doi:10.1046/j.1420-9101.2003.00656.x

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

U. ZECHNER,* W. SHI,*à M. HEMBERGER,* H. HIMMELBAUER,* S. OTTO,* A. ORTH,§ V. KALSCHEUER,* U. FISCHER,* R. ELANGO,– A. REIS,** W. VOGEL, H. ROPERS,* F. RU¨ SCHENDORF**àà &R.FUNDELE*à *Max-Planck-Institute for Molecular Genetics, Ihnestrasse, Berlin, Germany Institute for Human Genetics, Johannes Gutenberg-University Mainz, Langenbeckstr, Mainz, Germany àDepartment of Development and Genetics, Uppsala University, Norbyva¨gen, Uppsala, Sweden §Laboratory of Genomes and Populations, University of Montpellier, Place Euge`ne Bataillon C.C., Montpellier, France –Discovery Bioinformatics, GlaxoSmithKline, Gunnels Wood Road, Stevenage SG1 2NY, UK **Molecular Genetics and Mapping Center, Max-Delbru¨ck-Center, Robert-Ro¨ssle-Strasse, Berlin, Germany Department of Human Genetics, University of Ulm, Ulm, Germany ààInstitute for Medical Biometry, Informatics and Epidemiology, Rheinische Friedrich-Wilhelms-University, Bonn, Germany

Keywords: Abstract hybrid dysgenesis; Interspecific hybridization in the rodent genera Peromyscus and Mus results in interspecies hybridization; abnormal placentation. In the Peromyscus interspecies hybrids, abnormal loss-of-imprinting; allelic interaction between an X-linked and the imprinted paternally Mus; expressed Peg3 locus was shown to cause the placental defects. In addition, Peromyscus; loss-of-imprinting (LOI) of Peg3 was positively correlated with increased placentation. 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 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- 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.

Introduction Correspondence: Reinald Fundele, Department of Development and New species arise when populations diverge sufficiently to Genetics, Uppsala University, Morbyva¨gen 18A, 75236 Uppsala, Sweden. become reproductively isolated. This isolation is initiated Tel.: +46 18 471 5769; Fax: +46 18 471 2684; by mechanisms acting before the union of the gametes E-mail: [email protected] Present address: Myriam Hemberger, Department of Biochemistry and (‘prezygotic’ mechanisms, such as mate discrimination Molecular Biology, University of Calgary, 3330 Hospital Drive, NW, and gametic incompatibility) and after the union of the Calgary, Alberta T2N 4N1, Canada gametes (‘post-zygotic’ mechanisms, including hybrid

