Developmental progression of Gpd expression from the inactive X chromosome of the virginia opossum

Metatherian (marsupial) mammals possess a non-random form of X-chromosome inactivation in which the paternally-derived X is always the one inactivated. To examine the progression of X-linked gene expression during metatherian development, we compared relative levels of the maternally and paternally encoded Gpd gene products in heterozygous female Virginia opossums (Didelphis virginiana) across a moior portion of the developmental period. Panels of tissues obtained from fetuses, newborns, and pouch young were examined via polyacrylamide gel electrophoresis of the G6PD protein. As in adults, G6PD phenotypes in these developmental stages were highly skewed in favor of the maternal allele product, but in some tissues there was a marked increase in paternal allele expression with advancing developmental age. However, even by 42 days of post-partum development, expression of the paternal Gpd allele had not attained the adult, tissue-specific activity pattern. Our findings indicate remarkable developmental changes in the activity of the paternal allele in several tissues/organs continuing well into mid pouch-life stages and beyond. Specifically we found that 1) a substantially repressed paternal Gpdgene is present in the cells of female stage 29 fetuses and later developmental stages, 2) the activity state of the paternal Gpd gene is not fixed during early embryonic development in this species, 3) maior changes in paternal Gpd expression occur in advanced developmental stages and comprise a maturation of the gene expression pattern during ontogeny, and 4) alterations of paternal Gpd allele activity during development occur in a tissue-specific manner. © 1995 Wiley-Liss, Inc.     

Marsupial--mouse cell hybrids containing fragments of the marsupial X chromosome.

Hybrids were obtained from fusions of HPRT-deficient mouse fibroblasts and marsupial lymphocytes. These hybrids retained no identifiable marsupial chromosomes, but all expressed the marsupial form of HPRT. Half the clones also expressed marsupial PGK-A, and half of these also marsupial G6PD; no other marsupial allozyme markers were detected. Since G6PD is known to be sex linked in these species, we conclude that Hpt and Pgk-A are also located on the X chromosome and the markers lie in the order Hpt-Pgk-A-Gpd.     

X-linked gene expression in metatherian fibroblasts: Evidence from theGpd andPgk-A loci of the Virginia opossum and the red-necked wallaby

Fibroblasts cultured from ear pinna biopsies of Virginia opossums (Didelphis virginiana) and red-necked wallabies (Macropus rufogriseus) were examined electrophoretically to determine the relative expression levels of the maternally and paternally derived alleles at X-linked, enzyme-coding loci. Only the maternally derived allele was expressed at thePgk-A locus in fibroblasts of heterozygousD. virginiana (M. rufogriseus not examined), but fibroblasts of both species exhibited evidence of paternal allele expression a t theGpd locus. Furthermore, the heterozygous G6PD phenotypes in both species were skewed in favor of the maternal gene product, as expected if the paternal allele is only partially (incompletely) expressed. ForM. rufogriseus this result is contrary to a previous finding which suggested equal expression of bothGpd alleles in cultured fibroblasts of this species. The present results suggest that X-linked genes in metatherian fibroblasts are subject to the same kind of determinate, paternal allele inactivation, incomplete at some loci, described previously for X-linked genes in adult tissues and that the pattern of paternal X-linked gene expression in these cells is independent of the patterns in the tissues from which the fibroblasts are derived.The work was supported in part by grants from the National Institutes of Health (Biomedical Research Support Grant RR-05519) and the National Science Foundation (DCB 8516949).     

Lack of correlation between Gpd expression and X chromosome late replication in cultured fibroblasts of the kangaroo Macropus robustus

Cultured fibroblasts and lymphocytes from M. robustus females heterozygous for the X-linked Gpd gene were examined electrophoretically and cytologically. Gpd expression in lymphocytes was restricted to the maternal allele while in fibroblasts there was also partial expression of the paternal allele. The Gpd gene is thought to be located on the long arm of the X chromosome. However, in fibroblasts the long arm of the paternal X chromosome showed no indication of an early replicating segment.     

Parental influences on expression of glucose-6-phosphate dehydrogenase, G6pd, in the mouse; a case of imprinting

Glucose-6-phosphate dehydrogenase (G6PD) activity was measured in blood from heterozygotes for the normal allele G6pda and the low activity allele G6pda-mlNeu. In adult mice lower activity was found in G6pda/G6pda-mlNeu than in the reciprocal heterozygote G6pda-mlNeu/G6pda (the maternal allele being listed first). Thus, either the paternally derived allele was over-expressed or the maternally derived allele was under-expressed. By contrast, in younger mice the difference in G6PD activity in reciprocal crosses was less marked. The findings are interpreted in terms of differential imprinting of maternally and paternally inherited information. The explanation offered for age related differences is that, as a consequence of imprinting, either the paternal X-chromosome is preferentially reactivated, or cells in which the paternally derived allele is active are at a selective advantage, and proliferate better than those in which the maternally inherited allele is active.     

