Screening for novel ENU-induced rhythm, entrainment and activity mutants.

Chemical mutagenesis has provided an opportunity to develop and expand the repertoire of behavioural mutants for gene function studies. With this in mind, we have established a screen in mice for mutations affecting circadian rhythms, entrainment to light and other wheel-running parameters. The screen consists of an assessment of mouse wheel-running activity in a 12:12 h light/dark cycle for 7-10 days followed by assessment in constant darkness for up to 20 days. Responses to light are assessed using two protocols; a 15 minute light pulse given at circadian time 16 on the tenth day in constant darkness and an additional 12 h of light upon transition from light/dark conditions to constant darkness. To date, approximately 1300 progeny of chemically mutagenised mice have been screened. Computer-aided assessment of wheel-running parameters has helped in identifying abnormal phenotypes in approximately 5% of all animals screened. Inheritance testing of mice with abnormal phenotypes has confirmed the number of robustly inherited mutant phenotypes to be 1% of the total screened. Confirmed mutants including those affecting free-running period, light-responsiveness and wheel-running endurance have been identified. Thus far, low-resolution map positions have been established for four mutants by completing genome scans in backcross progeny. Mutant loci do not correspond with those previously associated with wheel-running behaviour. This result confirms that phenotype-driven approaches such as this should continue to provide material for mammalian gene function studies.

Molecular phenotyping of the mouse ky mutant reveals UCP1 upregulation at the neuromuscular junctions of dystrophic soleus muscle.

The ky mutant mouse displays a muscular dystrophy that affects almost exclusively slow type muscles in which persistent muscle regeneration, neuromuscular junction instability and an absence of the hypertrophic response are prominent features. In order to gain insights into the pathogenesis of this muscular dystrophy we have undertaken RNA profiling of the extensor digitorum longus, a fast unaffected muscle, and the highly pathological soleus slow muscle, followed by further expression studies to validate the results. In dystrophic soleus, there is a coordinated change in the expression level of genes encoding energy transducing mitochondrial proteins and an increase in the expression of stretch response genes. Upregulation of uncoupling proteins 1 and 2 is a unique molecular signature of the ky muscular dystrophy and was further characterised at the protein level. Our results show a spatial and temporal association between disorganisation of acetylcholine receptor clusters and upregulation of uncoupling protein 1. There is also evidence of a breakdown of neuromuscular junction muscle-specific kinase-dependent signalling in adult mutant soleus. Sarcolemma-associated proteins implicated in muscular dystrophies revealed no differences on microarrays and were confirmed as normally distributed by immunofluorescence. Altogether, the data presented suggest that the ky muscular dystrophy develops by a distinctive pathogenic mechanism.