Biology Department, Texas A&M University, College Station, TX 77843-3258, USA
* Author for correspondence (e-mail: briley{at}mail.bio.tamu.edu)
Accepted 9 August 2002
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SUMMARY |
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Key words: Inner ear, Hair cell, FGF signaling, ace, Morpholino, val, kreisler, pax5, Zebrafish
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INTRODUCTION |
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Much less is known about the role played by hindbrain signals in later
stages of inner ear development. Experiments in chick embryos show that
rotation of the early otic vesicle about the anteroposterior axis reorients
gene expression patterns in a manner suggesting that proximity to the
hindbrain influences differentiation of cells within the otic vesicle
(Wu et al., 1998;
Hutson et al., 1999
). In
zebrafish, Xenopus, chick and mouse embryos, Fgf3 continues
to be expressed in the hindbrain after otic placode induction
(Mahmood et al., 1995
;
Mahmood et al., 1996
;
McKay et al., 1996
;
Lombardo et al., 1998
;
Phillips et al., 2001
). This
raises the question of whether this factor also helps regulate subsequent
development of the otic placode or otic vesicle.
Analysis of the valentino (val) mutant in zebrafish
provides indirect evidence that hindbrain signals are necessary for normal
development of the otic vesicle (Moens et
al., 1996; Moens et al.,
1998
). val encodes a bZip transcription factor that is
normally expressed in r5 and r6. val/val mutants produce an abnormal
hindbrain in which the r5/6 anlagen fails to differentiate properly and gives
rise to a single abnormal segment, rX, which shows confused segmental
identity. Although the val gene is not expressed in the inner ear,
val/val mutants produce otic vesicles that are small and malformed.
As otic induction appears to occur normally in val/val mutants
(Mendonsa and Riley, 1999
), we
infer that altered hindbrain patterning perturbs signals required for later
aspects of otic development. Mice homozygous for a mutation in the
ortholologous gene, kreisler (Mafb Mouse Genome Informatics),
also show later defects in development of the otic vesicle
(Deol, 1964
;
Cordes and Barsh, 1994
). The
inner ear defects in kreisler mutants are thought to result from insufficient
expression of Fgf3 in the hindbrain
(McKay et al., 1996
). In
contrast to zebrafish, mouse Fgf3 is initially expressed at moderate
levels in the hindbrain from r1 through r6. As development proceeds,
expression downregulates in the anterior hindbrain but upregulates in r4
(Mahmood et al., 1996
). After
formation of the otic placodes, Fgf3 expression also upregulates in
r5 and r6. This upregulation fails to occur in kreisler mutants, possibly
accounting for subsequent patterning defects in the inner ear
(McKay et al., 1996
).
To examine the relationship between hindbrain and otic vesicle development in zebrafish, we have examined patterning of these tissues in wild-type and val/val mutant embryos. We find that val/val mutants produce excess and ectopic hair cells at virtually any position in the epithelium juxtaposed to the hindbrain. Expression of the anterior otic markers nkx5.1 (hmx3 Zebrafish Information Network) and pax5 is also seen ectopically throughout this region of the otic vesicle. Conversely, expression of the posterior marker zp23 (pou23 Zebrafish Information Network) is ablated in val/val embryos. Analysis of hindbrain patterning shows that fgf3 is misexpressed in the rX region of val/val mutants. Disruption of fgf3 function by injection of an antisense morpholino oligomer blocks formation of ectopic hair cells and suppresses AP patterning defects in the otic vesicle of val/val mutants. By contrast, fgf8 is expressed normally in val/val embryos, and loss of fgf8 does not suppress the inner ear defects caused by the val mutation. These data indicate that the expanded domain of fgf3 plays a crucial role in the etiology of inner ear defects in val/val mutants and suggest that Fgf3 secreted by r4 normally specifies anterior fates, suppresses posterior fates and stimulates hair cell formation in the anterior of the otic vesicle.
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MATERIALS AND METHODS |
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Identification of mutant embryos
Live val/val homozygotes were reliably identified after 19 h by
the small size and round shape of the otic vesicle. In addition, fixed
val/val embryos stained for pax2.1, pax5 or zp23
showed characteristic changes in posterior hindbrain patterning. At earlier
stages, val/val mutants were identified by loss of krox20
(egr2 Zebrafish Information Network) staining in rhombomere 5
(Moens et al., 1996). Live
ace/ace (fgf8/fgf8 Zebrafish Information Network)
mutants were readily identified after 24 h by the absence of a
midbrain-hindbrain border and enlarged optic tectum
(Brand et al., 1996
). In
addition, ace/ace specimens that were fixed and stained for
pax2.1 or pax5 showed no staining in the midbrain-hindbrain
border. At earlier stages (14 h), ace/ace mutants were identified by
loss of fgf3 expression in the midbrain-hindbrain border.
