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Congenital vertebral malformations caused by embryonic segmentation defects are relatively common in humans and domestic animals. Although reverse genetics approaches in mice have provided information on the molecular mechanisms of embryonic somite segmentation, hypothesis-driven approaches cannot adequately reflect human dysmorphology within the population. In a N-ethyl-N-nitrosourea (ENU) mutagenesis project in Kyoto, the Oune mutant rat strain was isolated due to a short and kinked caudal vertebra phenotype. Skeletal staining of heterozygous rats showed partial loss of the cervical vertebrae as well as hemivertebrae and fused vertebral blocks in lumbar and sacral vertebrae. In homozygous embryos, severe displacement of the whole vertebrae was observed. The Oune locus was genetically mapped to rat chromosome 1 using 202 backcross animals and 50 genome-wide microsatellite markers. Subsequently, a miss-sense mutation in the Tbx6 gene was identified in the critical region. Although the mutation is located within the T-box domain near a predicted dimmer-interface, in vitro experiments revealed that the Tbx6 variant retains normal DNA binding ability and translational efficiency. However, the variant has decreased transcriptional activation potential in response to Notch-mediated signaling. Recently, it was reported that a dominant type of familial spondylocostal dysostosis is caused by a stoploss mutation in TBX6. Thus, we propose that partial dysfunction of Tbx6 leads to similar congenital vertebral malformations in both humans and rats. The Oune strain could be a unique animal model for dominant spondylocostal dysostosis and is useful for molecular dissection of the pathology of congenital vertebral malformations in humans.

Inflammation is a complex cellular and humoral response against trauma and infection, and its presence leads to destruction of tissue in humans. The mechanisms that initiate inflammatory diseases remain largely unknown because of complex interactions between multiple genetic and environmental factors during pathogenesis. Animal models for human diseases offer dissection of complex pathogenesis by inbred genetic backgrounds and controlled circumstances. In this article we report a chemically induced new mutation, Ali18 (Abnormal limb), as a mouse model for inflammatory arthritis and dermatitis. Ali18/+ mice exhibit rubor and swelling of footpads in hindlimbs in adults. In Ali18/Ali18 mice, the digits in forelimbs and hindlimbs and tails were necrotic and/or deformed by severe swelling. Histologic analysis revealed infiltration of mixed populations of inflammatory cells into bone marrow, peripheral joints, and skin in the affected areas of Ali18/Ali18 mice. In addition, generalized osteoporosis-like phenotypes were confirmed by dual energy X-ray absorptiometry (DXA), microcomputed tomography (muCT), and peripheral quantitative computed tomography (pQCT) in homozygous animals. Whereas the Ali18 mutation was mapped to a single locus, the phenotype presentation was altered by complex modifier effects from other inbred genetic backgrounds. Detailed analysis of the Ali18 phenotype and identification of the mutation and its modifier genes may provide molecular insights into the complex nature of inflammatory diseases and the relationship between inflammation and bone metabolism.

Conversion of the cellular prion protein (PrPC) into the pathogenic isoform (PrPSc) is the fundamental event underlying transmission and pathogenesis of prion diseases. To control the expression of PrPC in transgenic (Tg) mice, we used a tetracycline controlled transactivator (tTA) driven by the PrP gene control elements and a tTA-responsive promoter linked to a PrP gene [Gossen, M. and Bujard, H. (1992) Proc. Natl. Acad. Sci. USA 89, 5547-5551]. Adult Tg mice showed no deleterious effects upon repression of PrPC expression (>90%) by oral doxycycline, but the mice developed progressive ataxia at approximately 50 days after inoculation with prions unless maintained on doxycycline. Although Tg mice on doxycycline accumulated low levels of PrPSc, they showed no neurologic dysfunction, indicating that low levels of PrPSc can be tolerated. Use of the tTA system to control PrP expression allowed production of Tg mice with high levels of PrP that otherwise cause many embryonic and neonatal deaths. Measurement of PrPSc clearance in Tg mice should be possible, facilitating the development of pharmacotherapeutics.

Dehydration stress in the drought-tolerant resurrection plant Craterostigma plantagineum is accompanied by the accumulation of a large number of desiccation stress proteins (Dsp). One abundant class of these is represented by the dehydrin-related Dsp16 protein which contains 15 amino acid conserved lysine-rich repeats and a stretch of eight serine residues providing extremely hydrophilic characteristics. Recombinant Dsp16 from Craterostigma plantagineum has been cloned and expressed in Escherichia coli. The protein was purified and characterized regarding its physicochemical properties. Irrespective of successful crystallization experiments, dilute aqueous buffer solutions do not display a well-defined three-dimensional structure in terms of the canonical secondary structural elements. 1H-NMR (nuclear magnetic resonance) spectra in aqueous solution are characterized by a small chemical shift dispersion typical for an unfolded protein; however, the observed line-widths are not typical for a highly mobile random coil structure. Instead they indicate an equilibrium between conformational states with preferentially extended substructures. As a consequence of its loose structure, Dsp16 is extremely sensitive towards proteolysis unless its structure is stabilized by structure-making additives such as trifluoroethanol. Denaturants such as guanidinium chloride do not induce cooperative structural transitions. pH-dependent fluorescence changes reflect protonation/deprotonation rather than conformational changes. Sedimentation/diffusion experiments confirm the predicted molecular mass of 16 kDa. Due to the high serine/threonine content and its loose structure, Dsp16 is accessible to phosphorylation, supporting the idea that in situ the structurally relatively undefined protein may be involved in both water binding and phosphorylation.