RESEARCH ARTICLE 3977
Development 138, 3977-3987 (2011) doi:10.1242/dev.066639
© 2011. Published by The Company of Biologists Ltd
Fgf and Hh signalling act on a symmetrical pre-pattern to
specify anterior and posterior identity in the zebrafish otic
placode and vesicle
Katherine L. Hammond and Tanya T. Whitfield*
SUMMARY
Specification of the otic anteroposterior axis is one of the earliest patterning events during inner ear development. In zebrafish,
Hedgehog signalling is necessary and sufficient to specify posterior otic identity between the 10 somite (otic placode) and 20
somite (early otic vesicle) stages. We now show that Fgf signalling is both necessary and sufficient for anterior otic specification
during a similar period, a function that is completely separable from its earlier role in otic placode induction. In lia–/– (fgf3–/–)
mutants, anterior otic character is reduced, but not lost altogether. Blocking all Fgf signalling at 10-20 somites, however, using
the pan-Fgf inhibitor SU5402, results in the loss of anterior otic structures and a mirror image duplication of posterior regions.
Conversely, overexpression of fgf3 during a similar period, using a heat-shock inducible transgenic line, results in the loss of
posterior otic structures and a duplication of anterior domains. These phenotypes are opposite to those observed when
Hedgehog signalling is altered. Loss of both Fgf and Hedgehog function between 10 and 20 somites results in symmetrical otic
vesicles with neither anterior nor posterior identity, which, nevertheless, retain defined poles at the anterior and posterior ends
of the ear. These data suggest that Fgf and Hedgehog act on a symmetrical otic pre-pattern to specify anterior and posterior otic
identity, respectively. Each signalling pathway has instructive activity: neither acts simply to repress activity of the other, and,
together, they appear to be key players in the specification of anteroposterior asymmetries in the zebrafish ear.
INTRODUCTION
Inner ears of jawed vertebrates are highly asymmetrical about all
three body axes, particularly the anteroposterior (AP) axis, along
which the main vestibular and auditory receptors, the sensory
maculae, are arrayed. Asymmetrical expression of marker genes
about the AP axis arises early, and is evident at placode stages in
both fish and amniotes. Hmx3, for example, is expressed in a
distinct anterior otic domain from as early as 16 hours post
fertilisation (hpf) (14 somite stage) in zebrafish and E8.5 in the
mouse (Hadrys et al., 1998; Adamska et al., 2000). Rotation and
ablation experiments have shown that induction from surrounding
tissues is important in establishing these asymmetries, and that at
least two factors must be required to specify AP otic identity
(Harrison, 1936; Wu et al., 1998; Waldman et al., 2007; Liang et
al., 2010).
We have previously shown that Hedgehog (Hh) signalling from
the notochord and floorplate is necessary and sufficient to specify
posterior domains in the zebrafish ear: severely reduced or
increased Hh signalling results in mirror image duplications of the
anterior or posterior halves of the otic vesicle, respectively
(Hammond et al., 2003; Hammond et al., 2010). Similar doubleanterior ears have been generated in the Xenopus embryo by
overexpression of the Hh inhibitor Hip (Waldman et al., 2007). Hh
signalling also appears to be required for the correct development
of hair cells and innervation of the zebrafish posterior (saccular)
macula (Sapède and Pujades, 2010).
MRC Centre for Developmental and Biomedical Genetics and Department of
Biomedical Science, University of Sheffield, Sheffield S10 2TN, UK.
*Author for correspondence ([email protected])
Accepted 1 July 2011
A good candidate for an anterior otic specification factor is
Fibroblast growth factor (Fgf). In the zebrafish val–/– (mafba–/–)
mutant, fgf3 expression, which is normally restricted to
rhombomere (r) 4 of the hindbrain, expands posteriorly into r5 and
r6. Concomitantly, expression of anterior markers expands
posteriorly in the otic vesicle, whereas posterior markers are
reduced (Kwak et al., 2002). Importantly, the otic phenotype can
be rescued by injection of an fgf3 morpholino, which also reduces
expression of anterior otic markers in wild-type embryos (Kwak et
al., 2002). A more severe gain of anterior otic character is seen in
hnf1ba–/– (vhnf1–/–) homozygotes, which also have expanded fgf3
expression in the hindbrain (Lecaudey et al., 2007). Previous
reports also suggest that anterior otic character is disrupted in lia–/–
(fgf3–/–) otic vesicles (Herzog et al., 2004; Kwak et al., 2006),
which we have analysed in detail here. Consistent with an anterior
otic specification role for Fgfs, Fgf receptors are expressed widely
in the head, including in the otic epithelium, from placode stages
(Thisse and Thisse, 2005; Thisse et al., 2008; Esterberg and Fritz,
2009; Rohner et al., 2009). Moreover, expression of pea3, spry4
and etv5b (erm), genes that are direct targets of Fgf signalling, is
concentrated in anterior parts of the otic placode and vesicle
(Raible and Brand, 2001; Roehl and Nüsslein-Volhard, 2001;
Thisse et al., 2001; Nechiporuk et al., 2005).
Confirmation that Fgfs act as otic anteriorising factors, however,
is not straightforward. The situation is complicated by their earlier
essential roles in otic placode induction (Phillips et al., 2001; Léger
and Brand, 2002; Maroon et al., 2002; Liu et al., 2003), which are
conserved across different vertebrate species (for reviews, see
Ohyama et al., 2007; Schimmang, 2007; Ladher et al., 2010). Here,
using conditional approaches to manipulate Fgf signalling after otic
placode induction is complete, we demonstrate that Fgf signalling
is necessary and sufficient to specify anterior otic identity in the
DEVELOPMENT
KEY WORDS: Fgf, Hh, Axial patterning, Otic vesicle, Zebrafish
3978 RESEARCH ARTICLE
zebrafish. The critical time window for this anteriorising function
is between the 10 and 20 somite stages, a time at which Hh is also
acting to posteriorise the ear. Using pea3 and ptc1 (ptch2 –
Zebrafish Information Network) expression as readouts of Fgf and
Hh activity, respectively, we show that neither factor affects
transduction of the other. In addition, when we inhibit Fgf
signalling in a Hh loss-of-function background, we see an additive
phenotype: both anterior and posterior otic identity are lost.