J. EVOL. BIOL. 17 (2004) 453–460 ª 2004 BLACKWELL PUBLISHING LTD 453 454 U. ZECHNER ET AL.

inviability and sterility). Post-zygotic isolation is assumed Materials and methods to be due to three types of genetic differences between the species: chromosomal differences, different ploidy levels Animals or allelic differences (Coyne & Orr, 1998). Allelic differences in genes are believed to be the most common The following mice were used: Mus spretus (MSP) strains cause of post-zygotic isolation in animals. The basis of SEG, SFM and SMZ, and the inbred M. musculus (MMU) post-zygotic isolation via allelic differences typically strains C57BL/6 (B6) and BALB/c. In some experiments involves interactions among alleles at different loci, alleles (see below), B6 · C3H F1 hybrids (B6C3) were used. All that yield high fitness in their usual genetic backgrounds. MSP strains were derived and are maintained in Dobzhansky and Muller both saw that post-zygotic Montpellier. Their origin and characteristics have been reproductive isolation could develop via incompatibilities described (Bonhomme & Gue´net, 1996). F1 animals were among genetic loci when two allopatric populations with generated by mating MMU females with MSP males. identical genotypes evolve independently at two ore more Female mice were checked for vaginal plugs and plug day 3genetic loci (Dobzhansky, 1937; Muller, 1940). Thus, was counted as day 1 (e1). Conceptuses were dissected on additive and epistatic interactions between several genetic e18 except for one Peg3 LOI experiment, where dissection loci have been discussed extensively as the causes of was carried out on e14. After removal of foetal mem- reproductive isolation in the genus Drosophila (Orr, branes, placentas were weighed and frozen in liquid N2 1997). Consistent with Haldane’s rule (Haldane, 1922), until further use. Foetuses were used for genotyping. sterility or inviability of the heterogametic males is often To analyse the segregation of a paternally inherited observed in interspecific Drosophila hybrids. Peg3 locus with placental dysplasia we obtained fertile In mammals, interspecific hybridization frequently males heterozygous at the Peg3 locus using the following results in post-zygotic reproductive isolation because of breeding strategy: A (B6 · SMZ)F1 female was back- male sterility, abnormal growth and placental dysplasia crossed with a B6C3 male. One BC1 female was crossed (Gray, 1971; Allen et al., 1993; Zechner et al., 1996; with a B6C3 male. A fertile BC2 male was then mated Vrana et al., 2000). In the rodent groups Peromyscus and with a B6C3 female to generate the BC3 males used for Mus, very similar placental hybrid phenotypes were this study. Genotyping for X and chromosome 7 specific described (Rogers & Dawson, 1970; Zechner et al., 1996). markers (see below) showed that these BC3 males were Depending on the direction of the crosses and backcros- MMU over the whole length of the X and MMU/MSP ses, in both instances the placental phenotype manifested heterozygous over the proximal part of chromosome 7 to itself in increased or decreased growth of the placenta 18.0 cM (D7Mit 310) but homozygous MMU/MMU at and specifically the spongiotrophoblast layer. However, 26.4 cM (D7Mit 18). (BALB/c · SEG)F1 and (BALB/ different effects on foetal growth were observed (Rogers c · SMZ)F1 were mated with these BC3 males. & Dawson, 1970; Kurz et al., 1999). This conservation of placental phenotypes suggested that the same genes Allele-specific expression cause placental dysplasia in Peromyscus and Mus. Recently, Vrana et al. (2000) reported that genetic and RNA isolation from placental tissue and reverse transcrip- epigenetic incompatibilities underlie the parent-of-ori- tion (RT) were carried out using Trizol Reagent (Gibco) gin-effects on both embryonic and placental growth in and M-MLV reverse transcriptase (Promega). Prior to RT, hybrids between Peromyscus polionotus (PPO) and RNA samples were treated with RNAse-free DNAseI (1 U/ P. maniculatus (PMA). Thus, placental dysplasia was lg RNA) for 30 min at 37 °C. DNAseI digestion was linked to maternally inherited X-chromosomal loci with terminated by adding RQ1 DNAse stop solution (Prome- the Plp locus showing the most significant linkage (Vrana ga). Polymerase chain reaction (PCR) reactions were et al., 2000). This was also observed in murine interspe- performed on all RNA samples without prior RT to exclude cific hybrid placental dysplasia (IHPD) (Zechner et al., contamination with genomic DNA. Peg3 specific RT-PCR 1996). Also, in the PPO · (PMA · PPO) backcross (BC) analysis was performed exactly as described before (female always shown first) strong linkage of placental (Kuroiwa et al., 1996). To assess expression of X-linked hyperplasia to the PMA derived paternally expressed Peg3 genes in F1 hybrids, RNA was prepared from brain, liver, locus was detected. In addition, it was shown that loss-of- heart, spleen and eye, isolated from 4 (MMU · MSP) and 1 imprinting (LOI) with expression from the maternal Peg3 (MSP · MMU) F1 adult hybrid females. G6pdx and U2af1 allele and increasing Peg3 transcript levels were positively were amplified using the primers: 5¢-TCCACAAGAC- correlated with degree of placental hyperplasia (Vrana CTGAATCACC-3¢ and 5¢-TCACATAGGTGTCCTTGACC- et al., 2000). Here we present evidence that this gene is 3¢;and5¢-AGGCCAAAGAGTATGCCAGA-3¢ and 5¢-CCAT- not involved in generating placental dysplasia observed CAAAGCAATATGGC-3¢, respectively. PCR conditions in Mus interspecific hybrids indicating that in closely were 94 °C for 3 min; 35 cycles of 94 °C for 30 s, 55 °C related groups divergent molecular mechanisms may be for 30 s, 72 °C for 30 s; and 72 °C for 3 min. Single-strand recruited to produce phenotypically similar post-zygotic 4conformational polymorphism (SSCP) analysis was per- barriers against hybridization. formed as described before (Blagitko et al., 1999).

