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
* Author for correspondence (e-mail: dabdouba{at}nidcd.nih.gov)
Accepted 11 February 2003
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SUMMARY |
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Key words: Planar polarity, Organ of Corti, Sensory epithelium, Mechanosensory hair cells, Mouse
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INTRODUCTION |
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The Wnt family encodes secreted glycoproteins that are ligands for the
frizzled (Fzd) family of seven-transmembrane receptors and the low density
lipoprotein receptor-related protein family of co-receptors
(Wodarz and Nusse, 1998;
Tamai et al., 2000
;
Pinson et al., 2000
). At
present, the Wnt family includes approximately 15 to 20 genes per species
(reviewed by Miller et al.,
1999
). Wnt proteins can elicit various responses in the same, and
different, tissues via binding to different Fzd receptors and activation of
distinct intracellular signaling pathways (reviewed by
Wodarz and Nusse, 1998
;
Bejsovec, 2000
).
Wnt proteins can signal through canonical ß-catenin-dependent pathways
and through ß-catenin-independent (non-canonical) pathways including the
Wnt/Ca2+ pathway and the Jun-N-terminal kinase (JNK) pathway
(reviewed by Huelsken and Birchmeier,
2001). Results have demonstrated a role for the JNK pathway in
planar cell polarity (PCP) in some invertebrate systems and convergent
extension in vertebrate ones (reviewed by
Mlodzik, 2002
).
Recent studies have demonstrated that Drosophila homologs of two
deafness genes, protocadherin 23 (Fat in Drosophila) and myosin VIIa
(Myosin VIIa/crinkled in Drosophila), are components of the
Drosophila planar polarity pathway
(Weil et al., 1995;
Bolz et al., 2001
;
Bork et al., 2001
;
Di Palma et al., 2001
;
Wilson et al., 2001
;
Winter et al., 2001
;
Wada et al., 2001
;
Yang et al., 2002
). Moreover,
stereociliary bundle formation is defective in mice with mutations in either
protocadherin 23 (DiPalma et al.,
2001
) or myosin VIIa (Self et
al., 1998
), suggesting that the planar polarity pathway may play a
similar role in the cochlea. Finally, mice containing a mutation in
Fzd4 are deaf, although there are no obvious defects in the cochlea,
and Fzd10 has been identified as a candidate gene for non-syndromic
deafness DFNA41 (Wang et al.,
2001
; Blanton et al.,
2002
). Based on these results, and others, Lewis and Davis
(Lewis and Davies, 2002
)
recently suggested that the ear might provide evidence for parallels between
flies and vertebrates in terms of planar polarity. We have investigated the
potential role of Wnt signaling in the development of stereociliary bundles
and present evidence that disruption of Wnt signaling leads to defects in
stereociliary bundle orientation.
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MATERIALS AND METHODS |
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After fixation, sensory epithelia were exposed and developing kinocilia
were labeled using a monoclonal anti-acetylated-tubulin antibody (Sigma) and
an Alexa 568-conjugated anti-mouse secondary antibody (Molecular Probes). The
`Mouse-On-Mouse Kit' (Vector Laboratories) was used to reduce nonspecific
labeling. Stereocilia were labeled with either Alexa 488-conjugated phalloidin
(Molecular Probes) or with a biotin-conjugated Griffonia
simplicifolia lectin (Vector Laboratories)
(Lanford et al., 1999).
Binding of G. simplicifolia was detected using the ABC Elite Staining
Kit (Vector Labs).
Outer hair cell stereociliary bundles are comprised of a V-shaped group of actin-based stereocilia with a single tubulin-based kinocilium located at the vertex of the crescent (Figs 1 and 2). The orientation of each bundle was determined by extending a straight line from the single kinocilium to create an arrow with the stereociliary bundle as the head (Fig. 1). The orientation of this line was then determined relative to a line perpendicular to the row of pillar cells and passing through the center of the same cell (Fig. 1B). Bundles that were exactly aligned with the perpendicular line and oriented towards the outer edge of the epithelium were assigned an orientation of 0°. Orientations were plotted either as absolute degrees of deviation from an orientation of 0° regardless of direction or as a distribution plot between 180° and +180°, with 0° corresponding exact alignment with the perpendicular line and rotation towards the apex considered as positive.
|
Analysis of Wnt7a mutants
Cochleae from P0 and adult mice homozygous for a deletion of Wnt7a
with obvious limb patterning defects (Parr
and McMahon, 1995) were selected for analysis, isolated and fixed.