Nevertheless, initial sensory differentiation proceeds, but in a
pattern that is symmetrical about the AP axis. This suggests that
both Fgf and Hh have independent, instructive activity, imparting
anterior and posterior identity to the poles of the otic placode. Our
data support a model in which Hh and Fgf act on an AP
symmetrical ‘pre-pattern’ to trigger the development of AP
asymmetries in the zebrafish ear.
MATERIALS AND METHODS
Development 138 (18)
RESULTS
lia–/– (fgf3–/–) otic vesicles display a partial loss of
anterior otic character
Fgf3 has a redundant role in zebrafish otic placode induction
(Phillips et al., 2001; Léger and Brand, 2002; Maroon et al., 2002).
Previous studies have also suggested a role in later otic
development (Kwak et al., 2002; Herzog et al., 2004; Kwak et al.,
2006). To clarify the role of fgf3 in otic patterning, we characterised
the otic phenotype of the fgf3 mutant lim absent (liat21142–/–), which
is known to have otic defects (Herzog et al., 2004). The threedimensional structure of lia–/– otic vesicles was grossly normal at
3 days post-fertilisation (dpf): there were no obvious defects in
semicircular canal pillar or crista development (Fig. 1A-G,J).
However, the anterior otolith was fused to the posterior otolith and
positioned medially, resembling the normal position of the posterior
otolith (Fig. 1A-F) (Herzog et al., 2004). Staining with FITCphalloidin, to reveal the actin-rich stereociliary bundles of the
Animals
Zebrafish strains were AB (wild type), liat21142 (Herzog et al., 2004),
smob577 (Varga et al., 2001), Tg[hsp70:fgf3] (Lecaudey et al., 2008) and
ptc1–/–;ptc2–/– (Koudijs et al., 2008). Embryonic stages are given as hours
post-fertilisation (hpf) at 28.5°C or somite stages (S): 10S⬵14 hpf;
20S⬵19 hpf (Kimmel et al., 1995; Westerfield, 2000). All experiments
with animals were performed under UK Home Office licence and
conformed to UK regulatory standards.
In situ hybridisation
Whole-mount in situ hybridisation was carried out as described (Hammond
et al., 2003) using probes designed against eya1, fgf8, fst1 (fsta – Zebrafish
Information Network), hmx3, otx1, pax2a, pax5, ptc1 (Hammond et al.,
2003), fgf3 (Kwak et al., 2002), hmx2 (Feng and Xu, 2010), hoxb1 (Prince
et al., 1998), egr2b (krox20) (Oxtoby and Jowett, 1993), neurod (Blader et
al., 1997), pea3 (Münchberg et al., 1999) and mafba (Moens et al.,
1998).
Genotyping
ptc1–/–;ptc2–/–, 20S+ smo–/– and 72 hpf+ lia–/– embryos were identified
morphologically. Other mutant embryos were genotyped. Genomic DNA
was prepared as described (Westerfield, 2000). liat21142 primers were: 5⬘TGTCCAGTCATGAATGTCAAAG-3⬘ and 5⬘-CCATCTCATGGTCCTTGTTG-3⬘. The resulting 320 bp fragment was digested with NsiI,
producing 40 bp, 82 bp and 198 bp fragments from wild-type DNA and 40
bp and 280 bp fragments from liat21142 DNA. smob577 primers were: 5⬘CTATACTGGCCAATTCACAG-3⬘ and 5⬘-ATGGAAAACAATGTCATAACC-3⬘. The resulting 325 bp PCR band was sequenced: wild-type
DNA has a G at position 160, whereas smob577 DNA has a T.
FITC-phalloidin and anti-acetylated tubulin antibody staining
Staining was carried out as described (Haddon and Lewis, 1996).
hsp70:fgf3 transgenic and sibling embryos from a hsp70:fgf3/+ ⫻
hsp70:fgf3/+ cross were incubated at 39°C for 2 hours. Transgenic and
sibling embryos were distinguished morphologically. Control embryos
were kept at 28.5°C.
SU5402 treatment
Embryos were treated with 10 mM SU5402 (Merck) in 0.5% DMSO or
with 0.5% DMSO alone in embryo medium from 10S and were washed
after 3, 5 or 7 hours.
Microscopy
Microscopy was carried out as described (Hammond et al., 2003).
Acridine Orange staining
Embryos were treated with 5 mg/ml Acridine Orange in embryo medium
from 38.5 hpf for one hour, washed in embryo medium and imaged at 40
hpf.
Fig. 1. Anterior otic character is reduced in lia–/– (fgf3–/–)
homozygotes. (A-F)Live 72 hpf lia–/– and sibling (sib) zebrafish inner
ears. (G-L)Confocal z-stacks of 84 hpf ears stained with FITC-phalloidin
to mark sensory hair cells. (M)The anterior macula of a 5 dpf lia–/–
embryo stained with anti-acetylated tubulin antibody (kinocilia; red)
and FITC-phalloidin (stereocilia; green). (N,O)Typical polarity maps for
wild-type maculae. (P)Hair cell polarity map obtained from the
specimen shown in M. (Q,R)Polarity maps from two further lia–/–
specimens. A,B,D,E,G,H,J,K: Lateral views; anterior to left, dorsal to top.
A,D,G,J: Lateral focal plane. B,E,H,K: Medial focal plane. C,F,I,L: Dorsal
views; anterior to left, medial to top. am, anterior macula; ao, anterior
otolith; c, cristae; pm, posterior macula; po, posterior otolith. Asterisks
indicate semicircular canal pillars. Scale bars: 50mm.
DEVELOPMENT
Heat shock
sensory hair cells, showed that the underlying anterior macula was
positioned more medially than normal, abutting the posterior
macula (Fig. 1H,I,K,L).
The phenotype of the lia–/– anterior macula was variable, ranging
from nearly normal in size and shape, to reduced in size and
resembling the posterior macula in shape (Fig. 1M-R). However,
analysis of hair cell polarity patterns, by double staining for tubulin
in the hair cell kinocilia and actin in the stereociliary bundles,
suggested that the anterior macula retained anterior character: hair
cell polarities radiated in a fan-like pattern, resembling the normal
anterior macula, rather than pointing away from a central midline
as in wild-type posterior maculae (Fig. 1M-R).
This suggested that some, but not all, anterior otic character was
lost in lia–/– embryos. To confirm this, we performed in situ
hybridisation to lia–/– and sibling embryos with a panel of anteriorand posterior-restricted otic markers (Fig. 2). Expression of pax5,
hmx2, hmx3 and fgf3 itself, which were strongly expressed at the
anterior of the otic vesicle in phenotypically wild-type sibling
embryos at 26 hpf, were all severely reduced in lia–/– embryos (Fig.
