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Regular Article |
a Glycobiology Program, The Burnham Institute, La Jolla, California 92037
b Department of Pediatrics and Molecular and Integrative Physiology, University of Kansas Medical Center, Kansas City, Kansas 66160-7338
c Basic Mouse Cancer Genetics Program, National Cancer Institute-Frederick, Frederick, Maryland 21702-1201
d Institute of Organ Transplants, Reconstructive Medicine and Tissue Engineering, Shinshu University Graduate School of Medicine, Matsumoto, Japan
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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early development, embryo, female reproductive tract, implantation, placenta, pregnancy, trophoblast, uterus
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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More than 90% of human trophinin protein consists of decapeptide repeats unique to this protein. Within these repeats are hydrophobic domains, which allow this protein to be integrated in the plasma membrane. The decapeptide repeats are considered to be key structural features for trophinin-mediated homophilic cell adhesion on the cell surface. Tastin and bystin are cytoplasmic proteins required for trophinin to function as an efficient cell adhesion molecule by organizing trophinin in plasma membranes. The human trophinin gene maps to the short arm of the X chromosome [6].
At the initial stage of implantation, the previously nonadhesive apical surfaces of the trophectoderm of blastocysts and the endometrial surface epithelia become adhesive to each other. The embryo-uterus adhesion not only supports the physical junction between the embryo and the uterus but also generates the intracellular signal cascades necessary for functional changes in embryonic and maternal cells. To our knowledge, the mechanisms underlying this initial attachment and subsequent invasion of trophoblasts into maternal tissues have been analyzed mainly in rodents [7–11]. These studies established the existence of cross-talk between blastocysts and the luminal endometrium during apposition [12], and they identified molecules mediating the initial attachment in mouse blastocyst implantation [12–14]. What we learned from these studies is that a molecule involved in embryo implantation is tightly regulated both spatially and temporally. For example, heparin-binding epidermal growth factor (EGF)-like growth factor (HB-EGF) is expressed exclusively in the endometrial luminal epithelium surrounding an implanting blastocyst [14]. The most likely receptor for HB-EGF in the mouse blastocyst is the neuregulin receptor ErbB4, one of the EGF receptors, which is strongly expressed on the apical surface of the mouse blastocyst at the time of implantation [12].
Our previous studies show that, in humans and primates, trophinin is expressed in cells involved with implantation [1, 3, 5]. In the monkey blastocyst, trophinin is strongly expressed in the trophectoderm cells at the embryonic pole where the blastocyst adheres to the uterine wall. In the human endometrium, strong trophinin expression was detected in the restricted region of luminal epithelium at the early secretory phase. The restricted, but strong, expression of trophinin in human endometrium during the "implantation window" suggests a specific role of trophinin during implantation in humans. In the mouse, ovarian hormones regulate trophinin expression by the uterus, and trophinin expression coincides with the timing of implantation [15]. However, trophinin was not particularly restricted to cells involved with implantation in the mouse. Trophinin was found in the entire uterine luminal and glandular epithelia, regardless of the presence or absence of implanting blastocysts.
