Biology Department, Texas A&M University, College Station, TX 77843-3258, USA
* Author for correspondence (e-mail: briley{at}mail.bio.tamu.edu)
Accepted 27 October 2003
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SUMMARY |
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Key words: Otic induction, Hindbrain patterning, pax8, foxi1, erm, dickkopf, Preplacodal domain, Zebrafish
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Introduction |
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A number of studies now point to members of the Fgf family of peptide
ligands as the best candidates for otic-inducing factors produced by periotic
tissues. In particular, Fgf3 appears to play a highly conserved role in otic
induction. In all vertebrates examined to date, Fgf3 is expressed in the
hindbrain directly between the developing otic anlage during mid-late
gastrulation (Wilkinson et al.,
1989; Mahmood et al.,
1995
; Mahmood et al.,
1996
; McKay et al.,
1996
; Lombardo et al.,
1998
; Phillips et al.,
2001
), and misexpression studies in chick and Xenopus
show that Fgf3 can induce formation of otic placodes in ectopic locations
(Vendrell et al., 2000
,
Lombardo et al., 1998
). Loss
of Fgf3 function does not prevent otic induction in either mouse or zebrafish,
although later otic development is clearly impaired
(Mansour et al., 1993
;
Phillips et al., 2001
;
Maroon et al., 2002
;
Leger and Brand, 2002
;
Kwak et al., 2002
). The reason
for continued otic induction is that other Fgf homologs provide redundancy in
the inductive pathway. In zebrafish, fgf8 is coexpressed with
fgf3 in the hindbrain, and loss of both leads to complete failure of
otic induction (Phillips et al.,
2001
; Maroon et al.,
2002
; Leger and Brand,
2002
; Liu et al.,
2003
). Fgf8 does not play a comparable role in tetrapods, but it
is likely to regulate later stages of otic development (reviewed by
Riley and Phillips, 2003
).
Instead, other Fgfs provide redundancy. In the mouse, Fgf10 is
expressed in mesoderm just beneath the preplacode, and loss of both Fgf3 and
Fgf10 ablates otic development (Wright and
Mansour, 2003
). The above studies do not exclude a role for other
inductive signals but, taken together, they indicate that Fgf signaling is
both necessary and sufficient for otic induction.
By contrast, an alternative model was proposed recently in which Fgf must
cooperate with another factor, Wnt8, to induce the otic placode
(Ladher et al., 2000a). In
chick, Fgf19 is expressed initially in subjacent mesoderm and is
found later in hindbrain between prospective otic placodes. By itself, Fgf19
does not induce expression of any otic markers in explants of uncommitted
ectoderm but it does induce expression of the hindbrain factor Wnt8c, the
chick ortholog of Wnt8 (Schubert et al.,
2000
). Exogenous Wnt8c weakly induces a subset of otic markers in
explant cultures, whereas Fgf19 plus Wnt8 strongly induce the full range of
otic markers. Thus, it was proposed that Fgf19 in the mesoderm induces
expression of Wnt8c in the hindbrain, and that the two factors synergyze to
induce the otic placodes. This model has not been tested previously in vivo.
In addition, a complication of the model is that Wnt8c and Fgf19 also strongly
induce expression of Fgf3, which may have played a direct role in inducing the
full range of otic markers. Because FGF19 has no known ortholog in zebrafish,
we addressed the question of whether known zebrafish otic inducers, Fgf3 and
Fgf8, are sufficient to induce otic tissue or must cooperate with Wnt8. Our
data demonstrate that Fgf signaling is both necessary and sufficient for otic
induction whereas Wnt8 is neither necessary nor sufficient. Expression of Fgf
and Wnt reporter genes indicates that Fgf, but not Wnt, signals directly to
the otic anlage. Instead, Wnt8 appears to be indirectly involved in otic
induction by virtue of its requirement for timely hindbrain expression of Fgf
genes.
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Materials and methods |
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In situ hybridization
Embryos were fixed in MEMFA [0.1 M MOPS (pH 7.4), 2 mM EGTA, 1 mM
MgSO4, 3.7% formaldehyde]. In situ hybridizations
(Stachel et al., 1993) were
performed at 67°C using probes for pax2.1
(Krauss et al., 1991
),
fgf8 (Reifers et al.,
1998
), pax8 (Pfeffer
et al., 1998
), TOPdGFP
(Dorsky et al., 2002
),
erm (Roehl and Nusslein-Volhard,
2001
; Raible and Brand,
2001
), wnt8 ORF2
(Lekven et al., 2001
),
foxi1(Solomon et al.,
2003
) and krox-20
(Oxtoby and Jowett, 1993
)
transcripts. The fgf3 construct was generated by amplifying the
coding sequence of fgf3 (GenBank Accession Number NM 131291) and
ligating it into the ClaI and EcoRI sites of pCS2+.
