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 23 April 2003
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SUMMARY |
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Key words: Fibroblast growth factor, Otic, Placode, Inner ear, Induction, Mouse mutant
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INTRODUCTION |
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Transplantation studies in amphibia and avians have established that the
region of surface ectoderm competent to form an otic vesicle is initially
quite large (for reviews, see Torres and
Giraldez, 1998; Baker and
Bronner-Fraser, 2001
; Noramly
and Grainger, 2002
). When quail ectoderm from the midbrain or
somitic region in 1-6 somite embryos is grafted in place of presumptive chick
otic ectoderm, it responds to inductive signals by expressing otic markers and
by forming an ectopic vesicle. This competency declines rapidly with age and
by 10 somites neither midbrain nor somitic ectoderm is competent to express
otic markers or to contribute to the developing otic placode. Only the
ectoderm near the hindbrain maintains these abilities
(Groves and Bronner-Fraser,
2000
). Therefore, as development proceeds, the region of otic
competency becomes progressively restricted and the placodal tissue adjacent
to the hindbrain becomes specified for an otic fate.
Tissue recombination experiments as well as genetic depletion and ablation
studies in zebrafish and mice suggest that placodal development is directed by
signals arising from the underlying mesenchyme and the adjacent neurectoderm
(Baker and Bronner-Fraser,
2001; Kiernan et al.,
2002
). Co-culture of chick stage 7 mesendoderm that will underlie
the presumptive otic placode with stage 5 anterior cephalic ectoderm induces
the expression of otic markers in the ectoderm. By stage 9+, the
equivalent mesoderm only induces otic markers when adjacent neurectoderm is
also included in the culture (Ladher et
al., 2000
). Furthermore, there are many examples of mouse and
zebrafish mutants with hindbrain abnormalities that also have inner ear
abnormalities. For example, the kreisler mutant mouse and the
valentino mutant zebrafish, which carry mutations in orthologous
hindbrain-expressed transcription factors, have otic defects that are
secondary to disruption of r5 and r6
(Frohman et al., 1993
;
Cordes and Barsh, 1994
;
McKay et al., 1994
;
Moens et al., 1998
).
The molecular identities of signals responsible for otic placode induction
are the subject of intense interest. In the chick, mesodermal Fibroblast
growth factor (Fgf)19 and neurectodermal Wnt8c have
the spatio-temporal expression patterns appropriate for otic inducers.
Simultaneous application of these factors to cultured chick anterior ectoderm
elicits expression of a variety of otic markers, including Fgf3
(Ladher et al., 2000). Mouse
Fgf15, the presumed ortholog of chick and human FGF19
(Ornitz and Itoh, 2001
),
however, is not expressed in the mesenchyme underlying the otic placode and
Fgf15 mutants do not have otic abnormalities, suggesting that this
FGF is likely not to function as a uniquely necessary otic inducer in mice
(T.J.W. and S.L.M., unpublished).
Fgf3, which in mice and chicks is normally expressed in a
hindbrain domain that narrows to r5 and r6, and also in prospective otic
ectoderm (Wilkinson et al.,
1988; Mahmood et al.,
1995
; Mahmood et al.,
1996
; McKay et al.,
1996
), has also been proposed as an otic inducer
(Represa et al., 1991
).
Indeed, ectopic expression of Fgf3 in chick embryos induces the
formation of small otic-like vesicles
(Vendrell et al., 2000
;
Adamska et al., 2001
),
suggesting that Fgf3 expression may be sufficient to promote otic
vesicle formation.
Genetic depletion and ablation studies in zebrafish and mice reveal a more
complex picture of the requirement for Fgf genes in otic development.
Depletion of FGF3 by injection of Fgf3 morpholinos into wild-type
zebrafish embryos causes a reduction in otic vesicle size very similar to that
seen in ace (Fgf8) mutants
(Leger and Brand, 2002).
Simultaneous depletion of both FGF3 and FGF8 by injection of both morpholinos
into wild-type embryos or injection of Fgf3 morpholinos into
ace mutants blocks otic vesicle formation in most treated embryos,
demonstrating that these two FGFs have redundant roles in zebrafish otic
placode induction (Phillips et al.,
2001
; Leger and Brand,
2002
; Maroon et al.,
2002
). In this species, however, both Fgf3 and
Fgf8 are expressed in r4 and the otic defects seen in embryos lacking
both FGFs are accompanied by severe abnormalities of hindbrain patterning
(Maves et al., 2002
;
Walshe et al., 2002
). Thus it
is not clear whether FGF3 and FGF8 both signal directly to the prospective
otic placode, or whether one or both factors are instead required for
expression of the otic inducer(s) by the hindbrain. As Fgf8 is not
expressed in the mouse hindbrain (Crossley
and Martin, 1995
) (T.J.W. and S.L.M., unpublished) its function
(if any) with respect to otic placode induction is likely to be different to
that of zebrafish Fgf8. Unfortunately, mouse Fgf8 null
mutants die of severe gastrulation defects prior to the initiation of otic
development (Sun et al.,
1999
). Therefore, potential roles for Fgf8 in mouse otic
development have not yet been established.