Whole-mount immunofluorescent staining
Embryos were fixed in MEMFA (0.1 M MOPS at 7.4, 2 mM EGTA, 1 mM
MgSO4, 3.7% formaldehyde) and stained as previously described
(Riley et al., 1999). Primary
antibodies used in this study were: polyclonal antibody directed against mouse
Pax2 (Berkeley Antibody Company, 1:100 dilution), which also recognizes
zebrafish pax2.1 (Riley et al.,
1999
); Monoclonal antibody directed against acetylated tubulin
(Sigma T-6793, 1:100), which binds hair cell kinocilia
(Haddon and Lewis, 1996
).
Secondary antibodies were Alexa 546 goat anti-rabbit IgG (Molecular Probes
A-11010, 1:50) or Alexa 488 goat anti-mouse IgG (Molecular Probes A-11001,
1:50).
Whole-mount in situ hybridization
Whole-mount in situ hybridization was performed as described
(Stachel et al., 1993) using
riboprobes for fgf3 (Kiefer et
al., 1996a
), fgf8
(Reifers et al., 1998
),
dlA (Appel and Eisen,
1998
; Haddon et al.,
1998b
), pax5 (Pfeffer
et al., 1998
), dlx3 and msxc
(Ekker et al., 1992
),
nkx5.1 (Adamska et al.,
2000
), otx1 (Li et
al., 1994
), and zp23
(Hauptmann and Gerster, 2000
).
Two-color in situ hybridization was performed essentially as described by
Jowett (Jowett, 1996
) with
minor modifications (Phillips et al.,
2001
).
Morpholino oligomer injection
fgf3-specific morpholino oligomer obtained from Gene Tools was
diluted in Danieaux solution [58 mM NaCl, 0.7 mM KCl, 0.4 mM MgSO4,
0.6 mM Ca(NO3)2, 5.0 mM HEPES, pH 7.6] to a
concentration of 5 µg/µl as previously described
(Nasevicius and Ekker, 2000;
Phillips et al., 2001
).
Approximately 1 nl (5 ng fgf3-MO) was injected into the yolk cell at the one-
to two-cell stage.
Mis-expression of val
Wild-type val was ligated into pCS2 expression vector by Andrew
Waskiewicz (Cecilia Moens' laboratory) and was kindly provided as a gift. RNA
was synthesized in vitro and 1 ng of RNA was injected into the yolk of
cleaving embryos at the one- to four-cell stage.
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RESULTS |
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Altered anteroposterior patterning in val/val mutants
We next examined expression of various otic markers to further characterize
altered patterning in val/val embryos. Expression of pax5 is
first detectable in the inner ear at 17.5-18.0 h
(Pfeffer et al., 1998). This
expression domain is normally restricted to the anterior part of the otic
vesicle adjacent to r4 and is maintained through at least 30 h
(Fig. 3A,C). In
val/val embryos, pax5 expression extends along the entire
length of the medial wall of the otic vesicle
(Fig. 3B,D). Another anterior
marker, nkx5.1, is also expressed throughout the medial wall of the
otic vesicle in val/val mutants
(Fig. 3F). By contrast,
zp23 is normally expressed in posterior medial cells adjacent to r5
and r6 in the wild type but is not detectably expressed in val/val
embryos (Fig. 3G,H). Otic
patterning is not globally perturbed, however. Mutant embryos show a normal
pattern of dlx3 expression in the dorsomedial epithelium
(Fig. 4F). Similarly,
otx1 is expressed normally in ventral and lateral cells of
val/val mutants (Fig.
