The transmembrane inner ear (tmie) gene contributes to vestibular and lateral line development and function in the zebrafish (Danio rerio)

Abstract

The inner ear is a complex organ containing sensory tissue, including hair cells, the development of which is not well understood. Our long-term goal is to discover genes critical for the correct formation and function of the inner ear and its sensory tissue. A novel gene, transmembrane inner ear (Tmie), was found to cause hearing-related disorders when defective in mice and humans. A homologous tmie gene in zebrafish was cloned and its expression characterized between 24 and 51 hours post-fertilization. Embryos injected with morpholinos (MO) directed against tmie exhibited circling swimming behavior (approximately 37%), phenocopying mice with Tmie mutations; semicircular canal formation was disrupted, hair cell numbers were reduced, and maturation of electrically active lateral line neuromasts was delayed. As in the mouse, tmie appears to be required for inner ear development and function in the zebrafish and for hair cell maturation in the vestibular and lateral line systems as well.

Figure 1

Tmie proteins are conserved across vertebrate species. A: Zebrafish tmie gene structure and the positions of the primers used to obtain the sequence of tmie cDNA. The positions of MOtmie1 and MOtmie2 are also shown. Gray boxes indicate the open reading frame for the predicted tmie protein, and the white boxes indicate the untranslated regions. B: Alignment of Tmie proteins from four species is shown. Positions that exhibit amino acid identity across all four species are shaded in blue. Positions that are identical in at least two species are shaded in black. Positions with biochemically similar residues are shaded in gray. Bars indicate predicted transmembrane domains. Asterisks indicate arginine residues that are altered in humans with hearing loss due to mutations at the DFNB6 locus (Naz et al.,2002). Arrows indicate the positions of exon junctions in the corresponding genes.

Figure 1

Tmie proteins are conserved across vertebrate species. A: Zebrafish tmie gene structure and the positions of the primers used to obtain the sequence of tmie cDNA. The positions of MOtmie1 and MOtmie2 are also shown. Gray boxes indicate the open reading frame for the predicted tmie protein, and the white boxes indicate the untranslated regions. B: Alignment of Tmie proteins from four species is shown. Positions that exhibit amino acid identity across all four species are shaded in blue. Positions that are identical in at least two species are shaded in black. Positions with biochemically similar residues are shaded in gray. Bars indicate predicted transmembrane domains. Asterisks indicate arginine residues that are altered in humans with hearing loss due to mutations at the DFNB6 locus (Naz et al.,2002). Arrows indicate the positions of exon junctions in the corresponding genes.

Figure 2

Expression of tmie mRNA in the brain, and ear. A: RT-PCR results from various stages of embryonic development: −, no reverse transcriptase; +, with reverse transcriptase. B,C: Lateral view of 26-hpf embryos with tmie antisense probe (B) and sense probe (C). D,E: Lateral view of 36-hpf embryos with tmie antisense probe (D) and sense probe (E). F,G: Lateral view of 51-hpf embryos with tmie antisense probe (F) and sense probe (G). Signals were found in the developing otic vesicle at all stages observed. There is also staining in the brain. ot, otic vesicle; sc, spinal cord. Scale bar = 100 µm.

Figure 3

Morpholinos directed against tmie decrease levels of normal transcripts. A: Injection of an anti-tmie morpholino (MOtmie1) directed against the splice donor site of exon 3 resulted in a decreased level of intact transcripts and a substantial increase in shorter tmie transcripts that lacked exon 2 (arrow) at 36 hpf. Very little of the shorter transcript was detected at 36 hpf in uninjected embryos (wt) or in embryos injected with a control morpholino (MOcon). B: Injection of an anti-tmie morpholino (MOtmie2) directed against the splice donor site of exon 2 resulted in decreased levels of intact tmie transcripts at 36 hpf (products amplified from two independent RNA preparations are shown), relative to those present in embryos injected with a control morpholino. Levels of Gapdh transcripts are shown in the bottom panel. Size standards are indicated in kb.

Figure 3

Morpholinos directed against tmie decrease levels of normal transcripts. A: Injection of an anti-tmie morpholino (MOtmie1) directed against the splice donor site of exon 3 resulted in a decreased level of intact transcripts and a substantial increase in shorter tmie transcripts that lacked exon 2 (arrow) at 36 hpf. Very little of the shorter transcript was detected at 36 hpf in uninjected embryos (wt) or in embryos injected with a control morpholino (MOcon). B: Injection of an anti-tmie morpholino (MOtmie2) directed against the splice donor site of exon 2 resulted in decreased levels of intact tmie transcripts at 36 hpf (products amplified from two independent RNA preparations are shown), relative to those present in embryos injected with a control morpholino. Levels of Gapdh transcripts are shown in the bottom panel. Size standards are indicated in kb.