Histochemical differences in expression of X-linked glucose-6-phosphate dehydrogenase between ectoderm- and endoderm-derived embryonic and extra-embryonic tissues

We examined the activity of X-linked glucose-6-phosphate dehydrogenase (G6PD) in concepti of the enzyme-deficient mutant and wild-type C3H mice. By using different crosses between the G6PD-deficient homozygous, heterozygous, or wild-type females and hemizygous or wild-type males, we confirmed the inactivation of one of the two X chromosomes in female concepti by a histochemical method. With this technique, a dual (G6PD + or -) cell population could be observed in the tissue sections. We demonstrate that the paternal X chromosome is inactivated in the endoderm of parietal and visceral yolk sac and in the trophoblast, whereas in the embryo and in the yolk sac mesoderm this inactivation is random. Our results confirm biochemical observations showing that only the maternal X chromosome is expressed in all derivatives of trophectoderm and primitive endoderm, whereas derivatives of the primitive ectoderm show random X chromosome expression.     

Sex chromosome elimination,X chromosome inactivation and reactivation in the southern brown bandicoot Isoodon obesulus (Marsupialia: Peramelidae)

Cytogenetic studies have shown that bandicoots (family Peramelidae) eliminate one X chromosome in females and the Y chromosome in males from some somatic tissues at different stages during development. The discovery of a polymorphism for X-linked phosphoglycerate kinase (PGK-1) in a population of Isoodon obesulus from Mount Gambier, South Australia, has allowed us to answer a number of long standing questions relating to the parental source of the eliminated X chromosome, X chromosome inactivation and reactivation in somatic and germ cells of female bandicoots. We have found no evidence of paternal PGK-1 allele expression in a wide range of somatic tissues and cell types from known female heterozygotes. We conclude that paternal X chromosome inactivation occurs in bandicoots as in other marsupial groups and that it is the paternally derived X chromosome that is eliminated from some cell types of females. The absence of PGK-1 paternal activity in somatic cells allowed us to examine the state of X chromosome activity in germ cells. Electrophoresis of germ cells from different aged pouch young heterozygotes showed only maternal allele expression in oogonia whereas an additional paternally derived band was observed in pre-dictyate oocytes. We conclude that reactivation of the inactive X chromosome occurs around the onset of meiosis in female bandicoots. As in other mammals, late replication is a common feature of the Y chromosome in male and the inactive X chromosome in female bandicoots. The basis of sex chromosome loss is still not known; however later timing of DNA synthesis is involved. Our finding that the paternally derived X chromosome is eliminated in females suggests that late DNA replication may provide the imprint for paternal X inactivation and the elimination of sex chromosomes in bandicoots.     

Analysis of mechanisms regulating the expression of parental alleles at the GPD locus in mule erythrocytes

Erythrocyte glucose-6-phosphate dehydrogenase (G6PD) was examined by 13% starch gel electrophoresis in 74 mules (42 females and 32 males), 35 donkeys, and ten horses. The quantitative expression of the parental alleles at the Gpd locus varies greatly in female mules from the hemizygous expression of the maternal allele to that of the paternal. The data obtained indicate that the X chromosomes are randomly inactivated in female mules. No selective advantage of a cell population with a maternally (or paternally) derived X active was found in female mule erythrocytes. It is suggested that the phenotypic variability in the expression of the parental Gpd alleles is related to the random proportions established between cells having either a maternal or paternal X active in an initiator (stem) cell group giving rise to erythroid tissue. Initiator cell numbers estimated for erythroid tissue (six or seven) are close to those reported for human females and intergeneric fox hybrids. These numbers may vary depending on the duration of the time of determination and the division rate of initiator cells at determination.     

The myoblast defect identified in Duchenne muscular dystrophy is not a primary expression of the DMD mutation

Summary We previously proposed the hypothesis that the primary expression of the defect in X-linked Duchenne muscular dystrophy (DMD) occurred in the myoblast, or muscle precursor cell. This was based on the observation that the number of viable myoblasts obtained per gram DMD muscle tissue was greatly reduced and those that grew in culture had decreased proliferative capacity and an aberrant distended flat morphology. Here we test that hypothesis by determining whether the expression of the myoblast defect is X-linked. Muscle cells were obtained from five doubly heterozygous carriers of two X-linked loci, DMD and glucose-6-phosphate dehydrogenase (G6PD), and compared with those from five sex-and age-matched controls heterozygous for G6PD only. A total of 1,355 individual clones were determined to be muscle and evaluated at the single cell level for proliferative capacity, morphology, and G6PD isozyme expression. The results demonstrate that the proportion of defective myoblast clones is significantly increased in DMD carriers. However, since this cellular defect does not consistently segregate with a single G6PD phenotype in the myoblast clones derived from any of the carriers, it is unlikely to be the primary expression of the DMD mutant allele.     

