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First published online February 13, 2009
Journal of Experimental Biology 212, 639-647 (2009)
Published by The Company of Biologists 2009
doi: 10.1242/jeb.022798
Plasma membrane calcium ATPase required for semicircular canal formation and otolith growth in the zebrafish inner ear
1 Institute of Fisheries Science, College of Life Science, National Taiwan
University, Taipei, Taiwan
2 Institute of Oceanography, College of Science, National Taiwan University,
Taipei, Taiwan
3 Institute of Biological Chemistry, Academia Sinica, Nankang, Taipei,
Taiwan
4 Institute of Cellular and Organismic Biology, Academia Sinica, Nankang,
Taipei, Taiwan
* Authors for correspondence (e-mails: jcshiao{at}ntu.edu.tw; pphwang{at}gate.sinica.edu.tw)
Accepted 24 November 2008
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Summary |
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 TOP Summary INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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Key words: labyrinth, auditory system, deafness, vestibule, calcium transport, otolith
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INTRODUCTION |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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). Otoliths of fish and otoconia of other vertebrates
function as mechanoreceptors. The inner ear is stimulated by differential
movement between the dense otolith and less dense sensory epithelium, which
causes bending of hair bundles on the epithelium. This stimulation allows the
inner ear to detect sound and motion
(Popper and Lu, 2000).
Some key proteins are known to be involved in defining the morphological
and molecular basis of the sensory organ in the inner ear
(Bryant et al., 2002). For
instance, an active calcium transporter known as plasma membrane calcium
ATPase isoform 2 (ATP2B2) is of physiological importance in the development of
the inner ear in mammals (Kozel et al.,
1998). ATP2B2 was suggested to be the major isoform in the
mammalian inner ear based on high and restricted expression in several cell
types, such as cochlear out hair cells and spiral ganglion cells
(Kozel et al., 1998). Thus
ATP2B2 is considered to be of specialized physiological importance in auditory
systems compared to other isoforms (Furuta
et al., 1998). In addition, Stauffer et al.
(Stauffer et al., 1995) showed
that ATP2B1 and four isoforms in mammals are the house-keeping genes,
responsible for cellular homeostasis. However, little is known about the
physiological role of Atp2b2 and other isoforms in the development of teleost
inner ear (Shull, 2000).
Teleost sagittal otoliths are mostly composed of the aragonite form of
calcium carbonate. A composition of 99.8% CaCO3 in the otolith of
adult trout (Borelli et al.,
2001) illustrates the high involvement of calcium for otolith
daily growth. Driven by thermodynamic controls, aqueous calcium
(Ca2+) and carbonate ions (CO 2–3) in
the endolymphatic fluid bind together to form calcium carbonate, which is
deposited on the otolith (Romanek and
Gauldie, 1996). Proteins extracted from tilapia (Oreochromis
niloticus) otolith chambers reveal high calcium-binding capacity, which
promotes otolith calcification (Sasagawa
and Mugiya, 1996).
Otoliths grow continually throughout the life of a fish
(Campana and Neilson, 1985),
but the otoconia size in other vertebrates is fixed after the individual
matures. This suggests a higher and ongoing requirement for calcium transport
to the inner ear of fish in comparison to mammals. Based on the study of
calcium transport by Mugiya and Yoshida
(Mugiya and Yoshida, 1995),
cytosolic calcium is extruded to the apical side of saccular cells proximal to
the otolith by active intracellular pathways. Using mannitol, a marker for
paracellular ion traffic, Mugiya and Yoshida
(Mugiya and Yoshida, 1995)
suggested that intercellular junctions were too tight to allow the marker to
penetrate into the endolymph, thus negating the possibility of a paracellular
pathway. Conversely, Payan et al. (Payan
et al., 2002) revealed a linear endolymph
45Ca2+ gradient by perfusion proximal to the saccular
epithelium in trout, suggesting passive diffusion through a paracellular
pathway. In addition, Ibsch et al. (Ibsch
et al., 2004) found pronounced calcium precipitates at the
proximal surface of the otolith between the sensory epithelium and the
otolith, and suggested that calcium incorporation takes place in this region.
