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First published online 12 September 2007
doi: 10.1242/dev.006627
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Department of Human Genetics, University of Utah, Salt Lake City, UT 84112-5330, USA.
* Author for correspondence (e-mail: suzi.mansour{at}genetics.utah.edu)
Accepted 27 July 2007
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Fibroblast growth factor, Mouse mutant, Endolymphatic duct and sac, WNT signaling, Hearing and balance
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Morton and Nance, 2006) and up
to 39% of sensorineural deafness is associated with inner ear malformations
(Mafong et al., 2002;
Wu et al., 2005).
In mice, inner ear formation initiates on embryonic day (E) 8.0 as a
placodal thickening of head ectoderm adjacent to hindbrain rhombomeres (r)
5-6. Subsequently, from E8.5-9.0, the placode invaginates, forming the otic
cup, from which neuroblasts of the eighth ganglion (GVIII) delaminate
anteroventrally and migrate medially. By E9.5, the cup closes and separates
from the surface ectoderm, forming the spherical otic vesicle, or otocyst.
During the next 6 days, this simple, fluid-filled epithelium acquires its
mature and complex morphology and begins to differentiate its multitude of
distinct sensory and non-sensory cell types. Morphogenesis of the vesicle
initiates at E10.5 with a dorsomedially directed outgrowth of the EDS anlage
and a ventrally directed outgrowth of the cochlear duct. At E11.5, the otic
epithelium evaginates dorsolaterally to form the vertical canal plate, the
precursor of the anterior and posterior semicircular canals, which are
separated by the common crus, and shortly thereafter evaginates laterally to
form the lateral canal plate, the precursor of the lateral semicircular canal.
The canals are formed at E12.5 by fusion of cells within two central regions
of the vertical plate and a single central region of the lateral canal plate,
followed at E13.5 by resorption of the fused cells into the epithelium. During
this period, there are additional evaginations of the central region to form
the more dorsally situated utricle and the more ventrally situated saccule. By
E15.5, the inner ear epithelium has acquired its mature morphology, but is
still undergoing cellular differentiation within the six sensory patches
(Kiernan et al., 2002;
Barald and Kelley, 2004;
Mansour and Schoenwolf, 2005;
Fritzsch et al., 2006).
The initial induction and subsequent morphogenesis and differentiation of
the otic epithelium involves signaling interactions within and between otic
and non-otic tissues. In particular, fibroblast growth factor (FGF) signals
from the endoderm induce a mesenchymal FGF that is required together with
hindbrain FGFs for expression of otic placode genes and for otic placode
induction and vesicle formation. Based on genetic data, these signals are
likely to be provided in mice by FGF8, FGF10 and FGF3, expressed by the
endoderm, mesenchyme and hindbrain, respectively
(Alvarez et al., 2003;
Wright and Mansour, 2003;
Ladher et al., 2005;
Zelarayan et al., 2007). WNT
signals, presumably from the hindbrain, are required to limit the region of
the ectoderm that forms the otic placode
(Ohyama et al., 2006). In
response to the inductive signals, the otic placode expresses several
transcription factor genes uniformly, including Pax2, Dlx5 and
Gbx2 (Wright and Mansour,
2003; Ladher et al.,
2005). By the otocyst stage, the expression domains of most otic
markers become confined to restricted domains of the vesicle specified to give
rise to different ear structures (Fekete
and Wu, 2002). This division of the otocyst into gene expression
domains required for normal morphogenesis also depends on signals from the
hindbrain. Removal or rotation of the neural tube in the vicinity of the
developing otic tissue or rotation of the vesicle disrupts otic vesicle
molecular patterning and development, particularly the outgrowth of the EDS
and cochlear duct (Hutson et al.,
1999; Bok et al.,
2005). Mutations in hindbrain-expressed genes, such as
Mafb (Kreisler) and Hoxa1, which cause defects in
the development of r5-6, also show aberrant patterning of the otic vesicle
that presages inner ear malformations
(Pasqualetti et al., 2001;
Choo et al., 2006). WNT
signaling from the hindbrain is sufficient to maintain the expression of some
dorsal otic genes, and Wnt1 and Wnt3a, which are expressed
in the dorsal hindbrain, are required redundantly for formation of the
vestibular structures (Riccomagno et al.,
2005).
FGF signaling is also implicated in otic vesicle morphogenesis. FGF3 and
FGF10 are expressed by prospective otic sensory tissues
(Wilkinson et al., 1988;
Mahmood et al., 1996;
McKay et al., 1996;
Pirvola et al., 2000;
Pauley et al., 2003), and
their major receptor, FGFR2b, is found in prospective non-sensory tissue
(Pirvola et al., 2000). Global
inhibition of FGF signaling by application of SU5402 to chick E2.5-3 otic
vesicles just prior to semicircular canal pouch evagination causes
dose-dependent inhibition of canal formation, suggesting that developing
sensory structures control morphogenesis of non-sensory structures
(Chang et al., 2004). Mouse
mutants support this concept. Mice lacking FGFR2b form otic vesicles that
subsequently develop dysmorphologies initiating at EDS outgrowth and include
failure of semicircular canal formation
(Pirvola et al., 2000). Most
Fgf10 mutants completely lack semicircular canals
(Pauley et al., 2003;
Ohuchi et al., 2005).
Fgf3-null mutants undergo normal otic vesicle formation, but then go
on to develop highly variable and incompletely penetrant inner ear
dysmorphologies that, like those of Fgfr2b mutants, appear to
initiate at the EDS outgrowth stage. These mutants also show a reduction in
the size of GVIII (Mansour et al.,
1993).
