Laboratory of Molecular Biology, National Institutes on Deafness and Other Communication Disorders, National Institutes of Health, Bethesda, MD 20850, USA
* Author for correspondence (e-mail: wud{at}nidcd.nih.gov)
Accepted 28 February 2005
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SUMMARY |
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Key words: Gbx2, Otx2, kreisler, Hindbrain signaling, Inner ear development, Otic vesicle, Mouse
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Introduction |
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Tissues surrounding the inner ear, such as the hindbrain, mesoderm and
endoderm, have been implicated in conferring signals required for inner ear
development (for reviews, see Fekete,
1999; Kiernan et al.,
2002
). The importance of the hindbrain in this process is evident
from analyses of mutant mice such as the Hoxa1 knockout and
kreisler (for a review, see
Kiernan et al., 2002
). Both
kreisler/Mafb and Hoxa1 are expressed in the hindbrain but
not the inner ear, yet inner ears of mice with these genes mutated are
abnormal. The inner ear defects of these mutant mice are attributed, in
particular, to defects in rhombomere 5 (r5), a region of the hindbrain
juxtaposing the developing otic placode
(Kiernan et al., 2002
).
Although the inner ear phenotypes in these mutants are variable, they often
include the absence of the endolymphatic duct and an enlargement of the
membranous labyrinth. The enlarged membranous labyrinth could be secondary to
the loss of the endolymphatic duct, which has been shown to be important in
maintaining fluid homeostasis within the membranous labyrinth
(Everett et al., 2001
;
Hulander et al., 2003
). In
addition to the kreisler and Hoxa1 mutants, knockout of
Raldh2 (retinaldehyde dehydrogenase 2) also results in otocyst
malformations that are attributed to a defective hindbrain
(Niederreither et al.,
2000
).
As both kreisler/Mafb and Hoxa1 are transcription
factors, their effects on inner ear development are likely to be mediated via
the regulation of signaling molecules. Several lines of evidence suggest that
FGF3 might be one of these hindbrain-derived signals that mediate inner ear
development. First, inner ears of Fgf3 knockout mice show a similar
phenotype to those of kreisler and Hoxa1 mutants
(Mansour et al., 1993).
Second, FGF3 and kreisler/Mafb are thought to positively regulate each other
in the hindbrain (Marin and Charnay,
2000
; Theil et al.,
2002
). Consistent with these results, the expression of
Fgf3 in r5 and r6 is absent in kreisler mutants, whereas
Fgf3 expression in the mutant inner ears is present
(McKay et al., 1996
). Third,
knockout of a receptor for FGF3, Fgfr2(IIIb), results in severe inner
ear malformations that include an absence of the endolymphatic duct
(Pirvola et al., 2000
).
However, FGF3 may not be the only signaling factor from r5 that mediates inner
ear development, as the inner ear phenotypes of Fgf3 knockout mice
are relatively milder and lower in penetrance when compared with inner ears of
kreisler, Hoxa1, Raldh2 and Fgfr2(IIIb) mutant mice.
Furthermore, another Fgf3 knockout mouse strain that was recently
generated has no apparent inner ear phenotype
(Alvarez et al., 2003
).
Therefore, additional signaling factors from the hindbrain including other
members of FGF family could be involved in mediating inner ear development.
Nevertheless, to date, specific downstream otic genes that are activated by
these signaling molecules from the hindbrain remain elusive.
