1 Institute of Neuroscience, University of Oregon, Eugene, OR 97403, USA
2 Gene Tools, LLC, 1 Summerton Way, Philomath, OR 97370, USA
Author for correspondence (e-mail:
monte{at}uoneuro.uoregon.edu)
Accepted 19 February 2003
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SUMMARY |
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Key words: dlx3b, dlx4b, Inner ear, Morpholino, Olfactory placode, sox9a, sox9b, Zebrafish
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INTRODUCTION |
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Although the precise molecular nature of the signals that induce cells to
form the otic placode is still unknown, several studies implicate Fgf3 and
Fgf8, members of the Fgf family of signaling peptides. In zebrafish, the genes
that encode these peptides are expressed in the presumptive hindbrain by late
gastrula stages and fgf3 is also expressed at this stage in the
underlying mesendoderm (Phillips et al.,
2001). Fgf3 and Fgf8 mediate inter-rhombomere signaling required
for hindbrain patterning (Maves et al.,
2002
). Loss of Fgf3 function in chick
(Repressa et al., 1991
) or of
both Fgf3 and Fgf8 functions together in zebrafish
(Phillips et al., 2001
;
Léger and Brand, 2002
;
Maroon et al., 2002
) is
sufficient for near or total ablation of otic tissue, and ectopic expression
of Fgf3 (Vendrell et al.,
2000
) or Fgf2 (Lombardo and
Slack, 1998
) results in the formation of ectopic otic vesicles in
frog and chick.
To understand how Fgf signals may specify cells to form the otic placode,
we studied the functions of four transcriptions factors expressed by otic
placode precursor cells in zebrafish, dlx3b (previously called
dlx3) (Ekker et al., 1994), dlx4b (previously called
dlx7) (Stock et al.,
1996; Ellies et al.,
1997
), sox9a (Chiang
et al., 2001
; Yan et al.,
2002
) and sox9b
(Chiang et al., 2001
;
Li et al., 2002
). The
dlx3b and dlx4b genes are closely linked, as are their
mammalian orthologues (Nakamura et al.,
1996
; Morasso et al., 1997), probably because of ancestral tandem
duplication in the lineage giving rise to vertebrates
(Stock et al., 1996
). We have
previously shown that early precursor cells of the otic placode express
dlx3b before any overt morphological signs of differentiation
(Ekker et al., 1992
;
Akimenko et al., 1994
);
dlx4b has a similar expression pattern
(Ellies et al., 1997
). By
prim-5 stage (24 hours), only a subset of cells in the otic vesicle still
expresses dlx3b (Ekker et al.,
1992
).
In humans, a small deletion in the DLX3 gene is thought to be
responsible for Trichodentoosseous syndrome (TDO)
(Price et al., 1998).
Individuals with TDO exhibit a variety of clinical problems, including ear,
tooth and skull defects (Shapiro et al.,
1983
), consistent with the expression pattern of Dlx3 in
mice (Robinson and Mahon,
1994
) and zebrafish (Akimenko
et al., 1994
). A knockout mutation of Dlx3 in mice
results in embryonic lethality due to placental insufficiency before the ear
forms (Morasso et al., 1999
).
Analysis of dlx3b and dlx4b in zebrafish suggested that they
may serve redundant roles in otic development
(Solomon and Fritz, 2002
).
We have previously shown that the zebrafish sox9a and
sox9b genes are duplicate orthologues of the human SOX9 gene
and that both genes are expressed in the otic placode
(Chiang et al., 2001). In
humans, SOX9 haploinsufficiency results in campomelic dysplasia,
characterized by abnormal development of the long bones and associated sex
reversal (Foster et al., 1994
;
Wagner et al., 1994; Hageman et al.,
1998
; Cameron et al.,
1996
; Huang et al.,
2001
; Vidal et al.,
2001
). Most patients die of respiratory distress during the
neonatal period, but one who survived through adolescence had hearing loss
(Houston et al., 1983
).
