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First published online 23 May 2007
doi: 10.1242/dev.000448
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1 Institute of Neuroscience, University of Oregon, Eugene, OR 97403, USA.
2 Biotechnology Center and Center of Regenerative Therapies, University of
Technology, Dresden, Germany.
* Author for correspondence (e-mail: monte{at}uoneuro.uoregon.edu)
Accepted 30 April 2007
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Competence, dlx3b, Danio rerio, fgf3, fgf8, foxi1, Inner ear, Morpholino, Otic induction, Otic placode, Retinoic acid, Zebrafish
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Couly et al.,
1993; Fritzsch et al.,
1997; Whitfield et al.,
2002; Barald and Kelley,
2004).
Previous studies support the primacy of fibroblast growth factors (Fgfs) in
the induction of the otic placode. Various Fgf family members from various
sources regulate otic induction in different species, with only
hindbrain-derived Fgf3 playing a conserved role (reviewed in
Fritzsch et al., 1997;
Torres and Giráldez,
1998; Whitfield et al.,
2002; Brown et al.,
2003). In zebrafish, Fgf3 and Fgf8 have been implicated to have
overlapping functions; loss of both fgf3 and fgf8 together
results in near or total ablation of otic tissue
(Phillips et al., 2001;
Maroon et al., 2002;
Léger and Brand, 2002).
In mouse, Fgf3 and Fgf10 act as redundant signals during otic induction
(Wright and Mansour, 2003;
Alvarez et al., 2003): Fgf3 is
expressed in the hindbrain abutting the preotic domain, whereas Fgf10 is
expressed in the mesoderm beneath it, and loss of both Fgf3 and Fgf10 results
in the complete ablation of otic development
(Wright and Mansour, 2003;
Alvarez et al., 2003).
Furthermore, Fgf8 has been shown to play a crucial role upstream of the Fgf
signaling cascade required for otic induction in this species
(Ladher et al., 2005). In
chick, Fgf3, Fgf8 and Fgf19 and, in amphibians, Fgf2 and Fgf3, have been
implicated in otic induction (Mahmood et
al., 1995; Ladher et al.,
2000; Ladher et al.,
2005; Song and Slack,
1994; Lombardo et al.,
1998).
Recently, studies have shown that Pax2a and Pax8 are the main effectors
downstream of Fgf-signaling and that cells need to express Foxi1 and Dlx3b
transcription factors to be competent to respond to Fgf signaling in zebrafish
(Hans et al., 2004;
Solomon et al., 2004). The
expression of foxi1 is restricted to bilateral domains, including the
preotic domain at late gastrula stages, and disruption of foxi1
expression leads to severe defects in otic placode formation and highly
variable ear phenotypes (Thisse et al.,
2005; Solomon et al.,
2003; Nissen et al.,
2003). The homeobox gene, dlx3b, is co-expressed with
dlx4b in late gastrula stage embryos in a stripe corresponding to
cells of the future neural plate border, and knockdown of dlx3b and
dlx4b together causes a severe loss of otic and olfactory tissues
(Akimenko et al., 1994;
Ekker et al., 1992;
Ellies et al., 1997;
Kudoh et al., 2001;
Solomon and Fritz, 2002;
Liu et al., 2003). Loss of
both Dlx3b and Foxi1 ablates all indications of otic induction even in the
presence of a fully functional or over-activated Fgf signaling pathway
(Hans et al., 2004;
Solomon et al., 2004;
Hans et al., 2007).
Retinoic acid (RA), a derivative of vitamin A, is required for proper
embryonic development. Embryos deficient in RA signaling show defects in the
circulatory system, limbs, trunk and hematopoietic system (reviewed by
Maden, 2002). RA plays a
crucial role in hindbrain patterning and rhombomere (r) identity, and the
hindbrain is known to regulate otic development (reviewed by
Gavalas and Krumlauf, 2000;
Romand, 2003). In amniote
embryos, RA is required in a concentration- and time-dependent manner for the
development of the posterior hindbrain, particularly r5-r7, and compromised RA
signaling leads to an expansion of anterior hindbrain at the expense of
posterior hindbrain (Dupé et al.,
1999; Dupé and Lumsden,
2001). Complete absence of RA signaling leads to a complete loss
of r5-r7 accompanied by an expansion of r3-r4, as observed in mouse embryos
mutant for Aldh1a2 (also known as Raldh2 - Mouse Genome
Informatics). Aldh1a2 is an aldehyde dehydrogenase that is responsible for the
majority of RA production in the early embryo
(Niederreither et al., 1999;
Niederreither et al., 2000).