Two-color in situ hybridization was performed essentially as described by
Jowett (Jowett, 1996
), with
several modifications. RNase inhibitor (100 units ml-1, Promega)
was added during antibody incubation steps to help stabilize mRNA. Fast Red
(Roche) was used in the first alkaline phosphatase reaction to give red color
and fluorescence. Afterward, alkaline phosphatase from the first color
reaction was inactivated by incubating embryos in a 4% formaldehyde solution
for 2 hours at room temperature and then heating for 10 minutes at 37°C.
NBT-BCIP (Roche) was used for the second alkaline phosphatase reaction to give
blue color. For sectioning, embryos were embedded in Immunobed resin
(Polysciences No. 17324) and cut into 4 µm sections.
Morpholino oligomer injections
Morpholino oligomers (Gene Tools Inc) were 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 concentrations of
2.5 µg µl-1 fgf3-MO, 2.5 µg µl-1
fgf8-MO, 1.25 µg µl-1 wnt8 ORF1-MO, 1.25
µg µl-1 wnt8 ORF2-MO. Filtered green food coloring
was added to a concentration of 3% to visualize fluid during injections.
Approximately 1-5 nl was injected into the yolk of one- to two-cell stage
embryos. Embryos were injected and maintained in Holtfreter's solution [60 mM
NaCl, 0.6 mM KCl, 0.9 mM CaCl2, 5 mM HEPES (pH 7.4)] with 50 units
ml-1 penicillin and 50 µg ml-1 streptomycin.
Morpholino used were: fgf3-MO
(Phillips et al., 2001);
fgf8-MO (Furthauer et al.,
2001
); wnt8 ORF1-MO; and wnt8
ORF2-MO (Lekven et al.,
2001
).
Misexpression
To misexpress Fgfs, we tried several approaches in which mRNA and DNA
(10-100 ng µl-1) were injected into embryos between one- and
16-cell stages. Two methods were used to achieve mosaic misexpression of Fgf
mRNA: injection at one-cell stage followed by blastomere transplantation into
uninjected hosts; and injection between four- and 16-cell stages. Both methods
resulted in embryos that were too severely dorsalized to study otic
development. Alternatively, pCS2+ plasmid DNA containing a constitutive
cytomegalovirus promoter upstream of the coding sequence of interest was
injected between one- and 16-cell stages. The method that resulted in the
greatest frequency of ectopic otic tissue was eight-cell injection of
fgf plasmid at a concentration of 30 ng µl-1. Mosaic
misexpression of Wnt8 was achieved by eight-cell injection of 30-40 ng
µl-1 of ORF1 or ORF2 plasmid. Global misexpression of Wnt8 was
achieved by one-cell injection of 80 ng µl-1 ORF1 or ORF2
plasmid. Global misexpression of Dkk1 was accomplished by one-cell injection
of either 40 ng µl-1 or 80 ng µl-1 plasmid. In all
cases, injection volume was 1-5 nl. Filtered, green food coloring was added to
a concentration of 3% to visualize fluid during injections.
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Results |
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Wnt8 regulates timely expression of fgf3 and fgf8 in the hindbrain
To clarify whether the delay in otic induction observed in Wnt8
loss-of-function embryos was caused by indirect effects, we examined
expression of previously identified otic inducers, Fgf3 and Fgf8, in embryos
lacking Wnt8 function. Normally, fgf3 is expressed in r4 by 90%
epiboly (9 hpf). However, the hindbrain domain of fgf3 was barely
visible at tailbud stage (10 hfp) in over half (71/128) of wnt8
morphants and is undetectable at this stage in Dfw8 mutants
(Fig. 2B,C). Strong r4
expression of fgf3 becomes evident by the six-somite stage (12 hpf)
in Dfw8 homozygotes (Fig.
2D). The hindbrain domain of fgf8 becomes evident by 75%
epiboly (8 hpf) in wild-type embryos but was only weakly expressed in most
(61/81) wnt8 morphants even as late as 90% epiboly (9 hpf).
