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First published online 18 July 2007
doi: 10.1242/dev.02874
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1 Section on Developmental Neuroscience, Porter Neuroscience Research Center, 35
Convent Dr, Room 2A-100, National Institute on Deafness and Other
Communication Disorders, National Institutes of Health, Bethesda, MD 20892,
USA.
2 University of Maryland College Park, Department of Biology, College Park, MD,
USA.
3 Genetics of Vertebrate Development Section, Cancer and Developmental Biology
Laboratory, National Cancer Institute, Frederick, MD, USA.
Author for correspondence (e-mail:
kelleymt{at}nidcd.nih.gov)
Accepted 29 May 2007
 |
SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Organ of Corti, Hair cell, Fgfr3, Mouse
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). The tunnel of Corti and the PCs that form this structure
are unique to the mammalian auditory system and are found in no other
vertebrate class. Defects in PC development result in significant hearing
impairment (Colvin et al.,
1996).
Despite their crucial role in cochlear function, the factors that regulate
PC formation are poorly understood. Previous work has demonstrated that
ongoing activation of one of the fibroblast growth factor receptors, Fgfr3, is
required for PC development (Colvin et al.,
1996; Mueller et al.,
2002). Ectopic activation of Fgfr3 in vitro by treatment with Fgf2
induces an overproduction of PCs, suggesting that the relative level of ligand
available for Fgfr3 activation plays a key role in regulating PC number and
position within the OC (Mueller et al.,
2002). Fgfr3 is one of four related receptors that bind to members
of the fibroblast growth factor family. All Fgf receptors are transmembrane
proteins that contain a tyrosine kinase (TK) domain in their intracellular
region. Fgfr activation is mediated through binding of one of at least 23
known Fgfs and a sulfated glycosaminoglycan such as heparin sulfate. Binding
of Fgf ligand and heparin leads to receptor dimerization, cross-activation of
the TK domains and downstream signaling through the MAP kinase signaling
pathway (Mohammadi et al.,
2005).
Within the developing cochlea, Fgfr3 is initially expressed at 
E16 in
a broad pool of progenitor cells located directly adjacent to developing IHCs
(Mueller et al., 2002;
Peters et al., 1993), the
first cells to differentiate within the epithelium
(Sobin and Anniko, 1984).
Based on the spatiotemporal pattern of expression, it seems likely that Fgfr3
is expressed in progenitors that will ultimately develop as PCs and OHCs, as
well as HeCs and DCs. As development proceeds, Fgfr3 is downregulated in
progenitors that develop as OHCs, HeCs and DCs, but is maintained in PCs
(Mueller et al., 2002;
Pirvola et al., 1995). RNA
expression analysis using quantitative PCR has suggested that the
Fgfr3c splice variant is the predominant isoform expressed in the
cochlea (Pickles, 2001). In
addition, ligand-binding assays indicate that the `c' isoform of Fgfr3 binds
to the Fgf8b isoform with high affinity
(MacArthur et al., 1995;
Olsen et al., 2006;
Ornitz et al., 1996). The
Fgf8b ligand has been shown to have important regulatory roles during pattern
formation, differentiation and cell growth throughout the developing embryo
and nervous system (Olsen et al.,
2006). Quantitative RT-PCR analysis has indicated that it is
expressed in the embryonic cochlear sensory epithelium
(Pickles, 2001). Here, we
demonstrate that Fgf8 is expressed in a pattern that is consistent
with an inductive role in PC development and that changes in the levels of
Fgf8, or in Fgfr3 activation, lead to corresponding changes in the number and
differentiation of PCs.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) for
Fgf8 and Fgfr3 on 12 µm frozen sections or whole organs
from cochleae isolated at E15, E16 and P0. A probe specific to exons 2 and 3
of Fgf8 (the region excised by Cre in the
Fgf8
2,3n/flox;
Foxg1cre/+ mutants) was also used on E16-18 cochleae from
Fgf82,3n/flox;
Foxg1cre/+ mutants and their wild-type littermates to
demonstrate excision of the targeted region.
