenin (DIG) RNA was performed with an alkaline phosphatase-conjugated anti-DIG antibody (Roche Diagnostics). The WISH experiment was performed as described [40] with a modified washing solution (2x SSC, 50% formamide, 1% SDS).

Immunocytochemistry
Anti-Tex19 mono- and polyclonal antibodies were prepared by the IGBMC facility using the peptide sequences given in supplemental online Table 3. Two hundred eight monoclonal antibody and control ascites were purified using caprylic acid and ammonium sulfate. For immunostaining, all steps were performed at room temperature. Cells were fixed for 15 minutes in 4% PFA in PBS. Then, samples were permeabilized in 0.25% (or 0.5% for mES cells) Triton X-100 in PBS for 10 minutes. For mES cells, a 30-minute step of saturation was added in a solution containing 5% normal goat serum, 0.2% bovine serum albumin (BSA), and 0.05% Triton X-100 in PBS. Primary antibody incubations were carried out for 2 hours with rabbit Flag, 205, or 208 (1/250) or overnight at 4°C for double staining with mouse 208 (1/2,000) and Oct4 (1/300; Santa Cruz Biotechnology Inc., Santa Cruz, CA, http://www.scbt.com) antibodies. Cells were washed three times in 0.1% Triton X-100 in PBS for 10 minutes. Secondary antibody incubations were performed 45 minutes with a fluorescent-conjugated secondary antibody (Cy3-conjugated goat anti-rabbit antibody or goat anti-mouse antibody [Jackson Immunoresearch Laboratories, West Grove, PA, http://www.jacksonimmuno.com] and Alexa488-conjugated goat anti-mouse [Invitrogen]). Then, samples were washed three times in 0.1% Triton X-100 in PBS for 10 minutes, and nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI; 1 µg/ml in PBS). Slides were mounted with AquaPoly/Mount (Polysciences Inc., Warrington, PA, http://www.polysciences.com).

Preimplantation Embryo Whole Mount Immunofluorescence
Embryos were collected by dissecting oviducts or flushing the uterus with PBS. Zona pellucida was retrieved using Thyrode's Acide at pH 2.3. Then, embryos were fixed in 4% PFA in PBS for 4 hours at 4°C. They were thoroughly rinsed with 0.1% Tween 20 in PBS (PBT). Permeabilization and saturation were performed for 1 hour at room temperature with the following blocking solution: 5% normal goat serum, 1% BSA, 0.5% Triton X-100 in PBS. Antibodies were diluted in the blocking solution (purified 208, 1/2,000; Oct4, 1/300; Santa Cruz Biotechnology) and were incubated overnight at 4°C. Three washes of 15 minutes each were performed with PBT. Secondary antibody incubation, washes, and DAPI staining were performed as described above.

	RESULTS

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Identification of Tex19 as a Potential Pluripotent Marker
To identify new genes involved in stem cell self-renewal and pluripotency, we started from the observation that most ES cell pluripotency markers are also detected in PGC and spermatogonia stem cells. Therefore, we began our study with genes described by Wang et al., who identified, by subtractive cloning, 25 genes specifically expressed by spermatogonia cells [25]. Among these 25 genes, 10 were found, by EST database searches, to be expressed in ES cells and genital ridges and were thus selected for further analysis for their PGC expression by RT-PCR and ISH (data not shown). This initial work allowed us to select one gene (Tex19) because of its early embryo, testis, amnion, and genital ridge restricted expression, supported by EST GenBank records and RT-PCR, and HIS 12.5 dpc genital ridge expression (Fig. 1A).

View larger version (55K): [in this window] [in a new window]   Figure 1. Tex19 selection and gene organization. (A): In situ hybridization on mouse 12.5-day post coitum embryos section stained with Oct4 and Tex19. Arrows indicates the specific staining in genital ridges. (B): Genomic organization of the mouse Tex19.1 and Tex19.2 paralog. Both genes are localized on chromosome 11 and are separated by an approximately 27.5-kb intergenic region. They each contain three exons (E1, E2, and E3) and two short introns. The coding sequences are fully carried by the third exon (black boxes). The ATG codons of translation initiations are indicated by arrows. Scale: each graduation is 100 base pairs. Abbreviations: E, exon; kb, kilobase.

