1-Genetic and parent-of-origin influences on X chromosome choice in Xce heterozygous mice

Genetic and parent-of-origin influences on X chromosome choice in Xce heterozygous miceLisa Helbling Chadwick,1,2Huntington F.Willard11Institute for Genome Sciences&Policy,Duke University,Box3382,101Science Drive,Durham,North Carolina27708,USA2Department of Genetics,Case Western Reserve University,Cleveland,Ohio,44106,USAReceived:19April2005/Accepted:1June2005AbstractX chromosome inactivation is unique among dosage compensation mechanisms in that the two X chro-mosomes in females are treated differently within the same cell;one X chromosome is stably silenced while the other remains active.It is widely believed that,when X inactivation is initiated,each cell makes a random choice of which X chromosome will be silenced.In mice,only one genetic locus,the X-linked X controlling element(Xce),is known to influence this choice,because animals that are het-erozygous at Xce have X-inactivation patterns that differ markedly from a mean of0.50.To document other genetic and epigenetic influences on choice, we have performed a population-based study of the effect of Xce genotype on X-inactivation patterns.In B6CAST F1females(Xce b/Xce c),the X-inactivation pattern followed a symmetric distribution with a mean of0.29(SD=0.08).Surprisingly,however,in a population of Xce b/Xce c heterozygous B6CAST F2 females,we observed significant differences in both the mean(p=0.004)and variance(p=0.004)of the X-inactivation patterns.This finding is incompatible with a single-locus model and suggests that addi-tional genetic factors also influence X chromosome choice.We show that both parent-of-origin and naturally occurring genetic variation at autosomal loci contribute to these differences.Taken together, these data reveal further genetic complexity in this epigenetic control pathway.IntroductionIn female mammals,most of the genes on one of the two X chromosomes are silenced in every somatic cell,so that males and females each have effectively one functional X chromosome(Lyon1961).Al-though other organisms with dimorphic sex chro-mosomes also undergo some form of dosage compensation(Meller2000),X inactivation is un-ique in that two otherwise identical chromosomes are treated differently within the same nucleus.At around the time of implantation,each cell must choose which X chromosome will be inactivated and which will be active.As the cell continues to divide, this choice is inherited epigenetically by its daughter cells,so that the adult female is essentially a mosaic of two cell types.In extraembryonic tissues,this choice is determined by parental origin,where the paternal X is preferentially inactivated(Takagi and Sasaki1975;Huynh and Lee2001).In contrast,the cells in the epiblast of eutherian mammals undergo random X inactivation,and each chromosome has an equal probability of becoming the inactive X(Xi), irrespective of parental origin(for reviews see Clerc and Avner2003;Takagi2003).Although the choice between X chromosomes in somatic cells is generally referred to as being ran-dom,genetic evidence in mice has suggested that,in certain situations the choice is predictably biased in favor of one over the other X chromosome.Some four decades ago,Cattanach identified an X-linked locus responsible for this bias,referred to as the X controlling element,Xce(Cattanach and Isaacson 1967).Three alleles of Xce have been characterized in inbred mouse strains:Xce a(BALB/cByJ,CBA/J, C3H/HeJ),Xce b(C57BL/6J and DBA/2J),and Xce c (CAST/Ei)(Cattanach et al.1969;West and Chap-man1978;Johnston and Cattanach1981).The Xce alleles vary in their strength or likelihood that each will be preferentially located on the active X(Xa), such that the Xce a allele is the weakest while the Xce c allele is the strongest(Xce a<Xce b<Xce c). When any of these alleles is present in a homozygousCorrespondence to:H.F.Willard;E-mail:hunt.willard@DOI:10.1007/s00335-005-0059-2 Volume16,691À699(2005) ÓSpringer Science+Business Media,Inc.2005691state,X inactivation proceeds in an unbiased man-ner;both X chromosomes have an equal probability of being inactivated(Krietsch et al.1982).In this case,a population of Xce homozygous female mice would be expected to have an average X-inactivation pattern(representing the proportion of cells that have made each choice)of around50:50.However,in animals that are heterozygous at Xce,the chromo-some carrying the weaker Xce allele is more likely to be inactivated,which results in skewed X-inactiva-tion patterns.The degree of skewing depends on the combination of alleles present.For example,a pop-ulation of Xce a/Xce c heterozygous females,who carry the weakest allele in combination with the strongest allele,have X-inactivation patterns that are more skewed