Furthermore, 10% of wnt8 morphants still had reduced expression at
tailbud stage (10 hpf, Fig.
2F). Expression of fgf8 was also delayed in Dfw8
homozygotes, in which expression cannot be detected in the hindbrain until
tailbud stage (10 hpf, Fig.
2G). Dfw8 mutants and wnt8 morphants show strong
fgf8 expression by the six-somite stage (12 hpf,
Fig. 2H and data not shown).
This indicates that Wnt8 is necessary for timely expression of both
fgf3 and fgf8 in the hindbrain. The delay in Fgf expression
correlates well with the delay in otic induction and indicates that the otic
defects observed in these embryos may be an indirect effect resulting from a
deficiency in Fgf signaling. The possibility remains, however, that Wnt
signaling regulates later aspects of otic development (see Discussion).
|
Misexpression of Fgf3 or Fgf8 induces ectopic otic tissue
Although loss-of-function studies indicate that Fgf3 and Fgf8 but not Wnt8
are necessary for otic induction, we sought to test whether any of these
factors are sufficient for otic induction. To misexpress either Fgf3 or Fgf8,
we injected at various stages either synthetic RNA or plasmid DNA containing
Fgf cDNA under the control of a constitutive promoter. We found that embryos
are extremely sensitive to Fgf misexpression because both mRNA and early stage
plasmid injection led to severe dorsalization and expansion of the neural
plate at the expense of epidermal and preplacodal ectoderm (data not shown).
This most likely reflects an early function of Fgf signaling in dorsal/ventral
patterning (Fürthauer et al.,
1997; Koshida et al.,
2002
). However, injection of plasmid into wild-type embryos at the
eight-cell stage resulted in belated, mosaic Fgf expression. With this
technique, some embryos still exhibited moderate dorsalization, but by
co-staining injected embryos for neural marker and Fgf expression, we
determined that the majority had only small, scattered patches of expressing
cells and did not show overt signs of dorsalization. Of the nondorsalized
class, 26% (30/118) of Fgf3-misexpressing embryos and 15% (14/94) of
Fgf8-misexpressing embryos showed ectopic patches of pax8 expression
and/or significant expansion of the endogenous preotic domain. Such expression
did not result from expansion of the otic-inducing portion of the hindbrain
because krox-20 expression was normal
(Fig. 3B). Instead, sites of
ectopic pax8 correlated with sites of Fgf3 or Fgf8 misexpression
(Fig. 3C,D and data not shown).
Furthermore, Fgf misexpression was able to induce ectopic domains of
expression of foxi1, which encodes an upstream regulator of
pax8 (Solomon et al.,
2003
) (Fig. 3F).
Fgf misexpression also led to ectopic or expanded expression of later preotic
markers pax2a and dlx3b
(Fig. 3G and data not shown).
When allowed to develop further, 9% (17/196 nondorsalized) of embryos injected
with fgf3 plasmid and 8% (37/464 non-dorsalized) of embryos injected
with fgf8 plasmid displayed ectopic vesicles containing
differentiated sensory patches and associated otoliths
(Fig. 3H-J,
Table 1). Formation of ectopic
vesicles was limited to the periphery of the anterior neural plate, although
during earlier developmental stages isolated pax8 expressing cells
were occasionally observed elsewhere, including the neural plate (not shown).
Importantly, co-injection of fgf8 and wnt8 plasmids did not
significantly increase the number of embryos displaying ectopic vesicles,
indicating that Wnt8 does not augment the ability of Fgf to induce otic
tissue.
|
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Wnt8 cannot induce ectopic otic tissue without Fgf
To test whether mosaic misexpression of Wnt8 is sufficient to induce
ectopic otic tissue, we injected wnt8 ORF1 or ORF2 plasmid into
wild-type embryos at the eight-cell stage. None of the embryos injected with
ORF2-plasmid showed ectopic otic vesicles (n=50). A small fraction
(5/249) of embryos injected with ORF1-plasmid produced supernumerary otic
vesicles. In these few cases, embryos appeared to be severely posteriorized;
they showed bilateral loss of nasal pits and eyes, and no morphological
development of the epiphysis or midbrain-hindbrain boundary (not shown). We
infer that these are phenotypes resulting from more widespread expression of
Orf1. To test the effects of increasing Wnt8 signaling, we doubled the
concentration of wnt8 plasmid and injected embryos at the one-cell
stage. Injection of ORF2-plasmid caused mild posteriorization in some embryos
but had no visible effect on otic development (n=118, data not
shown). By contrast, 73% (129/177) of embryos injected with ORF1-plasmid were
strongly posteriorized, and these included the 5-6% (10/177) of embryos that
produced supernumerary otic vesicles (Fig.
