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First published online 17 September 2008
doi: 10.1242/dev.026674
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1 Laboratory for Sensory Development, RIKEN Center for Developmental Biology,
2-2-3 Minatojima-Minamimachi, Chuo-ku, Kobe 650-0047, Japan.
2 Neurogenetics Group, Carl of Ossietzky University, Oldenburg 26111,
Germany.
* Author for correspondence (e-mail: raj-ladher{at}cdb.riken.jp)
Accepted 1 September 2008
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Inner ear, Epibranchial, Sensory placode, Fibroblast growth factor, Wnt
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). In both chick and mouse, the inner ear is
initiated by the action of localised fibroblast growth factor (FGF) signals
(Kil et al., 2005;
Ladher et al., 2005). In the
case of chick, the combined actions of Fgf3 and Fgf19 acting from the
mesoderm, initiate a cascade of events that ultimately result in the induction
of the inner ear. One important step in this cascade is the induction in the
overlying neural plate of the Wnt signalling protein Wnt8c
(Ladher et al., 2000). FGF and
Wnt signalling are then thought to induce otic fate in adjacent, non-neural
ectoderm. However, these studies do not resolve the temporal requirements for
these interactions nor do they assign individual roles to each signal.
The role of Wnt signalling in otic induction has been subject to
considerable speculation. Experiments from zebrafish seem to discount a direct
role for Wnt signalling in the early specification of the inner ear
(Phillips et al., 2004),
although data from the mouse contradict this view
(Ohyama et al., 2006).
Modulation of canonical Wnt signalling in the mouse suggests that this
signalling actually directs a fate choice between otic and epidermis tissue
within a progenitor field. This progenitor region, which was termed the
`pre-otic field', is marked by the expression of Pax2. However, data
from both genetic labelling in the mouse and vital dye labelling in the chick
show that the Pax2 expression domain should, more properly, be
considered to encompass the inner ear and the epibranchial placodes, a group
of neurogenic placodes that will give rise to the geniculate, petrosal and
nodose ganglia (Ohyama and Groves,
2004b; Streit,
2002). To reflect these derivatives, and to prevent any ambiguity
with a definition for `pre-otic' that could be understood as the region of the
embryo rostral to the otic placode, we have termed the
Pax2-expressing progenitor domain the otic-epibranchial progenitor
domain (OEPD). Although the mechanisms of OEPD induction are not fully
understood, in zebrafish an FGF signal has been proposed as being sufficient
and necessary for the induction of both otic and epibranchial placodes
(Nechiporuk et al., 2007;
Nikaido et al., 2007;
Sun et al., 2007). Such a
common precursor domain may also reflect a common evolutionary relationship
between the inner ear and epibranchial derivatives
(Baker et al., 2008).
Inner ear cells must be specified from the precursor domain, and the view
that otic fate restriction is progressive is suggested by careful examination
of the timing of particular genes within the chick otic placode. Pax2
is expressed at around the 4 somite stage (ss). However, the otic placode is
not actually morphologically visible until 8/9ss. At this stage, inner ear
markers, such as Nkx5.1, Soho1 and Bmp7, begin to be
expressed (Baker and Bronner-Fraser,
2001; Groves and
Bronner-Fraser, 2000). Experiments that isolate the otic placode
from potential extrinsic signals suggest a further dimension; the progressive
restriction of inner ear fate is due to changes in either the nature or the
duration of signalling interactions. Otic ectoderm isolated at 5ss and
cultured for 24 hours will express Pax2; however, it will not express
later markers, such as Bmp7. Only otic explants isolated after 7-8ss
express both Pax2 and Bmp7
(Groves and Bronner-Fraser,
2000). The relevance of such a restriction is not clear, and
indeed these experiments do not identify the nature of the signals or the
mechanisms by which they act.
In this report, we provide definitive evidence for the progressive restriction of developmental fate in inner ear induction. Overexpression and inhibition studies allow us to determine the clear functional significance of FGF and Wnt signalling during early inner ear induction. We find that an initial pulse of FGF establishes a mitotically active progenitor domain to both the inner ear and epibranchial placodes, the OEPD. Subsequently, the attenuation of FGF together with the action of canonical Wnt signalling allow the medial region of the OEPD to commit to an inner ear fate. Conversely, epibranchial placode differentiation is enhanced by continued FGF signalling but inhibited by canonical Wnt signalling. Thus, the progressive restriction of inner ear and epibranchial potential results from the interplay of the FGF and Wnt signals that form the basis of signalling checkpoints that determine the correct and orderly differentiation of the inner ear and the epibranchial placodes.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
Embryo dissections
Embryos at 1ss/HH7-16ss/HH12 were removed and washed in Ringer's solution.
