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Zona Pellucida Domain-Containing Protein b-Tectorin is
Crucial for Zebrafish Proper Inner Ear Development
Chung-Hsiang Yang
1,2
, Chia-Hsiung Cheng
2
, Gen-Der Chen
2
, Wei-Hao Liao
2
, Yi-Chung Chen
2
, Kai-Yun
Huang
1
, Pung-Pung Hwang
3
, Sheng-Ping L. Hwang
3
, Chang-Jen Huang
1,2
*
1Institute of Biochemical Sciences, National Taiwan University, Taipei, Taiwan, 2Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan, 3Institute of Cellular
and Organismic Biology, Academia Sinica, Taipei, Taiwan
Abstract
Background:
The zona pellucida (ZP) domain is part of many extracellular proteins with diverse functions from structural
components to receptors. The mammalian b-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. b-Tectorin is specifically expressed in the mammalian and avian inner ear.
Methodology/Principal Findings:
We identified and cloned the gene encoding zebrafish b-tectorin. Through whole-mount
in situ hybridization, we demonstrated that b-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 b-tectorin affected
the position and number of otoliths in the ears of morphants. Finally, swimming behaviors of b-tectorin morphants were
abnormal since the development of the inner ear was compromised.
Conclusions/Significance:
Our results reveal that zebrafish b-tectorin is specifically expressed in the zebrafish inner ear, and
is important for regulating the development of the zebrafish inner ear. Lack of zebrafish b-tectorin caused severe defects in
inner ear formation of otoliths and function.
Citation: Yang C-H, Cheng C-H, Chen G-D, Liao W-H, Chen Y-C, et al. (2011) Zona Pellucida Domain-Containing Protein b-Tectorin is Crucial for Zebrafish Proper
Inner Ear Development. PLoS ONE 6(8): e23078. doi:10.1371/journal.pone.0023078
Editor: Bruce Riley, Texas A&M University, United States of America
Received April 5, 2011; Accepted July 5, 2011; Published August 2, 2011
Copyright: ß2011 Yang et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This study was supported by a grant (AS-99-TP-B08) from Academia Sinica and a grant (97-2313-B-001-002-MY3) from National Science Council, Taipei,
Taiwan. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: cjibc@gate.sinica.edu.tw
Introduction
The zona pellucida (ZP) was first discovered as a glycoprotein
surrounding the plasma membrane of an oocyte, and it is
important in fertilization. The ZP domain is a sequence shared
by many extracellular proteins with diverse functions from
structural components to receptors. Among these proteins are
the mammalian ZP1, ZP2, and ZP3, and non-mammalian egg-
coating proteins, Tamm-Horsfall protein (THP), glycoprotein
(GP)-2, a- and b-tectorins, transforming growth factor (TGF)-b
receptor III, endoglin, deleted in malignant brain tumor (DMBT)-
1, no-mechanoreceptor potential-A (Nomp A), Dumpy, and
cuticlin-1 [1]. Each of these ZP-containing proteins is composed
of a signal sequence driving these proteins to the endoplasmic
reticulum (ER), and each possesses a ZP domain of approximately
260 amino acids long that is comprised of 8,10 conserved
cysteine residues, a C-terminal, a hydrophobic transmembrane-
like region, and a short cytoplasmic tail [2,3]. ZP domain-
containing proteins are highly conserved among all species and are
often glycosylated [4]. They are generally modified with a variable
number of high-mannose type, N-linked oligosaccharides in the
ER. These proteins can be further modified by the addition of O-
linked oligosaccharides and by processing of high-mannose-type,
N-linked oligosaccharides to the complex type when transferred
to Golgi apparatuses. ZP domain-containing proteins are often
present in filaments and/or matrices which play important roles in
protein polymerization [1].
In the inner ear organ of Corti, the tip of hair cell stereocilium
bundles is covered by a gel-like matrix called the tectorial
membrane. The mammalian tectorial membrane is formed by 3
different collagens (types II, V, and IX) combined with 3 non-
collagenous, glycosylated polypeptides, called a-tectorin, b-
tectorin, and otogelin [1]. The tectorial membrane is a particular
structure that deflects the stereocilia of hair cells during sound-
triggered vibrations of the basilar membrane, and hair cells
facilitate the transduction of sounds into neural signals. a- and b-
tectorins belong to the ZP domain-containing protein family, and
mutations in a-tectorin or b-tectorin were reported to result in
human nonsyndromic deafness. Studies of the human a-tectorin
gene, TECTA, showed that it is related to dominant forms of
prelingual, nonprogressive deafness: DFNA8 (MIM601543) and
DFNA12 (MIM601842) [5] or a recessive form at locus DFNB21
[6]. On the other hand, TECTB, which encodes b-tectorin, also
plays an important role in maintaining the normal function of the
tectorial membrane. Previous studies on knockout mice reported
that the structure of the striated-sheet matrix is disrupted and
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cochlear tuning is sharpened in TECTB
2/2
mice [7]. Together,
both types of tectorins are important for maintaining normal
auditory function of the inner ear.