Homozygous deletion of the Wnt7a gene was confirmed by PCR
(Hall et al., 2000
).
PCR for Wnt genes
Cochleae were dissected from E15, E17 and E18 mice. For E15, Trizol reagent
(Invitrogen) was used to extract total RNA from tissue that included both the
developing cochlear epithelial cells as well as associated mesenchymal cells
and developing neuronal and glial cells from the spiral ganglion. For E17 and
E18, the developing sensory epithelia and surrounding epithelial cells were
separated from mesenchymal and neural cell types using thermolysin (250
µg/ml, Sigma) (Montcouquiol and Corwin,
2001) prior to RNA extraction. Degenerate primers and PCR
reactions were as described by Gavin et al.
(Gavin et al., 1990
) and were
used to amplify Wnt genes from E15 or E17 cochlear RT-DNA. PCR products were
separated on agarose gels, cloned using the TA-cloning kit (Invitrogen) and
then sequenced. In order to examine more fully the spectrum of Wnt genes that
might be expressed in the developing cochlea, specific primer sets for
individual Wnt genes were used to amplify genes from E18 cochlear RT-DNA.
Cochlear explant cultures
Cochlear explant cultures from E13 embryos were established as described
previously (Kelley et al.,
1993) and treated after 18 hours in vitro. Explants were
maintained until hair cell stereociliary bundles had developed along the
length of the sensory epithelium a total of 6 days in vitro (DIV,
equivalent to P0). At the conclusion of each experiment, explants were fixed
in 4% paraformaldehyde for 45 minutes. Stereociliary bundles were labeled as
described for in vivo samples.
Modulation of Wnt signaling
Cultures were either exposed to medium conditioned with Wnt7a protein or to
medium conditioned by the parent cell line. Conditioned medium was generated
as described by Hall et al. (Hall et al.,
2000). For co-culture, Wnt7a expressing, or parent, RatB1a
fibroblast cells were grown to confluence in MatTek dishes
(Shimizu et al., 1997
).
Twenty-four hours prior to the start of each experiment, the growth media was
replaced with serum-free media containing 1.0 mM sodium butyrate to stimulate
expression of Wnt7a protein (also added to control media). After 24 hours in
serum-free media, RatB1a cells were scraped from a small region in the center
of each dish and a cochlear explant was placed in that position. Explants were
maintained in serum-free media with sodium butyrate for the duration of the
experiment (6 DIV).
Secreted frizzled-related protein 1 (Sfrp1; 50 µg/ml) was dissolved in
the culture medium along with 1µg/ml heparin
(Uren et al., 2000). The
culture medium was replaced with fresh Sfrp1-containing medium after 48 hours.
Control medium contained 1 µg/ml heparin.
Wnt inhibitory factor 1 (Wif1) was obtained in conditioned medium. Explant cultures were exposed to either Wif1-conditioned medium or control medium (Hsieh et al., 1998) as described.
Synthesis of heparan sulfate proteoglycans was inhibited by addition of 30
mM sodium chlorate in the medium (Kispert
et al., 1996).
In situ hybridization
Wnt7a whole-mount in situ hybridization was performed on cochleae
from mice at E14, E16, P0 and P3 as described previously
(Lanford et al., 1999).
Sections were obtained by embedding wholemounts and sectioning at 12 µm
using a cryostat.
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RESULTS |
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Multiple Wnt genes are expressed in the embryonic cochlea
To determine if Wnt genes are expressed in the developing cochlea, we
initially performed a degenerate PCR screen for Wnt genes in cochleae from E15
and E17 mice. Approximately 50 individual colonies were sequenced. Greater
than 50% of those colonies represented Wnt7a, and an additional
approximately 25% were Wnt5a. Other Wnts identified were
Wnt2 and Wnt10b. Specific primer sets were used to confirm
expression of Wnt7a, Wnt5a, Wnt2 and Wnt10b. In addition,
transcripts were also detected for Wnt4, Wnt7b, Wnt8 and
Wnt11.