The functions of many conserved genes in eukaryotes are exchangeable between species, as exemplified by the proteins involved in the cell cycle [16]. However, the molecules involved with implantation and placentation diverge significantly among species [7, 17]. Nonetheless, in the present study, due to the difficulties in analyzing human embryo implantation, the mouse has been used as a model for the analysis of trophinin function. Therefore, we took a targeted gene-disruption approach for testing the in vivo function of trophinin in the mouse. This paper describes the mouse trophinin gene with respect to chromosomal location, genomic organization, expression pattern, and loss of function.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A genomic clone of human trophinin was isolated from a human genomic P1 plasmid library by polymerase chain reaction (PCR) screening using the upstream forward oligonucleotide primer 5'-ATGGATATCGACTGCCTAAC-3' and the downstream reverse primer 5'-GAAGCTAATACTAGCACGAT-3'. A genomic library of 129/SvJ mouse strain constructed in the 
FIX II vector (Stratagene, La Jolla, CA) was plated on Escherichia coli XL-1-Blue MRX host cells and screened by plaque hybridization using a 2.5-kilobase (kb) fragment of the entire coding region of human trophinin cDNA as a probe. Phage lifts were prepared with Magna NT nylon filters (Micron Separations, Westboro, MA). The cDNA was labeled with [
-32P]dCTP using the Prime-It II kit (Stratagene). Filters were prehybridized at 35°C for 5 h in 6x SSPE (single strength: 150 mM NaCl, 10 mM NaH2PO4, and 1 mM Na/EDTA) containing 50% (w/v) formamide, 5x Denhardt solution, 0.1% (w/v) SDS, and 0.2 mg/ml of denatured salmon sperm DNA and then hybridized in the same solution containing 32P-labeled cDNA probe at 35°C for 20 h. The filters were washed 3 times at 35°C for 10 min in 6x SSPE containing 0.1% SDS and subjected to autoradiography. The DNA purified from the positive clone was digested with restriction enzymes, subcloned into the pBluescript II KS (+) plasmid (Stratagene), and sequenced.
Interspecific Mouse Backcross Mapping
Interspecific backcross progeny were generated by mating (C57BL/6J x Mus spretus) F1 females and C57BL/6J males as described previously [18]. A total of 205 N2 mice were used to map the trophinin locus, Tnn (see Results for details). The DNA isolation, restriction enzyme digestion, agarose gel electrophoresis, Southern blot transfer, and hybridization were performed essentially as described previously [19]. The probe, the 5.1-kb BamHI fragment of mouse genomic DNA, was labeled with [-32P]dCTP using a nick translation labeling kit (Boehringer Mannheim, Indianapolis, IN); washing was done to a final stringency of 1.0x SSC (0.15 M sodium chloride and 0.015 M sodium citrate) and 0.1% SDS at 65°C. A fragment of 10.5 kb was detected in HincII-digested C57BL/6J DNA, and a fragment of 6.3 kb was detected in HincII-digested M. spretus DNA. The presence or absence of the 6.3-kb HincII M. spretus-specific fragment was followed in backcross mice.
Descriptions of the probes and restriction fragment length polymorphisms (RFLPs) for the loci linked to Tnn, including Bruton agammaglobulinemia tyrosine kinase (Btk) and myelin proteolipid protein (Plp), have been reported previously [20]. Recombination distances were calculated as described by Green [21] using the computer program Spretus Madness. Gene order was determined by minimizing the number of recombination events required to explain the allelic distribution patterns.
Southern Blot Analysis of Genomic DNA
Southern blot analysis was performed as described by Ausubel et al. [22]. Briefly, each genomic DNA prepared from human placenta and mouse liver was digested with restriction enzymes, electrophoresed in a 0.7% (w/v) agarose gel, and transferred onto a Nytran Plus membrane (Schleicher & Schunell, Keene, NH). The DNAs were fixed under ultraviolet light (1200 µJ). The hybridization was performed as described above.
Antibodies
Rabbit antitrophinin antibody (no. 1845) was raised against glutathione-S-transferase fused to a peptide (mouse 553) or residues 976–1046 of mouse trophinin. A rabbit polyclonal antibody against human ribosomal protein S4X, which cross-reacts with mouse S4, was obtained from Riken (Tsukuba, Japan) [23]. A polyclonal antibody raised against a synthetic peptide (TSTDFSGGLNHNADFN) for mouse trophinin and a monoclonal antibody for human trophinin were described previously [5, 15].