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2,3n/flox; Foxg1cre/+ mutants and analysis of pillar cell defects2,3n/+;
Foxg1cre/+ males. Mice carrying these alleles have been
described previously (Meyers et al.,
1998; Hebert and McConnell,
2000). Mutant progeny of the genotype
Fgf82,3n/flox;
Foxg1cre/+ were visually identified based on obvious
defects in the development of the forebrain
(Storm et al., 2003). Siblings
were of the genotypes Fgf8+/flox;
Foxg1cre/+, Fgf8+/flox;
Foxg1+/+ or
Fgf82,3n/flox;
Foxg1+/+ and served as normal littermate controls.
Cochleae were dissected from mutants and littermate controls at E15.5, E16 and
E19, and fixed in either 4% paraformaldehyde (PFA) or 3% glutaraldehyde/2% PFA
overnight. Following fixation, the cochleae were dissected and the OC were
exposed and labeled with cell type-specific antibodies: anti-myosin VI
(Proteus Biosciences) 1:1000; anti-p75ntr (Chemicon) 1:1000;
anti-ß-actin (Sigma) 1:200. Secondary antibodies were conjugated to one
of the following: Alexa 350, Alexa 488, Alexa 546 or Alexa 633 (Molecular
Probes). In addition, filamentous actin was labeled using phalloidin at 1:200
conjugated to either Alexa 488 or Alexa 633 (Molecular Probes). Specimens were
then flat-mounted and the total length of the cochlear duct was measured. The
cochlea was then divided into four equal sections, each representing a quarter
of the total length of the cochlear duct, and the distances between the inner
hair cells and first row of outer hair cells (ITO distances) were determined
in each region (n=5 animals; greater than 50 cells counted per
region). All animal care and procedures were approved by the Animal Care and
Use Committee at NIH and complied with the NIH guidelines for the care and use
of animals.
Measurement of ITO distance
The inner-to-outer (ITO) distance is defined as the distance between the
lateral edge of the IHC and the medial edge of the first row OHC. This is the
distance encompassed by the inner pillar head. Digital images of the OC were
captured for each sample using a Zeiss 510 LSM confocal laser-scanning
microscope. Measurements of ITO distances were taken at three specific points
along the length of the cochlear duct of each sample, roughly at 25%, 50% and
75% of the distance from the most basal region and moving towards the apex. A
minimum of 15 ITO measurements were made at each of the three locations. Cell
counts were also taken of each cell type in the measured quadrants.
Histological sections
Temporal bones from control and
Fgf82,3n/+;
Foxg1cre/+ littermates were fixed in 3% glutaraldehyde/2%
paraformaldehyde, tissue was dehydrated in ethanol and then embedded in
Immunobed (Polysciences). Cochleae were oriented to generate mid-modiolar
sections, cut at 5 µm and stained with thionin.
Explant cultures
Explant cultures of embryonic cochleae were established as described
previously (Montcouquiol and Kelley,
2003) and maintained for 6 DIV. E13.5 explants were incubated for
24 hours before exposure to growth factors or antibodies that were diluted in
culture medium to the stated final concentrations along with 0.1% DMSO and 1
µg/µl heparin. Anti-Fgf8b, 75-150 µg/ml; anti-Fgf5, 75-150 µg/ml;
Fgf17, 300 ng/ml (all from R&D systems). Antibodies and proteins were used
at 100 times the ND50 and ED50, to ensure penetration
through the reticular lamina, a strong ionic barrier that exists at the
lumenal surface of the OC.
Electroporation
Full-length cDNA for murine Fgf8b was kindly provided by Elizabeth
Grove, University of Chicago
(Fukuchi-Shimogori and Grove,
2001). Fgf8b was excised from its original vector using
BamHI and then directly ligated into the pAM/CAG-IRES_EGFP vector at
the BamHI site. Orientation was determined by sequencing. Empty
pAM/CAG-IRES_EGFP vector and pAM/CAG-IRES_EGFP containing full-length
Fgf8b in the reverse orientation were used as controls.