Exposure to Wnt7a-conditioned medium inhibits stereociliary bundle
reorientation
As the results of our PCR screen indicated that Wnt7a was strongly
expressed within the developing cochlear duct, and, because of its reported
role in polarity (Parr and McMahon,
1995; Kengaku et al.,
1998
), we decided to investigate the effects of exogenous Wnt7a
protein on the development of polarity in cochlear explant cultures. As
reported previously (Kelley et al.,
1993
), explant cultures of embryonic cochleae from E13 animals
developed normally in vitro, including the formation of normal stereociliary
bundle orientation (all bundles oriented towards the outer border of the organ
of Corti; Fig. 3A). The overall
development of stereociliary bundle morphology on both IHC and OHC also
appeared normal, with the formation of characteristic curved or V shapes. In
cultures maintained in Wnt7a-conditioned medium, the overall development of
the sensory epithelium, including the development of IHC and OHC, appeared
unaffected. However, in the outer hair cell region, and in particular in the
second and third rows, many cells were observed with deviated bundle
orientations with some of the deviations equal to or exceeding 90°
(Fig. 3B). Similar results were
obtained for explants that were co-cultured with Wnt7a-expressing RatB1a cells
(data not shown). Wnt7a exposure did not seem to impede hair cell maturation,
as evidenced by the presence of stereociliary bundles, markers of maturation
(Sobin and Anniko, 1984
) and
by the development of mature bundle morphologies including a chevron shape and
asymmetrically located kinocilium (Fig.
3B). Furthermore, both control and treated cultures expressed
similar levels of prestin, a protein expressed exclusively in the plasma
membrane of postnatal OHC (data not shown)
(Belyantseva et al., 2000
).
|
In cultures exposed to Wnt7a-conditioned medium, the orientations of stereociliary bundles located at the mid-point of the sensory epithelium did not significantly differ from control (Fig. 3C). However, although the average deviation from 0° gradually decreased at more basal positions in control cultures, a similar decrease was not observed in cultures exposed to Wnt7a-conditioned medium. As a result, stereociliary bundles located in the basal regions of Wnt7a-treated cultures were significantly misoriented in comparison with controls (Fig. 3C). Moreover, in cultures exposed to Wnt7a-conditioned medium, the average deviation from 0° was approximately the same at each position along the basal half of the sensory epithelium.
The observation that there was a change in the average deviation from 0° in the presence of Wnt7a protein suggested that Wnt7a could play a role in either the reorientation of stereociliary bundles or in the overall orientation of the plane of cellular polarity within the outer hair cell region. If Wnt7a influences the overall orientation of cellular polarity, then treatment with Wnt7a protein would lead to a uniform deviation in bundle orientation. To examine this hypothesis, the spectrum of possible stereociliary bundle orientations (180° to +180°) was partitioned into 15° intervals. Individual bundle orientations were assigned to specific partitions to generate a histogram for the distribution of bundle orientations (Fig. 4). Analysis of the distribution of stereociliary bundle orientations along the basal half of the sensory epithelium indicates that treatment with Wnt7a protein does not lead to a uniform deviation in stereociliary bundle orientation (Fig. 4A,B). Rather, the relatively broad initial distribution of bundle orientations is maintained at positions along the length of the sensory epithelium, suggesting that the effect of treatment with Wnt7a protein is to inhibit stereociliary bundle reorientation.
|
Blocking endogenous Wnt Signaling with Sfrp1 or Wif1 inhibits
stereociliary bundle reorientation
Recent studies have identified a secreted family of molecules that are
related to the Frizzled family of Wnt receptors
(Rattner et al., 1997;
Leyns et al., 1997
;
Finch et al., 1997
).
