Search

Proline rich 15 (Prr15), which encodes a protein of unknown function, is expressed almost exclusively in postmitotic cells both during fetal development and in adult tissues, such as the intestinal epithelium and the testis. To determine if this specific expression is lost in intestinal neoplasias, we examined Prr15 expression by in situ hybridization (ISH) on mouse intestinal tumors caused by different gene mutations, and on human colorectal cancer (CRC) samples. Prr15/PRR15 expression was consistently observed in mouse gastrointestinal (GI) tumors caused by mutations in the Apc gene, as well as in several advanced stage human CRCs. In contrast, no Prr15 expression was detected in intestinal tumors derived from mice carrying mutations in the Smad3, Smad4, or Cdkn1b genes. These findings, combined with the fact that a majority of sporadic human CRCs carry APC mutations, strongly suggest that the expression of Prr15/PRR15 in mouse and human GI tumors is linked, directly or indirectly, to the absence of the APC protein or, more generally, to the disruption of the Wnt signaling pathway. (C) 2010 Wiley-Liss, Inc.

Male infertility is one possible consequence of a group of disorders arising from dysfunction of cilia. Ciliopathies include primary ciliary dyskinesia, polycystic kidney disease, Usher syndrome, nephronophthisis, Bardet-Biedl syndrome, Alstrom syndrome, and Meckel-Gruber syndrome as well as some forms of retinal degenerations. Mutations in the retinitis pigmentosa GTPase regulator gene (RPGR) are best known for leading to retinal degeneration but have also been associated with ciliary dysfunctions affecting other tissues. To further study the involvement of RPGR in ciliopathies, transgenic mouse lines overexpressing RPGR were generated. Animals carrying the transgene in varying copy numbers were investigated. We found that infertility due to aberrant spermatozoa correlated with increased copy numbers. In animals with moderately increased gene copies of Rpgr, structural disorganization in the flagellar midpiece, outer dense fibers, and fibrous sheath was apparent. In contrast, in animals with high copy numbers, condensed sperm heads were present, but the flagellum was absent in the vast majority of spermatozoa, although early steps of flagellar biogenesis were observed. This complexity of defects in flagellar assembly suggests a role of RPGR in intraflagellar transport processes.

The LIM homeobox containing genes of the LIM-3 group, Lhx3 and Lhx4, are critical for normal development. Both genes are involved in the formation of the pituitary and the motoneuron system and loss of either gene causes perinatal lethality. Previous studies had shown that Lhx3 is overexpressed in hyperplastic placentas of mouse interspecies hybrids. To determine the role of LHX3 in the mouse placenta, we performed expression and function analyses. Our results show that Lhx3 exhibits specific spatial and temporal expression in the mouse placenta. However, deletion of Lhx3 does not produce a placental phenotype. To test whether this is due to functional substitution by Lhx4, we performed a phenotype analysis of Lhx3/; Lhx4/ double-mutant placentas. A subset of Lhx3/; Lhx4/ placentas exhibited abnormal structure of the labyrinth. However, absence of both LIM-3 genes did not interfere with placental transport nor consistently with expression of target genes such as Gnrhr. Thus, LHX3 and LHX4 appear to be dispensable for placental development and function. Developmental Dynamics 237:1517–1525, 2008. © 2008 Wiley-Liss, Inc.

Maternal diabetes causes placental and foetal abnormalities in both rat and humans; however, its effect is less well documented in the mouse. We used a standard approach to induce manifest diabetes in pregnant mice and assessed morphology, function and gene expression in the placentas isolated from these females. We found that diabetic placentas exhibit a consistent abnormal phenotype characterized by increased junctional zone cross sectional area. Lipid profiling of diabetic foetuses and placentas showed that the placental phenotypes do not compromise the lipid transport function of this organ. In a genome-wide survey of mRNA expression by using cDNA micro-arrays, we identified 118 ESTs, corresponding to 59 annotated genes, with differential expression in the diabetic placentas. A significant proportion of these known is involved in metabolism, immunity and defence, and signal transduction. In addition, we found two imprinted genes, Igf2 and Gatm, which exhibited altered expression. The expression of other imprinted genes, Peg1, Gtl2, Peg3, Igf2r and Grb10, was determined by quantitative RT-PCR. For all of these genes, slight changes in gene expression were observed between diabetic placentas and control placentas. Our study thus provides the basis for future work that will address gene action in the diabetic mouse placenta.

Interspecific hybridization in mammals causes hybrid dysgenesis effects, such as sterility and abnormal placentation. Here, we describe a novel obesity syndrome caused by interspecific hybridization in the genus Mus and show that this obesity, appearing sporadically in F1 littermates derived from inbred strains, has an epigenetic basis. Mus hybrids from various strains of M. musculus and M. spretus were generated and the sporadic obese phenotype was confirmed through assessment of physiological and biochemical parameters in littermates. To understand the underlying mechanisms, large-scale and candidate gene expression assays, global DNA methylation assays and allelic expression analysis were performed. Studies showed that obese hybrids are similar to other known models of obesity. While increased axial growth indicated a defect in POMC pathway, comparison of global gene expression patterns in brain of obese F1 and obese Pomc mutant mice showed little similarity. In F1 obese mice many genes involved in the maintenance of epigenetic states, as well as several imprinted genes, were differentially expressed. Global DNA methylation analysis in brain showed that increased methylation levels were associated with obesity. The imprinted gene Gnasxl, known to be important in lipid homeostasis, was found over expressed in the obese hybrids. Allelic expression and methylation analysis of Gnasxl showed that alterations of epigenetic marks underlying F1 obesity are probably many and multi-factorial. Conclusions: This model of obesity, which is both spontaneous and epigenetic, may be a useful tool to address the epigenetic aspects of clinical obesity.

HP1 proteins are thought to be modulators of chromatin organization in all mammals, yet their exact physiological function remains unknown. In a first attempt to elucidate the function of these proteins in vivo, we disrupted the murine Cbx1 gene, which encodes the HP1-β isotype, and show that the Cbx1−/−-null mutation leads to perinatal lethality. The newborn mice succumbed to acute respiratory failure, whose likely cause is the defective development of neuromuscular junctions within the endplate of the diaphragm. We also observe aberrant cerebral cortex development in Cbx1−/− mutant brains, which have reduced proliferation of neuronal precursors, widespread cell death, and edema. In vitro cultures of neurospheres from Cbx1−/− mutant brains reveal a dramatic genomic instability. Our results demonstrate that HP1 proteins are not functionally redundant and that they are likely to regulate lineage-specific changes in heterochromatin organization.