Two types of explant were performed. In both cases, the inner ear region was
dissected by making two transverse cuts, just rostral to the first somite and
just caudal to rhombomere 3. Presumptive otic explants were dissected away
from neural ectoderm and underlying paraxial mesendoderm. Presumptive otic
regions are similar but were just bisected at the midline. The explanted
tissue was transferred immediately to a chilled 10-µl drop of collagen.
Once set, collagen drops were flooded with pre-warmed medium [DMEM+10%
knockout serum replacement (KSR; Invitrogen, CA]. Otic regions were
additionally incubated in media supplemented with 100 ng/ml recombinant human
DKK1 (R&D Systems). Explants were grown for 1 or 7 days in a humidified
CO2 incubator, at which time they were rinsed in PBS and fixed in
4% PFA for one hour.
Collagen drops were processed for in situ hybridisation and
immunohistochemistry as described previously
(Wright et al., 2004).
DNA constructs and electroporation
Knockdown of Fgf3 and Fgf19 was performed using
shRNA-encoding constructs. Briefly, putative shRNA sequences designed against
20 bases starting from +251 and +324 of Fgf3 and Fgf19,
respectively were inserted into pSilencer 1.0 (Ambion). In addition, a
scrambled oligonucleotide based on the Fgf19 +324 sequence was
inserted in pSilencer (shScrambled) to act as a control for
non-specific effects. To overexpress Fgf3 and Fgf19, coding
regions were cloned downstream of the Ef1
promoter. A stabilised, and
thus, constitutively active β-catenin and a construct expressing mouse
Dickkopf1 (Dkk1) were the kind gift of Drs Fumi Kubo and Shinichi Nakagawa
(RIKEN ASI, Wako, Japan).
DNA was unilaterally co-electroporated with a tracer (mCherry) into either
the anterior streak of HH4 embryos or the ectoderm of HH5 embryos that were
cultured ex ovo (Uchikawa et al.,
2003). The embryos were then incubated in a humidified
CO2 incubator at 37°C for 10-49 hours.
In situ hybridisation and immunochemistry
Embryos were fixed in 4% paraformaldehyde and rinsed in PBS. The following
probes were used for whole-mount in situ hybridisation: Fgf3, Fgf19,
Nkx5.1, Pax2, Phox2b and Soho1. These probes were as described
previously (Begbie et al.,
2002; Ladher et al.,
2000). A probe recognising Foxi2 was obtained through the
BBSRC chick EST database (ChEST 884m4)
(Boardman et al., 2002). In
situ hybridisation was performed as described previously
(Ladher et al., 2005). Some
stained embryos were cryosectioned.
Double-fluorescent in situ hybridisation was based on published protocols
(Denkers et al., 2004).
Digoxigenin-labelled probes were detected using an alkaline
phosphatase-conjugated antibody and revealed using VectorRed (Vector
Laboratories). The second probe was labelled using fluorescein. This was
detected using a peroxidase-conjugated antibody, and revealed using a
fluorescein-tyramide kit (Perkin Elmer).
The following antibodies were used: anti-hair-cell-antigen (a kind gift of Prof. Guy Richardson, University of Sussex, UK), anti-phospho-histone H3 (Upstate) and anti-Ds-Red (Living Colors), which was used to detect mCherry protein.
Statistical analysis
Collagen cultures were analysed after in situ hybridisation for
Soho1 and immunohistochemistry for hair-cell antigen (HCA). Explants
were grouped based on stage and the proportion of positive staining. We used
statistical tests (Smith's statistical package) for two samples with two
possible probabilities (positive or negative) to determine the earliest stage
at which the expression of Soho1 and the development of inner ear
hair cells were autonomous.