Genetic ablation of Fgf3 expression in mice does affect ear
development, but the reported effects initiate after formation of the otic
vesicle and are confined to the later stages of vesicle morphogenesis. The
defects, moreover, have incomplete penetrance and variable expressivity,
suggestive of redundancy in the FGF signalling system during otic development
(Mansour et al., 1993). In
support of this idea, disruption of Fgf10, which is expressed in the
developing otic cup and its neuronal derivatives
(Pirvola et al., 2000
), also
causes morphogenetic and innervation abnormalities of otic development
(Ohuchi et al., 2000
;
Pauley et al., 2003
).
Furthermore, ectopic expression of a secreted, dominant-negative form of the
IgIIIb isoform of FGF receptor 2 (FGFR2b), which is the high-affinity receptor
for FGFs-3, -7 and -10 (Ornitz et al.,
1996
; Igarashi et al.,
1998
), has effects on otic vesicle development that appear to be
more severe than those of either Fgf3 or Fgf10 single
mutants (Celli et al., 1998
).
Finally, specific elimination of the FGFR2b isoform by targeted mutagenesis of
the exon encoding the IgIIIb splice variant causes highly penetrant otic
abnormalities that are similar to those expected from an additive combination
of the Fgf3 and Fgf10 mutant phenotypes
(Pirvola et al., 2000
).
We show here that mouse Fgf10 is expressed in the mesenchyme underlying the prospective otic placode. To uncover potential redundancy between Fgf3 and Fgf10 during early otic development we generated double mutant embryos. These embryos lacked otic vesicles and had aberrant patterns of otic placode marker gene expression, suggesting that FGF3 and FGF10 signals are required redundantly for otic placode induction and that these signals emanate from both the hindbrain and mesenchyme. These signals are likely to act directly on the prospective otic ectoderm, as double mutant embryos showed normal patterns of gene expression in the hindbrain. There were no major effects on cell proliferation or survival in double mutant embryos, suggesting that the major role of FGF signalling in otic induction is to establish appropriate patterns of gene expression in the placode. In addition, examination of otic vesicles in embryos carrying three of four possible mutant Fgf alleles revealed intermediate phenotypes that could be distinguished both from each other as well as from embryos carrying two or four mutant alleles. We suggest that an FGF3 gradient may explain the quantitative and unequal requirement for these two FGFs in otic development.
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MATERIALS AND METHODS |
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In situ hybridisation
Embryos were isolated on the indicated days following detection of a
vaginal plug. Controls demonstrating the standard expression patterns of
Fgf3, Fgf10, Fgfr2IgIIIb and Fgfr1 were performed using
wild-type CD-1 embryos. Control embryos for otic marker gene expression
studies came from the intercross litters and were matched to the mutant
embryos by somite number. Digoxigenin-labelled probes were prepared,
hybridised to the embryos and detected as described
(Henrique et al., 1995). cDNAs
used to prepare probes for Fgf3
(Manley and Capecchi, 1995
),
Fgf10 (Xu et al.,
1998
), Fgfr2IgIIIb
(Orr-Urtreger et al., 1993
),
Pax2 (Dressler et al.,
1990
), Dlx5 (Depew et
al., 1999
), Gbx2
(Wassarman et al., 1997
),
Pax8 (Plachov et al.,
1990
), Hoxb1
(Carpenter et al., 1993
),
Krox20 (Carpenter et al.,
1993
) and kr (Cordes
and Barsh, 1994
) have been described in the cited publications. A
probe for the 3' UTR of Fgfr1 was generated by cloning a
PCR-amplified DNA fragment (bp 2408-2910 of cDNA clone 3830408H21, GenBank
accession number AK028354). The sequences of the PCR primers were: 5'
primer, 5'-ACCCTGTCCCCAGTTTTCTCC-3'; 3' primer,
5'-ACCAGGCAGGTATTTGGTCA-3'. The product was cloned into pCRII
(Invitrogen) and an antisense probe was generated by digesting the clone with
Xho I and transcribing with Sp6 RNA polymerase.
Otic vesicle development was analysed at E9.5 using the marker genes Dlx5 and Pax2 (n=3 double mutants; n=4 Fgf3-/-;Fgf10+/-; n=4 Fgf3+/-;Fgf10-/-; n=5 Fgf3-/-; n=4 Fgf10-/-) and otic placode induction was analysed at E8.5 using Dlx5, Pax2, Pax8 and Gbx2 (n=6 double mutants). Hindbrain development was analysed using the molecular markers Krox20, MafB/kr and HoxB1 (n=4 double mutants).
Whole mount detection of mitosis and apoptosis
To detect proliferating cells, embryos (n=2 controls, n=2
double mutants) were prepared and stained with an antibody directed against
phosphorylated histone H3 as described
(Gavalas et al., 2001). Whole
mount detection of apoptosis was performed using the TUNEL method as described
previously (n=3 controls, n=3 double mutants)
(Maden et al., 1997
;
Graham, 1999
). Following
staining and observation of whole mounts, embryos were cryosectioned and
sections containing the otic tissues were identified using anatomical markers.
