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

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.


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]. In contrast, all homozygous Tw mice are born with cleft palate and die soon after birth [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 also located on proximal chromosome 18, raising the possibility that these phenotypes are allelic [3]. However, complementation testing is inconclusive [3]. Similarly, the Irxl1 gene, located within a broad critical map interval for Tw and expressed in developing palate, has also been ruled out as a candidate for Tw [4].
Zeb1 is also located on proximal chromosome 18 and encodes a transcription factor, Zeb1, that binds E-box-like elements to either repress [5,6], or activate transcription [7][8][9]. Mice that are homozygous for a targeted deletion of exon 1 of Zeb1 (Zeb1 DEx1 ) die soon after birth with cleft palate, limb defects and other skeletal abnormalities, and T-cell deficiency [10], whereas heterozygous Zeb1 DEx1/+ mice are viable and adult females show increased adiposity [11]. This partial phenotypic overlap with Twirler does not include stereotypic vestibular behavior or inner ear malformations, although these were likely not examined in Zeb1 DEx1 mice. Ectopic expression of Zeb1 in neoplastic epithelium has been implicated in the epithelial-to-mesenchymal transition (EMT) leading to local tumor invasion and metastasis [12]. In normally developing mesenchymal tissue, Zeb1 is thought to repress epithelial-specific genes such as E-cadherin and activate mesenchyme-specific genes such as collagen, smooth muscle actin and myosin [9]. Genome-wide expression profiling reveals a probable similar role for Zeb1 in the regulation of gene expression in developing mouse inner ear mesenchyme [13]. In humans, heterozygous mutations of ZEB1 cause posterior polymorphous corneal dystrophy, characterized by an epithelial transition and abnormal proliferation of corneal endothelium [14]. Zeb1 DEx1/+ mice also show corneal abnormalities and further implicate Zeb1 in the suppression of an epithelial phenotype [15].
In the current study we show that Twirler is caused by a noncoding point substitution in the first intron of Zeb1. The mutation does not affect splicing, but does disrupt a consensus binding site sequence for Myb proteins [16]. The maintenance of inner ear mesenchyme-and epithelium-specific gene expression is disrupted in Twirler inner ears [13], demonstrating a novel mutation and developmental mechanism for the pathogenesis of hearing or balance disorders.

Obesity and metabolic phenotype of Twirler mice
There was no significant difference in average body weight between Tw/+ and wild type littermates of either sex until 12 weeks of age ( Figure 1A and 1B). Beginning at seven weeks of age, Tw/+ mice consumed approximately 15 to 20% more food than wild type littermates ( Figure 1C and 1D). There was a significant increase in the percentage of body fat and slightly reduced lean body mass in Tw/+ mice of both sexes (Table 1), indicating that fat accounts for the increased body mass. Body weight-adjusted energy expenditure, estimated from oxygen consumption, revealed a reduced metabolic rate in Tw/+ mice that did not reach statistical significance (Table 1). Tw/+ mice had normal serum glucose levels but elevated levels of serum free fatty acids, triglycerides, insulin, leptin, corticosterone and adiponectin (Table 1). Insulin and glucose tolerance tests of 15-week-old females showed insulin resistance and slight glucose intolerance in Tw/+ mice ( Figure 1E and 1F), consistent with data for other obese mice with hyperinsulinemia [17].

Inner ear phenotype of Twirler mice
We evaluated the morphology of mutant inner ears using the paint-filling technique ( Figure 2). The Tw/+ inner ears had grossly intact semicircular canals and neurosensory cristae ampullaris, but the contours of the canals were irregular due to small bulges and projections ( Figure 2B). The most anatomically consistent malformation was found at the non-ampullated end of the lateral canal where it normally narrows to join the vestibule in wild type ears ( Figure 2D). In contrast, the non-ampullated ends of Tw/+ lateral canals were irregular or constricted ( Figure 2E). Tw/Tw inner ears have more severe malformations that include absence of the lateral semicircular canal, truncation of the posterior semicircular canal, and shortening of the cochlear duct ( Figure 2C, 2F and 2I).
The average length of Tw/+ cochlear ducts ( Figure 2H) was 91% (65%) that of wild type ears (P,0.00002; Figure 2G). Binaural average ABR thresholds were elevated for Tw/+ mice in comparison to wild type controls at one month of age (3361.6 dBSPL vs. 5565.3 dBSPL at 8 kHz, p,0.0006; 3361.8 dBSPL vs. 4664 dBSPL at 16 kHz, p,0.01; 2961.9 dBSPL vs. 3963.6 dBSPL at 32 kHz, p,0.023; Figure 2J). Tw/+ mice showed no significant change in ABR thresholds measured at three months of age in comparison to thresholds measured at one month of age (not shown).

