Deletion of Y chromosome sequences located outside the testis determining region can cause XY female sex reversal.

An approach designed to map and generate mutations in the region of the short arm of the mouse Y chromosome, known to be involved in sex determination and spermatogenesis, is described. This relies on homologous Yp-Sxra pairing and asymmetrical exchange which can occur at meiosis in XY males carrying Sxra on their X chromosome. Such exchange potentially generates deficiencies and duplications of Yp or Sxra. Three fertile XY females were found out of about 450 XY offspring from XSxra/Y x XX crosses. In all three, despite evidence for deletion of Y chromosomal material, the Sry locus was intact. Each deletion involved a repeat sequence, Sx1, located at a distance from Sry. Since expression of Sry was affected these results suggest that long range position effects have disrupted Sry action.

A systematic, genome-wide, phenotype-driven mutagenesis programme for gene function studies in the mouse.

As the human genome project approaches completion, the challenge for mammalian geneticists is to develop approaches for the systematic determination of mammalian gene function. Mouse mutagenesis will be a key element of studies of gene function. Phenotype-driven approaches using the chemical mutagen ethylnitrosourea (ENU) represent a potentially efficient route for the generation of large numbers of mutant mice that can be screened for novel phenotypes. The advantage of this approach is that, in assessing gene function, no a priori assumptions are made about the genes involved in any pathway. Phenotype-driven mutagenesis is thus an effective method for the identification of novel genes and pathways. We have undertaken a genome-wide, phenotype-driven screen for dominant mutations in the mouse. We generated and screened over 26,000 mice, and recovered some 500 new mouse mutants. Our work, along with the programme reported in the accompanying paper, has led to a substantial increase in the mouse mutant resource and represents a first step towards systematic studies of gene function in mammalian genetics.

Lymphoid development in mice congenitally lacking T cell receptor alpha beta-expressing cells.

Vertebrate T cells express either an alpha beta or gamma delta T cell receptor (TCR). The developmental relatedness of the two cell types is unresolved. alpha beta + T cells respond to specific pathogens by collaborating with immunoglobulin-producing B cells in distinct lymphoid organs such as the spleen and Peyer's patches. The precise influence of alpha beta + T cells on B cell development is poorly understood. To investigate the developmental effects of alpha beta + T cells on B cells and gamma delta + T cells, mice homozygous for a disrupted TCR alpha gene were generated. The homozygotes showed elimination of alpha beta + T cells and the loss of thymic medullae. Despite this, gamma delta + T cells developed in normal numbers, and there was an increase in splenic B cells.

Neuropilin-2 is required in vivo for selective axon guidance responses to secreted semaphorins.

Neuropilins are receptors for class 3 secreted semaphorins, most of which can function as potent repulsive axon guidance cues. We have generated mice with a targeted deletion in the neuropilin-2 (Npn-2) locus. Many Npn-2 mutant mice are viable into adulthood, allowing us to assess the role of Npn-2 in axon guidance events throughout neural development. Npn-2 is required for the organization and fasciculation of several cranial nerves and spinal nerves. In addition, several major fiber tracts in the brains of adult mutant mice are either severely disorganized or missing. Our results show that Npn-2 is a selective receptor for class 3 semaphorins in vivo and that Npn-1 and Npn-2 are required for development of an overlapping but distinct set of CNS and PNS projections.

X chromosome inactivation and the Xist gene.

X chromosome inactivation in mammals was first described over 30 years ago. The biological problem is how to achieve gene dosage equivalence between XX females and XY males; the solution is to genetically silence one whole X chromosome in each cell of the early developing female embryo. The molecular mechanism by which this is achieved, however, remains a mystery. Recently, through the discovery of the Xist gene, it appears that we may be on the brink of learning how this unique phenomenon is mediated. Here, I discuss the developmental regulation of X inactivation and the candidacy of Xist as the X chromosome inactivation centre, with particular reference to its possible role in the initiation, spread and maintenance of X inactivation.

Functional genomics: going forwards from the databases.

Extrapolating systematically from gene sequence to function is undoubtedly the major challenge facing industry and academia alike as we approach the end of the millennium. Many electronic and laboratory approaches are being developed to meet this challenge but the rate of evolution of these is not keeping pace with the speed of sequence generation.

Genomics: saviour or millstone?

The Keystone Symposium on the impact of Genomics Development was held in Santa Fe, New Mexico, from 2 to 7 February 2001.

Regulatory elements in the minimal promoter region of the mouse Xist gene.

The Xist gene plays a central role in regulating X chromosome inactivation and Xist transcription has recently been shown to be necessary for X inactivation in mouse. We are currently analysing regulation of the Xist gene in order to determine the mechanisms underlying initiation of Xist expression and X inactivation. Sequence comparisons indicate that a region of approximately 0.4 kb upstream of the the major transcriptional start site comprises the Xist minimal promoter. Analysis of reporter constructs demonstrates that the minimal promoter region is active both in embryonic stem (ES) cells and in differentiated derivatives, indicating that sequences either further upstream or downstream are required for appropriate developmental control of Xist transcription. We have examined the minimal promoter region in detail, and in addition to common promoter elements have identified two previously uncharacterised transcription-factor binding sites. Mutation of these sites in reporter constructs indicates that they are functionally important.

