Divergent genetic and epigenetic post-zygotic isolation mechanisms in Mus and Peromyscus

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.     

Widespread disruption of genomic imprinting in adult interspecies mouse (Mus) hybrids

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.     

Genetic dissection of X-linked interspecific hybrid placental dysplasia in congenic mouse strains.

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.     

Genetic and developmental analysis of X-inactivation in interspecific hybrid mice suggests a role for the Y chromosome in placental dysplasia

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.     

Towards identification of individual homologous chromosomes: comparative genomic hybridization and spectral karyotyping discriminate between paternal and maternal euchromatin in Mus musculus x M. spretus interspecific hybrids

We have developed an in situ technique to label individual euchromatic chromosome arms in interspecific crosses between Mus musculus (MMU) and M. spretus (MSP). The MMU and MSP genomes diverged 2-3 million years ago and show an overall sequence divergence of approximately 1%. Comparative hybridization of MMU versus MSP DNA and subsequent spectral analysis of the euchromatic hybridization profiles discriminated between maternal (MMU) and paternal (MSP) chromosomes in F(1) hybrids. Dispersed repetitive DNA elements were the preferred hybridization target of MMU DNA on maternal chromosomes and of MSP DNA on paternal chromosomes. Differences in centromeric satellite DNAs were detected by conventional fluorescence in situ hybridization and served as internal controls. Our experiments suggest that it is possible, in principle, to discriminate between paternal and maternal chromosomes on the basis of sequence differences.     

Maternal–foetal genomic conflict and speciation: no evidence for hybrid placental dysplasia in crosses between two house mouse subspecies

Interspecific hybridization between closely related mammalian species, including various species of the genus Mus, is commonly associated with abnormal growth of the placenta and hybrid foetuses, a phenomenon known as hybrid placental dysplasia (HPD). The role of HPD in speciation is anticipated but still poorly understood. Here, we studied placental and foetal growth in F₁ crosses between four inbred mouse strains derived from two house mouse subspecies, Mus musculus musculus and Mus musculus domesticus. These subspecies are in the early stage of speciation and still hybridize in nature. In accordance with the maternal–foetal genomic conflict hypothesis, we found different parental influences on placental and foetal development, with placental weight most affected by the father's body weight and foetal weight by the mother's body weight. After removing the effects of parents’ body weight, we did not find any significant differences in foetal or placental weights between intra‐subspecific and inter‐subspecific F₁ crosses. Nevertheless, we found that the variability in placental weight in inter‐subspecific crosses is linked to the X chromosome, similarly as for HPD in interspecific mouse crosses. Our results suggest that maternal–foetal genomic conflict occurs in the house mouse system, but has not yet diverged sufficiently to cause abnormalities in placental and foetal growth in inter‐subspecific crosses. HPD is thus unlikely to contribute to speciation in the house mouse system. However, we cannot rule out that it might have contributed to other speciation events in the genus Mus, where differences in the levels of polyandry exist between the species.     

DNA methylation in placentas of interspecies mouse hybrids

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.     

Immune presensitization and local intrauterine defences as determinants of success or failure of murine interspecies pregnancies

Putatively immuno-incompetent Mus musculus females exhibited failure to support pregnancy of Mus caroli embryos. These results for M. musculus females (i.e. treated by cyclosporine A, of the nu/nu genotype, and as an interspecies chimaera) can be explained in immunological terms. Mus musculus females possessed pre-sensitized cytotoxic T cells against Mus caroli antigen. Nu/nu mice possessed activated NK cells and macrophages, and selectively discriminated against Mus caroli embryos early in pregnancy unlike normal +/+ females; the requirement for T cells to activate non-specific cytotoxic effector mechanisms was bypassed in nu/nu mice. Mus caroli are not inbred, and interspecies chimaeras which are tolerant of the antigens on the Mus musculus donor strain were not tolerant of cells from unrelated Mus caroli. Interspecies chimaeras also behaved as if they were pre-sensitized to Mus caroli. Our results show that Mus caroli embryos recruit fewer active suppressor cells even when gestating in Mus caroli decidua as compared to Mus musculus embryos in Mus musculus decidua and that the ability of Mus caroli placental cells to directly inhibit cytotoxic effector cell killing was inherently less than the inhibitory activity of placental cells from Mus musculus. Mus caroli embryos therefore appear to be less well defended against maternal immune attack even when gestating in a uterus possessing compatible Mus caroli decidual tissue.     