on average than those of Xce b/Xce c females(Johnston and Cattanach1981;de La Casa-Esperon et al.2002).Although the molecular basis of the Xce pheno-type is not yet understood,most models posit that it serves as a binding site for trans-acting factors that regulate other loci in the X-inactivation center,such as Xist,Tsix,or Xite(Borsani et al.1991;Brockdorff et al.1991;Lee et al.1999;Ogawa and Lee2003).In support of this hypothesis,mutagenesis screens have uncovered three autosomal loci that suppress the skewing of X chromosome choice normally seen in Xce heterozygotes(Percec et al.2002,2003).If the Xce genotype were the sole genetic influ-ence on X chromosome choice,one would predict that all populations of mice with a given Xce geno-type would have similar distributions of X-inactiva-tion patterns.However,suggestive data from several studies have raised the possibility that this may not be the case(Forrester and Ansell1985;Fowlis et al. 1991;Bittner et al.1997;Plenge et al.2000),indi-cating that parental origin of the X chromosomes may influence choice to some degree,even in the embryo itself.In all of these cases,the paternal X has a slight tendency toward becoming the Xi,which is reminiscent of the imprinted form of X inactivation (Huynh and Lee2001).In addition to imprinting, genetic variation at autosomal loci may also influence choice in embryonic tissues,although the nature of these loci and their role(s)in the X-inacti-vation pathway are unclear.Previously,we observed a slight difference be-tween the distribution of X-inactivation patterns of F1and F2females with the same Xce genotype using an allele-specific RT-PCR assay to measure the X-inactivation patterns in a variety of inbred mouse crosses(Plenge et al.2000).Although parental origin may explain the differences seen in these crosses(as the inheritance of the Xce c alleles were of opposite parental origin in F1versus F2females in that study),an alternative hypothesis is that genetic background can influence X chromosome choice.To investigate this further,we have performed a systematic study of X-inactivation patterns in larger F1,F2,and back-crossed populations,all of which are Xce hetero-zygotes.In this report,we present evidence that the X-inactivation patterns in both backcrossed and in-tercrossed populations differ significantly in both mean and variance from what is observed in the F1 cross,providing strong evidence that the Xce phe-notype can be modified by additional genetic factors. To explore the basis for these effects,we investigated the role of both parental origin and genetic back-ground in influencing X chromosome choice and demonstrate conclusively that each of these factors contributes to the differences we have observed be-tween these populations.Materials and methodsMice and mouse crosses.C57BL/6J(B6),BALB/cByJ (BALB),and CAST/Ei(CAST)mice used in these crosses were purchased from The Jackson Laboratory (Bar Harbor,ME).Mice were housed in accordance with IACUC guidelines.F1crosses were carried out using B6or BALB females and CAST males. F1progeny were then intercrossed to generate F2females.In addition,we backcrossed B6CAST F1 females to B6males and continued backcrossing for two to eight generations,selecting for the presence of CAST X chromosome alleles at each backcross generation.However,after the first backcross gen-eration both males and females carrying CAST al-leles were used.Ear biopsies were collected at weaning for later RNA isolation.RNA isolation and cDNA synthesis.RNA was isolated from ear biopsies and whole mouse embryos using the RNeasy Miniprep kit(Qiagen,Valencia, CA),according to the manufacturerÕs recommenda-tions.For ear RNA,the modified fibrous tissue pro-tocol was used.cDNA synthesis was carried out as described(Percec et al.2003),using random primers.Allele-specific expression assays.The Pctk1 expression assay was carried out as described previ-ously(Plenge et al.2000),except that products were separated and analyzed on an ABI3100capillary sequencer(Applied Biosystems,Foster City,CA).We designed a similar assay to Idh3g for use with animals that were heterozygous at Xce but not informative at Pctk1.This Idh3g assay was performed as the Pctk1 assay using the primers5¢-AACTATGGCCATGTGT ATGC-3¢and5¢-CTCCAATATCTGGGGTATGC-3¢,and using Taq a I for digestion of products.Genotyping.All genotyping was carried out by PCR amplification of microsatellite markers,and PCR products were separated on an ABI3100capillary sequencer.Genetic location of markers and primer sequences were taken from the Mouse Genome Database(Blake et al.2003).Mice were genotyped at markers across the X chromosome(DXMit55, DXMit165,DXMit75,DXMit42,DXMit41,DXMit97, DXMit151,DXMit156)to identify regions of hetero-zygosity and at flanking markers