Phosphohistone H3-expressing cells or apoptotic cells were counted in the otic
ectoderm, neurectoderm and the mesenchyme underlying the otic ectoderm of
double heterozygote and double mutant embryos. As a control, mitotic or
apoptotic cells were counted in the heart fields, which were unaffected in
double mutant embryos. No consistent differences were identified between
genotypes, and the sections shown in Fig.
5 illustrate the presence of mitotic or apoptotic cells in all the
tissues relevant to otic induction.
|
Photography and size measurements
Whole embryos were photographed using a Zeiss SV-11 dissecting microscope
fitted with a digital camera (Kodak MDS120 or MDS240). Sections were
photographed using a Zeiss Axioscop fitted with DIC optics and a digital
camera (AxioCam).
To compare the relative sizes of otic vesicles between embryos of different genotypes, we first found the central section taken through each vesicle of three E9.5 embryos of each genotype (n=6 ears and eyes) and then measured the areas of both the otic and the optic vesicles. To account for differences in staging of the embryos, we calculated the ratio of the otic vesicle area to the optic vesicle area (which is not affected by the Fgf mutations). To compare the positions of the otic vesicles in different embryos, the vertical distance from the dorsal surface of the neural tube to the dorsal surface of the otic vesicle was measured and compared to the dorsoventral length of the neural tube. All areas and lengths were determined by using the measurement functions in the AxioCam software package (Zeiss).
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RESULTS |
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If FGF3 and/or FGF10 signal to the otic ectoderm, an appropriate receptor
should be present in that tissue. Of the seven major FGF receptor isoforms,
both FGF3 and FGF10 bind with highest affinity to and signal most strongly
through the IgIIIb isoform of FGFR2
(Ornitz et al., 1996;
Igarashi et al., 1998
).
Therefore, we determined the early expression pattern of Fgfr2b by
hybridising an isoform-specific probe to whole embryos
(Fig. 2A-H). At 3 and 6
somites, Fgfr2b transcripts were found in the neurectoderm, extending
along most of the anteroposterior axis of the embryo
(Fig. 2A-D). To confirm that
these transcripts were expressed in the hindbrain adjacent to presumptive otic
ectoderm, we hybridized 2-8 somite embryos with a mixture of the probes for
Fgfr2b and Pax2, a marker of otic ectoderm. In all cases,
transverse sections exhibiting Pax2 expression in the ectoderm also
showed Fgfr2b expression in the neurectoderm (data not shown).
Beginning at 8 somites, and coincident with ectodermal thickening,
Fgfr2b transcripts were detected throughout the otic placode
(Fig. 2E,F). This expression
persisted through otic cup invagination in embryos with 16 somites
(Fig. 2G,H). At this stage,
Fgfr2b transcripts in neurectoderm were restricted to the most dorsal
region (Fig. 2H). By E9.5,
Fgfr2b transcripts in the otic vesicle and the neurectoderm were
restricted to the dorsal domain (data not shown).
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Embryos homozygous for null mutations in both Fgf3 and Fgf10 do not
develop otic vesicles
To determine whether Fgf3 and Fgf10 play redundant roles
in otic placode induction, embryos lacking both Fgf3 and
Fgf10 were generated by intercrossing mice that were heterozygous for
null alleles of both genes. One-thousand two-hundred and sixty-nine embryos
were harvested between E8 and E10.5 and all genotypes, including the double
mutant, were obtained in the numbers expected for segregation of two unlinked
recessive mutations (Table 1).
Thus, early lethality did not compromise the analysis of the double mutant
phenotype. Compared with double heterozygote control embryos at E10.5, double
mutant embryos lacked limbs and had short dorsally curved tails
(Fig. 3A,C), characteristic of
Fgf10 and Fgf3 single mutants, respectively
(Mansour et al., 1993;
Min et al., 1998
;
Sekine et al., 1999
).
Strikingly, the double mutant embryos also appeared to lack otic vesicles
(Fig. 3C). Comparison of
transverse sections of the control and double mutant embryos revealed
bilateral microvesicles at the position expected for otic vesicles
(Fig. 3B,D). Other double
mutant embryos had either a unilateral microvesicle or lacked any sign of
vesicle formation. Of 15 double mutant embryos, or 30 ears, analysed between
E9.5 and E10.5, a microvesicle was identified in 15 cases (50%).
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Hindbrain patterning is not affected in
Fgf3-/-;Fgf10-/- embryos
Genes encoding FGF receptors, including Fgfr2b, are expressed in
the developing hindbrain (Fig.
2 and data not shown)
(Yamaguchi et al., 1992) and
FGFs play important roles in neural induction and patterning
(Marin and Charnay, 2000
).
