Institute of Neuroscience, University of Oregon, Eugene, OR 97403, USA
* Author for correspondence (e-mail: monte{at}uoneuro.uoregon.edu)
Accepted 9 July 2004
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SUMMARY |
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Key words: dlx3b, fgf3, fgf8, foxi1, Inner ear, Morpholino, Otic placode, pax8, pax2a, sox9a, sox9b, Zebrafish
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Introduction |
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Studies in various species suggest that signals from the underlying
mesoderm and adjacent hindbrain induce ectodermal cells to form the otic
placode (reviewed by Fritzsch et al.,
1997; Torres and
Giráldez, 1998
; Baker
and Bronner-Fraser, 2001
;
Whitfield et al., 2002
). In
zebrafish, Fgf3 and Fgf8 appear to have overlapping functions in otic placode
induction. The genes are expressed in the future hindbrain by late gastrula
stages, and fgf3 is also expressed at this stage in the underlying
mesendoderm. Loss of either fgf3 or fgf8 leads to a
reduction in ear size and loss of both fgf3 and fgf8
together results in near or total ablation of otic tissue
(Phillips et al., 2001
;
Maroon et al., 2002
;
Leger and Brand, 2002
).
Furthermore, Fgf signaling is sufficient and necessary for otic induction,
indicating a direct role for Fgf3 and Fgf8
(Phillips et al., 2004
). In
the mouse, Fgf3 and Fgf10 act as redundant signals during otic induction
(Wright and Mansour, 2003
;
Alvarez et al., 2003
). We have
previously shown that Fgf signals are required for the preotic expression of
some, but not all, of the transcription factors involved in otic induction
(Liu et al., 2003
). For
example, the four transcription factors Dlx3b
(Ekker et al., 1992
), Dlx4b
(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
) are all
required for otic placode specification. sox9a and to some extent
sox9b require Fgf signaling for their proper expression in the
preotic region, whereas dlx3b and dlx4b do not. When Fgf3
and Fgf8 functions are removed, dlx3b and dlx4b gene
expression is unaffected during induction and early patterning stages. Later
expression of dlx3b and dlx4b in the otic anlagen is reduced
when Fgf signals are blocked, but this is probably an indirect effect caused
by the loss of sox9a function
(Liu et al., 2003
).
Fate-mapping experiments at mid-gastrula stages indicate that precursors of
cranial placodes are arranged in an anteroposterior order at the lateral
border of the prospective anterior neural plate
(Kozlowski et al., 1997). We
have previously shown that dlx3b and dlx4b are both
expressed in late gastrula stage embryos in this same region, a stripe
corresponding to cells of the future neural plate border; expression of both
genes becomes restricted to cells of the future olfactory and otic placodes by
the beginning of somitogenesis (Akimenko et
al., 1994
; Ekker et al.,
1992
; Ellies et al.,
1997
). We also showed by fate mapping that a subset of cells in
the anterior part of the dlx3b stripe later contribute to the
olfactory placodes (Whitlock and
Westerfield, 2000
). Knockdown of dlx3b and dlx4b
causes a severe loss of otic tissue even in the presence of functional Fgf
signaling (Solomon and Fritz,
2002
; Liu et al.,
2003
), indicating that these genes are required to specify the
competence of cells to form the ear. Expression of the forkhead class winged
helix transcription factor, foxi1, is progressively restricted at
late gastrula stages to bilateral domains, including the presumptive otic
placode, and, subsequently, foxi1 expression is downregulated prior
to placode formation. Disruption of foxi1 leads to severe defects in
otic placode formation and highly variable ear phenotypes
(Solomon et al., 2003
;
Nissen et al., 2003
),
suggesting that foxi1 also influences otic competence.
