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First published online 1 March 2006
doi: 10.1242/dev.02306
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Centre for Developmental and Biomedical Genetics, Department of Biomedical Science, University of Sheffield, Sheffield S10 2TN, UK.
* Author for correspondence (e-mail: t.whitfield{at}sheffield.ac.uk)
Accepted 1 February 2006
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Lamprey, Zebrafish, Agnathan, Gnathostome, Inner ear, Otic vesicle, Macula, follistatin, ptc, otx, Evolution, Placode, Hair cell, Planar polarity pattern
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Gnathostome vertebrates and the few remaining extant agnathan (jawless)
vertebrates (lampreys and hagfish) are thought to have evolved from a common
agnathan ancestor. Inner ear morphology underwent a significant change at the
agnathan/gnathostome transition. The inner ears of adult agnathan fish are, at
least superficially, more symmetrical about the anteroposterior (AP) axis than
those of gnathostomes (Fig. 1).
Adult lamprey inner ears have only two semicircular canals, each with a
tricuspid crista, and a single ventrally positioned macula communis that
covers the floor of two joined, symmetrical, ciliated chambers
(de Burlet and Versteegh, 1930
;
Lowenstein et al., 1968;
Thornhill, 1972;
Avallone et al., 2005). A
similar pattern, with two semicircular canals, is found in the abundant
agnathan fossil record, making it likely that this represents the ancestral
condition (Mazan et al.,
2000). Hagfish also have a single macula communis on the ventral
floor of the inner ear, but differ in having a single toroidal semicircular
canal, which nevertheless is associated with two cristae, and is probably a
derived characteristic (Lowenstein and
Thornhill, 1970) (for a review, see
Lowenstein, 1971). Although
the general appearance of the inner ear is symmetrical about the AP axis in
adult agnathan fishes, the macula communis can be subdivided into at least
three morphologically distinct regions, the anterior horizontal, vertical and
posterior horizontal regions (Lowenstein
et al., 1968; Lowenstein and
Thornhill, 1970; Hagelin,
1974), and there have been several different interpretations of
the homology of these regions to the various gnathostome maculae
(de Burlet and Versteegh, 1930;
Lowenstein et al., 1968;
Thornhill, 1972;
Hagelin, 1974). Several
studies describe hair cell planar polarity patterns in the late larval and
adult lamprey and hagfish macula communis as roughly symmetrical about the AP
axis (Lowenstein et al., 1968;
Lowenstein and Thornhill,
1970; Thornhill,
1972), and Hagelin (Hagelin,
1974) tentatively proposes that both the anterior and posterior
ends of the lamprey macula correspond to the gnathostome utricular macula.
Although these homologies are not completely resolved, the relative symmetry
of the adult agnathan ear suggested to us that the asymmetric gnathostome ear
might have evolved from a more primitive symmetric form.
Although the gnathostome otic vesicle shows distinct asymmetry from early
stages, studies in amphibian, mouse and zebrafish embryos have suggested that
the anterior and posterior ends of the otic vesicle are initially
equipotential, and that the ear is capable of developing as a more symmetric
structure following physical or genetic manipulation of the embryo
(Harrison, 1936;
Harrison, 1945;
Waldman et al., 2001;
Léger and Brand, 2002;
Raft et al., 2004). We have
shown that disruptions of Hedgehog (Hh) signalling in the zebrafish embryo
give rise to an ear that is more symmetric about the AP axis. Specifically, a
loss of Hh signalling in the embryo results in an enantiomorphic (mirror
image) ear with double anterior character, whereas overactivity of the Hh
pathway results in a double posterior ear
(Hammond et al., 2003). In
both cases, a single mirror-image, twinned macula can be produced, reminiscent
of the single macula communis in the agnathan ear.
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). Such an interpretation rests upon the assumption
that the relative symmetry of the adult agnathan ear is reflected in its
development. A prediction of this idea is that the agnathan otic vesicle
develops in a symmetric manner, lacking the asymmetric expression of marker
genes seen in the zebrafish. It might also be expected that the agnathan otic
vesicle is unresponsive to polarising signals known to be important in
zebrafish, such as Hh signalling. To test these ideas, we have examined ear
development in the lamprey, at both morphological and molecular levels. We
have concentrated on the morphology and planar polarity patterns of the
developing macula communis, and the expression of genes for which the
zebrafish homologue is expressed in a polarised fashion in the otic vesicle.
Contrary to our expectation, we find that the otic vesicle of the lamprey
displays many similarities to that of zebrafish, including considerable
morphological and molecular asymmetry about the AP axis; thus the apparent
symmetry of the adult agnathan ear belies an asymmetric developmental
programme. We also show that the lamprey otic vesicle expresses a homologue of
patched, suggesting that it is responsive to Hh signalling, and
indicating that a new role for Hh signalling in the ear probably did not
account for the evolution of additional complexity in inner ear morphology at
the agnathan/gnathostome transition.