View larger version (68K): [in this window] [in a new window]   Figure 2. Multiple alignment of complete Tex19 protein sequences. The alignment is composed of 13 mammal proteins; all proteins are full-length from the N-terminal methionine to the C terminus except the wgs_Spermo sequence, which might be incomplete. Boldface protein names indicate UniProt database identification. Protein names beginning with either wgs or htg represent polypeptide sequences that were deduced from tblastn homology searches in the HTG and WGS genomic sequence divisions of GenBank with q99mv2_mouse as a query. Amino acid clustering was as follows: (W, Y, F), (L, V, I, M), (K, R), (D, E), (T, A, C, S), (H, N), P, G, and Q. Three-level conservation shading is as follows: invariant residues, black background; 100% conserved clusterized residues, dark gray background; 80% conserved clusterized residues, light gray background. Brackets indicate unreported C-terminal residues. Parentheses indicate unreported residues of the variable region. Arrows: domain delineations. Secondary structure predictions (H or L) reliability indices of secondary structure predictions are reported. *, stop codon. Lowercase letters represent residues that have been translated in silico on the 3' of a stop codon. Abbreviations: Atelerix, Atelerix albiventris; Bos, Bos taurus; H, helix; L, loop; Macaca, Macaca mulatta; Pan, Pan troglodytes; Rattus, Rattus norvegicus; Sorex, Sorex araneus; Spermo, Spermophilus tridecemlineatus; Tupaia, Tupaia belangeri.

In the three available primate sequences, we observed a conserved TAG stop codon that ends the coding sequence leading to a protein of 164 amino acids. Thus, primate Tex19 is significantly shorter than the other sequences. In Homo sapiens and Pan troglodytes mRNA sequences, a second TAG stop codon is observed 93 nucleotides 3' to the first one; a third TAG stop codon is detected after another 64 nucleotides, but only in chimpanzee. Strikingly, the translation of the primate sequence 3' of the TAG stop codons completely restores the rest of the protein, whose sequence seems phylogenetically consistent by comparison with other species (Fig. 2). The stop codons that are present in the three primate Tex19 ORF sequences are always TAG, except the last stop codon, which is a TAA. These stop codons were confirmed by sequencing the human TEX19 cDNA (data not shown).

Tex19 Protein Contains Two Well-Conserved and Novel Domains
A multiple alignment was constructed with the 13 collected Tex19 homologs (Fig. 2). All Tex19 proteins shared two highly conserved domains separated by a variable region. The first conserved domain localized on the N-terminal boundary of the protein is 58 residues long (hereafter called the MCP domain) and begins with an invariant MCPPVS motif. A secondary helix-loop-helix structure was predicted in this domain. The second conserved domain (hereafter called the VPTEL domain) begins with an invariant VPTEL motif and is approximately 38 residues long. No structural prediction could be obtained for this latter domain. The amino acid identity percentage of these domains calculated for each pair of sequences ranged between 58% and 100% for the MCP domain and between 61% and 100% for the VPTEL domain (supplemental online Table 5). No functional predictions have been made by the Protein Family database on the Tex19 conserved domains, suggesting that these domains might be distinct from other functionally or structurally characterized domains.

Finally, a slightly conserved third domain can be delineated at the C terminus of Tex19 proteins. This domain is absent in primates (hereafter called the non-primate-specific domain). The amino acid identity percentage, calculated for each pair of nonprimate sequences in this domain, ranged between 33% and 96% (supplemental online Table 5).

Synteny of Tex19 Genes in Human and Mouse Genomes
In both human and mouse, Tex19 genes are linked to CD7, Sectm1, and UTS2R genes (Fig. 3). Human TEX19 and murine Tex19.1 are oriented in the same direction according to the centromere, and both genes are separated from the UTS2R locus by an equivalent length of approximately 18 kb. Thus, considering the synteny between human and mouse genomic regions, the human TEX19 appears more closely related to the murine Tex19.1.

View larger version (27K): [in this window] [in a new window]   Figure 3. Scheme of human and mouse genomic synteny in the vicinity of Tex19 genes. Chromosome nucleotide scales (kilobases) refer to Homo sapiens genome build 36.2 and Mus musculus genome build 36.1 (Map Viewer). The syntenic region contains UTS2R (Urotensin 2 receptor), Tex19 (Testis expressed 19), Sectm1 (Secreted and transmembrane 1), and CD7 (Cluster of differentiation 7). The NM_027622 mRNA has been annotated as a hypothetical protein coding transcript, but we show in this study that it is coding for mouse Tex19.2 protein. Tex19 homologs are in boldface. Small arrows indicate gene orientations. RefSeq database cDNA accession numbers are reported in parentheses. – – –, syntenic genes; · · ·, putative synteny of human Tex19 and murine Tex19.1.