Secreted-frizzled-related-proteins (Sfrps) have been shown to bind to multiple
Wnt proteins and to prevent bound Wnt from binding to Frizzled receptors
(Wang et al., 1997
;
Xu et al., 1998
;
Bafico et al., 1999
;
Uren et al., 2000
). As a
result, Sfrps can be used to inhibit a broad range of Wnt signals, including
Wnt7a (Hall et al., 2000
;
Yoshino et al., 2001
;
Bergwitz et al., 2001
). To
investigate the effects of inhibition of endogenous Wnt signaling on the
development of bundle orientation, cochlear cultures were established and
exposed to medium containing Sfrp1. Sfrp1 induces a disruption in OHC
stereociliary bundle reorientation that appears comparable to the effects of
Wnt7a (Fig. 5A), supporting a
role for endogenous Wnt signaling in stereociliary bundle reorientation.
|
Disruption of endogenous Wnt diffusion inhibits reorientation of
stereociliary bundles
Previous studies have demonstrated that heparan sulfate proteoglycans
(HSPGs) play a key role in regulating Wnt/Wg signaling, and in particular, in
the extracellular distribution of Wnt/Wg
(Tsuda et al., 1999;
Lin and Perrimon, 1999
;
Baeg et al., 2001
;
Dhoot et al., 2001
;
Giráldez et al., 2002
).
To determine whether HSPGs play a role in bundle reorientation, cochlear
explant cultures were exposed to 30 mM NaClO3, a concentration that
has been shown to be sufficient to inhibit the sulfation of polysaccharides
and therefore to prevent the production of HSPGs
(Kispert et al., 1996
).
Treatment with NaClO3 lead to significant defects in stereociliary
bundle orientations at both the 5% and 12.5% positions when compared with
controls (Fig. 5B).
Expression pattern of Wnt7a in the embryonic cochlea
Based on the results of exposure to Wnt7a protein, the cellular
distribution of Wnt7a was determined in whole-mount in situ on
cochleae from E14, E16, P0 and P3. At E14, Wnt7a is expressed
throughout a wide region of the developing cochlear epithelium
(Fig. 6A). However, the
relative intensity of expression appears to vary along both the
basal-to-apical and inner-to-outer axes of the epithelium. In the relatively
undeveloped apex of the cochlea at E14, Wnt7a is expressed broadly in
the inner half of the epithelium (Fig.
6B). However, its expression ends abruptly in a region of the
epithelium that correlates with the position of the spiral vessel in the outer
half of the epithelium (Fig.
6B). As the spiral vessel is located beneath the region of the
epithelium that will develop as the pillar cells, the edge of Wnt7a
expression appears to correlate with the mid-point of the developing organ of
Corti.
|
At E16, Wnt7a is strongly expressed in a narrow band of cells that extends along the length of the cochlea in a position that is consistent with the developing sensory epithelium (Fig. 6D). Weak expression also persists in the apical region in some cells located in the inner half of the cochlear duct (Fig. 6D). Cross-sections of the base of the cochlea at E16 indicate that the strong band of Wnt7a expression correspond to developing pillar cells (Fig. 6E). By P0, expression is restricted to the pillar cells (Fig. 6F). Expression of Wnt7a in pillar cells extends at least through P3, the oldest stage analyzed (data not shown).
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DISCUSSION |
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Cochlear stereociliary bundles reorient during development
The factors that play a role in the determination of specific stereociliary
bundle orientations in hair cells are largely unknown. The results of two
previous studies in chick cochlea and mouse vestibular system have
demonstrated that orientation apparently arises as a two-step process
(Cotanche and Corwin, 1991;
Denman-Johnson and Forge,
1999
). During the first step, stereociliary bundles develop with
an initial, non-random orientation that is biased towards the final
orientation of each cell. Following initial development, individual
stereociliary bundles gradually reorient to obtain their final orientation.
Our observations suggest that hair cells within the mammalian organ of Corti
develop in a similar manner.
In the proposed two step process of bundle orientation, a general
specification of polarity must occur prior to bundle formation, and a second
phase of orientation is required to achieve a final uniform polarity. A
similar series of specifications appears to occur during the development of
wing hair polarity in Drosophila (reviewed by
Adler and Lee, 2001). In both
vertebrate and invertebrate systems, it appears that uniform planar polarity
develops through a series of interactions in which an initial overall polarity
is determined possibly through the perception of a gradient and subsequent
refinements are made based on the gradient and signals produced through
cell-cell signaling.