Mammals have three HP1 protein isotypes HP1β (CBX1), HP1γ (CBX3) and HP1α (CBX5) that are encoded by the corresponding genes Cbx1, Cbx3 and Cbx5. Recent work has shown that reduction of CBX3 protein in homozygotes for a hypomorphic allele (Cbx3 hypo) causes a severe postnatal mortality with around 99% of the homozygotes dying before weaning. It is not known what the causes of the postnatal mortality are. Here we show that Cbx3 hypo/hypo conceptuses are significantly reduced in size and the placentas exhibit a haplo-insufficiency. Late gestation Cbx3 hypo/hypo placentas have reduced mRNA transcripts for genes involved in growth regulation, amino acid and glucose transport. Blood vessels within the Cbx3 hypo/hypo placental labyrinth are narrower than wild-type. Newborn Cbx3 hypo/hypo pups are hypoglycemic, the livers are depleted of glycogen reserves and there is almost complete loss of stored lipid in brown adipose tissue (BAT). There is a 10-fold reduction in expression of the BAT-specific Ucp1 gene, whose product is responsible for non-shivering themogenesis. We suggest that it is the small size of the Cbx3 hypo/hypo neonates, a likely consequence of placental growth and transport defects, combined with a possible inability to thermoregulate that causes the severe postnatal mortality.

Carboxypeptidase E (CPE) has important functions in processing of endocrine pro-peptides, such as pro-insulin, pro-opiomelanocortin, or pro-gonadotropin-releasing hormone, as evidenced by the hyper-pro-insulinemia, obesity, and sterility of Cpe mutant mice. Down-regulation of Cpe in enlarged placentas of interspecific hybrid (interspecies hybrid placental dysplasia (IHPD)) and cloned mice suggested that reduced CPE enzyme and receptor activity could underlie abnormal placental phenotypes. In this study, we have explored the role of Cpe in murine placentation by determining its expression at various stages of gestation, and by phenotypic analysis of Cpe mutant placentas. Our results show that Cpe and Carboxypeptidase D (Cpd), another carboxypeptidase with a very similar function, are strictly co-localized in the mouse placenta from late mid-gestation to term. We also show that absence of CPE causes a sporadic but striking placental phenotype characterized by an increase in giant and glycogen cell numbers and giant cell hypertrophy. Microarray-based transcriptional profiling of Cpe mutant placentas identified only a very small number of genes with altered expression, including Dtprp, which belongs to the prolactin gene family. Concordant deregulation of Cpe and Cpd in abnormal placentas of interspecies hybrids before the onset of IHPD phenotype and recapitulation of some phenotypes of IHPD hyperplastic placentas in Cpe mutant placentas suggests that these two genes are causally involved in IHPD and may function as speciation genes in the genus Mus.

The extracellular matrix protein fibulin-1 (FBLN1) is an important component of blood vessel walls, as shown by the lethality of mice with homozygous targeted deletion of the Fbln1 gene. Here, we show that a murine placental overgrowth phenotype is associated with elevated Fbln1 transcript levels, suggesting that the gene and its product have a functional role in placentation. Fbln1 exhibits a specific expression pattern in the mouse placenta. Transcripts could not be detected prior to day 12. In subsequent stages, Fbln1 was expressed strongly in the spongiotrophoblast. Other sites of expression were endothelia of large fetal blood vessels, a tissue type reported to not express this gene. In addition, a subset of giant cells expressed the gene. This giant cell specific expression was strongly increased in hyperplastic placentas. Analysis of the placentation in fibulin null mice did not show any abnormality. Attempts to rescue the placental phenotypes of a congenic model of interspecies hybrid placental dysplasia (IHPD) by normalizing expression of Fbln1 proved that Fbln1 alone is not the key cause of phenotypes in these models of placental hyperplasia.

Different causes, such as maternal diabetes, cloning by nuclear transfer, interspecific hybridization, and deletion of some genes such as Esx1, Ipl, or Cdkn1c, may underlie placental overgrowth. In a previous study, we carried out comparative gene expression analysis in three models of placental hyperplasias, cloning, interspecies hybridization (IHPD), and Esx1 deletion. This study identified a large number of genes that exhibited differential expression between normal and enlarged placentas; however, it remained unclear how altered expression of any specific gene was related to any specific placental phenotype. In the present study, we focused on two genes, Car2 and Ncam1, which both exhibited increased expression in interspecies and cloned hyperplastic placentas. Apart from a detailed expression analysis of both genes during normal murine placentation, we also assessed morphology of placentas that were null for Car2 or Ncam1. Finally, we attempted to rescue placental hyperplasia in a congenic model of IHPD by decreasing transcript levels of Car2 or Ncam1. In situ analysis showed that both genes are expressed mainly in the spongiotrophoblast, however, expression patterns exhibited significant variability during development. Contrary to expectations, homozygous deletion of either Car2 or Ncam1 did not result in placental phenotypes. However, expression analysis of Car3 and Ncam2, which can take over the function of Car2 and Ncam1, respectively, indicated a possible rescue mechanism, as Car3 and Ncam2 were expressed in spongiotrophoblast of Car2 and Ncam1 mutant placentas. On the other hand, downregulation of either Car2 or Ncam1 did not rescue any of the placental phenotypes of AT24 placentas, a congenic model for interspecies hybrid placentas. This strongly suggested that altered expression of Car2 and Ncam1 is a downstream event in placental hyperplasia.

Mammalian interspecies hybrids exhibit parent-of-origin effects in that offspring of reciprocal matings, even though genetically identical, frequently exhibit opposite phenotypes, especially in growth. This was also observed in hybridization with the genus Mus. These parent-of-origin effects suggested that imbalance in the expression of imprinted genes, which are expressed differentially, depending on their transmission through the maternal or paternal germline, and/or differential loss-of-imprinting (LOI) could underlie these opposite growth phenotypes in reciprocal mammalian hybrids. Here we report that tissue-specific LOI occurs in adult Mus hybrids. Contrary to expectations, LOI patterns were not consistent with a direct influence of altered expression levels of imprinted genes on growth. Bisulfite sequencing revealed that reactivation of maternal alleles of Peg3 and Snrpn in specific tissues was accompanied by partial demethylation at their potential imprinting control regions. We propose that abnormal reprogramming after fertilization and during preimplantation development is in part responsible for hybrid dysgenesis, for which a strong epigenetic basis has been demonstrated.