In addition to these genes implicated in otic development, some Pax genes
that encode paired box transcription factors are expressed at the right time
and place to be involved in otic specification. The Pax gene family is
subdivided into four distinct classes based on sequence similarities
(Noll, 1993;
Mansouri et al., 1996
). The
Pax2, Pax5 and Pax8 genes constitute one such class and
encode highly related transcription factors with similar biochemical
activities (Pfeffer et al.,
1998
). Pax2-Pax5-Pax8 genes have important roles in
embryonic development and organogenesis of the eye, ear, kidney and thyroid
(Dressler et al., 1990
;
Nornes et al., 1990
;
Plachov et al., 1990
). The
best-studied Pax2-Pax5-Pax8 gene function is in development of the
midbrain-hindbrain boundary (isthmus). In the mouse, Pax2 or
Pax2 and Pax5, depending on the genetic strain, tops a
hierarchy of genes that function together to form the isthmus. Pax2 and Pax5
act at multiple stages in this process and are also required for maintenance
of Pax2 (Urbanek et al.,
1994
; Torres et al.,
1996
; Mansouri et al.,
1998
). Implantation of Fgf8-soaked beads into chick embryos showed
further that Fgf8 acts in this positive feedback loop maintaining
Pax2 expression in the isthmus
(Martinez et al., 1999
). In
zebrafish, pax2a has been shown to function in this process; loss of
pax2a leads to failed formation of the isthmus
(Brand et al., 1996
;
Lun and Brand, 1998
). Similar
to amniote embryos, maintenance but not induction of zebrafish pax2a
depends on both pax2a and fgf8
(Lun and Brand, 1998
;
Reifers et al., 1998
). Gene
replacement in the mouse has shown that Pax5 can functionally
substitute for Pax2, indicating that the Pax2-Pax5-Pax8 proteins are
interchangeable (Bouchard et al.,
2000
). Combined, redundant gene function has also been shown for
Pax2 and Pax8 during development of the mouse urogenital
system (Bouchard et al.,
2002
).
The relative roles of Pax2 and Pax8 in otic specification are still
somewhat unclear. Pax8 is one of the earliest known markers of otic
cells in vertebrates, showing onset of otic expression before Pax2
(Pfeffer et al., 1998;
Heller and Brandli, 1999
;
Hutson et al., 1999
;
Groves and Bronner-Frasier,
2000
). In mouse, loss of Pax8 does not prevent expression
of Pax2 or proper inner ear formation
(Mansouri et al., 1998
) and
loss of Pax2 has no effect on otic induction but variably affects
formation of the cochlea (Torres et al.,
1996
). Zebrafish have two Pax2 genes, pax2a and
pax2b, both expressed in the preotic region with pax2a
expressed at higher levels several hours earlier than pax2b
(Pfeffer et al., 1998
). Loss
of pax2a, pax2b or both alters hair cell development but does not
hinder otic placode induction (Riley et
al., 1999
; Whitfield et al.,
2002
). Cells of the zebrafish otic placode also express
pax8 (Pfeffer et al.,
1998
); functional studies have not previously been described.
We show that Pax8-depleted zebrafish embryos, like pax2a mutants, have only mild ear defects. By contrast, Pax8 depleted pax2a mutants fail to form a differentiated otic vesicle or inner ear, although a few residual cells express genes characteristic of otic fate. These data demonstrate that Pax2a and Pax8 have similar functions required for the correct expression of sox9a, sox9b and pax2a, and to a much lesser extent, dlx3b. However, pax8 and pax2a are regulated by two independent factors, Foxi1 and Dlx3b, respectively; removal of both factors is required to block the establishment of otic fate. Our results integrate pax2a and pax8 gene functions into the previously known genetic pathways that regulate otic placode induction; Foxi1 and Pax8 mediate early Fgf dependent otic specification, whereas Dlx3b and Pax2a mediate later Fgf signaling required for maintained development.
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Materials and methods |
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Genes and markers
Approved gene and protein names that follow the zebrafish nomenclature
conventions
(http://zfin.org/zf_info/nomen.html)
are used.
