1 The Jackson Laboratory, 600 Main Street, Bar Harbor, ME 04609 USA
2 Institut für Molekularbiologie, Medizinische Hochschule, 30625 Hannover,
Germany
3 Departments of Molecular Biology and Pharmacology and Medicine, Washington
University School of Medicine, St Louis, MO 63110, USA
* Author for correspondence (e-mail: gridley{at}jax.org)
Accepted 18 July 2005
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SUMMARY |
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Key words: Notch signaling, Lateral inhibition, Hair cell differentiation, Cochlea
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Introduction |
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Because both the Dll1 and Jag2 genes are expressed in
nascent hair cells (Lanford et al.,
1999; Morrison et al.,
1999
), the prospect of redundancy was a real possibility, as both
ligands may be necessary to fully deliver a lateral inhibitory signal. To test
whether the Dll1 gene also plays a role in lateral inhibition and to
investigate potential genetic interactions with Jag2 mutations, we
generated embryos that carried various combinations of Jag2 and/or
Dll1 mutant alleles. Our results show that Dll1 functions
synergistically with Jag2, demonstrating that both ligands are
required to regulate the numbers of hair cells that form in the mammalian
cochlea. Using conditional gene inactivation, we also show that both the JAG2
and DLL1 ligands are likely to signal through the NOTCH1 receptor. Consistent
with the proposed lateral inhibition model, most supernumerary hair cells in
the Dll1/Jag2 double mutant cochleae did not arise through excess
proliferation, suggesting instead a switch in cell fate. However, supporting
cells did exhibit abnormal proliferation, implicating a novel role for the
Notch pathway in regulating cellular proliferation in the ear.
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Materials and methods |
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Immunocytochemistry
For whole-mount preparations, inner ears were dissected and fixed overnight
in 4% paraformaldehyde. The bony shell and the stria vascularis were removed,
and the ears were incubated with a biotinylated lectin (Griffonia simplifonia
I, Vector Laboratories) diluted 1:100. A FITC-labeled avidin secondary reagent
was used to visualize the hair cells. All other immunocytochemistry was
performed on standard 7-µm paraffin-embedded sections. Antibodies used
included anti-myosin VIIa (1:1000, a gift from Drs A. EL-Amraoui and C. Petit,
Institut Pasteur, Paris, France), anti-p27kip1 (1:100, Neomarkers)
and anti-BrdU (1:500, Roche). For anti-p27kip1 labeling, an
antigen-retrieval step was performed by boiling the sections for 10 minutes in
10 mM citric acid. For BrdU labeling, two different protocols were used: in
the first, antigen retrieval was performed as described previously for
p27kip1, followed by pepsin digestion (100 µg/ml for 20 minutes
at 37°C) and acid treatment (2N HCl for 30 minutes at 37°C). For
doubly labeled sections, heat-activated antigen retrieval was performed
followed by DNAse I digestion (5 U/ml for 30 minutes at 37°C). Cell death
was examined by TUNEL staining of paraffin-embedded sections, using the TMR in
situ cell detection kit (Roche).
Cell counts
Hair cell counts from lectin-stained wholemounts Hair cell counts were
performed on mid-basal regions of the lectin-stained cochleae, extending
between 800 and 1400 µm. Each genotype contained counts from three or four
different embryos. After capture of high-resolution images of the cochleae,
counts and measurements were performed using Zeiss Axiovision software.
Hair and supporting cell counts from myosin VIIa/p27kip1 immunostained sections
For each ear, images from 32 sections through the mid-modiolar region of
the cochlea that were triply labeled for 4'-6-Diamidino-2-phenylindole
(DAPI; to stain nuclei), myosin VIIa (to label hair cells) and
p27kip1 (to label supporting cells) were captured using Zeiss
Axiovision software. Nuclei from cells that were doubly labeled with DAPI and
either myosin VIIa (hair cells) or p27kip1 (supporting cells) in
the basal and middle turns of the organ of Corti were counted.
