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First published online 29 March 2007
doi: 10.1242/dev.000760
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1 National Institute on Deafness and other Communication Disorders, Rockville,
MD 20850, USA.
2 Department of Genetics, University of Pennsylvania School of Medicine,
Philadelphia, PA, USA.
3 Institute for Animal Developmental and Molecular Biology, Heinrich-Heine
University, Düsseldorf, Germany.
* Author for correspondence (e-mail: wud{at}nidcd.nih.gov)
Accepted 20 February 2007
 |
SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Inner ear, Gli, Sonic hedgehog, Shh, Activator, Repressor, Mouse
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Fekete and Wu, 2002;
Torres and Giraldez,
1998).
Shh, a member of the Hedgehog family of secreted signaling proteins, is
essential for many aspects of vertebrate development
(Ingham and McMahon, 2001).
Mutations in the Shh gene or inappropriate regulation of the Shh
signaling pathway causes cancers in humans
(Pasca di Magliano and Hebrok,
2003; Taipale and Beachy,
2001), as well as developmental defects in a variety of organs in
both humans and mice (Bale,
2002; Hammerschmidt et al.,
1997; McMahon et al.,
2003; Riccomagno et al.,
2002).
Inner ear analyses of Shh-knockout mouse embryos, as well as
midline ablation experiments in chicken embryos, demonstrated that Shh
emanating from the floor plate or notochord is required for the specification
of ventral inner ear structures such as the saccule and cochlear duct
(Bok et al., 2005;
Riccomagno et al., 2002). The
expression patterns of Patched1 (Ptc1, also known as
Ptch1 - Mouse Genome Informatics; the receptor for Shh) and
Gli1 (a transcriptional mediator of Shh signaling) within the otic
epithelium and periotic mesenchyme, indicate that these cells respond directly
to Shh (Riccomagno et al.,
2002). However, it was not clear how Shh mediates formation of the
various inner ear structures. Deciphering the molecular mechanisms underlying
Shh signal transduction in the ear is of fundamental importance considering
that mutations in genes associated with this pathway, including SALL1
and GLI3, are known to cause deafness in humans
(Kohlhase et al., 1998) (E. C.
Driver and M. W. Kelley, personal communication).
Cubitus interruptus (Ci), is the major mediator of Hedgehog (Hh) signaling
in Drosophila (Aza-Blanc and
Kornberg, 1999; Ingham and
McMahon, 2001). The three vertebrate Ci homologs, Gli1, Gli2 and
Gli3, are also thought to regulate most, if not all, of the transcriptional
responses to Hh signaling (Ingham and
McMahon, 2001). Gli1 contains a transcriptional activator domain
and was shown to positively regulate expression of Hh target genes
(Ingham and McMahon, 2001).
However, Gli1-null mutants are viable and do not show any obvious
developmental defects (Bai et al.,
2002; Park et al.,
2000). Gli2, by contrast, is required for the proper formation of
several organs including the lung and CNS
(Ding et al., 1998;
Matise et al., 1998;
Motoyama et al., 1998).
Despite the presence of both transcriptional activator and repressor domains,
Gli2 is thought to act predominantly as a positive regulator of Hh target gene
expression (Bai and Joyner,
2001).
Gli3 functions as either a transcriptional activator or repressor,
depending on the availability of Hh protein
(Wang et al., 2000). In the
absence of any Hh input, the C-terminal region of Gli3 is cleaved, and the
truncated N-terminal domain serves to repress the transcription of Hh target
genes (Wang et al., 2000). In
the presence of Hh, Gli3 is not cleaved and can operate as a transcriptional
activator (Dai et al., 1999;
Bai et al., 2004). The
Hh-dependent regulation of Gli3 processing is essential for many different
aspects of embryonic development, including limb digit number and identity,
dorsoventral patterning of the neural tube and somites, as well as various
aspects of renal, bone and mammary gland development
(Ahn and Joyner, 2004;
Buttitta et al., 2003;
Hatsell and Cowin, 2006;
Hu et al., 2006;
Koziel et al., 2005;
Litingtung and Chiang, 2000;
Litingtung et al., 2002;
McDermott et al., 2005;
Persson et al., 2002;
Rallu et al., 2002).