Linkage mapping and positional cloning of Tw
We screened 1679 [(C57BL/6J-Tw/+ x CAST/Ei)F1-Tw/+ x C57BL/6J]N2 progeny for recombinations. Recombination locations were refined with additional markers to narrow the Tw interval to 814 kb between D18Nih6 and D18Nih42 ( Figure 3A). This interval was five Mb proximal to the Tg9257 transgene insertion site [3]. The Tw interval contained three genes: Zeb1, Zeb1os (Zeb1 opposite strand transcript, annotated in MGI as predicted gene Gm10125) and Zfp438 ( Figure 3A). Zeb1 encodes a transcription factor with two zinc finger motifs and one homeobox motif. Zeb1os is predicted to encode a long noncoding RNA of unknown function. It is located on the opposite strand of Zeb1 where the two overlapping genes share parts of their first introns. Finally, Zfp438 is predicted to encode a zinc finger protein whose biological function is unknown [18].
Zeb1 was a good candidate for the gene mutated in Tw based upon the phenotype associated with a targeted deletion allele, Zeb1 DEx1 . Homozygous Zeb1 DEx1 mice are born with cleft palate, skeletal and thymus abnormalities, and die shortly after birth [10]. We observed that Zeb1 DEx1/+ heterozygotes have inner ear morphology and hearing thresholds that are indistinguishable

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.
from those of wild type littermates, whereas Zeb1 DEx1/DEx1 homozygotes have a subtle constriction of the midportion of the lateral semicircular canal that differs in location and severity from that observed in Tw/+ mice ( Figure S1). This difference is probably not due to genetic background since both lines were congenic on a C57BL/6J background.
To determine if Tw and Zeb1 DEx1 can complement to form a normal palate or inner ear, we crossed heterozygous Tw and heterozygous Zeb1 DEx1 mice. We observed an approximate Mendelian ratio of genotypes: five +/+, five Tw/+, seven Zeb1 DEx1/+ and eight Tw/Zeb1 DEx1 . All Tw/Zeb1 DEx1 mice were born with normal palates and developed into adults with circling behavior typical of Tw/+ mice. The lateral semicircular canals resembled those of Tw/+ mice ( Figure S1). These results suggest these mutations exert their effects via different genes or mechanisms. While the Zeb1 pathway may be altered in Twirler mice, it is unlikely to be due to a loss-of-function allele of Zeb1.
To identify the Tw mutation, we first used 59-RACE and 39-RACE to identify novel exons of Zeb1, Zeb1os and Zfp438. 59-RACE revealed Zeb1 transcripts with each of five additional alternative first exons (designated 1b, 1c, 1d, 1e and 1f) between exon 1 (heretofore termed exon 1a) and exon 2 ( Figure S2). We amplified and sequenced all novel and annotated exons of Zeb1, Zeb1os and Zfp438 from genomic DNA of Tw/Tw, Tw/+ and wild type mice. We also amplified and sequenced cDNA transcripts of these genes from embryonic mRNA. All major transcripts of these genes were amplified from mice with each genotype. We found no sequence differences in the cDNAs or genomic exons. Sequence analysis of the 192-bp region of overlap of Zeb1 and Zeb1os revealed a single nucleotide substitution (G.A) 181 bp downstream of Zeb1 exon 1 and 12 bp downstream of Zeb1os exon 1 in Tw ( Figure 3B). We designated this Tw variant as c.58+181G.A, which was the only sequence variation we detected. The wild type variant c.58+181G was conserved among 13 normal control inbred mouse strains as well as other vertebrate species ( Figure 3B). . Body weight, daily food intake, and insulin and glucose tolerance of Twirler mice. Body weight was measured weekly in males (A) and females (B). (n = 6 for each sex and each genotype). Food intake was measured weekly to calculate average daily intake for males (C) and females (D). Tw/+ males and females show increased body weight after 12 and 13 weeks of age, respectively. Both Tw/+ males and females show increased food intake at most tested ages. Serum glucose levels are shown for Tw/+ (n = 8) and wild type (n = 6) female mice injected with insulin (E) or glucose (F) at 15 weeks of age. Mean body weights were 28.5 g and 22.4 g for Tw/+ and wild type mice, respectively. Tw/+ mice show insulin resistance (E). In the glucose tolerance test, the area under the curve (AUC) is significantly higher in Tw/+ mice compared with wild type mice (p = 0.039). The vertical bars indicate the standard error of the mean (SEM In silico analyses (NNsplice, GeneSplicer, Net2Gene) predict that c.58+181G.A does not affect splicing of the adjacent splice donor site for exon 1 of Zeb1os. Sequence analysis of Zeb1 and Zeb1os cDNA transcripts confirmed no effect of c.58+181G.A on splicing.
Electrophoretic mobility shift assay of Tw DNA c.58+181G.A disrupts a predicted site for Myb protein binding ( Figure 3B) [16]. To test if this change can alter the binding of a Myb protein, recombinant mouse C-Myb was expressed and purified for an electrophoretic mobility shift assay (EMSA) of its binding to oligonucleotide probes containing either c.58+181G or c.58+181A and the flanking genomic sequences. There was a shift of the mobility of the wild type DNA probe in the presence of C-Myb, while the Tw DNA probe mobility was unchanged ( Figure 4A). The binding of C-Myb to wild type DNA was inhibited by both the wild type probe and a mim-1 control probe which has been shown to interact with C-Myb [19], but not by the Tw probe ( Figure 4B). These data provide in vitro evidence that the Tw mutation can disrupt binding of a Myb protein (C-Myb) to the mutated first intronic sequence of Zeb1.