Mutations in the Norrie disease pseudoglioma gene (NDP) are known to cause X-linked recessive Norrie disease. In addition, NDP mutations have been found in other vasoproliferative retinopathies such as familial exudative vitreoretinopathy, retinopathy of prematurity, and Coats disease, suggesting a role for Norrin in vascular development. Here we report that female mice homozygous for the Norrie disease pseudoglioma homolog (Ndph) knockout allele exhibit almost complete infertility, while heterozygous females and hemizygous males are fertile. Histological examinations and RNA in situ hybridization analyses revealed defects in vascular development and decidualization in pregnant Ndph−/− females from embryonic day 7 (E7) onwards, resulting in embryonic loss. Using RT-PCR analyses we also demonstrate, for the first time, the expression of Ndph in mouse uteri and deciduae as well as the expression of NDP in human placenta. Taken together, these data provide strong evidence for Norrin playing an important role in female reproductive tissues. genesis 42:253–262, 2005. © 2005 Wiley-Liss, Inc.

Interspecific hybridization in the rodent genera Peromyscus and Mus results in abnormal placentation. In the Peromyscus interspecies hybrids, abnormal allelic interaction between an X-linked locus and the imprinted paternally expressed Peg3 locus was shown to cause the placental defects. In addition, loss-of-imprinting (LOI) of Peg3 was positively correlated with increased placental size. As in extreme cases this placental dysplasia constitutes a post-zygotic barrier against interspecies hybridization, this finding was the first direct proof that imprinted genes may be important in speciation and thus in evolution. In the Mus interspecies hybrids, a strong role of an X-linked locus in placental dysplasia has also been detected. However, here we show by backcross and allele specific expression analyses that neither LOI of Peg3 nor abnormal interactions between Peg3 and an X-linked locus are involved in generating placental dysplasia in Mus hybrids, although the placental phenotypes observed in the two genera seem to be identical. In contrast to this, another dysgenesis effect common to Peromyscus and Mus hybrids, altered foetal growth, is caused at least in part by the same X-chromosomal regions in both genera. These findings first underline the strong involvement of the X-chromosome in the genetics of speciation. Secondly, they indicate that disruption of epigenetic states, such as LOI, at specific loci may be involved in hybrid dysgenesis effects in one group, but not in another. Thus, we conclude that even in closely related groups divergent molecular mechanisms may be involved in the production of phenotypically similar post-zygotic barriers against hybridization.

Contains fulltext : 59117.pdf (Publisher’s version ) (Closed access) Choroideremia (CHM) is a hereditary eye disease caused by mutations in the X-linked CHM gene. Disruption of the Chm gene in mice resulted in prenatal death of Chm-/Y males and Chm-/Chm+ females that had inherited the mutation from their mothers. Male chimeras and Chm+/Chm- females with paternal transmission of the mutation were viable and had photoreceptor degeneration reminiscent of human choroideremia. Here, we show that Chm-/Y males and Chm-/Chm+ females were retarded at e7.5 and died before e11.5 due to multiple defects of the extra-embryonic tissues. Mutant embryos exhibited deficiency of diploid trophoblasts associated with overabundance of giant cells. In yolk sac and placenta, severe defects in vasculogenesis were obvious. Chm-/Y males exhibited more pronounced phenotypes than Chm-/Chm+ females. The lethal genotypes could be rescued by tetraploid aggregation. Chm-/Chm+ females, but not Chm-/Y males, could also be rescued when their Chm+/Chm- mothers were mated with Mus spretus males. Backcross analysis suggested that the viability of interspecies hybrid Chm-/Chm+ females may be due to expression from the Chm allele on the M. spretus X-chromosome rather than a modifier effect. Our results demonstrate that Chm is essential for diploid trophoblast development and plays a role in the vascularization in placenta and yolk sac.

Mice of the XO genotype with a paternally derived X chromosome (XpO) have placental hyperplasia in late pregnancy, although in early pregnancy the ectoplacental cone, a placental precursor, is smaller in XpO mice than in their XX sibs. This early size deficiency of the ectoplacental cone is apparently a consequence of Xp imprinting, because XmO embryos (with a maternally derived X chromosome) are unaffected. In the present study we sought to establish whether XpO placental hyperplasia in late pregnancy is also a consequence of Xp imprinting. Placental weight data were first collected from litters that included XpO or XmO fetuses and XX controls. Comparison of XO placentae with XX placentae showed that XpO and XmO placentae are hyperplastic. This finding suggested that the hyperplasia might be an X dosage effect, and this hypothesis was supported by the finding that XY male fetuses from the same crosses also had larger placentae than their XX sibs. Further analysis of a range of sex-chromosome variant genotypes, including XmYSry-negative females and XXSry transgenic males, showed that mouse fetuses with one X chromosome consistently had larger placentae than littermates with two X chromosomes, independent of their gonadal/androgen status.

TBX22 belongs to the T-box family of transcription factors and was originally found in an in silico approach designed to identify new genes on the human Xq12-q21 region. Mutations in TBX22 have been reported in families with X-linked cleft palate and ankyloglossia (CPX), but the underlying pathogenetic mechanism remained unknown. We have identified mouse Tbx22 and analyzed its expression during embryogenesis by reverse transcriptase-polymerase chain reaction and in situ hybridization. In mouse embryos, it is expressed in distinct areas of the head, namely the mesenchyme of the inferior nasal septum, the posterior palatal shelf before fusion, the attachment of the tongue, and mesenchymal cells surrounding the eye anlage. The localization in the tongue frenulum perfectly correlates with the ankyloglossia phenotype in CPX. Furthermore, we identified positionally conserved binding sites for transcription factors, two of which have been implicated previously in palatogenesis (MSX1, PRX2).