Currently, only mammalian and chicken tectorins have been
identified and cloned [8]. Little is known about zebrafish b-
tectorin and its role during embryonic development. Zebrafish
have many benefits as a model animal for studying genes involved
in inner ear development: first, zebrafish eggs develop in vitro;
second, the zebrafish ear is transparent for the first few weeks of
life; and third, forward genetic screens and antisense technology
are well-established [9]. The zebrafish inner ear system consists of
semicircular canals, otoliths, and different sensory patches that
are formed by special type of cells, called hair cells. The
semicircular canals are attached to the sensory epithelium called
cristae which are located at the base of each canal. Cristae are
important for sensing the position of the head and angular
acceleration. Polycrystalline masses called otoliths are connected
to 2 or more macular organs, called saccules and utricles [9].
Zebrafish is considered to be a fish with excellent hearing in the
teleost family due to the function of Weberian ossicles which
connect the swim bladder to the saccule allowing sound
amplification [10,11]. Although, the structure of the zebrafish
inner ear greatly differs from that of the mammalian inner ear
due to the lack of a cochlea, the convenience in handling it and
the evolutionarily conserved molecular mechanisms of inner ear
development make zebrafish a good animal model for studying
development of the inner ear.
In order to investigate the role of ZP domain-containing
proteins in zebrafish inner ear development, we predicted and
identified a zebrafish ZP domain-containing protein, zebrafish b-
tectorin, through a bioinformatics method, and designed a
specific morpholino to knock down the expression of zebrafish
b-tectorin. We also characterized the temporal and spatial
expressions of zebrafish b-tectorin by whole-mount in situ
hybridization, and demonstrated that it is specifically expressed
in the zebrafish inner ear in the early stages of development.
Morpholino knockdown of b-tectorin expression in zebrafish
resulted in abnormal inner ear development. Otoliths of b-
tectorin morphants showed delocalization or a fused pattern.
Taken together, we propose that the zebrafish b-tectorin plays an
important role in the proper formation of zebrafish inner ear
components, and therefore a lack of it will cause serious defects in
development of the inner ear.
Results
Cloning of b-tectorin from zebrafish
The overall deduced amino acid sequences of zebrafish b-
tectorin respectively showed 49%, 50%, 50%, and 49% identities
to those of human, mouse, chicken, and Xenopus b-tectorin (Fig. 1).
The zebrafish b-tectorin protein contains a conserved ZP domain,
which has highly conserved cysteines of C1 to C8, and Cx, Cy, Ca,
and Cb. A signal peptide of 16-amino acids, MAAVGLF-
FILLPVTWA, in the NH
2
-terminal was predicted by the online
software, SignalP 3.0 (http://www.cbs.dtu.dk/services/SignalP/),
which likewise occurs in mammalian b-tectorin proteins. Mam-
malian b-tectorin proteins have a signal peptide of 17 amino acids.
A hydrophobic C-terminus characteristic of proteins that are
membrane bound via a putative GPI-anchor was reported in avian
and mammalian b-tectorins [12,13]. Moreover, human b-tectorin
is a glycoprotein and contains 4 N-linked sugar chains on Asn80,
Asn104, Asn116, and Asn145. Similarly, the zebrafish b-tectorin
protein contains 4 N-linked glycosylation sites of
77
NHS,
104
NDS,
116
NYT, and
145
NGS (Fig. 1).
Genomic structure of the zebrafish b-tectorin gene
We then used the 1542 bp of zebrafish b-tectorin cDNA (with
accession no. FJ374270) to perform an online BLAST search of
the GenBank database. The zebrafish b-tectorin cDNA matched
10 non-contiguous regions of a 92,355-bp zebrafish BAC clone,
CH73-92E20 (GenBank accession no. CU462848). Subsequently,
a BLAST 2-sequence comparison of BAC CH73-92E20 with the
zebrafish b-tectorin cDNA indicated that b-tectorin cDNA
contained 10 putative exons and 9 introns spanning at least
8.6 kb (Fig. 2). Using these putative exons as a model, a sequence
alignment was produced such that each intron concurred with the
GT/AG intron donor/acceptor site rule [14]. Exon 1 contained
the 59-UTR, while exon 2 contained the putative translation
initiation site. Exon 2 contained 9 bp of the 59-UTR and 66 bp of
the first coding sequences of b-tectorin cDNA. Exon 10 contained
the last 69 bp of the coding sequences and 362 bp of 39-UTR. The
size of the introns considerably varied, ranging 81 (intron 8) to
2419 bp (intron 9).
Comparison of the exon-intron organization of zebrafish and
mouse b-tectorin genes indicated that their genomic structures were
similar with 10 exons and 9 introns. The mouse b-tectorin gene
spanned approximately 15.4 kb. In addition, the average intron
size of the mouse b-tectorin gene (1411 bp) was larger than that of
the zebrafish b-tectorin gene (799 bp) (Fig. 2).
Expression profiles of zebrafish b-tectorin messenger
(m)RNA in adult tissues and embryos at different
developmental stages
Expression levels of zebrafish b-tectorin transcripts in adult tissues
and embryos from different developmental stages were examined
by an RT-PCR analysis. A pair of primers was used to amplify a
DNA fragment that spanned exons 2 to 4 to avoid genomic DNA
interference in the PCRs. The amplified product of this pair of
primers was about 1208 bp long. As shown in Fig. 3A, a high level
of b-tectorin expression was detected in the brain, with moderate
expression in the kidneys and less in the intestines.
During embryogenesis, b-tectorin transcripts were not detected at
1 day post-fertilization (dpf), and their expression levels constantly
increased thereafter; however, a significant decrease in the
expression level was observed at 5 dpf and thereafter (Fig. 3A).
