13
Otolith matrix proteins OMP-1 and Otolin-1 are necessary for normal otolith growth and their correct anchoring onto the sensory maculae Emi Murayama a,b, * , Philippe Herbomel b , Atsushi Kawakami c , Hiroyuki Takeda c , Hiromichi Nagasawa a a Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo, 113-8657 Tokyo, Japan b Unite ´ Macrophages et De ´veloppement de l’Immunite ´, De ´partement de Biologie du De ´veloppement, URA2578 du CNRS, Institut Pasteur, 25 rue du Dr Roux, 75724 Paris cedex 15, France c Department of Biological Science, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo, 113-0033 Tokyo, Japan Received 19 October 2004; received in revised form 2 March 2005; accepted 21 March 2005 Available online 11 May 2005 Abstract Fish otoliths are highly calcified concretions deposited in the inner ear and serve as a part of the hearing and balance systems. They consist mainly of calcium carbonate and a small amount of organic matrix. The latter component is considered to play important roles in otolith formation. Previously, we identified two major otolith matrix proteins, OMP-1 (otolith matrix protein-1) and Otolin-1, from salmonid species. To assess the function of these proteins in otolith formation, we performed antisense morpholino oligonucleotide (MO)-mediated knockdown of omp-1 and otolin-1 in zebrafish embryos. We first identified zebrafish cDNA homologs of omp-1 (zomp-1) and otolin-1 (zotolin-1). Whole-mount in situ hybridization then revealed that the expression of both zomp-1 and zotolin-1 mRNAs is restricted to the otic vesicles. zomp-1 mRNA was expressed from the 14-somite stage in the otic placode, but the zOMP-1 protein was detected only from 26- somite stage onwards. In contrast, zotolin-1 mRNA expression became clear around 72 hpf. MOs designed to inhibit zomp-1 and zotolin-1 mRNA translation, respectively, were injected into 1–2 cell stage embryos. zomp-1 MO caused a reduction in otolith size and an absence of zOtolin-1 deposition, while zotolin-1 MO caused a fusion of the two otoliths, and an increased instability of otoliths after fixation. We conclude that zOMP-1 is required for normal otolith growth and deposition of zOtolin-1 in the otolith, while zOtolin-1, a collagenous protein, is involved in the correct arrangement of the otoliths onto the sensory epithelium of the inner ear and probably in stabilization of the otolith matrix. q 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Biomineralization; Matrix protein; Morpholino; Otolith; Zebrafish 1. Introduction The hair cell-containing epithelia of the vertebrate inner ear are the sensory endorgans of the vestibular and auditory systems. Otoliths are dense crystals composed of calcium carbonate and an organic matrix that are primarily involved in gravity sensing by the vestibular hair cells (Lowenstein, 1971). In teleosts, three otoliths, sagitta, lapillus and asteriscus, are contained in each of three inner ear sacs, sacculus, utriculus and lagena, respectively. Each otolith is anchored to a corresponding sensory epithelium, or macula, via a gelatinous layer called otolithic membrane. By virtue of its inertial mass, the displacement of the otoliths relative to the sensory macula is able to generate the hair cell depolarization by deflecting the sensory hair bundles (Manning, 1924). In zebrafish, the otic vesicle is formed by cavitation of the otic placode around the 18-somite stage (18 h post- fertilization—hpf). Right from this stage, two otoliths, the future lapillus and sagitta, start to form at the anterior and posterior poles of the otic vesicle by localized accretion of Mechanisms of Development 122 (2005) 791–803 www.elsevier.com/locate/modo 0925-4773/$ - see front matter q 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.mod.2005.03.002 * Corresponding author. Address: Institut Pasteur, 25 rue du Dr Roux, 75724 Paris cedex 15, France. Tel.: C33 1 45 68 86 21; fax: C33 1 45 68 89 21. E-mail address: [email protected] (E. Murayama).

Otolith matrix proteins OMP-1 and Otolin-1 are necessary for normal otolith growth and their correct anchoring onto the sensory maculae

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Page 1: Otolith matrix proteins OMP-1 and Otolin-1 are necessary for normal otolith growth and their correct anchoring onto the sensory maculae

Otolith matrix proteins OMP-1 and Otolin-1 are necessary for normal

otolith growth and their correct anchoring onto the sensory maculae

Emi Murayamaa,b,*, Philippe Herbomelb, Atsushi Kawakamic,

Hiroyuki Takedac, Hiromichi Nagasawaa

aDepartment of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences,

The University of Tokyo, 1-1-1 Yayoi, Bunkyo, 113-8657 Tokyo, JapanbUnite Macrophages et Developpement de l’Immunite, Departement de Biologie du Developpement,

URA2578 du CNRS, Institut Pasteur, 25 rue du Dr Roux, 75724 Paris cedex 15, FrancecDepartment of Biological Science, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo, 113-0033 Tokyo, Japan

Received 19 October 2004; received in revised form 2 March 2005; accepted 21 March 2005

Available online 11 May 2005

Abstract

Fish otoliths are highly calcified concretions deposited in the inner ear and serve as a part of the hearing and balance systems. They consist

mainly of calcium carbonate and a small amount of organic matrix. The latter component is considered to play important roles in otolith

formation. Previously, we identified two major otolith matrix proteins, OMP-1 (otolith matrix protein-1) and Otolin-1, from salmonid

species. To assess the function of these proteins in otolith formation, we performed antisense morpholino oligonucleotide (MO)-mediated

knockdown of omp-1 and otolin-1 in zebrafish embryos. We first identified zebrafish cDNA homologs of omp-1 (zomp-1) and otolin-1

(zotolin-1). Whole-mount in situ hybridization then revealed that the expression of both zomp-1 and zotolin-1 mRNAs is restricted to the otic

vesicles. zomp-1 mRNA was expressed from the 14-somite stage in the otic placode, but the zOMP-1 protein was detected only from 26-

somite stage onwards. In contrast, zotolin-1 mRNA expression became clear around 72 hpf. MOs designed to inhibit zomp-1 and zotolin-1

mRNA translation, respectively, were injected into 1–2 cell stage embryos. zomp-1 MO caused a reduction in otolith size and an absence of

zOtolin-1 deposition, while zotolin-1 MO caused a fusion of the two otoliths, and an increased instability of otoliths after fixation. We

conclude that zOMP-1 is required for normal otolith growth and deposition of zOtolin-1 in the otolith, while zOtolin-1, a collagenous protein,

is involved in the correct arrangement of the otoliths onto the sensory epithelium of the inner ear and probably in stabilization of the otolith

matrix.

q 2005 Elsevier Ireland Ltd. All rights reserved.