Immunocytochemistry
Antibody staining was carried out as described previously
(Westerfield, 2000) with some
modifications. Primary antibodies were used in the following concentrations:
-Pax2 (Covance), 1:100;
-Dlx3b
(Liu et al., 2003
), 1:50;
rabbit polyclonal IgG
-Myc (Santa Cruz Biotechnology), 1:500. The
following secondary antibodies were used: goat
-mouse Alexa Fluor 488
(Molecular Probes), goat
-mouse Alexa Fluor 568 (Molecular Probes),
1:100; goat
-rabbit Alexa Fluor 488 (Molecular Probes), 1:100. Embryos
were analyzed using a Zeiss Axiophot 2 microscope.
In situ hybridization and mRNA synthesis
cDNA probes that detect the following genes were used: dlx3b
(Ekker et al., 1992);
sox9a (Chiang et al.,
2001
); sox9b (Chiang
et al., 2001
; Li et al.,
2002
); egr2b (previously krox20)
(Oxtoby and Jowett, 1993
);
cldna (Kollmar et al.,
2001
); and pax2a
(Krauss et al., 1991
). For
detection of pax2a in dnpax2a-myc-injected embryos, the
5' and 3' UTRs of pax2a were amplified by PCR, subcloned
into pBluescript, linearized with NotI and EcoRI,
respectively, and transcribed with T7 RNA polymerase. Probe synthesis and
single or double-color in situ hybridization was performed essentially as
previously described (Thisse et al.,
1993
; Jowett and Yan,
1996
; Whitlock and
Westerfield, 2000
). We purified the in vitro synthesized mRNA and
probes using an RNeasy mini column (Qiagen GmbH). In vitro mRNA synthesis was
performed using an SP6 RNA synthesis kit (Ambion). The construct encoding
dnPax2a-myc was generated by PCR amplification of the pax2a gene
coding for the first 295 amino acids, which were fused in-frame with six
Myc-epitopes and cloned into the CS2+ vector
(Turner and Weintraub, 1994
).
For RNA injections, 1-3 nl of a 300 ng/µl solution was delivered into the
cytoplasm of one cell at the two-cell stage.
Morpholinos (MOs)
We have described the dlx3b-MO, fgf3-MOs and
fgf8-MOs previously (Liu et al.,
2003; Maves et al.,
2002
). Splice-blocking pax8-MOs were: E2/I2,
5'-GTGTGTGTTCACCTGCCCAGGATCT; E3/I3, 5'-GTGTGTACCGGTTGATGGAGCTGAC;
E4/I4, 5'-CACAGCACTTACTCAGTGTGTGTCC; E5/I5,
5'-TTTCTGCACTCACTGTCATCGTGTC; and E9/I9,
5'-ACCGGCGGCAGCTCACCTGATACCA. About 1-3 nl of MO-solution was injected
into the cytoplasm of one-cell stage embryos. The concentration of the
pax8 splice-blocking MOs was 1 µg/µl each for
pax8-E5/I5 and pax8-E9/I9.
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Results |
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We find that knockdown of Fgf3 and Fgf8 in wild-type embryos or knockdown
of Fgf3 in fgf8 mutants significantly reduces
pax8 expression in the preotic region, although weak residual
expression can still be detected (Fig.
1I,J). This finding is similar to the observations of Maroon et
al. (Maroon et al., 2002), but
contrasts with the results of Phillips et al.
(Phillips et al., 2001
) and
Leger and Brand (Leger and Brand,
2002
) who reported complete loss of pax8 when Fgf3 and
Fgf8 are knocked-down by antisense morpholino oligonucleotide (MO) injection.
Although the discrepancies between these studies might be explained by
incomplete effectiveness of the MOs, the Fgf receptor blocking drug SU5402
also led to differing results: Leger and Brand
(Leger and Brand, 2002
)
reported a complete loss of pax8 expression whereas Maroon et al.