Spatial and temporal expression patterns of zebrafish b-tectorin
were further analyzed by whole-mount in situ hybridization.
During different stages of development, expression of the b-tectorin
transcript was specifically detected in the anterior and posterior
maculae on both sides of the zebrafish from 48,120 h post-
fertilization (hpf) (Fig. 3B, panels a,l). It was interesting to note
that the expression of b-tectorin mRNA in the anterior macula was
much weaker than that of the posterior macula in 48-hpf embryos.
The signals of b-tectorin in situ hybridization are restricted to the
macula of the inner ear, no signals in other parts of the embryos
can be detected in various stages (Fig. 3B, panels c, f, i and l).
Through longitudal section of the zebrafish inner ear at 72 hpf, b-
tectorin is both expressed in the hair cells and supporting cells of
macula, and the expression pattern resembles the early expression
of pax5 in the macula [15]. The overall expression pattern of
zebrafish b-tectorin in the inner ear was quite similar to that of the
Starmaker gene, which is also expressed in the anterior and posterior
maculae on both sides of the inner ear [16].
Abnormal otolith formation in b-tectorin morphants
Morpholino (MO)-mediated knockdown of genes in zebrafish
embryos has become a routine and efficient method to provide
information about gene function in vivo [17]. To examine the
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function of b-tectorin in vivo, we designed zebrafish b-tectorin MOs,
which targeted the sequence located at the 59-UTR of b-tectorin
mRNA, to specifically knock down the translation of endogenous
b-tectorin mRNA.
To determine the specificity of the MO used, a control
approach was used. The 25-bp target sequence of the b-tectorin
MO was cloned upstream of the green fluorescent protein (GFP)
ORF into a pCMV backbone expression vector (bTec-GFP). As
for the control, a target sequence containing 5 mismatches was
used (MM b-Tec-GFP). The MM b-Tec-GFP or bTec-GFP
plasmid was injected into zebrafish embryos in the absence or
presence of a 9-ng b-tectorin MO. Co-injection of bTec-GFP RNA
and the b-tectorin MO completely blocked GFP expression
(n= 22/24; Fig. S1A). Conversely, GFP expression was not
affected when 5 bp of the target sequence was exchanged (MM b-
Tec-GFP) (n= 36/36; Fig. S1B) indicating the spe cificity of the b-
tectorin MO.
After injecting 4 ng/embryo of b-tectorin MO, we observed
abnormal otolith morphology in embryos at 120 hpf. There are 2
otoliths on each side in wild-type (WT) zebrafish embryos; the
one in the anterior has a flattened-oval shape, whereas the other
one in the posterior, is larger, and has a round shape (Fig. 4A,
panel a). b-Tectorin morphants displayed 2 different phenotypes
including the fusion of 2 otoliths (Fig. 4A, panel b, n= 50/168,
29%; Fig. 4B) as well as a single otolith (Fig. 4A, panel c, n=5/
168, 2.9%; Fig. 4B). Changes in morphology of the otoliths were
correlated with the irregular formation of the vestibular system in
the inner ear; development of semicircular canals seemed to be
affected in b-tectorin morphants when observed at 72,120 hpf. b-
Tectorin morphants with either fused or single otoliths were
Figure 1. Zebrafish b-tectorin amino acid sequence alignment with those of other species. Deduced amino acid sequences of zebrafish
b-tectorin were aligned with those of human, mouse, chicken, and Xenopus. All b-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 b-tectorin from different species are listed below: human (XM_521604), mouse (X99806), chicken (AAA92461),
and Xenopus (CAJ82963).
doi:10.1371/journal.pone.0023078.g001
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observed with abnormal semicircular canal outgrowth (Fig. 4A,
panels e, h, f, and i, arrows), whereas the control showed 2
normal otoliths and outgrowth of the semicircular canals (Fig. 4A,
panels d and g).
We also generated splice MOs to block splicings of the b-
tectorin sequence within exon 2 and exon 3 (supplementatry
Fig. 2A). The splice MO 1 which blocks a splicing donor site
locates at the boundry of Exon 2 and intron 2 while the splice
MO 2 targets the acceptor site at the boundry of intron 2 and
Exon 3. Each of the splice MOs was used individually,
producing abnormal phenotypes similar to those of the ATG
MOs, differing only in efficiency (data not shown). The
combination of the two splice MOs is more efficient in
generating abnormal phenotypes that include both the fused
and single otolith phenotype like the ATG MO. The efficiency of
the splice MO mixture was analyzed by RT-PCR (supplemen-
tary Fig. 2B). After injecting a mixture of splice MOs 2 ng each,
we observed the appearance of the fusion of 2 otoliths (n = 113/
287, 39.37% ; Fig. 4B) and single otolith (n = 7/287, 2.43%;
Fig. 4B) in the resulting morphants. There were no other severe
morphological defects in the ATG MO (supplementary Fig. 3).
Gradient increases in the amount of ATG MO injected into the
embryos also showed increases in the abnormal phenotypes in a
dosage-dependent manner (Fig. 4B).
To rule out the possibilities that the phenotypes of these
morphants are the results of off-target effect of the morpholino
used [18], the p53 MO was used in co-injecting with ATG MO
into embryos and the phenotypes of these embryos were observed.