Gbx2 is a homeobox gene that is related to Drosophila
unplugged (Chiang et al.,
1995). The expression of Gbx2 in the midbrain-hindbrain
junction of vertebrates is conserved among several species
(Bouillet et al., 1995
;
Shamim and Mason, 1998
;
Su and Meng, 2002
;
von Bubnoff et al., 1996
), and
knockout of Gbx2 in mice affects the normal positioning of this
junction in the brain (Wassarman et al.,
1997
). Gbx2 is also expressed in the otic placode of
several species (Bouillet et al.,
1995
; Shamim and Mason,
1998
; Su and Meng,
2002
). In mice, the expression of Gbx2 in the otic
placode is correlated with proper otocyst formation, but its specific role in
inner ear development is not known (Wright
and Mansour, 2003
). In this study, we analyzed the inner ears of
Gbx2 knockout mice and show that Gbx2 is a key molecule in patterning
both vestibular and auditory components of the inner ear. Based on comparisons
of inner ear phenotypes and gene expression analyses between Gbx2 and
some of the hindbrain mutants, in particular, kreisler, we postulate
that Gbx2 is an important downstream target of hindbrain
signaling.
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Materials and methods |
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Paint fill and in situ hybridization
Paint-fill analyses and in situ hybridization experiments were performed as
described (Morsli et al.,
1998). A total of 50 Gbx2 homozygous mutant embryos
between 9.5 and 15.5 dpc were used for in situ hybridization analyses, and a
total of 40 Gbx2-/- embryos between 8.5 and 10.5 dpc were
processed for whole-mount in situ hybridization. Heterozygous and homozygous
Gbx2 embryos from 8.5 to 9.0 dpc for whole-mount in situ
hybridization were age matched based on the total number of somite pairs. RNA
probes for bone morphogenetic protein 4 (Bmp4), lunatic fringe
(Lfng), neurofilament protein 68 kDa (NF68; Nef1
Mouse Genome Informatics) and orthodenticle 2 (Otx2) were prepared as
described (Morsli et al.,
1999
). RNA probes for Eya1
(Xu et al., 1997
),
Gata3 (Karis et al.,
2001
), Gbx2 (Bouillet
et al., 1995
), Myo15a
(Anderson et al., 2000
),
Neurod1 (Ma et al.,
1998
) and Pax2
(Dressler et al., 1990
) were
prepared according to cited references.
Cell proliferation and apoptosis assays
Cell proliferation and apoptotic assays were performed as described
(Burton et al., 2004).
Apoptotic cells were identified using terminal dUTP nick-end labeling (TUNEL)
method (ApopTag).
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Results |
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In addition, Gbx2 transcripts are detected in the mid-hindbrain
junction at 9.5 dpc (Fig. 1A,
arrowheads) (Bouillet et al.,
1995), as well as longitudinal columns in the dorsal and
intermediate regions of the hindbrain and spinal cord
(Fig. 1A,C, asterisks).
Paint-fill analysis of Gbx2 mutant inner ears
Next, we investigated the gross anatomy of Gbx2-/-
inner ears at 15.5 dpc using the paint-fill technique
(Fig. 2). A total of 19
homozygous mutant embryos were analyzed, and a repertoire of phenotypes was
observed. We divided the specimens into four categories (I, II, III and IV),
based on the severity of the phenotype
(Fig. 2,
Table 1). Overall,
Gbx2 mutant inner ears are usually missing the endolymphatic duct
(Table 1, n=18/19),
with an enlarged membranous labyrinth (Fig.
2A). By contrast, the lateral canal and ampulla are usually
present (Table I,
n=17/19). Type I, the mildest phenotype, shows an enlarged membranous
labyrinth, and three out of the four specimens are missing the endolymphatic
duct (Table 1). In the Type II
category, most of the inner ears are missing the common crus
(Fig. 2A, asterisk;
n=5/7), in addition to the absence of the endolymphatic duct
(n=7/7). The utricle and saccule are not easily discernible, and the
saccule is often fused with the cochlear duct. In the Type III inner ears, the
anterior and posterior semicircular canals are also missing but the lateral
canal is present (n=3). The cochlear ducts of Type III specimens are
more malformed than those of Type I and Type II, and have less than one coil.
Inner ears categorized as Type IV are the most severe; they are cystic without
any discernible structures except for the presence of the lateral canal in
some cases (n=3/5). Taken together, the lack of Gbx2
function affects inner ear structures such as canals and cochlear duct that do
not express Gbx2 (Fig.