Cell counts for phospho-histone H3 (PHH3)-positive cells were performed after imaging whole embryos, using Adobe Photoshop software. Briefly, the OEPD was first defined as being an area just rostral to the first somite adjacent to the hindbrain, and that extended rostrally for three somite lengths. All subsequent counts were made of positive cells within this area. PHH3-positive cells were counted and the normal difference between the left and right sides of 10 control embryos between 10ss and 13ss calculated. Such values provide a strong control for differences among embryos. The difference in PHH3-positive cells between left, control sides and right, electroporated sides of six pEF-Fgf electroporated embryos were similarly calculated. P-values were calculated using Student's t-test.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). To
understand individual roles that FGF and Wnt play during inner ear
development, the particular steps in the developmental program of the inner
ear required clarification. Studies had hinted at multiple states during inner
ear induction. Explants of presumptive otic tissue are competent to express
Pax2, but not Bmp7 when isolated at 5ss; explants can
express both when isolated at 7-8ss
(Groves and Bronner-Fraser,
2000). We hypothesised that these studies reflected a progressive
commitment to otic differentiation. Thus, to assess whether presumptive otic
ectoderm, isolated at 5ss, is committed to a definitive inner ear fate, we
tested the ability of this tissue to express the otic marker Soho1
and to develop inner ear hair cells, the mechanosensory receptor of the inner
ear (Fig. 1A)
(Bartolami et al., 1991;
Deitcher et al., 1994). Few
(18%) presumptive otic explants isolated at 5ss and cultured for 24 hours
expressed Soho1 (Fig.
1B,C). Similarly, few presumptive otic explants were able to
undergo differentiation, as judged by immunoreactivity to HCA after seven days
of culture (Fig. 1B,D). These
numbers were not statistically significant. Thus, we concluded that, although
5ss explants are able to express Pax2, they lack the ability to
develop definitive otic character. By contrast, a significant number of
presumptive otic explants isolated at 7-8ss (HH9) showed expression of
Soho1 and could develop hair cells
(Fig. 1B,E,F).
Attenuation of FGF expression is necessary for otic differentiation
The fibroblast growth factor family members Fgf3 and Fgf19 act from caudal
cephalic paraxial mesoderm in the chick to induce otic ectoderm. We have
previously shown that Fgf19 is downregulated from the mesoderm and
neuroectoderm at 8ss (Ladher et al.,
2000). Soon after, at 9ss, we find that mesodermal Fgf3
expression is also downregulated. The coincidence of the ability of
presumptive otic ectoderm to differentiate with this downregulation led us to
suspect that the lowering of FGF expression was a pre-requisite for otic
differentiation. To test the necessity of FGF attenuation for otic commitment,
we introduced constructs of Fgf3 and Fgf19 driven by the
Ef1 promotor (pEF-Fgf3 and pEF-Fgf19 respectively,
collectively called pEF-Fgf). This promotor acts constitutively to
drive sustained FGF expression in the early chick embryo.
We first verified the function of the pEF-Fgf constructs by
assessing their effects on Pax2 expression. Several groups have
identified a role for FGF signalling in the induction of Pax2
expression in the OEPD of zebrafish
(Nechiporuk et al., 2005;
Nikaido et al., 2007;
Sun et al., 2007). We thus
unilaterally introduced pEF-Fgf3, pEF-Fgf19, or both, into
the anterior streak region of HH4 embryos by electroporation
(Fig. 2A-C). Such
electroporation targets the mesoderm as well as the neural ectoderm adjacent
to the presumptive otic region (Iimura et
al., 2007; Psychoyos and
Stern, 1996). Overexpression of pEF-Fgf constructs caused
expansion of the normal OEPD Pax2-expression domain
(Fig. 2D-I). This expansion was
seen as early as 8-14 hours after electroporation, when embryos were at
9-16ss. The effect of electroporating either pEF-Fgf3 or
pEF-Fgf19 alone was indistinguishable; however, introducing both
constructs caused a larger expansion and stronger expression; we therefore
presumed that the two ligands are additive during OEPD induction. These
results are consistent with those reported in zebrafish.
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). Like Soho1, expression of Nkx5.1 was
similarly reduced in response to sustained FGF signalling
(Fig. 2P-S). These results
strongly suggest that definitive otic specification was inhibited in embryos
when FGF signalling was sustained, and that, during normal development, FGF
attenuation is necessary for otic differentiation.
The expansion of the OEPD and the block in otic commitment caused by
sustained FGF expression could be the result of the maintenance of
proliferation within the OEPD; the failure to exit cell cycle in this case may
result in a block in commitment. To test this, we used an antibody to the
serine-10 phospho-form of histone H3 (PHH3), which labels mitotic cells
between late G2 to anaphase (Hans and
Dimitrov, 2001; Hendzel et
al., 1997). In normal, non-electroporated embryos, there is a
slight difference in the number of PHH3-positive cells between the left and
right sides of the embryo. On average there are 3% fewer PHH3-positive cells
on the right side of non-electroporated embryos. By contrast, embryos
unilaterally electroporated with pEF-Fgf constructs, have 23% more
mitotic cells on the right, electroporated, side than on the left,
non-electroporated side (Fig.