J. EVOL. BIOL. 17 (2004) 453–460 ª 2004 BLACKWELL PUBLISHING LTD Divergent genetic and epigenetic post-zygotic isolation mechanism 455

reactions by the random oligo labelling method. Label- Genetic mapping and linkage analysis ling reactions were incubated 60 min at 37 °C and Genomic DNA was prepared from e18 backcross embryos denatured by adding 10 lL 0.5 M NaOH. Unpurified derived from matings between (B6 · SEG)F1, probes were added directly into the hybridization (B6 · SFM)F1, and (B6 · SMZ)F1 females and B6 males solution containing the filters with the spotted PCR using the mammalian Genomic DNA Extraction Kit products. Hybridizations were performed in 10 mL of 5(Cambridge Molecular Technologies, Cambridge, UK). Church Buffer [0.5 M NaHPO4, 5% sodium dodecyl DNAs were typed using simple sequence length sulphate (SDS), 1 mM ethylenediaminetetraacetic acid] polymorphism markers (SSLPs) and interspersed repet- at 65 °C in 15 mL Falcon tubes in a water bath. Filters itive sequence markers (IRS). For SSLPs, a published were washed once at 65 °C in 2x SSC, 0.1% SDS for subset of markers was used as described previously 60 min and once at 65 °C in 0.2 · SSC, 0.1% SDS for (Schalkwyk et al., 1999; Table 1). Genotyping via IRS 60 min, and exposed to film for 4–14 days with inten- markers is based on the species-specific amplification of sifying screens. IRS-PCR products, owing to divergence in the sequence The MAPMAKER/QTL 1.9 computer package was or presence or absence of repetitive elements surround- applied for linkage analysis (Lander & Botstein, 1989). ing a single-copy DNA sequence (McCarthy et al., 1995). Order of loci was obtained from the Jackson Laboratory In our case, genotyping was accomplished by hybrid- Chromosome Committee Reports (http://www.informa izing genetically mapped MSP strain-specific IRS probe tics.jax.org/ccr/). MAPMAKER/EXP 3.0b was used to DNAs to filters of gridded IRS products of individual make the setup of genetic maps for all , backcross fetuses as described previously (Elango et al., searching for genotyping errors and controlling of loci 1996). The presence of a hybridization signal at a order. MAPMAKER/QTL 1.9 uses the technique of particular position on the filter grid indicated a mater- 8‘interval mapping’ for the calculation of LOD (likelihood nally inherited MSP allele of the corresponding back- of disequilibrium) scores. Interval mapping uses the cross foetus at the probe DNA locus, the absence of a power of a complete genetic map to discern weak effects hybridization signal a maternally inherited MMU allele from genetic distance between marker locus and putative of the corresponding backcross foetus at the probe DNA QTL (phenotype: non-transformed placenta weights). locus. The production of IRS-PCR genotyping filters and LOD scores were calculated in steps of 0.5 cM. To hybridizations were carried out as follows: 100 ng of genotype the BC3 males and their offspring, the follow- DNA from each backcross foetus was amplified in ing X and chromosome 7 specific SSLP markers were 100 lL reactions with 50 pmoles of primer B1MvsCH used: DXMit 54–3.8 cM; DXMit 57–5.9 cM; DXMit 50– and five units of Taq Polymerase (Amersham Biosciences, 14.1 cM; DXMit 143–26.0 cM; DXMit 8–32.0 cM; DXMit 6Freiburg, Germany) under the following conditions: 114–40.2 cM; DXMit 65–48.5 cM; DXMit 37–56.0 cM; 5 min at 94 °C followed by 40 cycles of 10 s at 94 °C; DXMit 28–65.7 cM; and D7Mit 306–1.7 cM; Peg3 - 30 s at 50 °C; 60 s at 72 °C followed by 5 min at 72 °C. 6.5 cM; D7Mit 310–18.0 cM; D7Mit 18–26.4 cM; D7Mit All PCRs were performed in a Perkin Elmer 9600 151–65.5 cM. thermal cycler in 96 well plates. The reactions were analysed by running 5 lL of the PCR products in 1.5% Histology 7agarose gel in 1 · TBE (90 mM Tris, 90 mM borate, 20 mM EDTA, pH 8.0) visualized under UV lamp. The Placentas were fixed in Serra’s fixative overnight at 4 °C PCR products were spotted as previously described and then processed for routine paraffin histology. Sec- (Hunter et al., 1994). Genetically mapped MSP strain- tions were cut at 7 lm on a Leica rotary microtome, specific marker probe DNAs were labelled in 25 lL rehydrated and stained with haematoxylin and eosin.