Quantitative RT-PCR analysis of Zeb1 Tw and Zeb1 DEx1 transcripts We analyzed mRNA expression levels of Zeb1, Zeb1os and Zfp438 from inner ears of Tw/Tw, Tw/+ or wild type mice at E13.5. We performed the same analysis with Zeb1 DEx1 heterozygotes, homozygotes, and wild type littermates. We designed primer pairs to specifically amplify Zeb1 transcripts starting from each of exons 1a, 1b, 1c, 1d, 1e or 1f. One primer pair for constitutively spliced exons 2 and 3 was designed to amplify all Zeb1 transcripts. The levels of Zeb1 transcripts containing exon 1b, 1c, 1d, 1e, or 1f, as well as the Zeb1os and Zfp438 transcripts, were too low to be reliably quantified by RT-PCR. The levels of transcripts containing exons 1a and 2, as well as exons 2 and 3, were significantly increased from the Tw allele of Zeb1 (Zeb1 Tw ) in comparison to wild type Zeb1 ( Figure 5A). In contrast, Zeb1 DEx1 expressed no Zeb1 transcripts containing exons 1a and 2, and nearly non-detectable levels of any other Zeb1 transcripts containing other exons ( Figure 5B). Transcripts levels for the closely related Zeb2 gene were unchanged among all three Zeb1 genotypes ( Figure 5B). These results indicate that Zeb1 DEx1 is a loss-of-function allele whereas Zeb1 Tw is likely to act via gain-offunction. The overall structure of Tw/+ inner ears was intact, but the contours of the semicircular canals were irregular due to small bulges and projections along the canals (indicated by asterisks in B). There were irregularities and constrictions of the non-ampullated ends of lateral canals (E). Shortened cochlear ducts are consistently observed in Tw/ + ears (H). Tw/Tw inner ear anatomy is disrupted but recognizable (C, F, I). Tw/Tw semicircular canals and cochlear ducts were either discontinuous or ruptured. Average ABR thresholds for all wild type ears (n = 24) are shown as white circles and Tw/+ ears (n = 24) as black circles with the standard error of the mean (SEM). Results are shown for click, 8-, 16-and 32-kHz pure-tone stimuli (J). Scanning electron microscopy showed no difference in otoconia between wild type (K) and Tw/+ saccules (L) at P6. Tw/+ utricles (N) had giant otoconia. a, anterior semicircular canal; c, cochlear duct; es, endolymphatic sac; l, lateral semicircular canal; p, posterior semicircular canal; s, saccule; u, utricle. doi:10.1371/journal.pgen.1002307.g002 A mouse knockin of the Zeb1 Tw intron 1 sequence variant recapitulates the Twirler phenotype To confirm the pathogenic effect of c.58+181G.A, we generated two knockin mouse lines: KI A segregates the Tw variant c.58+181A and KI G segregates the wild type variant c.58+181G ( Figure 6). Compound heterozygous KI G /KI A mice consumed more food and grew heavier with increased adiposity in comparison to KI G /KI G control males and females ( Figure 7A-7D, Table 2). The energy expenditure and circulating hormone levels in KI G /KI A mice recapitulated the Tw/+ phenotype ( Table 2). The reduction in body weight-adjusted energy expenditure reached statistical significance in KI G /KI A female mice, whereas it did not in Tw/+ females (Table 1). Insulin and glucose tolerance tests showed insulin resistance and slight glucose intolerance in KI G /KI A mice ( Figure 7E and 7F). Although KI G /KI A mice showed neither circling behavior nor constricted semicircular canals, the semicircular canals were irregular ( Figure 8B) and the utricles contained giant otoconia ( Figure 8N). Average ABR thresholds for KI G /KI A and KI G /KI G mice were not significantly different ( Figure 8J). KI A /KI A and KI A / Tw inner ears displayed the same malformations as Tw/Tw ears ( Figure 8C, 8F, 8I, and Figure 9B, 9D, 9F). KI G /KI A average spleen weight was decreased by 15% (P,0.05) but average thymus weight did not differ relative to KI G /KI G littermates ( Table 2). We observed cleft palate with or without cleft lip in KI A /KI A and KI A /Tw mice with 50% and 90% penetrance, respectively (not shown). We did not observe cleft palate or cleft lip in KI G /KI A , KI G /KI G or KI G /Tw mice, indicating that the recapitulation of the Tw phenotype is specific.
The different phenotypic severity and penetrance of KI A in comparison to Tw could result from genetic background differences, since Tw arose on a different undefined stock. However, we serially backcrossed Tw to wild type C57BL/6J for over 30 generations, and KI A was generated from C57BL/6-derived Bruce4 ES cells and maintained on an isogenic C57BL/6J background. Therefore the differences in severity and penetrance could result from closely linked sequence variation, the residual loxP site in KI A , or a combination of these effects.