It is important to consider that there were marked differences in the uniformity of initial stereociliary bundle orientations between hair cells located in different regions of the organ of Corti in vivo. Initial orientations for IHC and first row OHC were fairly uniform (average deviation of 15°) and comparable. However, there was a progressive increase in the average deviation of the initial orientations of second (20°) and third row (30°) OHC. One explanation for this result would be that a diffusible cue plays a role in the determination of initial bundle orientation and that the source of this cue is located closer to the IHC and first row OHC. Therefore, greater distance from the source could lead to a greater degree of inaccuracy as a result of an asymptotic diffusion gradient.
A role for wnt signaling in bundle reorientation
Our results strongly support a role for Wnt signaling during stereociliary
bundle reorientation. At least eight different Wnt genes are present in the
developing cochlear epithelium and treatment with Wnt7a leads to a disruption
in bundle reorientation. Similarly, inhibition of Wnt signaling using factors
that either bind directly to Wnts (Sfrp1, Wif1) or that prevent the diffusion
of Wnts (NaClO3) also inhibit bundle reorientation. However, the
specific mechanism(s) of Wnt signaling in bundle orientation have not been
determined. Wnt signaling is mediated through binding to Fzd receptors,
leading to activation of at least three downstream signaling pathways.
Although the specific downstream pathways that are activated by Wnts in the
cochlea have not been identified, the effects on bundle orientation would seem
most consistent with the JNK/PCP pathway. If this is the case, then
differential activation of Fzd receptors across developing outer hair cells
could serve as an instructive cue for the re-orientation of the stereociliary
bundle (Tomlinson et al.,
1997). Preliminary results indicate that mRNAs for multiple
frizzled genes are expressed in the developing cochlear epithelium (A.D. and
M.W.K., unpublished), and mRNAs for at least five frizzled genes are expressed
in the adult rat cochlea (Daudet et al.,
2002
). However the cellular localization of these genes has not
been determined in either the embryonic or adult cochlea.
The dynamics of Wnt-dependent Fzd activation across outer hair cells has
not been determined. One possibility, which will be discussed below in more
detail, is that Wnts act as a soluble molecule that diffuses across the
developing outer hair cells, leading to differential activation of Fzds based
on distance from the Wnt source. Alternatively, in Drosophila
although wg is necessary for the development of ommatidial polarity,
it apparently serves a permissive, rather than instructive, role
(Adler, 2002). Based on these
results, it has been suggested that polarizing signals may arise through the
differential localization of Fzd receptors to one side of the cell
(Adler and Lee, 2001
). If a
similar mechanism exists in the organ of Corti, then Fzd receptors should be
differentially localized to one edge of each developing OHC. However, at
present there are no data regarding the cellular distribution of Fzd proteins
in developing hair cells. Finally, it is important to consider that the
effects of exogenous Wnt7a protein on stereociliary bundle orientation are not
obviously consistent with a purely permissive role for Wnts.
Interestingly, the orientation of stereociliary bundles located on IHC did not seem affected by treatment with Wnt7a-conditioned medium or Sfrp1. One possible explanation would be that orientation of IHC stereociliary bundles could occur at an earlier developmental stage than OHC. This is supported by the finding that IHC are already differentiated at E14, at least 2 days prior to OHC as determined by the expression of the hair cell specific marker myosin-VI (M.M. and M.W.K., unpublished).
A gradient could play a role in stereociliary bundle
reorientation
The effects of treatment with Wnt7a, Sfrp1, Wif1 and sodium chlorate, as
well as the pattern of expression for Wnt7a, are consistent with a
role for Wnt-signaling in the re-orientation of OHC bundles. Wnt7a is
expressed in developing pillar cells, creating a potential line source that
could result in the formation of a gradient leading to an asymmetric
distribution of the soluble protein across a plane of epithelial cells.
Individual cells within the plane would be able to detect this gradient and to
generate a uniform cellular polarity that would correspond with the direction
of the gradient. The Wg/Wnt families of signaling proteins have been shown to
act as morphogens during development of various structures in
Drosophila (Zecca et al.,
1996; Neumann and Cohen,
1997
; Strigini and Cohen,
2000
) and as has been discussed, a gradient in the activation of
Fzd receptors determines planar orientation in both the Drosophila
eye and wing (reviewed by Mlodzik,
1999
).