In addition to extrinsic patterning information, transcription factors
expressed within the otic epithelium have cell autonomous functions in otic
patterning. A role for Otx1 has already been implicated in the appearance of
the third, horizontal semicircular canal and crista in the gnathostome ear
(Tomsa and Langeland, 1999;
Mazan et al., 2000). An
otx gene is not expressed in the lamprey ear
(Ueki et al., 1998;
Tomsa and Langeland, 1999). In
gnathostomes, however, Otx1 is expressed in a region that gives rise
to the horizontal semicircular canal and crista, and is critical for their
formation; in mice homozygous for a targeted disruption of Otx1, the
horizontal canal and crista are absent, and the utricle fails to separate
properly from the saccule (Acampora et al.,
1996; Morsli et al.,
1999; Fritzsch et al.,
2001). We show that a knockdown of Otx1 function in the zebrafish
embryo has similar effects on the ear to those seen in the mouse, confirming
that the acquisition of otic otx1 expression at the
agnathan/gnathostome transition was likely to have been responsible, not only
for the appearance of the horizontal canal and crista, but also for the
separation of distinct areas of sensory tissue from an ancestral (but already
polarised) macula communis.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Live
observation was carried out as for zebrafish
(Westerfield, 1995).
Dechorionated embryos were fixed overnight at 4°C in 4% paraformaldehyde
in 1xPBS, washed several times in PBS, and either stored at 4°C in
1xPBS, 0.1% sodium azide for FITC-phalloidin staining and
immunohistochemistry, or dehydrated through a methanol series and stored at
-20°C in 100% methanol for in situ hybridisation. Fixed Petromyzon
marinus embryos, collected at the Hammond Bay Biological Station, MI,
USA, were kindly provided by Dr David McCauley, Caltech
(McCauley and Bronner-Fraser,
2002).
Zebrafish
WIK embryos were cultured as previously described
(Westerfield, 1995).
Observation of live embryos and fixation were carried out as for lamprey
embryos.
Photography
Examination and photography of live and fixed samples were carried out
using an Olympus BX51 compound microscope, Olympus Camedia (C-3030Z00M) camera
and AnalySIS software (Olympus). Images were assembled with Adobe
Photoshop.
FITC-phalloidin staining and immunohistochemistry
Fixed embryos were stained whole with FITC-phalloidin (Sigma) as described
by Haddon and Lewis (Haddon and Lewis,
1996), except that lamprey embryos were permeabilised in 2% Triton
in 1xPBS for five days at 4°C. Double staining using FITC-phalloidin
and anti-acetylated tubulin antibody (Sigma) was performed essentially as
described by Haddon and Lewis (Haddon and
Lewis, 1996), but lamprey ears were dissected out and
permeabilised for 30-60 minutes in 1% Triton at room temperature before
staining. Zebrafish embryos were permeabilised in 1% [24-30 hours
post-fertilisation (hpf)] or 2% (72 hpf +) Triton at room temperature for
three hours and stained whole. Samples were mounted in Vectashield (Vector
Laboratories) and imaged on a Leica SP confocal microscope.
Isolation and sequencing of lamprey cDNA
A 747 bp ptc fragment was amplified from a 6- to 10-day-old P.
marinus embryo oligo (dT)-primed lambda ZapII cDNA library (a gift from
J. Langeland, Kalamazoo College) by degenerate PCR, using the primers
5'-tcacccctctggactgcttytgggargg-3' (forward) and
5'-gcacctgggtggtggcngcrttraa-3' (reverse), followed by a
semi-nested PCR reaction with an internal reverse primer,
5'-cggacaagcacaccagcarnacnccngc-3', and the forward primer from
the first reaction. A 301 bp follistatin fragment was amplified from
L. fluviatilis oligo (dT)-primed stage 22/23 cDNA by degenerate PCR
(primers: forward, 5'-acacagtgcaggctggaaaytgytggyt-3'; reverse,
5'-tgttggagcagtcaggagcrcanacrca-3'). RNA was extracted from stage
22/23 L. fluviatilis embryos using Trizol (Sigma). cDNA was
transcribed using a Superscript III first strand synthesis kit (Invitrogen)
and PCR was carried out using Diamond Taq polymerase for GC-rich DNA
(Bioline). PCR fragments were cloned into pGEM T-Easy using a T-Easy cloning
kit (Promega) and used to screen the P. marinus cDNA library.