Studies in mouse have shown that Sox9 is expressed in the developing
otic capsule (Kanzler et al.,
1998
). Heterozygous Sox9 mutant mice show phenotypes
similar to individuals with campomelic dysplasia and die at birth
(Bi et al., 2001
). Our analysis
of zebrafish mutants demonstrated that sox9a is required for
cartilage development (Yan et al.,
2002
).
To analyze the potential functions of dlx3b, dlx4b and
sox9a in otic development, we characterized a deficiency mutation we
previously isolated [Df(LG12)dlx3bb380, called
Dfb380] (Fritz et al.,
1996) that lacks all three genes. We found that homozygous
Dfb380 mutants completely lack otic placodes and fail to
form a differentiated otic vesicle or inner ear, although a few residual cells
express genes characteristic of the developing inner ear. Knock down of all
three genes by injection of morpholino antisense oligonucleotides (MOs)
produces these phenotypes in wild-type embryos, and injection of wild-type
mRNAs from all three genes together is sufficient for phenotypic rescue of the
ear defects in homozygous Dfb380 mutants. The residual
cells fail to form if sox9b function is also blocked. We analyzed Fgf
function using fgf3-MOs and ace
(fgf8) mutants and found that sox9a, but
not dlx3b or dlx4b, expression depends on Fgf signaling, and
that the residual otic cells in homozygous Dfb380 mutants
fail to form if fgf8 function is also absent. Our results demonstrate
that Fgf3- and Fgf8-dependent (sox9a) and -independent
(dlx3b and dlx4b) transcription factors are required for
specification of the otic placode. Moreover, we found that the residual otic
cells form a small epithelial ball characteristic of the early otic vesicle in
Dfb380 mutants but not in the absence of Fgf3 and Fgf8
signaling, thus indicating a role of Fgfs in induction of both the otic
placode and the epithelial organization of the otic vesicle.
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MATERIALS AND METHODS |
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The wild-type line used was AB (University of Oregon, Eugene, OR). The
Df(LG12)dlx3bb380 strain name has been approved by the
zebrafish nomenclature committee
(http://zfin.org/zf_info/nomen_comm.html)
and we refer to homozygous mutants as Dfb380. The
acerebellarti282a (ace) line, a strong
hypomorphic allele of fgf8, has been described previously
(Brand et al., 1996); we refer
to the homozygous mutants as fgf8. Heterozygotes
for both Dfb380 and fgf8
genotypes were obtained by crossing Dfb380 and
acerebellarti282a carriers. Homozygous
Dfb380 mutants were primarily scored by their lack of
somites; homozygous ace mutant embryos were scored by their loss of
the cerebellum. Homozygous Dfb380;fgf8
embryos were scored with both criteria.
The jellyfish (jef) mutation (jef
hil134), which results from the insertion of a retrovirus
(Amsterdam et al., 1999),
disrupts sox9a. Exon 2 is skipped in the splicing in jef
hil134 mutants, leading to a truncated protein that lacks the
C-terminal half of the HMG domain (Yan et
al., 2002
). jef hil134 shows a more severe
otic phenotype than jef tw37 (data not shown), which in
theory should produce a truncated protein that lacks a large part of HMG and
the entire C terminus.
Genes, markers and mapping
Approved gene and protein names that follow the zebrafish nomenclature
conventions
(http://zfin.org/zf_info/nomen.html)
are used according to
http://zfin.org.
To map the deletion boundaries and the genes and markers missing from the
deficient region, we used primers for genes and markers on LG12 to amplify
expected bands from Dfb380 genomic DNA by PCR.
In situ hybridization, mRNA synthesis and rescue
cDNA probes that detected the following genes were used: dlx3b
(Ekker et al., 1992);
dlx4b (Stock et al.,
1996
); sox9a (Chiang
et al., 2001
); egr2 (krox-20)
(Oxtoby and Jowett, 1993
);
cldna (Kollmar et al.,
2001
); fn1 (Zhao et
al., 2001
) and pax2a
(Krauss et al., 1991
). Probe
synthesis and single or double-color in situ hybridization (whole-mount) were
performed essentially as previously described
(Thisse et al., 1993
;
Jowett and Yan, 1996
;
Whitlock and Westerfield,
2000
), except for minor modifications. We purified the in vitro
synthesized mRNA and probes using an RNeasy mini column (Qiagen GmbH). Instead
of NBT/BCIP (Boehringer), we used BM purple (Boehringer) to develop color at
room temperature for more than 40 hours. We usually removed the yolks from
young embryos using forceps. Embryos were mounted in phosphate-buffered saline
(PBS) and photographed using a Zeiss Axiophot 2 microscope.