Results Table 1 LOD scores of markers on mouse chromosome 7 for placental weight in the MSM BC. Allelic expression of Peg3 in BC1 placentas Number of To address the question whether autosomal loci contrib- Marker cM LOD scores conceptuses analyzed ute to IHPD, we performed a genome-wide polymorphic Peg3 6.5 0.071 147 marker screen of (MMU · MSP) · MMU (msm) back- D7Mit227 16.0 0.185 131 cross foetuses. This analysis provided no evidence for D7Mit145 26.4 0.261 129 genetic linkage between IHPD and proximal chromo- D7Hun22 37.0 0.466 161 some 7. None of the analysed markers on mouse D7Mit31 44.0 0.152 131 chromosome 7 exceeded the statistical significance level D7Mit238 53.0 0.254 132 of a LOD score >2.0 (Table 1). In the next step, we D7Hun25 59.0 0.482 161 performed a RT-PCR/RFLV analysis of 1 e14 and 13 e18 D7Mit46 69.0 0.606 55 Peg3 heterozygous, hyperplastic MSM placentas weighing

J. EVOL. BIOL. 17 (2004) 453–460 ª 2004 BLACKWELL PUBLISHING LTD 456 U. ZECHNER ET AL.

more than 160 mg to assess the parent-of-origin-depend- the weight of a normal e18 placenta. In Peromyscus, Peg3 ent expression of the Peg3 gene. Peg3 expression was LOI was observed in placentas with about twice the strictly paternal in all placentas analysed (Fig. 1a). Thus, weight of a normal placenta (Vrana et al., 2000). While no indication for Peg3 LOI was seen in MSM placentas. these combined results excluded Peg3 LOI as modifier of Placental weights were 287 mg on e14 and murine IHPD, they allowed no conclusion regarding the 261 ± 92.9 mg (mean ± SD) on e18. The largest of these role of interactions between Peg3 and X-chromosomal e18 placentas weighed 456 mg, approximately 4.5 times loci.

(a) MSM

1(106 mg) 2(87.6 mg) 3(88.3 mg) 4(287 mg) 5(153 mg) MSP MMU

(b) 1 23456789

Weight (mg) 151 90.1 111 430 147 293 91.5 86.6 159 Peg3 genotype het het Mmu het het het het het Msp Peg3 allelic expression Msp Mmu Mmu Msp Msp Mmu Msp Msp Msp 3.8 5.9 14.1 26.0 32.0 40.2 48.5 56.0

X-chromosome (cM) 65.7

Mmu Msp Heterozygous

(c)

(d)

J. EVOL. BIOL. 17 (2004) 453–460 ª 2004 BLACKWELL PUBLISHING LTD Divergent genetic and epigenetic post-zygotic isolation mechanism 457