DXMit62(34.6cM) and DXMit64(45.0cM)to determine Xce($42cM) genotype.Because the exact location of the Xce locus has not yet been identified,animals with crossovers between DXMit62and DXMit64were excluded from analysis because of uncertainty of their Xce genotype.Embryo dissections.Embryos were collected at 10.5days post coitum(dpc)according to established protocols(Hogan1994).Mating was ascertained by checking for vaginal plugs,with the first day after mating designated as0.5dpc.The sex of embryos was determined by PCR amplification of the Smcx and Smcy genes,as described previously(Mroz et al. 1999).Statistical analyses.Statistical analyses were performed using the R statistical software(Ihaka and Gentleman1996).StudentÕs t-tests were performed using WelchÕs correction because we could not as-sume equal variances in the distributions being compared.Estimation of precursor cell number.The number of precursor cells present at the time of X chromosome choice was estimated using data we obtained in B6CAST F1whole embryos.We used a statistical approach similar to that of McMahon et al.(1983):n=p(1)p)/v,where n is the number of precursor cells present and p and v are the mean and variance,respectively,of the X-inactivation patterns in the F1embryo population.ResultsSignificant differences in the X-inactivation pattern distributions of F1and F2Xce heterozygous females.Many previous analyses of Xce phenotypes have been carried out using assays such as coat color variegation or protein allozyme studies.Although these approaches have proven quite valuable in establishing our general understanding of Xce ef-fects,they are not sufficiently precise to detect subtle differences in X-inactivation patterns that might point toward additional genetic influences on X chromosome choice.We previously developed sensitive allele-specific expression assays(Plenge et al.2000;Percec et al.2002),which have allowed us to address the complexity of the genetic regula-tion of X chromosome choice in greater detail.In order to extend our earlier observations on a small number of animals(Plenge et al.2000),we generated populations of B6CAST F1females,which have an Xce b/Xce c heterozygous genotype,and BALBCAST F1females,which have an Xce a/Xce c heterozygous genotype.As expected,we observed symmetrically distributed patterns of skewed X-inactivation patterns in both F1populations(Fig.1), where the degree of skewing predictably correlated with the Xce genotype(B6CAST F1:n=79, mean=0.29,SD=0.08;BALBCAST F1:n=78, mean=0.26,SD=0.09).These patterns are similar to those reported previously by us and by others for these crosses(Johnston and Cattanach1981;Plenge et al.2000;de La Casa-Esperon et al.2002;Percec et al.2002).We hypothesized that if X chromosome choice was governed by only a single locus(i.e.,Xce),F2 females with the same Xce genotype would have X-inactivation pattern distributions similar to their F1 counterparts,even though the composition of their autosomal genomes differed.To address this,we generated B6CAST F2and BALBCAST F2mice and selected Xce heterozygous females for analysis.The X-inactivation pattern distributions of the F2popu-lations differed significantly from those observed in the F1s(Fig.1).The most striking differences were seen when comparing B6CAST F1and F2(n=75, mean=0.25,SD=0.11)populations;these differed significantly in both mean(t-test p=0.005)and variance(F-test p=0.004),while the BALBCAST F1 and F2(n=73,mean=0.21,SD=0.09)populations differed significantly only in mean(t-test p=0.002, F-test p=0.86).In each of these comparisons,the mean X-inactivation pattern was significantly more skewed in the F2than in the corresponding F1pop-ulation.We also backcrossed B6CAST F1females to B6males for two to eight generations and determined the X-inactivation patterns of Xce het-erozygous female progeny of these crosses(n=95, mean=0.31,SD=0.13)(Fig.1A).As in the B6CAST F1-F2comparison,we observed highly significant differences in variance when compared with the F1population(F-test p=0.00002),although the means were not significantly different in this comparison(t-test p=0.44).Because each of these comparisons involved two populations with identi-cal Xce genotypes,these data are inconsistent with a single-locus model and suggest that genetic factors in addition to Xce influence X chromosome choice in mice.Parental origin of X chromosomes partially accounts for differences in X-inactivation patterns between F 1and F 2populations.In limited studies,several groups have noted that the parent-of-origin of Xce alleles may affect the X-inactivation patterns in Xce heterozygotes (Papaioannou and West 1981;For-rester and Ansell 1985;Fowlis et al.1991).Because the parental inheritance of Xce alleles is opposite in the F 2cross relative to the F 1cross,it