Abstract
Using transposon-mediated gene-trap mutagenesis, we have generated a novel mouse mutant termed Blad (Bloated Bladder). Homozygous mutant mice die perinatally showing a greatly distended bladder, underdeveloped diaphragm and a reduction in total skeletal muscle mass. Wild type and heterozygote mice appear normal. Using PCR, we identified a transposon insertion site in the first intron of Nmnat2 (Nicotinamide mononucleotide adenyltransferase 2). Nmnat2 is expressed predominantly in the brain and nervous system and has been linked to the survival of axons. Expression of this gene is undetectable in Nmnat2blad/blad mutants. Examination of the brains of E18.5 Nmnat2blad/blad mutant embryos did not reveal any obvious morphological changes. In contrast, E18.5 Nmnat2blad/blad homozygotes showed an approximate 60% reduction of spinal motoneurons in the lumbar region and a more than 80% reduction in the sensory neurons of the dorsal root ganglion (DRG). In addition, facial motoneuron numbers were severely reduced, and there was virtually a complete absence of axons in the hind limb. Our observations suggest that during embryogenesis, Nmnat2 plays an important role in axonal growth or maintenance. It appears that in the absence of Nmnat2, major target organs and tissues (e.g., muscle) are not functionally innervated resulting in perinatal lethality. In addition, neither Nmnat1 nor 3 can compensate for the loss of Nmnat2. Whilst there have been recent suggestions that Nmnat2 may be an endogenous modulator of axon integrity, this work represents the first in vivo study demonstrating that Nmnat2 is involved in axon development or survival in a mammal.
Highlights
Genes required for axonal development and neuronal survival may provide targets for the treatment of neurodegenerative disorders and for the re-innervation of tissues after injury
It is a variant of the Sleeping Beauty transposon [23,24] modified to contain a proven splice acceptor at either end to ensure efficient gene trapping when inserted into an intron of a gene [19]
The chromosome 1 insertion into intron I of Nmnat2 created a null mutation and was 100% linked to the Blad phenotype in over 1,028 offspring studied
Summary
Genes required for axonal development and neuronal survival may provide targets for the treatment of neurodegenerative disorders and for the re-innervation of tissues after injury. They may be used to promote innervation of tissues and organs created using tissue engineering techniques. In Nmnat this region is palmitoylated at two cysteine residues and, when cleaved, the NAD+ synthesis activity of the enzyme increases significantly [4,10]. This provides a mechanism to increase the cytosolic pool of NAD+ quickly in response to a stimulus like cell stress
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