A paracellular calcium pathway was also suggested based on the observation of
calcium precipitates at macular junctions
(Ibsch et al., 2004). These
contradictory findings were based on kinetic, pharmacological and histological
techniques. To date, there are no molecular, biochemical or cellular data to
support any of these calcium transport models for the inner ear of fish.
It is believed that active and transcellular calcium transport in fish
gills is mediated by the epithelial calcium channel at the apical side and a
plasma membrane calcium ATPase (PMCA) and/or a sodium–calcium exchangers
(NCX) at the basolateral side (Hwang and
Lee, 2007). However, the role of active calcium transport in the
teleost inner ear is largely unknown. Six PMCA isoforms and seven NCX isoforms
of zebrafish were identified in our recent study
(Liao et al., 2007) and only
one PMCA isoform (atp2b1a) was expressed in the inner ear of larval
zebrafish (Liao et al., 2007).
This observation indicated that Atp2b1a isoform might be involved in the
development of the zebrafish inner ear. In the present study, the role of
Atp2b1a isoform was elucidated in zebrafish in order to clarify the role of
calcium transport in teleost inner ear development.
Abundant bioinformation and genetic tools are available for zebrafish,
facilitating the study of inner ear development in this species. The optical
clarity of larval zebrafish enables the direct observation of otic defects
induced by experimental manipulations during the early stages of development
(Whitfield et al., 2002).
Increasing knowledge of otolith formation and inner ear development in fish
may enhance our understanding of diseases and disorders affecting hearing and
balance in other vertebrates, including humans.
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MATERIALS AND METHODS |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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) to dissect and obtain adult inner ear tissue. Inner ear
total RNA was extracted from 30–50 adults with TRIZOL reagent
(Invitrogen, Carlsbad, CA, USA), following the standard protocol, and was used
for cDNA synthesis using the Invitrogen Superscript III (SSIII) kit.
RNA probe synthesis
The full-length atp2b1a cDNA was obtained by PCR (forward
5'-GGCTAACAACTCATACAGCGGGG-3' and reverse
5'-GGCGTGGTCAATTTCATCAAGGT-3') amplification and inserted into the
pGEM-T easy vector (Promega, Madison, WI, USA) for the synthesis of antisense
and sense RNA probes. Purified plasmids were then linearized by restriction
enzyme digestion, and in vitro transcription was performed with T7
and SP6 RNA polymerase (Roche, Penzberg, Germany), respectively, in the
presence of digoxigenin (dig)-UTP. Dig-labeled RNA probes were examined with
RNA gels and dot-blot assays to confirm quality and concentration. For
dot-blot assays, synthesized probes and standard RNA probes were spotted on
nitrocellulose membrane according to the manufacturer's instructions. After
cross-linking and blocking, the membrane was incubated with an alkaline
phosphatase-conjugated anti-dig antibody and stained with nitro blue
tetrazolium (NBT) and 5-bromo-4-chloro-3-indolyl phosphate (BCIP).