Keywords: Biomineralization; Matrix protein; Morpholino; Otolith; Zebrafish

1. Introduction

The hair cell-containing epithelia of the vertebrate inner

ear are the sensory endorgans of the vestibular and auditory

systems. Otoliths are dense crystals composed of calcium

carbonate and an organic matrix that are primarily involved

in gravity sensing by the vestibular hair cells (Lowenstein,

1971). In teleosts, three otoliths, sagitta, lapillus and

0925-4773/$ - see front matter q 2005 Elsevier Ireland Ltd. All rights reserved.

doi:10.1016/j.mod.2005.03.002

* Corresponding author. Address: Institut Pasteur, 25 rue du Dr Roux,

75724 Paris cedex 15, France. Tel.: C33 1 45 68 86 21; fax: C33 1 45 68

89 21.

E-mail address: [email protected] (E. Murayama).

asteriscus, are contained in each of three inner ear sacs,

sacculus, utriculus and lagena, respectively. Each otolith is

anchored to a corresponding sensory epithelium, or macula,

via a gelatinous layer called otolithic membrane. By virtue

of its inertial mass, the displacement of the otoliths relative

to the sensory macula is able to generate the hair cell

depolarization by deflecting the sensory hair bundles

(Manning, 1924).

In zebrafish, the otic vesicle is formed by cavitation of

the otic placode around the 18-somite stage (18 h post-

fertilization—hpf). Right from this stage, two otoliths, the

future lapillus and sagitta, start to form at the anterior and

posterior poles of the otic vesicle by localized accretion of

Mechanisms of Development 122 (2005) 791–803

www.elsevier.com/locate/modo

Page 2: Otolith matrix proteins OMP-1 and Otolin-1 are necessary for normal otolith growth and their correct anchoring onto the sensory maculae

E. Murayama et al. / Mechanisms of Development 122 (2005) 791–803792

precursor particles on the tips of kinocilia of specialized hair

cell precursors, hence called ‘tether cells’ (Riley et al.,

1997). This initial process of ‘otolith seeding’ occurs during

a period from 18 to 22 hpf (18- to 26-somite stage). Then,

precursor particles gradually disappear from the endo-

lymph, and otolith formation enters a phase of ‘otolith

growth’, from solute material. The first sensory hair cells of

the future maculae appear by 24 hpf close to the two

growing otoliths. By 72 hpf, the anterior otolith (lapillus)

exhibits an oval shape, whereas the posterior otolith

(sagitta) shows a disk-like structure. The third otolith, the

asteriscus, appears not before 9–17 days post-fertilization

(dpf), in an additional chamber of pars inferior, the lagena,

which individualizes from the sacculus at 15 dpf (Riley and

Moorman, 2000; Bever and Fekete, 2002). All chambers of

pars inferior connect until late stages, then the sacculus

becomes separated from the utriculus around 20 dpf

(Haddon and Lewis, 1996).

It is known that fish otoliths grow diurnally, and form

daily rings within their microstructure (Campana and

Neilson, 1985). Each ring is composed of an incremental

zone in which CaCO3 predominates, and a discontinuous

zone mainly composed of organic matrix (Watabe et al.,

1982). Thus, otolith growth is based on the alternate

deposition of calcium carbonate and proteins on the otolith

surface (Mugiya, 1987). Based on these findings, the

organic matrix has been considered to play important

roles in otolith formation. A number of proteins that

accumulate in otoliths or otoconia (the equivalent of otoliths

in tetrapods) have been identified from various animal

species. Otoconin-22 (Pote et al., 1993; Yaoi et al., 2003)

was identified in amphibian otoconia, while its homolog

Otoconin-90/95 was independently found in mammalian

otoconia (Wang et al., 1998; Verpy et al., 1999). Calbindin-

D28K was then identified in the otoconia of chick (Balsamo

et al., 2000) and lizard (Piscopo et al., 2003). Recently,

Sumanas et al. (2003) and Sollner et al. (2003) examined the

function of the chaperone protein GP96 and Starmaker

protein, respectively, in otolith formation in zebrafish.

Knockdown of GP96 resulted in an otolith seeding defect,

whereby the seeding particles did not adhere to the kinocilia

of the tether cells (Sumanas et al., 2003). In contrast, loss of

function of Starmaker protein led to a change in crystal

polymorph of the otoliths from aragonite to calcite, and thus

to a change in otolith morphology (Sollner et al., 2003).

We previously identified two major otolith matrix

proteins, OMP-1 (otolith matrix protein-1) and Otolin-1,

from the otoliths of two salmonid species. They are the

major components of EDTA-soluble and -insoluble matrix

protein fractions, respectively (Murayama et al., 2000,

2002). OMP-1 is a member of the transferrin family of

proteins and shows 40% similarity with the C-terminal half

of human melanotransferrin, a monomeric glycoprotein

identified in human melanoma cells and thought to play a

role in iron metabolism (Aisen and Leibman, 1972;

Woodbury et al., 1980). Otolin-1 is a collagenous protein

that belongs to the type VIII and X collagen family. So far,

these collagens were mainly reported to be present in non-

calcified tissues, such as basement membranes (type VIII;

Labermeier et al., 1983; Shuttleworth, 1997) and hyper-

trophic chondrocytes (type X; Schmid and Conrad, 1982).

We have previously shown by immunohistochemical

examination that OMP-1 and Otolin-1 proteins are co-

localized in otoliths of embryonic and adult rainbow trout

inner ears (Murayama et al., 2004). In addition, Otolin-1

was localized in fibrous material connecting otolith

primordia to the sensory epithelium, likely to be the otolith

membrane (Murayama et al., 2004). In the sacculus of the

adult inner ear, OMP-1 is synthesized by most epithelial

cells outside the sensory maculae, while Otolin-1 protein is

found in a restricted group of cylindrical cells located next

to the marginal zone of the sensory epithelium (Murayama

et al., 2004).

Here, in order to investigate the roles of OMP-1 and

Otolin-1 in otolith formation, we identified the homologous

genes from zebrafish and analyzed the knockdown pheno-

types in zebrafish embryos, using antisense morpholino

oligonucleotides (MO).

2. Results and discussion

2.1. Isolation of zebrafish cDNA homologs of omp-1

and otolin-1

In order to isolate zebrafish homologs of omp-1

(Murayama et al., 2000) and otolin-1 (Murayama et al.,

2002), we performed PCR using degenerate primers on

zebrafish inner ear cDNA (see Section 3). The PCR

produced cDNA fragments of approximately 400 bp for

zebrafish omp-1 (zomp-1) and 200 bp for zebrafish otolin-1

(zotolin-1), respectively. Sequence analyses revealed that

the predicted amino acid sequences had high degrees of

sequence identity with the rainbow trout OMP-1 (81.6%)

and the chum salmon Otolin-1 (84%), respectively. To

obtain the 5 0 end of each cDNA, we performed RACE

reactions and obtained additional sequence including a

translation initiation codon. The remaining sequences 3 0 to

the cloned cDNA fragments and the gene structures were

deduced using the zebrafish genome sequence (available at

http://www.ensembl.org/Danio_rerio/) and a GENSCAN

program (Burge and Karlin, 1997) (Fig. 1).