placentas exhibited increased weight and abnormal Size of placentas with both MSP derived paternal morphology. Placental weights were 190, 267, 388, and Peg3 and X-chromosome 430 mg (exemplified for one of these litters in Fig. 1b). For their experiments, Vrana et al. (2000) had used a BC This is well within the weight range of hyperplastic between PPO females and (PMA · PPO)F1 males in placentas with MSP X-chromosomes and MMU Peg3 in which PMA and PPO derived paternal Peg3 alleles the standard MSM BC (Zechner et al., 1996). Comparat- segregate (Vrana et al., 2000). This strategy allowed the ive histological analysis of this 430 mg (Fig. 1c) placenta detection of paternally, but not maternally, expressed with a standard MSM BC placenta of 434 mg (Fig. 1d) genes that segregate with placental dysplasia in Pero- showed no significant histological differences. Both myscus hybrids. However, (MMU · MSP)F1 hybrid placentas exhibited increased growth of spongiotropho- males are sterile. Thus, we were forced to use a BC blast layer and increased differentiation of spongiotro- between (MMU · MSP)F1 females and MMU males in phoblast to glycogen cells (Fig. 1c,d). which only the inactive MMU and MSP derived maternal Peg3 alleles segregate and the active paternal Peg3 allele is Determinants of foetal growth on the X-chromosome always derived from MMU. This in turn precluded variation of the paternal genome in the backcross and Vrana et al. (2000) showed that foetal and placental detection of linkage to paternally expressed genes except growth effects are in part because of maternally in the case of LOI. To circumvent this problem, we expressed X-linked PPO locus. Specifically, they demon- generated BC3 (MSMMM) males heterozygous at Peg3 strated that male hybrids display a body overgrowth and mated them with (MMU · MSP)F1 females. In the phenotype linked to a PPO genotype at the X-chromo- case of an allelic interaction between a maternally somal Plp locus. Female hybrids showed normal body inherited X-chromosomal locus and a paternally inher- growth. The authors explained this by skewing of X ited Peg3 locus the assumption was that the presence of inactivation in favour of an active PMA X-chromosome. an MSP-derived maternal X-chromosome together with To determine whether hybridization in the genus Mus an active paternal MSP-derived Peg3 allele (or other MSP has an influence on body growth, we performed a derived genes in close vicinity to Peg3) in the same statistical analysis of e18 embryonic body weights in placenta should lead to normal allelic interaction and no MSM hybrids. In contrast to Peromyscus hybrids, weights genetic incompatibilities. Consequently, the correspond- of both male and female hybrids exhibited strong ing placenta should exhibit normal weight and morphol- variability, either exceeding or falling below the parental ogy. In five such litters, four placentas were obtained that range (Fig. 2a). However, in males, this was more had an MSP-derived X-chromosome and expressed the pronounced. In both sexes, embryonic growth showed MSP Peg3 allele. Contrary to our assumption, all these significant linkage to the X-chromosome. In males, linkage extended over the whole length of the X-chromosome (Fig. 2c). In females, significant linkage Fig. 1 (a) Allelic expression of Peg3 in e14 MSM hybrid placentas. was detected for markers on the central and distal Allele-specific expression was determined by RT-PCR analysis of X-chromosome only (Fig. 2b). Increased body weight placental RNAs from three normal sized placentas with MMU was associated with MSP alleles, reduced weight with genotype at the maternally inherited DXMit8 allele (1–3) and two MMU alleles on the X-chromosome. In both sexes, the hyperplastic placentas that were MSP at the maternal DXMit8 allele most significant linkage was detected around DXMit8 at (4, 5). Peg3 genotyping had demonstrated that all foetuses except 3 32 cM (males: 5.404; females: 3.878) and in the region had been heterozygous for Peg3 (not shown). (b) Placental weights, between markers DXHun25 at 45 cM and DXMit37 at X and chromosome 7 genotypes and Peg3 allelic expression in nine 56 cM (males: 5.572; females: 3.398). For all X-chromo- conceptuses derived from one of the five (MMU · MSP) F1 · BC3 matings that were analysed. In this litter, placenta 4 was strongly somal markers tested, male weights showed a higher hyperplastic with 430 mg, although it was MSP over the whole LOD-score than female weights. length of the X, was MMU/MSP heterozygous at markers D7Mit 307 (1.7 cM), Peg3 (6.5 cM), and D7Mit 310 (18.0 cM), and expressed the X-inactivation in MS F1 hybrids MSP Peg3. In addition, placentas 2 and 8 had similar weights and were both MMU for the whole X, although 2 expressed MMU Peg3 Linkage of female overgrowth to the X-chromosome and 8 MSP Peg3. (c) Haematoxylin/eosin staining of paraffin section suggested that, in contrast to female Peromyscus hybrids, of e18 MSP X - MSPPeg3 placenta weighing 430 mg. The lighter female Mus hybrids display no skewed X-inactivation in staining areas indicated by arrow-heads are spongiotrophoblast favour of the MMU X-chromosome. This was determined derived glycogen cells that have ‘invaded’ through the labyrinth to by RT-PCR/SSCP analysis of allelic expression of the the chorionic plate. (d) Haematoxylin/eosin staining of paraffin X-linked genes (Fig. 2d) and (not shown) in section of an e18 MSM MSP X-MMU Peg3 placenta weighing G6pdx U2af1 434 mg. Glycogen cells again are indicated by arrow-heads. The female (MMU · MSP)F1 hybrids. The results provided different shapes of these placentas (‘kidney’-shaped for C, ‘pancake’- no indication for skewed X-inactivation in these F1 shaped for D) are both within the normal range for a MSM BC and females. For both X-chromosomal genes, all tissues can be observed within a single litter. showed equal expression levels from MMU and MSP