(Maroon et al., 2002
) found
that pax8 was unaffected. Thus, taken together, these results could
indicate either that pax8 is highly sensitive to Fgf signaling and
only complete loss of the Fgf signal leads to loss of pax8 expression
or that pax8 expression is regulated partly by some factor in
addition to Fgf. foxi1 mutants fail to initiate
pax8 expression (Solomon et al.,
2003
; Nissen et al.,
2003
), suggesting that this forkhead-related transcription factor
may be the other regulator of pax8 expression. To test this
interpretation, we examined whether foxi1 acts independently of Fgf
signaling. We found that knockdown of Fgf3 and Fgf8 in wild-type embryos or
knockdown of Fgf3 in fgf8 mutants has no
significant effect on foxi1 expression
(Fig.1K,L), supporting the
hypothesis that foxi1 expression is independent of Fgf signaling, but
required for cells to respond to Fgf signaling
(Nissen et al., 2003
).
Pax2a and Pax8 function synergistically in otic specification
Because pax8 mutants are not available, we used
MOs to knock down gene function in a gene-specific manner
(Nasevicius and Ekker, 2000).
In addition to their ability to block the translation of mRNAs in the
cytoplasm, MOs can inhibit pre-mRNA splicing
(Draper et al., 2001
), thus
interfering with the transport of transcript from the nucleus to the cytoplasm
(Yan et al., 2002
). This
inappropriate retention of transcripts in the nucleus can be used as an assay
for MO efficacy in the absence of an antibody to test for the production of a
translated product (Yan et al.,
2002
). We used this splice-blocking strategy for pax8
because neither a Pax8 antibody nor the sequence of the 5' terminus of
the pax8 mRNA (Pfeffer et al.,
1998
) was available. We determined the sequence of several
introns, designed specific splice-blocking MOs
(Fig. 2A), and injected the MOs
alone and in various combinations. As a control for the efficacy of the MOs,
we visualized the nuclear localization of pax8 messenger by in situ
hybridization; the most efficacious combination of MOs was used in this study
(Fig. 2A-C).
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Pax2a and Pax8 are required for maintenance of otic sox9a, sox9b and pax2a expression, but neither induction nor maintenance of dlx3b expression
To study the placement of pax2a and pax8 in the genetic
pathway regulating otic development, we examined the expression of several
otic markers at preplacodal, placodal and vesicle stages. We concentrated on
the transcription factor genes, sox9a, sox9b and dlx3b
(Yan et al., 2002;
Chiang et al., 2001
;
Akimenko et al., 1994
), that
are essential for formation of the ear
(Liu et al., 2003
). We also
examined pax2a expression that is reduced in
pax2a mutants at later stages
(Brand et al., 1996
).
Loss of Pax2a, together with Pax8 knockdown, affects sox9a and sox9b expression. sox9a is broadly expressed in the preotic region at the three-somite stage (Fig. 3A) and expression is maintained in the placode (Fig. 3C) and in the vesicle (not shown). In pax2a mutants injected with pax8-MOs, the expression of sox9a is reduced in extent and level (Fig. 3B). At the 12-somite stage, when the placode is morphologically visible in wild-type embryos, we detect no sox9a expression in pax2a mutants injected with pax8-MOs (Fig. 3D). The sox9a duplicate, sox9b, is also expressed from preplacodal to vesicle stages, although it is initiated later in development (Fig. 3E,G). Like sox9a, sox9b expression is compromised at preplacodal stages in pax2a mutants depleted of pax8 (Fig. 3E) and absent at the 12-somite stage (Fig. 3H).
|
Maintenance of pax2a expression depends strongly on Pax2a or Pax8.
In pax2a mutants, pax2a transcription
initiates normally and we cannot distinguish between wild-type,
pax2a mutants and pax2a
mutants injected with pax8-MOs at preplacodal stages (not shown).
However, there is severe downregulation of pax2a in
pax2a mutants injected with pax8-MOs by
the 12-somite stage (Fig. 3N)
and pax2a is completely lost by vesicle stages
(Fig. 3P). Labeling for
fgf3 or fgf8 expression and egr2b or mafb
(previously valentino) expression, both downstream targets of Fgf
signaling (Maves et al.,
2002), reveals that expression of fgf3 or fgf8
and patterning of the hindbrain occurs normally in
pax2a mutants depleted of Pax8
(Fig. 3N, and not shown). These
results indicate that Pax2a and Pax8 act synergistically downstream of Fgf3
and Fgf8; when Pax2a and Pax8 functions are both compromised, otic induction
is weaker and otic fate is not maintained, even in the presence of normal Fgf
signaling.
sox9a, sox9b and pax2a, but not dlx3b, are transcriptional targets of Pax8
Because we see some residual specification of otic cells in Pax2a and Pax8
knockdown embryos, we were concerned that some Pax protein function remained.