It has been shown previously that abnormal placental growth occurs in crosses and backcrosses between different mouse (Mus) species. In such crosses, late gestation placentas may weigh between 13 and 848 mg compared with a mean placental weight of approximately 100 mg in late gestation M. musculus intraspecific crosses. A locus on the X-chromosome was shown to segregate with placental dysplasia. Thus in the (M. musculus × M. spretus)F1 × M. musculus backcross, placental hyperplasia cosegregates with a M. spretus derived X-chromosome. Here we have investigated whether increased cell proliferation and aberrant expression of two genes that are involved in placental growth control, Igf2 and Esx1, may cause, or contribute to placental hyperplasia. Increased bromodeoxyuridine labeling of nuclei, reflecting enhanced proliferation, was indeed observed in hyperplastic placentas when compared with normal littermate placentas. Also, increased expression of Igf2 was seen in giant cells and spongiotrophoblast. However, when M. musculus × M. spretus F1 females were backcrossed with males that were heterozygous for a targeted mutation of the Igf2 gene, placentas that carried a M. spretus derived X-chromosome and were negative for a functional Igf2 allele exhibited an intermediate placental phenotype. Furthermore, in early developmental stages of placental hyperplasia, we observed a decreased expression of the X-chromosomal Esx1 gene. This finding suggests that abnormal expression of both Igf2 and Esx1 contributes to abnormal placental development in mouse interspecific hybrids. However, Esx1 is not regulated by IGF2. © 2002 Wiley-Liss, Inc.

In the normal diploid mouse embryo, active demethylation of the paternal genome but not of the maternal genome occurs within only a few hours and in a highly coordinated fashion as the zygote proceeds through the first G1 phase. This zygotic demethylation may be necessary to reprogram the sperm genome for somatic development. Immunofluorescence staining with an antibody against 5-methylcytosine shows that the cellular machinery of the fertilized egg cannot demethylate the second maternal genome in parthenogenetic, gynogenetic and triploid digynic embryos or remethylate the additional (already demethylated) paternal genome in androgenetic and triploid diandric embryos. This suggests that differential zygotic demethylation results from differences in the remodeling of paternal and maternal chromatin structures after fertilization, i.e. sperm nuclear decondensation and protamine-histone exchange. A proportion of embryos derived from normal matings display abnormal methylation patterns some of which are indistinguishable from those in androgenetic or gynogenetic embryos. We conclude that methylation reprogramming defects in mammalian zygotes contribute to the high incidence of early pregnancy failure.

It has been shown previously that abnormal placental growth, i.e., hyper- and hypoplasia, occurs in crosses and backcrosses between different mouse (Mus) species. A locus that contributes to this abnormal development has been mapped to the X chromosome. Unexpectedly, an influence of fetal sex on placental development has been observed, in that placentas attached to male fetuses tended to exhibit a more pronounced phenotype than placentas attached to females. Here, we have analyzed this sex dependence in more detail. Our results show that differences between male and female placental weights are characteristic of interspecific matings and are not observed in intraspecific Mus musculus matings. The effect is retained in congenic lines that contain differing lengths of M. spretus-derived X chromosome. Expression of the X-linked gene Pgk1 from the maternal allele only and lack of overall activity of two paternally inherited X-linked transgenes indicate that reactivation or lack of inactivation of the paternal X chromosome in trophoblasts of interspecific hybrids is not a frequent occurrence. Thus, the difference between male and female placentas seems not to be caused by faulty preferential X-inactivation. Therefore, these data suggest that the sex difference of placental weights in interspecific hybrids is caused by interactions with the Y chromosome.

We have studied the process of tspy gene silencing in murine evolution. We have isolated functional tspy sequences from Apodemus agrarius, A. sylvaticus, A. flavicollis, and Mus platythrix (subgenus Pyromys) and nonfunctional tspy sequences from species of the subgenus Mus. We present two alternative models as to how tspy may have lost its function in the murine lineage.

The placenta plays a pivotal role in fetal growth control and is considered a major site of genetic conflict between maternal and paternal genomes within the conceptus and, in addition, the genome of the mother. Accordingly, placental development is a strictly controlled process, and both placental and fetal weights do not vary much in intraspecific crosses of laboratory mice (Mus musculus). In mouse interspecific crosses and backcrosses [(M. musculus x M. spretus) x M. musculus], tremendous variation of placental, but not of fetal weight was observed. We have studied trophoblast cell type distribution and differentiation, and their effect on the associated placentas and fetuses in such backcrosses. Differentiation of spongious trophoblast, but not size of materno-fetal interface, correlated with fetal weight. Giant fetuses were observed only if less than one third of the spongiotrophoblast was formed by glycogen cells. Thus, placental efficiency was inversely related to the amount of glycogen cells. This influence of a trophoblast-derived cell type on fetal growth was not anticipated. We conclude that: (1) glycogen cells are able to negatively modulate fetal growth by an as yet unidentified mechanism; (2) correlation between fetal and placental weights is weak or absent in interspecific hybrids; (3) impaired control over placental and embryonic development in hybrids may contribute to post-mating isolation of species.

X-linked retinitis pigmentosa (XLRP) results from mutations in at least two different loci, designated RP2 and RP3, located at Xp11.3 and Xp21.1, respectively. The RP3 gene was recently isolated by positional cloning, whereas the RP2 locus was mapped genetically to a 5-cM interval. We have screened this region for genomic rearrangements by the YAC representation hybridization (YRH) technique and detected a LINE1 (L1) insertion in one XLRP patient. The L1 retrotransposition occurred in an intron of a novel gene that consisted of five exons and encoded a polypeptide of 350 amino acids. Subsequently, nonsense, missense and frameshift mutations, as well as two small deletions, were identified in six additional patients. The predicted gene product shows homology with human cofactor C, a protein involved in the ultimate step of beta-tubulin folding. Our data provide evidence that mutations in this gene, designated RP2, are responsible for progressive retinal degeneration.

The ‘Genes and Development’ workshop focused on recentadvances in our understanding of the molecular control ofplacental development, focusing on the mouse model. JayCross gave an introduction to mouse placental developmentthat outlined the major structures and cell types. He alsoreviewed the considerable progress that has been made indefining molecular pathways controlling development usingmutant and transgenic mice as well as increasing knowledge ofthe biology underlying implantation and establishment of themature placenta [1,2]. One of the important general findings toemerge in recent years is that development and function of thedifferent sub-structures of the placenta can be interdependentand able to adapt to defects in other parts. This can complicatethe analysis of a phenotype in that some types of changes cansimply represent secondary changes reflecting attempts tocompensate, and investigators need to take this into consider-ation before making conclusions. The remaining speakers wereselected to discuss different aspects of placental developmentand highlight the use of new experimental approaches forstudying mouse placental development and function.