Mutations in aldh1a2 in zebrafish are less profound and show only a
loss of r7 accompanied by a slight expansion of r5 and r6 similar to weak
vitamin A deficiency syndrome (VAD) in amniote embryos
(Begemann et al., 2001;
Grandel et al., 2002;
Maves and Kimmel, 2005).
However, a loss of r5-r7 accompanied by an expansion of r3 and r4 is observed
after knockdown of RA signaling by pharmacological treatment, suggesting that
RA is produced by something in addition to Aldh1a2
(Grandel et al., 2002;
Maves and Kimmel, 2005).
Treatment of vertebrate embryos with excess RA posteriorizes the anterior
neural plate with the transformation of r2-r3 to r4-r5 identity and expansion
of posterior hindbrain at the expense of presumptive fore- and mid-brain
structures (Marshall et al.,
1992; Kudoh et al.,
2002).
Otic defects generated by changes in RA signaling are considered mostly
secondary consequences due to changes in Fgf signaling, because hindbrain
patterning is disturbed in RA gain- and loss-of-function studies. In embryos
with no RA signaling, such as mouse Aldh1a2 mutants, expression of
Fgf3, which is specifically expressed in the presumptive r5-r6 in wild-type
embryos, is weak and not properly restricted, and otocysts are hypoplastic and
abnormally distant from the hindbrain
(Niederreither et al., 1999).
The same otic phenotype can be observed in zebrafish after knockdown of RA by
pharmacological treatment and it has been suggested, but not yet shown, that
RA is required for normal fgf3 expression in the presumptive r4 and
for the induction of the otic placode
(Perz-Edwards et al., 2001;
Whitfield et al., 2002). A
moderate loss of RA signaling, such as in weak quail or rat VAD embryos,
however, leads to the formation of supernumerary otic vesicles caudal to the
main otocyst, which correlates with the expansion of posterior hindbrain and,
in particular, with the caudal expansion of Fgf3 expression
(White et al., 2000;
Kil et al., 2005). In
zebrafish, the opposite result has been reported; excess, rather than reduced,
RA results in the formation of supernumerary and ectopic otic vesicles in an
Fgf-dependent manner (Phillips et al.,
2001). In zebrafish embryos treated with teratogenic doses (1
µM) of RA, hindbrain expression domains of fgf3 and fgf8
are greatly expanded into the anterior neural plate, inducing preotic
pax8 expression surrounding the anterior neural plate. Inactivation
of Fgf3 and/or Fgf8 by morpholino injection blocks the effects of exogenous RA
(Phillips et al., 2001).
Here, we show that the opposite otic phenotypes generated by more or less RA signaling are produced by different mechanisms. We used a 50-fold lower dose of RA than that of previous studies, and show that RA affects otic induction without expanding fgf3 and fgf8 expression in the hindbrain. Instead, this low dose of excess RA leads to an increase in otic competence due to expanded expression of foxi1 throughout the preplacodal domain. By contrast, reduced RA signaling results in lower induction and impaired maintenance of otic fates, due to compromised fgf3 and wnt8b expression in the hindbrain. We further show that fgf8 is the primary inducing factor in RA-deprived embryos and that the induction but not the maintenance phenotype can be rescued by an increase in Fgf-signaling.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) and staged
according to standard criteria (Kimmel et
al., 1995) or by hours post fertilization at 28°C (hpf). The
wild-type line used was AB. The lines acerebellarti282a (a
strong hypomorphic allele of fgf8); liat24149 (a
hypomorphic allele of fgf3); tcf2hi2169 (a null
or strong hypomorphic allele of tcf2) and the transgenic line
Tg(hsp:fgf8) (to misexpress fgf8) have been described
previously (Brand et al., 1996;
Herzog et al., 2004;
Sun and Hopkins, 2001;
Hans et al., 2007), and we
refer to the homozygous mutants as fgf8, fgf3 and tcf2
mutants, respectively. Homozygous mutants and double mutants were scored
either by their morphological phenotype and/or by PCR or loss of
pou1f1 expression (Herzog et al.,
2004; Sun and Hopkins,
2001). Heat-shock-treatment embryos were transferred, still in
their chorions, into fresh 37-39°C embryo medium in a 1.5 ml tube (20
embryos per tube) and maintained for 30 minutes in a heating block.