Electroporation of cochlear explants was carried out as previously described
(Jones et al., 2006);
n>30 for each vector type.
Luminosity measurements
Images of electroporated explants were obtained using a Zeiss LSM510
confocal microscope. All samples were obtained during the same session using
the same laser power and detection settings. To quantify the effects of
overexpression of Fgf8, a rectangle (225 µmx110 µm) was
oriented such that the short dimension of the rectangle was parallel with the
line of PCs in the region being measured. The rectangle was positioned so that
its strial edge aligned with third row OHCs. Thus, the rectangle included a
110 µm stretch of the OC with the adjacent region of the greater epithelial
ridge containing transfected cells. Control and experimental regions were
obtained and then thresholded for both green and red pixels. The total number
of pixels of each color was then determined as a percentage of the total of
number of pixels within the entire rectangle.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Pirvola et al.,
1995), it seemed likely that hair cells located adjacent to
developing PCs might act as a source of ligand for Fgfr3. Furthermore, Fgf8, a
high affinity ligand for Fgfr3, has been reported to be expressed by the IHCs
of the adult cochlea (Pirvola et al.,
2002; Shim et al.,
2005). To determine whether Fgf8 could play a role in PC
development, the expression pattern for Fgf8 in the embryonic cochlea
was compared with that of Fgfr3 by ISH. Cochlear development proceeds
in a spatially conserved pattern in which cells in the basal region mature
prior to those in the apical region, allowing one to visualize multiple
developmental stages within the same ear
(Sobin and Anniko, 1984). No
expression of Fgf8 was observed between E13 and E15 (data not shown)
or at E16 in the less mature apex (Fig.
2A). However, Fgf8 expression was observed in a single
cell in the more mature basal region at E16
(Fig. 2B). Fgfr3
expression was also first observed in the basal region of the cochlea at E16
in a group of cells that correlates with developing PCs, OHCs, HeCs and DCs
(Fig. 2D). Weak expression of
Fgfr3 was observed in the apex of the same E16 cochlea
(Fig. 2C), suggesting that its
onset might slightly precede that of Fgf8. By P0, Fgf8
expression was clearly present in the single IHC
(Fig. 2E,G), whereas expression
of Fgfr3 had largely become restricted to the developing PCs
(Fig. 2F). Some expression of
Fgfr3 persisted in the developing DC and HeC region
(Fig. 2F); however, based on
immunolocalization, this expression appears to be downregulated as development
continues (Mueller et al.,
2002). These expression patterns demonstrate that the timing of
the onset of Fgf8 expression correlates strongly with the onset of
Fgfr3 expression and subsequent differentiation of PCs.
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) and the
Foxg1cre line (Hebert
and McConnell, 2000) were used to delete Fgf8 in a subset
of tissues. It has been demonstrated that Cre-mediated excision of the second
and third exons of the Fgf8flox allele results in complete
inactivation (Meyers et al.,
1998). The Foxg1 promoter induces expression of
Cre in a relatively small number of tissues, including the developing
otocyst, beginning at E8.5. By E9, the expression of Cre is strong in
virtually all cells within the otocyst
(Hebert and McConnell, 2000),
well before the normal onset of Fgf8 expression.
Embryos that were
Fgf82,3n/flox;
Foxg1cre/+ (see Materials and methods for specific genetic
cross) die at birth as a result of defects in the development of the forebrain
(Storm et al., 2003); however,
overall development of the inner ear and cochlea appeared normal. To examine
the effects of inactivation of Fgf8 on PC development, cochleae were
obtained from mutants at E15 and E18.5. Consistent with the timing of the
onset of Fgf8, there were no obvious differences in cellular
patterning or in the expression of p75ntr (a marker that is
co-expressed with Fgfr3 during cochlear development; also known as Ngfr -
Mouse Genome Informatics) (Mueller et al.,
2002) between mutants and controls at E15 (data not shown).