Parthenogenetic cells are lost from fetal chimeras. This may be due to decreased proliferative potential. To address this question, we have made use of combined cell lineage and cell proliferation analysis. Thus, the incorporation of bromodeoxyuridine in S-phase was determined for both parthenogenetic and normal cells in several tissues of fetal day 13 and 17 chimeras. A pronounced reduction of bromodesoxyuridine incorporation by parthenogenetic cells at both developmental stages was only observed in cartilage. In brain, skeletal muscle, heart and intestinal epithelium, this reduction was either less pronounced or observed only at one of the developmental stages analysed. No difference between parthenogenetic and normal cells was observed in epidermis and ganglia. Our results show that a loss of proliferative potential of parthenogenetic cells during fetal development contributes to their rapid elimination in some tissues. The analysis of the fate of parthenogenetic cells in skeletal muscle and cartilage development demonstrated different selection mechanisms in these tissues. In skeletal muscle, parthenogenetic cells were largely excluded from the myogenic lineage proper by early post-midgestation. In primary hyaline cartilage, parthenogenetic cells persisted into adulthood but were lost from cartilages that undergo ossification during late fetal development.

To asses the potential of androgenetic cells to participate in post-midgestation fetal development we have made use of an in situ detectable cell lineage marker in the analysis of chimeric mouse fetuses containing an androgenetic cell lineage. Our results show conclusively that androgenetic cells participate in the formation of derivatives of all lineages and in some tissues may contribute the majority of the total cell population. However, the allocation or persistence of androgenetic cells was non-random. High contribution of androgenetic cells was observed in brown adipose tissue, mesenchyme, smooth muscle, perichondrium, peripheral nerves and epithelia of the intestinal tract and the trachea. Thus, androgenetic cells were able to efficiently populate mesodermal, ectodermal and endodermal derivatives. In contrast, there was a clear prejudice against androgenetic cells in the brain.

Diploid parthenogenetic or androgenetic mouse conceptuses produce characteristic and opposite mutant phenotypes and are non-viable, presumably due to different contributions from the maternal and paternal genomes. This is likely to be the result of the preferential expression of only one parent's copy of certain genes in the offspring. So far, four such endogenous imprinted genes are known: the paternal alleles of Igf2 and Snrpn and the maternal alleles of Igf2r and H19 are active, while their opposite parental alleles are inactive. Here we demonstrate that the expression patterns of the Igf2 and Igf2r genes in androgenetic and parthenogenetic conceptuses correlate with which parental alleles normally express them, implying that the imprint can be maintained in the absence of the other parent's genome for these genes. This also indicates that both types of uniparental conceptuses are lacking developmentally important gene products. We did find, however, that the H19 gene was highly expressed not only in the parthenogenetic conceptus, but also in giant trophoblasts and secondary giant cells in the androgenetic placenta, in spite of the imprinting of the H19 gene in normal mouse extra embryonic tissues. We discuss these observations with respect to the non-viability of uniparental conceptuses and the reciprocal imprinting patterns of the Igf2 and H19 genes.

The ability of parthenogenetic (pg) cells to contribute to proliferating stem cell populations of postnatal aggregation chimeras was investigated. Using DNA in situ analysis, pg participation was observed in highly regenerative epithelia of various regions of the gastrointestinal tract, e.g., stomach, duodenum and colon, in the epithelia of tongue and uterus and in the epidermis. Pg cells also contributed to the epithelium of the urinary bladder, which is characterized by a relatively slow cellular turnover. Using a sensitive proliferation marker to determine division rate of pg and normal (wt) cells in tissues of a 24-day-old chimera, no significant differences between pg and fertilized cells were observed. However, in colon and uterus of a pg↔wt chimera aged 101 days, a significant loss of proliferative capacity of pg cells was found. In the colon, this loss of proliferative potential was accompanied by an altered morphology of pg crypts. In general, they were situated at the periphery of the epithelium and lacked access to the lumen, with consequent cystic enlargement and flattened epithelium. No obvious morphological changes were observed in the pg-derived areas of the uterine epithelium of this chimera. Our results provide evidence that pg cells can persist as proliferating stem cells in various tissues of early postnatal chimeras. They suggest that pg-derived stem cells may cease to proliferate in restricted areas of the gastrointestinal tract and in the uterine epithelium of pg ↔wt chimeras of advanced age. However, no indications of such a loss of proliferative potential of pg cells could be observed in other areas of the digestive tract, e.g., in the stomach and duodenum, or in oral epithelium and in the epidermis. These findings argue for a high degree of specificity of selection against pg cells in postnatal life. They also suggest that an impaired proliferative capacity is not a general feature of pg cells. Hence, it is possible that, to explain the impaired growth of of pg↔wt chimeras and the overall stringent selection against pg cells in such chimeras during fetal development, additional and/or alternative causes apart from a decreased proliferative potential of pg cells have to be considered. Apart from their contribution to the endodermderived epithelia of stomach, gut and uterus, to the mesoderm-derived epithelium of uterus and to ectoderm-derived oral epithelium and epidermis, pg-derived cells differentiated into the mesoderm-derived mesenchymal cells of the stromal layers that lie between the epithelial cells and smooth muscle of stomach, gut, uterus and urinary bladder. Contribution to smooth muscle was also observed. Thus our results show that pg cells are able to differentiate into various cell types of different embryonic origin in tissues of composite structure.

Previous studies have revealed an enrichment of reproduction- and brain-related genes on the human X chromosome. In the present study, we investigated the evolutionary history that underlies this functional specialization. To do so, we analyzed the orthologous building blocks of the mammalian X chromosome in the chicken genome. We used Affymetrix chicken genome microarrays to determine tissue-selective gene expression in several tissues of the chicken, including testis and brain. Subsequently, chromosomal distribution of genes with tissue-selective expression was determined. These analyzes provided several new findings. Firstly, they showed that chicken chromosomes orthologous to the mammalian X chromosome exhibited an increased concentration of genes expressed selectively in brain. More specifically, the highest concentration of brain-selectively expressed genes was found on chicken chromosome GGA12, which shows orthology to the X chromosomal regions with the highest enrichment of non-syndromic X-linked mental retardation (MRX) genes. Secondly, and in contrast to the first finding, no enrichment of testis-selective genes could be detected on these chicken chromosomes. These findings indicate that the accumulation of brain-related genes on the prospective mammalian X chromosome antedates the divergence of sauropsid and synapsid lineages 315 million years ago, whereas the accumulation of testis-related genes on the mammalian X chromosome is more recent and due to adaptational changes.