The percentage of abnormalities in the b-Tectorin morphants
coinjected with p53 MO was approximately equal to that of b-
Tectorin morphants (Fig. 4B), suggesting that the phenotypes of
these morphants were not related to off-target effect of the
morpholino.
To further confirm the specificity of gene knockdowns by the b-
tectorin MO, mRNA rescue was performed. Full length b-Tectorin
mRNAs, which were synthesized in vitro and injected into
embryos in one- or two- cell stage, were used to investigate the
rescue of b-Tectorin morphants. Misexpressions of the b-Tectorin
mRNA have no effect on morphologies of the control embryos
without MO injection. Coinjection of 8 ng ATG MO with
,100 pg b-Tectorin mRNA into each embryo resulted in a
reduction in the percentage of the b-Tectorin morphant with
abnormal ear phenotypes, fused otoliths (n = 12/35, 34%) and
single otolith (n = 3/35, 8.5%), whereas injection of 8 ng ATG
MO alone showed a percentage of as high as 84% abnormalities in
the inner ear, fused otoliths (n = 35/45, 78%), single otolith (n = 3/
45, 6%) (Fig. 4B). These results demonstrated that the b-Tectorin
mRNA can rescue defects in b-Tectorin morphants, validating the
specificity of b-Tectorin MO.
Development of the inner ear was affected in the
b-tectorin MO morphants as shown by whole-mount in
situ hybridization
To further analyze inner ear defects observed in morphants,
whole-mount in situ hybridization was first performed with
Starmaker (stm), which was reported to regulate the growth, shape,
and crystal lattice of otoliths [15]. In control MO-injected
zebrafish, the stm transcript was expressed in anterior and
posterior maculae on both sides at 96 hpf (Fig. 5B, lateral, panel
a, dorsal panel a9). However, the stm transcript was less expressed
in the anterior macula of b-tectorin morphants either with fused
otoliths or a single otolith compared to control MO-injected
zebrafish (Fig. 5B, panels b, b9c, and c9; arrows). Otolith matrix
protein 1 (omp-1) is important for otolith growth and correct
anchoring of otoliths to the maculae [19]. The expression pattern
of omp-1 in b-tectorin morphants was also reduced in the anterior
macula compared to the control, and its distribution seemed
to differ from that of control MO-injected zebrafish (Fig. 5C).
On the other hand, another gene marker, zona pellucida-like
domain-containing protein-like 1 (zpDL1) (Genbank accession
no. XM_00192195), was used to label anterior, lateral, and
posterior cristae of otoliths of control MO-injected zebrafish at 96
hpf (Fig. 5D, panels a and b). In b-tectorin morphants with fused
otoliths, the zpDL1 signal was detected only in the anterior and
posterior cristae of the inner ear, and the signal in the lateral
crista was lost (Fig. 5D, arrow).
FM1-43, a fluorescent dye, is known for labeling hair cells of the
inner ear by entering the mechanotransduction channels [20].
This function of FM1-43 allows us to monitor the formation of
active hair cells in the morphants. For this purpose, zebrafish at
diffenerent stages were injected with FM1-43 dyes specifically in
Figure 2. Genomic organization of zebrafish and mouse
b-tectorin
genes. Coding regions are shown as filled boxes numbered from 1 to 10
in both zebrafish and mouse b-tectorin genes. The 59- and 39-untranslated regions are shown as open boxes, while introns and 59- and 39-flanking
regions are indicated by solid lines.
doi:10.1371/journal.pone.0023078.g002
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lumen of the otic vesicle through an injection tube. FM1-43 dyes,
which were taken up by macula and cristae, could be easily
observed under a confocal fluorescence microscopy [20]. ATG
MO-injected zebrafish embryos were further injected with FM1-
43 dyes at 72 hpf to investigate whether the functions of these hair
cells are affected. b-tectorin morphants with fused otoliths of 72
hpf had lost its lateral crista as compared to control zebrafish.
These results are consistent with the data gained from whole-
mount in situ hybridization, as signals of zpDL1 lost in the lateral
crista in the b-tectorin morphants.
Taken together, the altered expression patterns observed in ear-
marker genes indicated that b-tectorin may play an important role
in both otolith and inner ear formation during zebrafish
development.
Figure 3. Expression profiles of zebrafish
b-tectorin
mRNAs by RT-PCR and whole-mount
in situ
hybridization. (A) RT-PCR of the
b-tectorin transcript was performed using a pair of primers to produce a DNA fragment of 1208 bp. b-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 b-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.
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Figure 4. Abnormal otolith phenotypes in
b-tectorin
morphants. (A) The otolith phenotypes of b-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
b-tectorin morphants from 72 to 120 hpf (panels d to i). (B) The percentage of abnormal otolith phenotypes in zebrafish embryos injected with
different b-tectorin MOs or combined with b-tectorin mRNAs or p53 MOs. All samples are observed at 72 hpf. Bars, 50 mm.