[32P]dCTP-labelled probes were made from these PCR fragments and
the library screened as described previously
(Nehls et al., 1994). Phagemid
was excised from isolated plaques using ExAssist helper phage (Invitrogen). A
single 1502 bp partial ptc clone and two follistatin clones
(1227 bp and 1886 bp, both containing the full ORF) were obtained. A 538 bp
fgf8/fgf17 fragment was also amplified by PCR from L.
fluviatilis cDNA (primers: forward,
5'-caaactgaccgtggaaacgg-3'; reverse,
5'-ccctccctcctgcggcgtc-3'). Sequencing was performed by the
Genetics Core Facility, University of Sheffield, using an ABI 3730 capillary
sequencer and analysed using the The Biology WorkBench
(http://workbench.sdsc.edu).
Sequences for P. marinus patched, P. marinus follistatin (long), P. marinus follistatin (short) and L. fluviatilis fgf8/fgf17 have been deposited in GenBank with Accession numbers DQ370170, DQ370171, DQ370172 and DQ370173, respectively.
In situ hybridisation
Digoxigenin-labelled probes were synthesised using a DIG labelling kit
(Roche). Antisense and sense riboprobes were made from linearised full-length
L. fluviatilis tbx1 cDNA
(Sauka-Spengler et al., 2002),
our P. marinus follistatin cDNA and the partial P. marinus
ptc cDNA, together with an antisense probe to our L. fluviatilis
fgf8/fgf17 fragment. In situ hybridisation to lamprey embryos was carried
out essentially as described by Tomsa and Langeland
(Tomsa and Langeland, 1999).
For photography, stained lamprey embryos were dehydrated through an ethanol
series and cleared and mounted in Murray's clear (2:1 benzyl benzoate:benzyl
alcohol). In situ hybridisation to zebrafish embryos was carried out as
described previously (Hammond et al.,
2003). Zebrafish embryos were cleared and mounted in glycerol and
photographed as above. All sense hybridisations were negative.
Morpholino injections to zebrafish embryos
An antisense morpholino (GeneTools, sequence
5'-taacatatagcctacctgaactcgg-3') was targeted to the exon 3/intron
3-4 junction of the zebrafish otx1 gene (GenBank Accession number NM
131250/Ensembl reference ENSDARG00000043643, Zv5). Approximately 5 nl of 0.66
mM or 1.00 mM morpholino was injected into one- or two-cell embryos using a
Narishige microinjection rig. To test for disruption of splicing, RT-PCR was
performed (primers: exon 2 forward, 5'-atggacctactacacccgtc-3';
exon 4 reverse, 5'-gtgctgagcctggagaaatg-3'). RNA was extracted
from pools of 25 24 hpf, 50 hpf, 74 hpf or 4 days post-fertilisation (dpf)
embryos (either uninjected, or injected with 5 nl of 1 mM morpholino) using
Trizol (Sigma), and cDNA was transcribed using a Superscript III first strand
synthesis kit (Invitrogen). PCR was carried out using Taq polymerase
(Promega). The 447 bp band represents correctly spliced otx1 mRNA;
the 471 bp and 408 bp bands represent aberrantly spliced forms, where splicing
has occurred from cryptic donor sites 39 bases 5' or 24 bases 3'
of the correct exon 3 donor site. This was confirmed by sequencing the PCR
products using internal primers (exon 3 forward,
5'-agaggactacatttacgcgct-3'; exon 4 reverse,
5'-ctgtgttattggtggaggaag-3'). PCR was also carried out using
gene-specific primers (forward, 5'-aagcaggagtacgatgagtctg-3';
reverse, 5'-ggtaaacgcttctggaatgac-3') to amplify actin cDNA as a
positive control.
Fluorescent labelling of the otic vesicle lumen
Embryos at 4 dpf were anaesthetised, immobilised in 1.0% low melting point
agarose and fluorescein-conjugated morpholino injected into the lumen of the
ear using a Narishige microinjection rig. Injected ears were imaged on a Leica
SP confocal microscope within a few minutes of injection.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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At stage 24 (approximately 8 dpf), the lamprey inner ear consists of a simple epithelial vesicle; a small dorsal outpocketing that will form the endolymphatic duct is just visible (Fig. 2A,A'), and anteroventral cells are beginning to delaminate to form the statoacoustic (VIIIth) ganglion (Fig. 2A',D). By stage 25 (10-11 dpf), hair cells develop in two discrete regions at the anterior and posterior ends of the vesicle, forming first at the posterior and then a short time later at the anterior (Fig. 2B,C,C'). These are the first AP asymmetries seen within the otic vesicle. By stage 26 (11-12 dpf), a well-defined group of hair cells is present at each end of the vesicle and the endolymphatic duct is obvious in the dorsal roof of the ear (Fig. 2D,D'). The statoacoustic ganglion can be visualised at stages 26-27 with anti-acetylated tubulin antibody (Fig. 2E,E'), and remains in a similar anteroventral position with respect to the vesicle until at least stage 29.