is possible that the differences in X-inactivation pattern distributions that we have observed can be attributed entirely to differences in the parental origin of the Xce alleles in these two populations.To address this,we compared the X-inactivation patterns of backcross females with paternal inheritance of Xce b (n =51,mean =0.29,SD =0.13)with the X-inactivation patterns of back-cross females with maternal inheritance of Xce b (n =44,mean =0.35,SD =0.13)(Fig.2).Indeed,the mean X-inactivation pattern in these two populations differed significantly (t -test p =0.02),suggesting that parental origin contributes to the difference in means we observed between the F 1and F 2populations and indicating that this factor imposes a bias on X chro-mosome choice in addition to the Xce genotype.In contrast,however,parent-of-origin did not signifi-cantly affect the variance in X-inactivation patterns (F -test p =0.78),indicating that genetic factors other than parent-of-origin,such as the effects of naturally occurring genetic variation at autosomal loci that were not segregating in the backcross,must account for the overall differences observed between the F 1and F 2X-inactivation patterns.Genetic background affects X chromosome choice.To address the influence of genetic back-ground on X chromosome choice,we next compared populations with the same parent-of-origin of Xce alleles but different genetic backgrounds.We com-pared the X-inactivation patterns of B6CAST F 1fe-Fig.1.X-inactivation patterns (expressed as inbred strain allele expression/total expression)in F 1,F 2,and backcrossed mice.A .X-inactivation patterns were determined in samples from B6CAST F 1females (n =79),B6CAST F 2Xce het-erozygous females (n =75),and B6CAST backcross Xce heterozygous females (n =95).B .X-inactivation patterns were determined in samples from BALBCAST F 1females (n =78)and BALBCAST F 2Xce heterozygous females (n =73).Mean and SD are indicated for each group.Under a single-gene model,the mean and SD would be expected to be similar in the various B6CAST or BALBCASTpopulations.Fig.2.Effect of parental origin on the X-inactivation pat-tern.B6CAST backcrossed females were divided into two groups,according to whether X chromosome B6alleles were inherited from their father (Xp,n =51)or their mo-ther (Xm,n =44).The mean X-inactivation pattern is indicated for each group.The means differ significantly,indicating that parent-of-origin of the X chromosomes influences the X-inactivation pattern.males to backcrossed females (2-8generations of backcrossing:$75%À99%homozygous for B6alleles at autosomal loci)with maternal inheritance of Xce b ,and of B6CAST F 2females to backcrossed fe-males with paternal inheritance of Xce b (Fig.3).In the maternal comparisons,both mean and variance were significantly different (t -test p =0.01,F -test p =0.00005).The paternal comparisons showed the same trends,but did not reach significance (t -test p =0.08,F -test p =0.20),perhaps because fewer backcross animals were available.Alternatively,this may reflect the fact that the genetic backgrounds are more different in the maternal comparisons (the autosomal genome of the F 1is 100%hybrid,while the backcross is largely homozygous for B6alleles)than in the paternal comparisons,where both crosses have a significant degree of homozygosity for B6alleles at autosomal loci.These data indicate that genetic background significantly impacts the X-inactivation pattern;thus,naturally occurring ge-netic variation at autosomal modifier loci may also influence X chromosome choice and explain differ-ences in the distribution of X-inactivation patterns seen between the F 1and F 2populations,either through a primary influence on X chromosome choice or via secondary effects.Adult vs.embryo distributions.The differences we have observed in the mean and variance of the F 1and F 2populations could result from a primary effect on X chromosome choice;alternatively,segregating modifier loci in the F 2population may alter the X-inactivation pattern observed in adult ear samples through later,secondary effects unrelated to X inactivation per se ,such as tissue-specific selection at a particular allele on one or the other X chromo-some.To address this,we isolated RNA from B6CAST F 1and B6CAST F 2Xce heterozygous em-bryos.Ideally,one would assess primary vs.sec-ondary effects on choice in embryos at a time point closer to that of the initial choice ($5.5dpc).How-ever,earlier in postimplantation development,it can be difficult to ensure that all extraembryonic tissues have been removed before RNA extraction from embryonic cells.Because these tissues undergo an imprinted choice where the paternally inherited X chromosome is preferentially inactivated (Takagi and Sasaki 1975),any contamination of extraem-bryonic