Piglet and placental traits at term in relation to the estrogen receptor genotype in gilts

Liveborn piglets from gilts with estrogen receptor (ESR) genotype AA (95 AA-AA and 91 AA-AB piglets), AB (88 AB-AA, 118 AB-AB, and 37 AB-BB piglets), and BB (97 BB-AB and 89 BB-BB piglets) were compared after farrowing, to examine whether piglet ESR genotype (ESRp) nested within maternal ESR genotype (ESRm) affected placental traits at term, piglet birth weight, and growth until weaning. Furthermore, the relation of birth weight to various placental traits and the relations between placental traits were evaluated relative to ESR genotype. For this study, 62 Large White x Meishan F2 crossbred gilts (18 AA, 24 AB, and 20 BB) were used. The gilts belonged to a population in which the A allele is favorable for litter size. ESRp nested within ESRm did not affect placental length, weight, surface area and number of areolae. ESRp nested within ESRm affected amnion weight (AA-AA amnions were heavier than AA-AB, AB-AA and BB-AB amnions), placental weight after including placental surface area in the model (AA-AB placentae were lighter than AA-AA, AB-BB and BB-AB placentae), placental efficiency calculated as birth weight divided by placental weight (AB-AA placentae were less efficient than AA-AB placentae), and the relations of birth weight to placental weight and birth weight to number of areolae. The found differences imply an interaction of maternal and fetal ESR genotype on placental traits (especially weight and number of areolae) during fetal development. Furthermore, the found effects on placental and amnion weight might be the result of a difference in thickness or vascularization or both. The favorable ESR allele for litter size, i.e. the A allele, appears to be the unfavorable allele for pre-weaning piglet growth. Therefore, further research on ESR in relation to vascularization, weight and thickness of placentae. uterine size, endometrial gland development, and piglet growth is recommended.     

Perturbation of DNA repair gene expression due to interspecies hybridization

The effect of interspecies hybridization on gene regulation was examined using real-time polymerase chain reaction (RT-PCR) to measure the expression of five base-excision repair genes in brain, eye, gill, liver, and tailfin tissues from Xiphophorus parental species and F(1) hybrids. Relative mRNA levels of uracil N-glycosylase (Ung), Apurinic/apyrimidinic endonuclease (Ape1), polymerase-beta (Polb), flap endonuclease (Fen1), and DNA ligase (Lig1) were measured in three parental Xiphophorus species (X. maculatus Jp 163 B, X. helleri Sarabia, and X. andersi andC) and in two interspecies F(1) hybrids, the Sp-helleri hybrid (X. maculatus Jp 163 BxX. helleri Sarabia) and the Sp-andersi hybrid (X. maculatus Jp 163 BxX. andersi) to identify genes that undergo changes in expression levels upon interspecies hybridization. Significant differences in gene expression were observed between parental animals and their respective F(1) hybrids in both interspecies crosses. Generally, marked increases in DNA repair gene mRNA levels were observed across all tissues in F(1) hybrid animals from the Sp-helleri cross compared to either X. maculatus or X. helleri parents. In contrast, the Sp-andersi F(1) hybrid animals generally exhibited decreased base-excision repair gene expression, although this trend was more specific to individual tissues than observed for Sp-helleri hybrids.     

Antibody-Dependent Transplacental Transfer of Malaria Blood-Stage Antigen Using a Human Ex Vivo Placental Perfusion Model

Prenatal exposure to allergens or antigens released by infections during pregnancy can stimulate an immune response or induce immunoregulatory networks in the fetus affecting susceptibility to infection and disease later in life. How antigen crosses from the maternal to fetal environment is poorly understood. One hypothesis is that transplacental antigen transfer occurs as immune complexes, via receptor-mediated transport across the syncytiotrophoblastic membrane and endothelium of vessels in fetal villi. This hypothesis has never been directly tested. Here we studied Plasmodium falciparum merozoite surface protein 1 (MSP1) that is released upon erythrocyte invasion. We found MSP1 in cord blood from a third of newborns of malaria-infected women and in >90% following treatment with acid dissociation demonstrating MSP1 immune complexes. Using an ex vivo human placental model that dually perfuses a placental cotyledon with independent maternal and fetal circuits, immune-complexed MSP1 transferred from maternal to fetal circulation. MSP1 alone or with non-immune plasma did not transfer; pre-incubation with human plasma containing anti-MSP1 was required. MSP1 bound to IgG was detected in the fetal perfusate. Laser scanning confocal microscopy demonstrated MSP1 in the fetal villous stroma, predominantly in fetal endothelial cells. MSP1 co-localized with IgG in endothelial cells, but not with placental macrophages. Thus we show, for the first time, antibody-dependent transplacental transfer of an antigen in the form of immune complexes. These studies imply frequent exposure of the fetus to certain antigens with implications for management of maternal infections during pregnancy and novel approaches to deliver vaccines or drugs to the fetus.     