Non-random X-chromosome inactivation in mouse X-autosome translocation embryos--location of the inactivation centre.

X-chromosome inactivation was investigated cytologically using the modified Kanda method which differentially stains inactive X-chromosome material at metaphase in balanced 13 1/2-day female embryos heterozygous for four X-autosome rearrangements, reciprocal translocations T(X;4)37H, T(X;11)38H and T(X;16)16H (Searle's translocation) and the insertion translocation Is(7;X)1 Ct (Cattanach's translocation). In all cases non-random inactivation was found. In the reciprocal translocation heterozygotes only one translocation product ever showed Kanda staining. In addition in a proportion of cells from T(X;4)37H, T(X;11)38H and Is(7;X)1Ct the Kanda staining revealed differential staining of X-chromosome material and attached autosomal material within the translocation product. In a study of 8 1/2-day female embryos doubly heterozygous for Searle's translocation and Cattanach's translocation two unbalanced types of embryo were found. In one type of unbalanced female embryo of the karyotype 40(X(7)/X16;16/16) no inactivated X-chromosomal material is found. A second unbalanced type of female embryo, of the presumptive karyotype 40(X(7)/XN;16X/16) was found in which two inactivated chromosomes were present in the majority of metaphase spreads. A simple model for the initiation of X-chromosome inactivation based on the presence of a single inactivation centre distal to the breakpoint in Searle's translocation explains these findings.

Interaction between the Xce locus and imprinting of the paternal X chromosome in mouse yolk-sac endoderm.

In female eutherian mammals preferential inactivation of the paternally derived X chromosome (XP) takes place in certain extra-embryonic tissues such as mouse yolk-sac endoderm, chorionic ectoderm and trophoblast and has been demonstrated both biochemically and cytologically. This is thought to be due to the paternal X chromosome being 'imprinted', that is, somehow marked as different, during either male gametogenesis or fertilization, causing primary nonrandom X-inactivation in tissues that differentiate early, such as trophectoderm and primitive endoderm, from which yolk-sac endoderm is derived. Different alleles of the X-chromosome controlling element, Xce locus, centrally located on the mouse X chromosome, also cause primary nonrandom X-chromosome inactivation in embryonic tissues which would otherwise show random inactivation. The work reported here was designed to elucidate whether the nonrandom inactivation of the imprinted XP in yolk-sac endoderm could be modified, or even overridden, by the effect of different Xce alleles. Using the modified Kanda method we have therefore studied the proportion of cells at metaphase with the XP inactive in separated yolk-sac endoderm and mesoderm of mouse embryos heterozygous for a marker X chromosome (Cattanach's translocation) carrying different Xce alleles on XP and XM. The results show that the extreme Xcec allele, when present on the paternally derived X, can significantly reduce the proportion of inactive XP seen in yolk-sac endoderm compared with controls. This is the first evidence that imprinting of XP is not an 'all or none' event but can be modified by a 'strong' allele at the Xce locus, and is another indication that the Xce locus may represent the inactivation centre.

Parental source of chromosome imprinting and its relevance for X chromosome inactivation.

In imprinting, homologous chromosomes behave differently during development according to their parental origin. Typically, paternally derived chromosomes are preferentially inactivated or eliminated. Examples of such phenomena include inactivation of the mammalian X chromosome, inactivation or elimination of one haploid chromosome set in male coccids, and elimination of paternal X chromosomes in the fly Sciara. It has generally been thought that the paternal chromosomes bear an imprint leading to their inactivation or elimination. However, alteration of the parental origin of chromosomes, as in the study of parthenogenotes in mammals and coccids, shows that passage of chromosomes through a male germ cell or fertilization is not essential for inactivation or elimination. It appears that neither chromosome set is programmed to resist or undergo inactivation. Instead the two sets differ in relative sensitivity, and the question is whether the maternal set have an imprint for resistance, or the paternal set one for susceptibility. Very early in development of mammals both X chromosomes are active. This makes it simpler to envisage the maternal X bearing an imprint for resistance to inactivation, which persists through the early developmental period. Similar considerations also apply in coccids and Sciara. Thus, imprinting should be regarded as a phenomenon conferred on the maternal chromosomes in the oocyte. This permits simpler models for the mechanism of X-inactivation, and weakens the case for evolution of X-inactivation from an earlier form of inactivation during male gametogenesis. One may speculate whether imprinting affects timing of gene action in development.

The search for the mouse X-chromosome inactivation centre.

The phenomenon of X-chromosome inactivation in female mammals, whereby one of the two X chromosome present in each cell of the female embryo is inactivated early in development, was first described by Mary Lyon in 1961. Nearly 30 years later, the mechanism of X-chromosome inactivation remains unknown. Strong evidence has accumulated over the years, however, for the involvement of a major switch or inactivation centre on the mouse X chromosome. Identification of the inactivation centre at the molecular level would be an important step in understanding the mechanism of X-inactivation. In this paper we review the evidence for the existence and location of the X-inactivation centre on the mouse X-chromosome, present data on the molecular genetic mapping of this region, and describe ongoing strategies we are using to attempt to identify the inactivation centre at the molecular level.