tissues in the whole-embryo RNA tested would artifactually suggest parent-of-origin effects.To overcome this,we used E10.5embryos,a stage where extraembryonic tissues were easily dissoci-ated.While secondary selection in response to ge-netic imbalance for large chromosomal segments can be detected by this time (Disteche et al.1979;Takagi et al.2002),it seems unlikely that selection for or against one or a small number of X-linked allelic differences would be so rapid.We collected 71B6CAST F 1and 78B6CAST F 2Xce heterozygous female embryos and usedtheFig.3.Effect of genetic background on the X-inactivation pattern.We compared crosses where parental inheritance of theX chromosomes was identical but the autosomal genetic composition differed.Maternal (B6X chromosome maternally inherited)comparison is in the left panel,paternal (B6X chromosome paternally inherited)comparison is the right panel.Mean and SD are indicated for each group.The means differ significantly in each comparison;the variance differs significantly in the maternal comparison only.entire embryo for RNA isolation(Fig.4A).We ob-served highly significant differences in the mean X-inactivation patterns of the F1and F2embryos(0.3 in the F1vs.0.25in the F2,t-test p=0.0001),even compared with the difference between adult popu-lations(adult F1vs.F2,t-test p=0.005).However, the difference in variance fell just under the95% significance level(F-test=0.055)and was not as great as the difference detected between the adult F1and F2populations.This suggests that parent-of-origin differences(affecting the mean X-inactiva-tion pattern)result from very early effect(s)on X chromosome choice,while genetic background ef-fects observed in adult populations(affecting the phenotypic variation observed in populations with segregating autosomal alleles)may result from a combination of primary and secondary effects on the X-inactivation pattern.X-inactivation patterns are known to be rela-tively consistent within the tissues of an individual mouse,but do vary slightly(Nesbitt1971;Johnston and Cattanach1981;Krietsch et al.1986;Plenge et al.2000)because each tissue is derived from an independent sampling event in progenitor cells.We considered the possibility that sampling whole-em-bryo RNA rather than adult tissue samples could reduce the variation in X-inactivation patterns ob-served not directly attributable to the initial choice between chromosomes,making them a more suit-able system for studies of X chromosome choice.To test this hypothesis,we compared the X-inactivation patterns of F1adults to those of F1embryos.As shown in Fig.4B,the distribution of X-inactivation patterns in F1whole embryos was significantly tighter than we observed in the F1adult ear biopsies; the mean did not change significantly,but the vari-ance was significantly reduced in the whole embryos (F-test p=0.007).Although this finding may not be surprising given that the whole embryo is a more representative sample than a specific tissue with respect to X inactivation,it does suggest that em-bryos may be an ideal system for future studies of X chromosome choice.Estimating precursor cell population from whole embryos.The variance observed in X-inacti-vation patterns can be used to estimate the approx-imate number of cells present at the time of X chromosome choice,as in previous studies(McMa-hon et al.1983;Baader et al.1996).The whole-embryo data are a useful sample with which to readdress this question because the variance in pat-terns is attributed to just one sampling event (choosing between the two chromosomes),rather than two(choosing a subset of X-inactivated cells that will then give rise to the tissue in question). Using an approach outlined previously(McMahon et al.1983;see Materials and methods),we used the F1whole-embryo data to determine the number of precursor cells present.Our data indicate that approximately70cells were present at the initial choice(95%CI,48À96cells),suggesting that X chromosome choice in the cells of the embryo proper occurs between 4.5and 5.5days post coitum,in agreement with previous estimates(Monk and Harper1979;Rastan et al.1980;McMahon et al. 1983;McMahon and Monk1983;Baader et al.1996) Fig.4.A.X-inactivation patterns in F1(n=71)and F2 (n=79)E10.5embryos.Mean and SD are indicated for each distribution.B.X-inactivation patterns in B6CAST F1 adults(n=79),using ear biopsy RNA,and in E10.5 B6CAST F1whole embryos(n=71).Mean and SD are indicated for each distribution.DiscussionX chromosome choice is a complex genetic trait regulated by both autosomal factors and parent-of-origin effects.Since the X inactivation hypothesis was first proposed over40years ago(Lyon1961), only a few factors,Xce,Xist,Tsix,and Xite,all of which are tightly linked in the X-inactivation center on