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The secreted molecule fibroblast growth factor 9 (FGF9) plays a critical role in testis determination in the mouse. In embryonic gonadal somatic cells it is required for maintenance of SOX9 expression, a key determinant of Sertoli cell fate. Conditional gene targeting studies have identified FGFR2 as the main gonadal receptor for FGF9 during sex determination. However, such studies can be complicated by inefficient and variable deletion of floxed alleles, depending on the choice of Cre deleter strain. Here, we report a novel, constitutive allele of Fgfr2, hobbyhorse (hob), which was identified in an ENU-based forward genetic screen for novel testis-determining loci. Fgr2hob is caused by a C to T mutation in the invariant exon 7, resulting in a polypeptide with a mis-sense mutation at position 263 (Pro263Ser) in the third extracellular immunoglobulin-like domain of FGFR2. Mutant homozygous embryos show severe limb and lung defects and, when on the sensitised C57BL/6J (B6) genetic background, undergo complete XY gonadal sex reversal associated with failure to maintain expression of Sox9. Genetic crosses employing a null mutant of Fgfr2 suggest that Fgr2hob is a hypomorphic allele, affecting both the FGFR2b and FGFR2c splice isoforms of the receptor. We exploited the consistent phenotype of this constitutive mutant by analysing MAPK signalling at the sex-determining stage of gonad development, but no significant abnormalities in mutant embryos were detected. [ABSTRACT FROM AUTHOR]

We employed a random mutagenesis approach to identify novel monogenic determinants of type 2 diabetes. Here we show that haplo-insufficiency of the histone methyltransferase myeloid-lineage leukemia (Mll2/Wbp7) gene causes type 2 diabetes in the mouse. We have shown that mice heterozygous for two separate mutations in the SET domain of Mll2 or heterozygous Mll2 knockout mice were hyperglycaemic, hyperinsulinaemic and developed non-alcoholic fatty liver disease. Consistent with previous Mll2 knockout studies, mice homozygous for either ENU mutation (or compound heterozygotes) died during embryonic development at 9.5–14.5 days post coitum. Heterozygous deletion of Mll2 induced in the adult mouse results in a normal phenotype suggesting that changes in chromatin methylation during development result in the adult phenotype. Mll2 has been shown to regulate a small subset of genes, a number of which Neurod1, Enpp1, Slc27a2, and Plcxd1 are downregulated in adult mutant mice. Our results demonstrate that histone H3K4 methyltransferase Mll2 is a component of the genetic regulation necessary for glucose homeostasis, resulting in a specific disease pattern linking chromatin modification with causes and progression of type 2 diabetes, providing a basis for its further understanding at the molecular level. [ABSTRACT FROM AUTHOR]

Planar cell polarity (PCP) is a process in which cells develop with uniform orientation within the plane of an epithelium. To begin to elucidate the mechanisms of PCP in vertebrates, the localization of the protein Vangl2 (Van Gogh-like) was determined during the development of the mammalian cochlea. Results indicate that Vangl2 becomes asymmetrically localized to specific cell- cell boundaries along the axis of polarization and that this asymmetry is lost in PCP mutants. In addition, PDZ2 (postsynaptic density/Discs large/zona occludens 1), PDZ3, and PDZ4 of the PCP protein Scrb1 (Scribble) are shown to bind to the C-terminal PDZ binding domain of Vangl2, suggesting that Scrb1 plays a direct role in asymmetric targeting of Vangl2. Finally, Fz3 (Frizzled), a newly demonstrated mediator of PCP, is also asymmetrically localized in a pattern that matches that of Vangl2. The presence and asymmetry of Fz3 at the membrane is shown to be dependent on Vangl2. This result suggests a role for Vangl2 in the targeting or anchoring of Fz3, a hypothesis strengthened by the existence of a physical interaction between the two proteins. Together, our data support the idea that protein asymmetry plays an important role in the development of PCP, but the colocalization and interaction of Fz3 and Vangl2 suggests that novel PCP mechanisms exist in vertebrates. [ABSTRACT FROM AUTHOR]