In vitro mRNA synthesis was performed using an RNA synthesis kit (Ambion).
A partial dlx3b cDNA with a complete ORF (990 bp) was subcloned into
pXT7 (Dominguez et al., 1995)
at the EcoRI and SpeI sites. The plasmid was restricted with
XbaI, and the linear DNA served as a template to generate
dlx3b mRNA using T7 RNA polymerase. The complete dlx4b ORF
was amplified by PCR and cloned into the pCRT7/CT-TOPO vector (Invitrogen).
The resulting plasmid was linearized with PmeI and used as a template
to synthesize dlx4b mRNA using T7 RNA polymerase. The recognition
site of dlx4b-MO (see Morpholinos) on dlx4b mRNA was
eliminated. This dlx4b mRNA could partially restore dlx4b
activity if co-injected with dlx4b-MO, as judged by a restoration of
dlx4b-MO-induced reduction of the median fin fold (data not shown). A
full-length sox9a cDNA was cloned into pCDNA3 vector (Clontech).
After linearizing with ApaI, the plasmid was used as a template to
synthesize sox9a mRNA with T7 RNA polymerase.
For rescue experiments, we injected wild-type RNA into Dfb380 embryos. We also injected a YAC clone that contains both the dlx3b and dlx4b genes (a gift from Angel Amores) and a shorter cosmid clone that contains the dlx3b gene with 0.8 kb of 3' sequence. We obtained similar rescue as with dlx3b mRNA alone. The combination of the dlx3b-dlx4b YAC or cosmid DNA with a sox9a expression vector (driven by a CMV promoter/enhancer) resulted in similar rescue as obtained with injection of all three wild-type mRNAs (dlx3b, dlx4b and sox9a). For both DNA and RNA injections, we delivered about 1 nl of solution into the cytoplasm of one-cell stage embryos. The concentrations of the injection solutions were 50-100 ng/µl (DNA) and 200-500 ng/µl (RNA). For injection of all three mRNAs, we used no more than 750 ng/µl total mRNA.
Morpholinos
Morpholino antisense oligonucleotides (MOs) were obtained from Gene Tools
(Philomath, OR). Translation blocking MOs were: dlx3b-MO,
5'-ATGTCGGTCCACTCATCCTTAATAA-3'; dlx4b-MO,
5'-GCCCGATGATGGTCTGAGTGCTGC-3'; sox9a-MO,
5'-TCAGGTAGGGGTCGAGGAGATTCAT-3'; fgf3B,
5'-GGTCCCATCAAAGAAGTATCATTTG-3'; and fgf3C,
5'-TCTCGCTGGAATAGAAAGAGCTGGC-3'. Splice blockers were:
sox9bE1I1, 5'-GTGTGTTTCTGACGAGTTTGCCGAG-3';
sox9bI2E3, 5'-GCCCTGAGACTGACCTGCACACACA-3'; and
fgf8E2I2, 5'-TAGGATGCTCTTACCATGAACGTCG-3';
fgf8E3I3, 5'-CACATACCTTGCCAATCAGTTTCCC-3'. We described
the sox9a-MOs that block pre-mRNA splicing previously
(Yan et al., 2002). A
combination of both fgf8 splice-blocking MOs (fgf8-MO; 2.5
ng/each) was used to inject one- to two-cell stage embryos
(Draper et al., 2001
). We
blocked pre-mRNA splicing by the injection of a combination of both
sox9b-MO oligonucleotides (at 1 µg/µl each). As a control for
the sox9b-MO, we visualized the nuclear localization of
sox9b messenger by in situ hybridization (Y.Y. and D.L.,
unpublished). The concentration of single translation blocking MOs was
3µg/µl for dlx3b, dlx4b and sox9a. Combinatory MO
injections were as follows: dlx3b+dlx4b-MOs, 3+1.5
µg/µl; dlx3b+dlx4b+sox9a-MOs, 3+1.5+1.5
µg/µl; and sox9a-MOs (splicing blockers) 5+5 µg/µl. To
generate fgf3-MO;fgf8- embryos, fgf3B and
fgf3C-MOs at 1 and 0.25 µg/µl, respectively, were used
(Maves et al., 2002
). About
1-3 nl of MO solution was injected into the cytoplasm of one-cell stage
embryos. We have previously provided data to demonstrate the efficacy of the
fgf3-MOs (Maves et al.,
2002
).
