Altered expression of insulin-like growth factor-1 and insulin like growth factor binding proteins-2 and 5 in the mouse mutant Hypodactyly (Hd) correlates with sites of apoptotic activity.

Insulin-like growth factor-I (IGF-I) mediated signalling has been implicated to be of significant importance during vertebrate embryonic development. IGF-I signalling has also been shown to be modulated by a number of IGF binding proteins that are thought to act as either agonists or antagonists of IGF activity. IGF-I has been implicated in a number of cellular processes, including cell division and programmed cell death (apoptosis). We have used the mouse mutant Hypodactyly (Hd) as a tool to determine the role of IGF-I and two key IGF binding proteins (IGFBP-2 and IGFBP-5) during embryonic development. The Hd mutant is a good model with which to study developmental cascades, since it has a distinct phenotype in the limb where cellular and molecular circuits have been thoroughly investigated. The distinctive pointed limb buds observed in Hd mutant embryos have been shown to be the result of a massive increase in apoptosis. We show that all three genes, IGF-1, IGFBP-2 and IGFBP-5, display restricted expression patterns during limb development. Indeed, IGFBP-5 shows a remarkable similarity to the expression of Engrailed-1, which is the vertebrate homologue of the Drosophila selector gene Engrailed. We show that there is downregulation in the expression of IGFBP-2 in the entire apical ectodermal ridge (AER) in homozygous Hd/Hd limb buds, whereas IGFBP-5 is downregulated in specific regions in the mutant AER. IGF-I expression is downregulated in Hd limb buds in regions undergoing high levels of cell death, consistent with its proposed role as an anti-apoptotic factor, while IGFBP-5 is found at higher levels in regions of cell death, consistent with reports of its association with apoptosis in adult tissues. We propose that these three components of the IGF axis could be involved in the manifestation of the mutant phenotype in Hypodactyly, and that this is probably a result of their ability to regulate cell survival and cell death.

Shh, Fgf4 and Hoxd gene expression in the mouse limb mutant hypodactyly.

The semidominant mouse mutation hypodactyly (Hd), caused by a deletion within the Hoxa13 gene, results in reduced digits; heterozygotes lack digit I in the hindlimb and homozygotes have only one digit on each limb. We investigated expression of Shh and Fgf4 signaling molecules involved in digit specification in mutant limb buds. Shh and Fgf4 are expressed in the posterior part of the limb buds as normal but expression may be slightly prolonged. The extent of digit reduction in hypodactyly is much more severe than in the Hoxa13 deficient mouse and resembles that in the Hoxa13(-/-)/Hoxd13(-/-) double mutant mouse. We found that the pattern of Hoxd13 and Hoxd11 transcripts was not markedly different in the mutant compared with the normal limbs even though the mutant limbs are narrower. Therefore Hoxd genes are transcribed as normal in the mutant. This makes it likely that the severe digit reductions in hypodactyly are caused by interference with Hoxd13 function at the protein level. Similar interactions between mutant and normal HOX gene products have been suggested to occur in the human semidominant disorder, synpolydactyly, caused by mutations in HOXD13.

Cellular analysis of limb development in the mouse mutant hypodactyly.

The limb defect in the mouse Hypodactyly (Hd) affects only the distal structures. Heterozygotes (Hd/+) lack all or part of the distal phalanx and the terminal claw of digit on the hindlimbs; mice homozygous (Hd/Hd) for the mutation have just one digit on each of the four limbs. Early limb development in the mutant appears normal and a change in morphology can only be detected later. Limb buds of Hd/+ and Hd/Hd embryos become reduced in width, with Hd/Hd buds becoming very pointed instead of rounded. This change in bud shape is correlated with an increase in cell death anteriorly in Hd/+ hindlimbs and both anteriorly and posteriorly in Hd/Hd fore- and hindlimb buds. The apical ectodermal ridge is very pronounced in pointed Hd/Hd limb buds. Mesenchyme cells from the Hd/Hd mutant in culture show a cell-autonomous change in behaviour and less cartilage differentiates.

