A transgenic insertion upstream of sox9 is associated with dominant XX sex reversal in the mouse

In most mammals, male development is triggered by the transient expression of the Y-chromosome gene, Sry, which initiates a cascade of gene interactions ultimately leading to the formation of a testis from the indifferent fetal gonad. Several genes, in particular Sox9, have a crucial role in this pathway. Despite this, the direct downstream targets of Sry and the nature of the pathway itself remain to be clearly established. We report here a new dominant insertional mutation, Odsex (Ods), in which XX mice carrying a 150-kb deletion (approximately 1 Mb upstream of Sox9) develop as sterile XX males lacking Sry. During embryogenesis, wild-type XX fetal gonads downregulate Sox9 expression, whereas XY and XX Ods/+ fetal gonads upregulate and maintain its expression. We propose that Ods has removed a long-range, gonad-specific regulatory element that mediates the repression of Sox9 expression in XX fetal gonads. This repression would normally be antagonized by Sry protein in XY embryos. Our data are consistent with Sox9 being a direct downstream target of Sry and provide genetic evidence to support a general repressor model of sex determination in mammals.     

Sox9 induces testis development in XX transgenic mice.

Mutations in SOX9 are associated with male-to-female sex reversal in humans. To analyze Sox9 function during sex determination, we ectopically expressed this gene in XX gonads. Here, we show that Sox9 is sufficient to induce testis formation in mice, indicating that it can substitute for the sex-determining gene Sry.     

Subspecific origin and haplotype diversity in the laboratory mouse

Here we provide a genome-wide, high-resolution map of the phylogenetic origin of the genome of most extant laboratory mouse inbred strains. Our analysis is based on the genotypes of wild-caught mice from three subspecies of Mus musculus. We show that classical laboratory strains are derived from a few fancy mice with limited haplotype diversity. Their genomes are overwhelmingly Mus musculus domesticus in origin, and the remainder is mostly of Japanese origin. We generated genome-wide haplotype maps based on identity by descent from fancy mice and show that classical inbred strains have limited and non-randomly distributed genetic diversity. In contrast, wild-derived laboratory strains represent a broad sampling of diversity within M. musculus. Intersubspecific introgression is pervasive in these strains, and contamination by laboratory stocks has played a role in this process. The subspecific origin, haplotype diversity and identity by descent maps can be visualized using the Mouse Phylogeny Viewer (see URLs).     

Identification of Stk6/STK15 as a candidate low-penetrance tumor-susceptibility gene in mouse and human

Linkage analysis and haplotype mapping in interspecific mouse crosses (Mus musculus x Mus spretus) identified the gene encoding Aurora2 (Stk6 in mouse and STK15 in human) as a candidate skin tumor susceptibility gene. The Stk6 allele inherited from the susceptible M. musculus parent was overexpressed in normal cells and preferentially amplified in tumor cells from F(1) hybrid mice. We identified a common genetic variant in STK15 (resulting in the amino acid substitution F31I) that is preferentially amplified and associated with the degree of aneuploidy in human colon tumors. The Ile31 variant transforms rat1 cells more potently than the more common Phe31 variant. The E2 ubiquitin-conjugating enzyme UBE2N was a preferential binding partner of the 'weak' STK15 Phe31 variant form in yeast two-hybrid screens and in human cells. This interaction results in colocalization of UBE2N with STK15 at the centrosomes during mitosis. These results are consistent with an important role for the Ile31 variant of STK15 in human cancer susceptibility.     

The human pseudoautosomal GM-CSF receptor alpha subunit gene is autosomal in mouse.

The gene encoding the granulocyte macrophage colony stimulating factor receptor alpha subunit (CSF2RA) has previously been mapped to the pseudoautosomal region of the human sex chromosomes. In contrast, we report that the murine locus, Csf2ra, maps to an autosome in the laboratory mouse. By in situ hybridization and genetic mapping, Csf2ra maps at telomeric band D2 of mouse chromosome 19. This first instance of a pseudoautosomal locus in human being autosomal in mouse, indicates incomplete conservation between the human and mouse X chromosomes and suggests that the genetic content of the pseudoautosomal region may differ between species of eutherian mammals due to chromosomal rearrangements.     