Genes and markers
Approved gene and protein names that follow the zebrafish nomenclature
conventions
(http://zfin.org/zf_info/nomen.html)
are used.
In situ hybridization
cDNA probes that detect the following genes were used: cldna
(Kollmar et al., 2001);
dlx3b (Ekker et al.,
1992); fgf3 (Phillips
et al., 2001); fgf8
(Reifers et al., 1998);
foxi1 (Solomon et al.,
2003); otx2 (Li et
al., 1994); pax8
(Pfeffer et al., 1998);
pou1f1 (Nica et al.,
2004); stm
(Söllner et al., 2003);
and wnt8b (Kelly et al.,
1995). 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).
Morpholinos (MOs) and pharmacological treatments
The dlx3b-MO, foxi1-MO and wnt8b-MO have been
previously described (Liu et al.,
2003; Solomon and Fritz,
2002; Kim et al.,
2002). For pharmacological treatments, the following stock
solutions were made and stored at -80°C: 100 mM
4-(Diethylamino)-benzaldehyde (DEAB; Sigma) in DMSO and 1 mM all-trans
retinoic acid (RA; Sigma) in DMSO. For embryo treatments, dilutions of these
chemicals were made in embryo medium as follows: DEAB, 10 µM; and RA, 10,
20 and 1000 nM. Prior to gastrulation (30% epiboly or 4.7 hpf) embryos were
removed from their chorions and transferred into Petri dishes containing the
treatment solution. Treatments with a teratogenic dose of RA were carried out
as described previously (Kudoh et al.,
2002); dechorionated embryos at 40% epiboly stage were treated
with 1 µM RA for 80 minutes followed by thorough rinses with embryo medium.
For control treatments, sibling embryos were incubated in corresponding
dilutions of DMSO. All incubations were conducted in the dark.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Phillips et al., 2001). To
study the role of RA signaling in otic development, we analyzed the expression
of several otic markers at the vesicle, placodal and preplacodal stages. To
interfere with RA signaling, we used the pharmacological inhibitor
4-(Diethylamino)-benzaldehyde (DEAB), a potent retinaldehyde dehydrogenase
inhibitor (Russo et al.,
1988). Compared with the morphology of controls at 50 hpf, loss of
RA signaling led to strongly reduced otic vesicles with only one small otolith
and impaired semicircular canal protrusions, whereas excess RA at a
concentration of 10 nM led to the formation of enlarged otic vesicles
containing three otoliths and multiple semicircular canal protrusions
(Fig. 1A-C). Assessed at 22 hpf
by expression of the otic marker starmaker (stm), which was
expressed throughout the entire epithelium of control otic vesicles, otic
vesicle size was reduced by up to 50% in RA-signaling-depleted embryos, but
increased up to 40% in the presence of 10 nM RA
(Fig. 1D-F). At the 12-somite
stage, when the placode was outlined by cldna expression in control
embryos, the otic placode was reduced in size in RA-signaling-depleted
embryos, whereas excess RA led to a size increase
(Fig. 1G-I). Use of
pax8 expression, which is initiated in the otic anlagen prior to the
formation of the placode, showed that the opposite phenotypes generated by
excess or deficient RA signaling are already evident at preotic stages. In
RA-signaling-depleted embryos, the preotic expression of pax8 is
reduced, whereas, in RA-treated embryos, the preotic pax8 expression
domain is enlarged and weak expression of pax8 surrounding the entire
anterior neural plate can be detected (Fig.