Cell proliferation
To examine cell proliferation, pregnant female mice were injected with a
BrdU solution (10 mg/ml in PBS; final dose, 50 µg per gram of body weight),
three times daily (at 4 hour intervals), between E14.5 and E17.5. Animals were
euthanized and half heads were fixed and embedded for paraffin sectioning.
Electron microscopy
Inner ears were prepared for scanning electron microscopy, as described
previously, using a version of the osmium tetroxide-thiocarbohydrazide (OTOTO)
method (Kiernan et al., 1999).
Specimens were examined with a Hitachi 3000N scanning electron microscope.
In situ hybridization
For sample preparation, inner ears were dissected from the head and fixed
overnight in 4% paraformaldehyde. After washing in PBS, the bony shell and
stria were removed from the cochleae and the samples were dehydrated in
methanol. In situ hybridization was performed as described
(Stern, 1998), with the
exception of the post-hybridization washes, which were done according to Rau
et al. (Rau et al., 1999
).
After the reactions were judged to be complete, cochleae were flat mounted on
glass slides in 70% glycerol. Probes for
-tectorin and ß-tectorin
(gifts from Drs K. Legan and G. Richardson, University of Sussex, UK) were as
described (Rau et al.,
1999
).
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Results |
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Cochleae that lacked both copies of Jag2, and that carried either a single null allele of Dll1 (Dll1+/- Jag2-/-) or a null allele combined with the Dll1 hypomorphic allele (Dll1hyp/- Jag2-/-), showed even larger increases in hair cell numbers than Jag2-/- cochleae (Fig. 1G,H). In Dll1hyp/- Jag2-/- cochleae, two to four rows of inner hair cells were present and four to six rows of outer hair cells could be identified. However, unlike the Dll1+/- Jag2+/- or Jag2-/- cochleae, the rows of hair cells were more disorganized and the hair cells were very densely packed, making accurate determination of hair cell numbers difficult in whole-mount preparations.
We used scanning electron microscopy to examine in more detail the
morphological defects in the double mutant cochleae
(Fig. 2). Dll1hyp/- Jag2-/- mutant cochleae were
extremely disorganized, both in the patterning of the sensory cell rows and,
at a single hair cell level, in the loss of polarity and disorganization of
many hair cell stereocilia bundles (Fig.
2F). When compared with Jag2-/- and
Dll1+/- cochleae (Fig.
2A-D), it is clear the amount of disorganization correlates with
the amount of Notch ligand that is present, as some disorganization is present
even in Jag2-/- cochleae. However, it is not clear from
these data whether the Notch pathway plays a direct role in planar polarity
signaling (Barald and Kelley,
2004) and/or patterning of the hair cells, or whether the observed
disorganization may be a secondary effect of the increased numbers of hair
cells present in the mutant cochleae.
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p27kip1 immunostaining of the
Dll1hyp/-Jag2-/- mutant and control cochleae
indicated that many of the missing cells resided beneath the outer hair cells
and were presumably Deiter's cells (Fig.
3C,D). In order to assess which supporting cell populations were
decreased, Dll1/Jag2 double mutant and control cochleae were
processed for whole-mount in situ hybridization using probes that mark
specific populations of supporting cells, -tectorin and
ß-tectorin (Rau et al.,
1999
). Expression of
-tectorin in wild-type
cochleae marks the supporting cell populations surrounding the organ of Corti
proper (including Koelliker's organ), the supporting cells surrounding the
inner hair cells (inner phalangeal cells), and Hensen's cells. In
Dll1+/- Jag2-/- cochleae,
-tectorin expression appeared to be similar to in controls,
suggesting that none of these surrounding supporting cell populations were
converted to hair cells (Fig.
3E,F). ß-Tectorin marks several supporting cell regions that
lie in the organ of Corti proper, including the inner and outer pillar cells,
the last row of Deiter's cells, and a smaller domain of cells in Koelliker's
organ. In Dll1+/- Jag2-/- cochleae, most of
these expression domains appeared to be similar to those in controls, with the
exception of the Deiter's cell expression domain
(Fig. 3G,H). Here, the
ß-tectorin expression was punctate rather than being maintained in a
continuous stripe, and appeared to be reduced, suggesting that many of the
missing supporting cells were derived from the Deiter's cell population.