In situ hybridization
In situ hybridization was performed on whole larvae and on the
lagenar chamber of adult zebrafish as previously described
(Pan et al., 2005). Fish were
anesthetized with buffered MS-222 before dissection and fixation. Zebrafish
embryos of each stage and lagenar chambers dissected from adult zebrafish were
fixed with 4% paraformaldehyde overnight, and then washed several times with
phosphate-buffered saline (PBS). Fixed samples were rinsed with PBST (0.2%
Tween 20, 1.4 mmol l–1 NaCl, 0.2 mmol l–1
KCl, 0.1 mmol l–1 Na2HPO4, 0.002 mmol
l–1 KH2PO4; pH7.4), and incubated with
hybridization buffer (HyB) containing 60% formamide, 5xSSC, and 0.1%
Tween 20 for 5 min at 65°C. Prehybridization was performed in
HyB+, which is the hybridization buffer supplemented with 500 µg
ml–1 yeast tRNA and 25 µgml–1 heparin for
2 h at 65°C. After prehybridization, samples were incubated in 100 ng of
the RNA probe in 300µl of HyB+ at 65°C overnight for
hybridization. Samples were then washed at 65°C for 10 min in 75% HyB and
25% 2xSSC, 10 min in 50% HyB and 50% 2xSSC, 10 min in 25% HyB and
75% 2xSSC, 10 min in 2xSSC, and twice for 30 min in 0.2xSSC
at 70°C. Further washes were performed at room temperature for 5 min in
75% 0.2xSSC and 25% PBST, 5 min in 50% 0.2xSSC and 50% PBST, 5 min
in 25% 0.2xSSC and 75% PBST, and 5 min in PBST. After serial washings,
samples were incubated in blocking solution containing 2% sheep serum and 2 mg
ml–1 BSA in PBST for 2 h and then incubated in 1:10,000
alkaline phosphatase-conjugated anti-dig antibody in blocking solution at
4°C overnight. After incubation, samples were washed with PBST and
transferred to the staining buffer. The staining reaction was held with NBT
and BCIP in staining buffer until the signal was sufficiently strong. The
staining reaction was terminated by several washings in DEPC-PBST. Then the
samples were fixed with 4% paraformaldehyde for 20 min and washed twice with
PBST for 5 min each before storage at 4°C in a dark box.
Zebrafish atp2b1a knockdown by morpholino oligonucleotide
An antisense morpholino oligonucleotide (MO)
5'-GCTGTATGAGTTGTTAGCCATGTC-3' (nucleotides –3 to 22) was
designed by Gene Tools (Philomath, OR, USA) and directed against the
atp2b1a start codon to block protein translation. The same morpholino
with five nucleotides changed (mismatched MO) was used for the control group
(5'-GgTGaATGAcTTGTTAGCgATcTC-3'). To obtain a large morphant
sample size, we injected the MO (resuspended in 1x Danieau buffer: 58
mmol l–1 NaCl, 0.7 mmol l–1 KCl, 0.4 mmol
l–1 MgSO4, 0.6 mmol l–1
Ca(NO3)2, 5 mmol l–1 Hepes, pH7.6) into
embryos at the one- to four-cell stage. Dosage of 6 ng was found (range:
2–12 ng) to be the most efficient and caused less non-specific
abnormality in preliminary experiments. All the embryos were incubated under
the constant temperature of 29°C.
To further confirm the specificity of antisense MO, we inserted the atp2b1a partial sequence spanning the target site of antisense MO sequence into a pCS2 vector with a green fluorescent protein (GFP) construct. The pCS2_atp2b1a:GFP was sequenced to confirm the construct. Using the construct, synthesized capped mRNAs (cRNAs) by SP6 mMessage mMachine kit (Ambion, Austin, TX, USA) were generated. Capped mRNAs at 300 pg per embryo were co-injected with or without the antisense MO (6 ng) at one- to two-cell stages. Then, the embryos were incubated at 29°C for further observations.
Morphological development, including otolith and semicircular canal formation, was observed from the first 24 h up to 7 days post-fertilization (dpf) using a stereomicroscope (Olympus SZX 12, Tokyo, Japan) and a compound light microscope (Zeiss Axioplan 2, Oberkochen, Germany). Photographs of the sagittal otoliths of 80 morphants (injected with 6 or 12 ng MO) and 40 wild-type fish were taken at 2–7 dpf. The maximal diameters of sagittal otoliths were measured using the software Image-Pro plus (Media Cybernetics 1994, Silver Spring, MD, USA). If the atp2b1a morphants had fused otoliths, the sagittae and lapilli still could be identified and the maximal diameters of sagittal otoliths were measured from the otolith edge that was not connected to the lapilli. To investigate the behavior of atp2b1a morphants, zebrafish embryos were injected with 6, 3 and 2 ng MO and the fish were observed under the stereomicroscope at 6 dpf. The atp2b1a morphants that could not maintain their balance either at rest or while moving were considered balance-defective individuals and both abnormal and normal fish were counted.