The genetic basis of XX-XY differences present before gonadal sex differentiation in the mouse.

There is now a substantial body of data showing that in eutherian mammals (mouse, rat, cow and man) XY conceptuses are developmentally more advanced (and consequently larger) than XX conceptuses of equivalent gestational age. This developmental difference is already discernible in the preimplantation period and it has been suggested that the more advanced development of XY embryos may be a consequence of the preimplantation expression of Y chromosomal genes such as Sry or Zfy. In the present paper sex-chromosomally variant mice were used to analyse the genetic basis of XX-XY differences as manifest at 10.5 days post coitum. The results show that the XX-XY difference is due to a combination of a Y chromosome effect and an effect of the difference in X chromosome constitution (2X v 1X). The Y effect is not dependent on the presence of Sry. In the light of this and other studies, it is concluded that the Y chromosome of most mouse strains carries a factor which accelerates preimplantation development and that the resulting developmental advantage is carried over into the postimplantation period. The retarding effect of two X chromosomes is then superimposed on this Y effect subsequent to the blastocyst stage but prior to 9.5 days post coitum.

Y353/B: a candidate multiple-copy spermiogenesis gene on the mouse Y chromosome.

There is evidence from Y Chromosome (Chr) deletion mapping that there is a gene on the long arm of the mouse Y Chr that is needed for the normal development of the sperm head. Since mice with partial Y long arm deletions show incomplete penetrance of the sperm head defect, whereas mice with no Y long arm show complete penetrance, it has been suggested that the 'spermiogenesis' gene may be present in multiple copies. A Y-specific genomic DNA sequence (Y353/B) has previously been described that is present in multiple copies on the long arm of the mouse Y and identifies testis-specific transcripts. We have suggested that Y353/B could be the proposed multiple copy 'spermiogenesis' gene. In support of this suggestion, we show here that mice with a partial Y long arm deletion associated with a 3.5-fold increase in the frequency of abnormal sperm heads have a marked reduction in genomic Y353/B copies and a corresponding reduction in Y353/B-related transcripts. Thus, the incompletely penetrant phenotype correlates with a reduction in Y353/B-related transcription. Furthermore, by in situ hybridization with a Y353/B riboprobe to testis sections, we show that the Y353/B-related transcripts are confined to the round spermatid stage of spermiogenesis, just prior to the shaping of the sperm head. The transcripts sediment with the fraction of cytoplasmic RNA in adult testis that is loaded on polysomes, suggesting that the transcripts are actively translated.

Recombination between the X and Y chromosomes and the Sxr region of the mouse.

The Sxr (sex-reversed) region that carries a copy of the mouse Y chromosomal testis-determining gene can be attached to the distal end of either the Y or the X chromosome. During male meiosis, Sxr recombined freely between the X and Y chromosomes, with an estimated recombination frequency not significantly different from 50% in either direction. During female meiosis, Sxr recombined freely between the X chromosome to which it was attached and an X-autosome translocation. A male mouse carrying the original Sxra region on its Y chromosome, and the shorter Sxrb variant on the X, also showed 50% recombination between the sex chromosomes. Evidence of unequal crossing-over between the two Sxr regions was obtained: using five markers deleted from Sxrb, 3 variant Sxr regions were detected in 159 progeny (1.9%). Four other variants (one from the original cross and three from later generations) were presumed to have been derived from illegitimate pairing and crossing-over between Sxrb and the homologous region on the short arm of the Y chromosome. The generation of new variants throws light on the arrangement of gene loci and other markers within the short arm of the mouse Y chromosome.

Mouse models of human single gene disorders. I: Nontransgenic mice.

Mouse models of human genetic disorders provide a valuable resource for investigating the pathogenesis of genetic disease and for testing potential therapies. The high degree of resolution of linkage mapping in the mouse allows mutant phenotypes to be mapped precisely which, combined with the accurate definition of areas of homology between the mouse and human genomes, greatly facilitates the identification of mouse models. We describe here mouse models of human single gene disorders dividing them into three categories depending on the information available; phenotypic similarities, comparative mapping and identification of the underlying genetic lesion.