Mutations in GLI3 are responsible for several human disorders
including Pallister-Hall syndrome (PHS)
(Kang et al., 1997), Greig
cephalopolysyndactyly syndrome (GCPS)
(Vortkamp et al., 1991) and
postaxial polydactyly type A (Radhakrishna
et al., 1997). Several mouse models with mutations in
Gli3, similar to those reported in human subjects, are available. For
example, Extra-toesJ (XtJ) mutants, in
which Gli3 encodes a non-functional transcript
(Hui and Joyner, 1993;
Maynard et al., 2002), exhibit
characteristic defects similar to human GCPS patients
(Hui and Joyner, 1993;
Vortkamp et al., 1991).
Another mouse mutant, Gli3
699, which only expresses
the truncated N-terminal region of Gli3, was modeled after mutations
found in PHS patients (Böse et al.,
2002).
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699. Our results show that several mechanisms are
involved in mediating Shh signal transduction in the inner ear. For
development of the distal region of the cochlear duct, which is positioned
closest to the source of Shh in the notochord and floor plate, Gli2 and Gli3
activator (A) functions are required. Formation of the intermediate regions of
the ear including the proximal cochlear duct and saccule were found to be
dependent on the removal of Gli3 repressor (R). Interestingly, the vestibular
defects observed in Shh-/- embryos could be rescued by
removing one but not both alleles of Gli3, suggesting that Gli3R
functions in a dose-dependent manner to mediate vestibular development. Taken
together, our data suggest that reciprocal gradients of GliA and GliR mediate
the responses to Shh signaling along the dorsoventral axis of the inner
ear. |
MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Matise et al., 1998;
Maynard et al., 2002;
Böse et al., 2002).
Paint-fill and in situ hybridization
Paint-fill analyses and in situ hybridization experiments were performed as
described previously (Morsli et al.,
1998). The lengths of cochlear ducts were determined from ventral
views of paint-filled cochlear ducts by measuring their outer contours using
the ImageJ program (National Institutes of Health). The lengths of the mutant
cochlear ducts were compared with those of
Gli2+/-;Gli3+/- littermates. Riboprobes were
prepared as previously described: Shh
(Echelard et al., 1993),
Ptc1 (Milenkovic et al.,
1999), Gli1, Gli2 and Gli3
(Hui and Joyner, 1993),
Otx1, Otx2 and Bmp4
(Morsli et al., 1999), and
Msx1 (Satokata and Maas,
1994).
Cell proliferation and programmed cell death assays
Cell proliferation (BrdU labeling) and TUNEL (terminal dUTP nick-end
labeling) assays were performed as described
(Burton et al., 2004).
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Riccomagno et al.,
2002). By contrast, both Ptc1 and Gli1, which
are transcriptional readouts of Shh signaling
(Bai et al., 2002;
Dai et al., 1999;
Goodrich et al., 1996),
displayed graded expression patterns in the otocyst: stronger in the
ventral-medial region closest to the source of Shh and weaker in the
dorsal-lateral region from 9.5 to 12.5 dpc
(Fig. 1A-B' and see Fig.
S1 in the supplementary material). These expression patterns of Ptc1
and Gli1 indicate that the otic epithelium responds directly and
differentially to Shh signaling. The expression domains of Gli2 and
Gli3 were not limited to the medial-ventral region but also included
the lateral portion of the otocyst as well as the surrounding mesenchyme
(Fig. 1C,D). In addition, the
expression of Gli2 or Gli3 in the otic epithelium does not
appear to be regulated by Shh signaling (see Fig. S2 in the supplementary
material).