To gain insight into the mechanism of Fgf3 function in otic
morphogenesis, we determined the three-dimensional structure of Fgf3
mutant ears and the changes in their molecular patterning. We show that
homozygous mutants have variably penetrant and expressive inner ear
morphologies that are characterized most typically by loss of the EDS and
common crus, dilatation of the remaining epithelium and poor coiling of the
cochlea, phenotypes that are very similar to those of Hoxa1, Mafb and
Gbx2 mutants (Pasqualetti et al.,
2001; Lin et al.,
2005; Choo et al.,
2006) as well as to some classes of hearing-impaired patients
(Manfre et al., 1997;
Kim et al., 2004;
Wu et al., 2005). The initial
molecular patterning of Fgf3 mutant otocysts was normal, but by
E10-10.5 these ears lacked or had reduced domains of dorsal otic genes,
suggesting a loss of the cells fated to form the EDS. Ventrally expressed
genes important for cochlear development were not affected, but markers of the
developing vestibular sensory domains, particularly those expressed in the
posterior otic vesicle, were downregulated or absent and GVIII was reduced and
displaced dorsomedially. Finally, hindbrain expression of Wnt3a was
ventrally expanded in Fgf3 mutants. Taken together, our results show
that Fgf3 is required for dorsal otic patterning and that its primary
role is to sustain and focus the dorsal otic gene expression induced by WNT
signals. We suggest that Fgf3 also serves to focus inductive WNT
signals on the dorsal otic vesicle, highlighting a new example of cross-talk
between the two signaling systems.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). A new
Fgf3 allele (Fgf3
2),
which has a deletion of exon 2, was generated by first targeting loxP sites on
either side of exon 2 to generate a conditional allele and then deleting the
intervening DNA using CRE recombinase (X.W. and S.L.M., unpublished).
Fgf32 transcripts contain exon
1 spliced directly to exon 3, which is in a different frame. Thus, they encode
only the first 73 of 245 FGF3 amino acids, including only the first 28 of 137
amino acids of the FGF core homology domain.
Both Fgf3 alleles were studied on a mixed genetic background
containing hybrid 129 and C57Bl/6 alleles. Genotypes of heterozygous
intercross offspring were determined by PCR amplification of yolk sac or tail
DNA using three-primer mixes that distinguish the mutant and wild-type
alleles. The Fgf3neo genotyping assay was performed as
described previously (Wright and Mansour,
2003). The Fgf32
genotyping mix, containing primers 455C
(5'-CTGCCTATGTGCTATATCCATGG-3'), 456C
(5'-GTAGATGACTGAGTGTGTAGG-3') and 485B
(5'-GGTTCCTCGATCAAACTCTGG-3'), was amplified in an MJR
thermocycler for 35 cycles of 30 seconds at 94°C, 20 seconds at 60°C
and 1 minute at 72°C and produced a wild-type band of 250 bp and a mutant
band of 600 bp.
Paint-filling and RNA in situ hybridization
E15.5 paint-filled ears were prepared as described
(Morsli et al., 1998). Otic
and hindbrain marker gene expression was detected in somite-stage matched
E9.5-10.5 (the day of vaginal plug detection was regarded as E0.5) specimens
by whole-mount in situ hybridization with digoxigenin-labeled antisense cRNA
probes as described (Henrique et al.,
1995). Hybridization probes were generated from plasmids
containing: Pax2 (Dressler et
al., 1990), Dlx5
(Depew et al., 1999),
Gbx2 (Wassarman et al.,
1997), Hmx3 (Nkx5.1)
(Merlo et al., 2002),
Lfng (Morsli et al.,
1998), Fgf10 (Xu et
al., 1998), Wnt1 and Wnt3a
(Parr et al., 1993),
Wnt2b (Grove et al.,
1998), Bmp4 (Jones et
al., 1991), Otx2
(Simeone et al., 1993)
Mafb (Cordes and Barsh,
1994) and Gata3
(George et al., 1994). Stained
embryos were cryoprotected in sucrose and cryosectioned as described
(Stark et al., 2000). Two to
six embryos of each genotype were analyzed with each probe.
Auditory brainstem response threshold measurements
Mice were anesthetized using 0.02 ml/g avertin. Auditory brainstem response
(ABR) thresholds for click stimuli (47 µsecond duration, 29.3/second)
presented to each ear individually, were determined using high-frequency
transducers controlled and analyzed by SmartEP software (Intelligent Hearing
Systems) according to Zheng et al. (Zheng
et al., 1999).
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Here we present a more detailed analysis of the inner ear
dysmorphology in Fgf3 mutants and an analysis of otic marker genes
using the previously described Fgf3neo allele and a newly
targeted Fgf32 allele
(Fig. 1A).
Fgf3neo and Fgf32 adult homozygotes have similar survival and gross skeletal and otic phenotypes
To determine whether the new
Fgf32 allele was similar to the
Fgf3neo allele, we first compared the phenotypes of adult
homozygotes. Offspring of Fgf3+/neo and
Fgf3+/2 intercrosses
were observed and genotyped at weaning. In both cases, although all three
genotypes were recovered, the genotypic distribution deviated significantly
from the normal mendelian expectation (Fig.
1B). Only 10% of Fgf3+/neo and 9% of
Fgf3+/2 intercross
offspring were homozygous mutant, showing that there is significant and
similar lethality associated with both alleles. In addition, all homozygous
mutants exhibited the characteristic short, curly tails described previously
(Fig. 1B)
(Mansour et al., 1993). Thus,
the two Fgf3 alleles behave similarly at the overt phenotypic
level.
|
).
Therefore, we measured auditory brainstem response (ABR) thresholds in each
ear in 6-week-old animals of all three
Fgf32 genotypes. No significant
differences in auditory thresholds were observed between wild-type
(n=7) and heterozygous (n=11) animals and all of these
controls had normal behavior. By contrast, only one
Fgf32/2
animal had normal thresholds in both ears and eight of nine
Fgf32/2
animals had ABR thresholds that were significantly elevated (>2 standard
deviations above same-side controls) in one (n=3) or both
(n=5) ears (Fig. 1C).
All five bilaterally affected homozygous mutants showed circling behavior,
whereas the three unilaterally affected animals had only a slight head tilt.