4A-D). Based on studies in mouse, the dorsal and lateral domains
of dlx3 (dlx3b Zebrafish Information Network) and
otx1 probably help regulate development of the semicircular canals
and sensory cristae (Depew at al.,
1999
; Krauss and Lufkin,
1999
; Morsli et al.,
1999
; Mazan et al.,
2001
). It has previously been reported that formation of
semicircular canals is totally disrupted in val/val mutants
(Moens et al., 1998
). However,
we find that this is a highly variable phenotype, ranging from grossly
abnormal morphogenesis to nearly normal patterning at day 3
(Fig. 4G-I). Morphology
typically becomes increasingly aberrant with time, possibly resulting from
improper regulation of endolymph, as seen in kreisler mutant mice
(Deol, 1964
;
Brigande et al., 2000
) (see
Discussion). Regardless of whether semicircular canals develop properly, all
three sensory cristae are produced and express msxc (data not shown).
Thus, some aspects of axial patterning are relatively normal in
val/val embryos at early stages, and the only consistent defect is
that medial cells abutting the hindbrain all show anterior character. This is
consistent with the hypothesis that factors locally expressed in the hindbrain
regulate anterposterior fates in the medial wall of the otic vesicle, and that
such factors are misregulated in the rX region of val/val mutants.
Such misexpression could also explain the abnormal pattern of hair cells
produced in val/val mutants.
|
|
Expression of fgf3 and fgf8 in the val/val
hindbrain
Fgf3 and Fgf8 are both expressed in the r4 anlagen during late gastrulation
and cooperate to induce the otic placode
(Phillips et al., 2001). We
hypothesized that persistent expression of one or both of these factors in r4
plays a later role in patterning the otic placode and vesicle. In both
wild-type and val/val embryos, fgf8 is expressed at high
levels in r4 at 12 h (Fig.
5A,B) but is downregulated by 14 h (not shown). This argues
against a role for Fgf8 in the etiology of the inner ear phenotype in
val/val embryos. By contrast, fgf3 expression shows a
consistent difference between val/val and wild-type embryos. In the
wild type, hindbrain expression of fgf3 is restricted to r4 and is
maintained through at least 18 h when the otic vesicle forms
(Fig. 5C,E, and data not
shown). In val/val mutants, fgf3 shows similar developmental
timing but is expressed in an expanded domain extending from r4 through rX
(Fig. 5D,F). Within rX, the
level of expression falls off gradually towards the posterior such that there
is no clear posterior limit of expression. Ectopic expression of fgf3
in val/val embryos is first detectable at 10 h, corresponding to the
time when val normally begins to function in the r5/6 anlagen (data
not shown). Initially, ectopic expression of fgf3 in rX is much
weaker than in r4. Expression in rX subsequently increases to a level similar
to that seen in r4 by 12 h (Fig.
5D). These data suggest that expansion of the domain of
fgf3 in the hindbrain could play a role in misexpression of AP
markers and production of ectopic hair cells in the inner ear.
|
The above data also suggest that val normally functions, directly
or indirectly, to exclude fgf3 expression from r5/6. To explore this
more fully, we examined the effects of val mis-expression by
injecting val RNA into wild-type embryos. In more than half (55/98)
of val-injected embryos, hindbrain expression of fgf3 was
dramatically reduced or ablated (Fig.
6A,B). Similar effects were seen at 10, 12 and 14 h (data not
shown). At 24 h, otic vesicles were usually small (15/64) or totally ablated
(36/64) (Fig. 6C,D). Disrupting
fgf3 by itself impairs, but does not ablate, otic tissue
(Phillips et al., 2001;
Vendrell et al., 2001
;
Maroon et al., 2002
). This
indicates that val mis-expression affects other processes in addition
to fgf3 expression. Indeed, ubiquitous mis-expression of val
frequently caused truncation of the trunk and tail (46/64,
Fig. 6C) and could therefore
impair mesendodermal signals on which otic development relies (reviewed by
Whitfield et al., 2002
).
However, even among embryos with normal axial development, about half showed
partial loss of fgf3 expression (5/10) and impaired otic development (18/34).
In many of these cases, these defects were limited to one side of the embryo
(Fig. 6E,F), possibly resulting
from variation in the amount of RNA inherited by early cleavage stage
blastomeres. In contrast to fgf3, expression of fgf8 was
relatively normal in most (82/85) val-injected embryos, even those
with axial truncations (Fig.