Zeb1 protein expression in Twirler inner ears
To determine if Zeb1 protein is expressed from the Tw allele, we stained inner ears of Tw/Tw mice with anti-Zeb1 antibodies ( Figure 10A and 10B). We observed Zeb1 expression in nonepithelial (mesenchymal) cells surrounding Tw/Tw inner ears in which epithelial and mesenchymal tissue compartments could be microanatomically differentiated ( Figure 10B). Other Tw/Tw inner ears had poorly preserved microarchitecture, precluding a differentiation of epithelium versus mesenchyme ( Figure 10C). We conclude that Zeb1 protein is expressed in Tw/Tw ears, consistent with the result of real-time RT-PCR.
To determine if Zeb1 protein levels are altered by Tw, we performed a western blot analysis of inner-ear or whole-head protein extracts from E13.5 mice. We compared Tw/Tw, Tw/ + and wild type littermates, as well as KI G /KI G , KI G /KI A and KI A /KI A littermates. We were unable to detect Zeb1 in innerear protein extracts, but were able to reliably detect it in samples from whole heads. Total Zeb1 protein levels appeared to be slightly increased by Tw in comparison to wild type littermates ( Figure 10D). This difference was not significant (ANOVA, P.0.05), possibly due to small numbers of animals and the degree of variation of Zeb1 band intensities within genotypes ( Figure 10F). In contrast, Zeb1 protein levels in KI G / KI A and KI A /KI A mice were 2-to 3-fold higher than in KI G /KI G littermates ( Figure 10E). The variation within knockin genotype groups was smaller, resulting in differences between knockin genotype groups that were significant (P,0.05) ( Figure 10G).

