doi: 10.1242/10.1242/dev.00448
Wnt signaling mediates reorientation of outer hair cell stereociliary bundles in the mammalian cochlea
Alain Dabdoub1,*,
Maura J. Donohue1,
Angela Brennan2,
Vladimir Wolf3,
Mireille Montcouquiol1,
David A. Sassoon4,
Jen-Chih Hseih5,
Jeffrey S. Rubin3,
Patricia C. Salinas2 and
Matthew W. Kelley1
1 Section on Developmental Neuroscience, NIDCD, National Institutes of Health,
Rockville, MD 20850, USA
2 Department of Biological Sciences, Imperial College of Science, Technology and
Medicine, Exhibition Road, London SW7 2AZ, UK
3 Laboratory of Cellular and Molecular Biology, Division of Basic Sciences, NCI,
National Institutes of Health, Bethesda, MD 20892, USA
4 Department of Biochemistry and Molecular Biology, Mount Sinai School of
Medicine, New York, NY 10029, USA
5 Department of Biochemistry and Cell Biology, State University of New York at
Stony Brook, Stony Brook, NY 11794, USA
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Fig. 1. Cellular structure of the organ of Corti and determination of orientation
of stereociliary bundles. (A) The apical surface of the organ of Corti at P0.
The organ of Corti extends in a spiral along the basal-to-apical axis (arrow)
of the cochlear duct. A single row of IHC (black) is located closest to the
modiolus (inner edge). Adjacent to the IHC is a row of pillar cells (PC,
yellow). On the opposite side of the pillar cells, nearer to the stria
vascularis (outer edge) are three or four rows of OHC1-OHC3 (black). In both
the inner and outer hair cell regions, each hair cell is separated from
neighboring hair cells by non-sensory supporting cells (gray). On the apical
surface of each hair cell is a stereociliary bundle. At P0 this bundle is
comprised of a single tubulin-based kinocilium (red) and a group of
actin-based stereocilia (green dots). Stereocilia bundles are uniformly
oriented towards the outer edge, and are arranged into a curved (IHC) or
V-shaped (OHC) pattern with the kinocilium located at the vertex of the
bundle. (B) The orientation of stereociliary bundles (illustrated as in A) was
determined relative to a line (broken white line) extending perpendicular to
the row of pillar cells (PC, yellow). If the vertex of the bundle aligned
exactly with the perpendicular line, then the bundle was assigned an
orientation of 0°. Deviations towards the apex of the cochlea were
assigned a positive value and deviations towards the base were assigned a
negative value. The OHC on the left has a relatively small deviation of
15° from the perpendicular line, while the OHC on the right has a larger
deviation of 70°.
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Fig. 2. Stereociliary bundles reorient during development. (A,B) Stereocilia
(green) and kinocilia (red) were labeled in cochleae from P0, E17 and P10 (not
shown). (A) In the basal region of the cochlea at P0, a typical chevron-shaped
stereociliary bundle with a kinocilia located at the vertex of the bundle is
present on each hair cell. Orientations for hair cells located in the boxed
region are illustrated on the right. The locations of the single row of IHC
and three rows of OHC (numbered) are indicated on the left. (B) In the
mid-basal region of the cochlea at E17, chevron-shaped or flattened
stereociliary bundles with asymmetric kinocilia can be identified for IHC and
for OHC located in the first two rows (1,2). Bundles are present on third row
OHC (3) but specific orientations could not be determined. As in A,
orientations for cells located in the boxed region are illustrated on the
right. Scale bar: in A 10 µm for A,B. (C) Average orientations of
stereociliary bundles were determined for IHC and OHC in basal and apical
regions of the cochlea at E17, P0 and P10. Distinct orientations could not be
determined for stereociliary bundles located on second and third row OHC in
the apical region of the cochlea at E17. Average deviation in stereociliary
bundle orientation decreased with development for hair cells in all rows of
the organ of Corti, indicating that orientation improves towards 0° over
time. The average initial orientation for stereociliary bundles located in the
third row of outer hair cells (asterisk) was significantly greater
(P=0.02) than the initial orientation for bundles located in the row
of IHC or in the first row of OHC.
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Fig. 3. Wnt7a protein influences stereociliary bundle orientation. (A) Surface view
of labeled stereociliary bundles in a cochlear culture established on E13
after 6 days in vitro (DIV). Stereociliary bundles on both IHC and all rows of
OHC (1-4) have developed with appropriate orientation. Orientations for the
outer hair cells located in the boxed region are illustrated on the right. (B)
Surface view of a cochlear culture established on E13 and maintained for 6 DIV
in medium conditioned with Wnt7a protein. There are marked bundle
misorientations in all rows of outer hair cells (1-4). Orientations of IHC
appear unaffected. Orientations for the boxed region are illustrated on the
right. (C) Average stereociliary bundle orientations were determined for outer
hair cells at specific positions (determined as a percent of the total length)
along the basal-to-apical axis of the cochlea in control and Wnt7a-treated
cultures. In control cultures, average bundle orientation decreased
progressively towards the basal end of the cochlea. This pattern is consistent
with in vivo data (see Fig. 2)
and suggests that bundles reorient in vitro. Exposure to Wnt7a protein did not
alter the orientation of stereociliary bundles located at the 45% or 37.5%
positions along the cochlea; however, there were significant changes in
average stereociliary bundle orientation at the 25%, 12.5% and 5% positions
(asterisks; P<0.003). Results are from three pairs of control and
Wnt7a-treated cochlear explants. Error bars are s.e.m. (at least 12
stereociliary bundles were measured per point per culture). Scale bar in A
(same in B), 10 µm.