Indeed, zebrafish embryos depleted of both FGF3 and FGF8 have severe hindbrain
patterning defects (Maves et al.,
2002
; Walshe et al.,
2002
). This raised the possibility that the otic defects we
observed in Fgf3/Fgf10 double mutants could be a secondary
consequence of hindbrain abnormalities. To address this issue, we examined the
expression patterns of three hindbrain marker genes, Mafb/kreisler,
HoxB1 and Krox20. As expected, double heterozygote control
embryos at 9-13 somites expressed HoxB1 in r4, Mafb/kreisler
in r5 and r6 and Krox20 in r3 and r5
(Fig. 4Q,S,U). Expression of
these genes was unaffected in similarly staged double mutant embryos
(Fig. 4R,T,V), suggesting that
these embryos had grossly normal hindbrains. Furthermore, gross examination
and microscopic observation of coronal sections of E9-10.5 double mutant
embryos revealed normal rhombomeric divisions of the hindbrain (data not
shown). Therefore, the abnormalities in otic development seen in double mutant
embryos are probably a direct consequence of the loss of FGF3 and FGF10
signals to the otic ectoderm.
Cell proliferation and survival in the surface ectoderm of
Fgf3-/-;Fgf10-/- embryos are not significantly
altered
Loss of both Fgf3 and Fgf10 clearly affects molecular
patterning of the otic placode-forming region of the surface ectoderm. To
determine whether there were additional effects of the loss of these two genes
on cell proliferation, we labelled control and double mutant embryos at the 6
and 8 somite stages with an antibody directed against phosphohistone H3. No
differences between embryos of different genotypes in the distribution of
labelled cells were apparent upon examination of whole embryos. Furthermore,
examination of cryosections from these embryos revealed that mitotic cells
could be found in all tissues, including the dorsal region of the presumptive
otic ectoderm, of both control and double mutant embryos
(Fig. 5A-D). These data suggest
that loss of Fgf3 and Fgf10 does not lead to a block in cell
proliferation in these tissues. In addition, we investigated whether excessive
cell death occurred in the tissues involved in otic development in double
mutant embryos. Examination of TUNEL whole mount staining and cryosections
revealed no major differences between 7 somite embryos of different genotypes
in the number and distribution of apoptotic cells
(Fig. 5E-H). Apoptotic cells
could be found in all tissues of both control and double mutant embryos.
Therefore, absence of both Fgf3 and Fgf10 was not associated
with major changes in either mitogenic or survival signals within the otic
region.
Fgf3 and Fgf10 play quantitative and unequal roles in otic
development
Observations of E9.5
Fgf3-/-;Fgf10+/- and
Fgf3+/-;Fgf10-/- embryos suggested
that these embryos had otic vesicle abnormalities that were distinguishable
from each other and intermediate between those of Fgf3-/-
or Fgf10-/- mutant embryos and those of double mutant
embryos. To examine the morphology and patterning of these mutant vesicles,
embryos with three mutant alleles were stained with Pax2 and
Dlx5 and compared with the previously described control and double
mutant embryos as well as with Fgf3-/- and
Fgf10-/- embryos (Fig.
6). By comparison with control embryos
(Fig. 3F,J) or with embryos
homozygous for a single Fgf mutation
(Fig. 6B,D,J,L), embryos with
either combination of three mutant alleles appeared to have otic vesicles that
were smaller (Fig. 6F,H,N,P).
This phenotype was more extreme in the
Fgf3-/-;Fgf10+/- embryos
(Fig. 6H,P) than in the
Fgf3+/-;Fgf10-/- embryos
(Fig. 6F,N). Quantitative
comparisons between the ratio of the area of the central otic vesicle section
to the area of the central eye section in
Fgf3+/-;Fgf10+/-,
Fgf3+/-;Fgf10-/- and
Fgf3-/-;Fgf10+/- embryos detected
statistically significant differences between the three genotypes
(Fig. 7A). The otic to optic
area ratio of the Fgf3+/-;Fgf10-/- and
Fgf3-/-;Fgf10+/- samples were
approximately 72% (P=0.005) and 47% (P=0.001) respectively
of that of the Fgf3+/-;Fgf10+/-
controls. As the otic vesicle is roughly spherical at this stage, these
differences in area probably reflect even larger differences in volume.
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Alterations in otic marker gene expression were also apparent in these E9.5 embryos. Whereas the localisation of Pax2 to the ventromedial region of the otic vesicle was similar in control (Fig. 3F), Fgf3+/+;Fgf10-/- (Fig. 6B), Fgf3-/-;Fgf10+/+ (Fig. 6D) and in Fgf3+/-;Fgf10-/- (Fig. 6F) embryos, Pax2 otic expression expanded both dorsally and laterally in Fgf3-/-;Fgf10+/- embryos (Fig. 6H). Dlx5 expression was found in the dorsolateral region of otic vesicles of all combinations of Fgf mutant genotypes (Fig. 3J, Fig. 6J,L,N,P) except the double mutant, which does not have otic vesicles. In addition, there appears to be an expansion of Dlx5 towards the ventral and medial regions of the otic vesicle in the Fgf3-/-;Fgf10+/+ embryo (Fig. 6L). Compared with control embryos (Fig. 3J), the level of Dlx5 expression in the otic vesicle relative to that seen in the branchial arches, forebrain and limbs appeared markedly reduced in Fgf3+/-;Fgf10-/- and Fgf3-/-;Fgf10+/- embryos (Fig. 6N,P). This reduced level of Dlx5 expression made it difficult to determine whether the domain of expression was altered. Taken together, these data suggest that there is a quantitative requirement for FGF signalling to promote normal otic development and that loss of FGF3 has a more significant effect on otic development than does loss of FGF10.