Figure 4

Behavioral assessment of fish. A: Swirl test of embryos at 96 hpf with 2.5 µg/l control MO (n = 66), 2.5 µg/l MOtmie1 (n = 293), 3.5 µg/l MOcon (n = 45), and 3.5 g/l MOtmie1 (n = 152). Development of balance (B) and development of motor coordination (C) in control embryos (MOcon, n = 113), tmie morphants with 2.5 ng of MOs (n = 107), tmie morphants with 2.5 ng MOs plus 100 ng (n = 96) or 200 ng (n = 90) tmie RNA. Data show the means and standard errors of two independent experiments.

Figure 4

Behavioral assessment of fish. A: Swirl test of embryos at 96 hpf with 2.5 µg/l control MO (n = 66), 2.5 µg/l MOtmie1 (n = 293), 3.5 µg/l MOcon (n = 45), and 3.5 g/l MOtmie1 (n = 152). Development of balance (B) and development of motor coordination (C) in control embryos (MOcon, n = 113), tmie morphants with 2.5 ng of MOs (n = 107), tmie morphants with 2.5 ng MOs plus 100 ng (n = 96) or 200 ng (n = 90) tmie RNA. Data show the means and standard errors of two independent experiments.

Figure 4

Behavioral assessment of fish. A: Swirl test of embryos at 96 hpf with 2.5 µg/l control MO (n = 66), 2.5 µg/l MOtmie1 (n = 293), 3.5 µg/l MOcon (n = 45), and 3.5 g/l MOtmie1 (n = 152). Development of balance (B) and development of motor coordination (C) in control embryos (MOcon, n = 113), tmie morphants with 2.5 ng of MOs (n = 107), tmie morphants with 2.5 ng MOs plus 100 ng (n = 96) or 200 ng (n = 90) tmie RNA. Data show the means and standard errors of two independent experiments.

Figure 5

Staining of actin (phalloidin staining, red) and acetylated tubulin (green) in hair cell patches in the inner ear showed defects in tmie morphants. A,B: Double staining of actin (stereocilia) and tubulin (kinocilia) of the three cristae (arrowheads). C,D: Double staining of stereocilia and cristae in the maculae (arrows). E,F: Actin staining showing the epithelial pillars (ep), which form hubs of the developing semicircular canals. Arrows indicate the junction of the ventral bulge (b) and ventral projection (p). In the control ear, the junction has formed and fused (E), but in the tmie MO-treated fish, there is still a gap between the bulge and the projection. G–J: Phalloidin staining of the anterior macula (arrowhead) at 5 dpf (G, H) and 6 dpf (I, J). The patch is smaller in tmie morphants, especially at 6 dpf. K: Numbers of hair cells labeled by phalloidin at 4 dpf (n = 5 for wt, and n = 4 for MOstmie), and as illustrated in G–H at 5 dpf (n = 3 for wt, n = 6 for control, and n = 13 for MOstmie) and 6 dpf (n = 2 for wt, n = 2 for control, and n = 4 for MOstmie). L–O: Acetylated tubulin staining of the posterior macula.

Figure 6

The development of lateral line was affected in MOstmie morphants. A–D: DASPEI staining of lateral line neuromasts. There is no or very little staining in MOstmie at 72 hpf (B) compared to the control (A) at the same stage. The lateral staining is weaker at 5 hpf in the MOstmie morphants (D) but present compared to the control larvae (C). E–L: Whole mount in situ hybridization with cxcr4b (E, F, I, J) and ath1 probes (G, H, K, L). Arrows, the migrating lateral line primordium. Arrowheads, the neuromasts with atoh1a signal. M: DASPEI score of the lateral line. After DASPEI staining, the larvae were divided into 3 groups: strongly stained, weakly stained, and not stained. Each fish was assigned a score after the level of staining in all the fish was first observed. Each larva in the strongly stained group was given 2 points, while 1 point was given to the weakly stained larvae, and 0 points to larvae, which were not stained. The number of fish in each group was recorded. The cumulative scores were then calculated and the percentage of fish in each group following each treatment determined. The higher the score (and the closer to 2.0), the stronger the staining of DASPEI observed after each treatment.

Figure 6

The development of lateral line was affected in MOstmie morphants. A–D: DASPEI staining of lateral line neuromasts. There is no or very little staining in MOstmie at 72 hpf (B) compared to the control (A) at the same stage. The lateral staining is weaker at 5 hpf in the MOstmie morphants (D) but present compared to the control larvae (C). E–L: Whole mount in situ hybridization with cxcr4b (E, F, I, J) and ath1 probes (G, H, K, L). Arrows, the migrating lateral line primordium. Arrowheads, the neuromasts with atoh1a signal. M: DASPEI score of the lateral line. After DASPEI staining, the larvae were divided into 3 groups: strongly stained, weakly stained, and not stained. Each fish was assigned a score after the level of staining in all the fish was first observed. Each larva in the strongly stained group was given 2 points, while 1 point was given to the weakly stained larvae, and 0 points to larvae, which were not stained. The number of fish in each group was recorded. The cumulative scores were then calculated and the percentage of fish in each group following each treatment determined. The higher the score (and the closer to 2.0), the stronger the staining of DASPEI observed after each treatment.