Vestibular morphology is altered in Gli3-/- embryos
Given the expression patterns of the three Gli transcription factors in the
otocyst, we postulated that Shh function in inner ear patterning is mediated
through the Gli factors. Since Gli1-/- mice are normal
(Bai et al., 2002;
Park et al., 2000), we focused
our attention on Gli2 and Gli3 mutant embryos. The inner
ears of Gli2-/- embryos did not display any obvious
morphological defects as assessed by paint-fill analyses (see below). In
examining the ears of Gli3-/- embryos, a number of
vestibular defects were observed, including the loss of the lateral
semicircular canal (Fig. 2B,C,
red asterisks; n=30) and, in 87% of the embryos examined, truncation
or absence of the anterior semicircular canal
(Fig. 2B,C, white asterisks;
n=26/30). The lateral and anterior ampullae and associated sensory
organs were still present in Gli3-/- embryos
(Fig. 2B,C,E,F; data not
shown). The posterior semicircular canal was usually present, although its
diameter was often narrower (Fig.
2; n=29/30). The ventral inner ear structures of
Gli3-/- embryos were largely normal, except for a slight
misshaping of the saccule and the most proximal tip of the cochlear duct
(Fig. 2B, arrowhead). These
results are consistent with the histological analyses published previously
(Johnson, 1967).
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Gli3R is required for dorsal vestibular development
To examine whether Shh-dependent regulation of Gli3 activities is also
important for inner ear development, we analyzed the ears of embryos carrying
various combinations of Shh and Gli3 mutant alleles. The
inner ears of Shh-/- embryos lacked ventral otic
derivatives, including the cochlear duct and saccule
(Fig. 3B, asterisk), and were
missing dorsal vestibular structures such as the lateral semicircular
canal/ampulla and the endolymphatic duct
(Fig. 3B)
(Riccomagno et al., 2002).
Even though Gli3 expression in the inner ear is not regulated by Shh
(see Fig. S2 in the supplementary material), when one copy of the
Gli3 gene is mutated on a Shh-/- background
(Shh-/-;Gli3+/-), the morphology of the
vestibule was completely rescued to its proper integrity
(Fig. 3). By contrast, only a
rudimentary knob-like structure was evident ventrally
(Fig. 3C, arrow).
We next analyzed the inner ears of
Shh-/-;Gli3-/- double mutants. Since all of
these embryos exhibited exencephaly, they were compared with exencephalic
Gli3-/- single mutants
(Fig. 2C). The dorsal
vestibular structures that were rescued in
Shh-/-;Gli3+/- embryos were malformed in
Shh-/-;Gli3-/- double mutants (n=6),
and resembled the dorsal otic defects observed in Gli3-/-
single mutants (compare Fig. 2C
with Fig. 3D). For example, as
with the Gli3-/- inner ears
(Fig. 2C), the
Shh-/-;Gli3-/- double mutants lacked the
lateral and anterior semicircular canals
(Fig. 3D, asterisks), but
retained their associated sensory organs, the ampullae and cristae
(Fig. 3D; data not shown).
Moreover, a thinner posterior semicircular canal was also a common phenotype
for Gli3-/- and
Shh-/-;Gli3-/- mutants
(Fig. 2C and
Fig. 3D). Ventrally, the
saccule and the proximal portion of the cochlear duct, which fail to form in
Shh-/- mutants, were rescued in
Shh-/-;Gli3-/- embryos
(Fig. 3B,D). The partial rescue
of inner ear structures in Shh-/-;Gli3+/- and
Shh-/-;Gli3-/- mutants is likely to be the
result of a dose dependency in the amount of Gli3R that is needed for
vestibular and proximal cochlear development, similar to what has been
reported for other tissues (Litingtung and
Chiang, 2000; Litingtung et
al., 2002; Rallu et al.,
2002). Our results suggest that vestibular development is
particularly sensitive to the level of Gli3R, with embryos that possess too
much (Shh-/-) or too little (Gli3-/-)
succumbing to defects, which can be rescued in a genetic context that restores
the proper amount (Shh-/-;Gli3+/-).
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699 embryos, a mouse model of human PHS699 mutants, which only express the N-terminal,
repressor form of Gli3 (Fig.