Although development of IHCs and OHCs appeared normal at E18.5
(Fig. 3A,B), there was a marked
decrease in the size and number of PCs as well as a decrease in the overall
levels of expression of p75ntr, both in the PCs and HeCs
(Fig. 3C,D). The effects of
deletion of Fgf8 were quantified by determining the distance between
IHCs and OHCs, referred to as the ITO distance
(Mueller et al., 2002), at
different positions along the basal-to-apical axis of the OC. The developing
pillar heads expand as the cells differentiate and thus larger ITO distances
are reflective of more advanced PC development
(Mueller et al., 2002).
Deletion of Fgf8 resulted in a significant decrease in ITO distances
along the length of the cochlea (Fig.
3G), similar to that seen in Fgfr3-knockout mice (B.E.J.,
C. Puligilla and M.W.K., unpublished).
Rather than a complete absence of p75ntr-positive PCs, as seen
when Fgfr3 is pharmacologically inhibited
(Colvin et al., 1996;
Mueller et al., 2002), some
p75ntr-positive cells were clearly present in the PC space in
Fgf82,3n/flox;
Foxg1cre/+ mutants
(Fig. 3D). Therefore, semi-thin
plastic cross-sections of the cochlea from
Fgf82,3n/flox;
Foxg1cre/+ mice were examined at E15 (n=4) and
E18.5 (n=5). As expected based on the timing of onset of
Fgf8 expression, overall structure of the epithelium and putative
developing PCs appeared normal in cross-sections from E15 (data not shown). By
contrast, at E18.5, two cells were present in the region of the epithelium
between the IHCs and OHCs (Fig.
3E,F), but these cells had either weak lumenal projections or no
projections at all. In some sections, the IHCs and OHCs appeared to be in
direct contact with eachother (Fig.
3F, magnified in inset with a red line to show the IHC boundary
and a green line to show the OHC boundary). PCs with weak or no lumenal
projections were observed along the entire length of the cochlea with no
region-specific variations. To ensure that Cre-mediated excision of
Fgf8 exons 2 and 3 was complete in these mutants, ISH was performed
on Fgf82,3n/flox;
Foxg1cre/+ mutants and their control littermates
(n>3 for each genotype analyzed) at E16-18 using a probe generated
from exons 2 and 3 of the Fgf8 gene. Control and mutant cochleae were
processed together and the colorization step was deliberately extended to
ensure detection of any residual Fgf8. A single row of
Fgf8-positive IHCs was clearly present in control cochleae; however,
no expression of Fgf8 was observed in the mutant cochleae
(Fig. 3H). These results
indicate a complete deletion of Fgf8 in
Fgf82,3n/flox;
Foxg1cre/+ mutants.
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2,3n/flox;
Foxg1cre/+ mutants, compared with the complete absence of
these cells in cochlear explants in which Fgfr3 activation has been inhibited
(Mueller et al., 2002),
suggests possible residual Fgfr3 activity in
Fgf82,3n/flox;
Foxg1cre/+ mutants. This could be the result of functional
compensation within the Fgf8-deficient mutant cochleae, whereby another
endogenously expressed Fgf may bind to and activate Fgfr3 when no Fgf8 ligand
is present. Therefore, we sought to inhibit Fgf8 signaling at the protein
level using an Fgf8-function-blocking antibody on cochlear explant cultures.
As a control, similar explants were exposed to an antibody that specifically
blocks the function of Fgf5, a ligand not reported to be endogenously
expressed within the OC. Explants were established on E13, exposed to
anti-Fgf8 beginning after 24-36 hours, maintained for 4 to 6 days in vitro
(DIV), then fixed and stained to examine PC development. Exposure to anti-Fgf8
resulted in a complete loss of p75ntr labeling, a lack of obvious
pillar heads (Fig. 4C) and ITO
distances approaching zero (Fig.
4D), indicating a complete inhibition of PC development. This
effect phenocopies that observed in explants exposed to the Fgfr antagonist
SU5402 (Mueller et al., 2002).