Maternally transmitted severe glucose 6-phosphate dehydrogenase deficiency is an embryonic lethal

Mouse chimeras from embryonic stem cells in which the X-linked glucose 6-phosphate dehydrogenase (G6PD) gene had been targeted were crossed with normal females. First-generation (F(1)) G6PD(+/-) heterozygotes born from this cross were essentially normal; analysis of their tissues demonstrated strong selection for cells with the targeted G6PD allele on the inactive X chromosome. When these F(1) G6PD(+/-) females were bred to normal males, only normal G6PD mice were born, because: (i) hemizygous G6PD(-) male embryos died by E10.5 and their development was arrested from E7.5, the time of onset of blood circulation; (ii) heterozygous G6PD(+/-) females showed abnormalities from E8.5, and died by E11.5; and (iii) severe pathological changes were present in the placenta of both G6PD(-) and G6PD(+/-) embryos. Thus, G6PD is not indispensable for early embryo development; however, severe G6PD deficiency in the extraembryonic tissues (consequent on selective inactivation of the normal paternal G6PD allele) impairs the development of the placenta and causes death of the embryo. Most importantly, G6PD is indispensable for survival when the embryo is exposed to oxygen through its blood supply.     

Molecular studies of marsupial X chromosomes reveal limited sequence homology of mammalian X-linked genes

To explore the extent to which the X chromosome has been conserved during mammalian evolution, we compared six loci that are X-linked in the human genome with the corresponding genes of the North American marsupial, the Virginia opossum (Didelphis virginiana). Our analysis shows that in the opossum genome there are sequences highly homologous to those of human cDNAs for housekeeping genes, glucose-6-phosphoribosyltransferase (HPRT), phosphoglycerate kinase A (PGK1), and alpha-galactosidase A (GLA). However, ornithine transcarbamylase and blood clotting Factor IX--tissue-specific genes that are X-linked in eutherians mammals--have no highly conserved homologs in the marsupial genome. By cloning opossum G6PD and HPRT, we found that these genes are X-linked in the opossum and that homologous sequences are limited to coding regions. As all genomic fragments hybridizing with the human GLA probe show dosage effects, it is likely that the opossum counterpart is X-linked. Finally, the pattern of hybridization suggests that the autosomal pseudogenes of HPRT and PGK1 in the opossum have remained highly homologous to the human X-linked genes.     

X-chromosome inactivation patterns are unbalanced and affect the phenotypic outcome in a mouse model of rett syndrome

Rett syndrome (RTT), a neurodevelopmental disorder affecting mostly females, is caused by mutations in the X-linked gene encoding methyl-CpG-binding protein 2 (MeCP2). Although the majority of girls with classic RTT have a random pattern of X-chromosome inactivation (XCI), nonbalanced patterns have been observed in patients carrying mutant MECP2 and, in some cases, account for variability of phenotypic manifestations. We have generated an RTT mouse model that recapitulates all major aspects of the human disease, but we found that females exhibit a high degree of phenotypic variability beyond what is observed in human patients with similar mutations. To evaluate whether XCI influences the phenotypic outcome of Mecp2 mutation in the mouse, we studied the pattern of XCI at the single-cell level in brains of heterozygous females. We found that XCI patterns were unbalanced, favoring expression of the wild-type allele, in most mutant females. It is notable that none of the animals had nonrandom XCI favoring the mutant allele. To explore why the XCI patterns favored expression of the wild-type allele, we studied primary neuronal cultures from Mecp2-mutant mice and found selective survival of neurons in which the wild-type X chromosome was active. Quantitative analysis indicated that fewer phenotypes are observed when a large percentage of neurons have the mutant X chromosome inactivated. The study of neuronal XCI patterns in a large number of female mice carrying a mutant Mecp2 allele highlights the importance of MeCP2 for neuronal viability. These findings also raise the possibility that there are human females who carry mutant MECP2 alleles but are not recognized because their phenotypes are subdued owing to favorable XCI patterns.     

Self-imposed silence: parental antagonism and the evolution of X-chromosome inactivation

A model is proposed for the evolution of X-chromosome inactivation (XCI) in which natural selection initially favors the silencing of paternally derived alleles of X-linked demand inhibitors. The compensatory upregulation of maternally derived alleles establishes a requirement for monoallelic expression in females. For this reason, XCI is self-reinforcing once established. However, inactivation of a particular X chromosome is not. Random XCI (rXCI) is favored over paternal XCI because rXCI reduces the costs of functional hemizygosity in females. Once present, rXCI favors the evolution of locus-by-locus imprinting of X-linked loci, which creates an evolutionary dynamic in which different chromosomes compete to remain active.     