1J-L). An increase of RA to 20 nM amplifies the pax8
expression surrounding the anterior neural plate
(Fig. 2A), consistent with
results published previously (Phillips et
al., 2001). However, in contrast to Phillips et al., who reported
that 20-30% of embryos treated with teratogenic doses (1 µM) of RA produce
ectopic otic vesicles at the anterior limit of the head, we observed only
randomly distributed small patches of ectopic otic cells in embryos treated
with 20 nM RA (Fig. 2B-D). To
demonstrate that our RA treatment did not posteriorize the anterior neural
plate in the same manner as typical teratogenic doses of RA
(Phillips et al., 2001;
Kudoh et al., 2002), we
treated embryos with either 20 nM RA or a teratogenic dose of RA and compared
them to controls (see Fig. S1 in the supplementary material). Subsequent
marker analysis of the anteroposterior axis showed that teratogenic doses, but
not 20 nM, of RA significantly altered the expression of early
anteroposterior-axis-specific genes. In comparison to controls, expressions of
six3a and otx2 was unaffected by 20 nM RA but are completely
abolished in the presence of a teratogenic dose (see Fig. S1A-F in the
supplementary material). Expression of the RA-controlled gene hoxb1b
(Alexandre et al., 1996) in the
caudal hindbrain of control embryos was mildly expanded in embryos treated
with 20 nM RA, but it was strongly misexpressed throughout the entire neural
plate of embryos treated with a teratogenic dose of RA (see Fig. S1G-I in the
supplementary material). In embryos treated with 20 nM RA, expression of
gbx1 in the rostral hindbrain was indistinguishable from control
embryos, whereas it was misexpressed in the anterior neural plate of embryos
treated with a teratogenic dose of RA (see Fig. S1J-L in the supplementary
material). Together, our results show that the opposite phenotypes generated
by gain or loss of RA signaling can be traced back to otic induction and that
ectopic otic induction occurs with low doses of RA that do not apparently
affect other aspects of embryonic development.
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; Maroon et al.,
2002; Léger and Brand,
2002). At 95% epiboly, fgf3 was expressed strongly in the
r4 primordium of control embryos, but its expression was severely reduced in
embryos depleted of RA signaling (Fig.
3A,B). Because the complete loss of RA signaling led to a loss of
r5-r7 accompanied by an expansion of r3-r4, we consistently observed enlarged
fgf3 and fgf8 expression domains in DEAB-treated embryos at
the three-somite stage, although the level of fgf3 expression
appeared to still be reduced in comparison with control embryos
(Fig. 3D,E,G,H). By contrast,
expression of both fgf3 and fgf8 was completely unaffected
in embryos treated with 10 or 20 nM RA
(Fig. 3C,F,I and data not
shown). We and others recently proposed that Foxi1 and Dlx3b provide
competence for cells to form the inner ear
(Hans et al., 2004;
Solomon et al., 2004). In
comparison to control embryos, foxi1 expression was unchanged in the
absence of RA signaling, whereas in embryos treated with 20 nM RA,
foxi1 was misexpressed in a stripe surrounding the anterior neural
plate (Fig. 3J-L). By contrast,
we observed no change in dlx3b expression after the complete loss or
ectopic activation of RA signaling (Fig.
3M-O). These results show that the complete loss of RA signaling
impairs early fgf3 expression in the developing hindbrain, which
cannot be compensated for by expanded hindbrain-specific domains of
fgf3 and fgf8 expression at later stages. Excess RA
signaling, on the other hand, causes an expansion of foxi1 expression
throughout the entire preplacodal ectoderm.
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;
Kudoh et al., 2002).
Subsequently, other placode-derived structures might be affected. To examine
this possibility, we tested molecular markers for the formation of anterior
pituitary, olfactory, lens, trigeminal, lateral line and epibranchial
placodes. Except for the lens, all of these structures formed in embryos
treated with 20 nM RA (data not shown). Compared with controls at 50 hpf,
embryos treated with 10 nM RA developed a much smaller lens and embryos
treated with 20 nM RA completely failed to show any morphological sign of a
lens (Fig. 4A-C). Consistent
with this, expression of connexin 44.1, which is expressed only in
the ocular lens (Cason et al.,
2001), was changed. In comparison to controls at 24 hpf, embryos
treated with 10 or 20 nM RA showed severe downregulation or no expression of
connexin 44.1, respectively (Fig.
4D-F). Labeling for pitx3, which is expressed in the
ventral and posterior diencephalon, in the lens and in the pituitary placode
at 24 hpf in wild-type embryos (Dutta et
al., 2005), corroborated our results, showing expression changes,
after RA treatment, specifically in the lens placode without affecting other
expression domains (data not shown). At early segmentation stages,
pitx3 expression defines an equivalence domain for the lens and
anterior pituitary placodes (Dutta et al.,
2005), and we found that its expression was unaffected in the
presence of 10 or 20 nM RA (data not shown). By contrast, expression of
pax6b, a specific lens placode marker
(Thisse et al., 2001), was
downregulated or almost absent in embryos treated with 10 or 20 nM RA,
respectively, in comparison to control embryos
(Fig. 4G-I). Together, our
results show that increasing amounts of RA impair lens placode formation,
which subsequently leads to a loss of the lens.