The deduced amino acid sequence of zebrafish OMP-1

(zOMP-1) shows 84% overall similarity with rainbow trout

OMP-1 (rtOMP-1, Fig. 1A). All the cysteine residues and N-

linked glycosylation sites are conserved between zOMP-1

and rtOMP-1. In addition, both proteins also show about

50% similarity to the C-terminal lobe (C-lobe) of human

melanotransferrin (hMTf, Fig. 1A). In particular, 12 out of

15 cysteine residues are conserved between zOMP-1 and the

C-lobe of hMTf, suggesting that the two proteins may

have similar folding and tertiary structure. It is known that

Page 3: Otolith matrix proteins OMP-1 and Otolin-1 are necessary for normal otolith growth and their correct anchoring onto the sensory maculae

Fig. 1. Schematic representation of zOMP-1 and zOtolin-1 and their similarity to other known proteins. (A) Exon/Intron organization of the zebrafish omp-1

gene with the corresponding protein domain structure of zebrafish OMP-1 (zOMP-1), rainbow trout OMP-1 (rtOMP-1) and human melanotransferrin (hMTf);

white boxes, cDNA sequence determined in this study; light gray, sequence from zebrafish genome data. Inside the boxes depicting protein structure: green,

signal peptide; blue and blue-outlined bars, conserved and non-conserved amino acid residues involved in iron binding, respectively; red bars, potential N-

linked glycosylation sites; yellow bars, zinc binding consensus sequences. The similarities of rtOMP-1 and the C-lobe of hMTf (bolded) with zOMP-1 are

indicated by percentage. (B) Genome structure of the zebrafish otolin-1 gene, and the corresponding domain structure of zebrafish Otolin-1 (zOtolin-1), chum

salmon Otolin-1 (csOtolin-1), bluegill sunfish saccular collagen (bsSC), human C1q (hC1q), human type VIII (hCOL VIII) and X (hCOL X) collagens. White

boxes, cDNA sequence determined in this study; light gray, sequence from zebrafish genome data. Inside the boxes depicting protein structure: green,

signal peptide; bolded, collagenous domain (COL). Red bars indicate potential N-linked glycosylation sites. The similarity of all these proteins to zOtolin-1 in

the C-terminal non-collagenous (C-NC) domain is indicated by percentage. N-NC, N-terminal non-collagenous domain. (C) Multiple sequence alignment of

the C-NC domains. Identical and similar amino acid residues are shown in blue and gray bold types, respectively. The amino acid residues involved in calcium

binding are shown in red.

E. Murayama et al. / Mechanisms of Development 122 (2005) 791–803 793

the C-lobe of hMTf is unable to bind iron because of

changes in two of five iron-binding residues relative to

transferrin (Baker et al., 1992). zOMP-1 and rtOMP-1

conserve only one of these amino acid residues, indicating

that these molecules play a role other than iron transport

(Fig. 1A). On the other hand, both zOMP-1 and rtOMP-1

contain the consensus sequence for zinc binding, HEXXH,

found in many zinc-carrying metalloproteases such as

thermolysin (Jongeneel et al., 1989) (Fig. 1A).

The predicted amino acid sequence of zebrafish Otolin-

1 (zOtolin-1) shows 84% overall similarity with that of

chum salmon Otolin-1 (csOtolin-1; Murayama et al.,

2002) and 86% with that of bluegill sunfish saccular

collagen (bsSC; Davis et al., 1995), a probable homolog

of Otolin-1 in this species (Fig. 1B). Two potential

N-glycosylation sites are conserved among zOtolin-1,

csOtolin-1 and bsSC. These molecules belong to a family

of collagenous proteins that contains collagens type VIII

and X (Muragaki et al., 1991; Apte et al., 1991; Thomas

et al., 1991) and C1q (Reid, 1985). The N-terminal non-

collagenous (N-NC) domain is not significantly similar

among these proteins, neither among zOtolin-1, csOtolin-1

and bsSC, nor to other proteins thus far identified. In

contrast, their C-terminal non-collagenous (C-NC)

domains share about 60% similarity. Type VIII and X

collagens are non-fibrillar short chain collagens found in

Page 4: Otolith matrix proteins OMP-1 and Otolin-1 are necessary for normal otolith growth and their correct anchoring onto the sensory maculae

E. Murayama et al. / Mechanisms of Development 122 (2005) 791–803794

basement membranes (type VIII; Kapoor et al., 1988) and

hypertrophic chondrocytes (type X; Schmid and Conrad,

1982). Their N-NC and C-NC domains form nodes, which

are important for the helical chain association (Chan et al.,

1995), while the collagenous (COL) domain forms the

interconnecting spacers. These molecules oligomerize

supramolecularly into a three-dimensional, hexagonally

arranged lattice (Sawada et al., 1990; Kwan et al., 1991).

The size of the collagenous domain of zOtolin-1 is about

half of that of type VIII and X collagens (Fig. 1B),

suggesting that the lattice size is smaller in the otolith than

in the basement membranes and the hypertrophic

chondrocytes.

Type X collagen is believed to provide a temporal

pericellular matrix during endochondral ossification, in

which cartilage is replaced by trabecular bone (Chan and

Jacenko, 1998). Although type X collagen is found at later

stages only in the context of fracture repair and osteoarthritis

(Grant et al., 1987; von der Mark et al., 1992), previous

reports indicate that type X collagen plays a role in

calcification at least at the level of cartilage-bone conver-

sion (Chan and Jacenko, 1998; Sutmuller et al., 1997).

Another link to calcification is the calcium binding property

of type X collagen (von der Mark et al., 1992; Kirsch and

von der Mark, 1991; Bogin et al., 2002). The amino acid

Fig. 2. Developmental expression of zebrafish omp-1 (zomp-1) and otolin-1 (zoto

zomp-1 transcripts are detected only in the otic vesicles (arrowheads). (C) Magnifi

ventro-posterior part (arrowhead). (D) zomp-1 in situ hybridization at 72 hpf. The e

and pm, outlined by dotted lines) than in the non-sensory parts of the epithelium (

vesicle of a 26-somite embryo labeled with an anti-OMP-1 antibody. Both otoliths

anti-OMP-1 antibody. The ventro-lateral epithelium posterior to the anterior macul

three increasingly deeper focal planes of the same ear; zotolin-1 transcripts are not

and ventral sides of the latter (I, arrowheads); am, anterior macula; pm, posteri

juxtaposed for focusing the two otoliths. All panels show lateral views, anterior to t

residues involved in Ca-binding are conserved between

zOtolin-1 and type X collagen (Fig. 1C), suggesting that

zOtolin-1 may act as a mediator for the interaction of

organic matrix with inorganic materials in otolith formation.

Interestingly, like type X collagen, MTf is also expressed in

chondrocytes in mouse and rabbit (Kawamoto et al., 1998;

Nakamasu et al., 1999). It is notable that the two proteins,

respectively, related to OMP-1 and Otolin-1 share a cellular

source and potential function in chondrogenesis.