By contrast, exposure to anti-Fgf5 had no apparent effect on PC development
(Fig. 4B,D). If the addition of
anti-Fgf8 was delayed until the equivalent of E17, reductions in
p75ntr staining and ITO distance were observed, but their magnitude
was decreased compared with those observed upon exposure to anti-Fgf8 for 6
DIV (data not shown). Similarly, a 72-hour transient exposure to anti-Fgf8
beginning at E14.5 resulted in a decrease in ITO distances that was consistent
with a delay in the onset of PC differentiation (data not shown). Based on
these results, there appears to be an ongoing requirement for Fgf8 throughout
the 5-day period of embryonic PC development. The complete inhibition of
pillar head formation in these explants, in contrast to the small residual
development in Fgf82,3n/flox;
Foxg1cre/+ mutants, suggests that either Fgf8 was
not completely deleted in the
Fgf82,3n/flox;
Foxg1cre/+ mutants, or that the anti-Fgf8 antibody
recognizes and inhibits other Fgfs within the epithelium that also activate
Fgfr3. The first explanation seems less likely, considering that ISH indicated
no expression of Fgf8 mRNA at E16.5. By contrast, Fgf10 is
expressed in the developing inner sulcus
(Pauley et al., 2003), and
preliminary results indicate that Fgf17 is also expressed in the
cochlea (B.E.J., K. L. Mueller and M.W.K., unpublished).
Ectopic Fgf8 expression results in overexpression of pillar cell markers
It has been shown that addition of exogenous Fgf2 results in the formation
of additional rows of PCs (Mueller et al.,
2002), suggesting that the amount of Fgf within the epithelium
could be a limiting factor in PC formation. Therefore, the effects of
increased Fgf8 within the cochlea were determined by transfecting cochlear
explants with an Fgf8 expression vector containing EGFP as
an independent transcript to identify transfected cells
(Fukuchi-Shimogori and Grove,
2003; Jones et al.,
2006; Zheng and Gao,
2000). For controls, explants were electroporated with a vector
that expressed either EGFP alone or EGFP with Fgf8
in the reverse orientation. Electroporated explants typically contained large
clusters of transfected cells in Kolliker's organ, a population of epithelial
cells located adjacent to the developing OC
(Fig. 5A,B). In control
electroporated explants, no changes in PC number, as determined by expression
of p75ntr, were observed in regions of the OC located adjacent to
large clusters of transfected cells (Fig.
5A,C). By contrast, there was a marked increase in the number of
p75ntr-positive cells, and a decrease in the number of OHCs, in
regions of the sensory epithelium located adjacent to large clusters of
Fgf8-transfected cells (Fig.
5B,D and data not shown). PCs located at a distance from large
clusters of Fgf8-expressing cells were unaffected, suggesting that
Fgf8 has a limited diffusion radius within the cochlear epithelium
(Fig. 5B). However, it is also
possible that the gradient of Fgf8 is more rapidly decreased because of
diffusion into the culture media.
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Ectopic activation of Fgfr3 increases the expression of pillar cell markers at the expense of outer hair cells
The results presented above are consistent with the hypothesis that an
increased level of Fgfr3 activation leads to a greater number of
Fgfr3-positive progenitors becoming committed to develop as PCs. Therefore, we
sought to fully activate Fgfr3 throughout the developing OC by exposing
cochlear explants to Fgf protein. Treatment with Fgf8 protein had no apparent
effect on the development of the OC. Although the basis for this is unknown,
we were able to obtain a strong effect with Fgf17 protein. To initially
confirm that Fgf17 activates Fgfr3 in cochlear explants, explants were exposed
to the anti-Fgf8 function-blocking antibody and Fgf17 protein. The presence of
Fgf17 was sufficient to rescue PC development in these explants (data not
shown).
Treatment with Fgf17 resulted in a conversion of the OHC region of the OC into a band of cells that were positive for p75ntr (Fig. 6A-D). Absence of expression of the hair cell marker myosin VI (Fig. 6E,F) and lack of stereocilia (Fig. 6C,D) indicated that these p75ntr-positive cells were inhibited from developing as OHCs. When a few OHCs were present in Fgf17-treated explants, each was surrounded by a group of p75ntr-negative cells, suggesting that the presence of an OHC was sufficient to cause a local downregulation of p75ntr, even in the presence of Fgf17 (Fig. 6G-I). The effects of Fgf17 treatment appeared to be restricted to the PC/OHC region as myosin VI-positive IHCs were still present in all explants (data not shown). In addition, HeCs located lateral to the OHC domain were also present in Fgf17-treated explants.
