Heterozygous Twirler (Tw) mice develop obesity and circling behavior associated with malformations of the inner ear, whereas homozygous Tw mice have cleft palate and die shortly after birth. Zeb1 is a zinc finger protein that contributes to mesenchymal cell fate by repression of genes whose expression defines epithelial cell identity. This developmental pathway is disrupted in inner ears of Tw/Tw mice. The purpose of our study was to comprehensively characterize the Twirler phenotype and to identify the causative mutation. The Tw/+ inner ear phenotype includes irregularities of the semicircular canals, abnormal utricular otoconia, a shortened cochlear duct, and hearing loss, whereas Tw/Tw ears are severely malformed with barely recognizable anatomy. Tw/+ mice have obesity associated with insulin-resistance and have lymphoid organ hypoplasia. We identified a noncoding nucleotide substitution, c.58+181G>A, in the first intron of the Tw allele of Zeb1 (Zeb1 Tw). A knockin mouse model of c.58+181G>A recapitulated the Tw phenotype, whereas a wild-type knockin control did not, confirming the mutation as pathogenic. c.58+181G>A does not affect splicing but disrupts a predicted site for Myb protein binding, which we confirmed in vitro. In comparison, homozygosity for a targeted deletion of exon 1 of mouse Zeb1, Zeb1 ΔEx1, is associated with a subtle abnormality of the lateral semicircular canal that is different than those in Tw mice. Expression analyses of E13.5 Twirler and Zeb1 ΔEx1 ears confirm that Zeb1 ΔEx1 is a null allele, whereas Zeb1 Tw RNA is expressed at increased levels in comparison to wild-type Zeb1. We conclude that a noncoding point mutation of Zeb1 acts via a gain-of-function to disrupt regulation of Zeb1 Tw expression, epithelial-mesenchymal cell fate or interactions, and structural development of the inner ear in Twirler mice. This is a novel mechanism underlying disorders of hearing or balance.
Kurima, K., Hertzano, R., Gavrilova, O., Monahan, K., Shpargel, K. B., Nadaraja, G., … Griffith, A. J. (2011). A noncoding point mutation of Zeb1 causes multiple developmental malformations and obesity in Twirler mice. PLoS Genetics, 7(9). https://doi.org/10.1371/journal.pgen.1002307