2A-H). However, expression of neurod, which normally marks
delaminating neuroblasts and nascent neurons of the statoacoustic
ganglion in an anteroventral domain beneath the otic vesicle, was not
significantly reduced (data not shown). Expression of two posterior
markers, fst1 and otx1, was unaltered, suggesting no gain of posterior
character at the anterior of the otic vesicle (Fig. 2I-L). These data
suggest that Fgf3 is required to specify anterior otic domains, but that
loss of Fgf3 alone is not sufficient to re-specify anterior as posterior.
Fig. 2. Anterior otic markers are reduced in lia–/– homozygotes;
posterior markers are expressed as normal. (A-H)In situ
hybridisation to anterior otic markers. (I-L)In situ hybridisation to
posterior otic markers (arrows and brackets highlight the posterior otic
domains). A-H,K,L: Dorsal views; anterior to left, medial to top. I,J:
Lateral views; anterior to left, dorsal to top. Scale bars: 50mm.
RESEARCH ARTICLE 3979
Inhibition of all Fgf signalling between the 10 and
20 somite stages leads to a complete loss of
anterior otic character and a mirror image
duplication of posterior domains
Although the liat21142 allele is thought to be a null or very strong
loss-of-function allele (Herzog et al., 2004), the ears of liat21142–/–
homozygotes retain some anterior character. Another factor must,
therefore, be involved in specification of anterior otic identity. To
Fig. 3. Treatment with SU5402 from 10 to 20S leads to a
complete loss of anterior otic structures and a mirror image
duplication of posterior otic structures. (A-H)Live ears of 84 hpf
zebrafish embryos treated with 10mM SU5402 and DMSO-treated
controls. (I-P)Confocal z-stacks of 84 hpf SU5402-treated and control
ears stained with FITC-phalloidin to mark sensory hair cells. (I-L)Medial
focal planes showing the maculae. (M-P)Lateral focal planes showing
the cristae. (Q)The macula of an 86 hpf SU5402-treated embryo
stained with anti-acetylated tubulin antibody (kinocilia; red) and FITCphalloidin (stereocilia; green). (R)Hair cell polarity map obtained from
the macula shown in Q. (S)Polarity map obtained from a further
SU5402-treated specimen. (T-W)In situ hybridisation to the anterior
otic markers pax5 (T,U) and hmx2 (V,W). (X-Z⬘) In situ hybridisation to
the posterior otic markers fst1 (X,Y) and otx1 (Z,Z⬘). (A⬘,B⬘) In situ
hybridisation to neurod, which marks the statoacoustic ganglion.
(C⬘,D⬘) In situ hybridisation to atoh1a, which marks the first hair cells.
A-P,X,Y: Lateral views; anterior to left, dorsal to top. T-W,Z-D⬘: Dorsal
views; anterior to left, medial to top. am, anterior macula; ao, anterior
otolith; c, cristae; pm, posterior macula; po, posterior otolith. Asterisks
in A indicate semicircular canal pillars; arrow in C indicates reduced
lateral semicircular canal pillar. Dots in Z-D⬘ delineate the otic vesicle.
Scale bars: 50mm.
DEVELOPMENT
Axial patterning in the fish ear
3980 RESEARCH ARTICLE
Development 138 (18)
Table 1. Heat shock of an hsp70:fgf3 line from 9 to 10S causes a loss of posterior otic domains and a mirror image duplication of
anterior regions
Heat-shocked from:
Wild type
Duplicated separate patches
Duplicated fused patches
Total
4 somites
9-10 somites
12-13 somites
15-16 somites
sib
hsfgf3
sib
hsfgf3
sib
hsfgf3
sib
hsfgf3
sib
21
–
–
21
7
–
–
7
16
–
–
16
6
–
–
6
20
50
70
14
–
–
14
18
13
–
31
11
–
–
11
24
–
–
24
10
–
–
10
test whether this is another Fgf, we used the inhibitor SU5402,
which blocks all Fgf signalling (Mohammadi et al., 1997; Raible
and Brand, 2001). An early block of Fgf signalling using SU5402
or lia–/–;ace–/– (fgf3–/–;fgf8–/–) double mutants, however, results in
a complete absence of the otic placode, due to the essential role of
Fgfs in otic induction (Phillips et al., 2001; Léger and Brand, 2002;
Maroon et al., 2002; Liu et al., 2003). We therefore took a
conditional approach and applied 10 mM SU5402 in 0.5% DMSO
to wild-type embryos from the 10 somite stage (10S) (14 hpf) for
3, 5 or 7 hours. Longer treatments resulted in gross morphological
abnormalities and early death of embryos (data not shown). Control
embryos were treated with 0.5% DMSO for an equivalent time,
which had no effect on ear development.
Wild-type (wt) embryos treated with SU5402 for three hours
(10-16S, n6) had well-formed ears with well-developed
semicircular canal pillars. However, the otoliths were fused and
positioned medially, resembling the wild-type posterior otolith (Fig.
3B,F). Phalloidin staining revealed that the anterior macula was
absent; the otoliths sat over a single, medial, bow tie-shaped
macula (Fig. 3J). This suggested that anterior otic structures had
been lost and posterior structures duplicated in their place in a
mirror image fashion. Three cristae were present, as in untreated
embryos (Fig. 3N).
Ears of embryos treated for 5 hours [10-20S, n8 (wt), n42
(phenotypically wt smo siblings)] were similar except that the
single medial macula underlying the fused otoliths was circular
rather than bow tie-shaped, the lateral canal pillar was absent or
reduced (arrow, Fig. 3C), the lateral crista was absent and ears were
slightly reduced in size (Fig. 3C,G,K,O). This suggested a more
extreme loss of anterior character than in embryos treated with
SU5402 for 3 hours, with a mirror image duplication of posterior
structures around a more posterior axis of duplication. The single
circular macula originated as two separate domains of hair cells at
the anterior and posterior poles of the vesicle, as in wild-type
embryos (see Fig. S1 in the supplementary material; n4/4). Even
as early as 30 hpf, however, both groups of hair cells were
positioned medially, resembling those that form the posterior
macula in untreated embryos.
Ears of wild-type embryos treated for 7 hours (10-24S, n16)
were similar to those treated for 5 hours, but very reduced in size
and less well formed (Fig. 3D,H,L,P). These embryos were
generally very unhealthy, with large amounts of necrosis and
oedema. Further analysis was therefore performed on embryos
treated for 5 hours.
