The transmembrane inner ear (Tmie) protein is essential for normal hearing and balance in the zebrafish

Abstract

Little is known about the proteins that mediate mechanoelectrical transduction, the process by which acoustic and accelerational stimuli are transformed by hair cells of the inner ear into electrical signals. In our search for molecules involved in mechanotransduction, we discovered a line of deaf and uncoordinated zebrafish with defective hair-cell function. The hair cells of mutant larvae fail to incorporate fluorophores that normally traverse the transduction channels and their ears lack microphonic potentials in response to vibratory stimuli. Hair cells in the posterior lateral lines of mutants contain numerous lysosomes and have short, disordered hair bundles. Their stereocilia lack two components of the transduction apparatus, tip links and insertional plaques. Positional cloning revealed an early frameshift mutation in tmie, the zebrafish ortholog of the mammalian gene transmembrane inner ear. The mutant line therefore affords us an opportunity to investigate the role of the corresponding protein in mechanoelectrical transduction.

Fig. 1.

Defective hair-cell function. (A) In a WT (Upper) and a mutant (Lower) larva at 6 dpf, arrowheads indicate neuromasts labeled by Tg(pou4f3:gap43-mGFP) (green). (B) Upon exposure to FM4–64 (red), the WT specimen (Upper) shows robust incorporation of the fluorophore into hair cells but the mutant (Lower) shows none. (C) In the inner ears of WT (Upper) and mutant (Lower) animals at 5 dpf, Tg(pou4f3:gap43-mGFP) (green) marks the anterior (AC), medial (MC), and posterior cristae (PC). (D) Pressure-injection of FM4–64 (red) into the otic cavities labels hair cells in the cristae of WT (Upper) but not mutant (Lower) fish. (E) Averaged extracellular recordings of microphonic potentials measured at 5–7 dpf from the inner ears of eight WT larvae show a clear response at twice the stimulus frequency, whereas the record from eight mutant larvae shows only a stimulus artifact. (Scale bars, 200 μm in A and B; 20 μm in C and D.)

Fig. 2.

Absence of gross morphological and developmental defects. In each panel, a WT larva (Upper) is contrasted with a mutant (Lower). (A, A′) Mutants display a normal body shape and length at 4 dpf. (B, B′) In a mutant, the semicircular canals develop normally and the anterior (AO) and posterior otoliths (PO) are normal. (C, C′) Labeling of hair cells by Tg(pou4f3:gap43-mGFP) (green) reveals no obvious differences in their number, shape, or localization in the anterior macula. (D, D′) In lateral-line neuromasts at 3 dpf, the hair bundles of mutants display normal planar cell polarity. The kinocilium of each hair bundle is marked by a black notch denoting the absence of actin staining by phalloidin. At 22 hpf (E, E′), 2 dpf (F, F′), and 3 dpf (G, G′), mutants display normal migration of the lateral-line primordium and formation of neuromasts, here labeled by Tg(cldnB:lynGFP) (green). OV, otic vesicle; arrowheads indicate the leading edges of the primordia. (H and I) Supporting cells labeled by Tg(cldnB:lynGFP) (green) are normally arranged in mutants in the anterior (H, H′) and posterior lateral lines (I, I′) of 5-dpf larvae. (J, J′) Fifty hours after treatment with Cu²⁺, a 7-dpf mutant larva shows normal regeneration of hair cells labeled by Et(krt4:GFP)^sqet4 (green). (K, K′) Phalloidin staining shows that regenerated hair cells display planar cell polarity in a mutant. (Scale bars, 500 μm in A, F, and G; 50 μm in B; 5 μm in C; 2 μm in D and K; 100 μm in E; 10 μm in H–J.)

Fig. 3.

Identification of the mutation in tmie. (A) A genetic (Upper) and a physical map (Lower) depict the genomic region on chromosome 2 containing the mutation. (B) Partial chromatograms of the DNA sequence of tmie in WT (Left), homozygously mutant (Middle), and heterozygously mutant (Right) fish delineate a mutation affecting the thirteenth codon (boxed in red). (C) The gene structure of zebrafish tmie includes four coding exons, E1–E4. The short alternative transcript of tmie contains the more proximal E4a as the fourth coding exon. The asterisk denotes the location of the mutation. (D) The predicted amino acid sequence suggests a single-pass transmembrane protein after the cleavage of a signal peptide near the amino terminus (arrowhead). C, cytoplasmic surface; E, extracellular surface; M, membrane. (E) An alignment of protein sequences of Tmie from the zebrafish, mouse, and human depicts completely conserved residues in green and those identical in three instances in yellow.

Fig. 4.

Confirmation of the mutation. (A) Exposure of a WT 4-dpf larva to 4-Di-2-ASP (yellow) labels neuromasts. (B) Labeling is absent in a mutant. (C) Injection of capped RNA coding for normal Tmie partially rescues a mutant, restoring labeling in some neuromasts (arrowheads). (D) Injection of a WT embryo with a morpholino against tmie spares fluorophore incorporation in some neuromasts of the anterior lateral line but not in those of the posterior lateral line. (Scale bar, 500 μm.) (E) The morpholino (MO) targets the splice junction between exon 2 and intron 2. The arrows show the locations of the primers used to amplify the splicing products in RT-PCRs. (F) The RT-PCR products amplified from one WT and three morphant fish (1-3) confirm that the injected morpholino disrupts normal splicing of tmie (Left) and of its alternative transcript (Right), producing various aberrant products. L, DNA ladder in kilobases. (G) Endogenous expression of Tmie is sparse. The relative expression of tmie at different developmental stages was measured by qRT-PCR with RNA extracted from whole larvae. The results are presented as the mean and standard deviation of the ratio of each measurement to that at 24 hpf, the lowest value observed. Expression peaks at 38 hpf, declining sharply thereafter and remaining low through 120 hpf.

Fig. 5.

Ultrastructural features of mutant and normal hair cells. (A) A scanning electron micrograph of a midtail neuromast from the posterior lateral line of a mutant at 6 dpf shows a dozen long kinocilia emerging from short clusters of stereocilia. (B) A neuromast from a similar position in an age-matched, homozygously mutant fish displays shrunken kinocilia and a diminished number of hair bundles. The semilunar periderm cells surrounding the neuromast are abnormally smooth. (C) A low-power transmission electron micrograph of a neuromast in a 6-dpf control larva shows parts of seven hair cells, some contacting large nerve terminals. (D) A mutant neuromast is characterized by several lysosomes (arrowheads) both within them and in supporting cells. A representative lysosome is enlarged fivefold in the inset. (E) A high-power micrograph from a control animal demonstrates a pair of sterocilia connected by a tip link (arrowhead) that terminates at its upper end in a prominent insertional plaque. (F) In a mutant larva of the same age, the stereocilia are narrow and display little basal tapering. Neither tip links nor insertional plaques are evident, and the shorter stereocilia lack dense material beneath their tips. The magnifications are identical for A and B, for C and D, and for E and F.