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).
Misexpression of fgf8 at late gastrulation stages led to ectopic otic
induction and caused expanded marker expression, such as that of pax8,
pax2a and sox9a, within the preotic region in comparison to
non-transgenic siblings (Fig.
6A,B) (Hans et al.,
2007). Subsequently, many more cells embarked on the otic
development pathway and much larger otic vesicles formed
(Fig. 6E,F)
(Hans et al., 2007). By
contrast, we also observed an expanded pax8 expression domain after
misexpression of fgf8 at late gastrulation stages in embryos depleted
of RA signaling, but the subsequent formation of enlarged otic vesicles was
impaired (Fig. 6C,D,G,H). In
embryos depleted of RA signaling, otic vesicles were only slightly bigger
after ectopic activation of Fgf signaling at late gastrulation stages than in
non-transgenic siblings depleted of RA signaling, and they were still much
smaller than in untreated controls. We also frequently observed small ectopic
otic vesicles located anteriorly and posteriorly to the endogenous otic
vesicle after misexpression of fgf8 at late gastrulation stages in
RA-depleted embryos (data not shown), but, in general, otic specification was
always reduced in comparison to control embryos. Taken together, our results
demonstrate that, in the absence of RA signaling, cells in the preotic region
can undergo ectopic otic induction, similar to control embryos, after the
ectopic activation of Fgf signaling at late gastrulation stages. However, otic
fate cannot be properly maintained in the absence of RA signaling.
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; Hernandez
et al., 2004). In strong tcf2 mutant alleles, the r5-r6
region of the developing hindbrain is lost and partially transformed into an
r4 identity (Hernandez et al.,
2004). Although otic induction is unaffected at early stages in
these mutants (Sun and Hopkins,
2001), otic vesicles are reduced in size at later stages
indicating that maintenance of otic fate is compromised
(Fig. 7A,D)
(Sun and Hopkins, 2001). In
comparison to either wild type or tcf2 or fgf3 single
mutants, combined loss of tcf2 and fgf3 resulted in otic
vesicles that formed only one otolith (n=10 out of 10
fgf3;tcf2 double mutants), similar to RA-depleted embryos
(Fig. 7A-E). Semicircular canal
formation, however, which was severely impaired in RA-signaling-depleted
embryos, was less severely affected in fgf3;tcf2 double mutants,
indicating that, similar to mouse (Romand,
2003), RA signaling is involved in pattern formation of the
zebrafish otic vesicle. Recent studies in mouse have shown that Wnt signaling
is required early for the maintenance of otic fate and later in the otic
vesicle for expression of dlx5 and for the acquisition of a dorsal
fate (Ohyama et al., 2006;
Riccomagno et al., 2005). We
found that expression of dlx3b in the dorsal portion of the otic
vesicle was completely lost in RA-depleted embryos at 22 hpf
(Fig. 7F,G), suggesting that
Wnt signaling may be affected. Wnt8b is a likely candidate because it is
expressed strongly in r1, r3 and r5 from mid-somitogenesis onwards
(Kelly et al., 1995). Compared
to controls, both tcf2 mutants and RA-depleted embryos showed a loss
of wnt8b expression specifically in r5
(Fig. 7H-J). To test whether
wnt8b acts to maintain otic fate, we compromised Wnt8b function using
morpholino injection into wild type. Furthermore, we compromised Wnt8b
function in fgf3 mutants to test whether loss of fgf3 and
wnt8b together mimics the otic vesicle size reduction of
RA-signaling-depleted embryos, which have defects in fgf3 and
wnt8b expression (Fig.
7K-O). Loss of Fgf3 or Wnt8b function alone led to the formation
of smaller otic vesicles, but the size reduction was not as profound as in
embryos depleted of RA signaling (Fig.
7K-N). However, compromised Wnt8b function in fgf3
mutants showed an additive effect, producing smaller otic vesicles similar to
vesicles in RA-depleted embryos (n=14 out of 18)
(Fig. 7L,O). Depletion of RA
signaling and Wnt8b function together resulted in a slight further reduction
in otic vesicle size (data not shown). However, because of the expansion of
r3-r4, otic vesicles were located closer to r3 in RA-depleted embryos (data
not shown), and expression of wnt8b in r3 might partly compensate for
the loss of wnt8b from r5 in these embryos. Together, our results
demonstrate that the hindbrain-derived factor Wnt8b from r5 maintains otic
fate, and that compromised Fgf3 expression and loss of Wnt8b function together
mimics the size reduction of the otic vesicle observed in RA-depleted
embryos. |
DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Kudoh et al., 2002).