2.2. Expression of zomp-1 and zotolin-1

We examined the distribution of zomp-1 and zotolin-1

transcripts in developing zebrafish embryos. zomp-1 mRNA

was first detected at the 14-somite stage (16 hpf) in the otic

placode (Fig. 2A), which forms at about that stage (Haddon

and Lewis, 1996). At 24 hpf, zomp-1 mRNA was detected

throughout the otic epithelium (Fig. 2B) with more intense

expression in the ventro-posterior side (Fig. 2C). At 72 hpf,

zomp-1 expression was most intense in a region outside the

maculae (Fig. 2D, arrowheads). zomp-1 transcripts were

never detected in any other tissues through the stages

examined in this study. In contrast to the intense expression

of zomp-1 mRNA from the onset of otocyst development,

the zOMP-1 protein was first immunodetected at

lin-1). zomp-1 in situ hybridization at 14-somite stage (A) and 24 hpf (B).

cation of the otic vesicle at 24 hpf. zomp-1 expression is more intense in the

xpression level of zomp-1 is lower at the anterior and posterior maculae (am

arrowheads). A dotted line indicates the outline of the otic vesicle. (E) Otic

are slightly labeled. (F) At 35 hpf, both otoliths are heavily stained with the

a is also labeled (arrowhead). (G–I) zotolin-1 in situ hybridization at 72 hpf;

detected in the anterior (G) nor in the posterior macula (H), but at the dorsal

or macula. In panels (E) and (F), two photos at different focal planes are

he left, dorsal to the top. Scale bar in (C) indicates 25 mm in panels (C) to (I).

Page 5: Otolith matrix proteins OMP-1 and Otolin-1 are necessary for normal otolith growth and their correct anchoring onto the sensory maculae

E. Murayama et al. / Mechanisms of Development 122 (2005) 791–803 795

the 26-somite stage (22 hpf) in both anterior and posterior

otoliths (Fig. 2E), suggesting that the translation of the

zomp-1 mRNA is blocked until the 26-somite stage. At 27

and 35 hpf, the otoliths were intensely labeled with the anti-

OMP-1 antibody, and the ventro-lateral part of the

epithelium just posterior to the anterior macula was also

labeled (Figs. 5B,2F). The absence of zOMP-1 immunola-

beling of the precursor particles and nascent otoliths before

the 26-somite stage suggests that zOMP-1 is mainly

involved not in otolith seeding, but in the subsequent

phase of otolith growth from solute material, which begins

by that stage (data not shown).

In contrast to zomp-1, zotolin-1 mRNA was first detected

faintly at 48 hpf in a restricted, postero-medial part of the

otic vesicle (data not shown), and became clear by 72 hpf,

although still at a low level, essentially at the ventral and

dorsal edges of the posterior macula (Fig. 2G,H,I). The

zOtolin-1 protein was immunodetected in the otoliths at the

same stage (Fig. 5F). This result is reminiscent of our

previous immunohistochemical study using chum salmon,

in which Otolin-1 expression was only detected in

transitional epithelial cells just adjacent to the sensory

epithelium (Murayama et al., 2002). Like zomp-1

expression, zotolin-1 expression was never observed in

any other tissues through the stages examined in this study.

2.3. zomp-1 and zotolin-1 knockdown phenotypes

To probe the function of zOMP-1 and zOtolin-1, we used

morpholino antisense oligonucleotides (MO) to block

translation of their mRNA in zebrafish embryos. Injection

of zomp-1 MO or zotolin-1 MO into 1–2 cell embryos

reproducibly induced phenotypes of abnormal otolith

morphology. Otolith seeding appeared normal in both

zomp-1 and zotolin-1 MO-injected embryos (Fig. 3A–C).

The kinocilia of the tether cells were formed normally in all

morphants and the nascent otoliths formed at their tips (Fig.

3D–F), indicating that the otolith seeding phase was not

affected by the absence of zOMP-1 or zOtolin-1. This is

consistent with our previous study on the rainbow trout,

which revealed that OMP-1 and Otolin-1 were not present in

the core of the embryonic otoliths (Murayama et al., 2004).

zomp-1 MO-injected embryos displayed a phenotype

marked mainly by the smaller size of both otoliths

(‘small’ phenotype). The difference with control embryos

increased with time, indicating a reduced otolith growth rate

(Fig. 3I,M,Q). This phenotype was observed in both otic

vesicles of each embryo (Table 1).

We examined this otolith growth defect more closely by

measuring the longest linear dimension of the otoliths

(otolith length) in the live embryos at several stages of their

development (Fig. 4 and supplemental Table S1). In control

embryos, the anterior and posterior otoliths grew at the same

rate until 35 hpf. After this stage, the growth rate of the

anterior otolith became much slower than that of the

posterior otolith (Fig. 4A). In zomp-1 MO-injected embryos,

the growth of both otoliths proceeded slowly in contrast to

control embryos (Fig. 4B). At the 28-somite stage, the

otolith length was only 6% smaller than in controls for

the anterior (P!0.05) and 14% smaller for the posterior

(P!0.001) otolith. This relative difference increased to

26% (P!0.001) for the anterior, and 44% (P!0.001) for

the posterior otolith at 72 hpf (Table S1). In terms of otolith

volume, this translates into a 4-fold lower growth rate for

the anterior, and a 20-fold lower growth rate for the

posterior otolith between 35 and 72 hpf.

Injection of zotolin-1 MO mainly resulted in a fusion of

the two otoliths. Typically, at 4 ng of injected MO, the two

otoliths became closer to each other by 35 hpf (Fig. 3J),

came into contact by 55 hpf (‘contact’ phenotype, Fig. 3N),

and then were fused by 72 hpf (‘fused’ phenotype, Fig. 3R).

On an average, this progression towards fusion occurred

faster upon increasing the MO dose (between 1 and 8 ng).

Thus, at 72 hpf, 53.5% of embryos injected with 4 ng MO

displayed fused otoliths and 21.7% showed coalescent

otoliths, while only 28.7% of those injected with 1 ng MO

were already fused, and 33.9% still contact (Table 1).

Whatever the MO dose and developmental time, the stage in

this phenotypic progression was always similar in both otic

vesicles of each embryo, strengthening the conclusion that

variations in the timing of this phenotypic progression

reflected variations in the actual MO dose present in the

embryo’s cells. In zotolin-1 MO-injected embryos, the

posterior otolith was also significantly smaller than in the

controls, mainly due to a slower growth rate between 35 and

55 hpf (Fig. 4A,C and Table S1).

In both zomp-1 and zotolin-1 MO-injected embryos,

beside the otolith phenotypes, the development of support-

ing cells and hair cells appeared normal through the stages

examined, both in the maculae (Fig. 3G,K) and cristae (data

not shown). In contrast, the otic vesicle of both zomp-1 MO

and zotolin-1 MO-injected embryos appeared slightly

smaller, and rounder with time, than that of control

embryos, and the semicircular canals did not form; an

incomplete protrusion of anterior and posterior canals was

present by 72 hpf, while the lateral protrusion did not occur,

even past 5 dpf (data not shown). No abnormalities were

found in zomp-1 MO and zotolin-1 MO-injected embryos

outside the otocysts.