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Discussion |
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The standard model of lateral inhibition predicts that, if supernumerary
sensory cells are produced via a cell fate switch, a concomitant loss of
nonsensory supporting cells should be observed along with the increase in
sensory hair cells. However, an examination of supporting cell populations
revealed only a modest loss of p27kip1-positive supporting cells
that was significantly different from the increase in hair cell numbers (only
about a 1.2-fold loss in supporting cells compared with a 1.7-fold increase in
hair cells; Fig. 3I). This
affect was even stronger in Foxg1-Cre Notch1flox/- mutant
cochleae, where there was a dramatic 3-fold increase in hair cells accompanied
by only about a 1.6-fold drop in p27kip1-positive supporting cells
(Fig. 5E). Deiter's cells
appeared to be the most dramatically reduced supporting cell population,
suggesting a cell fate switch from Deiter's cells to outer hair cells. Cell
death does not appear to account for the missing supporting cells, as the
number of apoptotic profiles was not increased in either Dll1/Jag2
double mutant cochleae at E15.5 or Foxg1-Cre Notch1flox/-
mutant cochleae at E18.5, as determined using the TUNEL assay (data not
shown). BrdU incorporation studies in Dll1/Jag2 double mutant
cochleae revealed ectopic proliferation of supporting cells with very few
labeled hair cells in the double mutant cochleae. Interestingly, pillar cell
numbers were neither decreased nor increased in any of the Notch mutants
described here, despite the fact that they were frequently observed to be
dividing. These data suggest that pillar cells may have unique stem cell-like
properties in the developing organ of Corti. Stem cell-like cells have been
identified in the adult mammalian utricle
(Li et al., 2003), although it
is not known whether similar cells are present in the cochlea. Taken together,
these data indicate that the majority of the supernumerary hair cells arise
via a cell fate switch, and not through continued cell proliferation. However,
continued proliferation of the remaining nascent supporting cells compensates
for the loss of supporting cell precursors, resulting in only modest decreases
in the supporting cell population. These data support a role for the Notch
pathway in mediating lateral inhibition in the inner ear, and also reveal a
role for Notch signaling in the control of cell proliferation within the
developing organ of Corti (Fig.
6).
A previously described role for the Notch pathway in regulating cell
proliferation has been to maintain cells in an undifferentiated state, thereby
promoting cell proliferation in many contexts. However, it has become clear in
recent years that the effects of Notch signaling on cell proliferation are
complex and context dependent (Weng and
Aster, 2004). For example, an emerging role for the Notch pathway
in promoting differentiation and cell cycle withdrawal has been revealed by
studies in the skin and nervous system. Conditional Notch1 deletion
in mouse skin causes hyperplasia and deregulation of differentiation, leading
to the development of basal cell carcinoma-like tumors
(Rangarajan et al., 2001
;
Nicolas et al., 2003
).
Similarly, specific Notch1 downregulation is found in aggressive cervical
cancers (Talora et al., 2002
).
These results have led to the suggestion that, in some contexts, Notch
signaling may act as a tumor suppressor by promoting differentiation and cell
cycle withdrawal. In the nervous system, the Notch pathway has been implicated
in promoting the glial cell fate (Furukawa
et al., 2000
; Hojo et al.,
2000
; Morrison et al.,
2000
). Given that inner ear supporting cells share some
characteristics with glia, these results raise the possibility that the Notch
pathway plays an instructive role in supporting cell differentiation in the
cochlea. When Notch signaling is downregulated, as in this study, some
progenitor cells may fail to differentiate properly and continue dividing.