X-chromosome deletions in embryo-derived (EK) cell lines associated with lack of X-chromosome inactivation.

The predictions of a model for the initiation of X-chromosome inactivation based on a single inactivation centre were tested in a cytogenetic study using six different embryo-derived (EK) stem cell lines, each with a different-sized deletion of the distal part of one of the X-chromosomes. Metaphase chromosomes were prepared by the Kanda method from each cell line in the undifferentiated state and after induction of differentiation, and cytogenetic evidence sought for a dark-staining inactive X-chromosome. The results confirm the predictions of the model in that when the inactivation centre is deleted from one of the X-chromosomes neither X present in a diploid cell can be inactivated, and in addition considerably further localize the position of the inactivation centre on the X-chromosome.

X-chromosome inactivation may explain the difference in viability of XO humans and mice.

Only about 1% of human XO conceptuses survive to birth and these usually have the characteristics of Turner's syndrome, with a complex and variable phenotype including short stature, gonadal dysgenesis and anatomical defects. Both the embryonic lethality and Turner's syndrome are thought to be due to monosomy for a gene or genes common to the X and Y chromosomes. These genes would be expected to be expressed in females from both active and inactive X chromosomes to ensure correct dosage of gene product. Two genes with these properties are ZFX and RPS4X, both of which have been proposed to play a role in Turner's syndrome. In contrast to humans, mice that are XO are viable with no prenatal lethality (P. Burgoyne, personal communication) and are anatomically normal and fertile. We have devised a system to analyse whether specific genes on the mouse X chromosome are inactivated, and demonstrate that both Zfx and Rps4X undergo normal X-inactivation in mice. Thus the relative viability of XO mice compared to XO humans may be explained by differences between the two species in the way that dosage compensation of specific genes is achieved.

Molecular genetic analysis of the Ta25H deletion: evidence for additional deleted loci.

Seventeen linking clones sublocalized to the central region of the mouse X Chromosome (Chr) were screened against genomic DNA from male mice carrying the tabby-25H (Ta25H) deletion. Two of these linking clones, lambda EM131 and lambda EM169, were found to be deleted in Ta25H/Y animals. Genetic mapping through Mus musculus domesticus/Mus spretus interspecific backcross progeny, segregating for the original tabby (Ta) gene mutation, was utilized to order these markers and to define nearest flanking markers to the Ta25H deletion (lambda EM140 and lambda EM171). The size of the Ta25H deletion was thus estimated as up to 4.5 centiMorgans (cM). The order of markers, proximal to distal, was found to be lambda EM140/lambda EM131, mouse androgen receptor gene (Ar)/lambda EM169, Ta/lambda EM171. A putative CpG-rich island and a highly evolutionarily conserved DNA probe were isolated from the DXCrc169 locus which co-segregates with the Ta locus in this study.

Methylation status of CpG-rich islands on active and inactive mouse X chromosomes.

Single copy probes derived from CpG-rich island clones from Eag I and Not I linking libraries and nine rare-cutter restriction endonucleases were used to investigate the methylation status of CpG-rich islands on the inactive and active X chromosomes (Chr) of the mouse. Thirteen of the 14 probes used detected CpG-rich islands in genomic DNA. The majority of island CpGs detected by rare-cutter restriction endonucleases were methylated on the inactive X Chr and unmethylated on the active X Chr, but some heterogeneity within the cell population used to make genomic DNA was detected. The CpG-rich islands detected by two putative pseudoautosomal probes remained unmethylated on both the active and inactive X Chrs. Otherwise, distance from the X Chr inactivation center did not affect the methylation profile of CpG-rich islands. We conclude that methylation of CpG-rich islands is a general feature of X Chr inactivation.

Determination of a molecular map position for Hyp using a new interspecific backcross produced by in vitro fertilization.

We have established a Mus spretus/Mus musculus domesticus interspecific backcross segregating for two X-linked mutant genes, Ta and Hyp, using in vitro fertilization. The haplotype of the recombinant X chromosome of each of 241 backcross progeny has been established using the X-linked anchor loci Otc, Hprt, Dmd, Pgk-1, and Amg and the additional probes DXSmh43 and Cbx-rs1. The Hyp locus (putative homologue of the human disease gene hypophosphatemic rickets, HYP) has been incorporated into the molecular genetic map of the X chromosome. We show that the most likely gene order in the distal portion of the mouse X chromosome is Pgk-1-DXSmh43-Hyp-Cbx-rs1-Amg, from proximal to distal. The distance in centimorgans (mean +/- SE) between DXSmh43 and Hyp was 2.52 +/- 1.4 and that between Hyp and Cbx-rs1 was 1.98 +/- 1.39. Thus closely linked flanking markers for the Hyp locus that will facilitate the molecular characterization of the gene itself have been defined.