6H). These data support the hypothesis that val
specifically represses fgf3 expression in the hindbrain. This is in
sharp contrast to the function of the mouse homolog kreisler, which is
required to activate high level expression of Fgf3 in r5 and r6
(McKay et al., 1996
). Such
species differences may have been important for evolutionary changes in inner
ear structure and function (see Discussion).
|
Dependence of inner ear patterning on Fgf3
To test the role of Fgf3 in otic vesicle patterning, embryos were injected
with fgf3-MO, an antisense oligomer that specifically inhibits translation of
fgf3 mRNA (Nasevicius and Ekker,
2000; Phillips et al.,
2001
; Maroon et al.,
2002
). Injection of fgf3-MO into wild-type embryos results in a
range of defects with varying degrees of severity
(Phillips et al., 2001
). The
size of otic vesicle is usually reduced, and about half (42/86) of
Fgf3-depleted wild-type embryos show little or no pax5 expression in
the inner ear (Fig. 7A).
Expression of nkx5.1 is also reduced or ablated in the otic vesicle
and vestibulo-acoustic ganglion in about half (30/62) of injected wild-type
embryos (data not shown). By contrast, expression of zp23 often
expands anteriorly in the otic vesicle to include medial cells adjacent to r4
(21/32 embryos, Fig. 7D). Hair
cell production is reduced by up to 70% in severely affected embryos
(Fig. 7G;
Table 1, note range of data).
Injection of fgf3-MO into val/val mutants leads to further reduction
in the size of otic vesicle. Expression of pax5 is strongly reduced
in most cases: In one experiment, 37% (26/71) showed pax5 expression
limited to the anterior of the otic vesicle
(Fig. 7B) and 38% (27/71)
showed no detectable expression (Fig.
7C). Similarly, nkx5.1 is strongly reduced or ablated in
about half (16/30) of injected val/val embryos
(Fig. 7F). Most (12/15)
val/val embryos injected with fgf3-MO express zp23 in the
otic vesicle, including tissue adjacent to r4
(Fig. 7E). Hair cell production
is reduced to a level comparable with that seen in Fgf3-depleted wild-type
embryos (Table 1). In addition,
depletion of Fgf3 prevents formation of ectopic hair cells in the majority
(19/25) of val/val embryos (Fig.
7H,I). Thus, Fgf3-depletion prevents formation of excess and
ectopic hair cells as well as misexpression of AP markers in val/val
mutants. As the hindbrain is the only periotic tissue known to express
fgf3 at this time, we infer that the expanded domain of fgf3
in val/val mutants is crucial for generation of the above inner ear
defects.
|
Dependence of inner ear patterning on Fgf8
Although expression of fgf8 did not appear to correlate with
changes in inner ear patterning in val/val mutants, we sought to
characterize patterning defects in ace/ace mutants and examine
genetic interactions between ace and val. Defects in
ace/ace embryos are less variable than in embryos injected with
fgf3-MO (Phillips et al.,
2001). The otic vesicle in ace/ace mutants is reduced in
size but usually retains an oval shape at 24 h. Hair cell production is
reduced by more than half in the majority of ace/ace mutants
(Table 1), and more than a
third (7/19) of specimens produce no posterior hair cells at all
(Fig. 8E). In ace/ace;
val/val double mutants, the size of otic vesicle is further reduced
and the number of hair cells is comparable with that in ace/ace
single mutants (Fig. 8F;
Table 1). Hair cells often form
adjacent to r4 and/or rX in ace/ace; val/val double mutants and are
usually located in a more medial position than are hair cells in
ace/ace mutants (Fig.
8F). In addition, pax5 is expressed along the full length
of the anteroposterior axis of the ear
(Fig. 8D). Expression of
nkx5.1 is also expanded in acelace-val/val double mutants,
while zp23 is not expressed (data not shown). Thus, the ace
mutation strongly perturbs inner ear patterning, but loss of fgf8
function does not suppress the patterning defects associated with the
val mutation. This is probably because expression of fgf3 is
expanded in the hindbrain of ace/ace; val/val double mutants as in
val/val mutants (Fig.
8B). Together, these data indicate that val and
ace affect different developmental pathways, and that the early
patterning defects seen in the val/val mutant ear are not caused by
mis-regulation of fgf8 expression.
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DISCUSSION |
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The fact that any hair cells are produced at all in Fgf3-depleted embryos
indicates that additional hair cell-inducing factors must be present.
fgf8 is clearly required for normal hair cell formation and could
partially compensate for loss of fgf3
(Reifers et al., 1998;
Phillips et al., 2001
).