3). These data strongly suggest that one of the functions of FGF
during early OEPD induction is to maintain the proliferation of at least a
portion of these progenitors.
Fgf3 and Fgf19 downregulation prevents otic commitment by repressing OEPD induction
Many studies have described a role for FGF signalling in otic induction
(reviewed by Schimmang, 2007),
thus the finding that sustained FGF signalling actually inhibited otic
differentiation was surprising. In many of these studies, suppression of FGF
expression was used to show necessity for inner ear development. We
hypothesised that, in these studies, FGF suppression actually inhibited inner
ear development by blocking OEPD formation. To confirm this, we investigated
the effect of removing Fgf3 and Fgf19 on OEPD induction and
otic commitment. Constructs encoding short-hairpin interfering RNA were
designed to knockdown Fgf3 and Fgf19 (known as
shFgf3 and shFgf19, respectively, and, collectively, as
shFgf). Electroporation of either shFgf3 or shFgf19
caused a reduction of Fgf3 and Fgf19 expression,
respectively (Fig. 4A,B). Such
electroporated embryos were assessed after 12 hours for Pax2
expression (Fig. 4C-F) and
after 24 hours for Soho1 expression
(Fig. 4G-J). Knockdown of both
Fgf3 and Fgf19 substantially reduced the expression of both
Pax2 and Soho1 (Fig.
4F,J). Reducing the levels of either Fgf3 or
Fgf19 individually had some effect on OEPD induction
(Fig. 4D,E) and otic
development (Fig. 4H,I),
indicating that during normal development the two act redundantly. These data
show that FGF signalling is necessary for OEPD induction and, consequently,
otic fate.
FGF signalling does not affect otic/epibranchial patterning within the OEPD
The early expression of Pax2 defines the otic-epibranchial
precursor domain (OEPD), encompassing the otic and epibranchial placodes
(Ohyama and Groves, 2004b;
Streit, 2002). Even though
late otic markers are inhibited by sustained FGF signalling, Pax2 is
not. Thus, it is possible that sustained FGF signalling, as well as
maintaining proliferation, also altered specification within the OEPD by
converting the presumptive otic portion into non-otic cell types arising from
the OEPD. Thus, we assessed Foxi2 expression. In chick, as has been
described in mouse (Ohyama and Groves,
2004a), Foxi2 expression is excluded from the otic part
of the OEPD (Fig. 5A). At 22ss,
Foxi2 is detected in epibranchial placodes. Thus, we hypothesised
that early Foxi2 expression overlapped with Pax2 to label
non-otic OEPD derivatives. This was confirmed using double fluorescence in
situ hybridisation (Fig. 5D).
Such analysis identified regions of the ectoderm that are either Pax2
or Foxi2 positive, as well as a domain of Pax2 and
Foxi2 co-expression at the periphery of the normal Pax2
expression domain.
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Sustained FGF signalling does not repress epibranchial differentiation
We next asked whether, like the otic portion of the OEPD, sustained FGF
signalling also inhibited the differentiation of the non-otic portion of the
OEPD. To investigate this, we assessed the development of the
epibranchial-derived neurons. At 13ss, Pax2 expression begins to
partition into separate otic and epibranchial regions
(Fig. 5G). After
pEF-Fgf electroporation, epibranchial Pax2 expression was
expanded (Fig. 5I). We note
that segregation of Pax2 into an `otic' domain also occurred normally
after pEF-Fgf electroporation, despite the inhibition of otic
commitment. To further analyse the effect of sustained FGF signalling upon
epibranchial differentiation, we investigated the expression of
Phox2b. This homeodomain protein is expressed in the sensory neurons
of the epibranchial placodes, with expression apparent in all epibranchial
placodes by 23ss/HH14+ of development
(Begbie et al., 2002)
(Fig. 5J). In response to
sustained FGF signalling, epibranchial neurogenesis, as marked by
Phox2b expression, is increased
(Fig. 5L).