Dlx3b monoclonal antibody
We synthesized the complete dlx3b ORF using gene-specific primers
and the Herculase enhanced polymerase blend (Strategene) and ensured that it
is error-free by sequencing in both directions. We cloned the dlx3b
ORF DNA fragment into the pCRT7/CT-TOPO vector (Invitrogen). By following the
manufacturer's instructions, we introduced the resulting pCRT7dlx3b into host
BL21(DE3)pLysE and grew the bacteria in the presence of appropriate
antibiotics. SDS-PAGE electrophoresis analysis indicated strong expression of
soluble Dlx3b fusion protein with the predicted molecular weight.
After one round of nickel-charged Sepharose purification (PreBond), 95%
pure fusion protein was run on a native polyacrylamide gel. We eluted a small
amount of the single protein band directly from the gel and used 0.2 mg of the
purified fusion protein to raise monoclonal antibodies (University of Oregon
Monoclonal Antibody Facility). Out of 10 positive clones identified by ELISA,
we chose a clone (-Dlx3b) that recognized wild-type embryos but not
Dfb380 mutants by whole-mount immunocytochemistry. The
distribution of the antibody labeling is comparable with that of the mRNA in
situ probe for dlx3b.
Immunocytochemistry
Embryos were fixed in 4% paraformaldehyde in PBS overnight at 4°C.
After fixation, embryos were rinsed with PBS twice at room temperature for 20
minutes and then blocked in PBDTX (PBS with 1% bovine serum albumin, 1% DMSO,
pH 7.3, and 0.1% Triton X-100) with 2% normal goat serum (NGS) for 30 minutes.
Embryos were then incubated overnight at 4°C in primary antibodies at the
following dilutions in PBDTX with 2% NGS: -Pax2, 1:200 (BAbCO) and
-Dlx3b, 1:50. After PBDTX rinses to wash away the excess antibody,
embryos were incubated in secondary antibody goat anti-rabbit or anti-mouse
Alexa Fluor 488 (Molecular Probes) at 1:200 dilution in PBDTX with 2% NGS for
5 hours at room temperature or at 4°C overnight. Embryos were then rinsed
in PBS and analyzed using a Zeiss Axiophot 2 microscope.
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RESULTS |
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The Dfb380 deficiency lacks the dlx3b,
dlx4b and sox9a genes and blocks formation of the ear
To study the potential functions of Dlx genes in specification of the otic
placode, we isolated a deficiency mutation,
Df(LG12)dlx3bb380 (Dfb380)
(Fritz et al., 1996), that
removes the dlx3b locus. We identified the mutation in a screen for
deficiencies based on multiplex PCR amplification of genomic DNA from haploid
offspring of females carrying
-ray induced mutations
(Fritz et al., 1996
). We
initially identified the Dfb380 mutation by the absence of
a PCR amplification product from the dlx3b gene. We subsequently
mapped the dlx3b gene to LG12 of the zebrafish genetic map and showed
that the Dfb380 mutation removed 21-24 cM of LG 12 that
also contains several other genes (Fig.
1N), including dlx4b
(Stock et al., 1996
) and
sox9a (Chiang et al.,
2001
).