Structure-function analyses have shown that proteins of the Pax2-Pax5-Pax8
family have overlapping biochemical activities; their DNA-binding
specificities are highly similar and they can substitute for each other
(Bouchard et al., 2000). Thus,
to block all Pax2-Pax5-Pax8 function, we generated a dominant-negative form of
Pax2a (dnpax2a-myc) by replacing the C-terminal
transactivation-inhibitory domain with six Myc epitope tags
(Fig. 4A). We injected mRNA
from this pax2a variant into wild-type embryos and analyzed
subsequent otic development. To assess the effectiveness of
dnpax2a-myc, we examined eng3 expression in injected
embryos. Expression of eng3, a downstream target of Pax2a in the
midbrain-hindbrain boundary region, is initiated at the one-somite stage in
wild-type embryos (Fig. 4B) but
is never activated in strong pax2a mutants
(Lun and Brand, 1998
). In
dnpax2a-myc mRNA-injected embryos, we identify regions expressing the
variant protein by the presence of the Myc-epitopes. In these regions,
eng3 transcription is severely reduced or completely absent
(Fig. 4C). The nuclear
localization of the Myc-epitopes and the severe downregulation of
eng3 show that this Pax2a variant enters the nucleus and competes
with the endogenous Pax2-Pax5-Pax8 proteins for binding sites. We expect that,
owing to its abundance, this construct is able to out compete the endogenous
proteins and act in a dominant-negative fashion.
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Pax8 and Fgf control otic expression of pax2a synergistically
Recent studies implicate combined functions of Fgf3 and Fgf8 in otic
pax2a induction (Phillips et al.,
2001; Maroon et al.,
2002
; Leger and Brand,
2002
). In addition, Pax2a and Fgf8 are both required to maintain
pax2a expression in the isthmus
(Lun and Brand, 1998
;
Reifers et al., 1998
). To test
whether Pax8 also acts together with Fgf signals to promote pax2a
expression, we injected pax8-MOs into fgf8
mutants. pax2a expression in fgf8 mutants
alone shows normal timing, but the size of the otic expression domain is
significantly reduced (Fig.
5A,B) (Phillips et al.,
2001
), and the otic vesicle is subsequently smaller
(Fig. 5D,E)
(Phillips et al., 2001
).
fgf8 mutants depleted of pax8 show an even
stronger reduction of pax2a expression in the preotic region
(Fig. 5C) and form an even
smaller ear (Fig. 5F). This
observation suggests that Fgf8 and probably Fgf3 act through and in parallel
with Pax8 to promote proper pax2a expression.
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|
Together, these results show that in the absence of Foxi1 function, and hence also in the absence of pax8 expression, only weak induction of otic sox9a and sox9b occurs, and otic pax2a expression is restricted to Dlx3b-positive cells.
Pax2a acts partially independently of Foxi1
Our analysis of foxi1 mutants, dominant-negative
Pax2a and pax2a mutants depleted of Pax8 suggest
that Pax2a may provide a Foxi1-independent pathway for otic specification. To
test this hypothesis, we generated
foxi1;pax2a double
mutants and analyzed them for otic specification. The ears of
pax2a single mutants are virtually
indistinguishable from wild-type embryos by morphology
(Fig. 2D,E,H,I) or by gene
expression (Fig. 2L,M,P,Q).
foxi1 single mutants are highly variable; some
mutants develop a small lumen with only one or no otolith, whereas others have
small split lumens, each with a single otolith
(Solomon et al., 2003;
Nissen et al., 2003
). The
foxi1 mutant allele we used typically forms a small lumen with one
otolith (Fig. 7B,G) and
consistently retains some expression of the otic markers Dlx3b, cldna
and fn1 (Fig. 7L,O;
not shown).