View larger version (39K): [in this window] [in a new window]   Figure 4. Expression of Tex19.1 in various pluripotent cells. For reverse transcription (RT)-polymerase chain reaction (PCR) experiments, β-actin and Gapdh were used as PCR internal control. – corresponds to an RT control performed without reverse transcriptase, and H2O is a PCR control where cDNA was replaced by water. (A): Expression profile of Tex19.1 and Tex19.2 analyzed by RT-PCR in various adult tissues. (B): Expression profile of Tex19 in human Pl and T. (C): RT-PCR on NFo, 1C, 2C, and 8/16C embryos and on B. T was a positive control. (D): Analysis of Tex19.1 and Tex19.2 expression by RT-PCR in nd and d mouse ES cells (ES P1 and ES D3) and mouse EG cells (EG1 and EG3). EG cells were established at different stages of development: 12.5 days post coitum (dpc) for EG1 (EG1(12.5)) and 8.5 dpc for EG3 (EG3(8.5)). Oct4 was used as a control marker for pluripotency. (E): Expression analysis of Tex19.1 and Tex19.2 in P19 and F9 mouse embryonic carcinoma cell lines and mouse fibroblast 3T3 cells. T was a positive control. (F): Confocal microscopy of whole mount immunofluorescence on 2C stage embryo (top line), eight-cell stage embryo (middle line), and on B (bottom line) stained with Oct-4 (1/300; Santa Cruz Biotechnology), Tex19.1 (purified monoclonal 208; 1/2,000) antibodies, and DAPI. The eight-cell embryo picture shows a maximal projection of 10-µm optical sections. The last image of each line corresponds to transmitted light picture of the embryo. (G): RT-PCR analysis of human tex19 (hTEX19) expression in nd and d human ES cells (VUB01 cell line). cDNA of J was used as a positive control. Abbreviations: 1C, 1-cell; 2C, 2-cell; 3T3, NIH3T3; 8/16C, 8–16-cell; B, blastocyst (D); B, brain (A); BM, bone marrow; d, differentiated; DAPI, 4,6-diamidino-2-phenylindole; EG, embryonic germ; ES, embryonic stem; H, heart; I, intestine; J, Jurkat cells; K, kidney; L, liver; Lu, lung; M, muscle; nd, undifferentiated; NFo, unfertilized eggs; O, ovary; Pl, placenta; S, spleen; St, stomach; T, testis; Thy, thymus; Ut, uterus.

Tex19.1 Transcripts Are Detected in Primordial Germ Cells
Our initial screening showed that Tex19 expression is limited to 12.5-dpc genital ridges. To further investigate the expression of Tex19.1 and Tex19.2 throughout embryonic development, we performed RT-PCR and WISH analyses. By RT-PCR, Tex19.1 transcripts were detected in postimplantation embryos at 7.5, 8.5, and 9.5 dpc (data not shown). By WISH, at 9.5 dpc, Tex19.1 expression was similar to that of Oct4, with a signal restricted to the developing hindgut, suggesting that Tex19.1 is specifically expressed in migrating germ cells at this stage (Fig. 5A). Then, by RT-PCR, we detected Tex19.1 transcripts in male and female gonads at 13.5 dpc (Fig. 5B). In male gonads, its expression remained constant until the adult stage and, by WISH, was shown to be similar to that of Oct4 and to stain testis cords (Fig. 5C). In female gonads, Tex19.1 expression was detected at 13.5 dpc and decreased between 14.5 dpc and 16.5 dpc (Fig. 5B) similar to Oct4 expression [41]. To confirm this, we performed WISH on female gonad-mesonephros complexes from 12.5 dpc to 15.5 dpc and compared Tex19.1 and Oct4 transcript localization (Fig. 5D). At 12.5 dpc, Tex19.1 staining was located in clusters of cells all over the gonad. Then, Tex19.1 expression appeared to decrease at the rostral end of the ovary, with expression at 14.5 dpc restricted to some caudal clusters of cells. At 15.5 dpc, Tex19.1 was no longer detected. After birth, Tex19.1 expression decreased and became almost undetectable in the adult ovary (Fig. 5B).