2.4. Immunostaining of the otoliths in control and MO-

injected embryos

To examine the localization of zOMP-1 and zOtolin-1 in

the otoliths of control embryos, and the extent of

suppression of zomp-1 and zotolin-1 expression by the

MOs, we stained the injected embryos with antibodies

previously raised against recombinant rtOMP-1 and csOto-

lin-1 (Murayama et al., 2004). In wild type embryos, the

anti-OMP-1 antibody first labeled both otoliths at the 26-

somite stage (22 hpf, Fig. 2E), and then more strongly at

27 hpf (Fig. 5B) and 35 hpf (Fig. 2F). It is notable that the

Page 6: Otolith matrix proteins OMP-1 and Otolin-1 are necessary for normal otolith growth and their correct anchoring onto the sensory maculae

Fig. 3. Otolith phenotypes observed in live control, zomp-1 and zotolin-1 MO-injected embryos by DIC videomicroscopy. Embryos were injected with 4 ng

MO. (A–C) 28-somite stage; otolith seeding occurs normally in control MO (A), zomp-1 MO (B) and zotolin-1 MO (C) injected embryos. (D–F) Magnification

of the anterior otolith and two tethering kinocilia shown in (A–C). (G–K) At 35 hpf, the otoliths of zomp-1 MO (I) and zotolin-1 MO (J) injected embryos are

slightly smaller, and closer to each other, respectively, than those of control MO-injected embryos (H); the anterior otolith of zotolin-1 MO-injected embryos

does not stay in contact with the anterior macula (arrowhead in J); (G, K) Anterior macula, otolith and tethering kinocilia of the zomp-1 MO ear shown in (I) and

zotolin-1 MO ear shown in (J). (L–N) At 55 hpf, zomp-1 MO and zotolin-1 MO injection cause a ‘small otoliths’ phenotype (M) and a ‘fused otoliths’

phenotype (N), respectively. (O) Magnification of the cilia of the anterior macula shown in (N). At 72 hpf (P–S), the otoliths of control MO (P) and zotolin-1

MO (R) injected embryos keep growing, especially the posterior otolith, while both otoliths of zomp-1 MO-injected embryos stay small (Q). (S) Magnification

of the cilia of the anterior macula shown in (R). In panels (A–C), (H), (I), (L), (M), (P) and (Q), the pictures of posterior otoliths are inlayed, from a deeper focal

plane, onto the main pictures, which are focused on the anterior otoliths. Through the panels, anterior is to the left, dorsal to the top. Scale bar in (A) indicates

25 mm in (A–C), in (D) indicates 10 mm in (D–F), in (H) indicates 25 mm in (H–J, L–N, P–R), in (G) indicates 10 mm in (G), (K), (O) and (S).

E. Murayama et al. / Mechanisms of Development 122 (2005) 791–803796

size of the otoliths observed at these stages by immunos-

taining of fixed embryos was larger than observed in live

embryos (Fig. 5A,B). At this stage, Otolin-1 was not

detected in the otoliths yet (Fig. 5C). At 72 hpf, the otoliths

of control MO-injected embryos (Fig. 5D) were intensely

immunostained with the anti-OMP-1 antibody (Fig. 5E).

They were also stained by the anti-Otolin-1 antibody,

although less strongly (Fig. 5F). In zomp-1 MO-injected

embryos (Fig. 5G), both otoliths were immunonegative with

the anti-OMP-1 antibody (Fig. 5H), demonstrating that the

zomp-1 MO effectively suppressed OMP-1 expression.

Similarly, the otoliths of zotolin-1 MO-injected embryos

Page 7: Otolith matrix proteins OMP-1 and Otolin-1 are necessary for normal otolith growth and their correct anchoring onto the sensory maculae

Table 1

Dose dependence of otolith phenotypes at 72 hpf

Dose of

MO

injected

Otolith phenotypes of normal embryos (%) Curved

body axis

(%)Normal Fusion Contact Small

Control MO

1 ng 96.8G0.6 0 0 1.9G0.8 1.4G0.2

4 ng 95.9G0.1 0 0 2.1G0.3 1.7G0.4

zomp-1 MO

1 ng 41.2G5.6 0 0 56.8G5.4 2.1G0.2

4 ng 20.9G1.1 0 7.2G3.3 68.1G3.7 3.9G1.2

8 ng 10.2G2.3 0 5.9G1.8 73.9G1.2 10.1G1.7

zotolin-1 MO

1 ng 24.6G8.8 28.7G2.5 33.9G3.2 5.8G2.1 7.2G1.1

4 ng 7.2G0.8 53.5G2.7 21.7G2.6 2.2G0.4 15.6G1.1

8 ng 3.2G0.8 64.1G6 14.8G0.6 3.15G0.8 16.5G4.2

The numbers shown are the averages of two independent experiments. In

each experiment, at least 50 embryos were phenotyped for each dose of

MO. Otolith phenotypes were always similar in both ears. About 5% of the

embryos had an additional otolith in one ear, irrespective of MO nature and

dose.

E. Murayama et al. / Mechanisms of Development 122 (2005) 791–803 797

were immunonegative with the anti-Otolin-1 antibody

(Fig. 5K). Surprisingly, the anti-Otolin-1 antibody also

failed to stain the otoliths of zomp-1 MO-injected embryos

(Fig. 5I). This result suggests that the presence of zOMP-1

in the growing otolith is a prerequisite for the deposition of

zOtolin-1. Our previous work showed that Otolin-1 and

OMP-1 were co-localized in the otoliths of adult rainbow

trout (Murayama et al., 2004), and that purified rainbow

trout OMP-1 and Otolin-1 proteins interacted with each

other in vitro (Murayama, 2002b). These earlier obser-

vations support the present results. In contrast, the fused

otoliths of zotolin-1 MO-injected embryos (Fig. 5J) were

strongly immunolabeled by the anti-OMP-1 antibody at

72 hpf (Fig. 5L). Intriguingly, the immunostained otoliths

appeared substantially larger than in the live embryos

(Fig. 5J,L), just as we had found before with control

embryos of 22–35 hpf (Figs. 2E,F,5A,B). In other words,

the otolith matrix appeared to swell during the immunos-

taining procedure only when zOtolin-1 was absent, either

because not present yet in the otoliths (22–35 hpf embryos)

or because suppressed by the zotolin-1 MO (such swelling

did not occur, e.g. in 72 hpf control embryos, Fig. 5D–F).

Fig. 4. Otolith growth rates in control, zomp-1 and zotolin-1 MO-injected embryos

of anterior (blue squares) or posterior (orange circles) otoliths measured at various

each viewed laterally. Error bars show SD. The numbers are given in Table S1.