Identification of autonomous IAP LTR retrotransposons mobile in mammalian cells

Mammalian genomes contain two main classes of retrotransposons, the well-characterized long and short interspersed nuclear elements, which account for approximately 30% of the genome, and the long terminal repeat (LTR) retrotransposons, which resemble the proviral integrated form of retroviruses, except for the absence of an envelope gene in some cases. Genetic studies confirmed mobility of the latter class of elements in mice, with a high proportion of phenotypic mutations consequent to transposition of the intracisternal A particle (IAP) family of LTR retrotransposons. Using the mouse genome sequence and an efficient ex vivo retrotransposition assay, we identified functional, master IAP copies that encode all the enzymatic and structural proteins necessary for their autonomous transposition in heterologous cells. By introducing mutations, we found that the three genes gag, prt and pol are all required for retrotransposition and identified the IAP gene products by electron microscopy in the form of intracellular A-type particles in the transfected cells. These prototypic elements, devoid of an envelope gene, are the first LTR retrotransposons autonomous for transposition to be identified in mammals. Their high rates of retrotransposition indicate that they are potent insertional mutagens that could serve as safe (noninfectious) genetic tools in a large panel of cells.     

On the subspecific origin of the laboratory mouse

The genome of the laboratory mouse is thought to be a mosaic of regions with distinct subspecific origins. We have developed a high-resolution map of the origin of the laboratory mouse by generating 25,400 phylogenetic trees at 100-kb intervals spanning the genome. On average, 92% of the genome is of Mus musculus domesticus origin, and the distribution of diversity is markedly nonrandom among the chromosomes. There are large regions of extremely low diversity, which represent blind spots for studies of natural variation and complex traits, and hot spots of diversity. In contrast with the mosaic model, we found that most of the genome has intermediate levels of variation of intrasubspecific origin. Finally, mouse strains derived from the wild that are supposed to represent different mouse subspecies show substantial intersubspecific introgression, which has strong implications for evolutionary studies that assume these are pure representatives of a given subspecies.     

Somatic integration and long-term transgene expression in normal and haemophilic mice using a DNA transposon system

The development of non-viral gene-transfer technologies that can support stable chromosomal integration and persistent gene expression in vivo is desirable. Here we describe the successful use of transposon technology for the nonhomologous insertion of foreign genes into the genomes of adult mammals using naked DNA. We show that the Sleeping Beauty transposase can efficiently insert transposon DNA into the mouse genome in approximately 5-6% of transfected mouse liver cells. Chromosomal transposition resulted in long-term expression (>5 months) of human blood coagulation factor IX at levels that were therapeutic in a mouse model of haemophilia B. Our results establish DNA-mediated transposition as a new genetic tool for mammals, and provide new strategies to improve existing non-viral and viral vectors for human gene therapy applications.     

Ciliary proteins link basal body polarization to planar cell polarity regulation

Planar cell polarity (PCP) refers to coordinated polarization of cells within the plane of a cell sheet. A conserved signaling pathway is required for the establishment of PCP in epithelial tissues and for polarized cellular rearrangements known as convergent extension. During PCP signaling, core PCP proteins are sorted asymmetrically along the polarization axis; this sorting is thought to direct coordinated downstream morphogenetic changes across the entire tissue. Here, we show that a gene encoding a ciliary protein (a 'ciliary gene'), Ift88, also known as Polaris, is required for establishing epithelial PCP and for convergent extension of the cochlear duct of Mus musculus. We also show that the proper positioning of ciliary basal bodies and the formation of polarized cellular structures are disrupted in mice with mutant ciliary proteins ('ciliary mutants'), whereas core PCP proteins are partitioned normally along the polarization axis. Thus, our data uncover a distinct requirement for ciliary genes in basal body positioning and morphological polarization during PCP regulation.     

R-spondin1 is essential in sex determination, skin differentiation and malignancy

R-spondins are a recently characterized small family of growth factors. Here we show that human R-spondin1 (RSPO1) is the gene disrupted in a recessive syndrome characterized by XX sex reversal, palmoplantar hyperkeratosis and predisposition to squamous cell carcinoma of the skin. Our data show, for the first time, that disruption of a single gene can lead to complete female-to-male sex reversal in the absence of the testis-determining gene, SRY.     