Dictyate oocytes of a kangaroo (Macropus robustus) show paternal inactivation at the X-linked Gpd locus

Purified samples of large numbers of dictyate oocytes from 13 M. robustus pouch young heterozygous for glucose-6-phosphate dehydrogenase type and six homozygous controls were examined electrophoretically to determine activity states at the Gpd locus. Like somatic cortical and medullary cells, oocytes expressed only the maternal phenotype irrespective of the direction of the cross. No evidence was found of reactivation of the inactive (paternal) allele or inactivation of both maternal and paternal alleles. It was therefore concluded that unlike eutherian dictyate oocytes, only a single (maternal) allele is active in each dictyate oocyte in M. robustus. The stage of reactivation of the paternal allele remains to be determined.     

Expression of PGK-A in the Australian brush-tailed possum,Trichosurus vulpecula (Kerr), consistent with paternal X inactivation

An extensive survey of erythrocytes of marsupials other than kangaroos for electrophoretic variation in X-linked enzymes revealed two rare PGK-A phenotypes in the phalangerid Trichosurus vulpecula and one in Trichosurus caninus. Four putatively heterozygous females expressed only the variant allelic isozyme in some tissues but expressed a trace of the normal isozyme in others. A putatively hemizygous male expressed only the variant isozyme in all tissues. The phenotypic patterns were consistent with those observed in kangaroos known to exhibit partial or complete paternal X inactivation in cells of females. Two of the T. vulpecula were a mother and her female pouch young, further suggesting that paternal X inactivation occurs in T. vulpecula. This peculiar mechanism of dosage compensation may not be restricted to kangaroos.This work was supported by grants to D. W. C. from the Australian Research Grants Committee and Macquarie University, J. L. V. was the recipient of a Fulbright-Hays Award from the Australian-American Educational Foundation and a postgraduate fellowship from General Motors-Holden.     

Preferential X-chromosome activity in human female placental tissues

Preferential inactivation of the paternally derived X chromosome in extraembryonic membranes of female rodents has been clearly demonstrated, but the mode of X-chromosome inactivation in the human placenta has not been so clearly defined. We examined A and B variants of the X-linked enzyme glucose-6-phosphate dehydrogenase (G6PD) in 42 informative placentae to investigate whether the earliest differentiating human female embryonic cells exhibit preferential inactivation of the paternally inherited X. Contamination of villi with fetal blood was eliminated through culture of villous tissues, and maternal cell contamination was eliminated by careful dissection. Both fresh and cultured amnion and chorion, as well as cultured villi, revealed preferential maternal allele expression.     

Nucleotide variability at G6pd and the signature of malarial selection in humans

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common enzymopathy in humans. Deficiency alleles for this X-linked disorder are geographically correlated with historical patterns of malaria, and the most common deficiency allele in Africa (G6PD A-) has been shown to confer some resistance to malaria in both hemizygous males and heterozygous females. We studied DNA sequence variation in 5.1 kb of G6pd from 47 individuals representing a worldwide sample to examine the impact of selection on patterns of human nucleotide diversity and to infer the evolutionary history of the G6PD A- allele. We also sequenced 3.7 kb of a neighboring locus, L1cam, from the same set of individuals to study the effect of selection on patterns of linkage disequilibrium. Despite strong clinical evidence for malarial selection maintaining G6PD deficiency alleles in human populations, the overall level of nucleotide heterozygosity at G6pd is typical of other genes on the X chromosome. However, the signature of selection is evident in the absence of genetic variation among A- alleles from different parts of Africa and in the unusually high levels of linkage disequilibrium over a considerable distance of the X chromosome. In spite of a long-term association between Plasmodium falciparum and the ancestors of modern humans, patterns of nucleotide variability and linkage disequilibrium suggest that the A- allele arose in Africa only within the last 10,000 years and spread due to selection.     

Gene mapping in marsupials and monotremes. II. Assignments to the X chromosome of dasyurid marsupials

Somatic cell hybrids have been obtained between HPRT Chinese hamster cells and cells from several dasyurid marsupial species. These hybrids show the extensive loss of marsupial chromosomes characteristic of the majority of marsupial-eutherian somatic cell hybrids. Although all of the hybrids expressed the selected marsupial marker, HPRT, the only other markers observed were PGK, GLA, and G6PD, consistent with the conservation of X-linked genes extending to this major group of marsupials. Counterselection confirmed the synteny of PGK and GLA with HPRT, whereas G6PD showed decreased concordance.