Although the lamprey otic vesicle forms by invagination of placodal
ectoderm (Thornhill, 1972;
Hagelin, 1974), in contrast to
the cavitation seen in zebrafish (Haddon
and Lewis, 1996), there are parallels between the two species at
these early otic vesicle stages. In both species, neuroblasts migrate from
ventral regions of the vesicle to form the statoacoustic ganglion
anteroventral to the ear, and hair cells develop in two distinct groups at the
anterior and posterior of the vesicle
(Haddon and Lewis, 1996)
(Fig. 2H,I). The endolymphatic
duct, however, does not begin to form until later in the zebrafish, and is
never as obvious as in the lamprey (L. Abbas, K.L.H., C. Mowbray and T.W.,
unpublished).
At stage 27 (13-15 dpf), hair cells start to develop medially between the two initial groups in the lamprey otic vesicle (Fig. 2F,F'). Subsequently, hair cell number continues to increase, forming a sensory patch that folds anteriorly onto the vertical anterior wall and posteriorly onto the ventromedial wall, demonstrating significant AP asymmetry within the otic vesicle (Fig. 2G,G', Fig. 3A-A''). Although this patch appears to be a single sensory region (a `macula communis') by stage 29 (18 dpf), hair cell density within it is variable: an anterior region of high cell density and a posterior region of high cell density are linked by a much more sparsely populated region (Fig. 3G,H). This is especially obvious at stage 28 (16-17 dpf), and is also reflected in the arrangement of otoconia that can be observed in live embryos (Fig. 3B-E). The otoconia sit over the macula, forming an otoconial mass, which at stage 28 - and to a lesser extent at stage 29 - is variably split at the region of lower hair cell density, creating two distinct otoconial masses overlying the anterior and the posterior portions of the macula (Fig. 3D,E). Taken together, these data suggest that although this sensory patch has been termed a macula communis, it consists of at least two distinct morphological regions, evident from early stages.
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); however, his observations were made at later stages than
ours, and it is conceivable that the otoconial masses later subdivide
(Hagelin, 1974). Lowenstein et
al., however, report only a single otoconial mass, even in the adult
(Lowenstein et al., 1968). We
believe that our observations of otoconia in the live embryo are likely to be
more reliable than those made on fixed material, but unfortunately such live
observations are limited to stages when the embryo is relatively optically
clear.
At stage 29, there is no obvious sign of semicircular canal formation or
crista development in the lamprey ear. However, at stage 30, we have observed
what we interpret to be the beginnings of crista formation in two stage 30
L. fluviatilis ears (Fig.
4); epithelial projections also begin to form the semicircular
canal system at this stage. We have not observed any hair cells that obviously
correspond to the dorsal macula in the lamprey
(Lowenstein et al., 1968;
Thornhill, 1972;
Hagelin, 1974).
Distinct regions of the lamprey macula communis correspond to the utricular and saccular maculae of zebrafish
Based on their initial appearance and position within the otic vesicle, the
anterior and posterior regions of high cell density in the developing lamprey
macula communis bear a distinct resemblance to the developing utricular and
saccular maculae, respectively, of the zebrafish ear
(Fig. 3F,I). To investigate
these putative homologies further, we set out to map hair cell polarity
patterns in the lamprey macula communis. Sensory patches within the ear have
stereotypical patterns of hair cell planar polarity, specific to both a given
species and a particular type of patch. A confocal optical section at the
level of the apical membrane of the hair cell reveals a single tubulin-rich
kinocilium (red, Fig. 5B,C)
surrounded by a crescent of shorter actin-rich stereocilia (green,
Fig. 5B,C); we define a
polarity vector for each cell as an arrow pointing from the stereocilia to the
kinocilium (Fig.
5C').
To map hair cell polarities within the macula communis, we stained
dissected stage 29 ears (25 dpf) with FITC-phalloidin and anti-acetylated
tubulin antibody to mark the stereocilia and kinocilia, respectively
(Fig. 5). The FITC-phalloidin
stain revealed that lamprey hair cells have a substantial infracuticular
network of actin penetrating into the hair cell body
(Fig. 5B), which may correspond
to the striated organelle described by Thornhill
(Thornhill, 1972). This is not
found in zebrafish hair cells, but a similar network has been described in
guinea pig hair cells (Carlisle et al.,
1988; Slepecky,
1989); its function is unknown. We also observed shorter cilia
projecting from all non-sensory cells of the otic epithelium, similar to those
found in early zebrafish otic vesicles
(Fig. 5C,D)
(Riley et al., 1997).
To visualise polarity patterns within the macula, we mounted dissected L. fluviatilis ears such that each region of the epithelium was flattened for observation (Fig. 5E',F'). It was not possible to flatten and visualise the entire macula communis in any one otic vesicle, so multiple ears were examined in overlapping units and the polarity maps combined, with overall 4x coverage. A summary of the pattern for L. fluviatilis is shown in Fig. 6. We also obtained a map of the anterior region of a single P. marinus embryo that is virtually identical to those obtained from L. fluviatilis (data not shown).