Histological sections of normal-hearing heterozygous inner ears showed typical
gross morphology and normal cochlear structure
(Fig. 1D,D'). By
contrast, inner ear sections of a deaf homozygote showed only rudimentary
partitioning into distended cochlear and vestibular chambers. Nevertheless,
the otic epithelium could be identified and showed evidence of sensory
differentiation, albeit abnormal (Fig.
1E,E'). Thus, as was concluded using morphologic studies of
Fgf3neo homozygotes
(Mansour et al., 1993),
Fgf32 homozygotes have auditory
and vestibular phenotypes that are incompletely penetrant and variably
expressive.
Most Fgf3 mutants show abnormal otic morphologies
To visualize more clearly the consequences of loss of Fgf3 to
inner ear morphogenesis, we filled E15.5 Fgf3 control
(Fig. 2A,B) and mutant
(Fig. 2C-I) inner ear epithelia
with latex paint. Both Fgf3 alleles were studied, but no obvious
differences between them were apparent, so the results for a total of 120
mutant ears were combined. At the gross anatomical level, 50/120 (42%) of
mutant ears had a normal morphology, consistent with incomplete penetrance of
the adult Fgf3 auditory and vestibular phenotypes
(Fig. 2C). The remaining 70
(58%) showed graded morphologic defects that were assigned to groups based on
the classification of Gbx2-null ears
(Lin et al., 2005).
Virtually all affected Fgf3 mutant ears had a distended membranous
labyrinth (n=65/70). In most cases, the swollen cochlear duct showed
incomplete partition and poor coiling. All of the expected structures could be
identified in type Ia ears, but the endolymphatic duct appeared truncated
dorsally, lacking the endolymphatic sac
(Fig. 2D, n=8/70).
Type Ib ears were similar to type Ia, but entirely lacked the EDS appendage
(Fig. 2E, n=12/70).
Type II was the most common phenotype seen in affected Fgf3 mutant
ears and included absence of the EDS as well as of the common crus, such that
the anterior and posterior canals appeared as a single continuous canal
(Fig. 2F, n=32/70). In
addition, the utricle and saccule were fused with the enlarged cochlear duct.
Smaller numbers of mutant ears were even more severely affected. Type III ears
had the EDS and common crus aplasia typical of type II ears, together with
absence or severe truncation of the anterior and posterior semicircular canals
(Fig. 2G, n=5/70).
Most of the associated ampullae could be identified, but one ear lacked the
posterior ampulla and another lacked the anterior ampulla. These ears all
retained the lateral canal and ampulla. The type III cochlear duct was
severely malformed and shortened, with less than one coil. The type IV
phenotype was the most severe, with defects in all inner ear structures. The
only discernible formation was a cochlea-like structure
(Fig. 2H, n=7/70).
Taken together, these data show that the abnormal morphologies of most
Fgf3 mutant ears included hypoplasia or aplasia of the EDS, swelling
of the remaining labyrinth and, in many cases, aplasia of the common crus.
These are similar to the phenotypes seen in Hoxa1, Mafb and
Gbx2 mutant ears (Pasqualetti et
al., 2001; Lin et al.,
2005; Choo et al.,
2006).
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;
Pauley et al., 2003;
Ohuchi et al., 2005) and are
similar to those of Fgf10 hypomorphs (X.W., T.J.W. and S.L.M.,
unpublished).
Fgf3 is expressed in tissues relevant for otic vesicle morphogenesis
To correlate Fgf3 expression with major steps in early otic
morphogenesis, we detected Fgf3 transcripts by in situ hybridization
of E8.5-10.5 embryos. Consistent with results described previously, at 3-4
somites Fgf3 was expressed weakly in a dorsoventrally oriented stripe
within the presumptive otic placode and strongly in the adjacent neurectoderm
(data not shown) (but see McKay et al.,
1996; Wright and Mansour,
2003). Ectodermal expression of Fgf3 disappeared as the
placode was induced and formed a cup (5-12 somites, data not shown), whereas
neurectodermal expression remained strong throughout the otic cup stage and
was restricted to r5-6 (Fig.
3A-D) (Mahmood et al.,
1996; McKay et al.,
1996; Wright and Mansour,
2003). As the cup closed to form the otic vesicle, hindbrain
Fgf3 became restricted to r6, expression was induced in an
anteroventrolateral region of the vesicle and in delaminating neuroblasts of
GVIII (Fig. 3E-H). EDS
outgrowth was first evident morphologically at 27 somites, just as r6
expression of Fgf3 diminished
(Fig. 3I-L). At E10.5,
Fgf3 expression was maintained in anteroventrolateral otic cells from
which both neuroblasts and sensory cells are derived
(Fig. 3J,K)
(Li et al., 1978;
Carney and Silver, 1983). The
neuroblasts of GVIII, however, no longer expressed Fgf3. Thus,
Fgf3 shows a dynamic pattern of expression in the hindbrain, GVIII
and prospective sensory domains during the transition from the otic cup stage
to the initiation of EDS outgrowth.
Dorsal otic gene expression is markedly affected in Fgf3 mutants
The disruption of EDS development in the majority of abnormal Fgf3
mutant ears suggested that molecular patterning of the dorsal otocyst could be
perturbed. Thus, we analyzed expression of several dorsal otic markers at
E9.5-10.5, the stage when otic vesicle morphogenesis is initiated by EDS
outgrowth and when Fgf3 expression is found in the hindbrain and
initiates in the prosensory domain (Fig.
3). Again, both Fgf3 mutant alleles were examined, but no
allele-specific differences were apparent, so the results were combined
(Fig. 4).