1; Table 1),
suggesting that Gbx2 has a non-cell autonomous role in inner ear
development. Despite the variable phenotypes among the Gbx2 mutants,
the two ears of a given specimen usually display similar phenotypes
(n=8/10).
|
|
Loss of endolymphatic duct markers in Gbx2 mutant inner ears
The most prevalent phenotype of the Gbx2 mutants, the absence of
the endolymphatic duct, was examined in more detail using gene expression
analyses. Wnt2b is normally expressed in the endolymphatic duct, and
its expression is initiated in the dorsal pole of the otocyst starting at 9.5
dpc (Fig. 3A)
(Riccomagno et al., 2002).
Wnt2b expression is not detected in otocysts of Gbx2 mutants
at 9.5 or 10 dpc, suggesting a failure of endolymphatic duct specification
(Fig. 3B; n=4).
|
Ganglion and sensory organ development in Gbx2 mutants
Despite the fact that Gbx2 is not normally expressed in the
Lfng-positive neurogenic and sensory competent region
(Fig. 1F,G), the variable and
sometimes severe phenotypes observed in Gbx2 mutants suggest that the
development of this region is also affected. We examined ganglion and sensory
organ formations in Gbx2 mutants at 15.5 dpc using in situ
hybridization. Owing to the variability of phenotypes, cryosections from each
Gbx2 specimen were partially reconstructed and categorized as Type I
to Type IV. A total of seven Gbx2 mutant ears were analyzed for the
presence of vestibular and spiral ganglia (Type I, n=2; Type II,
n=3; and Type IV, n=2) using RNA probes for Nf68
and Gata3 transcripts. The vestibular ganglion is present in the
mutant ears of all phenotypes examined
(Fig. 4A,B). The spiral
ganglion is present in most of the Type II specimens
(Fig. 4C,D; n=2/3) but
missing in both Type IV specimens.
|
The Lfng expression domain that encompasses the neurogenic/sensory region appears normal in the Gbx2 mutants at 10.5 dpc (Fig. 5A,B, area between the red lines; n=3). However, based on the 3-D reconstruction of serial sections, the otic region dorsal to the Lfng domain is smaller, suggesting that this region is underdeveloped in Gbx2 mutants (Fig. 5A,B).
|
|
At 10.5 dpc, Otx2 is normally expressed in the ventral
posterolateral region of the otic vesicle, and its expression is complementary
to the Lfng domain (Fig.
6A-D). In the Gbx2 mutants, the dorsal region of the
Otx2 expression domain is fairly normal, but ventrally its expression
expands medially into the Lfng domain
(Fig. 6E-H, brackets; six
otocysts from four embryos). The extent of the Otx2 expression domain
expansion is variable among specimens. One ear from one embryo shows medial
expansion of the entire Otx2 expression domain, while another
specimen shows a normal expression domain. Both of these specimens show
variability between left and right ears. This aberrant expression of
Otx2 in the ventromedial region is also observed at later stages,
even though the extent of Otx2 domain expansion varies between
specimens (Fig. 6I, double
arrows; Fig. 6J-L; arrows;
n=8 between 11.5 to 15.5 dpc). Interestingly, unlike the situation in
the mid-hindbrain region, the Gbx2 expression domain in a normal
mouse inner ear does not abut the Otx2 domain
(Fig. 6M-N). Similar expression
patterns have been reported in the chicken inner ear
(Hidalgo-Sanchez et al.,
2000).
|
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Discussion |
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The role of Gbx2 in patterning ventral inner ear structures
Gbx2 also has a non-cell autonomous role in patterning ventral
inner ear structures such as the saccule and cochlea. In the developing neural
tube, the rostral expression domain of Gbx2 and the caudal expression
domain of Otx2 form a sharp border that dictates the position of the
mid-hindbrain junction within the neural tube. Ectopic expression studies
indicate that these two genes antagonize the expression of one another to form
this sharp border (Broccoli et al.,
1999; Millet et al.,
1999
). A similar antagonistic relationship between Gbx2 and Otx2
could be occurring in the inner ear. However, unlike the mid-hindbrain
junction, the expression domains of Gbx2 and Otx2 in the
inner ear do not abut each other. Although Gbx2 is expressed in the
dorsal medial region of the otic vesicle, Otx2 is expressed in the
ventral posterolateral region. Sandwiched in between the two domains is the
Lfng-positive sensory competent region that is negative for both
Gbx2 and Otx2 (Fig.