Development in mammals is influenced by genome imprinting which results in differences in the expression of some homologous maternal and paternal alleles. This process, initiated in the germline, can continue following fertilization with interactions between oocyte cytoplasmic factors and the parental genomes involving modifier genes. Further epigenetic modifications may follow to render the ‘imprints’ heritable through subsequent cell divisions during development. Imprinting of genes can be critical for their dosage affecting embryonic growth, cell proliferation and differentiation. The cumulative effects of all the imprinted genes are observed in androgenones (AG) and parthenogenones (PG), which reveal complementary phenotypes with respect to embryonic and extraembryonic tissues. The presence of PG cells in chimeras causes growth retardation, while that of AG cells enhanced growth. AG cells apparently have a higher cell proliferation rate and, unlike PG cells, are less prone to selective elimination. However, the PG germ cells are exempt from cell selection. In chimeras, PG cells are more likely to be found in ectodermal derivatives such as epidermis and brain in contrast to AG cells which make pronounced contributions to many mesodermal derivatives such as muscle, kidney, dermis and skeleton. The presence of androgenetic cells in chimeras also results in the disproportionate elongation of the anterior-posterior axis and sometimes in the abnormal development of skeletal elements along the axis. Genetic studies highlight the influence of subsets of imprinted genes, and identify those that are critical for development.

The fate of parthenogenetic cells was investigated during development of fetal and early postnatal chimeras. On day 13 of embryonic development, considerable contribution of parthenogenetic cells was observed in all tissues of chimeric embryos, although selection against parthenogenetic cells seemed to start before day 13. Between days 13 and 15 of development, parthenogenetic cells came under severe selective pressure, which was most striking in tongue. The disappearance of parthenogenetic cells from tongue coincided with the beginning of myoblast fusion in this tissue. Severe selection against parthenogenetic cells was also observed in pancreas and liver, although in the latter, parthenogenetic cells were eliminated later than in skeletal muscle or pancreas. In other tissues, parthenogenetic cells may persist and participate to a considerable extent throughout the gestation period and beyond, although a significant decrease was observed in all tissues. Parthenogen-etic↔fertilized chimeras were significantly smaller than their non-chimeric littermates at all developmental stages. These results suggest that the absence of paternal chromosomes is largely incompatible with the maintenance of specific differentiated cell types. Furthermore, paternally derived genes seem to be involved in the regulation of proliferation of all cell types, as indicated by the drastic growth deceleration of parthenogen-etic↔fertilized chimeras and the overall decrease of parthenogenetic cells during fetal development. Chromosomal imprinting may have a role in maintaining a balance between cell growth and differentiation during embryonic development. The major exception to the selective elimination of parthenogenetic cells appear to be the germ cells; viable offspring derived from parthenogenetic oocytes were detected, sometimes at a high frequency in litters of female parthenogenetic↔fertilized chimeras.

An oncogenic D842V mutation in the platelet-derived growth factor (PDGF) alpha-receptor (Pdgfra) has recently been described in patients with gastrointestinal stromal tumors. In order to test if the same mutation would confer oncogenic properties to the homologous PDGF beta-receptor (Pdgfrb), the corresponding aspartic acid residue at position 849 of Pdgfrb was changed into valine (D849V) using a knock-in strategy. This mutation turned out to be dominantly lethal and caused death even in chimeras (from 345 transferred chimeric blastocysts, no living coat chimeras were detected). Experiments employing mouse embryonic fibroblasts (MEFs) indicated hyperactivity of the mutant receptor. The mutant receptor was phosphorylated in a ligand-independent manner and, in contrast to wild-type MEFs, mutant cells proliferated even in the absence of ligand. Knockout experiments have previously indicated a role for Pdgfrb in placental development. We therefore analyzed wild-type and Pdgfrb D849V chimeric placentas from different gestational stages. No differences were detected at embryonic days 11.5 and 13.5 (n=4). At embryonic day 17.5, however, chimeric placentas (n=3/4) displayed abnormalities both in the labyrinth and in the chorionic plate. The changes included hyper-proliferation of alpha-smooth muscle actin and platelet/endothelial cell adhesion molecule-1 positive cells in the labyrinth and cells in the chorionic plate. In addition, the fetal blood vessel compartment of the labyrinth was completely disorganized.

A picornavirus (Ljunganvirus [LV]) has recently been associated with disease during pregnancy in its natural rodent reservoir and in humans. A study of laboratory mice infected under controlled conditions was therefore undertaken.CD-1 female mice were infected gestational day two and subjected to varying regimes of stress.LV infection in combination with stress resulted in uterine resorptions, malformations, and neonatal death. A short delay in time to first pregnancy and births was observed in pairs infected in utero.LV is found in different species of native animals in both Europe and the United States and human epidemiological evidence connects LV and human reproduction, while the observations here indicate that LV is responsible for reproductive problems in a laboratory mouse model. The current findings suggest that the hypothesis that LV also causes disease in pregnant women and their offspring deserves further study.

To obtain a deeper insight into the genes and gene networks involved in the development of placentopathies, we have assessed global gene expression in three different models of placental hyperplasia caused by interspecies hybridization (IHPD), cloning by nuclear transfer, and mutation of the Esx1 gene, respectively. Comparison of gene expression profiles of approximately 13,000 expressed sequence tags (ESTs) identified specific subsets of genes with changed expression levels in IHPD, cloned, and Esx1 mutant placentas. Of interest, only one gene of known function and one EST of unknown function were found common to all three placentopathies; however, a significant number of ESTs were common to IHPD and cloned placentas. In contrast, only one gene was shared between IHPD and Esx1 mutant, and cloned and Esx1 mutant placentas, respectively. These genes common to different abnormal placental growth genotypes are likely to be important in the occurrence of placentopathy.

Higher eukaryotes are endowed with a diploid somatic genome. At fertilization the paternal and maternal genomes are combined, forming the new diploid organism. Nuclear transfer experiments in the 1980s (McGrath and Solter, 1984; Surani et al., 1986) demonstrated for the first time the functional non-equivalence of the two parental genomes in mammals. In contrast to many lower vertebrates, mammalian uniparental embryos cannot develop beyond mid-gestation. Androgenetic embryos, with two paternal genomes, are usually very stage-retarded but often have well developed mural trophoblast and yolk sac. Gynogenetic embryos, with two maternal genomes, can develop relatively normally to mid-term but are small and have very restricted trophoblast and yolk sac. In both cases there is no normal placental development. Because of opposing patterns of gene expression from maternal and paternal alleles of imprinted genes (Fundele and Surani, 1994; Tilghman, 1999), normal mammalian development requires the participation of both a maternal and a paternal genome. Genomic imprinting is thought to be a by-product of the unique fetal-maternal relationship in mammals to control the different parental interests on embryo development. Consistent with the parental conflict hypothesis (Moore and Haig, 1991), many paternally expressed genes enhance fetal growth, whereas maternally expressed genes inhibit growth.