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DISCUSSION |
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We found that in Fgf3/Fgf10 double mutant embryos at E8.5, all
tested markers of prospective otic ectoderm were either entirely eliminated
from the ectoderm (Pax2), or were excluded from the dorsal ectoderm
(Dlx5, Gbx2 and Pax8). In contrast, it did not appear that
cell proliferation or survival in the otic ectoderm of double mutant embryos
was significantly affected. Taken together, these results suggest that the
main role of FGF signalling in otic induction is to establish appropriate
patterns of gene expression in dorsal ectoderm. These data are consistent with
the finding that zebrafish Dlx5 responds to signals required for
placodal induction (Solomon and Fritz,
2002). This role is different from that proposed for FGF
signalling in the development of the midbrain, in which Fgf8 and
Fgf17 are required quantitatively to regulate cell proliferation
(Xu et al., 2000
). The role of
Fgf3 and Fgf10 in otic development also differs from that of
Fgf8 in neural crest development
(Abu-Issa et al., 2002
;
Frank et al., 2002
) and of
Fgf4 and Fgf8 in limb development
(Sun et al., 2002
), in which
the respective signals are required for cell survival.
Although the double mutant otic phenotype was fully penetrant, there remains some variable expressivity, as microvesicles were observed lateral to the hindbrain in 50% of cases between E9.5 and E10.5. None of the microvesicles expressed otic markers, suggesting that they are not likely to develop similarly to bona fide inner ears. Our observation, however, of a ventrally localised thickening of the ectoderm in some E8.5 double mutant embryos, accompanied in one case by a small invagination, which may be a precursor of a microvesicle, does suggest that double mutant embryos may still express a weak signal with vesicle-inducing properties. Whether this signal is an additional FGF normally involved in otic induction or another type of signal remains to be determined.
FGF3 and FGF10 are likely to induce ear development in a paracrine fashion
through their high-affinity receptor FGFR2b, the transcript for which is first
detectable in the prospective otic placode at approximately the eight-somite
stage. The simplest model for otic induction that is consistent with all of
the data is that FGF3 expressed from the hindbrain and FGF10 expressed from
the mesenchyme act directly to activate FGFR2b in the ectoderm. It could be
argued, however, that the timing of receptor gene expression in the ectoderm
is slightly later than might be expected if FGF signalling were the primary
means by which the ectoderm is induced. In avians, otic placode specification
is thought to be complete by the 4-6 somite stage and this cranial ectoderm is
committed to an otic fate by the 10 somite stage
(Groves and Bronner-Fraser,
2000). In mice, however, prior to the present studies, the timing
of otic induction had not been established by any criteria other than that of
placodal thickening, which in different accounts has been reported to occur
between 4 and 13 somites (Anniko and
Wikstrom, 1984
; Sulik and
Cotanche, 1995
; Rinkwitz et
al., 2001
; Kiernan et al.,
2002
). Our own observations suggest that thickening occurs between
7 and 8 somites (this report and T.J.W. and S.L.M., unpublished). Thus it is
possible that otic induction occurs slightly later in mice than in other
species. Furthermore, the in situ hybridisation method for detecting
Fgfr gene expression may not be sensitive enough to indicate the true
onset of FGF signalling in the ectoderm, which could occur as soon as the
first receptor transcripts are translated and the receptor is inserted within
the cell membrane, but before the Fgfr transcripts accumulate to
levels detectable by in situ hybridisation.
More complex models for FGF3 and FGF10 function in otic placode induction cannot be excluded at this time. For example, it is possible that FGF10, expressed in early somite stage mesenchyme, has two functions. It could signal first to FGFR2b in the hindbrain, activating Fgf3 expression, and later signal in combination with hindbrain FGF3 to FGFR2b in the ectoderm. At this point it is unclear whether the FGF3 expressed in the prospective placode itself also plays an important autocrine-signalling role. Tissue-specific ablation of Fgf3 will be required to address this point.