Western Blot Analysis
Mouse tissues were homogenized in 3 volumes of 1x Laemmli SDS-PAGE sample buffer and boiled for 10 min. Proteins were resolved in a 7.5% SDS-PAGE gel and transferred to an Immobilon membrane (Millipore, Bedford, MA). The blots were blocked in PBS containing 5% skim milk and 0.1% Tween 20 for 1 h and then incubated with primary antibodies diluted with PBS containing 0.5% skim milk for 1 h at room temperature. After washing with PBS containing 0.5% Tween 20, the blots were incubated with horse radish peroxidase-conjugated anti-rabbit immunoglobulin G (Bio-Rad, Hercules, CA) diluted with PBS containing 0.5% skim milk for 30 min. After washing with PBS containing 0.5% Tween 20, immunoreactive bands were detected by using an enhanced chemiluminescence kit (Amersham, Arlington, IL) and exposing the filter to an x-ray film. Western blot analysis of human trophinin was carried out as described previously [5].
Immunohistochemistry of Mouse Tissues and Embryos
Frozen uteri were sectioned (thickness, 12 µm) and mounted onto poly-L-lysine-coated glass slides, fixed in 4% (w/v) paraformaldehyde in PBS for 10 min, and washed in PBS. Preimplantation embryos were recovered from the uteri, placed on poly-L-lysine-coated slide glass, cytospinned for 1 min, and fixed with 4% paraformaldehyde in PBS for 10 min. Immunostainings of uteri or embryos were performed by incubating the sections with protein A purified antibody diluted to 1:2000 (protein, 2 µg/ml) for 1 h at room temperature. A Zymed-Histostain-SP kit for rabbit primary antibodies and an aminoethyl carbazole single solution of peroxidase substrate (Zymed Laboratories, San Francisco, CA) were used for detecting the sites of immunoreactive proteins, which were seen as red deposits. Sections were lightly counterstained with hematoxylin.
Targeted Disruption of the Mouse Trophinin Gene
A 0.8-kb PstI-BamHI fragment including the translation initiation site was replaced with the PGK-neor cassette, in which the neomycin resistance gene was driven by the phosphoglycerate kinase promoter, for positive selection. The HSV-TK cassette including the herpes simplex thymidine kinase gene was ligated at the 5' end of the targeting vector for negative selection. Mouse embryonic stem (ES) cells D3 (sex chromosomes, XY) derived from 129/SvJ [24] were cultured according to the method of Narisawa et al. [25]. The linearized vector (20 µg) was electroporated (400 V, 250 µF) into 3 x 107 D3 cells and selected with 150 µg/ml of geneticin (Gibco BRL, Gaithersburg, MD) and 2 µM ganciclovir for 2 days, followed by G418 selection for 9 days. Homologous recombinant ES clones confirmed by Southern blot analysis were injected into C57BL blastocysts and transferred to foster mothers to generate chimeric mice. Chimeras were mated with NIH black Swiss mice to test for germ-line transmission or with the 129/SvJ and C57BL/6 strains to obtain lines carrying mutated trophinin alleles. Mice were housed in the animal care facility at the Burnham Institute according to NIH and institutional guidelines for the care and use of laboratory animals.