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Behavioral defects in b-tectorin morphants
Altered swimming behaviors and a lack of balance are indices of
abnormal ear function, for example, swimming in a corkscrew or
circular path [21]. Those b-tectorin morphants with either fused
otoliths or a single otolith were further tested for their ability to
maintain balance and swim after stimulation. At 5 dpf, about 40%
of noninjected embryos (WT) and control MO-injected embryos
(N = 70) displayed floating in an upright position and sometimes
swam spontaneously in random directions. Another 45% of
control MO-injected embryos remained lying down on the bottom
Figure 5. Characterization of ear defects in
b-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 b-tectorin morphants. Stm signals in the anterior macula (am) of b-tectorin morphants decreased or even
disappeared in fishes with either fused (panels b and b9) or single otoliths (panels c and c9), as indicated by arrows. zpDL1 signals in the lateral crista
(lc) of b-tectorin morphants vanished. Black bars, 100 mm (D) Confocal microscopy image analysis of b-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 mm.
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of the Petri dish. In order to test whether these 5-dpf zebrafish
could respond to vibration, several short vibrations were created
by deploying an ultrasonic processor in the water, and all zebrafish
swam away immediately (Video S1). The remaining 15% of
control MO-injected embryos were further stimulated using a glass
tube to touch the head of a zebrafish, and they swam away in a
straight manner (Video S3). On the other hand, b-tectorin
morphants with single or fused otoliths showed a failure to
maintain balance, and all stayed at the bottom of the Petri dish.
About 10% of those b-tectorin morphants responded to short
vibrations created by the ultrasonic processor in the water with
short irregular movements (n= 70) (Video S2). The remaining b-
tectorin morphants with fused otoliths were stimulated on the head
with a glass tube about 5 times, and they either did not respond to
the stimuli or swam a very short range in a circular path (Fig. 6, B1
to B4) (Videos S4 and S5). b-Tectorin morphants with a single
otolith had similar behavioral defects as those with fused otoliths
described above. Some swam in a corkscrew path which implied
profound defects (Fig. 6, A1 to A4) (Videos S6 and S7).
Discussion
In this study, the zebrafish b-tectorin gene and its cDNA were
cloned and characterized. The cDNA encodes a protein of 336
amino acids, which displays 49% and 50% identities to human
and chick b-tectorins. RT-PCR analyses showed that zebrafish b-
tectorin mRNA was primarily expressed in the brain with moderate
expression in the kidneys. Whole-mount in situ hybridization
showed that expression of the b-tectorin transcript was specifically
found in the anterior and posterior maculae of the ear. Similar to
human b-tectorin, zebrafish b-tectorin contains 4 N-linked
glycosylation sites (Fig. 1). Knockdown of zebrafish b-tectorin
expression caused the fusion of 2 otoliths or there was only a single
otolith, both of which led to severe malfunction of the inner ear.
The predicted amino acid sequence of zebrafish b-tectorin
exhibited overall identities of 49%, 50%, 50%, and 50% to b-
tectorins from the human, mouse, chick, and Xenopus (Fig. 1).
However, chick and mouse b-tectorins were homologous with 75%
identity at the amino acid level [13,22]. The higher similarity in
identity of chick b-tectorin to the human and mouse compared to
zebrafish may be due to differences in habitat, terrestrial and
aquatic, respectively. This suggests that the environment may
influence the evolution of this molecule. If fish b-tectorins from
different species were compared, these fish b-tectorins might display
higher identities to each other. Indeed, zebrafish b-tectorin showed
higher identities of 74%, 76%, and 74%, with 85% similarity, to b-
tectorin from Tetraodon, fugu, and medaka (Fig. S4).
In this study, zebrafish b-tectorin contained a highly conserved
ZP domain, which was a sequence of approximately 260 amino
acid residues with 8 or 10 cysteine residues and was located at the
C-terminus (Fig. 1). Many ZP domain-containing proteins with
various functions were found in vertebrates [1]. Some of those
proteins constitute the extracellular coat of animal eggs, such as
ZP1, ZP2 and ZP3. They are responsible for egg/sperm
recognition as well as for blocking polyspermy [23]. Other
proteins like a- and b-tectorins are 2 major components of the
tectorial membrane, which is an extracellular matrix covering the
sensory epithelia of the cochlea of the inner ear [24]. In transgenic
mice with a specific mutation in a-tectorin, the structure of the
tectorial membrane is disrupted leading to hearing loss [25].
Similarly, mice lacking b-tectorin have sharpened cochlear tuning
leading to low-frequency hearing loss [7]. Interestingly, zebrafish
b-tectorin is not expressed as a tectorial membrane in the cochlea;
instead, it is expressed in anterior and posterior maculae of the
zebrafish ear (Fig. 3B), which is similar to the expression of
Starmaker mRNA [16]. MO knockdown of b-tectorin expression
affected otolith formation in zebrafish larvae (Fig. 4A). These b-
tectorin morphants showed a failure to maintain balance and float.
Only a few b-tectorin morphants (10%) were able to respond to a
vibration created by an ultrasonic processor in the water, while
most of them continued to lie on the bottom of the Petri dish
(Video S2). In addition to their responses to short vibrations, b-
tectorin morphants with either fused or single otoliths also showed
abnormal swimming patterns after tactile stimulation (Videos S5
and S7). These phenotypes suggest that zebrafish b-tectorin has
crucial roles in the development and function of the zebrafish
inner ear.
Some extracellular matrix proteins are reported to play
important roles in the development of the zebrafish inner ear.
