Vestibular defects in head-tilt mice result from mutations in Nox3, encoding an NADPH oxidase

Abstract

The vestibular system of the inner ear is responsible for the perception of motion and gravity. Key elements of this organ are otoconia, tiny biomineral particles in the utricle and the saccule. In response to gravity or linear acceleration, otoconia deflect the stereocilia of the hair cells, thus transducing kinetic movements into sensorineural action potentials. Here, we present an allelic series of mutations at the otoconia-deficient head tilt (het) locus, affecting the gene for NADPH oxidase 3 (Nox3). This series of mutations identifies for the first time a protein with a clear enzymatic function as indispensable for otoconia morphogenesis.

Figure 1.

Typical features of R96, R542, and vst mutant mice. (A) Tilted position of the head (affected mouse). (B) Unaffected control. In both A and B, the angle of the head is shown in the inset. (C) Unable to swim or float in forced swim test (affected mouse). (D) Unaffected control.

Figure 2.

(A–D) Kossa-staining of inner ear transversal sections. The utricle (U) and saccule (S) of +/+ animals (A,C) contain dark stained, Kossa-positive otoconia, which are absent in Nox3^–/Nox3^– mutant animals (B,D). (E–H) SEM view of transversely cut inner ear. The otoconia of the utricle (U) and saccule (S) are present in +/+ animals only (indicated by asterisks in E, labeled OC in G). In contrast, Nox3^–/Nox3^– mutant animals (F,H) are devoid of otoconia. Bars: A,B,E,F, 100 μm; C,D,G,H, 10 μm.

Figure 3.

BAC transgenic rescue of the Nox3 mutant phenotype. (A, top) Proximal Chr 17 showing the relative positions of significant SSLP markers, the het-critical region, and the rescuing BAC RP23-27N1. (Bottom) Enlarged view of RP23-27N1 showing the relative positions of candidate genes Racgef1, Tiam2, and Cldn20 and the causative gene Nox3. (B) Inner ear explant from a wild-type C57BL/6J mouse showing normal otoconia. (C) Inner ear explant from a C57BL/6J-het/het mouse showing complete absence of otoconia. (D) Inner ear explant from a het/het; TgN(RP23-27N1)/+ mouse showing complete rescue/restoration of otoconia.

Figure 4.

Genomic and amino acid structure of Nox3. The top portion of the figure shows the intron/exon structure of Nox3; the lower left portion shows the protein structure (model according to Wallach and Segal 1997). The location and nature of the R96, R542, het, and het^2J mutations are also shown.

Figure 5.

RT–PCR analysis of Nox3 in different stages of mouse development. (A) Embryo. Nox3 is already present by E12 and persists throughout development. (M) 100-bp marker; (1) E13.5 embryo head; (2) E13.5 without head; (3) E18.5 head; (4) E10–E12 pooled; (C) no template control. (B) Adult ear. Nox3 is also present in adult ear preparations. (M) 100-bp marker; (1) adult ear; (C) no template control.