J. EVOL. BIOL. 17 (2004) 453–460 ª 2004 BLACKWELL PUBLISHING LTD 458 U. ZECHNER ET AL.

Fig. 2 (a) Distribution of e18 embryonic body weights in MSM and C57BL/6 (maternal MMU strain) individuals. Red bars, MSM females (n ¼ 73); blue bars MSM males (n ¼ 85); green bars: C57BL/6 (B6) individuals (n ¼ 29). (b and c) Association between X-chromosomal markers and embryonic weight in the female (b) and male (c) MSM individuals. LOD score plot for embryonic weight (Left). Markers are depicted on the right according to their genetic distance from the centromere (top) to the telomere (bottom) of the X-chromosome. (d) Allelic expression of the X-linked G6pdx gene as determined by SSCP analysis in parental species (MMU, MSP) and tissues of three different (MMU · MSP)F1 mice (1–3). Similar results were obtained with G6pdx RT-PCR/SSCP analysis of RNAs isolated from one additional (MMU · MSP) and one (MSP · MMU) female and with U2af1 RT-PCR/SSCP analysis of samples from all five hybrid females. alleles. Thus, X-inactivation in the female F1 hybrids is conferring MMU X-chromosome in any given tissue random and the mosaic activity of both overgrowth explain the less pronounced over growth and lower LOD conferring MSP X-chromosome and the normal growth scores in female hybrids.

J. EVOL. BIOL. 17 (2004) 453–460 ª 2004 BLACKWELL PUBLISHING LTD Divergent genetic and epigenetic post-zygotic isolation mechanism 459

Discussion between the X-chromosome and the paternally derived Peg3 allele these conceptuses developed massively hyper- Intriguingly, interspecies hybridization and the associated plastic placentas, whereas the placentas of their hybrid dysgenesis effects in Mus and Peromyscus exhibit XmMMUXpMMA and XmMMUY littermates were slightly both similarities and dissimilarities. Thus, the genetic hypoplastic (Zechner et al., 1997). Thus, these data also effects of the X-chromosome on foetal and placental argue against both an abnormal allelic X-Peg3 interaction growth are observed in both groups, whereas the epi- and a strong role of imprinting disturbance in IHPD. genetic effects on X-inactivation are unique to Peromys- Our findings must be considered in the context of the cus. In this context it is important to note that the genetic conflict model. This model suggests that during involvement of X-linked loci in both groups is in agree- pregnancy a genetic conflict exists between maternal and ment with the ‘rule’ of speciation that genes affecting foetal genomes. The maternal genome tries to limit the reproductive isolation are typically found on the transfer of nutrients to the foetus whereas the foetal X-chromosome (Coyne & Orr, 1989). The most striking genome attempts to increase the transfer of nutrients difference is that in Peromyscus placental dysplasia follows from the mother. A similar conflict within foetal cells the general Muller and Dobzhansky model of speciation between paternally expressed growth-promoting genes with strong negative interactions between X-chromoso- and maternally expressed growth-inhibiting genes is mal loci and Peg3 or other paternally expressed genes close suggested to be causative for the development of genomic to Peg3 (Dobzhansky, 1937; Muller, 1940). This was not imprinting (Haig & Graham, 1991; Moore & Haig, 1991). observed in Mus. In the MSM BC, linkage to nonimprinted Genetic conflicts have a tendency to escalate, with each genes and maternally expressed imprinted genes should move matched by a countermove. Because of its pivotal have been visible, whereas detection of linkage to any role