3E). In Gli3699/699
embryos, the cochlear duct was shortened and missing the distal region
(Fig. 3E; n=3). Given
that the length of the cochlear duct was normal in Gli3-/-
embryos (Fig. 2B), these
findings suggest that the truncated Gli3 protein derived from the
Gli3699 allele might act as a competitive inhibitor
of at least some GliA function (see below). The normal vestibular morphology
demonstrated by Gli3699/699 embryos
(Fig. 3E) is consistent with
our previous findings that dorsal vestibular structures are dependent on Gli3R
rather than Gli3A function.
Requirement of GliA during distal cochlear duct development
Absence of the distal cochlear duct in Gli3699
mutants coupled with the lack of cochlear phenotypes in
Gli2-/- (Fig.
4B,F,I) and Gli3-/-
(Fig. 2) single mutants,
suggested that the N-terminal truncated Gli3 protein could be acting to
inhibit multiple Gli activators. We therefore asked whether GliA function is
indeed required for distal cochlear duct development. Since the majority of
Gli2-/-;Gli3-/- double mutants die by 14.5 dpc,
we first examined inner ear morphology in Gli2/Gli3 compound
mutants. Cochlear duct lengths were measured in each of the compound mutants
and compared with Gli2+/-;Gli3+/- double
heterozygous littermates, which exhibited normal cochlear ducts
(Fig. 4A,E,I). Compound mutant
embryos carrying one wild-type Gli3 allele
(Gli2-/-;Gli3+/-) displayed a modest yet
significant reduction in the length of their cochlear ducts
(Fig. 4C,G,I, arrow). By
contrast, embryos with only one wild-type Gli2 allele
(Gli2+/-;Gli3-/-) had normal cochlear duct
lengths (Fig. 4D,H,I). These
results suggest that Gli2 and Gli3 have redundant functions in cochlear duct
outgrowth, yet the activator role of Gli2 is more pronounced than that of
Gli3.
Through the course of our studies, a few viable Gli2-/-;Gli3-/- double mutants were recovered at 13.5 dpc (n=3). We compared the inner ears of these double mutants with those of Gli2+/-;Gli3+/- and other compound mutants (Fig. 5). Cochlear duct development was severely impaired in Gli2-/-;Gli3-/- embryos, with only the formation of a short proximal region comparable to what was observed in Shh-/-;Gli3-/- embryos (Fig. 5B,C). This result indicates that, with the exception of the most proximal region, cochlear duct development is dependent on Gli2/Gli3 activator function. With respect to the vestibule, the defects observed in Gli2-/-;Gli3-/- double mutants closely resembled those from Gli3-/- single and Shh-/-;Gli3-/- double mutants (Fig. 5B-D), emphasizing once again the importance of Gli3R over Gli3A in dorsal vestibular development.
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). Neither
Msx1 nor Otx2 was expressed in the otocysts of
Shh-/- embryos, consistent with the absence of ventral
inner ear structures in these mutants (Fig.
6B,G). Interestingly, the partial restoration of the proximal-most
region of the cochlea in Shh-/-;Gli3+/- and
Shh-/-;Gli3-/- embryos was coincident with the
presence of Otx2 expression (Fig.
6C,D). By contrast, Msx1 remained absent from the ventral
tip of these inner ears (Fig.
6H,I, red asterisks), indicating that the cochlear duct of
Shh-/-;Gli3-/- mutants is indeed missing the
distal region. Similar results were found in the inner ears of
Gli3699 and
Gli2-/-;Gli3-/- double mutants
(Fig. 6E,J,N,R). Despite
possessing a slightly shortened cochlea
(Fig. 4G,I), the ears of
Gli2-/-;Gli3+/- embryos expressed Msx1
(Fig. 6L,P). These results
indicate that the reduction in cochlear duct length displayed by the majority
of Shh/Gli compound mutants correlates with the specific loss of the distal
portion of the cochlear duct.
|
). In Gli3-/- mutants, the horizontal and vertical pouches did not form properly and were smaller in size (Fig. 7E,F). Consistently, the Otx1 expression domain was much smaller, and this is likely to be due to the rudimentary nature of the horizontal pouch (Fig. 7H, arrowheads). Regardless of these defects, the presumptive lateral crista was still present, as evident by the overlapping expression of Bmp4 and Otx1 (Fig. 7G,H, arrow). These observations indicate that the lateral canal defect in Gli3-/- mutants is already evident at 11.75 dpc, the stage of horizontal pouch outgrowth.
