Three Novel Pax6 Alleles in the Mouse Leading to the Same Small-Eye Phenotype Caused by Different Consequences at Target Promoters

PURPOSE. To characterize three new mouse small-eye mutants detected during ethylnitrosourea mutagenesis programs. METHODS. Three new mouse small-eye mutants were morphologically characterized, particularly by in situ hybridization. The mutations were mapped, and the candidate gene was sequenced. The relative amount of Pax6-specific mRNA was determined by real-time PCR. Reporter gene analysis used Crygf and Six3 promoter fragments in front of a luciferase gene and HEK293 cells as recipients. RESULTS. The new mutations-ADD4802, Aey11, and Aey 18-were mapped to chromosome 2; causative mutations have been characterized in Pax6 (Aey11: C→T substitution in exon 8, creating a stop codon just in front of the homeobox; ADD4802: G→A substitution at the beginning of intron 8 changes splicing and leads to an altered open reading frame and then to a premature stop codon; Aey18: G→A exchange in the last base of intron 5a leads also to a splice defect, skipping exons 5a and 6). Real-time PCR indicated nonsense-mediated decay in Pax6 Aey11 and Pax6 Aey18 mutants but not in Pax6 ADD4802 . This result is supported by the functional analysis of corresponding expression constructs in cell culture, where the Aeyl 1 and Aey18 alleles did not show a stimulation of the Six3 promotor or an inhibition of the Crygf promoter (as wild-type constructs do). However, the Pax6 ADD4802 allele stimulated both promoters. CONCLUSIONS. Together with functional analysis in a reporter gene assay and immunohistochemistry using Pax6 antibodies, it is suggested that the Pax6 Aey11 and Pax6 Aey18 alleles act through a loss of function, whereas ADD4802 represents a gain-of-function allele.