Genotyping of Mice and Blastocysts
Genotypings of mice were carried out by PCR using genomic DNA isolated during tail biopsies. Genotypings of blastocysts flushed from the uterus were carried out using DexPat (Pan-Vera, Madison, WI) as described previously [26]. Primers for the neo and SRY genes were also described previously [26, 27]. The PCR for the trophinin gene was performed as follows: Primers used for the first PCR were 5'-TAAGCTATCAGAGATGAAGT-3' (forward) and 5'-GCTAATACCAGCACCATCAC-3' (reverse), and those used for the nested PCR were 5'-CAGTTGAGATGGACATTCAG-3' (forward) and 5'-TTGGTTGAAGAGGACATTAG-3' (reverse). Amplification reactions were carried out in a Thermal Cycler (model 2400, Perkin Elmer, Foster City, CA) by denaturation at 94°C for 10 min followed by 35 cycles of denaturation at 94°C for 30 sec, annealing at 58°C for 30 sec, extension at 72°C for 45 sec, and further extension at 72°C for 7 min after the 35 cycles. Reaction products were separated in an agarose gel and stained with ethidium bromide. The DNA bands were visualized under ultraviolet light and photographed.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The chromosomal location of mouse trophinin (locus designation, Tnn) was determined by interspecific backcross analysis using progeny derived from matings of ([C57BL/6J x M. spretus] F1 x C57BL/6J) mice. This interspecific backcross mapping panel has been typed for more than 2000 loci that are well distributed among autosomes as well as the X chromosome [18]. The C57BL/6J and M. spretus DNAs were digested with several enzymes and analyzed by Southern blot hybridization for informative RFLPs using a mouse genomic DNA probe. The 6.3-kb HincII M. spretus RFLP (see Materials and Methods) was used to follow segregation of the Tnn locus in backcross mice. The mapping results indicated that Tnn is located on the distal region of the mouse X chromosome linked to Btk and Plp. Although 92 mice were analyzed for every marker and are shown in the segregation analysis (Fig. 1), up to 88 mice were typed for some pairs of markers. Each locus was analyzed in pairwise combinations for recombination frequencies using the additional data. The ratios of the total number of mice exhibiting recombinant chromosomes to the total number of mice analyzed for each pair of loci and the most likely gene order are centromere, Btk, 3:92, Plp, 7:89, and Tnn. The recombination frequencies (expressed as genetic distances in centimorgans [mean ± SEM]) are Btk, 3.3 ± 1.9 cM, Plp, 7.9 ± 2.9 cM, and Tnn.
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We have compared our interspecific map of the X chromosome with a composite mouse linkage map that reports the location of many uncloned mouse mutations (provided from Mouse Genome Database, The Jackson Laboratory, Bar Harbor, ME). Tnn mapped to a region of the composite map that lacks mouse mutations with a phenotype that might be expected for an alteration in this locus (data not shown).
The distal region of the mouse X chromosome is homologous to both the long and short arms of the human X chromosome (summarized in Fig. 1). In particular, the region just proximal of the pseudoautosomal segment is homologous to the short arm. Tnn likely resides in this interval, which maps to human Xp11.21–22 as reported previously [6].
Genomic Organization of Human and Mouse Trophinin Genes
Southern blot analyses of genomic DNAs from the human and mouse showed a single band in each restriction digest (Fig. 2, A and B), suggesting that genomic regions encoding trophinin exist as a single copy in each genome. These results also exclude the possible existence of pseudogene.
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We sequenced a part of the P1 clone containing the human trophinin gene and compared the genomic sequence with the cDNA sequences for trophinin. The upstream region of trophinin matches a cDNA encoding a putative protein (KIAA1114) expressed in the human brain [28]. Thus, the previously reported trophinin sequence [1] is encoded by the 12th to the 16th exons of the gene encoding KIAA1114 (Fig. 3A).
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We isolated the mouse trophinin gene from 129/SvJ mouse genomic libraries (see Materials and Methods) and compared the genomic sequence with the human trophinin gene. Reverse transcription (RT)-PCR of mouse oocytes also identified exons encoding an upstream region of the mouse trophinin gene (unpublished data; GenBank accession no. AB017108) and revealed an open reading frame, which is homologous to human KIAA1114 in the mouse (Fig. 3B).
Human and Mouse Trophinin Proteins Expressed In Vivo
When we originally identified human trophinin based on its cell adhesion activity, we determined that the cDNA encodes a 69-kDa protein composed of 749 amino acid residues [1]. Recently, Kikuno et al. [28] reported a nucleotide and predicted polypeptide sequence named KIAA1114, which is expressed in human brain. It appears that trophinin is a part of the KIAA1114 protein (Fig. 3A). We examined if this 138-kDa protein is expressed in HT-H cells, the cell line used for the construction of a cDNA library [1]. Western blot analysis of a HT-H lysate using monoclonal anti-human trophinin antibody [5] showed a single band corresponding to 69 kDa (Fig. 4A). No band was detected at 138 kDa (Fig. 3A).