Loss of Igf2 imprinting in monoclonal mouse hepatic tumor cells is not associated with abnormal methylation patterns for the H19, Igf2, and Kvlqt1 differentially methylated regions

IGFII, the peptide encoded by the Igf2 gene, is a broad spectrum mitogen with important roles in prenatal growth as well as cancer progression. Igf2 is transcribed from the paternally inherited allele, whereas the linked H19 is transcribed from the maternal allele. Igf2 imprinting is thought to be maintained by differentially methylated regions (DMRs) located at multiple sites such as upstream of H19 and Igf2 and within Kvlqt1 loci. Biallelic expression (loss of imprinting (LOI)) of Igf2 is frequently observed in cancers, and a subset of Wilms' and intestinal tumors have been shown to exhibit abnormal methylation at H19DMR associated with loss of maternal H19 expression, but it is not known whether such changes are common in other neoplasms. Because cancers consist of diverse cell populations with and without Igf2 LOI, we established four independent monoclonal cell lines with Igf2 LOI from mouse hepatic tumors. We here demonstrate retention of normal differential methylation at H19, Igf2, or Kvlqt1 DMR by all of the cell lines. Furthermore, H19 was found to be expressed exclusively from the maternal allele, and levels of CTCF, a multifunctional nuclear factor that has an important role in the Igf2 imprinting, were comparable with those in normal hepatic tissues with no mutational changes detected. These data indicate that Igf2 LOI in tumor cells is not necessarily linked to abnormal methylation at H19, Igf2, or Kvlqt1 loci.     

Yy1 Gene Dosage Effect and Bi-Allelic Expression of Peg3

In the current study, we tested the in vivo effects of Yy1 gene dosage on the Peg3 imprinted domain with various breeding schemes utilizing two sets of mutant alleles. The results indicated that a half dosage of Yy1 coincides with the up-regulation of Peg3 and Zim1, suggesting a repressor role of Yy1 in this imprinted domain. This repressor role of Yy1 is consistent with the observations derived from previous in vitro studies. The current study also provided an unexpected observation that the maternal allele of Peg3 is also normally expressed, and thus the expression of Peg3 is bi-allelic in the specific areas of the brain, including the choroid plexus, the PVN (Paraventricular Nucleus) and the SON (Supraoptic Nucleus) of the hypothalamus. The exact roles of the maternal allele of Peg3 in these cell types are currently unknown, but this new finding confirms the previous prediction that the maternal allele may be functional in specific cell types based on the lethality associated with the homozygotes for several mutant alleles of the Peg3 locus. Overall, these results confirm the repressor role of Yy1 in the Peg3 domain and also provide a new insight regarding the bi-allelic expression of Peg3 in mouse brain.     

Proteomic analyses of the Xiphophorus Gordon-Kosswig melanoma model

Interspecies hybridization between the platyfish X. maculatus Jp 163 A, and the swordtail X. helleri (Sarabia), generates F(1) hybrids with pronounced melanin pigmentation. Backcrossing of F(1) hybrids with the X. helleri parent results in 25% of progeny that will spontaneously develop melanoma. We have applied proteomic methods to this Gordon-Kosswig (G-K) melanoma model to identify candidate proteins that exhibit modulated expression in fin tissue due to interspecies hybridization and progression of hybrid tissues to spontaneous melanoma. Difference Gel Electrophoresis (DIGE) was used to minimize the variability commonly observed in quantitative analyses of comparative protein samples. Following identification of up- or down-regulated protein expression by DIGE, candidate protein spots were identified by mass spectrometric sequencing. Several protein expression differences displayed in interspecies hybrids were identified and compared to distinct differences that occur upon backcrossing and progression to melanoma. These studies are important for the identification of distinct biochemical pathways involved in the variety of Xiphophorus interspecies hybrid tumor models.