Discussion
This study demonstrates that the phenotype of Twirler is caused by a noncoding nucleotide substitution within a shared first intron of the Zeb1 and Zeb1os genes on mouse chromosome 18. This is a rare example of a Mendelian noncoding point mutation that does not affect a splice site or promoter. Our results demonstrate the potential for complex phenotypic effects of noncoding point variants, which are increasingly implicated in association studies of genetically complex traits. Our recombinant knockin mouse model and wild type knockin control for testing the pathogenic potential of the Tw mutation may be a useful paradigm to explore the effects of other noncoding variants of unknown pathogenic potential. The altered penetrance potentially associated with a residual loxP site in the Tw knockin line serves a cautionary note to include a wild type knockin control.
Although the initial study by Lyon [1] described abnormal development of the sensory neuroepithelium in the cristae ampullaris of some semicircular canals of Tw/+ mice, we have not observed the same alteration. Instead we observed a highly penetrant constriction of the non-ampullated end of the lateral semicircular canal that could impede or prevent the flow of endolymph and disrupt neurosensory detection of angular acceleration. A difference in strain background [1] may account for the different result. Moreover, Lyon reported that utricular otoconia were absent in Tw/+ ears whereas we observed giant utricular otoconia. This difference could also result from the strain background difference, loss of giant otoconia during the dissection process, or our use of scanning electron microscopy in addition to  light microscopy. Nevertheless, either of the described utricular phenotypes could impair the detection of linear acceleration by Tw/+ utricles. We conclude that our observed semicircular canal and utricular anomalies underlie the vestibular behaviors of Tw/+ mice, although we cannot estimate their relative contributions to the observed vestibular functional phenotype. Correlating mouse vestibular structural or functional abnormalities with behavior is difficult due to a complex interrelationship between vestibular behavior and anxiety that is also dependent upon strain background [20].
The cause of hearing loss observed in some Tw/+ mice also remains obscure. Postmortem examination of middle ears did not  (Table S1). The 39 Southern blot probe is indicated ( Figure S3). doi:10.1371/journal.pgen.1002307.g006 Figure 7. Body weight, daily food intake, and insulin and glucose tolerance of knockin mice. Body weight was measured weekly in males (A) and females (B) (n = 7 for each sex and each genotype). Food intake was measured weekly to calculate average daily intake for males (C) and females (D). KI G /KI A males and females show increased body weight after 14 (p,0.017) and 19 weeks (p,0.018) of age, respectively. Both KI G /KI A males and females show increased food intake after 11 (p,0.02) and 17 weeks (p,0.03) of age, respectively. Serum glucose levels are shown for KI G / KI A (n = 7) and KI G /KI G (n = 7) female mice injected with insulin (E) or glucose (F) at 25 weeks of age. Mean body weights were 36.8 g and 25.4 g for KI G / KI A and KI G /KI G mice, respectively. KI G /KI A mice show insulin resistance. In the glucose tolerance test, the area under the curve (AUC) is significantly higher in KI A /KI G compared with KI G /KI G mice (p = 0.004). The vertical bars indicate the standard error of the mean. Asterisks indicate P,0.05 in an unpaired Student's t test. doi:10.1371/journal.pgen.1002307.g007 reveal otitis media or developmental malformations of the external or middle ears that could account for the hearing loss. Although severe hearing loss has been observed in other mouse mutants with much shorter cochlear ducts [21], the severity of hearing loss in Tw/+ mice was highly variable but the degree of shortening of the cochlear duct was nearly constant. This lack of correlation leads us to conclude that associated physiologic defects or undetected structural anomalies underlie hearing loss in Tw/+ mice.
The Tw/+ phenotype includes hyperphagia with elevated levels of circulating corticosterone and adiponectin that are similar to those in a corticotropin-releasing factor (CRF) transgenic mouse model of Cushing syndrome [22,23]. Other phenotypic similarities of that model to Tw/+ include increased body weight and adiposity, alopecia, atrophy of the thymus and spleen, and muscle wasting. This may suggest that Tw disrupts, at least in part, the hypothalamus-pituitary-adrenal axis. This is consistent with expression of Zeb1 in the pituitary gland [24]. However, Zeb1 protein is also expressed in adipose tissue and increases during adipogenesis in cell culture [11]. Moreover, Zeb1 DEx1/+ mice develop obesity that is not associated with hyperphagia [11], unlike Tw/+ mice ( Figure 1C and 1D). Therefore different mechanisms or tissues may underlie obesity phenotypes associated with Zeb1 DEx1 and Zeb1 Tw . The pathogenetic mechanism for one or both of these Zeb1 alleles may also underlie a locus for susceptibility to obesity on human chromosome 10p11 [25][26][27][28], which includes the human ZEB1 gene.
The results presented here and in Hertzano et al. [13], in combination with the body of published data on Zeb1 in cancer and normal development, show that Zeb1 is a master regulator of mesenchyme-specific gene expression in the developing mouse ear. Twirler is a novel example of a disorder of hearing or balance caused by a disruption of mesenchymal-epithelial identities or interactions. A similar lateral semicircular canal phenotype is seen in other hyperactive circling mice, including epistatic circler mice [29] and mice segregating a gene-trap allele of Chd7 [30]. Chd7 encodes a chromodomain protein required for the development of multipotent migratory neural crest cells [31], which includes an epithelial-to-mesenchymal transition. An auditory-vestibular phenotype approximating that of Twirler and Chd7 mutant mice is also observed in human patients with CHARGE syndrome and mutations of the human CHD7 gene [32,33]. Semicircular canal formation is also known to require Bmp4 [34] and heterozygosity for a knockout allele of mouse Bmp4 primarily affects the lateral semicircular canal [35]. Bmp4 is a member of the transforming growth factor-b (TGF-b) super-family [36], providing another link to Zeb1 since Zeb1 and Zeb2 have been implicated in TGF-beta/ BMP signaling [8].
Why do Twirler mice have a different inner ear phenotype than Zeb1 DEx1 mice? Genetic background differences seem unlikely to account for this difference since Zeb1 DEx1 and Twirler were both maintained on a congenic C57BL/6J background. It is possible that other Zeb1 transcripts could compensate for the loss of exon 1 in Zeb1 DEx1 ears, but our quantitative RT-PCR and expression profiling results [13] render this hypothesis unlikely. Alternatively, the closely related Zeb2 gene may be able to compensate for the loss of Zeb1 expression in the inner ear, but not other affected tissues such as the palate or lymphoid system. However, our quantitative RT-PCR results revealed no compensatory change in Zeb2 transcript levels in the mutants. It is also possible that disruption of Zeb1os may contribute to the Tw phenotype. Ectopic expression of an analogous long noncoding antisense RNA in epithelial cells leads to altered Zeb2 RNA splicing, increased Zeb2 protein levels, and epithelial-to-mesenchymal transition [37]. However, Zeb1os RNA levels were too low for us to reliably detect and monitor by either qRT-PCR or Northern blot analyses to confidently address this possibility (data not shown). Finally, perhaps Twirler does not exert its pathogenic effect via Zeb1. This also seems unlikely since there is significant phenotypic overlap of Twirler with Zeb1 DEx1 , including abnormalities of the semicircular canals associated with both mutant alleles. Furthermore, the phenotypic effects of compound heterozygosity for Zeb1 DEx1 and Tw are consistent with the conclusion that Zeb1 DEx1 is an amorphic or hypomorphic allele whereas Twirler acts as a hypermorphic or neomorphic allele to misregulate Zeb1 expression.
Our electrophoretic mobility shift experiment ( Figure 4) suggests that Tw exerts its pathogenic effect by disruption of binding of C-Myb or other Myb proteins to the first intron of Zeb1. There are also published observations supporting the general hypothesis that loss of Myb protein binding leads to de-repression of Zeb1 Tw and inner ear malformations: First, C-Myb can function as either an activator or repressor of gene transcription [38] and is thought to function in regulation of epithelial-mesenchymal cell identity [39]. Second, a pathogenic effect of up-regulation of developmental transcription factors has been demonstrated for Pax6 in the eye [40] and Tbx1 in the inner ear [41]. In the inner ear, increased expression of Tbx1 can cause malformations that include incomplete coiling and reduced extension of the cochlear duct [41]. Furthermore, Tbx1 expression in the periotic mesenchyme is required for cochlear duct outgrowth [42], suggesting a potential link to the observed inner ear phenotype of Twirler. Taken together, these observations and our results support the hypothesis that Twirler disrupts inner ear development via mis-regulation of Zeb1.
The cell type-specific gene expression profiles of Twirler ears [13] suggest that a pathologic disruption of epithelial and mesenchymal cell identities underlies the inner ear malformations. This could arise from a loss of mesenchymal cell identity leading to mesenchymal-epithelial transition (MET), a loss of epithelial cell identity leading to epithelial-mesenchymal transition (EMT), or a combination of these mechanisms. Although the gene expression profiles [13] seem consistent with MET, it is difficult to conceive a simple MET pathway that does not invoke a loss-of-function mechanism in Tw mesenchyme. In contrast, EMT would involve a gain-of-function with ectopic expression of Zeb1 in Tw inner ear epithelium. Indeed, ectopic expression of Zeb proteins in other epithelial tissues has been shown to lead to EMT in other neoplastic and developmental processes [43]. Distinguishing among EMT and MET mechanisms may be difficult if they involve complex regulatory pathways mediated by Zeb1os, Zeb2, microRNAs or other genes. In summary, we have identified the pathogenic mutation of Twirler as a noncoding point mutation that leads to over-or mis-expression of Zeb1, pathologic alterations of gene expression [13], cell fate and interactions in the developing inner ear. The ultimate result is a gross alteration of the structure and function of the vestibular and auditory organs. Disruption of epithelial-mesenchymal identity or interactions may be a shared pathogenetic mechanism underlying phenotypes that primarily affect development of the lateral semicircular canals, extension of the cochlear duct, or both.