Thus, Notch signaling may play a dual role in sensory differentiation in the
inner ear, preventing adoption of the hair cell fate through a lateral
inhibitory mechanism while promoting cell cycle withdrawal and supporting cell
differentiation. Alternatively, the continued progenitor/supporting cell
proliferation may be an indirect effect caused by the loss of contact-mediated
inhibition due to the supporting cell fate conversion or other cellular
changes in the epithelium. Regeneration studies using ototoxic drugs or
acoustic trauma in the avian inner ear and in the mammalian vestibular regions
have shown that hair cell death triggers proliferation of the supporting cells
(Corwin and Cotanche, 1988
;
Ryals and Rubel, 1988
;
Warchol et al., 1993
;
Matsui et al., 2002
). This
suggests that, under normal circumstances, the hair cell exerts an
anti-proliferative effect on the surrounding support cells, although the
molecular identity of this signal is not known. It is interesting to note that
analysis of the zebrafish Mind bomb mutant, a mutation in a gene encoding a
ubiquitin ligase involved in Notch signaling, demonstrated a 10-fold excess
hair cells in the inner ear, far more than could be explained by a simple cell
fate conversion (Haddon et al.,
1998
). These results suggest that excess cell division may also
occur in these mutant ears, although proliferation was not specifically
examined. Taken together, these data suggest there may be a direct role for
Notch signaling in the control of cell proliferation in the organ of
Corti.
|
Both Dll1/Jag2 double mutant cochleae and Foxg1-Cre
Notch1flox/- cochleae displayed severely disorganized hair
cell rows, and loss of organization and polarity of the hair cell
stereociliary bundles. It is not clear whether this disorganization is a
direct consequence of reduced Notch signaling, or whether it is a secondary
event resulting from the abnormal cellular composition of the cochlea. Recent
work has shown that an evolutionarily conserved mechanism for generating cell
polarity within epithelial cell layers, termed planar cell polarity (PCP), is
involved in regulating the polarity of inner ear hair cells and the
orientation of their stereocilia bundles
(Barald and Kelley, 2004). A
role for Notch signaling in PCP has not been reported in any vertebrate
system. However, a role for the Notch pathway in planar polarity has been
shown during eye development in Drosophila, where Notch signaling
specifies the R4 photoreceptor cell fate
(McNeill, 2002
). Proper
specification of the R3 and R4 cell fates is essential for the loss of
symmetry within the ommatidial clusters and thus the proper genesis of PCP
within the eye. Similarly, correct specification of the hair cell and
supporting cell fates may be required to establish PCP within the inner ear.
Further insights into a possible role for Notch signaling in planar polarity
in vertebrates may come from gene expression or genetic interaction studies
with recently identified planar polarity genes in the inner ear
(Curtin et al., 2003
;
Montcouquiol et al., 2003
;
Lu et al., 2004
). However, it
should be noted that, unlike the previously identified PCP mouse mutants where
hair cell bundles are intact but disoriented, many of the hair bundles in the
Notch mutants appear to have lost their organization completely. This suggests
that reduced Notch signaling may affect a more basic level of bundle
organization than PCP. Moreover, similarly disorganized bundles have been
observed in mutant cochleae that lack the Rb gene, which exhibit a
large overproliferation of hair cells
(Mantela et al., 2005
;
Sage et al., 2005
). Taken
together, these data suggest that the disorganization may be an indirect
effect of having too many hair cells in the epithelium, leading to a
disruption in the signals that polarize and organize the stereocilia
bundles.
These results highlight the complexity of Notch signaling in the inner ear,
and demonstrate that the Notch pathway plays a dual role in regulating
cellular differentiation and patterning in the cochlea, acting both through
lateral inhibition and the control of cellular proliferation
(Fig. 6). Unlike birds and
amphibians (Corwin and Cotanche,
1988; Ryals and Rubel,
1988
; Corwin and Oberholtzer,
1997
), mammals demonstrate little regenerative potential in the
inner ear (Johnsson et al.,
1981
; Forge,
1985
), which may be related, at least in part, to a failure of
supporting cell proliferation after injury
(Roberson and Rubel, 1994
).
Our results suggest that modulation of the Notch pathway may represent an
important avenue for regeneration studies in the mammalian inner ear.
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ACKNOWLEDGMENTS |
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