ENU mutagenesis is a powerful tool for the identification of novel models of human disease. Haploinsufficiency of the insulin receptor (IR) and associated mild insulin resistance has been utilised to sensitise an ENU screen to identify novel mutations resulting in impaired glucose tolerance and diabetes. An intraperitoneal glucose tolerance test (IPGTT) was utilised in a phenotype driven screen to identify F1 individuals with impaired glucose tolerance. Animals deviating more than 2 SDs from an unmutagenised control population entered inheritance testing. Here we describe two dominant mutations in the highly conserved HMG box of the transcription factor Sox4, one identified in the phenotype driven screen and the other subsequently obtained from an embryonic screen. Sox4 has previously been associated with pancreas development and, as expected, homozygotes for either mutation or compound heterozygotes were embryonic lethal, suggesting both mutations affect gene function and are nulls. Heterozygous Sox4 adult mice had mildly impaired glucose tolerance, and insulin secretion assays in both whole animals and in isolated islets indicated a defect in insulin secretion. In association with IR+/- induced insulin resistance, this insulin secretory defect causes a more severe impaired glucose tolerance and represents a novel polygenic model of type 2 diabetes. Knockdown of Sox4 in INS1 cells with two different siRNAs, targeted to separate regions of the gene, abolished glucose-stimulated insulin release. Interestingly, we also observed a significant decrease in insulin release in response to tolbutamide in Sox4 siRNA treated cells. Tolbutamide stimulates insulin secretion in pancreatic beta cells by closing KATP channels in the plasma membrane, leading to membrane depolarisation and activation of voltage-gated calcium channels. This results in calcium influx and a rise in intracellular calcium ([Ca2+]i) that triggers insulin release. Changes in [Ca2+]i evoked by glucose in Sox4-siRNA treated cells, however, were normal, indicating that KATP channels close normally. Taken together with the impaired secretory response to tolbutamide, this suggests the insulin secretory defect produced by Sox4 knockdown lies downstream of calcium signalling, perhaps at the level of exocytosis. [ABSTRACT FROM AUTHOR]

Type 2 diabetes is a complex disease that is caused by genetic and environmental factors, of which the genetic factors are not all understood. Population studies and murine models are complimentary approaches to identifying such genetic factors. Our approach was to identify novel genetic mutations causative for diabetes in a murine model. Initially, BALB/c male mice were treated with the chemical mutagen ENU to create random mutations throughout the genome. These mice were mated to female C3H/HeH mice and the progeny screened for high flee fed plasma glucose. Here we describe the genetic and physiological characterization of one mutant where male mice were identified with high plasma glucose, buttermouse. After identification of the mutant mouse, the F1 male was continually backcrossed onto a C3H/HeH background. Successive phenotyping and genotyping identified a region of 350kbp of DNA on chromosome 7 to contain the mutation. Sequencing of candidate genes revealed a putative missense mutation in a transcription factor. Functional studies have begun to determine if this mutation is causative for the phenotype. Heterozygous matings of male and female buttermouse showed that the homozygous phenotype was embryonic lethal. An intraperitoneal glucose tolerance test was performed and glucose concentrations were measured at 0, 60 and 120 minutes. At all time points the glucose concentrations were significantly higher for male buttermouse than wild-type controls (10.2±2.7, 25.6±5.9, 20.5±6.1 compared to 7.7±1.9, 15.0±4.1, 10.2±2.8, p<0.01, n>60). Liver sections at 19 weeks of age indicated that male buttermouse developed hepatic steatosis and hepatocyte ballooning suggestive of mild non-alcoholic fatty liver disease. Dependent on functional gene studies, we have identified buttermouse as a novel genetic model for type 2 diabetes. We plan to continue to study buttermouse as a model to understand how alterations in gene expression can lead to type 2 diabetes. [ABSTRACT FROM AUTHOR]