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;
Lin et al., 2005) and/or were
expressed in the developing EDS (Wnt2b). Gbx2 expression,
which marks a broad dorsomedial region of the vesicle, including the
prospective EDS, as well as more anterior and posterior dorsal otic tissue,
was generally not affected in E9.5 Fgf3 mutants
(Fig. 4A-B'). Only one
such Fgf3 mutant showed a slight reduction in the extent of the
medial Gbx2 domain (2/8 mutant ears, data not shown). The dorsomedial
Gbx2 expression domain persisted in E10.5 control inner ears
(Fig. 4C,C'). By
contrast, otic Gbx2 expression was strongly affected by E10.5 in the
Fgf3 mutant otocysts, which were noticeably smaller than control
otocysts (Fig. 4D,D'). In
eight of 12 Fgf3 mutant ears, no Gbx2 staining was found in
the otic epithelium (Fig.
4D,D', right ear), and in the remaining four ears, weak
staining was detected in the dorsomedial wall of the otocyst; however, the
expression domain was severely reduced
(Fig. 4D', left ear). At
E10.5, Gbx2 was also expressed in the mid-hindbrain region
(Lin et al., 2005). This
Gbx2 domain was not affected in any Fgf3 mutant
(Fig. 4C-D'). At E9.5,
Dlx5 was found in the dorsomedial otocyst of both controls and
Fgf3 mutants (Fig.
4E-F'). At E10.5, control otocysts showed initiation of EDS
outgrowth and expression of Dlx5 in the dorsal half of the otocyst
(Fig. 4G,G'). However, in
Fgf3 mutants there was a slight reduction in the ventral boundary of
the Dlx5 domain, both medially and laterally, possibly owing to
physical loss of the prospective EDS domain (n=6/6 ears;
Fig. 4H,H').
Wnt2b was first detected in E10.5 control otocysts in a dorsomedial
domain, marking the outgrowing EDS (Fig.
4I,I'). By contrast, Wnt2b was entirely absent from
the dorsal otocyst in four of ten Fgf3 mutant ears, and was severely
reduced, showing only weak staining on the medial otocyst wall, in six of ten
mutant ears, suggesting a failure of EDS specification
(Fig. 4J,J').
In addition, we studied Hmx3 (Nkx5.1), a marker that
broadly identifies the vestibular domain and is required redundantly with
Hmx2 (Nkx5.2) for vestibular development
(Wang et al., 1998;
Wang et al., 2001;
Wang et al., 2004).
Hmx3 was expressed dorsolaterally in E10.5 control otocysts
(Fig. 4K,K'), marking the
prospective EDS and lateral canal plate regions. In Fgf3 mutants,
however, Hmx3 expression shifted medially (n=8/8 ears;
Fig. 4L,L'). Finally,
Gata3, which is expressed in both dorsolateral and ventromedial
otocyst domains (Lawoko-Kerali et al.,
2002; Lillevali et al.,
2004) and is required for both dorsal and ventral otic development
(Karis et al., 2001;
Lillevali et al., 2006), was
unaffected in E10 Fgf3 mutants (data not shown), suggesting that its
role in otic morphogenesis is independent of Fgf3.
|
;
Burton et al., 2004).
Pax2 expression was similar in control and Fgf3 mutant
embryos at E9.5 (Fig.
5A-B') and E10.5 (Fig.
5C-D'). At E10.5, Otx2 is normally expressed in a
posterior ventrolateral patch (Morsli et
al., 1998) (Fig.
5E,E'). In most Fgf3 mutant ears, Otx2
expression was unchanged (n=4/6 ears;
Fig. 5F,F'). However, in
one specimen, Otx2 was reduced in one ear and absent in the other
(data not shown). Finally, ventromedial Gata3 expression was
unaffected in Fgf3 mutants (data not shown). Thus, it is likely that
Fgf3 mutant cochlear phenotypes are largely secondary to disturbances
of dorsal patterning and morphogenesis.
Posterior sensory domain markers are downregulated in Fgf3 mutants
Expression of Fgf3 in the anteroventrolateral otocyst domain that
gives rise to sensory cells and GVIII neuroblasts, together with the reduced
size of GVIII in E11.5 Fgf3neo mutants, suggested roles
for Fgf3 in sensory organ and ganglion development
(Mansour et al., 1993).
Therefore, we assessed the expression of sensory markers, Lfng and
Bmp4, and of a sensory and ganglion marker, Fgf10, in
Fgf3 mutant otocysts.
Lfng is normally expressed in the anteroventral otocyst - in the
precursors of the cochlear, saccular and utricular sensory organs
(Morsli et al., 1998). No
significant differences in the distribution of Lfng-expressing cells
were detected between control (Fig.
6A-A'') and mutant (Fig.
6B-B'') specimens at E10.5. Bmp4 marks the
presumptive cristae (Morsli et al.,
1998). At E10.5, Bmp4 expression was detected in two
patches: one anterodorsolateral (Fig.
6C,C') and the other posteroventral
(Fig. 6C,C''). The
anterior Bmp4 patch contains precursors of the anterior and lateral
cristae, whereas the posterior Bmp4 domain
(Fig. 6C, arrowhead) houses
posterior crista precursors (Morsli et
al., 1998). The anterior Bmp4 domain was present in all
E10.5 Fgf3 mutant embryos (Fig.
6D,D'); however, there was a reduction in the extent of the
staining and a ventral shift of that domain. The posterior Bmp4
domain was not detected in four of six mutant inner ears
(Fig. 6D,D''). Thus, loss
of Fgf3 can affect posterior crista development.
To assess GVIII development together with the prospective otic sensory
domains, we examined Fgf10 expression. At E9.5, Fgf10 was
expressed throughout both control and Fgf3 mutant otocysts (data not
shown). At E10.5, Fgf10 was detected in the anterior pole of control
vesicles, which includes the prospective sensory domain, and in delaminating
neuroblasts of GVIII. Fgf10 was also expressed in a second, smaller
posterior patch, which gives rise to the posterior crista
(Fig. 6E-E'')
(Pauley et al., 2003). The
posterior Fgf10 domain appeared absent in eight of 12 Fgf3
mutant ears (Fig. 6F,
arrowhead), but examination of sections showed that it was shifted anteriorly
and medially (Fig. 6F'').