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In the mouse, 6.5-kb trophinin transcripts were detected in the uteri at implantation stages, and we found that mouse trophinin expressed in the uterus is 110 kDa [15]. The mouse brain expresses 6.5-kb trophinin transcripts [15]. Presumably, this transcript encodes a KIAA1114 homologue, a 196-kDa protein, in the mouse. Western blot analysis of adult mouse brain showed a single band at 110 kDa (Fig. 4B). These results suggest that, in both the human and mouse, trophinin proteins are produced by translation from the downstream ATG codon (Fig. 3).
Predicted Structure of Mouse Trophinin Protein
The mouse trophinin protein is predicted to consist of 2 regions, an amino-terminal region (residues 1–50), and a tandem decapeptide repeat region (residues 51–1160) (Fig. 5A). The TMpred program [29] predicts this molecule to be a transmembrane protein with the amino-terminal region in the cytoplasm, a topology similar to that of human trophinin [1]. Sequence comparison of mouse and human trophinins show two regions highly homologous to each other. One is the amino-terminal cytoplasmic region (identities, 83%; positives, 89%) (Fig. 5B). Within this region, three serine and threonine residues that are potential phosphorylation sites by protein kinases are predicted (the residues are indicated in bold and underlined/bold in Fig. 5B). Two of these potential phosphorylation sites are conserved between the mouse and human (shown by arrows in Fig. 5B). Another region of high homology is the third hydrophilic domain within the decapeptide repeats, designated 553 (identities, 82%; positives, 84%) (Fig. 5, A and C). This domain of mouse trophinin may be extracellular, as with human trophinin.
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Expression of Trophinin in Pre- and Peri-Implantation Embryos
To determine the expression of trophinin in preimplantation-stage mouse embryos, RT-PCR followed by Southern blot analysis was performed. Trophinin transcripts were detected in oocytes and fertilized eggs, were not detectable in the 2- to 8-cell-stage eggs, and were detected at the morula and blastocyst stages (Fig. 6). This expression pattern of trophinin in embryos resembles that seen in mouse uterus during normal pregnancy [15].
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Immunohistochemistry detected no trophinins in fertilized eggs at the 1-cell stage or from the 2- to 8-cell stages recovered from the oviduct from 1.5 and 2.5 days postcoitus (dpc), respectively (data not shown). Morulae and blastocysts recovered from uteri from 3.5 and 4.5 dpc were positively stained with the antibody (Fig. 7, A–D). The morula-stage eggs encapsulated in the zona pellucida (Fig. 7A) and blastocyst just after hatching (Fig. 7B) gave a punctate staining pattern; immunostaining of hatched and expanded blastocysts was found in both inner cell mass and trophectoderm (Fig. 7C). Immunohistochemistry with fully expanded blastocysts showed no positive reactions (Fig. 7D).
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In 3.5 dpc (Fig. 7E) and 4.5 dpc (Fig. 7F) uterus, trophinin proteins are strongly expressed in both luminal and glandular epithelia. The blastocysts are, however, negative for trophinin (labeled bl in Fig. 7, E and F). These findings indicate that trophinin proteins appear in the preimplantation embryo with its peak at the morula-blastocyst stage before implantation.
In 5.5 dpc uteri, trophinin proteins are found in the epiblast of implanted embryo (shown by an arrow and labeled ep in Fig. 7G). At this stage, endometrial luminal epithelium surrounding the embryos is positive for trophinin, whereas uterine epithelia distant from the embryo are negative for trophinin (Fig. 7G). Trophinin expression in the epiblast is transient, because epithelia derived from the epiblast and surrounded proamniotic cells are negative for trophinin at 6.5 dpc (data not shown) and 7.5 dpc (Fig. 7H).