For instance, MO knockdown of specific genes like omp-1 and
otolin-1, which respectively encode otolith matrix protein 1 and a
collagen-like protein [19], also showed abnormal otolith formation
and impaired swimming behaviors. The omp-1 MO resulted in a
reduced otolith size, while otolin-1 MO caused fusion of the 2
otoliths. Therefore, omp-1 was proposed to play important roles in
normal otolith growth, while otolin-1 is involved in stabilizing the
otolith matrix. In this study, b-tectorin morphants also showed
similar fused otoliths, but the zebrafish b-tectorin is not a collagen-
like protein. Therefore, both otolin-1 and b-tectorin may interact
with each other and polymerize into the otolith matrix.
In addition, the expression patterns of starmaker and b-tectorin
mRNAs were similar in the inner ear and in anterior and posterior
maculae of the ear as shown in Figs. 3B and 5A. The zebrafish
Starmaker protein is a 66-kD protein that is enriched in strongly
acidic amino acid residues and 35% proteins, and is also extremely
hydrophilic [16]. During zebrafish development, the Starmaker
protein is required for otolith biomineralization, and starmaker
morphants showed starry or chunky otoliths with improper
balance in freely swimming zebrafish larvae. Expression of the
starmaker transcript was slightly reduced in the anterior macula in
zebrafish b-tectorin morphants with fused otoliths. However, in
zebrafish b-tectorin morphants with a single otolith, a large portion
of the starmaker signal was lost in the anterior macula. Interactions
among omp-1, b-tectorin, and Starmaker proteins might be the
foundation of proper otolith formation.
We also studied the expression of zpDL1 mRNA in zebrafish b-
tectorin morphants. ZpDL1 can be used as a marker to label the 3
sensory cristae of zebrafish embryos at 4 dpf. As shown in Fig. 5C,
zpDL1 signals were lost in lateral cristae of zebrafish b-tectorin
morphants. These data suggest that zebrafish b-tectorin not only
regulates anterior macula formation but is also involved in the
morphogenesis of cristae. However, the underlying mechanisms
require further investigation.
Materials and Methods
Zebrafish care
Zebrafish embryos were raised at 28.5uC, and different
developmental stages were determined based on criteria described
in the Zebrafish Book [26]. All animal procedures were approved by
the Animal Use and Care Committee of Academia Sinica
(protocol #10-12-114).
Total RNA isolation and reverse-transcription polymerase
chain reaction (RT-PCR) analysis of zebrafish b-tectorin
mRNA
Total RNA was isolated from different developmental stages
and various tissues of adult zebrafish, using the RNAzol reagent
b-Tectorin in Zebrafish Inner Ear Development
PLoS ONE | www.plosone.org 8 August 2011 | Volume 6 | Issue 8 | e23078
(Tel-Test, Friendswood, TX, USA) according to the instructions of
the manufacturer. After treatment with RQ1 RNase-Free DNaseI
(Promega, Madison, WI, USA), 50,100 mg of total RNA was
subjected to the first-strand cDNA synthesis. PCR amplifications
were performed with the following zebrafish b-tectorin RT-PCR
primers (b-tectorin-RT-F, 59- GCT GCT GAA GAC CTA CAC
AGG AAC-39and b-tectorin-RT-R, 59-TGG ATG TAT GCA
TGC ATG CGT GTC-39). Zebrafish b-actin primers (zACT-F,
59-GTG CTA GAC TCT GGT GAT GGT GTG-39and zACT-
R, 59-GGT GAT GAC CTG ACC GTC AGG AAG-39) were
used for the internal control to amplify a DNA fragment using
cDNA as a template. Primers for examining the efficiency of the
splice MOs are as follow (P1, 59- GCT GCT GAA GAC CTA
CAC AGG AAC- 39; P2, 59-GGC TAA ACA CGG CGT TGT
TGA CCA- 39.)
Cloning of the full-length cDNA encoding zebrafish b-
tectorin
To identify zebrafish complementary (c)DNA related to the
human b-tectorin gene, we used the coding region of human b-
tectorin (accession no. XM_521604) to search GenBank for related
expression sequence tags (ESTs) using the tBLAST program and
Figure 6. Abnormal swimming behaviors of
b-tectorin
morphants. b-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: b-tectorin morphants with a single (panel
A) and a fused otoliths (panel B), and a control with normal otoliths (panel C). Swimming behaviors of b-tectorin morphants at 5 days post-fertilization
under stimulation were recorded with a digital video camera. b-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.
doi:10.1371/journal.pone.0023078.g006
b-Tectorin in Zebrafish Inner Ear Development
PLoS ONE | www.plosone.org 9 August 2011 | Volume 6 | Issue 8 | e23078
found some zebrafish EST clones (CN315850 and EG585664)
related to human b-tectorin. Using 59- and 39-RACE to obtain
the 59- and 39-untranslated regions (UTRs), we assembled all
sequences to obtain 1542-bp cDNA with an open reading frame
(ORF) of 1011 bp encoding a protein of 336 amino acid residues.
The complete sequence was deposited in GenBank with the
accession number of FJ374270.
The full-length cDNA encoding zebrafish b-tectorin was isolated
by PCR amplification using gene-specific primers with linkers (b-
tectorin-BamH1-F, 59-CC GGA TCC ATG GCA GCT GTT
GGC CTT-39, and b-tectorin-EcoR1-R, 59-GG GAA TTC AAA
AGT AAA GTA TCC TAA-39) according to the sequence
submitted with GenBank accession no. FJ374270. Full-length
zebrafish b-tectorin was subcloned into the BamH1 and EcoR1 sites
of pcDNA3.1-myc to generate pcDNA3.1-b-tectorin- myc. Full
length b-tectorin was then further subcloned to T7TS plasmid using
BamH1 and Xba1 sites.