Staining of hair cells, hair bundles and ionocytes
Zebrafish embryos at different stages were anesthetized with buffered
MS-222 and the fish fixed in 4% paraformaldehyde in 0.1 mol
l–1 phosphate buffer (PB; pH 7.4) for approximately 1–4
h at 4°C. Fixation was followed by washing in PBS several times, then by
treatment with 2% Triton X-100 (Sigma) for 1 h at room temperature and washed
again in PBS. Fish were immersed in 3% bovine serum albumin (BSA) at room
temperature for 30 min to block nonspecific binding, followed by incubation in
primary antibody in PBS for 1–2 nights at 4°C. Monoclonal antibody
HCS-1 (1:100) was kindly provided by J. T. Corwin
(Finley et al., 1997). HCS-1
can specifically label the cell bodies of hair cells in the inner ear of
zebrafish (Blasiole et al.,
2006). Samples were washed in PBS twice for 10 min and incubated
with PBS-diluted secondary antibody goat anti-mouse IgG conjugated with FITC
(1:300; Jackson Immunoresearch Laboratories, West Grove, PA, USA) for 2 h at
room temperature. After washing, FITC-conjugated phalloidin solution (30 nmol
l–1) was used to label the hair bundle of the hair cells for
10–30 min at room temperature.
DASPEI [2-(4-dimethylaminostyryl)-N-ethylpyridinium iodide], a
mitochondrial vital probe, was used to stain the hair cells in the neuromasts
(Harris et al., 2003) and
epidermal ionocytes (Hiroi et al.,
1999), which are mitochondria-rich cells. Live samples of both
morphants and wild-type fish were immersed in tap water with DASPEI (100 ppm)
for 30 min in a dark box.
Samples stained with fluorescent dyes were immediately examined under a confocal laser scanning microscope (TCS-NT, Leica Lasertechnik, Heidelberg, Germany).
Measurement of whole-body calcium contents and influx
Whole-body calcium content and influx were measured following the method of
Chen et al. (Chen et al., 2003)
with a minor modification. Calcium content of atp2b1a morphants
(injected with 6 ng MO) and of wild-type fish were measured at 2, 4 and 6 dpf
using an atomic absorption spectrophotometer (Hitachi Z-5000, Tokyo, Japan).
Each analysis contained ten replicates and each replicate had five larvae.
Larvae were anesthetized with MS-222 and digested in 13.1 mol
l–1 HNO3 at 60°C. Digested solutions were
diluted with double-deionized water and then measured using the atomic
absorption spectrophotometer. Standard solutions from Merck (Darmstadt,
Germany) were used to make standard curves.
Morphants (injected with 6 ng antisense MO) and the wild-type zebrafish
(five replicates, three larvae each) at 2, 4 and 6 dpf were incubated in tap
water containing 4 ml 45Ca2+ for 4 h. After incubation,
samples were washed with isotope-free tap water three times and anesthetized
with MS-222 before digestion. Samples were digested with tissue solubilizer
(Solvable, Packard, Meriden, CT, USA) in the counting vial at 50°C
overnight. After digestion, 1 ml counting solution was added (Ultima Gold,
Packard) and the radioactivity counted with a liquid scintillation
β-counter (LS6500, Beckman, Fullerton, CA, USA). The calcium influx rate
was calculated by the following formula:
 | (1) |
Statistical analysis
Values are presented as the mean ± standard deviation (s.d.).
One-way analysis of variance (ANOVA) was used to evaluate sagittal otolith
growth, calcium content and calcium influx between wild-type zebrafish and
atp2b1a morphants. Differences among groups were identified by
Tukey's pairwise multiple comparison test. The Mann–Whitney rank sum
test was used to evaluate the number of hair cells in the neuromast and in the
inner ear between wild-type zebrafish and atp2b1a morphants.
Significance was set at 
<0.05.