Tri-methylated lysine 20 on histone H4 (Me(3)K20H4) is a marker of constitutive heterochromatin in murine interphase and metaphase cells. Heterochromatin marked by Me(3)K20H4 replicates late during S phase of the cell cycle. Serum starvation increases the number of cells that exhibit high levels of Me(3)K20H4 at constitutive heterochromatin. Me(3)K20H4 is also present at the centromeric heterochromatin of most meiotic chromosomes during spermatogenesis and at the pseudoautosomal region, as well as at some telomeres. It is not present on the XY-body. During murine embryogenesis the maternal pronucleus contains Me(3)K20H4; Me(3)K20H4 is absent from the paternal pronucleus. On Drosophila polytene chromosomes Me(3)K20H4 is present in a `punctate pattern' at many chromosomal bands, including the chromocenter. In coccids it is present on the facultatively heterochromatinised paternal chromosome set. We also present evidence that Me(3)K20H4 is dependent upon H3-specific Suv(3)9 histone methyltransferase activity, suggesting that there may be `epigenetic cross-talk' between histones H3 and H4.

Interspecific hybridization in the genus Mus results in several hybrid dysgenesis effects, such as male sterility and X-linked placental dysplasia (IHPD). The genetic or molecular basis for the placental phenotypes is at present not clear. However, an extremely complex genetic system that has been hypothesized to be caused by major epigenetic changes on the X chromosome has been shown to be active. We have investigated DNA methylation of several single genes, Atrx, Esx1, Mecp2, Pem, Psx1, Vbp1, Pou3f4, and Cdx2, and, in addition, of LINE-1 and IAP repeat sequences, in placentas and tissues of fetal day 18 mouse interspecific hybrids. Our results show some tendency toward hypomethylation in the late gestation mouse placenta. However, no differential methylation was observed in hyper- and hypoplastic hybrid placentas when compared with normal-sized littermate placentas or intraspecific Mus musculus placentas of the same developmental stage. Thus, our results strongly suggest that generalized changes in methylation patterns do not occur in trophoblast cells of such hybrids.

G90 is a novel mouse gene that does not belong to any known gene family. It has previously been shown that this gene is expressed exclusively in post-mitotic cells of the adult mouse intestine and testis, therefore suggesting a role in the control of proliferation and/or differentiation. Here we report the detailed spatio-temporal expression pattern of G90 during mouse embryonic development. We found G90 expression in specific structures of the developing head, namely the brain, inner and middle ear, olfactory epithelium, vomeronasal organ, nasopharynx, oropharynx, papillae of the tongue and oral cavity, pituitary gland and epiglottis. Interestingly, there was a clear correlation between G90 expression and absence of proliferation in most of the cells showing expression of this gene during embryonic development; this finding supported our functional hypothesis.

It has been shown previously that abnormal placental growth occurs in crosses and backcrosses between different mouse (Mus) species. In such crosses, late gestation placentas may weigh between 13 and 848 mg compared with a mean placental weight of approximately 100 mg in late gestation M. musculus intraspecific crosses. A locus on the X-chromosome was shown to segregate with placental dysplasia. Thus in the (M. musculus x M. spretus)F1 x M. musculus backcross, placental hyperplasia cosegregates with a M. spretus derived X-chromosome. Here we have investigated whether increased cell proliferation and aberrant expression of two genes that are involved in placental growth control, Igf2 and Esx1, may cause, or contribute to placental hyperplasia. Increased bromodeoxyuridine labeling of nuclei, reflecting enhanced proliferation, was indeed observed in hyperplastic placentas when compared with normal littermate placentas. Also, increased expression of Igf2 was seen in giant cells and spongiotrophoblast. However, when M. musculus x M. spretus F1 females were backcrossed with males that were heterozygous for a targeted mutation of the Igf2 gene, placentas that carried a M. spretus derived X-chromosome and were negative for a functional Igf2 allele exhibited an intermediate placental phenotype. Furthermore, in early developmental stages of placental hyperplasia, we observed a decreased expression of the X-chromosomal Esx1 gene. This finding suggests that abnormal expression of both Igf2 and Esx1 contributes to abnormal placental development in mouse interspecific hybrids. However, Esx1 is not regulated by IGF2.

Imprinted genes in mammals are expressed from only one of the parental chromosomes, and are crucial for placental development and fetal growth1,2,3,4. The insulin-like growth factor II gene (Igf2) is paternally expressed in the fetus and placenta5. Here we show that deletion from the Igf2 gene of a transcript (P0)6,7 specifically expressed in the labyrinthine trophoblast of the placenta leads to reduced growth of the placenta, followed several days later by fetal growth restriction. The fetal to placental weight ratio is thus increased in the absence of the P0 transcript. We show that passive permeability for nutrients of the mutant placenta is decreased, but that secondary active placental amino acid transport is initially upregulated, compensating for the decrease in passive permeability. Later the compensation fails and fetal growth restriction ensues. Our study provides experimental evidence for imprinted gene action in the placenta that directly controls the supply of maternal nutrients to the fetus, and supports the genetic conflict theory of imprinting8. We propose that the Igf2 gene, and perhaps other imprinted genes, control both the placental supply of, and the genetic demand for, maternal nutrients to the mammalian fetus.

We show that methylated lysine 9 of histone H3 (Me9H3) is a marker of heterochromatin in divergent animal species. It localises to both constitutive and facultative heterochromatin and replicates late in S-phase of the cell cycle. Significantly, Me9H3 is enriched in the inactive mammalian X chromosome (Xi) in female cells, as well as in the XY body during meiosis in the male, and forms a G-band pattern along the arms of the autosomes. Me9H3 is a constituent of imprinted chromosomes that are repressed. The paternal and maternal pronuclei in one-cell mouse embryos show a striking non-equivalence in Me9H3: the paternal pronucleus contains no immunocytologically detectable Me9H3. The levels of Me9H3 on the parental chromosomes only become equivalent after the two-cell stage. Finally, we provide evidence that Me9H3 is neither necessary nor sufficient for localisation of heterochromatin protein 1 (HP1) to chromosomal DNA.