9A). Here, we show that the Lfng domain appears to form
normally in the Gbx2 mutants. However, the lack of Gbx2
results in an expansion of Otx2 domain medially and possibly affects
the subsequent development of the Lfng positive, sensory region
(Fig. 9A). This aberrant
expression of Otx2 in the ventral region is not observed in other
knockout mice with cochlear defects such as sonic hedgehog and Pax2
knockout mice (Burton et al.,
2004
; Riccomagno et al.,
2002
). Given the known functions of Otx2 in regional identity in
other systems (Acampora et al.,
1995
; Ang et al.,
1996
; Rhinn et al.,
1998
), the ectopic Otx2 expression in the inner ear of
Gbx2 mutants could be causal to the ventral defects observed.
Interestingly, the lack of the endolymphatic duct in mice is invariably
associated with defects in other parts of the inner ear
(Fekete, 1999
;
Kiernan et al., 2002
),
possibly owing to loss of Gbx2 expression. This suggests that a
disrupted relationship between Gbx2 and Otx2 expression
patterns could be a common molecular mechanism underlying other inner ear
defects in mouse mutants lacking an endolymphatic duct. This hypothesis needs
to be tested directly in other experimental paradigms.
|
The role of Gbx2 in the hindbrain
In the hindbrain of Gbx2 mutants, we observed a change in gene
expression patterns caudal to r3, in addition to defects described for the
anterior hindbrain (Wassarman et al.,
1997). We show that r5 is abnormal based on Krox20 and
Epha4 expression patterns. The size of r4 appears relatively normal,
but Fgf3 is ectopically expressed in this region. In addition, the
temporal expression patterns of both kreisler/Mafb and Fgf3
are prolonged in r6. These results suggest that Gbx2 is required for
normal development of the caudal hindbrain; it regulates the temporal
expression of kreisler/Mafb and Fgf3 and maintains the
expression of Krox20 and Epha4. However, many of these
affected genes are thought to regulate each other in the hindbrain
(Theil et al., 2002
;
Theil et al., 1998
). For
example, ectopic expression of kreisler/Mafb in r3 and r5 results in
an upregulation of Fgf3 in these rhombomeres
(Theil et al., 2002
). However,
in chicken, the loss of FGF signaling using an inhibitor of the FGF receptor
abolishes kreisler/Mafb expression in the hindbrain
(Marin and Charnay, 2000
).
These results suggest that kreisler/Mafb and FGF3 positively regulate each
other. Furthermore, ectopic expression of Mafb also represses the
expression of both Krox20 and Epha4 in the chicken hindbrain
(Giudicelli et al., 2003
).
Taken together, these results suggest that the downregulation of
Krox20 and Epha4 in Gbx2 mutants is mediated by the
upregulation of kreisler/Mafb and/or Fgf3.
Inner ear Gbx2 expression is downstream of hindbrain signaling
Signaling from the hindbrain plays an important role in inner ear
development. As Gbx2 is expressed in both the hindbrain and inner ear
itself, the inner ear phenotypes observed in the Gbx2 knockout mice
might be due to disrupted hindbrain formation and signaling and/or the lack of
Gbx2 activity within the inner ear.