Considering that expression of p75ntr is a marker for undifferentiated cells at very early stages of OC development. we wanted to determine whether the large number of p75ntr-positive cells in Fgf17-treated explants represented induction of ectopic PCs, maintenance of undifferentiated cells, or both. ß-actin is expressed strongly in PCs and more weakly in OHCs, DCs and HeCs at P0 (see Fig. S1A-F in the supplementary material), but is not expressed in any cell types within the OC prior to E16. Cells which are positive for both p75ntr and ß-actin can thus be classified as differentiated PCs. In control explants (established on E13 and maintained for 7 DIV), only the cells located directly adjacent to the IHCs were positive for both p75ntr and ß-actin (Fig. 6E). Treatment with Fgf17 resulted in a marked increase in the number of p75ntr-positive cells, with many located throughout the OHC region. However, expression of ß-actin was restricted to the cells directly adjacent to the IHCs (Fig. 6F,F') and to a band of cells located on the lateral edge of the OC, which normally develop as HeCs (Fig. 6F, arrows). The presence of strong p75ntr expression in the absence of ß-actin suggests that the effect of activation of Fgfr3 within the OHC region is to inhibit differentiation rather than to induce a PC fate. To confirm this, explants were treated with Fgf17 for 72 hours followed by a 72-hour recovery period. In contrast to the effect of continuous application of Fgf17 (see Fig. S1G in the supplementary material), the patterning of the OC developed normally in explants in which Fgfr3 had been transiently activated. However, OHC development in these explants appeared to be delayed by approximately 72 hours based on the differentiation of OHCs in the second and third rows (see Fig. S1H,I in the supplementary material). These results strongly suggest that activation of Fgfr3 inhibits progenitor cells from developing as hair cells.
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Treatment with Fgf17 also induced a marked increase in the expression of ß-actin (Fig. 6F) and p75ntr (Fig. 7B) in a lateral band of cells that would normally develop as HeCs, suggesting that these cells may assume a PC fate in response to increased activation of Fgfr3. Confocal analysis indicated a marked change in these cells, including increased height, decreased width and maintenance of expression of p75ntr (Fig. 7E,H). Many of these cells developed lumenal projections similar to those of PCs (Fig. 7I-L). Thus, it seems that increased activation of Fgfr3 induces progenitor cells that would normally have developed as HeCs to form as PCs instead.
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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), Fgf8
or Fgf17, deletion of the sprouty 2 gene, an Fgf signaling pathway antagonist
that is expressed in a similar domain to Fgfr3, results in an extra row of PCs
(Shim et al., 2005). These
results are consistent with the hypotheses that normal Fgfr3 activation is
limited to the cells located directly adjacent to the IHCs and that changes in
the spatial activation of Fgfr3, either through increased availability of
ligand or through decreased receptor antagonism, results in defects in
cellular patterning.
The results presented here coupled with previous findings suggest that
Fgfr3 mediates two different aspects of the development of the OC. First,
activation of Fgfr3 inhibits the differentiation of cells as OHCs. As
discussed, the presence of PCs creates a disruption in the normal alternating
cellular mosaic of hair cells and supporting cells. The developmental and
evolutionary mechanisms that generate this disruption are unknown, but the
data presented here suggest that inhibition of OHC formation through
activation of Fgfr3 could represent an import aspect of this regulatory
mechanism. This hypothesis is supported by the recent demonstration of an
increase in the number of OHCs in Fgfr3 mutants
(Hayashi et al., 2007;
Puligilla et al., 2007).
