Down regulation of msrb3 and destruction of normal auditory system development through hair cell apoptosis in zebrafish

Down-regulation of msrb3 and destruction of normal

auditory system development through hair cell apoptosis

in zebrafish

XIAOFANG SHEN

_{, FEI LIU}

_{, YINGZHI WANG}

_{, HUIJUN WANG}

_{, JING MA}

_{, WENJUN XIA}

_{, JIN ZHANG}

_,

NAN JIANG

_{, SHAOYANG SUN}

_{, XU WANG}

_{and DUAN MA*}

1_{Key Laboratory of Metabolism and Molecular Medicine, Ministry of Education, Department of Biochemistry and Molecular} Biology, Institute of Medical Sciences, School of Basic Medical Sciences, 2_{&KLOGUHQ·V+RVSLWDORI)XGDQ8QLYHUVLW\DQG}

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ABSTRACT Hearing defects can significantly influence quality of life for those who experience them. At this time, 177 deafness genes have been cloned, including 134 non-syndromic hearing-loss genes. The methionine sulfoxide reductase B3 (Ahmed et al., 2011)gene (also called DFNB74) is one such newly discovered hearing-loss gene. Within this genec.265 T>G and c.55 T>C mutations are associated with autosomal recessive hearing loss. However, the biological role and mechanism underlying how it contributes to deafness is unclear. Thus, to better understand this mutation, we designed splicing morpholinos for the purpose of down-regulating msrb3 in zebrafish. Morphants exhibited small, tiny, fused, or misplaced otoliths and abnormal numbers of otoliths. Down-regula-tion of msrb3 also caused shorter, thinner, and more crowded cilia. Furthermore, L1-8 neuromasts were reduced and disordered in the lateral line system; hair cells in each neuromast underwent apoptosis. Co-injection with human MSRB3 mRNA partially rescued auditory system defects, but mutant MSRB3 mRNA could not. Thus, msrb3 is instrumental for auditory system development in zebrafish and MSRB3-related deafness may be caused by promotion of hair cell apoptosis.

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Introduction

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*Address correspondence to: Duan Ma. Key Laboratory of Molecular Medicine, Ministry of Education, Shanghai Medical College, Fudan University, Shanghai 200032, China. Tel: 021-5423-7441. E-mail: [email protected]

#Note: The indicated authors contributed equally to this work.

Accepted: 28 May 2015.

ISSN: Online 1696-3547, Print 0214-6282

© 2015 UBC Press Printed in Spain

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Results

Two kinds of morpholinos can knock down the expression of msrb3

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Msrb3 is essential for the development of cilia in inner ear and lateral line neuromasts

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Decreased msrb3 caused disordered neuromasts and fewer hair cells

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Fig. 2. Otic developmental defects in Msrb3 morphants.(A) Overall morphology of mismatched controls (In2Con, a; Ex2Con, c) and msrb3 morphants (In2MO, b; Ex2MO, d) at 60 hpf indicated otolith abnormalities. Otic vesicles were visualized at 60 hpf using DIC microscopy. Scale bars: 50 +m. Unlike mismatch control (e; k), msrb3 morphants had tiny otoliths (f, l), abnormal otolith numbers (g, m), fused otoliths (h, n), misplaced otoliths (i, o), small otoliths (j, p), shrunken inner ears, and malformed semicircular canals (f–g, l–p). (B,C) Quantitative analysis of otolith defects of In2MO and Ex2MO at different concentrations at 60 hpf. Relative number of fish with otic developmental defects. (D,E) Statistical analysis of otolith abnormalities in controls and msrb3 morphants. Error bars are s.d. ***P<0.0001.

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Human MSRB3A mRNA can rescue otic defects in msrb3 morphants

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Fig. 3. Cilia damage in Msrb3 morphants.(A–D) Ste-reocilia in zebrafish inner ear marked with FITC-labeled phalloidin (confocal microscopy). Stereocilia in msrb3 morphants (In2MO, Ex2MO) were shorter and thinner than those of mismatch controls (In2Con, Ex2Con). In addition, msrb3 morphants had malformed semicircular canals. Scale bars: 30 +m. (E–H) Kinocilia in lateral line neuromasts of morphants were damaged (not see in controls). Scale bars: 10 +m. (I)Statistical analysis of in-ner ear stereocilia in controls and msrb3 morphants, “n” represents fish number. Error bars are s.d. ***P<0.0001. (J) Statistical analysis of neuromast kinocilia in controls and msrb3 morphants, “n” represents neuromasts number. Error bars are s.d. ***P<0.0001.

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Fig. 5. Msrb3 morphant apoptotic hair cells.(A,B) TUNEL assay data. In 3 dpf larvae, msrb3 morphants had more hair cell apoptosis than mismatch controls. Scale bars, 5 +m. (C) Statistical analysis of apoptotic hair cells per neuromast in controls and msrb3 morphants, “n” represents neuromasts number. Error bars are s.d. ***P<0.0001. (D,E) Zebrafish embryos injected with msrb3 morpholinos (In2MO, Ex2MO) or co-injected with msrb3 mor-pholinos and p53MO—AO staining to detect whole larval apoptosis signal at 24 hpf. There was no difference between msrb3 morphants and p53MO co-injected larvae. The p53 off-target effect was ruled out. Scale bars, 200 +m.

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Fig. 6. MSRB3 comparisons.(A) Amino acid sequence comparison of MSRB3A between Homo sapiens and Danio rerio (68.28% homology). (B) Human MSRB3A and MSRB3A mutants were obtained and transcribed into mRNA. (C,D) Overexpression of human MSRB3A mRNA rescued otic defects in morphants, but human MSRB3A mutant did not. Error bars are s.d. (E,F) Statistical analysis of otolith abnormality in different types of larvae. Human MSRB3A mRNA but not mutant MSRB3A mRNA rescued otic developmental defects. Error bars are s.d. ***P<0.0001.

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Materials and Methods

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Morpholino antisense oligonucleotides and microinjection

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