View larger version (46K): [in this window] [in a new window]   Figure 5. Tex19 expression during mouse development. (A): Whole mount in situ hybridization on embryos at 9.5 dpc stained with Oct4 (Aa) and Tex19.1 (Ab) antisense probes. (B): Analysis of Tex19.1 and Tex19.2 expression by reverse transcription (RT)-polymerase chain reaction (PCR) on male and female gonad-mesonephros complexes at different stages of development, from 13.5 to 15.5 dpc, and on isolated gonads at 16.5 and 18.5 dpc, at Po, 6d, and in A. Oct4 and Vasa were used as germ cell markers. β-Actin corresponds to the PCR internal control. For these experiments, – corresponds to an RT control performed without reverse transcriptase, and H2O is a PCR control where cDNA was replaced by water. (C, D): Whole mount in situ hybridization on male (C) and female (D) gonad-mesonephros complexes from 12.5–14.5 dpc and 12.5–15.5 dpc, respectively. Samples were stained with Oct4 and Tex19.1 antisense probes. Abbreviations: A, adult; 6d, 6 days after birth; dpc, days post coitum; Po, birth.

Tex 19.1 Is Specifically Expressed in Germ Cells, Whereas Tex19.2 Is Detected in the Somatic Lineages of the Male Gonad
To confirm the germ cell expression of both Tex19 genes, we took advantage of the Oct4-GOF-18 PE:GFP transgenic line in which green fluorescent protein expression is restricted to PGC [34]. Gonad-mesonephros complexes of 12.5 dpc and gonads of 18.5 dpc transgenic embryos were dissected and dissociated. Then, male and female GFP-positive and -negative cells were sorted by FACS, and Tex19 expression was assessed by RT-PCR. For each stage, Tex19.1 transcripts were detected only in GFP-positive cells and not in negative cells (Fig. 6A, 6B). A slight Tex19.1 expression was seen in female GFP-negative cells at 12.5 dpc; however, this signal was also observed for Oct4 and Vasa, suggesting some slight contamination by GFP-positive cells (Fig. 6A). Interestingly, in 18.5-dpc male gonads, Tex19.2 was detected mainly in GFP-negative cells, and only weak amplification was observed in GFP-positive cells (Fig. 6B). At the same stages of development, in female gonads, Tex19.2 was detected only in GFP-positive cells. These results indicate that Tex19.1 is specifically expressed in male and female germ cells, whereas Tex19.2 is detected in female germ cells, but it is present mainly in somatic cells in male gonads.

View larger version (35K): [in this window] [in a new window]   Figure 6. Analysis of Tex19.1 and Tex19.2 in germ cells and somatic cells. m and f gonad-mesonephros complexes at 12.5 days post coitum (dpc) (A) and 18.5 dpc (B) of transgenic embryos were dissociated, and cellular suspension was sorted by fluorescence-activated cell sorting using green fluorescent protein (GFP) expression. Then, expression profile of Tex19.1 and Tex19.2 was analyzed by reverse transcription (RT)-polymerase chain reaction (PCR) on m (m+) and f (f+) GFP-positive cells and m (m–) and f (f–) GFP-negative cells. m15 or adult T was used as positive control. For these experiments, – corresponds to an RT control performed without reverse transcriptase, and H2O is a PCR control where cDNA was replaced by water. Oct4 and Vasa was used as marker of germ cells, and P450ssc was used as a marker of Leydig cells. (C): Two pregnant f at 10.5 dpc were injected with busulfan (B1 and B2) or DMSO (D1 and D2), and embryos were dissected at 14.5 dpc. Then, m and f gonad-mesonephros complexes were taken and analyzed by RT-PCR for Tex19.1 expression. H2O is a PCR control. (D): Whole mount in situ hybridization on m gonad-mesonephros complexes of DMSO control (Da, Dc) and busulfan-treated (Db, Dd) embryos were stained with Oct4 (Da, Db) and Tex19.1 (Dc, Dd) antisense probes. Abbreviations: DMSO, dimethyl sulfoxide; f, female; m, male; m15, gonad-mesonephros complexes of m embryos at 15.5 days post coitum; T, testis.