There was also a dorsomedial shift of the Fgf10-expressing anterior
domain together with a corresponding positional shift of GVIII in six of 12
mutant ears (Fig.
6F').
Fgf3 negatively regulates Wnt3a in r5-6
WNT signaling from the dorsal neural tube is required for dorsal otocyst
patterning, with Wnt1 and Wnt3a identified as the specific
ligands (Riccomagno et al.,
2005). We therefore examined Wnt1 and Wnt3a
expression in Fgf3 mutants. As expected at E9.5, both Wnt1
and Wnt3a expression marked the dorsal-most region of the developing
neurectoderm in control embryos (Fig.
7A,A',C,C') (Parr
et al., 1993). Wnt1 expression was unchanged in all three
Fgf3 mutants examined (Fig.
7B,B'). Similarly, Mafb expression in r5-6 was
unaffected in Fgf3 mutants (data not shown). By contrast, all six
Fgf3 mutants examined showed a significant ventral expansion of
Wnt3a expression in r5-6 (Fig.
7D,D''), but not in hindbrain regions anterior
(Fig. 7D') or posterior
(Fig. 7D''') to the otic
vesicle. These results show that Fgf3 negatively regulates
Wnt3a, but not other genes required for otic development, in the
dorsal hindbrain.
|
DISCUSSION |
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|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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), we
found that both Fgf3 alleles examined here gave similar otic
phenotypes. The phenotypic differences between our alleles and those of
Alvarez and colleagues could arise either from differences in genetic
background, or from the particular nature of the mutations, the contributions
of which will require additional experiments.
We found that otic epithelia from two Fgf3 mutant alleles
exhibited a graded series of morphologies ranging from normal to complete loss
of all vestibular structures. Common to all affected Fgf3 mutant
ears, except for those few members of the type O group, was hypoplasia or
aplasia of the EDS, global swelling of the entire epithelium and poor cochlear
coiling. Since the expression of ventral otocyst genes required for cochlear
development was largely unperturbed, it is likely that the gross distortion of
cochlear development was secondary to the effects on EDS and dorsal
development. The association between abnormal EDS development and otic
epithelial swelling is characteristic of both syndromic and non-syndromic
human hearing loss (Wu et al.,
2005) and is common to mouse mutations that disrupt the hindbrain
signaling required for EDS induction (e.g. Mafb, Hoxa1, Wnt1/Wnt3a
double mutants) (Pasqualetti et al.,
2001; Riccomagno et al.,
2005; Choo et al.,
2006) or disrupt transcription factors that are expressed early in
the dorsal otic vesicle and are required for EDS formation (e.g. Gbx2,
Hmx2, Hmx3, Dlx5, Dlx5/6) (Wang et
al., 2001; Merlo et al.,
2002; Lin et al.,
2005). Furthermore, mutations in Foxi1, which encodes an
EDS-expressed transcription factor, or in its genetic target, Slc26a4
(encoding the anion transporter, pendrin), cause abnormal EDS function,
swelling of the entire epithelial space, loss of endocochlear potential and
deafness (Everett et al.,
2001; Hulander et al.,
2003). Indeed, mutations in SLC26A4 are a common cause of
syndromic deafness (Pendred syndrome) and have also been found in
non-syndromic deafness (DFNB4) (Morton and
Nance, 2006). These patients show an enlarged vestibular aqueduct
(the structure that houses the EDS) and may show the Mondini defect
(dilatation and incomplete partitioning of the cochlear duct), similar to the
Fgf3 type Ia ears described here
(Phelps et al., 1998;
Fitoz et al., 2007). Thus, it
appears that any disruption of EDS development, whether leading to EDS
hypoplasia or aplasia, as when hindbrain-expressed or early otic-expressed
genes are disrupted, or to overall dilatation of a labyrinth that retains an
EDS, as in the case of later-acting EDS-expressed genes, can cause deafness.
The EDS is thought to participate in regulation of endolymph homeostasis,
which is crucial for hearing function in the mature ear, but the phenotypes
exhibited by Fgf3 and the other mutants with similar dysmorphologies
suggest that EDS function might be required from the earliest stages of otic
morphogenesis. It is interesting to speculate that Meniere's disease, an adult
condition characterized by both auditory and vestibular symptoms and
associated with enlarged endolymphatic spaces, could be caused either by
mutations in EDS-expressed genes that function only after completion of
morphogenesis, or by mutations in `developmental' genes that, like
Fgf3, have a wide spectrum of penetrance and expressivity.
EDS aplasia and general membranous swelling together with common crus
aplasia was the most prevalent phenotypic complex (type II) observed in
affected Fgf3 mutant inner ears and has also been noted in some
Hoxb1, Mafb and Gbx2 mutants
(Pasqualetti et al., 2001;
Lin et al., 2005;
Choo et al., 2006). Such a
congenital phenotype has also been reported in patients
(Manfre et al., 1997;
Kim et al., 2004), but its
cause is unknown. If EDS aplasia and consequent membranous swelling is
explained by failure of FGF3-mediated signaling from the hindbrain to the
dorsal otocyst, how might common crus aplasia arise? Since the common crus is
formed when two distinct central regions of the vertical canal plate fuse and
then resorb to form distinct anterior and posterior canals, one possibility is
that Fgf3, expressed from the developing cristae, normally functions
relatively directly to limit the fusion boundaries or the process of cellular
resorption. Alternatively, it could be that the abnormal swelling of the
epithelium consequent to failed or incomplete induction of the EDS is itself
the cause of excessive cellular fusion and/or resorption in the vertical canal
plate. Given the diversity of mutant genes that cause common crus aplasia, and
the fact that most of these do not appear to be expressed in the otic
epithelium, the latter possibility seems more likely. Conditional ablation of
Fgf3 in the otic epithelium versus hindbrain (work in progress) could
help to address this issue.