It is striking that the polarity map of the stage 29 L.
fluviatilis macula shows a high degree of AP asymmetry: patterns in the
anterior and posterior regions of the macula are quite distinct
(Fig. 5E,F,
Fig. 6). In the anterior
region, hair cell polarities converge on a line that separates a smaller
anterior domain from a larger posterior domain; in the posterior region, hair
cell polarities diverge from a central line that bisects this region. In the
extreme anterior and posterior domains of the posterior region, hair cells
point anteriorly and posteriorly, respectively. Note that the map is flattened
out; in the embryo, the anterior region of the macula actually folds up the
anterior wall of the otocyst, so that the hair cells in this region actually
point approximately ventrally and dorsally instead of posteriorly and
anteriorly (see Fig. 5A for
reference points). Beyond stage 29, however, the anterior region flattens down
to lie on the ventral wall of the otocyst, so that eventually these hair cells
will indeed point anteriorly and posteriorly (our observations of two stage 30
embryos) (Hagelin, 1974). Our
data largely corroborate, but differ in detail to, the polarity patterns
described in the larval L. fluviatilis ear using transmission
electron microscopy (Thornhill,
1972).
In the adult L. fluviatilis macula, Lowenstein et al.
(Lowenstein et al., 1968)
report a similar pattern: most hair cells point anteriorly in the larger,
anterior horizontal region, diverge from a midline in the vertical portion of
the macula, and point posteriorly in the smaller, posterior horizontal region.
Close examination of their data also reveals a small, posterior-facing domain
in the anterior horizontal part of the macula. Thus hair cell polarity
patterns in the stage 29 macula resemble those in the adult, but AP
asymmetries in the pattern, particularly in the anterior portion of the
macula, are much more evident at larval stages.
It is also noteworthy that the hair cell polarity patterns within the
lamprey macula resemble those found in the embryonic zebrafish utricular and
saccular maculae (Fig. 6)
(Haddon et al., 1999). The
anterior (utricular) macula of zebrafish consists mostly of roughly
anterior-facing hair cells, with orientations fanning out from the medial side
of the macula, abutting a thin stripe of posterior-facing hair cells,
resembling the anterior region of the lamprey macula. The zebrafish posterior
(saccular) macula consists mainly of a region of divergent polarities, with
hair cells pointing dorsally and ventrally away from a central line,
resembling the posterior region of the lamprey macula. There is one notable
difference: the zebrafish embryonic saccular macula has an anterior extension
with an antiparallel pattern of hair cell orientations, which does not
correspond to any region of the lamprey pattern that we see. Overall, however,
our data suggest that the anterior and posterior regions of high hair cell
density in the embryonic lamprey macula communis correspond closely to the
utricular and saccular maculae, respectively, of the zebrafish. This
interpretation is different to that suggested by Thornhill
(Thornhill, 1972), that most
of the anterior region of the embryonic macula contributes to the anterior
crista.
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; Raft et al.,
2004). tbx1 expression has previously been reported in
similar posterior regions of the L. fluviatilis otic vesicle
(Sauka-Spengler et al., 2002).
We have confirmed this in both L. fluviatilis and P. marinus
(to which L. fluviatilis tbx1 cross-hybridises) at stage 24-25. As in
jawed vertebrate embryos, we see a clear boundary between anterior
non-expressing and posterior tbx1-expressing cells in the lamprey
otic vesicle, demonstrating that there is a very distinct asymmetry in the
expression of this marker in the developing lamprey ear
(Fig. 7B,D).
We also chose to examine follistatin expression in the lamprey
ear, for two reasons. First, in zebrafish, follistatin expression
marks a localised region of posterior otic cells from 24 hpf to at least 30
hpf (Fig. 7E)
(Mowbray et al., 2001).
Second, otic follistatin expression is lost in zebrafish embryos
lacking Hh signalling, and is delocalised and duplicated at the anterior of
the ear in embryos overexpressing Hh
(Hammond et al., 2003). If the
lamprey ear resembles these relatively symmetric ears generated by a
disruption of Hh signalling in the zebrafish, we might expect to see a similar
lack or duplication of follistatin expression in the lamprey otic
vesicle. We amplified a single 301 bp follistatin fragment from
L. fluviatilis stage 23-24 cDNA by degenerate PCR and then used this
fragment to screen a 6- to 10-day P. marinus library (J. Langeland,
Kalamazoo College). We obtained four positive plaques corresponding to two
separate follistatin clones, one 1227 bp (one plaque) and one 1886 bp
(three separate plaques). Both contain full-length open reading frames (ORFs)
and a small region of 5' UTR, with the larger also containing a large
region of 3' UTR. The predicted protein has 53.8% and 56.5% identity
with zebrafish and mouse Follistatin proteins, respectively. These identities
are based on the 1227 bp clone, as this contains an extra 81 bp that is not
present in the larger cDNA but is present in the reported zebrafish and mouse
genes (see Fig. S3 in the supplementary material).