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RESULTS |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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)
(Fig. 1). The analysis included
the entire amino acid sequence of zebrafish (z Atp2b1a), human (h ATP2B),
mouse (m ATP2B), rat (r ATP2B), bullfrog (b ATP2B), tilapia (t ATP2B) and
Caenorhabditis elegans (c ATP2B). The ATP2Bs were roughly clustered
into four groups with isoforms from each species forming a clustered group
1–4. C. elegans ATP2B was not classified into any vertebrate
ATP2B isoform and constituted a unique lineage. According to the phylogenetic
analysis, zebrafish Atp2b1a belongs to the ATP2B1 isoform family
(Fig. 1). This nomenclature was
approved by the Zebrafish Nomenclature Committee
(http://zfin.org/zf_info/nomen.html#Approval).
Zebrafish Atp2b1a shares high identities (70–80%) with ATP2B of mammals,
bullfrogs (AAK11272) and tilapia (AAK15034) and relatively lower identities
(49%) with invertebrate ATP2B (C. elegans: AAR00672).
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). atp2b1a mRNA was also traced to the hair cells of
the otolith chamber in adult zebrafish. The staining of numerous hair cells
was observed from the apical view of the otolith chamber after removing the
otolith to expose the sensory macula as shown in
Fig. 2F. Zebrafish
atp2b1a sense probe was used as a control and did not reveal any
signals either in the larval or adult inner ear
(Fig. 2G,H). By browsing the
Ensembl Genome database (zebrafish assembly version 6, searched with the
SSAHA2 algorithm), zebrafish atp2b1a is linked to the genome locus on
chromosome 4 at location 17,525,704–17,571,442 bp, where an annotated
PMCA, si:dkey-18o7.1 (Ensembl gene ID: ENSDARG00000012684) was found.
The si:dkey-18o7.1 is a temporary name derived from the zebrafish
genome sequencing and annotation project of the ZFIN database and its
expression pattern is available on the ZFIN web site
(http://zfin.org/cgi-bin/webdriver?MIval=aa-markerview.apg&OID=ZDB-GENE-030925-29).
The gene si:dkey-18o7.1 was specifically expressed in several
tissues, including, otic vesicle, lateral line system, brain and olfactory
placode. These expression patterns were consistent with our data.
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Some morphants showed no otolith formation. For most morphants, otoliths were formed but the seeding of the otolith precursors occurred several hours later than in wild-type larvae. atp2b1 morphants with only one otolith were frequently observed and this may be due to the seeding failure of the otolith precursor (Fig. 4A,B). The precursors of lapilli and sagittae may have seeded in the wrong location resulting in decreased distance between the two structures (Fig. 4C). As the lapilli and sagittae accreted, the two otoliths would eventually connect and fuse to become a single otolith. The fused otoliths were observed as early as 30 hpf although most fused otoliths occurred around 40–48 hpf.
Normally degradation of the injected capped mRNAs starts at 2 dpf as observed with decreased GFP signal in embryos with injected capped mRNA alone, therefore, data could be only gathered between 1–2 dpf. In the capped mRNA-GFP group the green fluorescence signal was widely spread in the embryos body from head to tail including some part of the yolk with differentiating or migrating cells (supplementary material Fig. S1). Autofluorescence in the yolk can be distinguished from GFP signal by comparison with the control group without injection. Based on the result, the antisense MO injection was specific for atp2b1a as co-injection of capped mRNAs with antisense MO completely blocked the GFP signal in 1–2 dpf embryos (supplementary material Fig. S1). Hence, the phenotype of MO-injected embryos is not due to MO cytotoxicity.
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Semicircular canal phenotypes
The phenotype of the semicircular canal was analyzed using the same group
of atp2b1a morphants (Table
1). The atp2b1a morphants showed abnormal semicircular
canal formation, with disrupted or even absent semicircular canal outgrowth
(Fig. 4D–F). The defects
of otolith formation and semicircular outgrowth always appeared simultaneously
in individuals.
Staining of hair cells in the lateral line and inner ear
At 5 dpf, atp2b1a morphants had a mean of 4.8 (±1.9;
N=20; Fig. 6A,B) hair
cells in the first neuromast of the posterior lateral line
(Metcalfe et al., 1985),
whereas wild-type zebrafish had a mean of 8.7 (±1.9; N=16;
Fig. 6C,E) hair cells. Hair
cells in the neuromast of atp2b1a morphants were significantly less
numerous than in wild-type zebrafish (P<0.001, Mann–Whitney
rank sum test). The vital mitochondrial dye DASPEI is used for convenient
visualization of different epidermal cells in both in vivo and in
vitro studies (Hiroi et al.,
1999). In some cases, neuromast hair cells were totally absent in
atp2b1a morphants although the epidermal ionocytes were stained
normally (Fig. 6D).