To confirm the loss of anterior otic character and mirror image
duplication of posterior otic domains in SU5402-treated embryos,
we mapped hair cell polarity patterns in the medial maculae as
above (Fig. 3Q-S; see Fig. S2 in the supplementary material). Hair
cells pointed away from a central midline throughout the medial
maculae of SU5402-treated embryos, indicating a duplication of
the posterior part of the posterior macula. We also performed in situ
hybridisation with a panel of otic AP-restricted markers, as above.
In SU5402-treated embryos, expression of the anterior markers
hmx2 and pax5 was absent (Fig. 3U,W), whereas expression of the
posterior marker fst1 was duplicated at the anterior of the ear (Fig.
3Y). Expression of otx1 became symmetrical: the posteroventral
part of its normal expression domain extended into anteroventral
regions of the otic vesicle, whereas the anterolateral part of its
normal expression domain was lost (Fig. 3Z⬘). Expression of
neurod was also reduced (Fig. 3A⬘,B⬘). Dorsoventral and
mediolateral axes of SU5402-treated otic vesicles did not appear to
be affected: pax2a, a medial marker, and eya1, a ventral marker,
were expressed normally (see Fig. S3 in the supplementary
material). Similarly, expression of atoh1a, a hair cell marker, was
unaltered (Fig. 3C⬘,D⬘).
Taken together, these data show that Fgf signalling is absolutely
required for specification of anterior otic domains during a short
critical time window (between 10 and 20S) that is separable from
its earlier role in otic induction. In the absence of Fgf signalling,
anterior otic domains develop as posterior.
Overexpression of fgf3 during a short critical time
window causes a loss of posterior otic domains
and a duplication of anterior otic structures in
their place
To determine whether Fgf signalling is sufficient to specify anterior
otic identity, we overexpressed fgf3 throughout the embryo using a
heat-shock inducible fgf3 (hsp70:fgf3) transgenic line (Lecaudey et
al., 2008). In this line, fgf3 expression was severely upregulated 3040 minutes after initiation of heat-shock; 4 hours later, fgf3
expression had declined to almost normal levels (data not shown)
(Lecaudey et al., 2008).
We heat-shocked hsp70:fgf3 transgenic and sibling (nontransgenic) embryos for 2 hours at tailbud stage, 4S, 9-10S, 12-13S
and 15-16S. Only heat-shocking transgenic embryos at 9-10S gave
a consistent otic phenotype, although this was shared with some
embryos in the 12-13S batch, presumably the youngest (Table 1).
Ears of other groups were largely normal. This confirmed the
existence of a critical, short time window when Fgf signalling can
pattern the AP axis of the ear.
Embryos heat-shocked from 10S lost all posterior otic character,
concomitant with a remarkably complete mirror image duplication
of anterior otic structures at the posterior of the ear. Ears of heatshocked embryos had two small lateral otoliths resembling the
wild-type anterior otolith (Fig. 4B,C), each sitting over a ventral
macula resembling the anterior macula (Fig. 4B,C,E,F,K,L). Hair
cell polarities in both ventral maculae radiated in a fan-like pattern,
resembling wild-type anterior maculae (Fig. 4P,Q; see Fig. S4 in
the supplementary material). In some specimens, two separate
anterior-like maculae were present on the ventral floor of the otic
DEVELOPMENT
Tailbud
hsfgf3
Axial patterning in the fish ear
RESEARCH ARTICLE 3981
posterior of the vesicle and sometimes expanded to encompass
the entire medial wall, whereas expression of the posterior marker
fst1 was lost from the posterior of the ear (Fig. 5A-F). Expression
of neurod was also duplicated at the posterior of the ear (Fig. 5GJ); neurod-positive cells appeared to delaminate from
posteroventral as well as anteroventral regions of the otocyst.
pax2a and eya1 were expressed in medial and ventral parts of the
otic vesicle, respectively, as normal (see Fig. S3 in the
supplementary material).
These data clearly demonstrate that Fgf signalling is sufficient
to specify anterior otic identity: excess fgf3 prevents expression of
the endogenous posterior otic specification programme and causes
anterior otic structures to form at the posterior of the ear.
Altered Fgf signalling does not affect Hh signal
transduction in the ear; altered Hh signalling does
not affect Fgf signal transduction in the ear
The mirror image duplicated anterior and posterior otic phenotypes
obtained when we alter Fgf signalling throughout the embryo are
similar to those that occur when Hh signalling is altered
(Hammond et al., 2003; Hammond et al., 2010): Fgf specifies
anterior otic identity, whereas Hh specifies posterior otic identity.
We wanted to know whether both factors are instructive, or
whether one acts purely to repress activity of the other.
vesicle (Fig. 4E,K); in others, these were fused to form a single
sensory patch (Fig. 4F,L; Table 1). There was also a remnant of the
posterior macula on the medial wall of the otic vesicle (Fig.
4E,F,K,L). In one of six phalloidin-stained specimens, there were
four cristae, presumed to represent duplicated anterior and lateral
cristae, rather than the normal three (Fig. 4H). Where only three
cristae were present (5/6 specimens), the lateral crista was
positioned centrally and was enlarged, as though the axis of
duplication transected this crista (Fig. 4I, arrow).
In situ hybridisation with a panel of otic AP markers confirmed
the loss of posterior otic identity and duplication of anterior otic
regions in heat-shocked embryos. The major, posteroventral, part
of the otic otx1 expression domain was lost in heat-shocked
embryos, whereas the anterolateral part of the otx1 domain was
duplicated at the posterior of the vesicle (Fig. 4M-O). Domains
of the anterior markers hmx2 and pax5 were duplicated at the
Fig. 5. Anterior otic markers are duplicated and posterior otic
markers lost in hsp70:fgf3 zebrafish embryos heat-shocked at
10S. (A-F)In situ hybridisation to anterior and posterior otic markers.
(G-J)In situ hybridisation to neurod, which marks delaminating
neuroblasts and nascent neurons of the statoacoustic ganglion. A-F,I,J:
Dorsal views; anterior to left, medial to top. G,H: Lateral views; anterior
to left, dorsal to top. Dots delineate the outline of the otic vesicle in I
and J. Scale bars: 50mm.