Both P. marinus clones show identical expression patterns in stage 23 to 29 P. marinus embryos; the shorter clone also has the same expression pattern in L. fluviatilis. The longer clone, however, did not cross-hybridise, presumably because of the presence of the long 3' UTR, which is likely to be less conserved than the ORF. There is 95.3% identity at the nucleotide level between the 301 bp L. fluviatilis fragment and the corresponding region of the shorter P. marinus clone, suggesting that the ORF has diverged relatively little between these two lamprey species.
Expression of P. marinus follistatin is seen in similar regions to zebrafish follistatin, in the branchial arches, somites and in the ear; a full expression pattern will be reported elsewhere. Expression in the vicinity of the otic vesicle begins at stage 23 when transcript is detected just posterior to the otic epithelium (Fig. 7G). At stage 24 and 25, strong P. marinus follistatin expression is seen throughout posterior regions of the otic epithelium, with a clear boundary between anterior non-expressing epithelium and the posterior follistatin-expressing area (Fig. 7F,H). No expression within the ear is seen at subsequent stages. The domain of P. marinus follistatin expression is similar to zebrafish follistatin: both genes mark posterior otic regions, confirming the molecular asymmetry of the lamprey otic vesicle. The expression of P. marinus follistatin is, however, more widespread in the posterior of the lamprey ear than the very discrete posterior follistatin domain seen in zebrafish (Fig. 7E). Nevertheless, we find no evidence of a duplicated pattern of expression, as in zebrafish embryos in which the Hh pathway has been over-activated.
Hh signal transduction is likely to be active in the lamprey otic vesicle
In the zebrafish, Hedgehog (Hh) signalling is crucial for posterior otic
specification: as described above, embryos in which Hh signalling is disrupted
can have mirror symmetric ears, consisting of two anterior or two posterior
ends (Hammond et al., 2003).
We initially hypothesized that the lamprey ear would be unresponsive to Hh
signalling. However, as markers of otic AP asymmetry are shared between
lamprey and zebrafish, we wanted to examine whether known otic AP patterning
mechanisms might also be shared between the two species. To test whether the
lamprey ear is responsive to Hh signalling we have examined ptc
expression in the otic vesicle. ptc not only codes for the receptor
for Hh signalling, but is also a transcriptional target of the Hh pathway;
areas of strong ptc expression therefore indicate areas where active
Hh signal transduction is occurring.
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).
Although this evidence is circumstantial and does not prove that Hh signalling
has a similar role in otic AP patterning of lamprey and zebrafish, it shows
that a Hh signal is at least being transduced by similar regions of the otic
vesicle in the two species.
Otx1 knockdown results in the fusion of the utricular and saccular maculae in the zebrafish inner ear
Having shown that otic vesicles in both lamprey and zebrafish are
responsive to Hh signalling, we sought other candidate genes that may account
for the differences in morphology between the lamprey and zebrafish ear.
Acquisition of a role for Otx1 in the inner ear has previously been implicated
in the evolution of the horizontal semicircular canal and crista in the
gnathostome lineage (Mazan et al.,
2000). Although the lamprey possesses an otx gene, it is
not expressed in the developing ear, which lacks these structures
(Ueki et al., 1998;
Tomsa and Langeland, 1999). In
the mouse, a loss of Otx1 function not only results in the loss of the
horizontal semicircular canal, but also in the lack of constriction of the
utriculo-saccular foramen, and incomplete segregation of the utricular and
saccular maculae (Acampora et al.,
1996; Morsli et al.,
1999; Fritzsch et al.,
2001). As our data suggest that regions of the lamprey macula
communis correspond to the utricular and saccular maculae of zebrafish, we
wanted to test whether a loss of Otx1 function in the zebrafish ear would
result in the fusion of the utricular and saccular maculae, resulting in a
lamprey-like macula communis.