Homozygous Dfb380 mutants fail to form an ear. We
observed no otic placode or vesicle in live mutant embryos using Nomarski
optics (Fig. 1K) and initial
expression of pax2a, a marker of otic precursor cells in this region,
is also absent (Fig. 1M). Other
sites of pax2a expression
(Püschel et al., 1992)
appear normal in Dfb380 mutants, including optic stalk,
mid-hindbrain junction and pronephros, although pax2a expression is
elevated in the branchial arches (data not shown). Homozygous
Dfb380 mutants also fail to form olfactory organs. Because
olfactory organs form from ectodermal placodes that, like the otic placodes,
express dlx3b and dlx4b, this associated phenotype may
indicate a common pathway mediated by dlx3b and dlx4b for
specification of these two sensory structures, as we
(Akimenko et al., 1994
) and
others (Torres and Giraldez,
1998
; Solomon and Fritz,
2002
) have previously suggested. Heterozygous
(Dfb380/+) individuals develop with no obvious
abnormalities.
Despite this apparent lack of otic induction, a few residual cells express
characteristics of otic cells in Dfb380 mutants. By prim-5
stage (24 h), 30% of Dfb380 mutant embryos form a
patch of 10-20 pax2a-expressing cells
(Fig. 2B) lateral to hindbrain
rhombomere 5 in the region where the otic vesicle normally develops in
wild-type embryos. This pax2a-positive patch of cells is apparent in
80% of embryos (n=43) by prim-15 (30 h) and in all embryos
(n=14) by the second day of development. Consistent with their
differentiation as otic cells, these cells express other otic markers,
including fn1 (Fig.
2E) and claudin a (cldna), a marker of the otic
epithelium (Fig. 2H). Although
cells in other regions of the embryo also express fibronectin 1
(fn1) (Zhao et al.,
2001
), cldna expression marks the ear
(Kollmar et al., 2001
) and the
posterior lateral line (not shown). In contrast to wild-type embryos, however,
these residual otic cells form only a tight cluster, resembling an epithelial
ball, and never produce a vesicle or other morphological features of the
ear.
|
Knockdown of all three transcription factors phenocopies the Dfb380 deficiency mutant phenotype. When dlx3b-MO, dlx4b-MO and sox9a-MO are injected in combination into wild-type embryos, formation of the otic placode fails and we observe only a few residual pax2a-expressing cells as in Dfb380 mutants (Fig. 2C). These residual cells form a small epithelial ball and also express fn1 and cldna (Fig. 2F,I), as do Dfb380 mutants (Fig. 2E,H). These results demonstrate that the combined loss of Dlx3b, Dlx4b and Sox9a functions is sufficient to account for the Dfb380 mutant otic phenotype. To study the requirements and individual roles of these three transcription factors in otic specification, we then injected MOs directed against single genes or combinations of two genes.
Dlx3b function is required for early and complete maturation of the otic placode and vesicle. Injection of dlx3b-MO morpholinos into wild-type embryos delays specification of the otic placode as indicated by delayed and reduced pax2a expression (Fig. 3A-D) and morphological maturation (Fig. 3G,H,K,L). The dlx3b-MO also affects later differentiation of the otic vesicle; the vesicle fails to achieve either its normal size or numbers of otoliths (Fig. 3H,L) and sensory hair cells (Fig. 3I,J). Formation of epithelial protrusions and subsequent semicircular canals also fails (Fig. 3K,L). To ensure that the dlx3b-MO effectively knocks down dlx3b function, we raised a monoclonal antibody specific for Dlx3b protein and demonstrated absence of labeling after dlx3b-MO injection (Fig. 3E,F). These results are consistent with an absence of detectable levels of protein and provide additional support for the conclusion that Dlx3b is required for otic development. However, because the effects of dlx3b-MO on the ear are less severe than the Dfb380 mutant phenotype, other genes missing in the Dfb380 deficiency must also be required for otic placode specification.