If detection of a gradient is an important factor in stereociliary bundle
reorientation, then the diffusion dynamics of that gradient should impact on
the absolute change in concentration that exists at different distances from
its source (Monteiro et al.,
2001). Assuming that the developing pillar cells act as a line
source for Wnts then the relative drop in concentration across the first row
of OHC should be greater than the drop in concentration across the third row
of OHC assuming an asymptotic gradient. Under these circumstances, it seems
reasonable to expect that stereociliary bundle reorientation in third row OHC
might be delayed or that there might be an overall lower level of uniform
orientation as a result of the relatively smaller overall change in
concentration across those cells. One way to examine the level of uniformity
of bundle orientation is to compare the change in average bundle deviation
between first row and third row OHC during reorientation. However, as third
row OHC develop with a greater initial average bundle deviation, it is
difficult to make comparisons regarding changes that occur during the period
of reorientation. An alternative approach would be to compare the changes in
the standard deviation of the distribution of bundle orientations between
first and third row OHC. Between E17 and P0 the standard deviation for bundle
orientations in first row OHC decreases by 80% while the standard deviation
for orientation in the third row decreases by only 52%. Although a decrease in
the relative change of a concentration gradient across each row of OHC is
certainly not the only explanation for these differences, the relative delays
in the formation of uniform orientation in second and third row OHC is
consistent with this hypothesis.
It is important to note that we do not present direct or conclusive
evidence that a Wnt gradient is necessary to generate polarization of the
stereociliary bundles in the organ of Corti. Wnt could be responsible for the
organization of secondary gradients of other PCP molecules that in turn
influence planar polarity in the cochlea
(Yang et al., 2002;
Tree et al., 2002
;
Ma et al., 2003
). Moreover,
further experiments are needed to investigate whether Wnt plays a direct role
in PCP signaling in the stereociliary bundles, permissively or
instructively.
Alternative pathways for stereociliary bundle orientation
The observation that stereociliary bundle orientation is normal in
Wnt7a mutant animals suggests that Wnt7a is not necessary for bundle
reorientation. This result suggests that either alternative mechanism(s) for
the determination of appropriate bundle orientation exist within the cochlea
or that deletion of Wnt7a leads to the activation of compensatory
mechanisms. As treatment with Sfrp1 or Wif1, generic inhibitors of Wnt
signaling, leads to inhibition of reorientation, it is possible that one or
more of the seven other Wnts expressed within the cochlea could be acting in
either a functionally redundant or compensatory fashion in Wnt7a
mutants. For example, Wnt7b like Wnt7a induces axonal remodeling and synapsin
I clustering in mossy fibers (A. Hall, A.B. and P.C.S., unpublished).
It is also possible that other signaling pathways could act to compensate
for the loss of Wnt7a signaling. Previous studies have suggested a
role for the Notch signaling pathway in the determination of stereociliary
bundle polarity in mechanosensory hair cell epithelia in both zebrafish and
mice (Haddon et al., 1999;
Lanford et al., 1999
).
Analysis of stereociliary bundle orientation in the zebrafish mutant
mindbomb, a mutation in an as yet undetermined gene that has been
shown to disrupt the Notch pathway, indicates consistent defects in the
development of a normal pattern of stereociliary bundle orientation
(Haddon et al., 1999
). Similar
effects were observed in the orientations of OHC stereociliary bundles in the
organ of Corti from mice in which Jag2 (a Notch ligand) had been
deleted (Lanford et al.,
1999
). Therefore, it seems likely that Notch signaling also plays
a role in the determination of stereociliary bundle orientation. These
results, taken together with our findings, suggest the intriguing possibility
that Notch and Wnt signaling may collaborate to regulate hair cell orientation
as observed in other developing systems
(Tomlinson and Struhl, 1999
).
In addition, Notch signaling could compensate for the loss of Wnt7a function
or it is possible that Wnt signaling may be complementary to the short-range
Delta/Notch signaling pathway.
In summary, we have presented the first study on the development of planar polarity in a mammalian system. The results presented here strongly implicate the Wnt signaling pathway in the development of the mammalian cochlea and suggest that the molecular basis of planar polarization has been conserved between vertebrates and invertebrates.
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ACKNOWLEDGMENTS |
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