Correlatively, the zOMP-1 immunostaining was always

more intense in these swelled otoliths (Figs. 2F,5B,L),

which may be due to increased accessibility of zOMP-1

epitopes within the swollen organic matrix. This obser-

vation suggests that the presence of zOtolin-1 may stabilize

the otolith organic matrix in a compact form. Another

observation suggests that it may also stabilize its association

with the mineral component. Upon fixation, otoliths are

known to decalcify spontaneously with time. We observed

that this process occurs clearly faster in otoliths of zotolin-1

MO-injected embryos than in control embryos (all fixed at

7 dpf; see supplemental Fig. S1). These various obser-

vations suggest that the incorporation of the collagenous

protein zOtolin-1 in the otoliths may stabilize both the

mineral and the organic matrix components of the otoliths.

In trout otoliths, we previously found Otolin-1 and OMP-

1 co-localized in the daily organic increment layers

(Murayama et al., 2004). In zebrafish embryos, the adult

mode of otolith growth by alternate daily increments

appears to start by 3 dpf (J.Y. Sire, personal communi-

cation), i.e. about when we start to detect Otolin-1 clearly in

the otoliths. So we suggest that the switch to this growth

mode requires the participation of zOtolin-1, possibly

forming a 3D-lattice similar to what the related proteins

collagen VIII and X do, and that the resulting layered

structure brings increased stability to the otolith.

2.5. Relationship of the fused otoliths to their respective

sensory maculae in zotolin-1 MO-injected embryos

The ‘fused otoliths’ phenotype observed in vivo with the

zotolin-1 MO suggests that the otolith matrix has self-

aggregation property, causing the fusion of otoliths when

they are in contact with each other. This recalls a common

observation done on wild type embryos. A fraction of

them (about 5% in the stocks used for the present study—see

Table 1—but it can be quite higher in occasional stocks)

initially make one additional otolith; in most cases, this

additional otolith fuses sooner or later with the anterior or the

posterior otolith (the closer one; when this additional otolith

is unanchored, it most often fuses with the posterior otolith).

More intriguing here is the progressive displacement of

. Each point represents the mean of longest linear dimension (otolith length)

stages of development. Size measurements were made from 15 specimens

Page 8: Otolith matrix proteins OMP-1 and Otolin-1 are necessary for normal otolith growth and their correct anchoring onto the sensory maculae

Fig. 5. Whole-mount immunostaining of the otoliths of wild type and MO-injected embryos. (A, D, G, J) Live embryos; all other panels show fixed,

immunostained embryos. (A–C) Wild-type embryos at 27 hpf; (B) zOMP-1 is detected in both otoliths and in the ventro-lateral epithelium posterior to the

anterior macula (arrowheads), but zOtolin-1 is not observed yet (C). (D–O) Embryos at 72 hpf. In control MO-injected embryos, anti-OMP-1 (E) and anti-

Otolin-1 (F) antibodies recognize both otoliths. (G–I) zomp-1 MO-injected embryos; zOMP-1 is effectively knocked down (H); the anti-Otolin-1 antibody fails

to detect zOtolin-1 in the small otoliths (I). (J–L) zotolin-1 MO-injected embryos; zOtolin-1 is not detected (K) while the anti-OMP-1 antibody reacts strongly

with the fused otoliths (L). (M–O) Pre-immune serum shows non-specific staining (arrowheads) of the sensory hair cells of the anterior macula (M), posterior

macula (N) and anterior and lateral cristae (O). In panels (A–G), the posterior otoliths were inlayed from a deeper focal plane onto the main pictures, which are

focused on the anterior otoliths. Dotted lines outline otoliths in panels (H), (I) and (K) and the posterior macula in (N). Through the panels, anterior is to the left,

dorsal to the top. Scale bar in (A) and (O) indicates 25 mm in (A–N) and (O), respectively.

E. Murayama et al. / Mechanisms of Development 122 (2005) 791–803798

the anterior and posterior otoliths of zotolin-1 MO-injected

embryos that bring them into contact with each other.

Initially, they nucleate normally on the tips of the kinocilia of

the tether cells (Fig. 3F), which lie in the middle of the

prospective maculae. However, by 35 hpf, the anterior

otolith has already moved somewhat away from the anterior

macula (the position of the posterior otolith relative to its

macula is less easy to evaluate in vivo from this stage, due to

the more medial position of the latter) and by 72 hpf this

distance has increased (Fig. 3R). Yet it seems that the otolith

is still connected to the corresponding macula, the kinocilia

are longer than normal (Fig. 3K,O,S). To clarify the

topographical relationship of the fused otoliths to their

respective maculae and associated stereo- and kino-cilia in

zotolin-1 MO embryos, we fixed them at 72 hpf and

performed triple immunofluorescence confocal analysis

through the depth of the otocyst, labeling the otolith with

OMP-1 antibody, the kinocilia with anti-acetylated tubulin

Page 9: Otolith matrix proteins OMP-1 and Otolin-1 are necessary for normal otolith growth and their correct anchoring onto the sensory maculae

Fig. 6. Relationship of the fused otoliths in zotolin-1 MO-injected embryos to the corresponding sensory maculae. Triple-labeling with anti-OMP-1 (red), anti-

acetylated tubulin (blue) antibodies, and phalloidin (green). Confocal fluorescence images at 72 hpf. Phalloidin (green) reveals the hair (stereocilia) bundles of

the sensory cells and the contours of cells through their actin cortex. Acetylated tubulin (blue, false color) is found in the kinocilia and in the apical cytoplasm of

the sensory hair cells (Riley et al., 1997). (A) Anterior macula and otolith of a control MO-injected embryo, lateral view. (B–D) zotolin-1 MO-injected embryo,

with fused otoliths. (B) and (C) are the same z-stacks (3D reconstruction available upon request). (B) Anterior macula and anterior otolith with the fused

posterior otolith; note the long kinocilia (arrowhead). (C) Projection of the confocal series along the z-axis (22 images, taken every 2 mm). Both maculae are

visible through their hair bundles, in relation to both otoliths. (D) Far-red (blue-coded in A–C) fluorescence channel extracted from the image shown in (C), to

show the acetylated tubulin immunostaining of kinocilia at both maculae. ao, anterior otolith; po, posterior otolith. Dorso-lateral view, anterior to the left,

dorsal to the top. Scale bars, 20 mm.

E. Murayama et al. / Mechanisms of Development 122 (2005) 791–803 799

antibody, and the stereocilia with phalloidin (Fig. 6). This

analysis confirmed that both maculae display well-organized

stereocilia and kinocilia. It also showed that each of the two

fused otoliths remains associated with its respective macula

(Fig. 6B,C; 3D reconstruction available upon request). They

did not appear distant from the maculae as in live embryos,

possibly due to the swelling of the otolith matrix discussed

above. However, the kinocilia between the macula and the

otolith seemed particularly long (Fig. 6A,B,D), as the in vivo

observation had suggested.