It is curious that the otic abnormalities of embryos lacking
Fgfr2b or expressing a dominant negative FGF receptor are much less
severe than those of the double ligand mutants described here. Embryos
homozygous for an Fgfr2b isoform-specific targeted deletion or
heterozygous for a secreted dominant negative form of FGFR2b have small otic
vesicles at E10 and E11 (Celli et al.,
1998; Pirvola et al.,
2000
). One possible explanation for the milder otic phenotypes
displayed by these mutant embryos is that there may be some redundancy at the
level of the placodal receptor that is provided by FGFR1b, the only other FGF
receptor thought to be activated by FGF3 and FGF10
(Ornitz et al., 1996
;
Beer et al., 2000
). Consistent
with this possibility, we find that there is some detectable expression of
Fgfr1, probably encoding the IgIIIb isoform, at the time when the
otic ectoderm assumes a placodal morphology. When FGFR2b is absent or
inhibited, it is possible that the low levels of FGFR1b could weakly transduce
the FGF3 and FGF10 otic-inducing signals.
A different Fgf, Fgf8, has been shown to be required redundantly
with Fgf3 for otic placode induction in zebrafish
(Phillips et al., 2001;
Leger and Brand, 2002
;
Maroon et al., 2002
). In this
case, however, the severe abnormalities of hindbrain patterning
(Phillips et al., 2001
;
Maroon et al., 2002
;
Maves et al., 2002
;
Walshe et al., 2002
) argue
that FGF8 may not act directly on the prospective otic placode, which does not
express its highest affinity receptor, FGFR4
(Ornitz et al., 1996
), but may
instead act indirectly through the hindbrain, which does express FGFR4 (T.J.
Wright and S.L. Mansour, unpublished). An alternative explanation that does
not exclude the first possibility is that FGF8 functions very early in
gastrulation on the mesoderm, and in its absence, the mesoderm is reduced
and/or does not express otic-inducing signals such as FGF10. A final
possibility is that there are species-specific differences in FGF identity and
their sites of action with respect to otic placode development. Studies of
mouse Fgf3/Fgf8 mutant combinations may help to address this
point.
The otic phenotypes identified in mice carrying three mutant alleles
suggest that there is a quantitative requirement for FGF signalling to promote
normal otic development and that loss of Fgf3 is more detrimental
than loss of Fgf10. In particular, in the absence of Fgf3,
genes with polarised domains of expression in the otic vesicle become less
polarised, whereas in the absence of Fgf10, polarised expression
appears to be maintained. This effect might be explained if the otic placode
experiences a dorsal (high) to ventral (low) gradient of FGF3 expressed from
the hindbrain. In contrast, the primary source of FGF10 is the mesenchyme
underlying the entire placode, and these cells may experience a constant
concentration of FGF10. This difference may explain why failure of
endolymphatic duct outgrowth, a dorsal structure, is the primary defect in
Fgf3 single mutants (Mansour et
al., 1993), whereas this process occurs normally in Fgf10
single mutants (Ohuchi et al.,
2000
). It would be interesting to determine the effects of
reversing the proposed FGF3 gradient.
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ACKNOWLEDGMENTS |
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REFERENCES |
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![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Abu-Issa, R., Smyth, G., Smoak, I., Yamamura, K. and Meyers, E.
N. (2002). Fgf8 is required for pharyngeal arch and
cardiovascular development in the mouse. Development
129,4613
-4625.
Adamska, M., Herbrand, H., Adamski, M., Kruger, M., Braun, T. and Bober, E. (2001). FGFs control the patterning of the inner ear but are not able to induce the full ear program. Mech. Dev. 109,303 -313.[CrossRef][Medline]
Anniko, M. and Wikstrom, S. O. (1984). Pattern formation of the otic placode and morphogenesis of the otocyst. Am. J. Otol. 5,373 -381.
Baker, C. V. and Bronner-Fraser, M. (2001). Vertebrate cranial placodes I. Embryonic induction. Dev. Biol. 232,1 -61.[CrossRef][Medline]
Beer, H. D., Vindevoghel, L., Gait, M. J., Revest, J. M., Duan,
D. R., Mason, I., Dickson, C. and Werner, S. (2000).
Fibroblast growth factor (FGF) receptor 1-IIIb is a naturally occurring
functional receptor for FGFs that is preferentially expressed in the skin and
the brain. J. Biol. Chem.
275,16091
-16097.
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.
Celli, G., LaRochelle, W. J., Mackem, S., Sharp, R. and Merlino,
G. (1998). Soluble dominant-negative receptor uncovers
essential roles for fibroblast growth factors in multi-organ induction and
patterning. EMBO J. 17,1642
-1655.
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.[Medline]
Crossley, P. H. and Martin, G. R. (1995). The
mouse Fgf8 gene encodes a family of polypeptides and is expressed in
regions that direct outgrowth and patterning in the developing embryo.
Development 121,439
-451.
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.
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.[Abstract]
Frank, D. U., Fotheringham, L. K., Brewer, J. A., Muglia, L. J.,
Tristani-Firouzi, M., Capecchi, M. R. and Moon, A. M. (2002).
An Fgf8 mouse mutant phenocopies human 22q11 deletion syndrome.
Development 129,4591
-4603.
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.
Gavalas, A., Trainor, P., Ariza-McNaughton, L. and Krumlauf,
R. (2001). Synergy between Hoxa1 and Hoxb1: the relationship
between arch patterning and the generation of cranial neural crest.