The placenta is a highly specialized organ essential for embryonic growth and development. Here, we have applied cDNA subtraction between extraembryonic tissues of early- (day 7.5 of gestation) and late-stage embryos (day 17.5) to generate stage-specific cDNA pools that were used for screening of high-density mouse UniGene cDNA arrays containing 25,000 clones. A total of 638 clones were identified, 488 with the e7.5-specific probe and 150 with the e17.5-specific probe. Importantly, 363/638 (56.9%) of the hybridizing clones were not known to be expressed during placental development before. Differential regulation was confirmed by Northern blot and in situ hybridization for a total of 44/44 of positive clones. Thus, this combination of cDNA subtraction and array hybridization was highly successful for identification of genes expressed and regulated during placental development. These included growth factors and receptors, components of the transcriptional and translational machinery, cell cycle regulators, molecular chaperones, and cytoskeletal elements. The extensive in situ hybridization analysis revealed extraembryonic structures with a high density of differentially expressed genes, most strikingly the ectoplacental cone and the spongiotrophoblast. This large-scale identification of genes regulated during placentogenesis is extremely useful to further elucidate the molecular basis of extraembryonic development.

Trophoblast invasion is a critical process in development of most mammals that shares similarities with the invasive behavior of tumor cells. In the present investigation, a cDNA subtraction library was constructed between invasive trophoblast at day 8 of murine development and mature noninvasive placenta at day 18 of gestation. One of the differentially expressed clones, Epcs26, was mapped to the X chromosome and revealed no homology to any known gene. It was predominantly expressed in parietal endoderm, undifferentiated cells of the ectoplacental cone, and a few trophoblast giant cells. Another gene, designated Epcs50, was mapped to chromosome 19. It exhibited homologies to the mouse Mps1 gene and, like Mps1, may have a distant relationship to the lyric protein perforin. High expression was detected in parietal endoderm cells and in a subset of secondary trophoblast giant cells. Two sequences, Epcs24 and Epcs68, exhibited an extensive open reading frame that shared the common features of the cysteine proteinase cathepsin L. Expression was confined to an undefined subpopulation of trophoblast giant cells. Both genes were mapped to chromosome 13 in close proximity to cathepsins L and J. The known functions of MPS1 and cathepsin L proteins indicate that the related proteins EPCS50, EPCS24, and EPCS68 participate in conferring invasive properties to the mouse trophoblast.

We have used two different experimental approaches to demonstrate topological separation of parental genomes in preimplantation mouse embryos: mouse eggs fertilized with 5-bromodeoxyuridine (BrdU)-labeled sperm followed by detection of BrdU in early diploid embryos, and differential heterochromatin staining in mouse interspecific hybrid embryos. Separation of chromatin according to parental origin was preserved up to the four-cell embryo stage and then gradually disappeared. In F1 hybrid animals, genome separation was also observed in a proportion of somatic cells. Separate nuclear compartments during preimplantation development, when extreme chromatin remodelling occurs, and possibly in some differentiated cell types, may be associated with epigenetic reprogramming.

Interspecific hybridization in the genus Mus results in male sterility and X-linked placental dysplasia. We have generated several congenic laboratory mouse lines (Mus musculus) in which different parts of the maternal X chromosome were derived from M. spretus. A strict positive correlation between placental weight and length of the M. spretus-derived part of the X chromosome was shown. Detailed analysis was carried out with one congenic strain that retained a M. spretus interval between 12.0 and 30.74 cM. This strain consistently produced hyperplastic placentas that exhibited an average weight increase of 180% over the weight of control placentas. In derived subcongenic strains, however, increased placental weight could no longer be observed. Morphometric analysis of these placentas revealed persistence of abnormal morphology. Fully developed placental hyperplasia could be reconstituted by recombination of proximal and central M. spretus intervals with an intervening M. musculus region. These results may suggest that placental dysplasia of interspecific mouse hybrids is caused by multiple loci clustered on the X chromosome that act synergistically. Alternatively, it is possible that changes in chromatin structure in interspecific hybrids that influence gene expression are dependent on the length of the alien chromosome.

We describe the cloning and characterization of the murine G90 gene, identified by subtractive hybridization based on the differential presence of its transcript in large and small intestine. The full-length cDNA and genomic sequences were cloned and found to produce a 1.5 kb transcript that is polyadenylated but has no open reading frame larger than 249 bp.The G90 gene was mapped to the proximal region of mouse chromosome 6. Expression analysis by Northern blotting showed that G90 is transcribed at very high levels in the small intestine and at lower levels in large intestine, testis and kidney of the mouse. In situ hybridization analysis on sections of small and large intestine and testis showed that G90 transcripts are present only in post-mitotic cells.

The von Hippel‐Lindau (VHL) tumour suppressor gene product is believed to be involved in the downregulation of transcriptional elongation by preventing the association of elongin B and C with the catalytic subunit elongin A. Alterations in the human VHL gene lead to VHL disease which is associated with various rare neoplasias, including haemangioblastoma of the central nervous system, retinal angioma, clear cell renal carcinoma and pheochromocytoma. Recently, a protein (VBP1) was isolated that was found to bind to the VHL protein in vivo. We have used the murine Vbp1 homologous cDNA to investigate the expression of the Vbp1 mRNA in the mouse by in situ hybridization and northern blot analysis. In fetal stages between days 9 and 18 of gestation, Vbp1 was expressed mainly in the central nervous system, retina and liver. In addition, at day 12, high expression was observed in the labyrinthine region of the placenta. In later stage placentas, Vbp1 expression was, however, considerably reduced. Northern blot analysis of adult mouse tissues showed that Vbp1 was ubiquitously expressed. In situ analysis on several adult tissues showed that in most tissues, transcripts were evenly distributed. In brain, eye, kidney and intestine, however, Vbp1 was expressed in specific cell types. Moreover, expression of the human VBP1 gene was investigated in cerebellum and in various tumours of VHL patients encompassing haemangioblastomas, renal cell carcinomas and pheochromocytomas. In all of these tissues, VBP1 was ubiquitously expressed at low levels. However, no consistent differences in VBP1 expression levels could be detected between tumours and normal tissue. Mapping of the murine Vbp1 gene revealed conserved chromosomal localization between mouse and human in a region homologous to human Xq28.