DEVELOPMENT
Fig. 4. Heat shock of hsp70:fgf3 embryos at 10S leads to a
complete loss of posterior otic domains and a mirror image
duplication of anterior otic structures. (A-C)Ears of live 68 hpf
hsp70:fgf3 and control wild-type zebrafish embryos heat-shocked at
10S. (D-L)Confocal z-stacks of 4 dpf ears stained with FITC-phalloidin
to mark sensory hair cells. (D-F)Lateral focal planes showing the
maculae. (G-I)Medial focal planes showing the cristae. (J-L)Dorsal
views. (M-O)In situ hybridisation to otx1. (P)The macula of an 86 hpf
heat-shocked hsp70:fgf3 embryo stained with anti-acetylated tubulin
antibody (kinocilia; red) and FITC-phalloidin (stereocilia; green). (Q)Hair
cell polarity maps obtained from the maculae shown in P. A-I: Lateral
views; anterior to left, dorsal to top. J-Q: Dorsal views; anterior to left,
medial to top. am, anterior macula; ao, anterior otolith; c, cristae; pm,
posterior macula; po, posterior otolith. Arrow in I marks the lateral
crista, which is enlarged. Scale bars: 50mm.
To test whether altering levels of Fgf signalling affects Hh
transduction within the otic vesicle, we used expression of ptc1, a
direct target of the Hh pathway, as a read-out of Hh signalling
activity (Fig. 6). Its levels are dramatically altered in the ear in
smob577–/– mutants, in which Hh signalling is severely reduced
(Aanstad et al., 2009; Varga et al., 2001), and in ptc1–/–;ptc2–/–
mutants, in which Hh signalling is maximal (Hammond et al.,
2003; Hammond et al., 2010; Koudijs et al., 2008) (Fig. 6Q,R). We
compared expression of ptc1 in embryos treated with SU5402 from
10 to 20S to remove Fgf signalling (n6), and in hsp70:fgf3
transgenic embryos heat-shocked at 10S to increase Fgf signalling
(n7), with expression in control siblings (n13). We found no
obvious difference in ptc1 expression for either treatment at 18S or
21S, although we cannot rule out subtle changes (Fig. 6A-H).
These time points were chosen to allow time for transcriptional
activation or degradation of ptc1 in response to changes in Fgf
signalling initiated at 10S, while still being within the critical time
window when Fgf is required for AP patterning.
To test whether altering levels of Hh signalling affects Fgf signal
transduction in the ear, we used pea3 expression as a readout of Fgf
signalling activity in smob577–/– embryos (n8) and ptc1–/–;ptc2–/–
double mutant embryos (n6). Phenotypically wild-type sibling
embryos (n15) were used as controls. Expression of pea3,
normally concentrated in anterior otic domains (Fig. 6I-P), was
completely absent from SU5402-treated embryos and severely
raised in heat-shocked hsp70:fgf3 embryos by 18S (Fig. 6S,T). We
detected no obvious changes in pea3 expression in ptc1–/–;ptc2–/–
embryos at 18S or in smo–/– embryos at either 18S or 21S (Fig.
6I,J,M-P). In ptc1–/–;ptc2–/– embryos at 21S, however, pea3,
although still strongly expressed at the anterior of the otic vesicle,
was reduced slightly along its ventral wall (bracket, Fig. 6K,L).
Development 138 (18)
This is likely to reflect alterations in the underlying branchial
arches, which normally express fgf3, and which were reduced in
ptc1–/–;ptc2–/– double homozygotes (Fig. 6K,L; see Fig. S5 in the
supplementary material). Hindbrain AP patterning in ptc1–/–;ptc2–/–
mutants appeared normal, as revealed by egr2b, mafb1, hoxb1 and
hoxb3, as well as by r4 expression of fgf3 itself (see Fig. S5 in the
supplementary material). Taken together, these data suggest that
from 10 to 20 somites, the Fgf and Hh pathways act independently
to pattern the otic anteroposterior axis.
Loss of both Fgf and Hh signalling from 10 to 20
somites results in an ear with neither anterior nor
posterior otic identity
To confirm that both Hh and Fgf signalling are instructive during
otic AP patterning, we treated smob577–/– embryos, in which Hh
signalling is severely reduced, with 10 mM SU5402 from 10 to 20S
to remove Fgf signalling (n27). If both Hh and Fgf have
instructive activity, we expected the resulting otic vesicles to have
neither anterior nor posterior identity. If Hh acts merely to repress
the effects of Fgf signalling, otic vesicles would resemble those
seen when Fgf is absent: a mirror image double-posterior ear. If Fgf
acts merely to repress the effects of Hh signalling, then the effects
of a loss of Hh would be epistatic to the effects of a loss of Fgf, and
the resulting otic vesicles would have two anterior poles.
As controls, we used smo+/– heterozygous and wild-type sibling
embryos treated with SU5402 (n42) and smo–/– and sibling
embryos treated with 0.5% DMSO [n32 (smo–/–), n42
(siblings)], from 10 to 20S. Ears of all control embryos developed
as expected. Sibling embryos treated with DMSO had
phenotypically wild-type ears (Fig. 7). Ears of DMSO-treated
smo–/– embryos displayed a loss of posterior otic domains and an
Fig. 6. Altering Fgf and Hh signalling in the
embryo does not affect otic expression of ptc1
and pea3, respectively. (A-H)ptc1 expression in
hsp70:fgf3 compared with wild-type sibling
zebrafish embryos heat-shocked at 10S, and in
10mM SU5402-treated embryos compared with
control DMSO-treated embryos treated from 10 to
20S. (I-P)pea3 expression in otic vesicles of
ptc1–/–;ptc2–/– and smo–/– embryos compared with
siblings. (Q,R)ptc1 expression in smo–/– and
ptc1–/–;ptc2–/–embryos. (S,T)pea3 expression in
10mM SU5402-treated and in hsp70:fgf3 embryos
heat-shocked at 10S. A-H,Q,R: Dorsal views;
anterior to left, lateral to top. I-P,S,T: Lateral views;
anterior to left, dorsal to top. Dotted lines delineate
otic vesicle outlines. Bracket in K marks low-level
pea3 expression, lost from 21S ptc1–/–;ptc2–/–
embryos (bracket, L).
DEVELOPMENT
3982 RESEARCH ARTICLE
Axial patterning in the fish ear
RESEARCH ARTICLE 3983
Fig. 7. Treatment of smo–/– embryos with SU5402 from 10 to
20S leads to a loss of anterior and posterior otic character.