Furthermore, teratogenic doses of RA completely transform forebrain and
midbrain into hindbrain and enlarge the preotic expression domain of
pax8 (Phillips et al.,
2001). Our results demonstrate that much lower concentrations of
RA that do not interfere with the patterning of the anterior neural plate are
sufficient to produce ectopic expression of foxi1, which expands the
domain of preotic pax8 expression. Consistent with recent reports
showing that preotic pax8 expression depends on Foxi1
(Solomon et al., 2003;
Nissen et al., 2003), we found
that the enlarged pax8 expression observed in 20 nM RA-treated
wild-type embryos was severely reduced to a small, residual domain of
expression at the anterior end in RA-treated foxi1 mutants
(Hans et al., 2007). Fate
changes after the application of 20 nM RA were observed only in the
preplacodal domain, but not within the anterior neural plate, whereas a
teratogenic dose of RA affected both tissues. This difference is presumably
due to the presence of RA-degrading enzymes of the Cyp26 class that are
expressed within the anterior neural plate but not in the preplacodal domain
(Dobbs-McAuliffe et al., 2004;
Kudoh et al., 2002;
Hernandez et al., 2006).
Consistent with this interpretation, a recent study has shown that 5 nM RA is
sufficient to posteriorize cyp26a1 mutants, whereas 200 nM RA is required to
obtain the same effect on wild-type embryos
(Hernandez et al., 2006).
Our observation that excess RA leads to ectopic expression of foxi1
throughout the entire preplacodal domain provides a consistent explanation for
the findings of Woo and Fraser (Woo and
Fraser, 1997). Transplantation of ventral and lateral germring
tissue into the prospective forebrain region can induce ectopic otic vesicles,
whereas the transplantation of dorsal germring tissue (the embryonic shield)
does not. Although germring grafts also lead to the induction of ectopic
hindbrain tissue, the direct transplantation of hindbrain into the prospective
forebrain region never induces ectopic otic vesicles
(Woo and Fraser, 1997). RA is
generated by the enzymatic activity of aldehyde dehydrogenases, which are
encoded by Aldh genes, and, during zebrafish gastrulation, aldh1a2 is
expressed in the ventral and lateral germring but not dorsally in the
embryonic shield (Begemann et al.,
2001; Grandel et al.,
2002). Thus, we propose that RA produced by the grafted ventral
and lateral germring increases the competence of anterior preplacodal cells to
respond to otic-inducing signals because of ectopic activation of
foxi1. The observed ectopic otic induction might also benefit from
ectopic Fgf signaling provided by the graft, because fgf3, fgf8,
fgf17b and fgf24 are all expressed in a dorsoventral gradient in
the zebrafish germ ring, with highest levels on the dorsal side
(Fürthauer et al., 1997;
Fürthauer et al., 2004;
Reifers et al., 1998;
Cao et al., 2004).
Although RA is sufficient to induce foxi1 expression, our data
also show that loss of RA signaling during zebrafish gastrulation has no
effect on the expression of foxi1. Thus, impaired otic induction in
RA-depleted embryos is not due to an effect on Foxi1 level and, hence, we
conclude that RA signaling is not required for the early events of otic
induction. The observed effect of excess RA on foxi1 expression might
rather reveal a regulatory mechanism employed during other aspects of
development. In zebrafish at 48 hpf, foxi1 is expressed in the
retina, in the pharyngeal pouches and in the dorsal septum of the otic vesicle
(Thisse et al., 2005), and RA
signaling has been implicated in the development of all of these tissues
(Drager et al., 2001;
Mark et al., 2004;
Romand, 2003). It is currently
unknown, however, whether foxi1 expression depends on RA signaling in
these tissues.
Our results further indicate that early fgf3 expression is crucial
during the relatively short time-window for otic induction in zebrafish.