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Foxi1 and Dlx3b mediate convergent pathways required for otic development
Our observation that a few residual cells express otic markers even in
foxi1;pax2a double
mutants suggests the possibility that an additional factor participates in
otic specification; Dlx3b is a likely candidate. To test this possibility, we
compromised Dlx3b function using morpholino injection. Reduction of Dlx3b in
wild-type embryos impairs complete maturation of the otic vesicle and most
embryos form a smaller vesicle with only one otolith
(Fig. 7D,I)
(Solomon and Fritz, 2002;
Liu et al., 2003
) and reduced
cldna expression (Fig.
7Q). By contrast, foxi1 mutants
injected with dlx3b-MO show no morphological signs of otic
specification (Fig. 7E,J) and
no otic expression of cldna (Fig.
7R). In wild-type embryos depleted of Dlx3b, otic specification is
delayed as indicated by delayed onset and reduced expression of pax2a
(Fig. 7V). However, in
foxi1 mutants injected with dlx3b-MO, otic
pax2a expression is undetectable
(Fig. 7W). Taken together,
these results show that removal of the two factors, Foxi1 and Dlx3b, leads to
a complete absence of otic specification.
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Discussion |
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Otic vesicle formation in pax2a mutants is
virtually the same as in wild-type embryos; pax2a
mutants show a weak neurogenic phenotype, probably owing to reduced Delta
signaling (Riley et al.,
1999). This result indicates that in the absence of Pax2a, Pax8 is
sufficient for most aspects of otic development, even though there are
differences in the expression patterns of these two Pax genes. Specifically,
pax8 is expressed prior to pax2a, and in contrast to
pax8, pax2a transcription continues after the otic placode becomes
morphologically visible (Fig.
1) until it is subsequently restricted to sensory hair cells
(Riley et al., 1999
). The
presence of the pax2a duplicate, pax2b, cannot account for
the absence of a more dramatic phenotype because depletion of Pax2a and Pax2b
together leads to no loss of otic structures
(Whitfield et al., 2002
).
These observations suggest either that Pax8 protein is stable and can provide
sufficient function at later stages after transcription has ended or that
Pax2-Pax5-Pax8 function is not required after placode formation. We are unable
to distinguish between these two interpretations because no antibody against
Pax8 is currently available.
The otic phenotype of pax2a mutants depleted of
Pax8 is similar to embryos depleted of both Fgf3 and Fgf8. Previous studies
have shown that knockdown of Fgf3 and Fgf8 in wild-type embryos or knockdown
of Fgf3 in fgf8 mutants causes a synergistic loss
of otic tissue, indicating that fgf3 and fgf8 encode
overlapping functions required for otic specification
(Phillips et al., 2001;
Maroon et al., 2002
;
Leger and Brand, 2002
;
Liu et al., 2003
). The failure
of otic tissue formation in the absence of Fgf function is preceded by a
strong reduction of pax2a and pax8 expression
(Phillips et al., 2001
;
Leger and Brand, 2002
).
Furthermore, compromising Fgf signals leads to an absence of sox9a
expression and significant reduction of sox9b expression in the
preotic region (Liu et al.,
2003
), similar to the Fgf dependence of Sox9 in Xenopus
(Saint-Germain, 2004). By contrast, early dlx3b
(Leger and Brand, 2002
;
Liu et al., 2003
) and
foxi1 (Fig. 1)
expression is less affected by loss of Fgf signaling and effects on
dlx3b expression are caused at least in part by reduced levels of
sox9a at early stages and by reduced levels of sox9a and
sox9b at later stages (Liu et
al., 2003
). Together, these observations lead to the conclusion
that pax2a and pax8 act downstream of Fgf3 and Fgf8, but
upstream of sox9a and sox9b, and that Pax2a and Pax8 are
mediators of Fgf signals during otic placode induction
(Fig. 8). Recent experiments in
Xenopus have led to the suggestion that Sox9 may act upstream of
Pax8, although the results reported do not rule out the possibility that Sox9
and Pax8 interact to maintain each other's expression
(Saint-Germain et al.,
2004
).
|
Foxi1-Pax8 and Dlx3b-Pax2a mediate two phases of otic specification
Our results are consistent with those of Nissen et al.