Ears of smo–/– and sibling zebrafish embryos treated with 10mM
SU5402 and DMSO, respectively, from 10 to 20S. (A,B)Antiacetylated tubulin antibody stain marking the kinocilia of the firstforming hair cells (arrows). (C-J)FITC-phalloidin stain marking cell
outlines and stereociliary bundles of the sensory hair cells. (CF)Views of the whole vesicle. (G,H)Medial views showing the
maculae. (I,J)Lateral views showing the cristae. (K-N)Inner ears of
live embryos. Arrows indicate otoliths. cp, semicircular canal
projection tissue. (O-T)In situ hybridisation to anterior and posterior
otic markers. Dots delineate the otic vesicle in O and P. O,P,S,T:
Dorsal views; anterior to left, medial to top. A-N,Q,R: Lateral views;
anterior to left, dorsal to top. Scale bars: 50mm.
centre. We confirmed this using a panel of AP-restricted otic
markers as above: hmx2, an anterior marker, and fst1, a posterior
marker, were both lost from SU5402-treated smo–/– otic vesicles,
and expression of otx1 was reduced to a central domain, having
apparently lost both anterior and posterior domains of its wild-type
expression pattern (Fig. 7O-T). Expression domains of pax2a
(medial) and eya1 (ventral) in SU5402-treated smo–/– otic vesicles
were normal (see Fig. S3 in the supplementary material). Levels of
both markers were, however, reduced. This might reflect a
synergistic role for Hh and Fgf in controlling expression of these
genes, but is more likely to be due to the general poor development
and increased cell death in these embryos. The effects of Hh and
Fgf signalling on the ear are, therefore, additive: neither is epistatic
to the other, suggesting that both Fgf and Hh signalling have an
instructive role in otic AP patterning, acting to impose anterior and
posterior identity, respectively, on an initially symmetrical otic prepattern.
DISCUSSION
Fgf signalling is necessary and sufficient to
specify anterior otic identity in the zebrafish
Specification of the otic anteroposterior (AP) axis is one of the
earliest patterning events to take place during inner ear
development. Rotation and transplantation experiments in chick
and amphibian embryos have shown that the otic AP axis is fixed
before the dorsoventral (DV) axis, in response to induction from
surrounding tissues (Harrison, 1936; Harrison, 1945; Wu et al.,
1998; Bok et al., 2005). In zebrafish, Hh signalling is necessary and
sufficient to specify posterior otic identity during a time window
from ~10 to 20S, after otic placode induction is complete
(Hammond et al., 2003; Hammond et al., 2010). Using conditional
inhibition of Fgf signalling with SU5402, and conditional
overexpression of Fgf3 using a heat-shock inducible line, we have
shown that Fgf signalling is necessary and sufficient to specify
anterior otic identity in zebrafish during a similar period (Fig. 8).
This anteriorising function is distinct and temporally separable
from the earlier role for Fgfs in otic induction.
DEVELOPMENT
incomplete duplication of anterior otic domains (data not shown),
identical to the smo–/– otic phenotype (Hammond et al., 2003).
SU5402-treated smo siblings developed mirror image posterior
duplicated ears as described above (data not shown).
Otic vesicles of SU5402-treated smo–/– embryos initially
developed relatively normally: tubulin-rich tether cells
differentiated at the anterior and posterior poles of the vesicle
(n3/3 tubulin-stained specimens), and small otoliths seeded over
these, as in control embryos (Fig. 7A,B; data not shown). By 30
hpf, however, otoliths remained small, granular and positioned
symmetrically at the poles of the vesicle, whereas otoliths of
control embryos had grown in size and were no longer located
symmetrically at the anterior and posterior poles (Fig. 7K,L). In
control DMSO-treated sibling embryos, anterior otoliths were now
positioned ventrolaterally whereas the posterior otolith was more
medial; control smo–/– DMSO-treated embryos looked like
untreated smo–/– embryos with two lateral, ventral otoliths, and
SU5402-treated siblings had two medial otoliths, as in SU5402treated wild-type embryos (Fig. 7L; data not shown). At this stage
there were two or three hair cells underlying each otolith in both
the SU5402-treated smo–/– embryos and controls; their positions
reflected the position of the overlying otoliths (n3/3 phalloidinstained for each category) (Fig. 7C,D; data not shown).
By 45 hpf, further macular hair cells developed in all control
embryos; however, in SU5402-treated smo–/– embryos, no further
hair cells were evident, even by 86 hpf (n9/9 phalloidin-stained at
45-86 hpf) (Fig. 7E-J). Increased cell death in the otic vesicle might
contribute to the lack of differentiated hair cells (see Fig. S6 in the
supplementary material). Otoliths of SU5402-treated smo–/–
embryos remained small, or were absent altogether (Fig. 7M,N).
Development of SU5402-treated smo–/– otic vesicles was, however,
not entirely arrested at an early vesicle stage: semicircular canal
projection tissue formed, although it was grossly abnormal (cp, Fig.
7M,N).
Taken together, these data suggest that SU5402-treated smo–/–
otic vesicles lose anterior and posterior identity but retain an initial
symmetrical pattern about the AP axis, with defined poles and a
3984 RESEARCH ARTICLE
Development 138 (18)
The requirement for Fgf in zebrafish anterior otic patterning was
revealed by the partial loss of anterior character in lia–/– (fgf3–/–)
mutants and a complete loss of anterior character in embryos treated
with SU5402 from 10 to 20S. This differs from the situation in the
mouse, in which a loss of Fgf3 function results in dorsal
(endolymphatic duct) defects and partially penetrant posterior otic
defects for some Fgf3–/– alleles (Hatch et al., 2007; Mansour et al.,
1993). These phenotypic differences might reflect differences in the
spatiotemporal expression of hindbrain Fgf relative to the otic vesicle
between zebrafish and mouse. In the zebrafish, fgf3 is expressed in
r4, anterior to the developing otic placode and vesicle (Maves et al.,
2002; Walshe et al., 2002). By contrast, in the mouse, Fgf3 is
expressed in r4-6 at otic placode stages, becoming restricted to r5/6
at otic cup stages and eventually to r6 (i.e. posterior to the ear) at otic
vesicle stages (Wilkinson et al., 1988; Mahmood et al., 1996; McKay
et al., 1996; Alvarez et al., 2003). In keeping with this, work in both
mouse and chick has suggested that hindbrain-derived signals do not
specify the AP axis of otic vestibular structures in these species (Bok
et al., 2005), and that mesodermal and surface ectodermal signals are
more important (Liang et al., 2010; Bok et al., 2011).