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Fig. 4. Wnt7a does not bias the direction of stereociliary bundle orientation. (A)
Frequency histograms for stereociliary bundle orientations at different
positions along the basal-to-apical axis in control cochleae. Orientations
were determined for bundles on outer hair cells and the resulting values were
used to generate a frequency histogram with a class interval of 15°. In
more immature regions of the cochlea (45% and 37.5% positions) there is a
broad distribution of bundle orientations. By contrast, in more mature regions
of the cochlea (25%, 12.5% and 5% positions), the distribution of bundle
orientations becomes tightly clustered. (B) Frequency histograms, generated as
in A, for stereociliary bundle orientations in cochleae exposed to Wnt7a. The
distribution of bundle orientations at the 45% position appears similar to
control. However, bundle orientations remain fairly broad at more basal
(mature) positions, suggesting that Wnt7a inhibits bundle reorientation rather
than affecting the overall polarity of the epithelium. The data in A and B
represent orientations for stereociliary bundles from cochlear explant
cultures established on E13 after 6 days in vitro in the presence of either
control or Wnt7a-conditiond medium.
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Fig. 5. Inhibition of the Wnt signaling prevents stereociliary bundle
reorientation. (A) Secreted frizzled-related protein 1 (Sfrp1) inhibits
stereociliary bundle reorientation. Average stereociliary bundle orientations
were determined for outer hair cells at specific positions along the
basal-to-apical axis of the cochlea in control and Sfrp1-treated cultures.
Exposure to Sfrp1 inhibited bundle reorientation with significant differences
in average stereociliary bundle orientation at both the 5% and 12.5% positions
(*P<0.02, **P<0.003). (B) Wnt inhibitory factor 1
(Wif1) inhibits stereociliary bundle reorientation. Average stereociliary
bundle orientations were determined for outer hair cells at the 5% and 12.5%
positions along the basal-to-apical axis of the cochlea in control and
Wif1-conditoned media cultures. Wif1-conditioned medium significantly
inhibited stereociliary bundle reorientation at both positions
(*P<0.001). (C) Sodium chlorate inhibits stereociliary bundle
reorientation. Diffusion of Wnt protein within cochlear explants was reduced
by inhibition of synthesis of HSPG with 30 mM sodium chlorate
(NaClO3). NaClO3-treated cultures exhibited significant
differences, as compared with control, in average bundle orientations at the
5%, and 12.5% positions (*P<0.004). Results for all three graphs
are from a minimum of three control and three experimental cochleae and
orientations were determined for a minimum of 12 stereociliary bundles at each
position in each sample. Error bars indicate s.e.m.
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Fig. 6. Wnt7a is expressed in the developing organ of Corti. (A)
Whole-mount in situ hybridization for Wnt7a in the cochlear duct at
E14. A band of Wnt7a expression is present in the basal region (base)
of the cochlea (arrow). In the apical turn (apex) of the cochlea,
Wnt7a expression spans most of the width of the floor of the cochlear
duct. Box near base indicates the region of the epithelium illustrated in B.
Box near apex indicates region illustrated in C. (B) Cross-section through the
apical turn of the cochlea at E14. For all cross-sections, inner is towards
the left (Mod in E), outer is towards the right (Str in E). There is broad
expression of Wnt7a throughout the epithelium. However, there is a
distinct boundary of Wnt7a expression (arrow) that correlates with
the spiral vessel (asterisk), an embryonic marker for the location of the
developing organ of Corti. (C) Cross-section through the basal region of the
cochlear duct at E14. Wnt7a expression persists in a band of cells
that extends from the boundary at the spiral vessel (arrow) towards the
inner-side of the duct (arrowhead). In addition, Wnt7a is still
expressed in some cells located at the inner edge of the duct. (D) Whole-mount
in situ hybridization for Wnt7a at E16. By this stage, expression of
Wnt7a is restricted to a band of cells that extends along the length
of the cochlea (arrows) from base to apex. Box indicates region illustrated in
E. (E) Cross-section through the basal region of the cochlear duct at E16.
Expression of Wnt7a is restricted to developing pillar cells (arrow)
located directly above the spiral vessel (asterisk). (F) Cross-section through
the cochlear duct at P0. Wnt7a expression persists in the inner and
outer pillar cells (arrow), with the possibility of a small amount of
expression in the Deiter's cell located underneath the first row OHC (1).
Second (2) and third (3) row OHC are numbered for reference. Scale bars: 100
µm in A,D; in F, 20 µm for B,C,E,F.
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© The Company of Biologists Ltd 2003