in foetal growth control the mammalian placenta is paternally expressed imprinted genes is impossible. How- regarded as a major site of genetic conflict (Haig, 1993; ever, in their Ppo · (Pma · Ppo) BC, Vrana et al. (2000) Constaˆncia et al., 2002). Placental morphology is charac- found no linkage to any other of the imprinted regions terized by a unique diversity between or even within apart from that containing Peg3 which was tested by us mammalian orders making the placenta more variable in through the use of matings between BC3 males heterozy- structure than any other mammalian organ (Mossmann, gous at Peg3 and (MMU · MSP) F1 females. Thus, the 91937; Gopalakrishna & Karim, 1979). Our results occurrence in such matings of fully hyperplastic placentas strongly support the view that the evolutionary dynamics with MSP X-chromosomes and active MSP Peg3 alleles of move and countermove intrinsic to the genetic conflict does not support a major role of allelic interaction between result in rapid evolution of the molecular mechanisms a maternally inherited MSP derived X-chromosomal locus governing placental development and consequently in a and a paternally inherited MMU derived Peg3 locus in Mus rapid evolution and morphological diversification of the IHPD. In addition, it should be noted that in a previous placenta. study, designed to test the role of imprinted genes in IHPD, In conclusion, our results, when compared with those which entailed the use of F1 interspecies Xp 0 foetuses, it of Vrana et al. (2000) suggest for the first time that should have been possible to detect the influence of different molecular mechanisms are involved in the paternally expressed genes (Xp stands for paternally generation of an almost identical hybrid dysgenesis derived X-chromosome, Xm for the maternal X-chromo- effect. This may indicate a further level of complexity some). In this study, we had made use of MMU females in the (putative) function of epigenetic mechanisms in carrying the X-chromosomal inversion In(X)1H that speciation that is demonstrated by the rapid evolution of causes the consistent appearance of Xp0 female offspring. regulation of imprinting (Vrana et al., 1998). When these females were mated with M. macedoni- pMMA mMMU pMMA mMMU cus(MMA) males, X 0, X X , and X Y Acknowledgements conceptuses were obtained. In the placentas of XmMMU XpMMA and XmMMUY conceptuses only the maternally This work was supported by grants from the Svenska inherited XmMMU is active, whereas in the placentas of Vetenskapsra˚det to R.F. and by the Max-Planck- XpMMA0 conceptuses only the paternally inherited XpMMA Gesellschaft. We are grateful to Horst Hameister and chromosome is present and therefore active. Concerning Jo¨rn Walter for helpful discussions and for reading the the constitution of the autosomal genome, XpMMA0, manuscript. XmMMUXpMMA or XmMMUY conceptuses are identical. Consequently, it can be assumed that allelic expression References of autosomal imprinted genes was also identical in the XpMMA0 placentas when compared with XmMMUXpMMA Allen, W.R., Skidmore, J.A. & Antczak, D.F. 1993. Effects of fetal and XmMMUY placentas. In the XpMMA0 placentas, a genotype and uterine environment on placental development paternally derived MMA X-chromosome and a paternally in equids. J. Reprod. Fert. 97: 55–60. derived MMAPeg3 allele were active in the same tropho- Blagitko, N., Schulz, U., Schinzel, A.A., Ropers, H.H. & blast cell. Despite the presence of full genetic compatibility Kalscheuer, V.M. 1999. Gamma2-COP, a novel imprinted

