Tekt3 Safeguards Proper Functions and Morphology of Neuromast Hair Bundles

Abstract

The inner ear and/or lateral line are responsible for hearing and balance of vertebrate. The otic sensory hair cells (HCs) employ cilium organelles, namely stereocilia and/or kinocilia, to mediate mechanical stimuli to electrical signal transition. Tektins (Tekts) are known as the cilium microtubule stabilizer and inner-space filler, and four Tekt(1-4)-encoding genes are identified in zebrafish HCs, but the subcellular location of Tekts in HCs remains unknown. In the present study, we first found that tekt3 is expressed in the inner ear and lateral line neuromast. Antibody staining revealed that Tekt3 is present in neuromast and utricular HCs. It is absent in the saccule, the authentic hearing end-organ of zebrafish and the crista of semi-circular canals. Furthermore, Tekt3 were enriched at the apical side of neuromast and utricular HCs, mainly in the cytosol. Similar subcellular distribution of Tekt3 was also evident in the outer HCs of mature mouse cochlea, which are not directly linked to the hearing sense. However, only neuromast HCs exerted morphological defect of kinocilia in tekt3 mutant. The disrupted or distorted HC kinocilia of mutant neuromast ultimately resulted in slower vital dye intake, delayed HC regeneration after neomycin treatment, and reduced startle response to vibration stimulation. All functional defects of tekt3 mutant were largely rescued by wild-type tekt3 mRNA. Our study thus suggests that zebrafish Tekt3 maintains the integrity and function of neuromast kinocilia to against surrounding and persistent low-frequency noises, perhaps via the intracellular distribution of Tekt3. Nevertheless, TEKT3/Tekt3 could be used to clarify HC sub-types in both zebrafish and mice, to highlight the non-hearing HCs.

Keywords: cilia; hair cell; regeneration; tekt3; zebrafish.

Figure 1

TEKT3/Tekt3 is evolutionarily conserved. (A) Amino acid sequences of zebrafish and a few mammalian TEKT3/Tekt3 are aligned, and the resulted phylogenetic tree (B) reveals that zebrafish Tekt3 is older than human TEKT3 in evolution. Zebrafish (Danio rerio) is highlighted in red color in (A,B).

Figure 2

tekt3 mRNA is specifically present in zebrafish HC-bearing organs. (A) The expression profiles of zebrafish tekt1-3 between 2 and 6 dpf. (B) Expression patterns of tekt3 were detected by the whole-mount in situ hybridization at 3 and 5 dpf. Red arrows point to neuromasts and light blue arrows point to the ear. HC: the hair cell.

Figure 3

TEKT3/Tekt3 is present in some HCs. (A) Neuromast and utricular HCs pf zebrafish larvae at 5 dpf are positive for anti-Tekt3 antibody staining while the crista HCs are negative. (B) TEKT3 is present in mature mouse cochlear HCs. The left row of cells are inner HCs (IHC, pointed by a white arrow) and the right three rows are outer HCs (pillar cell, pointed by a pink arrow). Note: The saccular and crista HCs are always negative.

Figure 4

The subcellular location of Tekt3 in zebrafish utricular HCs. Crossed with Tg(brn3c:mGFP), both homozygous and heterozygous tekt3 mutants showed weaker α-Tektin3 staining at the utricle (apical portion of HCs). Note: two colors overlap at the very tip portion of hair bundles, yet at the root portion, they never overlap.

Figure 5

tekt3 mutant exhibited only neuromast HC kinocilium defect. (A) The CRISPR/Cas9-mediated genome editing targeted the exon 2 of tekt3 locus; the gRNA core sequence is shown. (B) Two tekt3 mutant alleles were verified by sequencing. (C) The (Alphfold2.0) predicted protein structure of one mutated Tekt3 is shown side by side with a normal protein. (D) A typical genotyping result of tekt3 mutant. (E) A 6 mpf homozygous tekt3 mutant looked normal. (F) Significant reduction in tekt3 mRNA level can be seen in both homozygous and heterozygous tekt3 mutants. (G) The tekt1 mRNA level in 5 dpf fish were significantly up in both homozygous and heterozygous tekt3 mutants. (H) Tekt3 was decreased in neuromast HCs of both homozygous and heterozygous tekt3 mutants (5 dpf). The arrowheads are pointed to tekt3 positive cells. (I) The abnormal neuromast HC hair bundle or kinocilia of the 5 dpf tekt3 mutant. (J) Almost all mutant neuromasts (n = 78) showed abnormal kinoclium tips while wild-type (n = 112) rarely possessed the defect. * p < 0.05; ** p < 0.01; and *** p < 0.001.

Figure 6

Neuromast HCs of tekt3 mutant are neomycin resistant. (A) Live dye (YO-PRO1 and FM1-43) intake by neuromast HCs in 5 dpf WT and tekt3 mutant. (B) YO-PRO1 and FM1-43X staining. (C) Weaker dye-intaking of tekt3 mutant neuromast. (D) Resistance to neomycin treatment. (E) Neomycin dose-dependency. (F) Weaker TUNNEL signals of mutant neuromast after neomycin treatment. The arrowheads are pointed to apoptotic neuromast hair cells. ** p < 0.01; *** p < 0.001; and **** p < 0.0001.

Figure 7

Heterozygous tekt3 mutant has weaker response to soundwave stimuli. (A) Self-made equipment to measure neuromast function (B,C) showed that the 6 dpf heterozygous tekt3 mutant exerted weaker response upon 75 Hz (B) or 800 Hz (C) stimuli. (D) A diagram of a larval zebrafish in linear VOR test. (E) The 5 dpf tekt3 mutant had a normal VOR score. ** p < 0.01; and *** p < 0.001.

Figure 8

Neuromast HC regeneration is delayed in tekt3 mutant. (A) The summary of HC numbers in L1-L3 neuromasts, days after neomycin administration. (B) The HC regeneration. (C,D) PCNA staining shows neuromast cell proliferation defect of tekt3 mutant. (E) EDU staining shows neuromast cell proliferation defect of tekt3 mutant 6 h after neomycin treatment. ** p < 0.01; *** p < 0.001; and **** p < 0.0001.

Figure 9

The rescue of tekt3 mutant. (A) Comparison of tekt3 mRNA levels between control and experiment groups is shown. (B,C) The protein level could be brought back a bit in the injected groups. (D) Rescued neuromast and utricle of tekt3 mutant. (E) The rescued startle response in mRNA injection groups. (F) The YO-PRO1 and FM1-43 intakes become normal in mRNA injected groups. The white arrow is pointed to a kinocilium with positive tekt3 signals. * p < 0.05; and **** p < 0.0001.