4E). Analysis at earlier stages showed that 22% (10/45) of
ORF1-misexpressing embryos produced enlarged domains of pax8 wrapping
around the anterior neural plate (Fig.
4C). This correlates with expanded hindbrain domains of fgf3,
fgf8 and erm, a reporter of Fgf activity
(Fig. 4A,B and data not shown)
reminiscent of the patterns seen in embryos posteriorized with retinoic acid
(Phillips et al., 2001). When
fgf3-MO and fgf8-MO were coinjected with ORF1-plasmid at the
one-cell stage, preotic expression of pax8 was severely reduced or
ablated (n=150; Fig.
4D,F). At later stages, most embryos appeared posteriorized but
none produced any ectopic otic tissue (n=240). This finding was
highly significant (P<0.0005) compared to the moderate level of
ectopic ear formation in embryos injected with ORF1-plasmid alone. These data
indicate that Wnt8 cannot directly induce otic tissue in the absence of
Fgf.
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Discussion |
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A direct role for Fgf signaling in otic induction
Comparative studies in zebrafish, Xenopus, chick and mouse
indicate that Fgf, especially Fgf3, plays a broadly conserved role in otic
induction. However, these model systems have used different experimental
approaches, each of which only partially addresses the nature of Fgf function.
Misexpression studies in chick and frog show that Fgf signaling can induce
ectopic otic tissue (Vendrell et al.,
2000; Lombardo et al.,
1998
), but this need not reflect the normal function of the
specific ligands under study. Loss-of-function studies in zebrafish and mouse
confirm an essential role for Fgf3 and, in addition, show that Fgf8 and Fgf10
have partially redundant roles in otic induction
(Phillips et al., 2001
;
Maroon et al., 2002
;
Leger and Brand, 2002
;
Liu et al., 2003
;
Wright and Mansour, 2003
).
However, these studies did not address whether any of these ligands are
sufficient for otic induction. We show here that misexpression of either Fgf3
or Fgf8 can induce ectopic otic tissue in zebrafish
(Fig. 3), demonstrating for the
first time in a single species that Fgf is both necessary and sufficient for
otic induction.
Although we cannot exclude the possibility that Fgf3 and Fgf8 induce expression of another hindbrain signal that is directly responsible for otic induction, this seems unlikely for several reasons. First, the Fgf reporter gene erm is expressed in ectoderm adjacent to the hindbrain during late gastrulation, indicating that preotic cells receive and respond to Fgf signals (Fig. 5). Furthermore, preplacodal expression of erm is ablated in embryos depleted of Fgf3 and Fgf8 (our unpublished observations). Finally, mosaic misexpression of Fgf can induce ectopic otic development without inducing hindbrain markers such as krox20 and wnt8 (Fig. 3B and data not shown). The simplest interpretation of these data is that Fgf3 and Fgf8 act directly on preplacodal ectoderm to induce the otic placode.
The function of Fgf signaling is clearly context-dependent. Fgf
misexpression induced ectopic otic tissue only in ectoderm immediately
surrounding the anterior neural plate. This probably corresponds to the
preplacodal domain, a distinct domain of the ectoderm lying between neural and
epidermal ectoderm. The preplacodal domain is marked by expression of a number
of transcription factors genes, including Six, Msx, Dlx and
Eya-related homologs (reviewed by
Baker and Bonner-Fraser, 2001;
Whitfield et al., 2002
;
Riley and Phillips, 2003
). The
signaling interactions that regulate these genes are not well understood, but
BMP signaling from ventral tissue is required for expression of Msx
and Dlx genes, and signals from the organizer and/or neural plate are
also required (Feledy et al.,
1999
; Pera et al.,
1999
; Beanan et al.,
2000
; McClarren et al., 2003). A balance of these competing axial
signals may be crucial for establishing an uncommitted preplacodal region
along the neural non-neural interface, which is then subdivided into different
kinds of placodes by specific local cues. The hindbrain domain of Fgf3 and
Fgf8 appears to constitute an essential part of the local trigger for otic
development. It is interesting to note that Fgf3 and Fgf8 are also expressed
in more anterior tissues, including the prechordal plate and
midbrain-hindbrain boundary, but these sources do not normally trigger otic
development in more anterior locations. This might reflect insufficiency in
the level, timing and duration of Fgf signaling, and the presence of other
factors could modify the response to Fgf. In any case, locally augmenting Fgf
signaling can overcome the restrictions on otic development in more anterior
regions. It is also noteworthy that Fgf misexpression did not induce formation
of ectopic otic tissue in regions posterior to the endogenous otic placodes.
