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
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 double-
anterior 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).
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 138, 3977-3987 (2011) doi:10.1242/dev.066639
© 2011. Published by The Company of Biologists Ltd
MRC Centre for Developmental and Biomedical Genetics and Department of
Biomedical Science, University of Sheffield, Sheffield S10 2TN, UK.
*Author for correspondence (firstname.lastname@example.org)
Accepted 1 July 2011
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.
KEY WORDS: Fgf, Hh, Axial patterning, Otic vesicle, Zebrafish
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*
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
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.,
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). liat21142primers were: 5?-
TGTCCAGTCATGAATGTCAAAG-3? and 5?-CCATCTCATGGTCCTT -
GTTG-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 liat21142DNA. smob577primers were: 5?-
CTATACTGGCCAATTCACAG-3? and 5?-ATGGAAAACAATGTCA -
TAACC-3?. The resulting 325 bp PCR band was sequenced: wild-type
DNA has a G at position 160, whereas smob577DNA 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.
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 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
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 three-
dimensional 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 FITC-
phalloidin, to reveal the actin-rich stereociliary bundles of the
Development 138 (18)
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: 50mm.
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