To test the necessity of FGF signalling for epibranchial development, knockdown constructs were electroporated to reduce Fgf3 and Fgf19 expression. These resulted in a marked reduction in epibranchial placodes, as revealed by Pax2 and Phox2b expression (Fig. 5H,K). Together these data suggest that, like the otic placode, FGF signalling is necessary for epibranchial development, most likely through the induction of the common progenitor domain. However, in contrast to otic development, sustained FGF signalling stimulates epibranchial neurogenesis.
Wnt signalling inhibits epibranchial fate
FGF signalling is sufficient and necessary for the early expression of
Pax2 in the OEPD, but it has profound effects on the differentiation
of its derivatives, repressing Soho1 and Nkx5.1, markers of
otic commitment, and stimulating Phox2b, a marker for epibranchial
neurogenesis. However, as evidenced by the continued exclusion of
Foxi2 from the presumptive otic region
(Fig. 5C), sustained FGF
expression does not alter the patterning of the otic-epibranchial progenitor
domain. This suggests that additional signals are necessary to direct
definitive otic specification.
Wnt signalling is strongly implicated in otic development
(Jayasena et al., 2008;
Ohyama et al., 2006;
Riccomagno et al., 2005). To
test whether Wnt signalling partitions the OEPD into otic and epibranchial
fates, we used a stabilised mutant of β-catenin. Canonical Wnt signalling
is mediated through the repression of cytoplasmic β-catenin degradation.
Thus, the stabilised β-catenin acts as a constitutively active effector
of canonical Wnt signalling. We introduced this construct into the ectoderm of
HH5/HH6 chick embryos and then allowed these embryos to develop until
10-15ss.
Ectodermal overexpression of constitutively active (CA) β-catenin did not alter the expression of Soho1 (Fig. 6A,B). Similarly, the dorsal expression domain of Nkx5.1, an additonal inner ear marker, was unchanged (Fig. 6C,D). We investigated the effect of Wnt activation on OEPD development using Pax2 expression. At 7ss, Pax2 expression is unaffected by the overexpression of CA-β-catenin (Fig. 6E-G); however, by 13ss, the Pax2 expression domain was more rounded, without the typical lateral flares (Fig. 6H-J). We postulated that this lateral domain represented the non-otic part of the OEPD. This was confirmed after observing the reduction of Foxi2 expression in these embryos (Fig. 6K-M). The reduction of lateral Pax2 OEPD expression and the loss of Foxi2 expression suggests that ectopic Wnt signalling might also inhibit the differentiation of non-otic regions of the OEPD. Expression of the epibranchial-derived neuronal marker Phox2b was reduced 36 hours after the electroporation of constitutively active β-catenin (Fig. 6N,O).
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To understand the effect of Wnt inhibition on patterning within the OEPD, we next analysed Foxi2 expression. Foxi2 is normally excluded from the presumptive otic region. After Dkk1 overexpression, the region of Foxi2 exclusion was reduced, but Foxi2 never invaded the whole of this presumptive otic portion of the OEPD (Fig. 7L-N). To determine whether the expansion of Foxi2 expression also correlated with an expansion of epibranchial neurogenesis, we investigated the expression of Phox2b. We could detect no clear difference in Dkk1 overexpressing embryos, although in a few cases the domain of epibranchial neurogenesis was slightly larger (n=4/11) (Fig. 7P). When considered together, these data suggest that canonical Wnt signalling is not required for the initial induction of the OEPD; however, Wnt signalling is necessary for otic commitment.
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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FGF induces and maintains inner ear progenitors
A role for FGF signalling in early inner ear development has been firmly
established (Adamska et al.,
2001; Alvarez et al.,
2003; Hans et al.,
2007; Ladher et al.,
2005; Ladher et al.,
2000; Leger and Brand,
2002; Liu et al.,
2003; Maroon et al.,
2002; Phillips et al.,
2001; Vendrell et al.,
2000; Wright and Mansour,
2003; Zelarayan et al.,
2007). More recent data from zebrafish suggest that FGF signalling
additionally induces the epibranchial placodes
(Nechiporuk et al., 2007;
Nikaido et al., 2007;
Sun et al., 2007). This is
consistent with the data reported here, showing that FGF overexpression
expands the precursor domain of both. Conversely, knockdown using a shRNA
strategy results in repression of the OEPD and, consequently, a loss of both
committed otic and epibranchial precursors. The idea that only early FGF
signalling is sufficient for inner ear development is supported by studies
using pharmacological inhibition. Here, Pax2 expression is inhibited
if the inner ear is treated with the FGF inhibitor SU5402 prior to 5ss. If the
treatment is performed between 5-8ss, Pax2 expression is unaffected
(Martin and Groves, 2006).