This might be because retinoic acid, a posteriorizing agent that is
synthesized by posterior mesoderm, strongly modifies the response to Fgf
signaling (Kudoh et al.,
2002
).
An indirect role for Wnt8 in otic induction
Although wnt8 is expressed in the hindbrain by 75% epiboly - at
the right time and place to influence otic induction - it is neither necessary
nor sufficient for this process. Loss of all wnt8 activity delays but
does not block expression of the preotic marker pax8
(Fig. 2). The initial delay in
otic induction is probably caused by a similar delay in the expression of
fgf3 and fgf8 in the hindbrain. Most embryos knocked down
for wnt8 ORF1 and ORF2, and embryos that misexpress the Wnt
antagonist Dkk1, produce small, well differentiated otic vesicles containing
sensory maculae and associated otoliths
(Fig. 1). Misexpression of
wnt8 did occasionally lead to production of supernumerary otic
vesicles. However, all such embryos appeared severely posteriorized, failing
to develop any anterior sensory structures, midbrain-hindbrain border and
epiphysis. Analysis at earlier stages confirmed that misexpression of
wnt8 caused the hindbrain domains of fgf3 and fgf8
to shift almost to the anterior limit of the embryo
(Fig. 4). Moreover, the lateral
edges of the hindbrain domain extend forward to form a U-shaped arc of
staining that is complementary to an inverse arc of preotic pax8 that
wraps around the anterior limit of the neural plate. Knockdown of
fgf3 and fgf8 blocked preotic pax8 expression and
totally ablated formation of otic vesicles in all embryos injected with
wnt8-plasmid. These data support the conclusion that Wnt8 acts
indirectly in otic induction by influencing expression of fgf3 and
fgf8 in the hindbrain.
Additional evidence for an indirect role for Wnt8 is that expression of
TOPdGFP, a Wnt-inducible transgene, is not detected in preotic cells
during gastrulation (Fig. 5).
It should be pointed out that one limitation of this transgene is that it
reports only transcriptional activation by Lef1, a mediator of the canonical
Wnt pathway, but it does not reflect signaling via the alternate Wnt
mediators, Tcf3 and Tcf3b (Dorsky et al.,
2002). Analysis of Tcf3 and Tcf3b in zebrafish indicates that
these proteins normally act as transcriptional repressors that are inactivated
by Wnt signaling (Kim, 2000; Dorsky, 2003). As yet, no genes have been
identified that specifically report Wntmediated derepression of Tcf3 activity.
Despite this caveat, the failure to detect TOPdGFP expression shows
that Wnt8 signaling is not sufficient to strongly activate the Lef1-dependent
pathway in preotic cells. It is also worth noting that none of the known
Frizzled receptors examined in several vertebrate species are expressed at
appreciable levels in prospective otic ectoderm during late gastrulation, when
otic development is initiated (Deardorf and
Klein, 1999
; Stark et al.,
2000
; Momoi et al.,
2003
). Expression of multiple Frizzled genes is detected
later within the nascent otic placode, indicating that Wnt signaling could
play a role in later stages of otic development. Indeed, TOPdGFP
expression is first detected in prospective otic ectoderm between 12-13 hpf
(6-8 somites), just prior to morphological formation of the otic placode (data
not shown). This is also consistent with the observation that, in rat,
periotic accumulation of nuclear ß-catenin is first detected just after
formation of the otic placode (Matsuda and
Keino, 2000
). In addition, secreted Frizzled proteins, which are
induced by Wnt signaling, are expressed in chick otic tissue only after
formation of the otic placode (Baranski et
al., 2000
; Ladher et al.,
2000b
; Esteve et al.,
2000
; Terry et al.,
2000
). Although we found no evidence to support a direct role for
Wnt8 in otic induction, zebrafish embryos lacking Wnt8 function produce
smaller vesicles indicating that Wnt8 signaling might stimulate proliferation
in the developing otic placode. Thus, later Wnt signaling could also regulate
morphogenesis or differentiation of ear tissue during post-placodal
stages.