Msh2 deficiency prevents in vivo somatic instability of the CAG repeat in Huntington disease transgenic mice

Huntington disease (HD), an autosomal dominant, progressive neurodegenerative disorder, is caused by an expanded CAG repeat sequence leading to an increase in the number of glutamine residues in the encoded protein. The normal CAG repeat range is 5-36, whereas 38 or more repeats are found in the diseased state; the severity of disease is roughly proportional to the number of CAG repeats. HD shows anticipation, in which subsequent generations display earlier disease onsets due to intergenerational repeat expansion. For longer repeat lengths, somatic instability of the repeat size has been observed both in human cases at autopsy and in transgenic mouse models containing either a genomic fragment of human HD exon 1 (ref. 9) or an expanded repeat inserted into the endogenous mouse gene Hdh (ref. 10). With increasing repeat number, the protein changes conformation and becomes increasingly prone to aggregation, suggesting important functional correlations between repeat length and pathology. Because dinucleotide repeat instability is known to increase when the mismatch repair enzyme MSH2 is missing, we examined instability of the HD CAG repeat by crossing transgenic mice carrying exon 1 of human HD (ref. 16) with Msh2-/- mice. Our results show that Msh2 is required for somatic instability of the CAG repeat.     

Regulation of anterior/posterior patterning of the axial skeleton by growth/differentiation factor 11.

The bones that comprise the axial skeleton have distinct morphological features characteristic of their positions along the anterior/posterior axis. We previously described a novel TGF-beta family member, myostatin (encoded by the gene Mstn, formerly Gdf8), that has an essential role in regulating skeletal muscle mass. We also identified a gene related to Mstn by low-stringency screening. While the work described here was being completed, the cloning of this gene, designated Gdf11 (also called Bmp11), was also reported by other groups. Here we show that Gdf11, a new transforming growth factor beta(TGFbeta) superfamily member, has an important role in establishing this skeletal pattern. During early mouse embryogenesis, Gdf11 is expressed in the primitive streak and tail bud regions, which are sites where new mesodermal cells are generated. Homozygous mutant mice carrying a targeted deletion of Gdf11 exhibit anteriorly directed homeotic transformations throughout the axial skeleton and posterior displacement of the hindlimbs. The effect of the mutation is dose dependent, as Gdf11+/- mice have a milder phenotype than Gdf11-/- mice. Mutant embryos show alterations in patterns of Hox gene expression, suggesting that Gdf11 acts upstream of the Hox genes. Our findings suggest that Gdf11 is a secreted signal that acts globally to specify positional identity along the anterior/posterior axis.     

Alymphoplasia is caused by a point mutation in the mouse gene encoding Nf-kappa b-inducing kinase.

The alymphoplasia (aly) mutation of mouse is autosomal recessive and characterized by the systemic absence of lymph nodes (LN) and Peyer's patches (PP) and disorganized splenic and thymic structures with immunodeficiency. Although recent reports have shown that the interaction between lymphotoxin (LT) and the LT beta-receptor (Ltbeta r, encoded by Ltbr) provides a critical signal for LN genesis in mice, the aly locus on chromosome 11 is distinct from those for LT and its receptor. We found that the aly allele carries a point mutation causing an amino acid substitution in the carboxy-terminal interaction domain of Nf-kappa b-inducing kinase (Nik, encoded by the gene Nik). Transgenic complementation with wild-type Nik restored the normal structures of LN, PP, spleen and thymus, and the normal immune response in aly/aly mice. In addition, the aly mutation in a kinase domain-truncated Nik abolished its dominant-negative effect on Nf-kappa b activation induced by an excess of Ltbeta r. Our observations agree with previous reports that Ltbeta r-deficient mice showed defects in LN genesis and that Nik is a common mediator of Nf-kappa b activation by the tumour necrosis factor (TNF) receptor family. Nik is able to interact with members of the TRAF family (Traf1, 2, 3, 5 and 6), suggesting it acts downstream of TRAF-associating receptor signalling pathways, including Tnfr, Cd40, Cd30 and Ltbeta r. The phenotypes of aly/aly mice are more severe than those of Ltbr-/- mice, however, indicating involvement of Nik in signal transduction mediated by other receptors.