Spermatogenesis in XY, XYSxra and XOSxra mice: a quantitative analysis of spermatogenesis throughout puberty.

Adult XYSxra mice exhibit varying degrees of spermatogenic deficiency but are usually fertile, while XOSxra mice have severe spermatogenic failure and are always sterile. The present quantitative spermatogenic analysis documents when these anomalies first appear during puberty. The results demonstrate that in XYSxra mice there was increased degeneration of pachytene spermatocytes and, to a lesser extent, meiotic metaphase stages. On average, there were only one-half the number of spermatids compared with the XY controls. The defect in XOSxra mice appeared a little later, with an almost complete arrest and degeneration during the meiotic metaphases, so that the number of spermatids produced was only 3% of the control value. These results are discussed in relation to an hypothesis that links sex chromosome univalence during meiotic prophase with spermatogenic failure.

The MIC2 gene product: epitope mapping and structural prediction analysis define an integral membrane protein.

The MIC2 locus is located in the pseudoautosomal (pairing) region of human X and Y chromosomes (Goodfellow et al., Science 234, 740-743, 1986). Despite extensive molecular analysis of MIC2 (see Darling et al., Cold Spring Harb. Symp. quant. Biol. 51, 205-211, 1986), study of the gene product has been limited (Banting et al., EMBO J. 41, 1967-1972, 1985). Here we report the combined use of monoclonal antibodies, plasmid expression vectors and structural prediction analysis to define the MIC2 gene product as an integral membrane protein. Random overlapping fragments of a cDNA, corresponding to the MIC2 locus, were cloned into the plasmid expression vector pEX1 (Stanley and Luzio, EMBO J. 3, 1429-1434, 1984) to produce "epitope libraries". Six different monoclonal antibodies, known to recognize the extracellular region of the MIC2 gene product, were used to screen these libraries. Clones recognized by these antibodies were sequenced and their sequences aligned with one another and with the complete MIC2 cDNA sequence. All antibodies tested recognized adjacent and/or overlapping epitopes in the same region of the molecule. These results complement data from a hydropathy plot of a conceptual translation of the MIC2 sequence, which demonstrated the presence of a single long hydrophobic region in the mature protein. Since the antibodies recognize the extracellular portion of the molecule, we were able to determine the orientation in the plasma membrane. This method of analysis is generally applicable where antibodies and cloned cDNAs are available.

Absence of methylation of a CpG-rich region at the 5' end of the MIC2 gene on the active X, the inactive X, and the Y chromosome.

We have identified and characterized a Hpa II tiny fragment (HTF) island associated with the promoter region of the pseudoautosomal gene MIC2. The MIC2 HTF island is unmethylated on both the active and inactive X chromosome and is similarly unmethylated on the Y chromosome. Unlike the majority of genes borne on the X chromosome, MIC2 fails to undergo X chromosome inactivation. HTF islands associated with X chromosome-linked genes that are inactivated are highly methylated on the inactive or transcriptionally silent homologue. The failure of MIC2 to undergo X chromosome inactivation correlates with the lack of methylation of the HTF island at the 5' end of the gene. These results provide further evidence that DNA methylation plays an important role in the phenomenon of X chromosome inactivation.

Genetics of sex determination in man and mouse.

The cytological evidence has revealed a visible mechanical basis for the production of males and females in equal numbers and irrespective of external conditions (Wilson, 1909).

A pseudoautosomal gene in man.

The X/Y homologous gene MIC2 was shown to exchange between the sex chromosomes, thus demonstrating that it is a pseudoautosomal gene in man. MIC2 recombines with the sex-determining gene(s) TDF at a frequency of 2 to 3 percent. It is the most proximal pseudoautosomal locus thus far described and as such is an important marker for use in studies directed towards the isolation of TDF.

Homozygosity for a Y/22 chromosome translocation: t(Y;22) (q12;p12/13).