An unexpected finding is the suppression of otic commitment when FGF
expression is sustained. However, FGF signalling is under tight regulation;
the mesodermal expression of Fgf3 and Fgf19 is rapidly
downregulated at 7-8ss. Furthermore, sprouty2, an inhibitor of FGF
signalling is expressed in presumptive inner ear tissue from 8ss
(Chambers and Mason, 2000).
Thus, the observation that otic commitment is suppressed when FGF expression
is experimentally sustained is not so surprising. When considered with the
behaviour of isolates of otic ectoderm, a clearer understanding of the role of
FGF signalling during inner ear development emerges. Isolates express
Pax2, but can neither express Soho1 nor show inner ear hair
cell differentiation when removed from their embryonic environment at 5ss.
During this time, FGF signalling induces the OEPD. Otic commitment only takes
place at 7-8ss, concomitant with the downregulation of Fgf3 and
Fgf19 expression, and the suppression of FGF signals by
sprouty2.
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).
This raises the intriguing possibility that FGF attenuation might also be
required in mice for otic commitment to take place.
A role for FGF in blocking otic commitment has not been previously
described. Indeed, previous data appear to contradict this model. However, one
caveat is that in many cases only early OEPD markers have been investigated
(Leger and Brand, 2002;
Maroon et al., 2002;
Sun et al., 2007). Some
experiments have investigated the effect of FGF overexpression on otic
commitment and differentiation. In Xenopus, the implantation of
Fgf2-soaked beads resulted in ectopic otocysts
(Lombardo and Slack, 1998). In
chick, the implantation of Fgf2-soaked beads resulted in larger otocysts
(Adamska et al., 2001).
Similarly, treatment of chick ectodermal explants with Fgf19-soaked beads
resulted in the induction of Soho1 expression
(Ladher et al., 2000). In all
cases, it is likely that the position of the beads has shifted during
development, and, when considered with protein half-life, it is possible that
the tissue can adopt a committed otic fate when it escapes the influence of
exogenous protein. A more stable method of gene transfer was performed in
chick embryos using an Fgf3-expressing retrovirus
(Vendrell et al., 2000). In
these experiments, even though viral infection was widespread, ectopic
otocysts formed adjacent to only some infected cells. A similar study
overexpressed Fgf3 in the hindbrain adjacent to the neural tube.
Again, ectopic otocysts appeared adjacent to the region of overexpression
(Zelarayan et al., 2007). In
both studies, only a single FGF was overexpressed, and it is possible, as we
have shown in this study, that the sustained action of two or more Fgfs is
necessary for efficient repression of otic commitment. Finally, a zebrafish
transgenic line that expresses a heat-inducible Fgf8 construct showed larger,
well-patterned, otocysts only when Fgf8 was overexpressed at the late gastrula
stage (Hans et al., 2007).
Such experiments might indicate a difference in the action of particular FGF
molecules.
Wnt activity is necessary for otic differentiation
Similar to the effect of sustained FGF action, inhibition of Wnt signalling
also results in the suppression of otic commitment. However, there are
differences in the effect of each on Pax2 expression. As described,
FGF overexpression expands the early OEPD domain. At later stages, and despite
the inhibition of otic commitment, Pax2 expression is detected in the
otic placode. By contrast, Wnt inhibition causes only a reduction of the
medial portion of the Pax2 expression domain in the OEPD after 7-8ss,
at the onset of otic specification. This requirement for Wnt signalling may be
transient: treatment of otic regions with recombinant DKK1 protein does not
inhibit otic formation after 9/10ss (S.F. and R.K.L., unpublished). The
ectopic activation of canonical Wnt signalling using a stabilised,
constitutively active β-catenin does not overtly affect otic development
and differentiation; however, epibranchial development is affected, again only
after 7-8ss (see below). These data strongly suggest that Wnt signalling is
not necessary for OEPD induction, but is required for otic specification,
acting after FGF signalling has established the OEPD.
Using conditionally active or mutant lines of β-catenin, Ohyama et al.
proposed a model for mouse inner ear induction that strongly suggested an
involvement of Wnt signalling in OEPD patterning
(Ohyama et al., 2006). Similar
to our data in the chick, these authors found that the removal of canonical
Wnt signalling from the OEPD resulted in the loss of the inner ear; however,
the absence of a detailed time course of the changes to the OEPD meant that
the possibility that the expression of Pax2 and Pax8 (the
markers that define the mouse OEPD) was initially normal was not ruled
out.