In adult fish, the otoliths are bound to the sensory macula

via a gelatinous material called otolithic membrane

(Dunkelberger et al., 1980). Otolin-1 was also identified

as a component of the otolithic membrane in adult rainbow

trout (Murayama et al., 2002, 2004) and bluegill sunfish

(Davis et al., 1997), suggesting that Otolin-1 may be

involved in the positioning of the otoliths via the otolithic

membrane. It is tempting to link these data from adult fish

with the present observation that the otoliths of zotolin-1

MO-injected embryos do not stay in contact with the

corresponding maculae. The development of the otolithic

membrane has not been documented in the zebrafish (nor in

other fish to our knowledge). The fact that the otoliths are

already moving away from the maculae by 35 hpf in the

zotolin-1 MO-injected embryos would suggest that some

form of Otolin-1-containing otolithic membrane is already

present by that stage in control embryos, and that it

contributes to maintaining the otoliths close to their

respective maculae. Our failure to immunodetect such an

Otolin-1-containing structure would not be surprising given

the moderate affinity of our anti-chum salmon Otolin-1

antibody for the zebrafish protein.

2.6. Behavioral defects in the zomp-1 and zotolin-1

MO-injected embryos

Finally, we examined the behavioral changes caused by

the loss of zOMP-1 or zOtolin-1. Vestibular defects became

Page 10: Otolith matrix proteins OMP-1 and Otolin-1 are necessary for normal otolith growth and their correct anchoring onto the sensory maculae

Table 2

Behavioral defects at 4 dpf embryos injected with 4 ng MOs

MO Control MO zomp-1 MO zotolin-1 MO

Phenotype Normal otoliths Small otoliths Fused otoliths

No. of tested

animals

38 71 67

Swimming 21 (55.3%) 0 0

Laying down 17 (44.7%) 71 (100%) 67 (100%)

No reaction 0 22 (31.0%) 18 (26.8%)

Short

movements

17 (44.7%)a 22 (31.0%)b 20 (29.9%)b

Circular

movements

0 27 (38.0%) 29 (43.3%)

Stimulus: tapping the edge of the Petri dish with tweezers.a Linear movements.b Non-linear movements.

E. Murayama et al. / Mechanisms of Development 122 (2005) 791–803800

obvious after 4 dpf. At this stage, nearly half of the non-

injected wild type embryos or control MO-injected embryos

keep an upright position and swim spontaneously (Table 2).

In contrast, all zomp-1 and zotolin-1 MO-injected embryos

lay down on the bottom of the Petri dish and remained still.

When the edge of the Petri dish was tapped, the half of

control MO-injected embryos that were laying down were

very sensitive to the stimulus and started swimming in a

straight manner (Movie S1). In contrast, about 30% of

zomp-1 MO or zotolin-1 MO-injected embryos showed only

short movements in response to the stimulus, and never kept

an upright position (Movie S2). Another 40% of zomp-1 MO

or zotolin-1 MO-injected embryos exhibited circular move-

ments (Movie S3). The remaining 30% of zomp-1 MO or

zotolin-1 MO-injected embryos did not respond to the

tapping stimulus (Table 2), whereas they did show an escape

response upon stimulation by touch with a tweezer tip. Most

otolith mutants (Whitfield et al., 1996) and some sensory

hair cell mutants (Nicolson et al., 1998) show similar

phenotypes, such as circular movement including looping

and/or rolling motion. The normal development of vestib-

ular function in zebrafish larvae is dependent on stimulus by

otolith weight (Moorman et al., 1999). Since the develop-

ment of the sensory maculae appears normal in zomp-1 MO

and zotolin-1 MO-injected embryos, the observed beha-

vioral defects are most likely caused by the lack or reduction

of hair cell stimulation due to the malformation or

malpositioning of the otoliths.

This work addressed the molecular mechanisms of

otolith formation through the roles of two otolith matrix

proteins, zOMP-1 and zOtolin-1. zOMP-1 is especially

involved in otolith growth and appears to be required for the

deposition of zOtolin-1. In contrast, zOtolin-1 is required

for the correct anchoring of the otoliths on the sensory

maculae, and as a component of the otolith it seems to

stabilize the otolith matrix from about 72 hpf, presumably

by providing a collagenous scaffold. Thus, so far, three

proteins, OMP-1 (Murayama et al., 2000), Otolin-1

(Murayama et al., 2002) and Starmaker (Sollner et al.,

2003), are known to be components of fish otoliths. Future

analyses of the interaction of these proteins with each other

and with calcium carbonate should contribute further

insights into the process of biomineralization. In addition,

comprehensive studies of other components contained in the

otolith, such as minor components of the protein matrix,

proteoglycans (Borelli et al., 2003) and polysaccharides

(Pisam et al., 2002), as well as of the otolithic membrane

and endolymph, will be needed for a precise understanding

of the molecular mechanism of otolith formation.

3. Experimental procedures

3.1. Fish strains and maintenance

AB and Tu wild type strains of the zebrafish, Danio rerio,

were used throughout this study. Fishes were kept under a

photoperiod of 14 h light/10 h dark at 28.5 8C. Eggs were

obtained by random crosses and kept at the same

temperature. Embryos were staged as hours post-fertiliza-

tion (hpf) at 28.5 8C and using developmental landmarks

(Kimmel et al., 1995).

3.2. Cloning of zebrafish omp-1 and otolin-1 cDNAs

A cDNA was synthesized with a SMART RACE cDNA

Amplification kit (Clontech) according to the manufac-

turer’s instruction using 1 mg of total RNA isolated from the

inner ear of adult zebrafish. To amplify the cDNA fragments

encoding zebrafish omp-1 (zomp-1) and otolin-1 (zotolin-1),

the following sets of degenerate oligonucleotide primers

were used.

zomp-1-F (5 0-GARGCIGARGARCARAARTG-3 0)

zomp-1-R (5 0-NWSICKCTCRTGCATCTC-3 0)

zotolin-1-F (5 0-TAYAAYGGCGARGGICAYTG

GGA-3 0)

zotolin-1-R (5 0-GCYTGRTCDATRTCYTGICC-3 0)

These primers were designed based on the deduced

amino acid sequence of rainbow trout OMP-1 (Murayama

et al., 2000) and chum salmon Otolin-1 (Murayama et al.,

2002), respectively. PCR reactions were carried out as

described previously (Murayama et al., 2002) using

zebrafish inner ear cDNA as a template. Briefly, the reaction

mixtures were denatured at 94 8C for 3 min followed by 40

cycles of 94 8C for 30 s, 54 8C for 30 s, and 72 8C for 30 s.