A newborn girl, homozygous for a balanced Y/22 chromosome translocation is described. This unique karyotype was detected during prenatal chromosome studies in the first pregnancy of a 26-year-old woman. Amniocentesis was performed because of clinical evaluation of severe fetal growth retardation in the 28th week of gestation. The cytogenetic results were confirmed using a lymphocyte culture after birth in the 30th week. Subsequent chromosome studies of the parents were hampered by the fact that the pregnancy was thought to be the result of artificial insemination with donorsperm. Nevertheless both, consanguineous, parents were shown to be carriers of the same, singular, chromosome translocation and the spermdonor could be excluded from paternity by bloodgroup- and HLA studies. Distamycin-A-DAPI chromosome staining and DNA studies of the mother were used to confirm the involvement of the Y-chromosome in this translocation. The probanda is developing quite normally at the age of 21 months.

Cloning an expressed gene shared by the human sex chromosomes.

The existence of genes shared by mammalian sex chromosomes has been predicted on both evolutionary and functional grounds. However, the only experimental evidence for such genes in humans is the cell-surface antigen encoded by loci on the X and Y chromosomes (MIC2X and MIC2Y, respectively), which is recognized by the monoclonal antibody 12E7. Using the bacteriophage lambda gt11 expression system in Escherichia coli and immunoscreening techniques, we have isolated a cDNA clone whose primary product is recognized by 12E7. Southern blot analysis using somatic cell hybrids containing only the human X or Y chromosomes shows that the sequences reacting with the cDNA clone are localized to the sex chromosomes. In addition, the clone hybridizes to DNAs isolated from mouse cells that have been transfected with human DNA and selected for 12E7 expression on the fluorescence-activated cell sorter. We conclude that the cDNA clone encodes the 12E7 antigen, which is the primary product of the MIC2 loci. The clone was used to explore sequence homology between MIC2X and MIC2Y; these loci are closely related, if not identical.

Isolation and characterization of an alphoid centromeric repeat family from the human Y chromosome.

A collection of human Y-derived cosmid clones was screened with a plasmid insert containing a member of the human X chromosome alphoid repeat family, DXZ1. Two positive cosmids were isolated and the repeats they contained were investigated by Southern blotting, in situ hybridization and sequence analysis. On hybridization to human genomic DNAs, the expected cross-hybridization characteristic of all alphoid sequences was seen and, in addition, a 5500 base EcoRI fragment was found to be characteristic of a Y-specific alphoid repeat. Dosage experiments demonstrated that there are about 100 copies of this 5500 base EcoRI alphoid fragment on the Y chromosome. Studies utilizing DNA from human-mouse hybrids containing only portions of the Y chromosome and in situ hybridizations to chromosome spreads demonstrated the Y centromeric localization of the 5500 base repeat. Cross-hybridization to autosomes 13, 14 and 15 was also seen; however, these chromosomes lacked detectable copies of the 5500 base EcoRI repeat sequence arrangement. Sequence analysis of portions of the Y repeat and portions of the DXZ1 repeat demonstrated about 70% homology to each other and of each to the human consensus alphoid sequence. The 5500 base EcoRI fragment was not seen in gorilla, orangutan or chimpanzee male DNA.

Methods to analyse the human genome.

There is much interest in the use of gene-specific probes for the study of dysfunction in human pathology. For the haemoglobinopathies, globin gene recombinants (either prepared from DNA sequences complementary to messenger RNA, or from genomic DNA) are used to determine whether globin genes are present, whether they are expressed in the nucleus, and whether they are correctly processed to give functional mRNAs. This has not only allowed a fuller understanding of the molecular aetiology of the thalassaemias, but also permitted antenatal diagnosis both by direct analysis of the gene lesion, and by linkage analysis using adjacent genes. Similar approaches are being applied to many other single gene defects. There are, however, other possible ways to study human hereditary disease using recombinants. It is now feasible to use random human chromosome-specific sequences to establish a linkage map for the entire human genome. Such a map may then be used either to determine the chromosomal localisation of any "single gene" phenotype by linkage analysis, or to study the contribution of different genes to a complex phenotype determined by several genes, as in multifactorial disease.