Zona pellucida domain-containing protein β-tectorin is crucial for zebrafish proper inner ear development

Abstract

Background: The zona pellucida (ZP) domain is part of many extracellular proteins with diverse functions from structural components to receptors. The mammalian β-tectorin is a protein of 336 amino acid residues containing a single ZP domain and a putative signal peptide at the N-terminus of the protein. It is 1 component of a gel-like structure called the tectorial membrane which is involved in transforming sound waves into neuronal signals and is important for normal auditory function. β-Tectorin is specifically expressed in the mammalian and avian inner ear.

Methodology/principal findings: We identified and cloned the gene encoding zebrafish β-tectorin. Through whole-mount in situ hybridization, we demonstrated that β-tectorin messenger RNA was expressed in the otic placode and specialized sensory patch of the inner ear during zebrafish embryonic stages. Morpholino knockdown of zebrafish β-tectorin affected the position and number of otoliths in the ears of morphants. Finally, swimming behaviors of β-tectorin morphants were abnormal since the development of the inner ear was compromised.

Conclusions/significance: Our results reveal that zebrafish β-tectorin is specifically expressed in the zebrafish inner ear, and is important for regulating the development of the zebrafish inner ear. Lack of zebrafish β-tectorin caused severe defects in inner ear formation of otoliths and function.

Figure 1. Zebrafish β-tectorin amino acid sequence alignment with those of other species.

Deduced amino acid sequences of zebrafish β-tectorin were aligned with those of human, mouse, chicken, and Xenopus. All β-tectorin proteins contained a conserved zona pellucida (ZP) domain of approximately 260 amino acids, with 12 highly conserved cysteine residues (indicated by arrows). Identical residues in 4 or 5 proteins are highlighted. Signal peptide and putative GPI-anchored domains are heavily overlined. The putative N-linked glycosylation sites are indicated by dots (…). The accession numbers of each β-tectorin from different species are listed below: human (XM_521604), mouse (X99806), chicken (AAA92461), and Xenopus (CAJ82963).

Figure 2. Genomic organization of zebrafish and mouse β-tectorin genes.

Coding regions are shown as filled boxes numbered from 1 to 10 in both zebrafish and mouse β-tectorin genes. The 5′- and 3′-untranslated regions are shown as open boxes, while introns and 5′- and 3′-flanking regions are indicated by solid lines.

Figure 3. Expression profiles of zebrafish β-tectorin mRNAs by RT-PCR and whole-mount in situ hybridization.

(A) RT-PCR of the β-tectorin transcript was performed using a pair of primers to produce a DNA fragment of 1208 bp. β-Actin bands were also used to normalize the amount of cDNA prepared from different tissues and embryos at different developmental stages. (B) Whole-mount in situ hybridization with antisense β-tectorin at different developmental stages was performed. The images were taken from the dorsal (a, d, g, j) and the lateral view (b, e, h, k), and complete lateral view (c, f, i, l) with the anterior to the left and dorsal to the top. Longitudinal sections of the embryo were at 72 hpf with anterior to the left and dorsal to the top (panel m). The straight line in panel n represents the region of sections in panel m. hc, hair cell; sc, supporting cell.

Figure 4. Abnormal otolith phenotypes in β-tectorin morphants.

(A) The otolith phenotypes of β-tectorin morphants are classified into normal (normal, panel a), fused (fused, panel b) and single otoliths (single, panel c). Abnormal development of the vestibular system is shown by arrows in β-tectorin morphants from 72 to 120 hpf (panels d to i). (B) The percentage of abnormal otolith phenotypes in zebrafish embryos injected with different β-tectorin MOs or combined with β-tectorin mRNAs or p53 MOs. All samples are observed at 72 hpf. Bars, 50 µm.

Figure 5. Characterization of ear defects in β-tectorin morphants.

The expression levels of the following inner ear marker genes, such as starmaker (stm) (A), otolith matrix protein 1 (omp-1) (B), and zona pellucida-like domain-containing protein-like 1 (zpDL1) (C), were examined by whole-mount in situ hybridization in β-tectorin morphants. Stm signals in the anterior macula (am) of β-tectorin morphants decreased or even disappeared in fishes with either fused (panels b and b′) or single otoliths (panels c and c′), as indicated by arrows. zpDL1 signals in the lateral crista (lc) of β-tectorin morphants vanished. Black bars, 100 µm (D) Confocal microscopy image analysis of β-tectorin morphants injected with FM1-43 dyes into the otic vesicle at 72 hpf. After injection, hair cells in anterior crista (ac), lateral crista (lc), macula (m) and posterior crista (pc) of control MO-injected embryos can take up FM1-43 dyes. White bar, 50 µm.

Figure 6. Abnormal swimming behaviors of β-tectorin morphants.

β-Tectorin morphants were examined for their abilities to remain balance and react to a stimulus. Tactile stimulation was created by poking a zebrafish on the head with a glass tube: β-tectorin morphants with a single (panel A) and a fused otoliths (panel B), and a control with normal otoliths (panel C). Swimming behaviors of β-tectorin morphants at 5 days post-fertilization under stimulation were recorded with a digital video camera. β-Tectorin morphants with either single or fused otoliths failed to maintain their balance, tended to remain leaning on one side, remained on the bottom (panel A, B), and tended to swim in a corkscrew (panel A, A1 to A4) or circular manner (panel B, B1 to B4). Control zebrafish maintained their balance, had immediate responses to stimulation, and swam in a straight line (panel C, C1 to C4). The trails of the zebrafish movement were illustrated by dark arrows.