We postulate that Gbx2 expression in the inner ear is a primary
downstream target of hindbrain signaling
(Fig. 9B). Several lines of
evidence support this hypothesis. First, kreisler/Mafb expression in
the hindbrain at 7.5 dpc precedes Gbx2 expression in the inner ear at
8.5 dpc. Second, the downregulation of Gbx2 expression in the inner
ears of kreisler mice strongly supports the notion that expression of
Gbx2 in the inner ear is regulated by the kreisler/Mafb
pathway (D. Choo, personal communication). Third, hindbrain rotation
experiments in chicken indicate that the maintenance of otic Gbx2
expression is dependent on signaling from the hindbrain
(Bok et al., 2004). Fourth,
there is a strong resemblance in the inner ear phenotypes described here for
the Gbx2 mutants and those described for kreisler (D. Choo,
personal communication). In kreisler, the endolymphatic duct fails to
develop, and the lateral ampulla and canal are the least affected structures,
similar to the Gbx2 mutants. Finally, similar molecular changes such
as ectopic expression of Otx2 and downregulation of Dlx5 are
also observed in kreisler mutants (D. Choo, personal communication).
It remains to be determined, however, whether other hindbrain mutants such as
Hoxa1 knockout mice show similar gene expression changes as we have
observed for Gbx2 mutants.
Other evidence supports the idea that Gbx2 expression in the inner
ear is a more important requirement for inner ear development than
Gbx2 expression in the hindbrain. Fgf3 expression in the
hindbrain is prolonged in the Gbx2 mutants. It has been shown that
ectopic expression of Fgf3 in the hindbrain of chicken resulted in a
distended endolymphatic duct (Vendrell et
al., 2000). Therefore, the prolonged expression of Fgf3
in the hindbrain of Gbx2 mutants could have resulted in a larger
endolymphatic duct, but this is not the case. The lack of an endolymphatic
duct phenotype in Gbx2 mutants is more consistent with a loss rather
than a gain of Fgf3 function. These results, however, could be
reconciled if kreisler/Mafb and/or Fgf3 in the hindbrain
normally induce or maintain Gbx2 expression in the inner ear. Then,
the absence of Gbx2 within the inner ear would negate any phenotype
caused by the upregulation of either kreisler/Mafb or Fgf3
and would result in a phenotype that resembles a loss of kreisler
function.
Although we postulate that the majority of the inner ear phenotype is due
to the loss of Gbx2 expression within the inner ear, any of the gene
expression changes observed in the hindbrain of Gbx2 mutants could
also contribute to the inner ear phenotype. No inner ear malformations due to
the lack of Krox20 and Epha4 have been reported
(Dottori et al., 1998;
Sham et al., 1993
;
Swiatek and Gridley, 1993
).
Even though the role of Wnt2b in inner ear development is not known,
the absence of Wnt2b expression in Gbx2 mutants could be due
to gene expression changes at the level of the hindbrain because the
expression patterns of Wnt2b and Gbx2 within the inner ear
appear to be independently regulated
(Riccomagno et al., 2002
). A
tissue-specific knockout of Gbx2 only in the inner ear or hindbrain
should determine which source of Gbx2 is more important for inner ear
development.
Under the assumption that Gbx2 in the inner ear is a major
downstream target of signaling from the hindbrain, this Gbx2
expression domain probably mediates hindbrain signaling by maintaining
expression of genes such as Dlx5 to promote formation of dorsal
structures (Fig. 9). However,
its role in ventral patterning is inhibitory and possibly mediated by
restriction of Otx2 expression. This proposed inhibitory role of
Gbx2 is different from the postulated inductive role of sonic
hedgehog, another signaling molecule that emanates from the hindbrain and
notochord, and induces or maintains expression of genes such as Otx2
and Pax2 (Riccomagno et al.,
2002). Otx1, Otx2 and Pax2 are all required for
the normal development of the saccule and cochlear duct
(Burton et al., 2004
;
Cantos et al., 2000
;
Morsli et al., 1999
). Multiple
inductive and inhibitory molecules at the level of transcriptional control are
likely to be involved in achieving the formation of an intricate organ such as
the inner ear, and this paper presents insight into such a mechanism.
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ACKNOWLEDGMENTS |
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