(Nissen et al., 2003) and
Solomon et al. (Solomon et al.,
2004
) and suggest that Foxi1 is required for the initial
Fgf-dependent induction of pax8. In foxi1
mutants, early expression of sox9a and sox9b is also
severely affected (Fig. 6),
similar to the effects of Pax8 depletion in pax2a
mutants. However, unlike embryos lacking both Pax2a and Pax8,
foxi1 mutants later recover sox9a and
sox9b expression (Fig.
6). This recovery is due to Pax2a
(Fig. 7); once expression
begins, Pax2a protein maintains its own expression and activates downstream
sox9 target genes. Thus, it is likely that variability in the onset
of pax2a expression, in the absence of Foxi1 and, hence, Pax8
(Fig. 8), produces the highly
variable phenotype of foxi1 mutants. Supporting
this interpretation,
foxi1;pax2a double
mutants exhibit consistent, more severe reduction of otic tissue
(Fig. 7).
Our data indicate that Pax2a and Pax8 participate in the same otic
developmental pathway: Foxi1 and Pax8 mediate the initial Fgf dependent
induction that includes initiation of Dlx3b-dependent pax2a
expression. Pax2a subsequently maintains its own expression. This model
contrasts somewhat from previous suggestions
(Riley and Phillips, 2003)
primarily based on studies in mouse where loss of Foxi1
(Hulander et al., 1998
;
Hulander et al., 2003
) or Pax8
(Mansouri et al., 1998
) does
not prevent otic Pax2 expression or early patterning and
morphogenesis of the otic vesicle. This apparent discrepancy in Foxi1 function
between zebrafish and mouse may be due to temporal differences in development.
Otic induction in response to Fgf signals occurs over a much longer time
period in mice than in zebrafish, which provides more time for cells in
mammalian embryos to respond to Fgf signals, even in the absence of Pax8.
Analysis of Pax2;Pax8 double mutant mice will be necessary
to test this interpretation definitively.
Foxi1 and Dlx3b provide competence to respond to Fgf signals
Our results also provide further insight into Fgf-dependent and
-independent processes and the mechanisms underlying competence in otic
development. Previously, we have demonstrated that loss of either Fgf3 and
Fgf8 or loss of Dlx3b, Dlx4b and Sox9a results in nearly complete loss of otic
tissue, although a few residual cells express otic markers including
pax2a, fn1 and cldna
(Liu et al., 2003). Loss of
both Fgf signals, and all three of these transcription factors completely
blocks all indications of otic induction, suggesting that Fgf-dependent and
Fgf-independent processes of otic induction act synergistically. We propose
that induction of otic fate by Fgf signals takes place only when cells are
competent to respond, and that this competence is provided by Foxi1 and Dlx3b
(Fig. 8). A direct role for
Foxi1 and Dlx3b in competence needs to be demonstrated, for example by ectopic
expression and transplantation experiments. Foxi1 and Dlx3b function by
regulating pax8 and pax2a expression, respectively, in an
Fgf-dependent fashion. In Dlx3b-deficient embryos, expression of pax8
is indistinguishable from that in wild-type embryos, presumably owing to
normal Foxi1 and Fgf signaling. However, otic pax2a expression is
initiated only very late and weakly
(Solomon and Fritz, 2002
;
Liu et al., 2003
). By
contrast, otic pax8 expression fails and pax2a expression is
present although delayed in foxi1 mutants
(Solomon et al., 2003
;
Nissen et al., 2003
).
Inhibition of both factors, Foxi1 and Dlx3b, completely blocks otic
specification even in the presence of functional Fgf signaling
(Fig. 7). By activating Pax8,
Foxi1 thus provides competence to otic precursor cells to respond to early Fgf
signaling; Dlx3b and Pax2a subsequently maintain this competence
(Fig. 8).
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ACKNOWLEDGMENTS |
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