Impaired fgf3 expression in the developing hindbrain of
RA-signaling-depleted embryos delayed otic induction and could not be
compensated for by an expanded hindbrain domain of fgf3 and
fgf8 expression at later stages. Consistently, depletion of
fgf3 by morpholino injection severely reduces pax8
expression at the tailbud stage (Phillips
et al., 2001; Léger and
Brand, 2002). Based on experiments with a stable transgenic line
that expresses fgf8 uniformly under the control of the zebrafish
temperature-inducible hsp70 promoter, we recently proposed that otic
induction in zebrafish occurs between the end of gastrulation and the early
segmentation stages (Hans et al.,
2007). Using the same approach here, we show that even in the
absence of RA signaling, cells in the preotic region can undergo ectopic otic
induction after ectopic activation of Fgf signaling at late gastrulation
stages, similar to control embryos. Thus, cells are still competent to adopt
otic fate after exposure to a high uniform dose of Fgf8 but are unable to
respond to the endogenous Fgf3 and Fgf8 emanating from the developing
hindbrain after the loss of RA signaling. Incorrect spatial positioning of
inducing and induced tissue might also contribute to impaired otic induction,
because, in RA-signaling-depleted embryos, the r3 primordium is greatly
expanded, displacing the inducing r4 from the preotic region.
Wnt signaling during otic induction
Our analysis further demonstrates that defects in otic specification caused
by loss of RA signaling are a secondary consequence of changes in hindbrain
patterning. We found that embryos depleted of RA signaling formed small otic
vesicles because of compromised otic induction due to decreased fgf3
expression and because of compromised maintenance of otic fate due to loss of
wnt8b expression. Reduced otic induction due to weaker expression of
Fgf3 has been proposed from work in both mammals and zebrafish
(Niederreither et al., 1999;
Whitfield et al., 2002;
Phillips et al., 2001;
Léger and Brand, 2002),
and a recent study in mouse shows that Wnt signaling is required after
Fgf-dependent induction to maintain otic fate
(Ohyama et al., 2006). In the
latter study, Wnt signaling was detected using a Tcf/Lef reporter construct
within the preotic region in a subset of Pax2-positive cells after
Fgf-dependent induction but prior to the formation of the placode.
Furthermore, disruption of the canonical Wnt signaling pathway in preotic
Pax2-positive cells leads to an expansion of epidermal fate at the expense of
otic fate, and otic vesicles are significantly reduced in size
(Ohyama et al., 2006).
Conversely, constitutive activation of canonical Wnt signaling in preotic
Pax2-positive cells leads to the expansion of otic fate at the expense of
epidermal fate, suggesting that Wnt signaling mediates a placode-epidermis
fate decision by directing preotic cells to an otic fate
(Ohyama et al., 2006). So far,
it is unknown whether Wnt signaling plays the same role in zebrafish. It has
been reported that, in zebrafish, the Wnt reporter gene TOPdGFP, a
transgene consisting of a GFP-coding sequence downstream of a minimal promoter
and four Lef-binding sites, is not expressed during early stages of otic
induction (Phillips et al.,
2004), but nothing is known about its expression later in
development, prior to the formation of the otic placode. Consistent with a
similar role for Wnt signaling, we found that, in RA-depleted embryos, otic
induction can be rescued but not maintained. Furthermore, we have previously
found that expression of the endogenous Wnt reporter gene axin2 is
absent during the initial stages of otic induction but that axin2 is
expressed within the preotic region prior to the formation of the otic region
(our unpublished results). Cell-autonomous disruption or constitutive
activation of the canonical Wnt signaling pathway in mouse by conditional
knockout of ß-catenin or by the expression of a conditionally activated
form of ß-catenin within preotic Pax2-positive cells led Ohyama et al. to
propose that Wnt8a from r4 is the source of Wnt signaling
(Ohyama et al., 2006). Our
data, on the other hand, indicate that wnt8b expression in r5 is the
source, consistent with findings that Wnt8b signaling is strongly deficient in
the posterior hindbrain of mafb mutants and that inactivation of
Wnt8b by morpholino injection results in smaller otic vesicles that mimic the
size and patterning defects of mafb mutants (M. Brand, personal
communication). In zebrafish, wnt8a encodes a bicistronic message
encoding two complete open-reading frames, ORF1 and ORF2, and, at 75% epiboly
(8 hpf), ORF2 can be detected in r5-r6 adjacent to the otic anlagen
(Kelly et al., 1995;
Lekven et al., 2001). Loss of
Wnt8a function by morpholino injection results in greatly reduced otocysts
(Phillips et al., 2004),
supporting the model proposed by Ohyama et al.
(Ohyama et al., 2006).
However, proper expression of fgf3 and fgf8 is disturbed in
the hindbrain of these embryos, thus compromising otic induction
(Phillips et al., 2004) and
making it difficult to address later Wnt8a function during otic development.