Animals
Mice were maintained on a 12:12-h light-dark cycle. All experiments and procedures were approved by the Animal Care and Use Committees of the National Institute of Diabetes and Digestive and Kidney Diseases, National Institute of Neurological Disorders and Stroke and National Institute on Deafness and Other Communication Disorders. Twirler mice were a kind gift from Drs. Miriam Meisler and Siew-Ging Gong at the University of Michigan and were maintained on a C57BL/6J background by backcrossing heterozygous Tw males to C57BL/6J females for at least 30 generations. Zeb1 DEx1 mice [10] were a generous gift from Dr. Douglas Darling and were serially backcrossed to C57BL/6J to maintain the line.

Generation of knockin mice
Bacterial artificial chromosome (BAC) clone RP23-135A18 containing mouse genomic DNA encoding exon 1 of Zeb1 was digested with PacI/SphI and SphI to yield 7.6-kb and 2.6-kb homology arms, respectively, for targeting constructs (Figure 6). Each targeting construct included loxP sites flanking a splice acceptor site and internal ribosomal entry site (IRES) (pGT1.8Ir-esBgeo, provided by Austin Smith at University of Edinburgh) [44], E. coli lacZ, and a reverse-oriented pPGK-neomycin resistance cassette cloned into the pPNT plasmid [45] (Figure 6). The wild type (KI G ) and Twirler (KI A ) 7.6-kb PacI/SphI homology arms contained G and A at position c.58+181, respectively. Bruce4 embryonic stem (ES) cells [46] were electroporated with the KI G or KI A targeting constructs and grown in the presence of Figure 8. Inner ear morphology, hearing thresholds, and otoconia of wild-type (KI G ) and Tw (KI A ) knockin mice. Paint-filled inner ears of KI G /KI G , KI G /KI A and KI A /KI A mice at E14.5 are shown from lateral (A-C), medial (D-F) and ventral (G-I) views. KI G /KI G inner ear morphology appeared normal. KI G /KI A ears had irregular contours of the semicircular canals (B), but we did not observe abnormal constrictions at the non-ampullated ends of the lateral semicircular canals (E). KI A /KI A inner ear structure is very abnormal (C), with discontinuous or partially ruptured semicircular canals (F) and shorter cochlear ducts (I). Average ABR thresholds for all KI G /KI G ears (n = 10) are shown as white circles and KI G /KI A ears (n = 26) as black circles with the standard error of the mean (SEM). Scanning electron microscopy showed no difference in otoconia between KI G /KI G (K) and KI G /KI A saccules (L) at P6. KI G /KI A utricles (N) had giant otoconia. a, anterior semicircular canal; c, cochlear duct; es, endolymphatic sac; l, lateral semicircular canal; p, posterior semicircular canal; s, saccule; u, utricle. doi:10.1371/journal.pgen.1002307.g008 G418 and ganciclovir, using standard protocols at the University of Michigan Transgenic Animal Model Core [47]. G418-resistant ES clones were screened for homologous recombination by PCR and Southern blot analyses. At least three recombinant ES cell lines for each targeting construct were injected into C57BL/6 blastocysts. Chimeric males were mated with C57BL/6 females and offspring were analyzed by Southern blot and PCR analyses for germline transmission of KI G or KI A ( Figure S3). Table S1 shows PCR primer pairs used to genotype KI alleles before and after neomycin cassette removal ( Figure 6). Mice transmitting KI G or KI A in the germline were crossed to Cre recombinase-expressing mice (C57BL/6-TgN(Zp3-Cre)93Knw, Jackson laboratory, ME) to delete the IRES-lacZ-neomycin resistance cassette, leaving a single loxP site 606 bp downstream from Zeb1 exon 1.

Phenotype survey
A comprehensive gross anatomical, histological, and serological analysis of three 15-week-old Tw/+ and three wild type littermate males was performed as described [48]. Tissue sections from two Tw/Tw, two Tw/+ and two wild type mice at postnatal day 0 (P0) were analyzed.

Inner ear phenotype analyses
Heterozygous Tw males and females were mated. Pregnant females were identified by the presence of a vaginal plug and gestational stage was estimated by defining that morning as 0.5 days post-conception (dpc). Embryos at 14.5 dpc were harvested and processed for paint-filling as described [49]. The length of the cochlear duct was measured along its outer contour from a ventral view [50]. For scanning electron microscopy (SEM), whole-mounted inner ears were fixed in 2.5% glutaraldehyde in 0.1 M sodium cacodylate with 2 mM CaCl 2 for 90 min. The organ of Corti, saccule, utricle, and crista ampullaris were dissected free in water and dehydrated with a serial dilution series of acetone. Samples were critical point-dried and sputter-coated followed by visualization with a field-emission scanning electron microscope (S-4800, Hitachi). Auditory brainstem response (ABR) thresholds were measured in response to click or pure-tone stimuli of 8, 16, or 32 kHz as described [51].