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Whole-body calcium influx and contents
Calcium influx and content increased abruptly beginning at 2 dpf in both
wild-type zebrafish and atp2b1a morphants, indicating increased need
for calcium. However, whole-body calcium influx and content measurements were
similar in atp2b1a morphants and wild-type zebrafish at 2, 4 and 6
dpf (supplementary material Fig. S2). The analysis indicated that calcium
absorption was still normal in zebrafish atp2b1a morphants. Calcium
ions were presumably absorbed through the epithelial ionocytes
(Pan et al., 2005). DASPEI
staining revealed the normal development and function of epithelial ionocytes
on the skin of the atp2b1a morphants larvae until at least 6 dpf
(Fig. 6D). These results imply
that Atp2b1a was not obligatory for the whole-body calcium absorption but
might have specific functions in inner ear calcium transport.
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DISCUSSION |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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). The
Ca2+ that enters through transduction channels is essential to hair
bundle function. However, elevated Ca2+ concentration is toxic to
the cells and regulation of Ca2+ concentration is critical. Plasma
membrane Ca2+ ATPase (PMCA) has been shown to be localized in the
hair bundle of bullfrog hair cells (Yamoah
et al., 1998) and to be responsible for Ca2+ extrusion
from hair cell stereocilia, which control intracellular Ca2+
concentration (Yamoah et al.,
1998). In the present study, inhibition of atp2b1a
expression reduced the number of hair cells in the inner ear and in the
neuromast along the lateral line of zebrafish. Loss of plasma membrane
Ca2+ ATPase in the hair bundle of atp2b1a morphants may
cause the breakdown of cellular calcium homeostasis, triggering the death of
the hair cells. However, further studies are needed to justify these
speculations.
Early regulators of inner ear development have been extensively studied in
zebrafish; among these regulators are Fgf, Dlx5, Hmx, Fox and Pax genes
(Noramly and Grainger, 2002;
Mackereth et al., 2005).
Evidently, atp2b1a was not required for early otic induction but was
essential for semicircular canal morphogenesis. In the present study, we
demonstrated that atp2b1a knockdown reduced the number of hair cells
in the sensory cristae. The sensory cristae and semicircular canals develop
during the same period of 42–72 hpf. Mechanisms of semicircular canal
formation are conserved among vertebrates
(Haddon and Lewis, 1996). As
demonstrated in chickens by Chang et al.
(Chang et al., 2004), sensory
cristae are responsible for the formation of their non-sensory components, the
semicircular canals. Abnormal sensory cristae, with few hair cells, may lead
to defective development of the semicircular canals by reducing the secretion
of hyaluronan-rich matrix (Haddon and
Lewis, 1991). Moreover, neuronal calcium sensor (NCS) proteins are
also involved in semicircular canal formation in zebrafish
(Blasiole et al., 2005).
Although the function of NCS in regulating semicircular canal formation is
still unclear, NCS may play a role in maintaining endolymphatic calcium
homeostasis through interactions with ion transporters such as Atp2b1a
(Blasiole et al., 2005). The
breakdown of cellular calcium homeostasis by atp2b1a knockdown may
cause the dysfunction of NCS, influencing the development of the semicircular
canal.