It is very likely that several Wnt molecules are involved in otic fate
maintenance after Fgf-dependent induction. Expression of wnt1, wnt3a
and wnt10b has been reported in the zebrafish hindbrain in addition
to wnt8a and wnt8b, and, interestingly, expression of
wnt1 and wnt3a is severely reduced in the caudal hindbrain
of tcf2 mutants (Lekven et al.,
2003; Buckles et al.,
2004; Lecaudey et al.,
2006), as we have shown for wnt8b. Conditional and
combinatorial knockout will be necessary to address the role of particular Wnt
molecules in otic fate maintenance.
Absent or inappropriate amounts of Wnt signaling might also be responsible
for the reduced number of otic cells in RA-treated embryos at later stages.
Our treatment of embryos with 20 nM RA led to the massive ectopic expression
of pax8 around the anterior neural plate border, similar to the
results published by Phillips et al.
(Phillips et al., 2001).
However, in contrast to Phillips et al., who reported that 20-30% of
RA-treated embryos produce ectopic morphologically visible otic vesicles at
the anterior limit of the head, we observed only randomly distributed small
patches of ectopic otic cells. Phillips et al. showed that the anterior neural
plate is strongly posteriorized after treatment with a teratogenic dose of RA,
which leads to the anterior expansion of hindbrain fgf3 and
fgf8 expression domains. Wnt signaling is presumably also shifted
anteriorly in these RA-treated embryos, maintaining the fate of the ectopic
otic cells. In wild-type embryos, however, Wnt signaling is absent in the
early neural plate anterior to the future midbrain-hindbrain boundary
(Wilson and Houart, 2004).
Because our conditions to generate excess RA signaling (20 nM) did not
apparently change anterior neural plate fate, pax8-positive cells
anterior to the future midbrain-hindbrain boundary presumably do not receive
Wnt signaling, which is essential for the maintenance of otic fates after
induction. Furthermore, pathways to generate other placode-derived sensory
organs were presumably intact under our experiments, which might also
contribute to fewer ectopic otic cells in RA-treated embryos at later
stages.
Ectopic RA signaling and lens placode induction
We found that the application of 20 nM RA led to the expression of
foxi1 within the entire preplacodal domain. However, we did not
observe any expansion of the otic tissue at the expense of other sensory
organs, except for the lens. Interestingly, it was recently shown that the
entire preplacodal domain is initially specified as lens tissue, implying that
lens fate is a default state of the preplacodal territory and that subsequent
development requires repression of lens fate in prospective non-lens domains
(Bailey et al., 2006). By
promoting otic fate in response to Fgf signaling, Foxi1 in the preotic region
of wild-type embryos and expanded Foxi1 expression in RA-treated embryos might
be involved in lens fate repression. Consistent with this interpretation,
microarray analysis covering approximately 20,000 zebrafish genes showed that
only seven genes, including pax6b, are significantly repressed after
overexpression of Foxi1 (Yan et al.,
2006). However, it is also likely that the reduction or loss of
lens tissue is not due to misexpression of foxi1 or to a
posteriorization of the preplacodal domain, but rather due to the ectopic
expression of pax8. Reciprocal inhibition between the paired-domain
proteins Pax2 and Pax6 has been implicated in the specification of mammalian
eye territories and in formation of the di-mesencephalic boundary
(Schwarz et al., 2000;
Nakamura and Watanabe, 2005).
Other studies have shown that both Pax2 and Pax6 can be converted from
transcriptional activators to repressors via interaction with co-repressors of
the Groucho protein family (Eberhard et
al., 2000). Pax2 is highly related to Pax5 and Pax8, and gene
replacement in the mouse has shown that Pax5 can functionally
substitute for Pax2, indicating that Pax2, Pax5 and Pax8 proteins are
biochemically interchangeable (Pfeffer et
al., 1998; Bouchard et al.,
2000). Combined and redundant gene function has also been shown
for Pax2 and Pax8 during development of the mouse urogenital system and the
zebrafish inner ear (Bouchard et al.,
2002; Hans et al.,
2004; Mackereth et al.,
2005). Further investigation of the roles of Foxi1 and Pax2 or
Pax8 in otic induction and in the repression of other fates will help clarify
the functional similarities and differences among these factors.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/134/13/2449/DC1
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ACKNOWLEDGMENTS |
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