Unlike in the Gli3-/- embryos, the horizontal pouch in
Shh-/- mutants appeared morphologically normal at 11.75
dpc (Fig. 7J). This indicates
that the lateral canal defect in Shh-/- embryos has a
later onset than in Gli3-/- embryos
(Fig. 7F). Despite the
differences in the timing of these phenotypes, the expression of Otx1
at the level of the lateral crista, which is normally restricted to the
lateral region of the horizontal pouch
(Fig. 7D), was shifted to the
medial side of the otic vesicle in Shh-/- embryos
(Fig. 7L, arrowheads) and no
longer overlapped with Bmp4 (Fig.
7K). Otx1 is required for the formation of the lateral
crista and canal (Morsli et al.,
1999). In Otx1-/- mutants, Bmp4
expression in the presumptive lateral crista was initially normal but failed
to be maintained by 12 dpc. Therefore, we attribute the eventual loss of the
lateral crista and canal in Shh-/- mutants to this
misexpression of Otx1.
Evidence in further support of the view that the lateral canal phenotypes in Shh-/- and Gli3-/- mutants are etiologically different, comes from the analysis of cell proliferation and programmed cell death in the lateral canal region at 11.75 dpc. Normally (in Gli3+/-), there is robust cell proliferation (BrdU labeling) and little cell death (TUNEL) in the horizontal pouch area (Fig. 8A-C). In Gli3-/- mutants, we observed a decrease in cell proliferation and an increase in cell death in the lateral wall, where the horizontal pouch normally forms (Fig. 8D-F, arrowheads). By contrast, in Shh-/- mutants, there was no obvious change in cell proliferation and programmed cell death in the horizontal pouch region at this stage (Fig. 8G-I), although there was an increase in cell death in the medial and anterior regions of the otic vesicle (Fig. 8I,I', arrowheads). Together, these observations indicate that the lateral semicircular canal is sensitive to the dose of Gli3R, with too much (Shh-/-) or too little (Gli3-/-) causing an arrest in its development, and that there are temporal and etiological differences in these requirements.
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) and Myf5 in epaxial muscle progenitor cells
(McDermott et al., 2005).
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699/699 but not
Gli3-/- embryos supports our claim that the N-terminal
truncated Gli3 protein encoded by the Gli3699 allele
is acting in a competitive fashion to block other Gli activators in the
cochlear duct. Similar dominant-negative effects of Gli3R have been postulated
to occur in the gastrointestinal tract of Gli3699
mutants (Böse et al.,
2002).
Genes such as Otx2 and Pax2 are required for ventral
patterning of the inner ear (Burton et
al., 2004; Morsli et al.,
1999) and both of these genes are regulated by Shh signaling
(Riccomagno et al., 2002). It
has been shown in other systems that little, if any, activator function of Gli
proteins exists in Shh-/-;Gli3-/- double
mutants to induce Shh target genes
(Litingtung et al., 2002;
te Welscher et al., 2002). The
presence of Otx2 and Pax2 in
Shh-/-;Gli3-/- embryos suggests that these
genes do not require Gli activator function and can be induced by alleviating
the repressive action of Gli3R. The presence of Otx2
(Fig. 6) and Pax2
(data not shown) in Gli2-/-;Gli3-/- inner ears
is consistent with this notion.
