Expression and characterization of ZP domain-containing proteins in zebrafish ear
Date Issued
2011
Date
2011
Author(s)
Yang, Chung-Hsiang
Abstract
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 one component of a gel-like structure called tectorial membrane which is involved in transmitting sound wave into neuronal signals and is important for normal auditory function. β-tectorin is specifically expressed in mammalian and avian inner ear. We identified and cloned the gene encoding zebrafish β-tectorin. Through whole-mount in situ hybridization, we demonstrated that β-tectorin mRNA was expressed in the otic placode and in the specialized sensory patch of the inner ear during zebrafish embryonic stages. Analysis by western blot and immunofluorescence staining suggested that β-tectorin may undergo some posttranslational modifications. Morpholino knockdown of zebrafish β-tectorin affected the position and number of otoliths in the ear of morphants. Finally, the swimming behaviors of β-tectorin morphants were abnormal since the development of inner ear was compromised.
In addition to studies of β-tectorin, we had also studied another ZP domain containing protein, zpDL1. Through bioinformatics method, we predicted and identified zpDL1. Since zpDL1 has not been reported in any literature yet, we first characterized its expression by whole-mount in situ hybridization. The expression profile of zpDL1 displayed a specific pattern in the three crista of the inner ear, with an appearance correlated to the onset of crista emerging (about 48 hpf to 72 hpf ). A transgenic Tg(zpDL1:GFP) line was generated using 2 kilo bps promoter region upstream the start codon to drive GFP (green fluorescent protein) expression. Specific expression of GFP in the crista will be used to label the hair cells in the crista and further applied in hair cell ablations by intense laser beam.
Our results reveal that zebrafish β-tectorin is specifically expressed in the zebrafish inner ear, specifically in the macula, and is important for regulating the development of zebrafish inner ear. Lack of zebrafish β-tectorin caused severe defects of inner ear, formation of otoliths and function. Furthermore, zpDL1, another ZP domain containg protein, also showed expression pattern in hair cells related sensory organ, crista. Taken together, our works contributed to better knowledge of the role of ZP domain containing proteins in the zebrafish inner ear.
Subjects
ZP domain protein
zebrafish
inner ear development
developmental biology
Type
thesis
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