A Noncoding Point Mutation of Zeb1 Causes Multiple Developmental Malformations and Obesity in Twirler Mice

Kiyoto Kurima,Karl B Shpargel,Yoshiyuki Kawashima,Yujiro Higashi,Scott E Strome,Oksana Gavrilova,Ronna Hertzano,Andrew J Griffith,Taku Ito,Garani Nadaraja,Kelly Monahan,Kyu Yup Lee,David J Eisenman,Wayne N Frankel

doi:10.1371/journal.pgen.1002307

Abstract

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.

Highlights

Twirler (Tw) spontaneously arose in a crossbred stock of mice segregating multiple recessive mutant alleles [1]
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
We show that the underlying mutation affects the Zeb1 gene

Summary

Introduction

Twirler (Tw) spontaneously arose in a crossbred stock of mice segregating multiple recessive mutant alleles [1]. Heterozygous Tw mice develop obesity after three months of age, and exhibit stereotypic behavior that includes waltzing, spinning, and horizontal head-shaking [1]. This behavior is thought to result from malformed vestibular labyrinths that include hypomorphic or absent lateral semicircular canals, irregular contours of the anterior and posterior semicircular canals, and absent otoconia in the utricle and saccule [1]. Tw is located on proximal chromosome 18 but the causative mutation has not been identified [1,2]. A transgene insertional mutant, Tg9257, exhibits a similar inner ear phenotype and is located on proximal chromosome 18, raising the possibility that these phenotypes are allelic [3]. The Irxl gene, located within a broad critical map interval for Tw and expressed in developing palate, has been ruled out as a candidate for Tw [4]

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