Based on the above findings, we propose that cochlear duct morphogenesis is
dependent on two phases of Shh signaling regulated by different Gli
intermediaries. In the first phase, Shh secreted from the ventral midline
promotes ventral identity in the otic vesicle by antagonizing Gli3R, thus
leading to the activation of Pax2, Otx2 and other target genes. In
the second phase of signaling, Shh recruits Gli2/Gli3A function to mediate the
distal outgrowth of the cochlear duct. This second phase of Shh signaling
could be provided by other tissues, including the cochleovestibular ganglion
(Liu et al., 2002).
Interestingly, Msx1 was used in our study as a marker of the distal
cochlear duct region; however, it might actually be a Shh-responsive gene that
participates in the extension of the cochlear duct. Whereas the cochlear duct
is normal in Msx1-/- embryos
(Satokata and Maas, 1994), it
is shortened in Msx1-/-;Msx2-/- double mutants,
consistent with the idea that these genes are downstream targets of Shh and
Gli activation pathways (Yiping Chen and D.K.W., unpublished).
Formation of the saccule and proximal cochlear duct requires a balance of Gli activator and repressor functions
The saccule and the proximal-most region of the cochlear duct are located
further from the sources of Shh than other regions of the cochlea and might
therefore depend on lower levels of Shh for their development
(Fig. 9). Shh appears to
regulate the formation of the saccule and proximal cochlear duct by
antagonizing Gli3R. The initial indication that a principal function of Shh is
to alleviate Gli3R activity was demonstrated by the partial suppression of
Shh mutant phenotypes in
Shh-/-;Gli3-/- embryos
(Litingtung and Chiang, 2000;
Litingtung et al., 2002;
te Welscher et al., 2002). For
example, in the spinal cord, a number of ventral neuronal subtypes that are
absent in Shh-/- embryos are rescued in
Shh-/-;Gli3-/- double mutants
(Litingtung and Chiang, 2000).
By the same token, the restoration of the saccule and proximal cochlear duct
in Shh-/-;Gli3-/- embryos indicates that a
primary function of Shh in this region of the ear is to effect the release of
Gli3R from Shh target genes.
Surprisingly, the saccule and proximal part of the cochlear duct develop
normally in Gli3699 mutants, which possess a form of
Gli3R whose activity is thought to be independent of Shh regulation. How then
does the intermediate region of the inner ear form in the
Gli3699 mutants? Since Shh signaling persists in
Gli3699/699 embryos, we reason that
other Gli activators, such as Gli2 and/or Gli1, can compete with
Gli3699 to promote the formation of the saccule and proximal
cochlear duct. In support of this claim, we observe a rudimentary saccule and
the absence of a definite cochlear duct in
Shh-/-;Gli3699/-mutants, in which no GliA
function is present (n=3; see Fig. S3 in the supplementary material).
Although GliA may be able to override the repressive effects of Gli3R to
promote saccule and proximal cochlear duct development in
Gli3699/699 embryos, it is not clear
whether GliA normally plays any role in the formation of these structures.
|
699 mutants, highlighting the importance of Gli3R
over Gli3A in mediating normal vestibular development. The finding that dorsal
vestibular defects arise in embryos with an excess
(Shh-/-) or shortage (Gli3-/-) of
Gli3R suggests that the absolute amount of Gli3R generated in the dorsal
otocyst is crucial for vestibular development. This contention is best
supported by results showing that the dorsal vestibular phenotypes seen in
Shh-/- embryos are corrected in the presence of one, but
not two, mutated copies of Gli3. One conclusion that can be drawn
from these findings is that Shh signaling in dorsal otic tissue serves to
block the processing of a set amount of full-length Gli3 into Gli3R.