However, several observations indicate that the role of fgf8 is
distinct from that of fgf3. First, periotic expression of
fgf8 declines sharply just before the placode forms at 14 h, thereby
limiting its ability to influence later otic patterning. Second, expression
patterns of nkx5.1, pax5 and zp23 are not altered in
ace/ace embryos (Fig.
8C, and data not shown), indicating that AP patterning is
relatively normal. Third, loss of fgf8 inhibits hair cell formation
but does not prevent formation of ectopic hair cells in val/val
mutants. The latter are dependent on fgf3 instead. Thus, in contrast
to fgf3, there is little evidence to suggest that the r4 domain of
fgf8 regulates regional patterning in the otic placode. Instead,
fgf8 may play a more general role in stimulating hair cell competence
during the process of placode induction.
Paradoxically, anterior hair cells are not as severely impaired in
ace/ace mutants as are posterior hair cells. Posterior hair cells are
totally ablated in about 1/3 of ace/ace mutants. This is difficult to
explain based solely on the expression domain of fgf8, but may
reflect changes in the dimensions of the otic placode. In ace/ace
mutants, the otic placode is often reduced to a domain juxtaposed to r4 and r5
only. Thus, secretion of Fgf3 from r4 may be sufficient to induce some
anterior hair cells in the absence of Fgf8, whereas cells in the posterior
otic placode may lie too far from r6 to benefit from inductive factors
possibly secreted from there. No clear candidates for r6-specific inducers are
known, but the Fgf-inducible genes erm, pea3 and sprouty4
are expressed in r6 (Fürthauer et
al., 2001; Raible and Brand,
2001
; Roehl and
Nüsslein-Volhard, 2001
) (S.-J. K., B. T. P., R. H. and B. B.
R., unpublished), suggesting that at least one as yet unidentified Fgf homolog
is expressed there.
The reason for expanded expression of fgf3 in val/val
mutants is not clear, but there are several possibilities. First, this could
result from mis-specification of segment identity in the rX territory. Several
other genes normally expressed in adjacent segments, including hoxb1
in r4 and hoxb4 in r7, eventually come to be expressed in rX
(Prince et al., 1998).
However, these changes do not occur until 20 somites (19 h). By contrast,
expression of fgf3 in rX is first detected at 10 h in
val/val mutants, corresponding to the time when val normally
begins to function (Moens et al.,
1998
). This raises the alternative possibility that Val protein
normally acts to transcriptionally repress fgf3. In support of this,
mis-expression of val inhibits r4-expression of fgf3, but
not fgf8 (Fig. 6).
Direct support for transcriptional regulation by Val will require analysis of
the promoter/enhancer regions of fgf3.
Comparison of val and kreisler
In sharp contrast to val function in zebrafish, mouse kreisler is
required, directly or indirectly, for upregulation of Fgf3 in r5 and r6
(McKay et al., 1996). This
difference is notable because so many other aspects of early hindbrain and ear
development are conserved between these species. The high degree of sequence
identity leaves little doubt that the zebrafish genes are orthologous to
kreisler and Fgf3 (Kiefer et al.,
1996a
; Moens et al.,
1998
). There are, however, differences in the N- and C- terminal
regions of Fgf3 in zebrafish and mouse. These regions are thought to be
important for mediating the characteristic receptor binding preferences and
signaling properties of Fgf3. Nevertheless, these functional properties are
actually very similar between the fish and mouse proteins
(Kiefer et al., 1996b
). This,
combined with the broad similarities in their expression patterns and
involvement in early otic development, strengthen the notion that the fish and
mouse fgf3 genes are indeed orthologs. Because zebrafish often has
multiple homologs of specific tetrapod genes, it is possible that a second
fgf3 gene might be present in the zebrafish genome that shows an
expression pattern more like the mouse gene. If so, it will be important to
address its function as well. However, we have shown that the known
fgf3 ortholog plays an essential role in the etiology of the ear
phenotype in val/val embryos, as key aspects of the phenotype are
suppressed by injecting fgf3-MO. Morpholino oligomers are highly gene-specific
in their effects, and even though they do not totally eliminate gene function,
they generate phenotypes that are indistinguishable from those caused by known
null mutations (Nasevicius and Ekker,
2000
; Phillips et al.,
2001
; Raible and Brand,
2001
; Maroon et al.,
2002
). On balance, it appears that the general role of Fgf3 in
otic development has been conserved in mouse and fish but that differential
regulation in the hindbrain represents a real difference between these
species.
