The hyperspin mutation is a spontaneous 123.6 kb deletion that begins in intron 3 of Slc25a13 and ends in exon 17, an interval that includes an enhancer of Dlx5. This deletion includes the putative enhancer elements hs2313 and hs1642, and overlaps the regions corresponding to the predicted intervals of split-hand/foot malformation (SHFM) and split-hand/foot malformation with hearing loss (SHFM & HL) sub-regions of the SHFM1 locus, but not the split-hand/foot malformation with hearing loss and craniofacial abnormalities (SHFM & HL & CF) sub-region. Hyperspin homozygotes exhibit the phenotypes indicative of a vestibular defect, rapid circling, head shaking, and failure to orient in water, but null alleles in Slc25a13 have not been found to cause vestibular defects. At 4 to 5 weeks of age homozygotes were found to be deaf by ABR analysis. At 5 weeks of age the inner ears of homozygotes are small and malformed, the cochlea has too few turns and appears swollen, and the three semicircular canals are absent, reduced, or malformed. At embryonic day 15.5 the endolymphatic duct and sac and the superior and posterior semicircular canals are missing, the cochlear duct is wider and has fewer coils than normal, and most homozygotes also lack the lateral semicircular canal and utricle. Scanning electron microscopy of the basal and middle turns of the cochlea at 4 weeks of age revealed only occasional missing hair bundles, but the apical turn had an additional forth and sometimes fifth row of outer hair cells and abnormal hair bundle orientations. Only approximately 30% of female and 50% of male hyperspin homozygotes survive to wean age. Hyperspin homozygotes have reduced otocyst expression but not branchial arch expression of Dlx5, a gene more than 600 kb away on Chromosome 6, and null alleles of Dlx5 have been found to cause perinatal death, dysmorphic inner ears and other craniofacial abnormalities. While hyperspin heterozygotes have a normal phenotype fewer than 10% of Slc25a13hspn Dlx5+/Slc25a13+ Dlx5tm1(cre/ERT2)Zjh transchromosomal heterozygotes survive to wean age, a more severe phenotype than that of hyperspin homozygotes and further proof of the presence of a Dlx5 modifier within the hyperspin deletion. These double heterozygotes display circling and head bobbing, are deaf by ABR, and have malformed inner ears similar to that found in hyperspin homozygotes.
To identify the sequence within the hyperspin deletion that regulates Dlx5 otic expression, endonuclease-mediated deletions were generated of hs2313 in intron 14, hs1642 in intron 3, and intron 3 through 4. None of these caused the phenotype found in hyperspin homozygotes, even when Dlx5tm1(cre/ERT2)Zjh transchromosomal heterozygotes were generated.