Alternatively, the dorsal otic defects observed in
Shh-/- embryos might be caused indirectly through the
disruption of signals derived from the dorsal hindbrain. Several lines of
evidence prompted this notion. First, there is normally little expression of
Ptc1 and Gli1 in the dorsal region of the inner ear. Second,
dorsal structures, including the endolymphatic duct and lateral canal, are
induced properly in Shh-/- embryos but fail to develop
later in development when the juxtaposition of the neural tube and otocyst is
altered by the abnormal morphology and size reduction of the Shh
mutant hindbrain (Fig. 7)
(Riccomagno et al., 2002). In
this scenario, one could postulate that malformations of dorsal otic
structures in Shh-/- embryos are caused by the loss of
dorsal neural tube signals at a critical time of development when signaling
from the hindbrain is still required for inner ear patterning
(Riccomagno et al., 2005). If
this is the case, then hindbrain defects should be rescued in
Shh-/-;Gli3+/- mutants, in which the dorsal
otic structures are well formed. Even though there is no substantial
improvement in the morphology of the hindbrain in
Shh-/-;Gli3+/- mutants, the positioning of the
hindbrain with respect to the otocyst is better in
Shh-/-;Gli3+/- as compared with
Shh-/- mutants at 11 dpc when the primordial endolymphatic
duct and lateral canal structures are forming
(Litingtung and Chiang, 2000).
Distinguishing which of the two interpretations discussed above is correct
will necessitate the conditional inactivation of Shh signaling in the dorsal
otocyst.
Regardless of how Shh signaling is mediated in the dorsal vestibular
region, analyses of the lateral canal defects in the various mutants indicates
that two temporally distinct processes regulate its formation: an earlier
process that requires Gli3R and a later process that requires Shh. The role of
Shh in the latter case is to repress Gli3R, as indicated by the restored
lateral canal in Shh -/- mutants possessing only one
wild-type copy of Gli3. Notably, in
Gli3699/699 embryos, the lateral
canal forms normally, suggesting that neither of these processes is disrupted
in these mutants. Although the early requirement for Gli3R may not be
perturbed, it is somewhat surprising that no canal phenotype resembling that
seen in Shh-null mutants occurs in
Gli3699/699 embryos. However, unlike
Shh mutants, other Gli activators are still present in
Gli3699/699 mutants. These Gli
activators might override Gli3R (699) function, resulting in normal
lateral canal formation, as was described above for the saccule and proximal
cochlear duct region.
In summary, our study reveals that multiple Shh/Gli-mediated signaling
mechanisms are operating during inner ear development, with Gli3R required
dorsally for vestibular formation and GliA functioning ventrally to form the
cochlear duct (Fig. 9). The
distribution of the various Shh/Gli signaling mechanisms along the
dorsoventral axis of the ear is similar to what has been described for the
spinal cord, where Gli3R functions in dorsal regions of the neural tube to
restrict the dorsal limits of p1 and p0 interneuron subtypes, and Gli2/Gli3A
function redundantly to specify ventral neuronal cell fates
(Bai et al., 2004;
Lei et al., 2004;
Persson et al., 2002). What is
particularly novel about Shh/Gli signaling in the inner ear is the dosage
requirement of Gli3R in the vestibular region. Although partial restitution of
Shh-/- limb, neural tube and somite defects are observed
in Shh-/-;Gli3+/- embryos when the dose of
Gli3R is limiting (Litingtung and Chiang,
2000; Litingtung et al.,
2002; McDermott et al.,
2005; Rallu et al.,
2002), only the dorsal ear shows an absolute dependency on Gli3R
dosage as vestibular structures are completely rescued in
Shh-/-;Gli3+/- embryos.
Furthermore, despite the necessity of the cochlear duct for hearing, not
much is known about the regulation of its outgrowth and coiling. Our results
indicate for the first time that different regions of the coiling cochlear
duct are patterned by different mechanisms, and that this patterning is
established early at the otocyst stage primarily by Shh emanating from the
ventral midline (Bok et al.,
2005; Riccomagno et al.,
2002). The association of human deafness with mutations in genes
involved in the Shh/Gli signaling pathway warrants a more thorough
understanding of how these genes function in inner ear development. Future
studies will focus on identifying the downstream targets regulated by Shh and
Gli proteins.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/134/9/1713/DC1
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ACKNOWLEDGMENTS |
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REFERENCES |
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