Tex19.1 and Tex19.2 Are Nuclear Proteins
Antibodies specific to Tex19.1, Tex19.2, or both (supplemental online Table 3) were initially tested on NIH3T3 cells transfected with a tagged expression vector. These experiments allowed us to confirm the specificity of the antibodies but also to determine the main nuclear localization of both proteins (Fig. 7A). A slight Tex 19.2 staining was also detected in the cytoplasm of transfected cells (Fig. 7A). This result was confirmed by the anti-Flag antibody (Fig. 7A). Then, the predominant nuclear localization of endogenous Tex19.1 was confirmed, by double immunofluorescence staining of Oct4 and Tex19.1, on preimplantation embryos (Fig. 4F) and on ES cells (Fig. 7B).

View larger version (48K): [in this window] [in a new window]   Figure 7. Localization of TEX19.1 and TEX19.2 proteins. (A): Immunolocalization of TEX19.1 and TEX19.2 proteins on mouse NIH3T3-transfected cells with Tex19.1 expression vector (TEX19.1) or Tex19.2 expression vector (TEX19.2). 205 and 208 antibodies are raised against TEX19.2 and TEX19.1 proteins respectively, 209 antibody is able to recognize both forms. FLAG was used as a positive control. Negative control corresponds to untransfected cells stained with F and 205 or 208 antibodies. (B): Immunolocalization of TEX19.1 and OCT4 proteins in murine CK35 embryonic stem cells using purified 208 monoclonal antibody and polyclonal anti-OCT4. Control ascites (without specific antibody) was used as negative control. Abbreviations: DAPI, 4,6-diamidino-2-phenylindole; F, anti-Flag; FLAG, anti-Flag.

	Discussion

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Tex19 was initially cloned through a subtractive cloning strategy using mRNA from purified spermatogonia stem cells versus mRNA from 11 adult tissues [25]. At that time, no human ortholog could be found. An initial bioinformatic analysis allowed us to identify a human homolog, to show that the gene is duplicated in mouse, rat, and cow and that Tex19s are most probably specific to mammals. Indeed, we found 13 Tex19 homologs, all of them from mammalian species. Mammalian-specific proteins are notably described for pluripotency-associated genes, such as Gdf3 or Dppa3 [42]. Apart from mammals, Nanog is found only in chicken, but its expression is restricted to primordial germ cells, and no transcripts have been detected in epiblast, suggesting that it is not involved in pluripotency in chicken [42]. Recently, a new eutherian gene family encoding proteins characterized by the presence of an RNA-binding KH domain has been described. This family includes several members such as Khdc1/dppa5/ecat1/ooep, which are specifically expressed in oocytes and/or embryonic stem cells [43]. All these results suggest that most pluripotency genes are specific to mammals and thus reinforce our hypothesis that Tex19.1 could be a new factor involved in pluripotency.

In mouse, we showed that the Tex19.1 and Tex19.2 genes are located on chromosome 11, separated by an short intergenic region, and transcribed in opposite directions. As for the mouse, the duplicated rat Tex19.2 is located on the same chromosome as Tex19.1; however, sequencing of the Rattus norvegicus genomic region is not completed, impairing its precise position and orientation. Concerning the Bos taurus proteins, we also found two forms on the same contig where sequencing is in progress, and at the present time we cannot relate these proteins to the rodent Tex19.1 or Tex19.2. In human, the synteny of Tex19 genes suggests that human TEX19 is the ortholog of the murine Tex19.1.

By aligning Tex19 proteins, two highly conserved domains could be identified, an N-terminal domain (named the MCP domain) and a domain (named the VPTEL domain) located in the middle part of the protein. These two domains are present in all Tex19 sequences studied, even in the primates, despite the presence of a premature stop codon. Indeed, primate Tex19 protein sequences are half as long as other sequences, suggesting that the C-terminal non-primate-specific domain might not be essential to Tex19 function. Until now, we could not define the function of these two domains in silico since they do not present any similarity with any known domains. The predominant nuclear localization of the murine Tex19.1 and Tex19.2 proteins suggests that they could correspond to transcription factors or chromatin architecture proteins.