|
Knockdown of Sox9a function by morpholino (sox9a-MO) injection has
a relatively mild effect on otic specification, resulting in a slightly
reduced vesicle with a normal number of otoliths (data not shown). To ensure
that Sox9a function was blocked, we used morpholinos directed against splice
donor and acceptor sites that would be expected to interfere with splicing of
the sox9a pre-mRNA. Consistent with this interpretation, we found,
using mRNA in situ hybridization, that sox9a message localizes in
nuclei after sox9a-MO injection as we previously reported
(Yan et al., 2002), suggesting
that the morpholinos effectively block splicing. We obtained similar results
with sox9a translation blocking and mRNA splice blocking MOs. The
effect of sox9a-MO on otic development is more severe than the otic
phenotype of jef (sox9a) mutants (data not shown), probably
because of partial early function of this mutant allele
(Yan et al., 2002
).
Function of Dlx3b, Dlx4b and Sox9a transcription factors is
sufficient to rescue otic placode specification in Dfb380
mutants
If combined loss of the dlx3b, dlx4b and sox9a genes is
responsible for the absence of otic placode specification in
Dfb380 mutants, then restoring wild-type function of only
these genes should rescue the mutant phenotype. To test this prediction, we
injected wild-type mRNAs for each gene into Dfb380 mutant
embryos. Although neither dlx4b nor sox9a mRNA rescues the
mutant phenotype, dlx3b mRNA partially restores otic placode
specification as indicated by pax2a expression and morphology
(Fig. 4C,G). Injection of a
combination of all three mRNAs into Dfb380 mutants
produces a much more robust rescue; the size of the placode, the number of
pax2a-expressing cells and the level of pax2a expression are
comparable with wild-type values (Fig.
4D,H). Rescue of later otic development is variable and less
complete, probably because of degradation of the injected mRNAs, as we have
previously shown for injected DNA
(Westerfield et al.,
1992).
|
|
Reduction of Fgf8 function in fgf8 mutants
significantly reduces sox9a otic expression
(Fig. 6C), whereas knockdown of
Fgf3 by fgf3-MO injection, results in a more modest reduction of
sox9a otic expression (Fig.
6B). Injection of fgf3-MO into
fgf8 mutants results in complete absence of
sox9a expression in the region where the otic placode would normally
form (Fig. 6D). Thus, Fgf3 and
Fgf8 appear to act synergistically to support sox9a expression, just
as they apparently act together to support otic development (Philips et al.,
2001; Léger and Brand,
2002; Maroon et al.,
2002
). Reduction of Fgf signaling has a similar although somewhat
less significant effect on sox9b expression
(Fig. 6E-H). Residual
sox9b expression in the otic area, even when both fgf3 and
fgf8 are compromised, is consistent with Fgf-independent regulation
of sox9b.
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DISCUSSION |
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Fgf3- and Fgf8-dependent and -independent pathways of otic
development
Our results provide evidence for Fgf3- and Fgf8-dependent and -independent
pathways in otic development (Fig.
10). We found that loss of either Fgf3 and Fgf8 function or
functions of Dlx3b, Dlx4b and Sox9a transcription factors missing in the
Dfb380 deficiency mutation results in nearly complete loss
of otic tissue, although a few residual cells express otic markers. Loss of
both Fgf3 and Fgf8 function, and functions of the three transcription factors
completely blocks all indications of otic induction. The Fgf-dependent and
-independent pathways appear to act synergistically. The number of otic cells
is drastically reduced in Dlx3b-and Dlx4b-deficient or Fgf3- and
Fgf8-deficient embryos. Providing either Dlx3b and Dlx4b or Fgf3 and Fgf8
function produces only limited otic specification, much less than when both
pathways are active.
|
Consistent with previous studies that suggested Fgf3 and Fgf8 have
redundant functions in hindbrain (Maves et
al., 2002; Walshe et al.,
2002
) and otic development (Philips et al., 2001;
Léger and Brand, 2002
;
Maroon et al., 2002
), we found
that reduction of either Fgf3 or Fgf8 signaling produces a partial loss of
sox9a and sox9b expression
(Fig. 6B,C,F,G) and a smaller
or somewhat disorganized otic vesicle (Fig.