We set out to test this hypothesis by knocking down Otx1 function in zebrafish embryos using an antisense morpholino (MO) designed to block splicing at the exon 3/4 boundary. We injected approximately 5 nl of either 0.66 mM or 1.0 mM otx1 MO into one- to two-cell wild-type (WIK) zebrafish embryos. Similar otic phenotypes were obtained at both concentrations, although with a greater frequency in the higher concentration group [46% (n=74) and 97% (n=68) of ears, respectively]. All further experiments were performed at 1 mM. To measure the efficacy of the otx1 MO, we carried out RT-PCR across the exon 3/4 splice junction at 24 hpf, 50 hpf, 74 hpf and 4 dpf, using cDNA produced from morphant and uninjected control embryos. We also used a primer pair directed to the actin gene as a positive control, which amplified a similar band from all samples (Fig. 8Q,R). The 447 bp otx1 band amplified from wild-type samples represents the correctly spliced product. Initially, this is absent from otx1 morphant samples, but begins to reappear by 48 hpf and is obvious at 74 hpf, suggesting that the efficacy of the morpholino is beginning to diminish by this time. The 471 bp and 408 bp bands amplified from the morphant samples represent aberrantly spliced products, where splicing has occurred from cryptic donor sites 39 bases 5' or 24 bases 3' of the correct exon 3 donor site. This was confirmed by sequencing the PCR products using internal primers (data not shown). Although neither of the aberrantly spliced products is predicted to cause a frame shift, the disrupted region is within the homeobox and is therefore likely to create a non-functional protein.
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The second striking aspect of the morphant phenotype concerns the positioning of the maculae. The anterior and posterior otoliths are, to varying extents, closer together than normal, reflecting the underlying fusion of the anterior (utricular) and posterior (saccular) maculae (Fig. 8K-P). The two maculae are still morphologically identifiable within the fused sensory area, suggesting that they have retained their AP identity despite the lack of non-sensory epithelium separating them. These data therefore support a role for Otx1 in the evolution of separate maculae in the gnathostome ear from an ancestral agnathan macula communis.
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Thornhill, 1972). Hagelin
proposes that both the horizontal regions of the macula communis are
homologous to the utricular macula of gnathostomes
(Hagelin, 1974), suggesting a
double anterior ear. Our observations in the lamprey embryo, however, based on
overall morphology, gene expression and hair cell polarity patterns, suggest
that this is not the case: asymmetries in both the positioning of the
embryonic macula and hair cell polarity patterns are exaggerated in the embryo
compared with the adult. We have also shown that both tbx1 and
follistatin are expressed in a polarised fashion in both lamprey and
zebrafish otic vesicles, suggesting that similar AP patterning mechanisms may
function in the two species.
This pattern does not hold for all genes: fgf8 is expressed at the
anterior of the zebrafish otic vesicle, but the lamprey homologue,
fgf8/fgf17, is not expressed in the otic vesicle of the Japanese
lamprey, Lampetra japonica
(Shigetani et al., 2002). We
have cloned a similar fragment from L. fluviatilis and also see no
otic expression (data not shown). This could be because fgf8/fgf17 is
not the true homologue of fgf8; however, this is unlikely, as
phylogenetic analysis and other aspects of the fgf8/fgf17 expression
pattern suggest homology with zebrafish fgf8
(Shigetani et al., 2002).
Alternatively, it is possible that a different Fgf gene may perform the role
of fgf8 in the lamprey ear. It may be, however, that not all AP
patterning mechanisms are shared between the lamprey and the zebrafish.
Regions of the developing macula communis appear to correspond to the utricular and saccular maculae of gnathostomes
Comparison of our stage 29 embryonic data with those of Lowenstein et al.
(Lowenstein et al., 1968)
(adult L. fluviatilis), Haddon et al.
(Haddon et al., 1999)
(5-day-old zebrafish) and Platt (Platt,
1993) (adult zebrafish), suggests that the anterior and posterior
regions of the macula communis correspond to the utricular and saccular
maculae of zebrafish, respectively. It is likely that this can be extrapolated
to the utricular and saccular maculae of gnathostomes in general, as there are
extensive similarities between polarity patterns in the various groups,
although in most cases these have only been studied in the adult ear
(Baird, 1974;
Popper, 1978;
Popper and Northcutt, 1983;
Mathiesen and Popper, 1987;
Platt, 1993;
Denman-Johnson and Forge, 1999;
Platt et al., 2004). The
general pattern of polarity in the saccular macula, with hair cells pointing
away from a central midline, is grossly similar to the adult zebrafish pattern
in all adult gnathostomes examined, although the shape of this organ varies
dramatically between groups. In most gnathostome species, the adult utricular
macula also has a very similar polarity pattern to the adult zebrafish
pattern. The utricular macula of the Chondrichthyes differs, however,
consisting of intermingled hair cells of opposing polarity
(Baird, 1974).
|
;
Bang et al., 2001;
Bever and Fekete, 2002). Two
different areas of the adult lamprey macula have previously been proposed to
correspond to the lagenar macula. These are the central vertical region
(de Burlet and Versteegh, 1930;
Hagelin, 1974) and the
posterior horizontal region (Lowenstein et
al., 1968). Our work suggests that there is no direct equivalent
of the lagenar macula in the lamprey by stage 29; it is also unlikely to arise
later, as all areas of the adult macula polarity pattern seem to be present in
the stage 29 macula (Fig. 5)
(Lowenstein et al., 1968). In
the absence of saccular and lagenar macula-specific markers, however, it is
difficult to be certain about these homologies.