Rescue of defects in b-Tectorin morphant by injecting
b-Tectorin RNA
Full length b-Tectorin was cloned into T7TS plasmid and
synthesized in vitro. T7TS-b-Tectorin was linearized to synthesize
capping mRNA by using mMESSAGE mMACHINE T7 Kit
(Ambion, Foster City, CA, USA). ,100 pg of b-Tectorin RNA was
injected into embryos at the one- to two-cell stage.
Morpholino oligonucleotide (MO) injection
Antisense MOs were obtained from Gene Tools (Philomath,
OR, USA), and the sequence of zebrafish b-tectorin MO was as
follows: 59-GTG GCA GAA TCC AGA AGA AAT GTT G-39.
The sequence of the two splice MOs used were as follow: splice
MO 1 : 59- AAC CCA TCA AAC ATC TTA CCT CAG A-39
and splice MO 2 : 59-CCT CCT ACA TAC TGA AAA GAA
GGT A-39. The morpholinos were resolved to 24 mg/ml injection
stock, and stored in a 220uC refrigerator. The diluted morpholino
was injected into wild-type (WT) zebrafish embryos at the 1,2-cell
stage using a microinjection system consisting of an SZX9
stereomicroscope (Olympus, Tokoyo, Japan) and an IM300
Microinjector (Narishige, Tokoyo, Japan). The sequence of p53
MO was as follow (p53 MO: 59-AAA ATG TCT GTA CTA TCT
CCA TCC G-39) [27].
To confirm the specificities of the b-tectorin morpholino, several
pCMV-GFP reporter plasmids were created. The morpholino
targeted a 25-bp sequence of the PCR by the following primer pairs
for the perfect match, bTec-GFP (bTec-GFP-F, 59- GAT CCC
AAC ATT TCT TCT GGA TTC TGC CAC G-39and bTec-
GFP-R, 59-AAT TCG TGG CAG AAT CCA GAA GAA ATG
TTG G-39). For a 5-base exchanged mismatch, MM-b-Tec-GFP
was used (MM-b-Tec-GFP-F, 59-GAT CCG AAG ATT ACT
TCT GCA TTC TGG CAC G-39and MM-b-Tec-GFP-R,59-AAT
TCG TGC CAG AAT GCA GAA GTA ATC TTC G-39). The 59
region of the zebrafish b-tectorin mRNA was fused to the N-terminal
of the GFP protein. Either construct bTec-GFP or MM-b-Tec-GFP
was co-injected with zebrafish b-tectorin morpholino, and the
fluorescence was analyzed by a fluorescent microscope at 48 hpf.
Whole-mount in situ hybridization
Digoxigenin-labeled RNA probes (Roche, Penzberg, Germany)
were generated by in vitro transcription using the linearized
pGEM-T-easy plasmids (Promega, Madison, WI, USA) carrying
the 39-UTR of the following zebrafish genes. Whole-mount in situ
hybridization was performed following a previously described
protocol [28]. Specific primers for stm (stm-F, 59- GAA TCA ACT
GAG ACA GTC AAG ATA ACC -39and stm-R, 59- TGA GAG
TGG AGA GCG GGA ATT ATC TGC - 39), zpDL1 ( zpDL1-F,
59-GCG GGA CAT CAG TGT GTA TTG TGG AGT TCA -39
and zpDL1-R, 59- GCA AGC TGT GTG TTG TTG ACC AGG
TAT TCC -39), and omp-1 (zomp-1-F, 59- CAC ACT ACA GTC
TTT GAC AAC ATG - 39and zomp-1-R, 59- CAT CAG ATC
AAC ACA AAC CTT CAC - 39) were used to amplify the 39-
UTR of each gene. Primers used in the RT-PCR of zebrafish b-
tectorin were also used to make the zebrafish b-tectorin probe.
FM1-43 labeling of hair cells
Labeling the hair cells in the inner ear with 40 mM of FM1-43
(N-(3-triethylammoniumpropyl)-4(4-(dibutylamino)styryl) pyridi-
nium dibromide), (Invitrogen, Carlsbad, CA, USA) dissolved in
the extracellular solution. An injection tube was used to inject
FM1-43 into the otic vesicle following the protocols described
previously [29]. The formula of extracellular solution is described
as follow, 134 mM NaCl, 2.9 mM KCl, 1.2 mM MgCl
2
, 2.1 mM
CaCl
2
, 10 mM HEPES, and 10 mM glucose, and was adjusted to
pH 7.8.
Video recording of the swimming behavior of zebrafish
b-tectorin morphants
Zebrafish b-tectorin morphants were recorded with a digital
video camera (Sony DCR-PC120 digital camera, Tokyo, Japan) at
5 dpf to examine their reactions to short vibrations and tactile
stimulation. Short vibrations were created by a Hielscher Up50H
ultrasonic processor (Hielscher, Teltow, Germany), at 30 kHz and
50 W with an amplitude of 30% and 0.5 s per cycle. Tactile
stimulation was created using a glass tube to touch the head of a
zebrafish, and instantaneous responses were recorded with a
digital camera. Zebrafish b-tectorin morphants and control MO-
injected zebrafish were touched on the head at least 5 times for
each test. The swimming behavior of the zebrafish was observed
and defined by whether the fish swam in a straight or circular
manner.