Sufficiency of Fgf for anterior otic patterning was revealed by
the mirror image duplicated anterior otic phenotypes that resulted
from heat shock of hsp70:fgf3 embryos at 10S. This phenotype is
stronger than the otic phenotypes of two zebrafish mutants that
show posteriorly expanded fgf3 expression in the hindbrain: val–/–
(mafba–/–) and hnf1ba–/– (Kwak et al., 2002; Hernandez et al.,
2004; Lecaudey et al., 2007). In val–/– embryos, anterior otic
domains are expanded, but not duplicated (Kwak et al., 2002;
Whitfield and Hammond, 2007). This might be because hindbrain
fgf3 expression in val–/– does not extend far enough to influence the
posterior pole of the otic vesicle, which retains fst1 expression
(Lecaudey et al., 2007) (K.L.H., unpublished). In hnf1ba–/–
mutants, expansion of fgf3 expression in the hindbrain is more
extensive than in val–/– (Hernandez et al., 2004), and duplication of
anterior otic markers is seen in 25-50% of hnf1ba–/– mutants, with
expansion in others (Lecaudey et al., 2007). In our heat-shocked
embryos, fgf3 expression was activated in all cells, resulting in
anterior duplications that were complete and fully penetrant.
Additional hindbrain patterning defects in val–/– and hnf1ba–/–
embryos, including the reduction of wnt expression domains, and
DEVELOPMENT
Fig. 8. A three-step model for AP axial patterning in the zebrafish ear. (A)Step 1 (prior to 10S): Induction of a symmetrical otic placode and
establishment of equipotential poles. Fgf is required as a placode-inducing factor. Step 2 (10-20S): Direct response of the poles to instructive
anteriorising (Fgf, pink) and posteriorising (Hh, blue) factors, with expression of target genes in otic tissue. Step 3 (20S+): The poles of the ear act as
intrinsic organising centres, with different anterior (A) and posterior (P) identities. Signalling within the otic vesicle (curved arrow) reinforces initial
asymmetry. (B)In the absence of Fgf signalling from 10 to 20S, both poles acquire posterior identity, as both are under the influence of Hh
signalling from midline tissues. (C)In transgenic hsp70:fgf3 embryos heat-shocked from 10 to 20S, Fgf signalling is activated in all cells, and both
poles acquire anterior identity. The red (anterior) and dark blue (posterior) arrows indicate tissue polarity in each half of the otic vesicle.
prolonged disruption of Fgf expression, might account for the
presence of additional otic defects in these lines (Wiellette and
Sive, 2003; Hernandez et al., 2004; Riley et al., 2004; Hans and
Westerfield, 2007; Lecaudey et al., 2007).
The most likely source of Fgf for anterior otic patterning in the
zebrafish is r4 of the hindbrain, although there might also be a
contribution from the developing branchial arches. The anterior
position of r4 relative to the otic placode from 10S, and the parallel
expansion of hindbrain fgf3 expression and anterior otic identity in
val–/– and hnf1ba–/– mutants, support the notion that r4-derived Fgf
acts as an anteriorising signal for patterning the zebrafish ear. Note
also that the positioning of the otic rudiment with respect to the
hindbrain rhombomeres alters over time in the zebrafish embryo,
consistent with the proposed sequential roles of r4-derived Fgf
signalling in otic induction and AP patterning. At the tailbud/1S
stage, when otic induction is occurring, the otic rudiment (marked
by pax8 expression) develops in close association with r4 (Phillips
et al., 2001), which at this stage expresses both fgf3 and fgf8
(Maves et al., 2002; Walshe et al., 2002). By 10S, when Fgfmediated otic AP patterning begins, the otic placode is positioned
next to r5, with its anterior end opposite r4 (which retains fgf3
expression) and its posterior end opposite r6 (Maves et al., 2002).
The action of Fgfs on the otic placode is likely to be direct:
several Fgf target genes are expressed at the anterior of the
zebrafish ear from early placode stages (Raible and Brand, 2001;
Roehl and Nüsslein-Volhard, 2001; Thisse et al., 2001; Nechiporuk
et al., 2005). It is unlikely that a secondary signal, resulting from
AP hindbrain patterning defects, accounts for the otic anterior and
posterior duplications that we see when Fgf signalling is lost or
increased. There were no hindbrain AP patterning defects in 1020S SU5402-treated embryos, as assayed by mafba, hoxb1 and
egr2b expression (see Fig. S7 in the supplementary material),
although there was a slight expansion of r5 markers into r6 in 10S
heat-shocked hsp70:fgf3 embryos (see Fig. S7 in the supplementary
material).
At least three Fgf genes, fgf3, fgf8 and fgf10a, are also expressed
at the anterior of the developing zebrafish inner ear by otic vesicle
stages (fgf8 and fgf10a by 18S; fgf3 by 21S) (Kudoh et al., 2001;
Léger and Brand, 2002; Walshe and Mason, 2003; Nechiporuk and
Raible, 2008). Our data, however, show that Fgf is required for otic
AP patterning prior to this, suggesting that otic Fgf expression
might reinforce, rather than specify, anterior otic identity.
Consistent with this idea, when Fgf signalling is inhibited from 18S
in zebrafish, anterior otic pax5 expression is lost (Léger and Brand,
2002). Similarly, otic Fgf8 expression has been proposed to
regulate Sox3 expression and neurogenesis in an anterior otic
territory in the chicken embryo (Abelló et al., 2010).
Fgf and Hh act in an opposite, but independent,
manner to pattern the otic AP axis
The enantiomorphic twinned ear phenotypes that we have obtained
through Fgf manipulation are very similar, but opposite with
respect to the AP axis, to those seen when Hh signalling is altered
in zebrafish and Xenopus. Increased Fgf or a loss of Hh leads to
double-anterior ears, whereas a loss of Fgf or increased Hh leads
to double-posterior ears (Hammond et al., 2003; Waldman et al.,
2007; Hammond et al., 2010; Sapède and Pujades, 2010).