Although ORF1 and ORF2 show very close sequence homology, their functions are not identical. Knockdown of ORF1 alone has negligible effects on inner ear development whereas knockdown of ORF2 alone significantly delays otic induction and leads to production of small otic vesicles. These effects are not significantly worsened by knockdown of both ORF1 and ORF2, suggesting a more crucial role for ORF2. It is possible that this reflects the proximity of the hindbrain domain of ORF2 to r4, the site of expression of both fgf3 and fgf8. By contrast, misexpression of ORF2 had only mild effects and did not induce excess or ectopic otic tissue, whereas misexpression of ORF1 posteriorized the neural plate and led to production of supernumerary otic vesicles in 2-5% of embryos. This could reflect enhancement of an early posteriorizing function normally associated with the germring domain of Wnt8. It is not clear why global misexpression of ORF2 does not have similar effects, but sequence differences between the ligands could be critical for differential receptor binding.
Feedback between the Fgf and Wnt pathways
Although Wnt8 is required for normal expression of fgf3 and
fgf8 in the hindbrain, Fgf signaling is also required for proper
expression of wnt8-ORF2 in the r5/6 domain. It is not known whether
this mutual regulation is direct or indirect, but it could reflect the
activity of a positive-feedback loop operating within the hindbrain. The
purpose of such a feedback loop could be analogous to that of the
midbrain-hindbrain boundary, wherein an anterior domain of Wnt1 abuts
a posterior domain of Fgf8, and the two factors cooperate to organize
surrounding brain tissue (reviewed by
Wurst and Bally-Cuif, 2001).
Induction of both genes is under the control of several upstream regulators.
Both factors are required to maintain the midbrain-hindbrain boundary and,
therefore, indirectly they require each other. The r4 region of the hindbrain
appears to be a second signaling center that helps pattern the hindbrain.
Expression of fgf3 and fgf8 in the r4 domain is necessary to
establish the identities of r5 and r6
(Walshe et al., 2002
;
Maves et al., 2002
;
Wiellette and Sive, 2003
).
This could partly explain why Fgf signaling is required for proper expression
of wnt8 in the r5/6 region. The requirement for Wnt8 ORF2 on
hindbrain patterning has not been examined, but this domain may help to
establish and stabilize the r4 signaling center and thereby provide a
sustained source of Fgf3 and Fgf8 required for otic induction.
Whether a similar mechanism operates in other vertebrates remains to be
fully tested. In chick and mouse, Wnt8 is expressed in a domain in
the hindbrain, consistent with the role proposed in our study
(Hume and Dodd, 1993;
Bouillet et al., 1996
). The
only functional analysis of this domain in amniotes is a study by Ladher and
colleagues (Ladher et al.,
2000a
) examining the effects of Fgf19 and Wnt8c on gene expression
in chick explant cultures. From that study it was proposed that Fgf19 from
periotic mesendoderm induces expression of Wnt8c in the hindbrain,
and the two factors then induce otic development in adjacent ectoderm.
However, a key observation was that exogenous Wnt8c induced prospective otic
ectoderm to express Fgf3, which was interpreted as a marker of early
otic differentiation. This presents a conundrum because Fgf3 is not
expressed in the chick ear until well after formation of the otic vesicle, but
Wnt8c did not induce expression of any earlier markers of otic development. By
contrast, Fgf3 is expressed in the chick hindbrain by the one-somite
stage (Mahmood et al., 1995
),
raising the possibility that induction of Fgf3 by Wnt8c mimics an
early aspect of hindbrain development. In this scenario, Wnt8c could
facilitate a feedback loop that augments and maintains Fgf signaling long
enough to induce otic development. Thus, the ability to induce a full range of
early otic markers in cultures exposed to Fgf19 and Wnt8c might reflect the
additive effects of exogenous Fgf19 plus newly synthesized Fgf3. More complete
analysis of the relative roles of Fgf and Wnt signaling will require Wnt8
misexpression in vivo and loss-of-function studies using morpholinos in chick
(Kos et al., 2003
) and
gene-knockouts in mouse.
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ACKNOWLEDGMENTS |
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