The Tex19.1 expression profile parallels that of Oct4, which led us to support the hypothesis that it could be involved in the self-renewal or pluripotency of stem cells and in the totipotency of early embryos. The POU domain transcription factor Oct4 is one of the main markers essential for establishing and maintaining pluripotency of the ICM [14, 18]. Oct4 is expressed at early stages in the unfertilized oocyte, and then it is detected in the ICM, epiblast, and primordial germ cells [15]. Tex19.1 transcripts are also found in the unfertilized oocyte, zygote, and early embryo, implying that it is maternally inherited, as are Sox2 and Esg1, other factors essential for pluripotency [8, 44]. The detection of Tex19.1 transcripts and protein in the ICM of the blastocyst supports a potential role for Tex19 in pluripotency. Indeed, the ICM cells are pluripotent, give rise to the epiblast, and can be derived as ES cells, and so far, all pluripotency transcription factors (Oct4, Sox2, Nanog, Esg1, and FoxD3) have been shown to be expressed in ICM [8, 9, 11, 13, 15, 44]. It is noteworthy that Tex19.1 protein was found in few cells outside the ICM that were also positive for Oct4. Oct4 is first present in all cells of the embryo and later is downregulated in the trophectoderm, but it shows a transient upregulation in ICM cells differentiating into primitive endoderm [45], so colocalization of Tex19.1 in the resting Oct4-expressing cells suggests that both proteins may share a common regulatory pathway. Tex19.1 transcripts are specifically detected in male and female germ cells. Tex19.1 is widely expressed in male gonad, and its expression continues up to adult testis. In female germ cells, Tex19.1 is downregulated in a rostro-caudal wave between 14.5 and 16.5 dpc, as observed for Oct4, Sox2, Esg1, Dppa2, and Dppa4 [44, 46]. This downregulation is known to be due to the entry into meiosis of female germ cells in a rostro-caudal wave [41, 47].

In addition, Tex19.1 is expressed by different pluripotent cells (ES, EG, P19, and F9) and decreases after ES or EG cell differentiation by LIF withdrawal. Our finding in mouse ES cells is consistent with results from hES cells, since human TEX19 transcripts are detected in hES cells and expression decreased after hES cell differentiation.

One difference between Tex19.1 and Oct4 expression is seen in the placenta, where only Tex19.1 transcripts are detected. It is worth noting that an extraembryonic expression of other pluripotent markers (FoxD3 and Esg1) has also been described in trophectoderm [11, 44].

The expression of Tex19.2 differs from that of Tex19.1 in that it occurs later during development (14.5 dpc), and it is limited to somatic cells of the male gonad. This suggests that Tex19.2 could have a different function during mouse development and testicular physiology.

The transcriptional regulation of major pluripotency factors forms an interconnected autoregulatory network that regulates many downstream target genes to maintain ES cell pluripotency and self-renewal [48]. Sox2 acts cooperatively with Oct4 on promoters and activates the transcription of several genes, such as Fgf4 [49], Zfp206 [17], and Nanog [36, 50]. Oct4, Sox2, and Nanog are also bound to their own promoters to limit their expression levels [48]. No response element for Oct4 or Sox2 has been found on Tex19.1 promoter, but a Nanog-binding site was identified in the 3' proximal region of Tex19 (5,858 bp downstream) [21].

The presence of the Tex19.1 protein in the nuclei of ICM and ES cells suggests that it could be a new transcription factor regulating target genes responsible for pluripotency or an actor controlling the chromatin architecture of the pluripotent nucleus. Further investigation will be required to determine the exact function of Tex19.1 in pluripotent cells.

	DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST

Top Abstract Introduction Materials and Methods Results Discussion Disclosure of Potential... Acknowledgments References

 
The authors indicate no potential conflicts of interest.

	ACKNOWLEDGMENTS

Top Abstract Introduction Materials and Methods Results Discussion Disclosure of Potential... Acknowledgments References

 
We are grateful to Drs. Irwin Davidson, Manuel Mark, and Olivier Poch for critical reading of the manuscript. We thank Andree Dierich and Chantal Kress for supplying P1, D3, and CK35 ES cell lines, Drs. Schöler and Matsui for providing Oct4 DPE:GFP transgenic mouse, and Prs. Sermon and Tournaye for providing human ARN from hES and adult testis. We are grateful to common facilities of IGBMC, particularly Oula Mustapha and Gilles Duval for monoclonal and polyclonal antibody production, Jean Marie Garnier for technical advice, and Manuel Marks for help with in situ hybridization and the cell culture service. We also thank Valerie Skory for technical assistance. This work was supported by the French Centre National de la Recherche Scientifique, Institut National de la Santé et de la Recherche Médicale (INSERM) (Grant Avenir), the Ministère de l'Education Nationale, the l'Enseignement Supérieur et de la Recherche, the Louis Pasteur University of Strasbourg, and ACI IMPBIO 2004-78, project SeBIG. S.K. was supported by INSERM, and E.K. is a recipient of a grant from the French Ministère de la Recherche. S.K. and E.K. contributed equally to this work.

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