Development 128,3017
-3027.
Graham, A. (1999). Whole embryo assays for programmed cell death. In Molecular Embryology, Methods and Protocols (ed. P. T. Sharpe and I. Mason), pp.667 -672. Totowa, NJ: Humana Press.
Groves, A. K. and Bronner-Fraser, M. (2000).
Competence, specification and commitment in otic placode induction.
Development 127,3489
-3499.
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]
Igarashi, M., Finch, P. W. and Aaronson, S. A.
(1998). Characterization of recombinant human fibroblast growth
factor (FGF)-10 reveals functional similarities with keratinocyte growth
factor (FGF-7). J. Biol. Chem.
273,13230
-13235.
Kettunen, P., Karavanova, I. and Thesleff, I. (1998). Responsiveness of developing dental tissues to fibroblast growth factors: expression of splicing alternatives of FGFR1, -2, -3, and of FGFR4; and stimulation of cell proliferation by FGF-2, -4, -8, and -9. Dev. Genet. 22,374 -385.[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. Tam), pp. 539-566. San Diego, CA, USA: Academic Press.
Ladher, R. K., Anakwe, K. U., Gurney, A. L., Schoenwolf, G. C.
and Francis-West, P. H. (2000). Identification of synergistic
signals initiating inner ear development. Science
290,1965
-1967.
Leger, S. and Brand, M. (2002). Fgf8 and Fgf3 are required for zebrafish ear placode induction, maintenance and inner ear patterning. Mech. Dev. 119,91 -108.[CrossRef][Medline]
Maden, M., Graham, A., Gale, E., Rollinson, C. and Zile, M.
(1997). Positional apoptosis during vertebrate CNS development in
the absence of endogenous retinoids. Development
124,2799
-2805.
Mahmood, R., Kiefer, P., Guthrie, S., Dickson, C. and Mason,
I. (1995). Multiple roles for FGF-3 during cranial neural
development in the chicken. Development
121,1399
-1410.
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]
Manley, N. R. and Capecchi, M. R. (1995). The
role of Hoxa-3 in mouse thymus and thyroid development.
Development 121,1989
-2003.
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.
Marin, F. and Charnay, P. (2000). Hindbrain
patterning: FGFs regulate Krox20 and mafB/kr expression in the otic/preotic
region. Development 127,4925
-4935.
Maroon, H., Walshe, J., Mahmood, R., Kiefer, P., Dickson, C. and
Mason, I. (2002). Fgf3 and Fgf8 are required together for
formation of the otic placode and vesicle. Development
129,2099
-2108.
Maves, L., Jackman, W. and Kimmel, C. B. (2002). FGF3 and FGF8 mediate a rhombomere 4 signaling activity in the zebrafish hindbrain. Development 129,3825 -3837.[Medline]
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]
McKay, I. J., Muchamore, I., Krumlauf, R., Maden, M., Lumsden,
A. and Lewis, J. (1994). The kreisler mouse: a
hindbrain segmentation mutant that lacks two rhombomeres.
Development 120,2199
-2211.
McMahon, A., O'Neill, L. and Carroll, J. (1990). Proteolysis of the zona pellucida of mouse ova. Biochem. Soc. Trans. 18,340 -341.[Medline]
Min, H., Danilenko, D. M., Scully, S. A., Bolon, B., Ring, B.
D., Tarpley, J. E., DeRose, M. and Simonet, W. S. (1998).
Fgf-10 is required for both limb and lung development and exhibits striking
functional similarity to Drosophila branchless. Genes
Dev. 12,3156
-3161.
Moens, C. B., Cordes, S. P., Giorgianni, M. W., Barsh, G. S. and
Kimmel, C. B. (1998). Equivalence in the genetic control of
hindbrain segmentation in fish and mouse. Development
125,381
-391.
Noramly, S. and Grainger, R. M. (2002). Determination of the embryonic inner ear. J. Neurobiol. 53,100 -128.[CrossRef][Medline]
Ohuchi, H., Hori, Y., Yamasaki, M., Harada, H., Sekine, K., Kato, S. and Itoh, N. (2000). FGF10 acts as a major ligand for FGF receptor 2 IIIb in mouse multi-organ development. Biochem. Biophys. Res. Commun. 277,643 -649.[CrossRef][Medline]
Ornitz, D. M. and Itoh, N. (2001). Fibroblast growth factors. Genome Biol. 2,3005.1 -3005.12.
Ornitz, D. M., Xu, J., Colvin, J. S., McEwen, D. G., MacArthur,
C. A., Coulier, F., Gao, G. and Goldfarb, M. (1996). Receptor
specificity of the fibroblast growth factor family. J. Biol.
Chem. 271,15292
-15297.