Otolith formation, localization and growth
Several otolith phenotypes were observed in atp2b1a morphants. The
absence of one or both otoliths (Table
1) indicated the failure of initial otolith seeding and subsequent
biomineralization with CaCO3. Glycogen is one of the factors
responsible for the early formation of the otolith by allowing the insertion
of otolith precursors (Pisam et al.,
2002). After the initial otolith seeding process at 18 to 24 hpf
(Riley et al., 1997),
precursors of sagittae and lapilli normally lie in posterior and anterior
positions, respectively. Failure of glycogen secretion or accretion of otolith
precursor particles may explain the absence of one or both otoliths in
atp2b1a morphants, as shown in
Fig. 4A,B. As observed in many
atp2b1a morphants, fused otoliths were most probably formed after the
seeding process due to the misplacement of the sagittae and lapilli
precursors. Fewer cells and development of tether cells in the wrong location
may prevent the hair cells from fixing the otolith precursors in the proper
position in the macula. In agreement with previous knockdown of the zebrafish
chaperone protein GP96 (also known as Hsp90b1), only one otolith was formed in
the atp2b1a larvae since precursor particles did not adhere to the
kinocilia of the tether cells (Sumanas et
al., 2003). In addition, zebrafish
Na,+K+-ATPase 1a.1 knockdown has been shown not
to damage the tether cells but completely blocked otolith formation
(Blasiole et al., 2006).
Na,+K+-ATPase coordinates with other ion exchangers and
transporters, including Atp2b1a, to maintain endolymph homeostasis
(Mugiya and Yoshida, 1995;
Shiao et al., 2005).
Therefore, knockdown of zebrafish Na+,K+-ATPase
1a.1 may result in the failure of the ion exchange system, including
that for Ca2+ and HCO –3, and impair
otolith formation.
Calcium ion transportation in the inner ear
Otolith accretion by larval zebrafish was affected by atp2b1a
knockdown, possibly by affecting endolymph composition. However, our data
showed equal amounts of whole-body calcium in wild-type fish and
atp2b1a morphants during the very early stages. This can be
attributed to maternal effects, based on the previous study of Peng et al.
(Peng et al., 2003). The
control and the morphants group showed a linear increase in calcium levels
between days 2 and 6. This implies normal acquisition of calcium through the
ionocytes (Pan et al., 2005),
as supported by DASPEI staining of ionocytes and normal calcium influx among
morphants. However, the inner ear manifested abnormal phenotypes during the
early stages when calcium balance in the embryo was still normal. Hence
zebrafish Atp2b1a appears to be an active calcium transporter that is
essential for normal development and function in the inner ear of larval
zebrafish, but is not required for whole-body Ca+ uptake from
ambient waters.
The confirmation of an Atp2b1a isoform in zebrafish inner ear strengthens
and supports previous findings by Mugiya and Yoshida
(Mugiya and Yoshida, 1995) in
trout. They concluded that an active ATP-dependent calcium pump was involved
in the cytosolic calcium extrusion across the sacculus cell apical membrane
facing the otolith. Their observations were based on calcium deposition into
the otolith under different chemical inhibitors or blockers of calcium
transporters/channels. Although we conducted immunohistochemical staining in
this study, the antibodies against human ATP2B, i.e. 5F10 (Sigma)
(Yamoah et al., 1998) and
others (Stauffer et al., 1995)
failed to show specific binding to zebrafish Atp2b1a in both larval and adult
tissues (the results are not shown). Therefore, the distribution of Atp2b1a in
the related cells of the zebrafish inner ear needs further investigation.
However, since otolith growth did not completely cease without Atp2b1a, the
possible involvement of other Atp2b isoforms or the passive diffusion of
calcium into the endolymph cannot be completely excluded. Moreover, abnormal
inner ears in atp2b1a morphants may cause the tight junctions to be
leaky, providing enough local calcium availability for reduced otolith
growth.
The presence of other zebrafish Atp2b isoforms needs further study in zebrafish inner ear development. To complete the active transepithelial calcium transport model in the inner ear of zebrafish, further study is required to identify the calcium transporters/channels at the basolateral membrane of the otolith chamber, either from molecular, biochemical or cellular methods.
This study demonstrates atp2b1a expression and its major role in otolith formation and semicircular canal development of a teleost fish. Based on our results, zebrafish Atp2b1a has a basic role in the active plasma membrane calcium pump which may influence sensory organ development via control of calcium concentration homeostasis in either single hair cells or the whole otic chamber.
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Footnotes |
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Supplementary material available online at http://jeb.biologists.org/cgi/content/full/212/5/639/DC1
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References |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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