Determining the molecular basis of the mutation underlying the mouse neural tube closure mutant, Splotch

Splotch (Sp) is a semidominant mouse mutant which maps to the proximal portion of chromosome 1 and is phenotypically expressed as a pleiotropic defect during neurogenesis, resulting in spina bifida, exencephaly and dysgenesis of neural crest cell derivatives. To identify the aberrant gene underlying the defects observed in the Sp mouse mutant we initiated positional cloning strategy. Our preliminary efforts were directed at establishing the boundaries of a deleted chromosomal segment found in the Sp$ sp{r}$ allele, using nine gene probes that were assigned to that region of chromosome 1. Four of these genes, Vil, Des, Inha, and Akp-3, spanning a genetic distance of approximately 15 cM, were found to map within the Sp$ sp{r}$ deletion. In order to further delineate the subchromosomal location of the Sp gene, the proximal segment of mouse chromosome 1 was saturated with microclones isolated from a library of microdissected genomic fragments generated from this region. An additional eight markers were found to map within the confines of the Sp$ sp{r}$ deletion.During the course of this work a member of the paired box gene family, Pax-3, was described as a candidate for Sp. The striking similarity between the tissue distribution of Pax-3 mRNA in normal developing embryos, and the neural structures affected in Sp mice, together with the chromosome 1 location of Pax-3 led us to examine whether Pax-3 was mutated in three alleles at this locus Sp$ sp{r}$, Sp$ sp{2H}$ and Sp. The entire Pax-3 gene was determined to be deleted in the Sp$ sp{r}$ allele. Analysis of genomic DNA and cDNA clones constructed from RNA isolated from $Sp sp{2H}/Sp sp{2H}$ embryos identified a deletion of 32 nucleotides within the paired type homeobox and is predicted to produce a truncated protein as a result of a newly created termination codon at the deletion breakpoint. The original Sp allele was also characterized and found to contain an A to T transversion at position -2 in the third intron of Pax-3 which abrogates the normal splicing of this intron due to the loss of its natural 3$ sp prime$ splice acceptor. Taken together, these studies indicate that the severe defect in neural tube formation detected in Sp and its allelic variants is linked to the inactivation of the paired box gene Pax-3, and provides direct genetic evidence of a key role for Pax-3 in normal neural development