|
;
Frohman et al., 1993;
McKay et al., 1996;
Pasqualetti et al., 2001), and
both Mafb and Fgf3 are required for normal dorsal otic
expression of Gbx2 (Choo et al.,
2006) (this study), this places Fgf3 between
Mafb and Gbx2 in the EDS induction pathway. However, because
Gbx2 expression in Fgf3 mutants was only strongly affected
at E10.5, and not at E9.5, when Gbx2 is affected in
Wnt1/Wnt3a double mutants (which have no dorsal development at all)
(Riccomagno et al., 2005), we
suggest that WNTs provide the initiating signal for dorsal patterning of the
otic vesicle, and that FGF3 directly or indirectly sustains or reinforces
expression of dorsal otic genes. This might explain, at least in part, the
reduced penetrance and severity of Fgf3 otic morphologic and
molecular phenotypes relative to those of Gbx2.
Expression of other dorsal otic genes downstream of Gbx2 was also
perturbed in Fgf3 mutants. Wnt2b, the gene that most
specifically marks the developing EDS, but the otic function of which is
unknown, is absent from Gbx2 mutant ears
(Lin et al., 2005) and was
strongly reduced or absent from Fgf3 mutant ears, consistent with the
EDS defects in both mutants. Dlx5, which normally marks the entire
dorsal half of the otocyst and is required for both EDS and semicircular canal
development (Merlo et al.,
2002), was also affected in both mutants, but in slightly
different ways. In Gbx2 mutants, Dlx5 is lost from the
entire dorsomedial domain (Lin et al.,
2005), whereas in Fgf3 mutants, only the ventral-most
region of both the dorsomedial and dorsolateral Dlx5 domains were
absent. This suggests that Dlx5 might not be a direct target of FGF3
signaling, but is downstream of Gbx2 and in this instance is
revealing the loss of the dorsomedial EDS domain and the consequent dorsal
shifts of its most ventromedial and ventrolateral domains. Finally,
Hmx3 is expressed in the dorsolateral otocyst and subsequently in the
semicircular canals and is required for canal, but not for EDS, development
(Wang et al., 2004).
Hmx3 expression is unchanged in Gbx2 mutants
(Lin et al., 2005) and
slightly downregulated in Wnt1/Wnt3a double mutants
(Riccomagno et al., 2005). By
contrast, in Fgf3 mutants there is a medial expansion of
Hmx3 expression, suggesting that Fgf3 might normally
function directly to restrict expansion of Hmx3 into the medial
otocyst or might function indirectly by restricting the Wnt3a domain
dorsally.
Fgf3 does not have a unique role in ventral otic patterning
Despite abnormal cochlear development in Fgf3 mutants, expression
of Pax2 and Otx2, ventral otic genes downstream of SHH
signaling and necessary for cochlear development
(Morsli et al., 1999;
Riccomagno et al., 2002;
Burton et al., 2004), were
largely unaffected, except for one case of loss of Otx2 expression.
This was somewhat unexpected, given that both Mafb and Gbx2
mutants show a medial expansion of otic Otx2 expression
(Lin et al., 2005;
Choo et al., 2006). However,
since Fgf3 appears to be required to maintain rather than to initiate
Gbx2 expression, it is possible that the early phase of Gbx2
expression is sufficient to establish the Otx2 domain and that FGF3,
on its own, is not a major player in cochlear development. The possibility for
redundant roles with Fgf10, such as occurs during otic induction,
remains and will be tested using conditional mutants.
|
) and
further suggests that Fgf3 might participate in posterior sensory
domain specification. However, addressing this issue directly and assessing
the relative contributions of Fgf3 and Fgf10 to posterior
sensory and non-sensory development will require analysis of double
conditional mutants in which the hindbrain requirement for Fgf3 in
EDS induction is bypassed.
As in the original study of Fgf3neo mutants, GVIII, as
marked in this study by Fgf10 expression, was notably smaller in
affected mutant ears. We also consistently observed a dorsal shift of the
ganglion in affected ears, as if either the neurogenic domain itself or the
expression domain for an otocyst-derived attractant for delaminating GVIII
neuroblasts had shifted. It is interesting that a similar dislocation of GVIII
and the Fgf3 expression domain was noted in studies of Mafb
mutants (McKay et al.,
1996).
Cross-talk between WNTs and FGFs in inner ear development
Our results show that Fgf3 negatively regulates Wnt3a in
the region of r5-6, preventing a ventral expansion of Wnt3a
transcripts. This suggests an important role for Fgf3 not only in
reinforcing the inductive effect of WNT signals on the dorsal otocyst, but
also in regulating localization of WNT signals to the dorsal-most region of
the neural tube. Since WNT proteins are unlikely to diffuse over long
distances, this localization may serve to limit the effects of WNTs to the
dorsal otic vesicle.
FGF/WNT cross-talk is emerging as an important mechanism regulating various
biological processes (Dailey et al.,
2005), including brain, tooth and kidney development
(Moon et al., 1997). In some
cases, the parallel activation of FGF and WNT pathways causes developmental
changes different from the individual effects of each factor, as exemplified
by FGF/WNT interactions to specify neural and epidermal fate in the chick
epiblast (Wilson et al.,
2001). Evidence to support the role for FGFs in modulating WNT
signaling comes from mouse genetic experiments showing that cross-regulation
of FGF and WNT signaling is fundamental to normal skull development, when FGFs
inhibit WNTs and subsequently osteoblast differentiation
(Dailey et al., 2005). There
are additional roles for cross-talk between the WNT and FGF pathways in otic
development. During chick otic induction, mesodermal FGF19 induces
neurectodermal expression of Wnt8c
(Ladher et al., 2000). Both
types of signals also participate in otic placode induction
(Ladher et al., 2000;
Wright and Mansour, 2003;
Ladher et al., 2005), with WNT
signals needed for stabilizing the otic placode cell state by enhancing and
sensitizing the response of ectoderm to inductive FGF signaling
(Ohyama et al., 2006). Given
that WNT and FGF signaling pathway components continue to be expressed in the
inner ear epithelia during the later stages of otic morphogenesis and sensory
organ patterning, and that roles for each pathway are beginning to be defined
(Pirvola et al., 2002;
Pirvola et al., 2004;
Wang et al., 2006;
Hayashi et al., 2007), it will
not be surprising if further intersections between these pathways are
uncovered in the ear.