9F,G). Reduction of both Fgfs
(Fig. 6D,H; Fig. 9H) produces a greater
defect than reduction of either alone. However, recent studies suggest that
Fgf3 and Fgf8 have different downstream targets (Reifers et al., 2000). Loss
of Fgf8 function (in fgf8 mutants) consistently
produces a more severe phenotype than knockdown of Fgf3 function alone by
injection of fgf3 MO, suggesting that although Fgf3 and Fgf8 may have
overlapping functions, Fgf8 apparently plays a more significant role in otic
induction than Fgf3, perhaps because of its earlier and more widespread
expression (Reifers et al.,
1998
; Maves et al.,
2002
).
Genetic interactions of sox9a and sox9b with
fgf3, fgf8, dlx3b and dlx4b
Part of Fgf3 and Fgf8 function in otic development is mediated by Sox9a and
Sox9b. We found that in the otic region after Fgf3 and Fgf8 reduction,
sox9a expression is lost (Fig.
6D) and sox9b expression is reduced although not
completely eliminated (Fig.
6H). The dlx3b and dlx4b expression domain is
reduced by knockdown of sox9a expression, and there is a similar
reduction in sox9a expression by knockdown of dlx3b and
dlx4b (Fig. 8C-F).
Depletion of Fgf3 and Fgf8 signaling similarly reduces dlx3b and
dlx4b expression (Fig.
6H), presumably because of loss of sox9a function. By
contrast, reduction of sox9b expression has little effect on
dlx3b and dlx4b expression, although sox9b
expression depends on Dlx3b and Dlx4b (Fig.
8I,J). These results indicate that sox9a interacts
genetically with both the Fgf3- and Fgf8-dependent and -independent pathways.
Thus, sox9a apparently plays a central role in coordinating otic
development.
Previous studies have shown that pax8 expression is also reduced
or lost in fgf8 mutants injected with
fgf3-MO (Philips et al., 2001;
Léger and Brand, 2002)
(but see Maroon et al., 2002
).
In addition, pax8 expression persists in the absence of the
dlx3b, dlx4b and sox9a genes
(Solomon and Fritz, 2002
)
(D.L., H.C. and M.W., unpublished). Thus, Fgf3 and Fgf8 signaling appears to
act through at least two genetically distinct pathways in the otic placode:
one dependent upon Sox9a and the other mediated by something else, possibly
Pax8 or other, as yet unidentified factors. Although pax8 expression
is unaffected by reduction of sox9a, we have not yet determined
whether Pax8 acts in the same or different pathways with Sox9a and Sox9b.
Fgf3 and Fgf8 functions in otic induction and morphogenesis
Our results may provide insight into how Fgf3 and Fgf8 growth factors
function in otic development. Previous studies have indicated that FGF3
directs morphogenesis of the avian otic vesicle
(Vendrell et al., 2000). We
found that the residual otic cells in Fgf3- and Fgf8-deficient embryos failed
to form an epithelial structure (Fig.
7D,F; Fig. 9H),
whereas the residual otic cells in Dfb380 mutants, with
Fgf3 and Fgf8 signaling still intact, form small epithelial balls
(Fig. 2B,E,H;
Fig. 9B). This might indicate
that Fgf3-Fgf8 signaling that is normally localized to rhombomere 4
`organizes' the placode cells into an epithelium that subsequently forms the
otic vesicle. We have recently provided evidence for a similar Fgf3- and
Fgf8-dependent activity of rhombomere 4 in organizing posterior hindbrain
segments (Maves et al., 2002
).
Our previous fate map analysis of the nose demonstrated that cells from a
relatively large area at the lateral edge of the neural plate converge to form
the olfactory placode and subsequent epithelium
(Whitlock and Westerfield,
2000
). A similar mechanism may occur during otic placode
development. Perhaps in the absence of Fgf3 and Fgf8 signals from rhombomere
4, cells fail to converge properly to this organizing region and, hence,
cannot form the otic epithelium.
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ACKNOWLEDGMENTS |
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Footnotes |
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