Supporting Information
Figure S1 Control experiments for morpholino speci-
ficity. To determine the specificities of the morpholinos used,
pCMV-GFP reporter plasmids containing a perfect (bTec-GFP) or
mismatched (MM-b-Tec-GFP) MO target sequence were em-
ployed. Both bTec-GFP (A) and MM-b-Tec-GFP were co-injected
with the b-tectorin MO. All images were taken from zebrafish
embryos at 48 h post-fertilization.
(TIF)
Figure S2 The splice MO targeting and RT-PCR anal-
ysis of b-tectorin mRNAs of embryos injected with splice
MOs. (A) The exon-intron genomic structure from exons 1–4 was
shown. Splice MO 1 and MO 2 target the donor and acceptor
sites, respectively. (B) Total RNAs were extracted from control
MO (C) and splice MO1/MO2-injected (MO) embryos at 72 hpf,
then RT-PCR was performed. Primers (P1/P2) flanking the region
resulted in a single 500 bp band in the case of control embryos.
On the other hand, in the case of morphants, the level of this band
was strongly reduced and a second 1800 bp band was visible. The
second band resulted from the use of an alternative splice donor.
b-Actin bands were used to normalize the amount of cDNA
prepared from both embyos.
(TIF)
Figure S3 The morphology of b-tectorin morphants. The
ATG MO injected zebrafish embryos with fused (a, b), and single
b-Tectorin in Zebrafish Inner Ear Development
PLoS ONE | www.plosone.org 10 August 2011 | Volume 6 | Issue 8 | e23078
otoliths (c, d) appeared to be normal without obvious defects. All
photographs were taken at 72 hpf.
(TIF)
Figure S4 Zebrafish b-tectorin amino acid sequence
alignment with other fish species. The deduced amino acid
sequences of zebrafish b-tectorin were aligned with those from
Tetraodon, fugu, and medaka. Identical residues in 3 or 4 proteins
are highlighted. The accession numbers of each b-tectorin from
different fish species are listed below: Tetraodon (GenBank, accession
no: CAG06543), fugu (ensembl no: ENSTRUP00000021095), and
medaka (ensembl no: ENSORLP00000014650).
(TIF)
Video S1 Control MO-injected zebrafish responded to
vibrations made with an ultrasonic processor. Control
MO-injected zebrafish at 5 days post-fertilization (dpf) with normal
otoliths were placed in a Petri dish, and a transient vibration was
generated with a Hielscher Up50H ultrasonic processor, at 30 kHz
and 50 W with an amplitude of 30% and 0.5 s per cycle.
(WMV)
Video S2 The b-tectorin morphant responded to the
vibration made with an ultrasonic processor. The b-
tectorin morphant at 5 days post-fertilization with either single or
fused otoliths were placed in a Petri dish, and a transient vibration
was generated with a Hielscher Up50H ultrasonic processor.
(WMV)
Video S3 Response of control MO-injected zebrafish to
touch with a glass tube. Control MO-injected zebrafish at 5
days post-fertilization with normal otoliths was touched on the
head with a glass tube at least 5 times, and its swimming behavior
was observed. The bright-field videomicrograph was taken with an
Olympus IX70-FLA inverted fluorescence microscope equipped
with a Sony DCR-PC120 digital video camera.
(WMV)
Video S4 Response of a b-tectorin morphant with fused
otoliths to touch with a glass tube. The b-tectorin morphant
at 5 days post-fertilization with fused otoliths was touched on
the head with a glass tube at least 5 times. The bright-field
videomicrograph was taken with an Olympus IX70-FLA inverted
fluorescence microscope equipped with a Sony DCR-PC120
digital video camera.
(WMV)
Video S5 Swimming behavior of a b-tectorin morphant
with fused otoliths. The b-tectorin morphant at 5 days post-
fertilization (dpf) with fused otoliths was touched on the head with a
glass tube at least 5 times, and its swimming behavior was observed.
The video was taken with a Sony DCR-PC120 digital video camera.
(WMV)
Video S6 Response of a b-tectorin morphant with a
single otolith to touch with a glass tube. The b-tectorin
morphant at 5 days post-fertilization with a single otolith was
touched on the head with a glass tube at least 5 times. The bright-
field videomicrograph was taken with an Olympus IX70-FLA
inverted fluorescence microscope equipped with a Sony DCR-
PC120 digital video camera.
(WMV)
Video S7 Swimming behavior of a b-tectorin morphant
with a single otolith. The b-tectorin morphant at 5 days post-
fertilization with a single otolith was touched on the head with a
glass tube at least 5 times, and its swimming behavior was observed.
The video was taken with a Sony DCR-PC120 digital video camera.
(WMV)
Author Contributions
Conceived and designed the experiments: C-HY C-JH. Performed the
experiments: C-HY C-HC G-DC W-HL Y-CC K-YH. Analyzed the data:
C-HY C-HC G-DC Y-CC C-JH. Contributed reagents/materials/analysis
tools: C-HC G-DC P-PH S-PLH. Wrote the paper: C-HY C-JH.
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