|
ACKNOWLEDGMENTS |
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REFERENCES |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Alvarez, Y., Alonso, M. T., Vendrell, V., Zelarayan, L. C.,
Chamero, P., Theil, T., Bosl, M. R., Kato, S., Maconochie, M., Riethmacher, D.
et al. (2003). Requirements for FGF3 and FGF10 during inner
ear formation. Development
130,6329
-6338.
Barald, K. F. and Kelley, M. W. (2004). From
placode to polarization: new tunes in inner ear development.
Development 131,4119
-4130.
Bok, J., Bronner-Fraser, M. and Wu, D. K.
(2005). Role of the hindbrain in dorsoventral but not
anteroposterior axial specification of the inner ear.
Development 132,2115
-2124.
Burton, Q., Cole, L. K., Mulheisen, M., Chang, W. and Wu, D. K. (2004). The role of Pax2 in mouse inner ear development. Dev. Biol. 272,161 -175.[CrossRef][Medline]
Carney, P. R. and Silver, J. (1983). Studies on cell migration and axon guidance in the developing distal auditory system of the mouse. J. Comp. Neurol. 215,359 -369.[CrossRef][Medline]
Carpenter, E. M., Goddard, J. M., Chisaka, O., Manley, N. R. and Capecchi, M. R. (1993). Loss of Hox-A1 (Hox-1.6) function results in the reorganization of the murine hindbrain. Development 118,1063 -1075.[Abstract]
Chang, W., Brigande, J. V., Fekete, D. M. and Wu, D. K.
(2004). The development of semicircular canals in the inner ear:
role of FGFs in sensory cristae. Development
131,4201
-4211.
Choo, D., Ward, J., Reece, A., Dou, H., Lin, Z. and Greinwald, J. (2006). Molecular mechanisms underlying inner ear patterning defects in kreisler mutants. Dev. Biol. 289,308 -317.[CrossRef][Medline]
Cordes, S. P. and Barsh, G. S. (1994). The mouse segmentation gene kr encodes a novel basic domain-leucine zipper transcription factor. Cell 79,1025 -1034.[CrossRef][Medline]
Dailey, L., Ambrosetti, D., Mansukhani, A. and Basilico, C. (2005). Mechanisms underlying differential responses to FGF signaling. Cytokine Growth Factor Rev. 16,233 -247.[CrossRef][Medline]
Depew, M. J., Liu, J. K., Long, J. E., Presley, R., Meneses, J. J., Pedersen, R. A. and Rubenstein, J. L. (1999). Dlx5 regulates regional development of the branchial arches and sensory capsules. Development 126,3831 -3846.[Abstract]
Dressler, G. R., Deutsch, U., Chowdhury, K., Nornes, H. O. and
Gruss, P. (1990). Pax2, a new murine paired-box-containing
gene and its expression in the developing excretory system.
Development 109,787
-795.
Everett, L. A., Belyantseva, I. A., Noben-Trauth, K., Cantos,
R., Chen, A., Thakkar, S. I., Hoogstraten-Miller, S. L., Kachar, B., Wu, D. K.
and Green, E. D. (2001). Targeted disruption of mouse Pds
provides insight about the innerear defects encountered in Pendred syndrome.
Hum. Mol. Genet. 10,153
-161.
Fekete, D. M. and Wu, D. K. (2002). Revisiting cell fate specification in the inner ear. Curr. Opin. Neurobiol. 12,35 -42.[CrossRef][Medline]
Fitoz, S., Sennaroglu, L., Incesulu, A., Cengiz, F. B., Koc, Y. and Tekin, M. (2007). SLC26A4 mutations are associated with a specific inner ear malformation. Int. J. Pediatr. Otorhinolaryngol. 71,479 -486.[CrossRef][Medline]
Fritzsch, B., Pauley, S. and Beisel, K. W. (2006). Cells, molecules and morphogenesis: the making of the vertebrate ear. Brain Res. 1091,151 -171.[CrossRef][Medline]
Frohman, M. A., Martin, G. R., Cordes, S. P., Halamek, L. P. and Barsh, G. S. (1993). Altered rhombomere-specific gene expression and hyoid bone differentiation in the mouse segmentation mutant, kreisler (kr). Development 117,925 -936.[Abstract]
George, K. M., Leonard, M. W., Roth, M. E., Lieuw, K. H.,
Kioussis, D., Grosveld, F. and Engel, J. D. (1994). Embryonic
expression and cloning of the murine GATA-3 gene.
Development 120,2673
-2686.
Grove, E. A., Tole, S., Limon, J., Yip, L. and Ragsdale, C. W. (1998). The hem of the embryonic cerebral cortex is defined by the expression of multiple Wnt genes and is compromised in Gli3-deficient mice. Development 125,2315 -2325.[Abstract]
Hayashi, T., Cunningham, D. and Bermingham-McDonogh, O. (2007). Loss of Fgfr3 leads to excess hair cell development in the mouse organ of Corti. Dev. Dyn. 236,525 -533.[CrossRef][Medline]
Henrique, D., Adam, J., Myat, A., Chitnis, A., Lewis, J. and Ish-Horowicz, D. (1995). Expression of a Delta homologue in prospective neurons in the chick. Nature 375,787 -790.[CrossRef][Medline]
Hulander, M., Kiernan, A. E., Blomqvist, S. R., Carlsson, P.,
Samuelsson, E. J., Johansson, B. R., Steel, K. P. and Enerback, S.