J. EVOL. BIOL. 17 (2004) 453–460 ª 2004 BLACKWELL PUBLISHING LTD 460 U. ZECHNER ET AL.

gene on chromosome 7q32, defines a new imprinting cluster Kurz, H., Zechner, U., Orth, A. & Fundele, R. 1999. Lack of in the . Hum. Mol. Genet. 8: 2387–2396. correlation between placenta and offspring size in mouse Bonhomme, F. & Gue´net, J.-L. 1996. The and interspecific crosses. Anat. Embryol. (Berl). 200: 335–343. its wild relatives. In: Genetic Variants and Strains of Lander, E. & Botstein, D. 1989. Mapping Mendelian factors The Laboratory Mouse (M. F. Lyon, S. Rastan & S. D. M. underlying quantitative traits using RFLP linkage maps. Brown, eds), pp. 1577–1596. Oxford University Press, Genetics 121: 185–199. London. McCarthy, L., Hunter, K., Schalkwyk, L., Riba, L., Anson, S., Constaˆncia, M., Hemberger, M., Hughes, J., Dean, W., Ferguson- Mott, R., Newell, W., Bruley, C., Bar, I., Ramu, E., Housman, Smith, A., Fundele, R., Stewart, F., Kelsey, G., Fowden, A., D., Cox, R. & Lehrach, H. 1995. Efficient high resolution Sibley, C. & Reik, W. 2002. Placental-specific IGF2 is a genetic mapping of mouse IRS-PCR products, towards major modulator of placental and fetal growth. Nature. 417: integrated genetic and physical mapping of the mouse 945–948. genome. Proc. Natl. Acad. Sci USA 92: 5302–5306. Coyne, J.A. & Orr, H.A. 1989. Two Rules of Speciation. In: Moore, T. & Haig, D. 1991. in mammalian Speciation and its Consequences (D. Otte & J. Endler, eds), pp. development: a parental tug-of-war. Trends Genet. 7: 45–49 180–207. Sinauer Associates, Sunderland, MA. Mossmann, H.W. 1937. Comparative morphogenesis of the Coyne, J.A. & Orr, H.A. 1998: The evolutionary genetics of foetal membranes and accessory uterine structures. Contr. speciation. Phil.Trans. R. Soc. Lond. B 353: 287–305 Embryol. Carnegie Instn. 26: 127–146. Dobzhansky, T. 1937. Genetics and the Origin of Species. Columbia Muller, H.J. 1940. Bearing of the Drosophila work on system- University Press, New York. atics. In: The New Systematics (J. S. Huxley, ed.), pp. 185–268. Elango, R., Riba, L., Housman, D. & Hunter, K. 1996. Generation Clarendon, Oxford. and mapping of Mus spretus strain-specific markers for rapid Orr, H.A. 1997. Haldane’s rule. A. Rev. Ecol. Syst. 28: 195–218. genomic scanning. Mamm. Genome 7: 340–343. Rogers, J.F. & Dawson, W.D. 1970. Foetal and placental size in a Gopalakrishna, A. & Karim, K.B. 1979. Fetal membranes and Peromyscus species cross. J. Reprod. Fertil. 21: 255–262. placentation in Chiroptera. J. Reprod. Fert. 56: 417–429. Schalkwyk, L.C., Jung, M., Daser, A., Weiher, M., Walter, J. Gray, A.P. 1971. Mammalian Hybrids. Commonwealth Agricul- Himmelbauer, H. & Lehrach, H. 1999. Panel of microsatellite tural Bureau, Edinburgh. markers for whole-genome scans and radiation hybrid Haig, D. 1993. Genetic conflicts in human pregnancy. Quart. Rev. mapping and a mouse family tree. Genome Res. 9: 878–887. Biol. 68: 495–532. Vrana, P.B., Guan, X.J., Ingram, R.S. & Tighman, S.M. 1998. Haig, D. & Graham, C. 1991. Genomic imprinting and the Genomic imprinting is disrupted in interspecific Peromyscus strange case of the insulin-like growth factor II receptor. Cell. hybrids. Nat. Genet. 20: 362–365. 64: 1045–1046. Vrana, P.B, JFossella, J.A., Matteson, P., Del Rio, T., O’Neill, M.J. Haldane, J.B.S. 1922. Sex-ratio and unisexual sterility in hybrid & Tilghman, S.M. 2000. Genetic and epigenetic incompat- animals. J. Genet. 12: 101–109. ibilities underlie hybrid dysgenesis in Peromyscus. Nat. Genet. Hunter, K.W., Ontiveros, S.D., Watson, M.L., Stanton, V.P. Jr, 25: 120–124. Gutierrez, P., Bhat, D., Rochelle, J., Graw, S., Ton, C., Zechner, U., Reule, M., Orth, A., Bonhomme, F., Strack, B., Schalling, M., Aburatani, H., Brown, S.D.M., Seldin, M.F. & Gue´net, J.L., Hameister, H. & Fundele, R. 1996. An Housman, D. 1994. Rapid and efficient construction of yeast X-chromosome linked locus contributes to abnormal placental artificial chromosome contigs in the mouse genome with development in mouse interspecific hybrids. Nat. Genet. 12: interspersed repetitive sequence PCR (IRS-PCR): Generation 398–403. of a 5-cM. >5 megabase contig on mouse Chr. 1. Mamm. Zechner, U., Reule, M., Burgoyne, P., Schubert, A., Orth, A., Genome 5: 597–607. Hameister, H. & Fundele, R. 1997. Paternal transmission of Kuroiwa, Y., Kaneko-Ishino, T., Kagitani, F., Kohda, T., Li, L.L., X-linked placental dysplasia in mouse interspecific hybrids. Tada, M., Suzuki, R., Yokoyama, M., Shiroishi, T., Wakana, S., Genetics. 146: 1399–1405. Barton, S.C., Ishino, F. & Surani, M.A. 1996. Peg3 imprinted gene on proximal chromosome 7 encodes for a zinc finger Received 1 July 2003; accepted 10 September 2003 . Nat. Genet. 12: 186–190.

J. EVOL. BIOL. 17 (2004) 453–460 ª 2004 BLACKWELL PUBLISHING LTD