To isolate the 5 0-end regions, a 5 0 RACE reaction was

performed using gene specific primers, zomp-1-5R1 (5 0-

GTTGTCCGCGAAGGTTCCTGGCTGTGGC-3 0) and

zotolin-1-5R1 (5 0-AAGAGAGTCACGCGTTCGCAG-

TTTCCG-3 0). For zotolin-1, the PCR reaction was carried

out using NUP primer (Clontech) and zoto1-5R2 (5 0-

ACGTACAGTTATGTAGTATGAAAAGACG-3 0) as

described previously (Murayama et al., 2002). To obtain

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E. Murayama et al. / Mechanisms of Development 122 (2005) 791–803 801

the complete 5 0 end of zotolin-1 cDNA, an additional RACE

reaction was done using the following primers:

zotolin-1-5R3 (5 0-GTTCCATTCCAGCCAGGCT-

CTCCGCGC-3 0)

zotolin-1-5R4 (5 0-CGCGCTCCCCTGGGTCTC-

CTTTCAGCC-3 0)

The amplified cDNA fragments were subcloned into a

pCR2.1 TOPO vector (Invitrogen) according to the

manufacturer’s instructions. The nucleotide sequences

were determined using a Thermo Sequenase Cy5 dye

terminator cycle sequencing kit (Amarsham Biosciences).

The nucleotide sequences of zomp-1 and zotolin-1 were

submitted to DDBJ/EMBL/GenBank and have been

assigned the accession numbers AB124553 and

AB124554, respectively.

3.3. Whole-mount in situ hybridization

Whole-mount in situ hybridization was performed

according to the procedure described by Thisse et al.

(1993) with slight modifications, using the digoxygenin-

UTP labeled RNA probes of zomp-1 and zotolin-1. The

cDNA fragments of zomp-1 (nucleotides 137–436) and

zotolin-1 (nucleotides 1227–1408) were subcloned into a

pCR4-TOPO vector (Invitrogen). Both constructs were

digested with Not I and transcribed with T3 RNA

polymerase. For embryos older than 48 hpf, heads were

cut off before hybridization. Hybridization was performed

overnight at 70 8C with 150 ng DIG-labeled riboprobe in

200 ml hybridization buffer. After a series of washes with

PBT (0.1% Tween-20 in PBS), embryos were incubated

with preabsorbed anti-DIG-alkaline phosphatase Fab frag-

ments (1:4000) overnight at 4 8C.

3.4. Whole-mount immunostaining and phalloidin staining

Embryos were fixed with 4% paraformaldehyde/PBS

overnight at 4 8C. After washing with PBST (0.1% TritonX-

100 in PBS), the samples were dehydrated overnight with

methanol and stored at K20 8C. The samples were

rehydrated with PBST, the heads were cut off, then treated

with the blocking solution (5% sheep serum, 0.2% BSA in

PBST) for 2 h at room temperature. Primary and secondary

antibodies were diluted in blocking solution. Primary

antibodies were (1) anti-rtOMP-1 polyclonal antibody

(Murayama et al., 2004) at 1:1000 dilution, (2) anti-

csOtolin-1 polyclonal antibody (Murayama et al., 2004) at

1:500 dilution, or (3) anti-acetylated tubulin monoclonal

antibody (Sigma) at 1:500 dilution. The embryos were

incubated with these antibodies overnight at 4 8C. After

washing with PBST for at least 4 h, the embryos were

blocked again then incubated with the corresponding

secondary antibodies; (1 0) biotin-conjugated anti-rabbit

IgG antibody or (2 0) horseradish peroxydase linked

anti-rabbit IgG at 1:800 dilution (Amersham Biosciences).

The signals were detected with the ABC staining kit

(Vector) using 3,3 0-diaminobenzidine (Sigma) as a sub-

strate, or directly visualized with 3-amino-9-ethyl calbazole

(Sigma) as a substrate. For the confocal fluorescence

analysis, the following secondary antibodies were used:

(1 00) Cy3-conjugated anti-rabbit IgG Fab fragments at 1:800

dilution (Jackson laboratory) and (3 0) Cy5-conjugated anti-

mouse IgG Fab fragments at 1:800 dilution (Jackson

laboratory), mixed with Alexa Fluor 488-phalloidin

(1:200, Molecular Probes). They were applied overnight

in the dark at 4 8C. The embryos were thoroughly rinsed,

mounted in glycerol, and viewed under the 40! oil

objective of a Zeiss LSM 510 confocal fluorescence

microscope (Zeiss Axioskop 2FSM). Optical sections

were taken every 2 mm.

3.5. Microinjection of morpholino oligonucleotides

Morpholino oligonucleotides (MOs) were designed to

target the initiation codons of zomp-1 and zotolin-1 mRNAs.

zomp-1 MO (5 0-CAAGATGTCCTCCTGGAAGATC-

CAT-3 0)

zotolin-1 MO (5 0-TGAACGGGTGGAGAATATTGGG-

CAT-3 0)

GeneTools control MO (5 0-CCTCTTACCTCAGTTA-

CAATTATA-3 0)

These MOs were dissolved in distilled water at 10 mg/ml

and diluted to 8 or 1 mg/ml with 200 mM KCl, containing

phenol red (2.5 mg/ml) before the injection. MOs (0.5–1 nl)

were injected into the yolk of 1–2 cell stage embryos

together with rhodamine-dextran 10000 conjugate (Mol-

ecular Probes) as a fluorescent tracer to check for the

homogenous distribution of the injected material in the

resulting embryos. After injection, embryos were incubated

in Volvic water at 28.5 8C until desired developmental

stages. Embryos with non-homogenous rhodamine-dextran

distribution at 22 hpf were discarded.

3.6. Imaging and otolith measurements on live embryos

Differential Interference Contrast (DIC) video-

microscopy was performed on live embryos as described

previously (Herbomel et al., 1999). To determine the size of

otoliths in MO-injected embryos at successive develop-

mental stages, the embryos were anesthetized and then

observed and video-recorded at 28-somite stage (29 hpf),

35, 55 and 72 hpf, by DIC video-microscopy (stage 28-

somite), or under a Leica MZ16 stereomicroscope at

maximum magnification (all other stages). Otolith measure-

ments were made in a total of 15 pairs of inner ears for both

anterior and posterior otoliths. Differences between zomp-1

(or zotolin-1 MO) injected embryos and control embryos

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E. Murayama et al. / Mechanisms of Development 122 (2005) 791–803802

were tested using the Student’s t test at each developmental

stage.

3.7. Behavioral tests

The MO-injected embryos at 4 dpf were placed in a 96-

well plate (one embryo per well) and left without

stimulation for at least 30 s. After verifying their inert

status, the edge of the well was tapped with a forceps. Their

reaction was observed and video-recorded for 10 s follow-

ing stimulation.

Acknowledgements

We are grateful to Kazuki Horikawa for many valuable

discussions, and to Karima Kissa-Marin and Pascal Roux

for their help with the confocal microscopy. We also thank

the members of Unite Macrophages et Developpement de

l’Immunite for their kind support. This work was supported

by Grants-in-Aid for Creative Basic Research (#12NP0201)

and for Scientific Research (Nos. 12876025, 13660176 and

13660178) from the Ministry of Education, Culture, Sports,

Science and Technology of Japan. E. M. was supported by a

Research Fellowship of the Japan Society for the Promotion

of Science for Young Scientists.

Supplementary material

Supplementary data associated with this article can

be found, in the online version, at doi:10.1016/j.mod.2005.

03.002

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