Production and Characterization of Trophinin-Deficient Mutant Mice
To determine the in vivo role of trophinin in the mouse, the trophinin gene was disrupted by homologous recombination (Fig. 8A). The targeting vector was transfected into ES cells derived from 129/SvJ strain [24]. Because of the unusual translation of trophinin from the downstream AUG codon (Fig. 3), we disrupted the trophinin gene, including the 14th in-frame AUG codon in the full-length trophinin mRNA (Fig. 8A). Southern blot analysis (Fig. 8B) identified several independent ES clones resulting from homologous recombination. Because the ES D3 line has an XY karyotype, the homologous recombinant should be trophinin null, which was confirmed by RT-PCR (data not shown). No morphological or proliferative abnormalities were observed in these ES clones under normal cultural conditions.
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Trophinin-null ES clones were injected into C57BL/6 blastocysts. Chimeric males obtained were backcrossed with wild-type NIH black Swiss female mice. A cross between heterozygous females and wild-type males yielded trophinin-null male pups (Table 1 and Fig. 8C), which grew normally. No significant differences were seen in the size or weight of the null mutants compared to their wild-type littermates. Heterozygous females were mated with trophinin-null males to generate trophinin-null females. Crosses between trophinin-null females and males resulted in apparently normal pups, indicating no maternal defect resulting from this mutation. No disorders were detected in elderly mutant mice, suggesting no long-term degenerative defects. In summary, the mutant phenotype was not demonstrated under these experimental conditions.
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When the same chimeras were backcrossed with wild-type 129/SvJ female mice, production of pups was hampered. Wild-type 129/SvJ gave an average of 6 pups/litter, but the average litter size of these crosses was 2.7 pups/litter (n = 8 litters). When the resultant G1 heterozygous females were mated with wild-type males, G2 heterozygous females and null males were born (Table 1). However, when G2 heterozygous females were further crossed with wild-type males, the numbers of G3 null males decreased (Table 1).
Genotyping of preimplantation-stage blastocysts recovered from 3.5 dpc uteri showed the existence of trophinin-null blastocysts, as expected from the Mendel law (Table 1). No abnormal morphologies were revealed in trophinin-null blastocysts, indicating that embryonic lethality did not occur before implantation. Genotyping of E (embryonic day) 8.5 embryos revealed reduction of trophinin-null embryos, suggesting that partial lethality occurred between E3.5 and E8.5 (Table 1). Although a lethality of trophinin-null epiblast in the 5.5 dpc epiblast was suspected, characterization of trophinin-null epiblast was not accomplished in this study due to incomplete penetrance of this phenotype.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The mouse trophinin gene, Tnn, mapped to the distal region of the mouse X chromosome. A marker, DXHXd1, and genes, Fgd1 and Smcx, also mapped to the same region [33, 34]. All these genes localize to Xp11.21–22 in human chromosomes, showing a highly conserved physical map between the two species.
Both the human and the mouse have a single trophinin gene (Fig. 2). Although human and mouse trophinin mRNAs contain an open reading frame and encode a large protein, KIAA1114 (Fig. 3), we could not detect this protein. Rather, our Western blot analysis confirmed the existence of a previously reported trophinin protein (69 kDa) [1] in the human and of its homologue (107 kDa) in the mouse (Fig. 4). This evidence suggests that trophinin mRNA is translated at the downstream AUG codon (Fig. 3). Further analysis is needed to determine how this occurs and to define whether the KIAA1115 protein is produced in vivo.
We reported previously that trophinin is expressed specifically in cells involved in implantation and early placental development in humans [1, 4]. In human endometrium, presumably at implantation stages, trophinin proteins are found in the endometrial luminal epithelium within narrowly restricted regions. Trophinin expressions in human placenta are limited to the implantation site [5]. However, mouse endometrial epithelium at the implantation stage strongly expresses trophinin, regardless of its distance from the blastocyst (Fig. 7, E and F) [15]. This pattern contrasts with those of HB-EGF, amphiregulin, betacellulin, and epiregulin in the mouse uterus, all of which are found exclusively in the luminal epithelium surrounding blastocysts [9, 14, 35, 36]. In human placenta, trophinin continues to be expressed during early first trimester, suggesting that it plays a role in placental development, whereas in the mouse, trophinin is not found in the trophoblast (Fig. 7H). These results suggest different mechanisms for trophinin expression by the trophoblast in the human and in the mouse.