Considering the above differences in hindbrain signaling, one might expect
the ear phenotypes in val/val and Mafb/Mafb mutants to be
quite different. Instead, the phenotypes appear strikingly similar. In
Mafb/Mafb embryos, as in val/val embryos, development of the
otic vesicle is highly variable and defects can be seen in virtually all
regions of the labyrinth (Deol,
1964). In Mafb/Mafb mutants, formation of the wall of the
otic capsule is often incomplete, with large gaps through with membranous
epithelia protrude, and morphology of the labyrinth is usually grossly
abnormal. Such global disruption may be related to buildup of excess fluid
pressure due to failure of the endolymphatic duct to form in many or most
Mafb/Mafb mutants (Deol,
1964
; Brigande et al.,
2000
). Whether a similar problem occurs in val/val
mutants is not clear. The existence of an endolymphatic duct in zebrafish has
only recently been documented (Bever and
Fekete, 2002
), but it does not begin to form until around day 8.
Most val/val mutants die before this time, and they often begin to
show defects in morphogenesis (e.g. of the semicircular canals) by 72 h
(Fig. 4, and data not shown).
Although these early defects cannot be explained by the absence of an
endolymphatic duct, mutant ears often appear swollen and distended by day 3,
suggesting a buildup of endolymphatic pressure. It is possible that cellular
functions normally required to maintain a proper fluid balance in the early
vesicle are mis-regulated in val/val mutants. Thus, hydrops may be an
important contributing factor to the defects in both Mafb/Mafb and
val/val mutants.
Another similarity between Mafb/Mafb and val/val mutants
is that they both form ectopic patches of hair cells. However, this phenotype
has a completely different etiology in the two species. In tetrapod
vertebrates, sensory epithelia do not begin to differentiate until after the
various chambers of the labyrinth begin to form. Thus, formation of ectopic
hair cells in Mafb/Mafb mutants probably reflects the general
disorganization of, and chaotic protrusions from, the labyrinth
(Deol, 1964). By contrast,
sensory epithelia in zebrafish begin to differentiate much earlier. Macular
equivalence groups are already specified at 14 h when the placode first forms
(Haddon et al., 1998a
;
Whitfield et al., 2002
), and
the first hair cells (visualized by the presence of kinocilia) are evident as
soon as the lumen of the vesicle forms at 18.5 h
(Riley et al., 1997
). Thus,
formation of ectopic hair cells in val/val mutants reflects an early
defect in cell fate specification rather than a later defect in morphogenesis.
It is noteworthy that there have been no detailed molecular studies of otic
development in Mafb/Mafb mutants, so a direct comparison of early
pattern formation is not yet possible.
Evolutionary implications
It is interesting to consider that the altered pattern of fgf3
expression in the val/val mutant hindbrain closely resembles the
normal pattern of Fgf3 expression in chick and mouse embryos
(Mahmood, 1995; Mahmood, 1996; McKay et
al., 1996). Analysis of val/val mutants suggests that
misexpression of fgf3 in rX leads to development of excess and
ectopic hair cells in the otic vesicle. It is possible that evolutionary
changes that led to normal expression of Fgf3 in r5/6 in amniotes
were crucial for evolution of the cochlea, which has no known counterpart in
anamniote vertebrates (Lewis et al.,
1985
). In the mouse, development of the cochlea requires FGF
signaling at early otic vesicle stages
(Pirvola et al., 2000
). The
FGF receptor isoform FGFR-2(IIIb) is expressed in the otic epithelium
juxtaposed to the hindbrain. Targeted disruption of this isoform leads to
severe dysgenesis of the cochlea. Cochlear development is also impaired in
Fgf3-null and Mafb/Mafb mutant mice
(Deol, 1964
;
Mansour et al., 1993
). In
Xenopus, Fgf3 expression shows a pattern intermediate between that of
zebrafish and amniotes: The frog gene is initially expressed in r3 through r5
and only later becomes restricted to r4
(Lombardo et al., 1998
).
Although amphibians do not possess a cochlea, they do show modifications of
the posterior otic vesicle that give rise to the basilar and amphibian
papillae, auditory organs not found in fish (reviewed by
Lewis et al., 1985
). Thus,
expression of fgf3 in more posterior regions of the hindbrain
correlates with elaborations of the inner ear that may have been essential for
enhancing auditory function in terrestrial environments.
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ACKNOWLEDGMENTS |
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