(2003). Lack of pendrin expression leads to deafness and
expansion of the endolymphatic compartment in inner ears of Foxi1 null mutant
mice. Development 130,2013
-2025.
Hutson, M. R., Lewis, J. E., Nguyen-Luu, D., Lindberg, K. H. and Barald, K. F. (1999). Expression of Pax2 and patterning of the chick inner ear. J. Neurocytol. 28,795 -807.[CrossRef][Medline]
Jones, C. M., Lyons, K. M. and Hogan, B. L. (1991). Involvement of Bone Morphogenetic Protein-4 (BMP-4) and Vgr-1 in morphogenesis and neurogenesis in the mouse. Development 111,531 -542.[Abstract]
Karis, A., Pata, I., van Doorninck, J. H., Grosveld, F., de Zeeuw, C. I., de Caprona, D. and Fritzsch, B. (2001). Transcription factor GATA-3 alters pathway selection of olivocochlear neurons and affects morphogenesis of the ear. J. Comp. Neurol. 429,615 -630.[CrossRef][Medline]
Kiernan, A. E., Steel, K. P. and Fekete, D. M. (2002). Development of the mouse inner ear. In Mouse Development: Patterning, Morphogenesis, and Organogenesis (ed. J. Rossant and P. P. L. Tam), pp. 539-566. San Diego: Academic Press.
Kim, H. J., Song, J. W., Chon, K. M. and Goh, E. K. (2004). Common crus aplasia: diagnosis by 3D volume rendering imaging using 3DFT-CISS sequence. Clin. Radiol. 59,830 -834.[CrossRef][Medline]
Ladher, R. K., Anakwe, K. U., Gurney, A. L., Schoenwolf, G. C.
and FrancisWest, P. H. (2000). Identification of synergistic
signals initiating inner ear development. Science
290,1965
-1967.
Ladher, R. K., Wright, T. J., Moon, A. M., Mansour, S. L. and
Schoenwolf, G. C. (2005). FGF8 initiates inner ear induction
in chick and mouse. Genes Dev.
19,603
-613.
Lawoko-Kerali, G., Rivolta, M. N. and Holley, M. (2002). Expression of the transcription factors GATA3 and Pax2 during development of the mammalian inner ear. J. Comp. Neurol. 442,378 -391.[CrossRef][Medline]
Li, C. W., Van De Water, T. R. and Ruben, R. J. (1978). The fate mapping of the eleventh and twelfth day mouse otocyst: an in vitro study of the sites of origin of the embryonic inner ear sensory structures. J. Morphol. 157,249 -267.[CrossRef][Medline]
Lillevali, K., Matilainen, T., Karis, A. and Salminen, M. (2004). Partially overlapping expression of Gata2 and Gata3 during inner ear development. Dev. Dyn. 231,775 -781.[CrossRef][Medline]
Lillevali, K., Haugas, M., Matilainen, T., Pussinen, C., Karis, A. and Salminen, M. (2006). Gata3 is required for early morphogenesis and Fgf10 expression during otic development. Mech. Dev. 123,415 -429.[CrossRef][Medline]
Lin, Z., Cantos, R., Patente, M. and Wu, D.
(2005). Gbx2 is required for the morphogenesis of the
mouse inner ear: a downstream target of hindbrain signaling.
Development 132,2309
-2318.
Mafong, D. D., Shin, E. J. and Lalwani, A. K. (2002). Use of laboratory evaluation and radiologic imaging in the diagnostic evaluation of children with sensorineural hearing loss. Laryngoscope 112,1 -7.[Medline]
Mahmood, R., Mason, I. J. and Morriss-Kay, G. M. (1996). Expression of Fgf-3 in relation to hindbrain segmentation, otic pit position and pharyngeal arch morphology in normal and retinoic acid-exposed mouse embryos. Anat. Embryol. 194, 13-22.[Medline]
Manfre, L., Genuardi, P., Tortorici, M. and Lagalla, R. (1997). Absence of the common crus in Goldenhar syndrome. AJNR Am. J. Neuroradiol. 18,773 -775.[Abstract]
Mansour, S. L. and Schoenwolf, G. C. (2005). Morphogenesis of the inner ear. In Development of the Inner Ear (ed. M. W. Kelley, D. K. Wu, A. N. Popper and R. R. Fay), pp.43 -84. New York: Springer-Verlag.
Mansour, S. L., Goddard, J. M. and Capecchi, M. R. (1993). Mice homozygous for a targeted disruption of the proto-oncogene int-2 have developmental defects in the tail and inner ear. Development 117,13 -28.[Abstract]
McKay, I. J., Lewis, J. and Lumsden, A. (1996). The role of FGF-3 in early inner ear development: an analysis in normal and kreisler mutant mice. Dev. Biol. 174,370 -378.[CrossRef][Medline]
Merlo, G. R., Paleari, L., Mantero, S., Zerega, B., Adamska, M., Rinkwitz, S., Bober, E. and Levi, G. (2002). The Dlx5 homeobox gene is essential for vestibular morphogenesis in the mouse embryo through a BMP4-mediated pathway. Dev. Biol. 248,157 -169.[CrossRef][Medline]
Moon, R. T., Brown, J. D. and Torres, M. (1997). WNTs modulate cell fate and behavior during vertebrate development. Trends Genet. 13,157 -162.[CrossRef][Medline]
Morsli, H., Choo, D., Ryan, A., Johnson, R. and Wu, D. K.
(1998). Development of the mouse inner ear and origin of its
sensory organs. J. Neurosci.
18,3327
-3335.
Morsli, H., Tuorto, F., Choo, D., Postiglione, M. P., Simeone, A. and Wu, D. K. (1999). Otx1 and Otx2 activities are required for the normal development of the mouse inner ear. Development 126,233
