Cell Type–Specific Transcriptome Analysis Reveals a Major Role for Zeb1 and miR-200b in Mouse Inner Ear Morphogenesis

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 (Zeb1Tw). 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, Zeb1ΔEx1, 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 Zeb1ΔEx1 ears confirm that Zeb1ΔEx1 is a null allele, whereas Zeb1Tw 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 Zeb1Tw 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.
Author Summary Twirler (Tw) mice have a combination of abnormalities that includes cleft palate, malformations of the inner ear, hearing loss, vestibular dysfunction, obesity, and lymphoid hypoplasia. In this study, we show that the underlying mutation affects the Zeb1 gene. Zeb1 was already known to encode a protein normally expressed in mesenchymal cells, where it represses expression of genes that are uniquely expressed in epithelial cells. The Tw mutation is a rare example of a single-nucleotide substitution in a region of a gene that does not encode protein, promoter, or splice sites, so we engineered a mouse model with the mutation that confirmed its causative role. The Tw mutation disrupts a consensus DNA binding site sequence for the Myb family of regulatory proteins. We conclude that this mutation leads to abnormal expression of Zeb1, structural malformations of the inner ear, and a loss of hearing and balance function. A similar mechanism may underlie other features of Twirler, such as obesity and cleft palate.