Our trophinin gene knock-out experiments show that, under 129/SvJ background, numbers of trophinin-null males and heterozygous females were reduced in a generation-dependent manner. When male chimeras were backcrossed with wild-type 129/SvJ females, pups (G1) were produced with low efficiency. It appears that G1 heterozygous females were selected during this process. Crossing such females with wild-type males would allow trophinin-null phenotypes to become apparent in later generations.
Trophinin proteins are expressed strongly in the blastocysts before implantation (Fig. 7B). Genotyping of the preimplantation-stage embryos (Table 1) together with careful morphological inspections, however, did not reveal any abnormality associated with the trophinin-null mutation. Immunohistochemistry revealed no trophinin proteins in the mouse blastocyst during implantation (Fig. 7, E and F). This demonstrates that trophinin does not play a role during embryo implantation in the mouse. Shortly after implantation, trophinin is expressed strongly, but transiently, in the epiblast of E5.5 embryos (Fig. 7G). This suggests a possibility that trophinin plays an important role in the E5.5 epiblast. However, we could not determine the stage and cell types leading to an embryonic lethality due to incomplete penetrance of this phenotype.
Implantation and placentation processes vary significantly among different mammalian species, and these diversities have long been recognized at both morphological and molecular levels [7, 17, 37, 38]. Homologues of trophinin and the trophinin-assisting cytoplasmic protein tastin are found in mice but not in nonmammalian organisms. Bystin, another trophinin-associated cytoplasmic protein, homologues are found in Saccharomyces, in Caenorhabditis, and in Drosophila [4]. In yeast, bystin is a nuclear protein, ENP1, essential for the survival of yeast in vegetative growth [39]. This suggests that the function of bystin has changed through evolution from yeast to humans, whereas the functions of trophinin and tastin have been acquired in mammals. Bystin is strongly and transiently expressed in the epiblast of E5.5 mouse embryos, which coincides with the expression of trophinin. The bystin gene knock-out mouse shows an embryonic lethality shortly after implantation with 100% penetrance (unpublished results), suggesting an essential role of bystin in the epiblastic development in the mouse. Because human bystin directly binds to the cytoplasmic domain of human trophinin [4], it is possible that mouse bystin and trophinin associate with each other in the epiblast. The expression pattern of trophinin and bystin and the effect of bystin gene knock-outs suggest that trophinin and bystin together play an essential role in epiblastic development in the mouse.
The present study suggests that the in vivo role of trophinin has diverged between the mouse and the human. During evolution, embryo implantation may not have converged into a dominant form. Recent technical advances such as manipulation of primate ES cells [40] and gene targeting in mammals other than mice [41, 42] will provide additional genetic models for clarifying the molecular basis of embryo implantation generated by evolution.
Although the present study aimed at testing the potential involvement of trophinin in human embryo implantation, this effort was hampered by significant differences in the patterns of trophinin expression observed between the mouse and the human. Differences in the expression patterns of mouse and human trophinins noted in this and in previous studies [1, 5, 15] are consistent with the idea that the processes of embryo implantation vary significantly among mammalian species [7, 17, 37, 38]. Further study is needed to determine the function of human trophinin, which is expressed in the trophoblast and endometrial epithelial cells at, apparently, the embryo implantation site in the human placenta and uteri [1–3, 5].
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by NIH R01 HD34108 (M.N.F.), HD37934, and HD34394 (B.C.P); in part by the National Cancer Institute DHHS, and by the American Cancer Society (California Division) Senior Postdoctoral Fellowship (D.N.). 
2 Correspondence: Michiko N. Fukuda, The Burnham Institute, 10901 North Torrey Pines Road, La Jolla, CA 92037. FAX: 858 646 3193; michiko{at}burnham.org
Accepted: September 17, 2001.
Received: July 5, 2001.
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