Orr-Urtreger, A., Bedford, M. T., Burakova, T., Arman, E., Zimmer, Y., Yayon, A., Givol, D. and Lonai, P. (1993). Developmental localization of the splicing alternatives of fibroblast growth factor receptor-2 (FGFR2). Dev. Biol. 158,475 -486.[CrossRef][Medline]
Pauley, S., Wright, T. J., Pirvola, U., Ornitz, D. M., Beisel, K. W. and Fritzsch, B. (2003). Expression and function of FGF10 in mammalian inner ear development. Dev. Dyn. 227,203 -215.[CrossRef][Medline]
Phillips, B. T., Bolding, K. and Riley, B. B. (2001). Zebrafish fgf3 and fgf8 encode redundant functions required for otic placode induction. Dev. Biol. 235,351 -365.[CrossRef][Medline]
Pirvola, U., Spencer-Dene, B., Xing-Qun, L., Kettunen, P.,
Thesleff, I., Fritzsch, B., Dickson, C. and Ylikoski, J.
(2000). FGF/FGFR-2(IIIb) signaling is essential for inner ear
morphogenesis. J. Neurosci.
20,6125
-6134.
Plachov, D., Chowdhury, K., Walther, C., Simon, D., Guenet, J. L. and Gruss, P. (1990). Pax8, a murine paired box gene expressed in the developing excretory system and thyroid gland. Development 110,643 -651.[Abstract]
Represa, J., Leon, Y., Miner, C. and Giraldez, F. (1991). The int-2 protooncogene is responsible for induction of the inner ear. Nature 353,561 -563.[CrossRef][Medline]
Rinkwitz, S., Bober, E. and Baker, R. (2001).
Development of the vertebrate inner ear. Ann. New York Acad.
Sci. 942,1
-14.
Sekine, K., Ohuchi, H., Fujiwara, M., Yamasaki, M., Yoshizawa, T., Sato, T., Yagishita, N., Matsui, D., Koga, Y. and Itoh, N. et al. (1999). Fgf10 is essential for limb and lung formation. Nat. Genet. 21,138 -141.[CrossRef][Medline]
Solomon, K. S. and Fritz, A. (2002). Concerted
action of two dlx paralogs in sensory placode formation.
Development 129,3127
-3136.
Stark, M. R., Biggs, J. J., Schoenwolf, G. C. and Rao, M. S. (2000). Characterization of avian frizzled genes in cranial placode development. Mech. Dev. 93,195 -200.[CrossRef][Medline]
Sulik, K. K. and Cotanche, D. A. (1995). Embryology of the ear. In Hereditary Hearing Loss and its Syndromes (ed. R. J. Gorlin, H. V. Toriello and M. M. Cohen), pp.22 -42. Oxford, UK: Oxford University Press.
Sun, X., Mariani, F. V. and Martin, G. R. (2002). Functions of FGF signalling from the apical ectodermal ridge in limb development. Nature 418,501 -508.[CrossRef][Medline]
Sun, X., Meyers, E. N., Lewandoski, M. and Martin, G. R.
(1999). Targeted disruption of Fgf8 causes failure of cell
migration in the gastrulating mouse embryo. Genes Dev.
13,1834
-1846.
Torres, M. and Giraldez, F. (1998). The development of the vertebrate inner ear. Mech. Dev. 71, 5-21.[CrossRef][Medline]
Vendrell, V., Carnicero, E., Giraldez, F., Alonso, M. T. and
Schimmang, T. (2000). Induction of inner ear fate by FGF3.
Development 127,2011
-2019.
Walshe, J., Maroon, H., McGonnell, I. M., Dickson, C. and Mason, I. (2002). Establishment of hindbrain segmental identity requires signaling by FGF3 and FGF8. Curr. Biol. 12,1117 -1123.[CrossRef][Medline]
Wassarman, K. M., Lewandoski, M., Campbell, K., Joyner, A. L.,
Rubenstein, J. L., Martinez, S. and Martin, G. R. (1997).
Specification of the anterior hindbrain and establishment of a normal
mid/hindbrain organizer is dependent on Gbx2 gene function.
Development 124,2923
-2934.
Wilkinson, D. G., Peters, G., Dickson, C. and McMahon, A. P. (1988). Expression of the FGF-related proto-oncogene int-2 during gastrulation and neurulation in the mouse. EMBO J. 7,691 -695.[Abstract]
Xu, J., Liu, Z. and Ornitz, D. M. (2000).
Temporal and spatial gradients of Fgf8 and Fgf17 regulate proliferation and
differentiation of midline cerebellar structures.
Development 127,1833
-1843.
Xu, X., Weinstein, M., Li, C., Naski, M., Cohen, R. I., Ornitz, D. M., Leder, P. and Deng, C. (1998). Fibroblast growth factor receptor 2 (FGFR2)-mediated reciprocal regulation loop between FGF8 and FGF10 is essential for limb induction. Development 120,753 -765.
Yamaguchi, T. P., Conlon, R. A. and Rossant, J. (1992). Expression of the fibroblast growth factor receptor FGFR-1/flg during gastrulation and segmentation in the mouse embryo. Dev. Biol. 152,75 -88.[Medline]
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