Obesity and metabolic phenotype analyses
Six Tw/+ male, six Tw/+ female, six wild type male and six wild type female mice were housed individually with regular mouse chow and water provided ad libitum. Body weights were measured weekly from 5 weeks of age. Weekly food intake was measured from weeks 6 through 22 to calculate average daily food intake. At 23 weeks of age, mice were transferred to the NIDDK Mouse Metabolism Core Laboratory for measurement of oxygen consumption, carbon dioxide production and motor activity as described [52]. Body composition was measured using Echo3-in-1 NMR analyzer (Echo Medical Systems, Houston, TX). Tail vein blood was used for serologic analyses. Fifteen-week-old female mice (eight Tw/+, six wild type) were tested for glucose and insulin tolerance as described [52]. All data are expressed as a mean 6 SEM. Student's t-test was used to identify statistically significant differences between genotype groups.

Recombination mapping
We genotyped short tandem repeat (STR) markers on 337 DBA/2J N2 backcross progeny to identify two STR markers (D18Mit65, D18Mit64, D18Mit19 and D18Umi1) flanking each side of Tw. These markers were genotyped in the 1679 CAST/Ei N2 backcross progeny to identify recombinations in the Tw region. The Tw map interval was defined by genotypes of additional markers in the recombinants. We genotyped MIT markers between D18Mit65 and D18Umi1, as well as 40 novel STR markers (denoted D18Nih1 through D18Nih44; PCR primer sequences listed in Table S1) located between D18Mit19 and D18Mit219.

Mutation analyses
Genomic DNA of Tw/Tw, Tw/+ and wild type mice were isolated for PCR amplification as described [53]. The primers were designed to amplify and sequence all of the annotated exons Figure 9. Compound heterozygous KI G /Tw and KI A /Tw inner ear morphology. Paint-filled inner ears of KI G /Tw and KI A /Tw mice at E14.5 are shown from lateral (A and B), medial (C and D) and ventral (E and F) views. In KI G /Tw ears, the irregular contours of the semicircular canals, the constriction of the non-ampullated end of the lateral semicircular canal, and the short cochlear duct were all indistinguishable from similar findings in Tw/+ ears (see Figure 2). In KI A /Tw ears, the discontinuous or ruptured semicircular canals and cochlear ducts were indistinguishable from those of Tw/Tw ( Figure 2) and KI A /KI A ears ( Figure 8). a, anterior semicircular canal; c, cochlea duct; es, endolymphatic sac; l, lateral semicircular canal; p, posterior semicircular canal; s, saccule; u, utricle. doi:10.1371/journal.pgen.1002307.g009 of the Zeb1, Zeb1os (MGI predicted gene Gm10125) and Zfp438 genes in the Tw critical interval. Additional novel exons were identified by 59 and 39-RACE (59 and 39 rapid amplification of cDNA ends) of the Zeb1, Zeb1os and Zfp438 genes. This revealed multiple alternative first exons for Zeb1 that were also sequenced. Reverse transcription (RT)-PCR was performed to amplify and sequence full-length cDNA clones of the three genes using whole body mRNA collected from embryonic Tw/Tw, Tw/+ and wild type littermates.
PCR reaction conditions were modified to amplify and sequence the overlapping genomic region of Zeb1 and Zeb1os. Fifty-ml PCR reactions contained 50 to 100 ng of genomic DNA, 5 pmol each of forward and reverse primers, 200 mM each dNTP, 0.5 M betaine, 10% dimethyl sulfoxide (DMSO), 2.5 mM MgCl 2 , and 0.5 U of thermostable polymerase. Thermal cycling conditions were: 95uC for 1 min; 33 cycles of 20 s at 95uC, 20 s at 57uC, and 45 s at 72uC; and a final 2-min extension at 72uC. For sequencing, 50 ml PCR reaction products were purified with a QIAquick PCR purification kit (Qiagen, Hilden, Germany) and eluted with 30 ml elution buffer. Three ml of purified products were added to a 10-ml sequencing reaction containing 3.2 pmol primer, 0.25 ml Big Dye Terminator Ready Reaction mix (PE Biosystems), sequencing buffer and 10% DMSO. Cycling conditions were 96uC for 2 min and 33 cycles of 96uC for 10 s, 55uC for 10 s, and 60uC for 4 min. We also amplified and sequenced the overlapping genomic region of Zeb1 and Zeb1os Scale bar = 100 mm. Zeb1 was detected at similar levels in wild type and Tw/Tw mesenchymal cells. e, epithelial layer; c, mesenchymal layer. Inner ear structures are difficult to identify with disrupted mesenchymal-and epithelial-specific microarchitecture and gene expression in some Tw/Tw ears (C). (D-G) Representative western blot analyses of E13.5 mouse-head protein extracts from individual Tw (D) and knockin animals (E) and littermate controls. Each Zeb1 band intensity measurement was normalized to the b-actin band intensity for that sample. These Zeb1/b-actin band intensity ratios for Tw samples were then normalized to mean wild type (+/+) ratios calculated from the same gel. Mean normalized Zeb1 expression values are shown for +/+ (n = 3), Tw/+ (n = 2) or Tw/Tw (n = 2) mice (F) and KI G /KI G (n = 2), KI G /KI A (n = 2) or KI A /KI A (n = 6) mice (G). The indicated variation in the +/ + mean value in (A) but not (B) reflects the inclusion of two +/+ samples on one of the gels for the Tw (A) analysis but only one +/+ sample on each gel for the knockin analysis (B). ANOVA analysis revealed that the observed differences among +/+, Tw/+ and Tw/Tw mice were not significant (P.0.05), but the differences among KI G /KI G , KI G /KI A and KI A /KI A mice were significant (P,0.05). doi:10.1371/journal.pgen.1002307.g010 from normal mouse control strains 129/J, AKR/J, BALB/cJ, C3H/HeJ, C57BL/6J, C58/J, CBA/J, CE/J, DBA/2J, P/J, RF/ J, SEA/GnJ and SWR/J DNA.