the X chromosome,have been shown definitively to be involved in the genetic regulation of this pro-cess in mice(Cattanach and Isaacson1967;Penny et al.1996;Lee and Lu1999;Ogawa and Lee2003).In the current study,we have investigated whether X chromosome choice is governed by a single locus in normal,unmutagenized mouse strains.Our results suggest that choice is a complex trait,influenced by both autosomal factors and parent-of-origin effects, in addition to the Xce locus.Parent-of-origin effects in X inactivation have been suggested by several previous studies in which the paternally inherited X chromosome is preferen-tially inactivated(Forrester and Ansell1985;Fowlis et al.1991;Bittner et al.1997).While these sugges-tive studies indicated that parent-of-origin could be a factor in determining X chromosome choice in Xce heterozygous females,these studies generally could not rule out the influence of genetic background (Forrester and Ansell1985;Fowlis et al.1991).In most cases,the individuals tested were either F1 crosses between two different strains(Xce b/Xce c heterozygous)or they arose through only one gen-eration of backcrossing such that a significant degree of autosomal heterozygosity would remain.In fact, when congenic Xce heterozygotes(CBA/Ca-Pgk1a Xce c·CBA/Ca-Pgk1b Xce a)were analyzed,in which autosomal loci were completely homozygous,no significant difference was observed between re-ciprocal crosses(Forrester and Ansell1985).The current study investigated both possibilities and showed conclusively that genetic background and parent-of-origin are significant influences on X chromosome choice in Xce b/Xce c heterozygotes. Although we did not exhaustively analyze the BALBCAST(Xce a/Xce c)crosses to rule out genetic background effects on choice,the differences in mean X-inactivation patterns we observed between the BALBCAST F1and F2distribution suggest that these Xce alleles are also subject to parent-of-origin influences.It has been widely accepted that before X inacti-vation occurs,both X chromosomes are active in most or all cells(Adler et al.1977;Epstein et al.1978; Kratzer and Gartler1978).However,recent studies have suggested that before the initiation of random X inactivation in somatic cells,the paternally inherited X chromosome is in fact initially inactive before being reactivated and participating in random choice (Huynh and Lee2003;Mak et al.2004;Okamoto et al.2004;Sado and Ferguson-Smith2005).Although these studies disagree somewhat on the exact timing of pre-inactivation,they provide direct evidence that the maternal and paternal X chromosomes are not identical at an epigenetic level,even in the embryo proper.Furthermore,these studies provide a basis for considering the mechanism for parent-of-origin ef-fects in X chromosome choice and alternative hypotheses about the mode of action of Xce.For example,parent-of-origin effects could reflect differ-ential abilities of the Xce alleles to overcome pre-inactivation of the paternally inherited chromosome. Alternatively,these could suggest a completely dif-ferent mechanism of choice,recently proposed by Williams and Wu(2004),in which the choice be-tween chromosomes is dictated in part by an im-printed asymmetric mark on one DNA strand.Notably,we found that parent-of-origin effects alone did not explain all of the differences seen be-tween the F1and F2populations because we still observed significant differences in both mean and variance when we compared populations with iden-tical parental inheritance of X chromosomes but different autosomal genetic composition.This sug-gests that modifier alleles present in the parental strains influence X chromosome choice.Although it has always been assumed that trans-acting factors are critical to the choice process,there are only a few reports to support this.Previously,we identified three autosomal N-ethyl-N-nitrosourea(ENU)-in-duced mutations that influenced X chromosome choice,which provided the first direct evidence for such trans-acting factors(Percec et al.2002,2003). Although this screen proved quite useful in identi-fying some candidate loci,additional modifier loci may exist.Standard complex trait mapping tech-niques,such as quantitative trait mapping,may be usefully applied to X-inactivation phenotypes such as choice to identify further loci.Another possibility is to investigate other crosses,particularly with additional wild-derived strains.Wild strains capture a larger fraction of the genetic diversity available in M.musculus species than the classical inbred strains (Ideraabdullah et al.2004),and many such poly-morphisms could have functional consequences that may influence X chromosome choice. AcknowledgmentsThe authors are grateful to Weihong Jiang and Stephanie Merrett for their assistance with animal。