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
Shelly Cruz1,
Jen-Chieh Shiao2,*,
Bo-Kai Liao1,
Chang-Jen Huang3,4 and
Pung-Pung Hwang1,4,*
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
View larger version (7K):
[in this window]
[in a new window]
|
Fig. 1. Phylogenic analysis of ATP2B amino acid sequences. The consensus tree was
constructed using the neighbor-joining method with bootstrap of 1000 times and
poisson correction (Mega-3 software). Bootstrap values are shown at the
branches. The GenBank accession numbers of the sequences used are as follows:
P20020 (h ATP2B1), Q01814 (h ATP2B2), Q16720 (h ATP2B3), P23634 (h ATP2B4),
P11505 (r ATP2B1), P11506 (r ATP2B2), Q64568 (r ATP2B3), Q64542 (r ATP2B4),
NP_080758 (m ATP2B1), NP_033853 (m ATP2B2), NP_796210 (m ATP2B3), NP_998781 (m
ATP2B4), AAK11272 (b ATP2B1bx), AAK15034 (t ATP2B), AAR00672 (c ATP2B); b,
bullfrog; c, C. elegans; h, human; m, mouse; r, rat; t, tilapia; z,
zebrafish. The bar represents 5% replacement of amino acids per site.
|
|
View larger version (102K):
[in this window]
[in a new window]
|
Fig. 2. Zebrafish atp2b1a mRNA expression pattern revealed by in
situ hybridization. (A–C) Zebrafish atp2b1a mRNA is
localized in the inner ear (A) and lateral-line neuromasts (indicated by
arrows in B,C). The larval inner ear (2 dpf) shows ubiquitous staining with
prominent staining in the hair cells of sensory macula (SM), the sensory
crista and the protrusion of the semicircular canals (SC). (D,E) Basolateral
view of the lagenar chamber of an adult fish with the otolith inside the
chamber. atp2b1a expression was detected in numerous cells around the
sensory macula as indicated by the arrows in D. (E) Higher magnification of
the boxed area in D. (F) Apical view of the lagenar chamber showing the strong
staining of atp2b1a in the hair cells in the macula. (G,H) The sense
probe, as a control, does not reveal signals in the inner ear of the larvae
(G) or the lagenar chamber of the adult (H). A, anterior; D, dorsal; LIC,
lagenar ionocytes; Nv, hearing nerve; P, posterior; SIC, saccular ionocytes;
V, ventral. Scale bars, 200 µm (B), 100 µm (A,D,F,G,H), 20 µm (E), 10
µm (C).
|
|
View larger version (36K):
[in this window]
[in a new window]
|
Fig. 3. Zebrafish atp2b1a morphant at 5 dpf shows normal body morphology
(A) and abnormal inner ear with the fused otolith (B, indicated by the
arrow).
|
|
View larger version (148K):
[in this window]
[in a new window]
|
Fig. 4. Zebrafish inner ear phenotypes of wild-type and morphants with anterior to
the left in all panels. (A,B) Different focal planes of the same placode of a
zebrafish atp2b1a morphant at 35 hpf. A shows a normal lapillus has
formed but the sagitta failed to crystallize. B shows two otolith precursor
particles, indicated by the black arrows. (C) Another zebrafish
atp2b1a morphant (35 hpf) showing the fused otoliths, with the
sagitta wrongly located in the anterior area of the placode. (D–F)
Zebrafish atp2b1a morphants (4 dpf) with one, two or three fused
otoliths and disrupted or reduced outgrowth of semicircular canals. (G)
Wild-type larva (4 dpf) with two otoliths and normal outgrowth of the
semicircular canals in the inner ear. Semicircular canals are indicated by the
white arrows. Scale bars, 10 µm (A–C); 50 µm (D–G).
|
|
View larger version (7K):
[in this window]
[in a new window]
|
Fig. 5. Comparison of sagittal otolith accretion in atp2b1a morphants (MT)
and wild-type (WT) zebrafish. Otoliths of atp2b1 morphants were
significantly smaller than those of the wild-type larvae. Higher doses of
antisense morpholinos (MO) result in smaller otoliths. A, B and C indicate
significant difference among groups (N=40 for each group). The
vertical bar represents one standard deviation.
|
|
View larger version (40K):
[in this window]
[in a new window]
|
Fig. 6. Staining of hair cells in the neuromast. (A,B) DASPEI staining of zebrafish
atp2b1a morphants (5 dpf) reveal only two hair cells and less dye
absorption in the hair cells of the neuromasts. (C) Wild-type larvae have 10
hair cells in the neuromast and normal uptake of the dye. Arrows indicate the
third and fourth neuromasts on the trunk lateral lines. (D,E) The same area of
a morphant and normal fish, respectively. Both show normal DASPEI staining of
the epithelial ionocytes, indicated by the arrowheads. Scale bar, 25 µm
(A–C); 200 µm (D,E).
|
|
View larger version (101K):
[in this window]
[in a new window]
|
Fig. 7. Staining of hair cells in the inner ear. (A–H) The tether cells are
revealed by HCS-1 antibody staining in wild-type larvae (A,B) and
atp2b1a morphants (C–H) at 35 hpf. Morphants may lose the
tether cells or the tether cells develop in the wrong location. E,F and G,H
show the tether cells in the posterior region and middle area, respectively.
(I–L) FITC-phalloidin (green) and HCS-1 antibody (red) staining of the
hair cells in zebrafish inner ear at 4 dpf. There are fewer hair cells in the
macula and crista in atp2b1a morphants (J,L) than in the wild-type
(I,K). ac, lc, pc, anterior, lateral and posterior cristae; nm, neuromast; sm,
saccule macula; um, utricular macula. Scale bars, 20 µm for all panels.
|
|
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati 
Twitter What's this?
© The Company of Biologists Ltd 2009