Quantitative RT-PCR analyses
Inner ears with adjacent mesenchyme were microdissected from E13.5 offspring of Tw/+ x Tw/+ matings. Total RNA was isolated from inner ears using PicoPure (Applied Biosystems, Foster City, CA). Total RNA from 10 to 14 ears of the same genotype was pooled and purified with the RNAeasy MinElute Cleanup kit (Qiagen). RNA integrity was measured with an Agilent 2100 Bioanalyzer (Applied Biosystems). One mg of total RNA was reverse-transcribed with oligo(dT) primers and SuperScriptIII (Invitrogen, Carlsbad, CA, USA). For TaqMan real-time PCR, PCR primers were designed to amplify Zeb1 exons 1a to 2, 1b to 2, 1c to 2, 1d to 2, 1e to 2, 1f to 2, and 2 to 3, Zeb1os exons 1 to 2, and Zfp438 exons 3 to 4 with ZEN double-quenched probes containing a 59 FAM fluorophore, 39 IBFQ quencher, and an internal ZEN quencher (IDT, Coralville, IA). Sequences for the primers and probes are listed in Table S1.
Comparative TaqMan assays were performed in triplicate on an ABI 7500 real-time PCR system (Applied Biosystems). PCR reactions were performed in a 50-ml volume containing 5 ml cDNA, 5 ml primer mix (IDT), and 25 ml of Universal PCR Master Mix (Applied Biosystems). Cycling conditions were 50uC for 2 min, 95uC for 10 min, followed by 40 cycles of 15 s at 95uC and 1 min at 60uC. Relative expression was normalized as the percentage of b-actin expression, and calculated using the comparative threshold cycle method of 2 2DDCT . Data are presented as mean values 6 S.D. from six technical replicates. ANOVA was used to identify statistically significant differences between genotype groups (P,0.05).

Western blot analyses
Proteins were extracted from E13.5 mouse inner ears or whole heads with NE-PER Nuclear and Cytoplasmic Extraction Reagents (Pierce Biotechnology) in the presence of Halt Protease Inhibitor Cocktail (Thermo Fisher Scientific Inc.). Proteins were separated by SDS-PAGE in 4-20% NuPage Bis-Tris gels followed by transfer to PVDF membranes (Millipore Corp., Billerica, MA). Proteins were detected with primary antibodies for Zeb1 (ab64098, Abcam, 1:200) and b-actin (A2228, Sigma-Aldrich, 1:1000). Secondary antibodies were conjugated with Cy 3 or Cy 5 (GE Healthcare) and detected with a Typhoon Trio+ (GE Healthcare). Band density was measured using ImageQuant TL software. b-actin levels were used for normalization. ANOVA analysis of two to six biological replicates from each genotype group was used to identify statistically significant differences (P,0.05).

Immunohistochemistry
Mouse inner ear sections were harvested, processed and immunostained with anti-Zeb1 or anti-CD326 antibodies as described in Hertzano et al. [13]. CD326 is also known as epithelial cell adhesion/activating molecule (EpCAM) that serves as a specific antigenic marker for epithelial cells [13]. Figure S1 Inner ear morphology and hearing thresholds of Zeb1 DEx1 mice. Paint-filled inner ears of Zeb1 +/+ , Zeb1 DEx1/+ , Zeb1 DEx1/DEx1 and Zeb1 DEx1/Tw mice at E14.5 are shown from lateral (A-D), medial (E-H), ventral (I-L) and dorsal (M-P) views. Inner ears from Zeb1 +/+ and Zeb1 DEx1/+ mice appeared similar and normal. Zeb1 DEx1/DEx1 lateral semicircular canals had a subtle constriction (indicated by *) of the midportion of the canal that differed from those observed in Tw/+ mice ( Figure 2). The lateral semicircular canals of Zeb1 DEx1/Tw mice did not contain this abnormality and resemble those of Tw/+ mice. Zeb1 DEx1/+ mice have normal ABR thresholds (Q). a, anterior semicircular canal; c, cochlear duct; es, endolymphatic sac; l, lateral semicircular canal; p, posterior semicircular canal; s, saccule; u, utricle.  Figure 5. The probe hybridizes to 4.2-and 5.9-kb fragments before and after Cre-mediated excision of the lacZ-PGK-Neo R cassette, respectively. B. Nucleotide sequence confirmation of KI G and KI A at c.58+181G/A. (TIF) Table S1 PCR primers and probes for mapping, genotyping and real-time PCR. Sequences are shown for PCR primers: 40 novel short tandem repeats located between D18Mit109 and D18Mit201 on mouse chromosome 18; used to genotype knockin mice before (KI G:Neo and KI A:Neo ) and after (KI G and KI A ) Cremediated excision of the lacZ-PGK-Neo R cassette; quantitative RT-PCR analysis of Zeb1, Zeb1os, Zfp438 and Zeb2. Actb and Gapdh were included as controls to calculate relative expression. (TIF)