The Hh- and Fgf-induced otic mirror image duplication
phenotypes are not, however, identical. The otic duplications seen
when Fgf is increased or absent were remarkably complete,
whereas the duplications are only partial in Hh pathway loss-offunction mutants (Hammond et al., 2003; Sapède and Pujades,
RESEARCH ARTICLE 3985
2010). This might be explained as follows. In the absence of Fgf
signalling, Hh can effectively re-specify anterior otic domains as
posterior because it emanates from the ventral midline, which lies
adjacent to anterior as well as posterior parts of the otic
placode/vesicle. In the absence of Hh signalling, Fgf does not fully
re-specify posterior otic domains as anterior because the Fgf source
is located at the anterior of the ear. Posterior otic domains are thus
substantially removed from its influence. Overexpression of either
Fgf or Hh throughout the embryo is sufficient to re-specify the otic
poles accordingly. Note, however, that neither factor affects
transduction of the other in the ear, as assayed by expression of
direct pathway target genes (ptc1, pea3). Integration of the effects
of the two signalling pathways must occur at the level of shared
targets further downstream, which are likely to include the earliest
markers of asymmetry, such as hmx2 and hmx3.
Additional otic DV and mediolateral (ML) patterning defects are
seen in the posterior duplicated ears of ptc1–/–;ptc2–/– embryos,
which have severely increased levels of Hh signalling (Koudijs et
al., 2008; Hammond et al., 2010). This might reflect an additional
patterning role for Hh not shared with Fgf, or might be because Hh
signalling remains upregulated throughout development in
ptc1–/–;ptc2–/– embryos, whereas we only inhibited Fgf for a short
time. Interestingly, even when Fgf was upregulated throughout the
embryo, expression domains of hmx2 and pax5 remained medial.
This suggests that correct otic mediolateral patterning depends on
mechanisms that are independent and separable from the
anteriorising function of Fgf.
Hh and Fgf act on an initially symmetrical otic
placode with equipotential poles
The ability to generate mirror symmetrical double-anterior and
-posterior ears by altering Fgf or Hh signalling demonstrates that
the anterior and posterior halves of the otic rudiment are initially
equipotential. This was first shown in the 1930s when Harrison
produced double-anterior and -posterior ears by rotating the
salamander otic rudiment about its AP axis (Harrison, 1936;
Harrison, 1945). There have since been other reports of physical
and genetic manipulations resulting in duplicated ears (Hammond
et al., 2003; Waldman et al., 2007; Bok et al., 2011). This suggests
that prior to the action of axial patterning signals, the otic placode
is symmetrical about its AP axis (Fig. 8). Indeed, when we
inhibited both Fgf and Hh signalling pathways from 10 to 20S, an
AP symmetrical otic pre-pattern, with neither anterior nor posterior
identity, was revealed.
Several genes, including those of the delta and atoh families, are
expressed symmetrically at the anterior and posterior poles of the
otic placode by 10S, prior to the AP patterning activity of Fgf and
Hh (Haddon et al., 1998; Millimaki et al., 2007). Their expression
prefigures the symmetrical appearance of tether cells, which
differentiate into the first functioning hair cells at either end of the
ear; later hair cells are added asymmetrically (Haddon and Lewis,
1996; Riley et al., 1997; Millimaki et al., 2007; Sapède and
Pujades, 2010; Tanimoto et al., 2011). Tether cells are not only the
first hair cells to develop in the ear, but also have different genetic
requirements: they are dependent on the function of atoh1b,
whereas later hair cells require the function of atoh1a (Millimaki
et al., 2007). In our experiments, tether cells still differentiated
when Fgf and Hh were inhibited from 10 to 20S, implying that
tether cells need neither anterior nor posterior identity to
differentiate into hair cells. However, further hair cell addition did
not occur and development of the sensory patches did not progress
beyond a symmetrical stage.
DEVELOPMENT
Axial patterning in the fish ear
The absence of otic AP asymmetries when both Fgf and Hh
signalling were inhibited from 10 to 20S suggests that, in zebrafish,
Fgf and Hh are the major early otic AP patterning signals.
However, manipulations of retinoic acid (RA) signalling in the
chick have recently also been shown to result in AP mirror image
duplications of the whole inner ear (Bok et al., 2011). High RA
levels appear to specify posterior otic development (in particular
Tbx1 expression), and low levels are required for anterior gene
expression (including markers of neurogenesis) (Bok et al., 2011).
A similar positive regulation of otic tbx1 expression by RA (and
negative regulation of neurogenesis) has recently been reported in
the zebrafish, although mirror image duplications were not
observed (Radosevic et al., 2011). Manipulation of RA signalling
is also known to affect earlier stages of zebrafish otic development,
but only indirectly; notably, early RA inhibition delays the onset of
hindbrain fgf3 expression, and so interferes with otic induction
(Hans and Westerfield, 2007). Any interaction of RA with the Fgf
and Hh signalling pathways between the 10 and 20S stages in the
zebrafish remains to be determined.
Although Hh and Fgf appear to act instructively to specify
zebrafish anterior and posterior otic identity, extrinsic sources of
Fgf and Hh are unlikely to provide the polarity information for
subsequent patterning of the ear. This is because when Hh or Fgf
signalling is activated throughout the embryo (with no focal source
or gradation of signal), the result is a mirror image duplication of
the ear, with two correctly patterned mirror image halves, rather
than a single enlarged anterior or posterior domain. It is thus likely
that organising centres exist within the ear to impart polarity to the
sensory patches. Ablation experiments indicate that these centres
must reside at or very near the poles of the otic placode (Waldman
et al., 2007). It is thus tempting to speculate that the tether cells
themselves might play an active role in signalling to organise
polarity in the ear (Fig. 8).
In conclusion, we have shown that in zebrafish, between 10 and
20S, Fgf and Hh act on a symmetrical pre-pattern to specify otic
anterior and posterior identity, respectively. Both factors appear to
have instructive activity to assign identity to the otic poles, but act
permissively to allow polarised patterning of each half of the ear,
which is likely to rely on additional intrinsic patterning mechanisms.
Acknowledgements
This work was funded by the BBSRC (BB/E015875/1) and the Wellcome Trust
(092401). We are grateful to G. Cakan-Akdogan and D. Gilmour for providing
the hsp70:fgf3 line, and to H. Roehl for making this available to us in Sheffield.
We thank N. Monk for helpful discussion, members of the zebrafish
community for providing probes, and the aquarium staff for expert care of the
zebrafish. The MRC CDBG zebrafish aquaria and imaging facilities were
supported by the MRC (G0700091), with additional support from the EU FP6
(ZF-MODELS) and the Wellcome Trust (GR077544AIA). Deposited in PMC for
release after 6 months.
Competing interests statement
The authors declare no competing financial interests.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/lookup/suppl/doi:10.1242/dev.066639/-/DC1
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Axial patterning in the fish ear