Epibranchial differentiation
As described above, sustained expression of Fgf3 and
Fgf19 blocks otic differentiation. We suggest that otic
differentiation is blocked because these cells remain in a proliferative
state; however, in the absence of otic differentiation, does this region also
develop a non-otic-OEPD epibranchial fate? Our analysis of the expression of
Foxi2 and Phox2b suggests that it does not. Foxi2
is normally excluded from the region of the OEPD fated to the otic lineage,
and its co-expression with Pax2 marks the non-otic OEPD region. Even
when FGF signalling is sustained, and otic differentiation inhibited, this
region of Foxi2 exclusion is maintained. However, the region of
overlap between Foxi2 and Pax2 is slightly broader, and
epibranchial-derived neurogenesis (as marked by Phox2b) is slightly
enhanced. These data suggest that early mesodermal FGF signalling is not
involved in patterning the OEPD, instead a second phase of FGF signalling,
probably from the endoderm, might permit epibranchial fate. This is suggested
by recent data showing that endodermal Fgf3 is involved in zebrafish
epibranchial development (Nechiporuk et
al., 2005). It should be noted that additional signals also play a
role in the development of the epibranchial placodes. Endodermal Bmp7 has been
shown to stimulate neurogenesis of these precursors
(Begbie et al., 1999).
Our data also show a negative role for Wnt signalling in the development of
the epibranchial placodes. Constitutively active β-catenin overexpression
results in the repression of epibranchial derivatives. Ohyama and colleagues
have touched on the role of Wnt signalling in epibranchial development in the
mouse (Ohyama et al., 2006).
Similar to our data in chick, the stimulation of canonical Wnt signalling in
mouse results in a reduction of epibranchial-derived precursors. However, the
result of removing β-catenin in mouse is somewhat confusing: here it
results in a reduction of epibranchial development. It is possible that the
mouse and chick use different mechanisms for epibranchial development, and
that a low basal level of Wnt signalling may be required for mouse
epibranchial development. However, a TOP-gal reporter that detects canonical
Wnt signalling was not active within epibranchial precursors
(Ohyama et al., 2006;
Riccomagno et al., 2005). This
argues against canonical Wnt signalling acting within epibranchial placodes.
Alternatively, the known participation of β-catenin in tight junctions
could suggest that mutants in this gene have impaired epithelial integrity. In
such a situation, the epibranchial placodes might be induced normally in mouse
embryos lacking β-catenin; however, continued development and
morphogenesis might be aberrant.
Patterning the OEPD
Our data suggest a sequential role for first FGF signalling and then Wnt
signalling in the development of the inner ear. In this revised model, we
propose that FGF signalling, from subjacent mesoderm and adjacent neural
ectoderm, establishes a mitotically active progenitor domain, the OEPD. The
OEPD is then influenced by Wnt signals from the neural tube. By acting on the
OEPD, Wnt enables otic differentiation of a subset of OEPD cells, while
repressing epibranchial development in others. These data point to a crucial
role for canonical Wnt signalling in the lineage choice between otic and
non-otic portions of the OEPD. However, an important question is whether other
signals are also involved. The failure of constitutively active β-catenin
to expand the otic domain could suggest a lateral signal that antagonises the
pro-otic Wnt signal. Similarly, the failure of Wnt inhibition to cause the
complete conversion of putative otic precursors to non-otic
Foxi2-expressing cells, despite the downregulation of otic Pax2,
Nkx5.1 and Soho1, may suggest that the lateral `anti-otic'
signal regulates the expression of Foxi2, but that its limited range
renders it insufficient to cause complete conversion of the OEPD to
Foxi2-positive precursors. Thus, the progenitor region is the site of
competing signals: a medial otic-promoting/epibranchial-repressing Wnt signal
and a lateral, as yet unidentified, signal that promotes epibranchial
development and is likely to repress otic development
(Fig. 8). An important feature
of this model is that the inner ear and epibranchial placodes can be
positioned only at sites of intersecting permissive interactions. Furthermore,
by controlling the timing of these interactions, the transition between cell
states can be controlled, ensuring the correct balance between progenitor
proliferation and cell-type differentiation.
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ACKNOWLEDGMENTS |
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