Fgfr3 is also expressed in the developing chick basilar papilla
(cochlea) but is downregulated in developing hair cells
(Bermingham-McDonogh et al.,
2001). Fgfr3 expression is maintained in basilar
papilla-supporting cells throughout life, and only becomes downregulated
during a regenerative response to hair cell loss
(Bermingham-McDonogh et al.,
2001). Once hair cells have been replaced, Fgfr3
expression returns, suggesting that downregulation of Fgfr3 might be necessary
to allow new hair cell formation.
|
),
suggesting the possibility that these cells might retain a higher degree of
cellular plasticity and that ongoing activation of Fgfr3 might be required to
maintain PCs in a differentiated state. This hypothesis is supported by the
recent demonstration that, in comparison with other cells in the OC, PCs
appear to possess a comparatively higher level of plasticity
(Izumikawa et al., 2005;
Kiernan et al., 2005;
White et al., 2006), and may
even be able to differentiate into hair cells under some circumstances
(White et al., 2006). Based on
these results, it seems possible that inhibition of Fgfr3 activation in mature
PCs could cause these cells to revert to a less differentiated state. Under
some circumstances, it might then be possible to induce these cells to adopt
an alternative fate, such as differentiation as a hair cell. The second role of Fgfr3 appears to be to specify the fate and/or subsequent differentiation of PCs. Deletion of either Fgfr3 or its endogenous ligand Fgf8 leads to a disruption in PC differentiation, whereas increased availability of Fgfs enhances the pace of PC differentiation. The induction of ectopic PCs is apparently restricted to a band of cells located on the lateral edge of the OC. The reasons for this restriction are unclear; however, the position of these cells is somewhat similar to the position of the endogenous PCs in that they are located on a border of the OHC domain. Therefore, it seems possible that cells within the OHC domain might be prevented from developing as PCs. This hypothesis is supported by the observation that OHCs were capable of inducing a local decrease in p75ntr even in the presence of Fgf17, suggesting that OHCs or OHC progenitors might exert a local influence that is not compatible with PC development. This type of interaction, along with the limited expression of Fgf8, might play a role in ensuring that PCs only develop between the IHCs and first row OHCs.
The limited expression of Fgf8 in the single row of IHCs, along with the
demonstrated roles of Fgfs in the development of PCs and OHCs, suggest that
IHCs act as a global organizing center for the development of the OC. Cellular
differentiation proceeds in a gradient that begins with the IHCs and moves
laterally through the PCs and OHCs. A similar role for Fgf8 has been described
during neurogenesis of the chick spinal cord. During development of the neural
tube, Fgf8 signals arising from the caudal neural plate act to regulate the
timing of neural development within the spinal cord by inhibiting
differentiation (Diez del Corral et al.,
2002; Diez del Corral et al.,
2003), thus maintaining a balance between neuronal and glial cell
types. When the Fgf8 signal is removed or inhibited, precocious
differentiation of spinal cord neurons is observed
(Diez del Corral et al., 2002).
Based on the above results, it seems likely that Fgf8 signaling may prolong
the ability of PCs to switch fates and undergo mitosis. In addition, the
expression of multiple sprouty genes within the DCs
(Shim et al., 2005) can act to
inhibit Fgf8 signaling and, thereby, to also inhibit the ability of DCs to
assume a more plastic role within the OC. However, the HeC region never
expresses sprouty genes and thus, when exposed to ectopic Fgf8 or Fgf17, can
develop as PCs.
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Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/134/16/3021/DC1
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ACKNOWLEDGMENTS |
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Footnotes |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Bermingham-McDonogh, O., Stone, J. S., Reh, T. A. and Rubel, E. W. (2001). FGFR3 expression during development and regeneration of the chick inner ear sensory epithelia. Dev. Biol. 238,247 -259.[CrossRef][Medline]
Colvin, J. S., Bohne, B. A., Harding, G. W., McEwen, D. G. and Ornitz, D. M. (1996). Skeletal overgrowth and deafness in mice lacking fibroblast growth factor receptor 3. Nat. Genet. 12,390 -397.[CrossRef][Medline]
Diez del Corral, R., Breitkreuz, D. N. and Storey, K. G.
(2002). Onset of neuronal differentiation is regulated by
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