The lagena is much more variable between gnathostome groups than either the
saccule or the utricle. Polarity patterns differ, with some groups (e.g.
sturgeon, bichir and gar) having a variation on the theme of an antiparallel
array, and others (e.g. teleosts and bowfins) displaying a pattern similar to
that of the utricle (Popper,
1978; Popper and Northcutt,
1983; Mathiesen and Popper,
1987; Platt,
1993). In other instances, notably the holocephalans and
elasmobranchs, the lagena is absent or rudimentary, with varying degrees of
separation from the saccular macula (Baird,
1974). In many tetrapods, including mammals, the lagena seems to
have been lost altogether. Thus the evolution of this endorgan is not
straightforward, perhaps having evolved or been secondarily lost more than
once, as has previously been suggested
(Fritzsch, 1992).
Acquisition of a role for otx1 in the developing gnathostome inner ear can account for all major differences between agnathan and gnathostome inner ears
We now know that our Hh loss- and gain-of-function otic phenotypes in the
zebrafish (Hammond et al.,
2003) do not appear to resemble the lamprey ear. We also find that
a transcriptional target of the Hh pathway, ptc, is expressed in the
ventromedial otic vesicle of both lamprey and zebrafish, suggesting that an AP
patterning mechanism based on Hh signalling may be present in the lamprey.
Similarly, although a loss of Tbx1 function in the mouse gives rise
to a more symmetric ear (Raft et al.,
2004), conserved expression of tbx1 at the posterior of
the lamprey otic vesicle suggests that an otic AP patterning mechanism based
on Tbx1 function may be shared by both species.
The major difference between lamprey and gnathostome ears, therefore, is
not one of symmetry about the AP axis, but concerns the structural arrangement
of the semicircular canals and maculae. In addition to the lack of a
horizontal semicircular canal and crista, the sensory macula is undivided in
lampreys but split into two separate patches in zebrafish. It has been noted
that the utriculo-saccular duct is less constricted in mice lacking Otx1
function, and that the saccular and utricular maculae are incompletely
segregated (Morsli et al.,
1999; Fritzsch et al.,
2001). In the zebrafish Otx1 morphants, the utricular and saccular
maculae are also juxtaposed, forming a single macula communis. Notably,
however, AP polarity is not lost in these ears: the phenotype is quite
different to the `twinned' macula seen in our Hh loss- and gain-of-function
experiments (Hammond et al.,
2003). Thus it appears that the loss of Otx1 function in the
zebrafish, rather than a disruption of Hh signalling, results in an ear that
resembles that of the lamprey. Therefore, the acquisition of otx1
expression in the otic vesicle - a new domain of expression of an existing
transcriptional regulator - can account for both of the major differences in
inner ear morphology between lampreys and gnathostomes.
When did AP polarity in the ear arise?
Our analysis of the developing lamprey ear suggests that an otic vesicle
with distinct AP polarity, similar to that in the zebrafish, was already
present in the common ancestor of these species. Can we extrapolate this to
the more general assertion that the common ancestor of the gnathostomes and
the agnathans had an ear with a distinct otic AP polarity?
|
) (reviewed by Meyer and
Zardoya, 2003). We can therefore conclude that the common ancestor
of the gnathostomes and lamprey, but not necessarily of all vertebrates, was
likely to have had an otic vesicle with distinct otic AP polarity similar to
that seen in the zebrafish.
We cannot exclude the possibility that the common ancestor of extant
gnathostomes and agnathans had a mirror symmetric ear, and that the
enantiomorphic ears produced by genetic or physical disruptions of gnathostome
embryos are indeed atavistic phenotypes. It is also possible that hagfish
embryos have a more symmetrical otic vesicle than do lamprey embryos, and that
this represents the ancestral form. Nevertheless, hagfish appear to have many
uniquely derived characteristics, and, as such, may not be representative of a
common ancestor (Janvier,
1996; Mallatt and Sullivan,
1998). As hagfish are deep-sea fish, their embryos are extremely
difficult to obtain, and we have not been able to examine ear development in
this species.
No evidence of otic placodes has yet been found in the cephalochordates, a
non-vertebrate chordate group (Kozmik et
al., 1999; Streit,
2001). However, in ascidians, the basal non-vertebrate chordate
group, Mazet et al. (Mazet et al.,
2005) present persuasive molecular evidence that the atrial siphon
of Ciona and its associated cupular organs are homologous to the
inner ear of vertebrates. The homology of these structures was initially
proposed based on morphological criteria and was later corroborated by
observation of expression of a pax2/pax5/pax8 homologue in larvae in
regions destined to form the atria
(Jefferies, 1969;
Bone and Ryan, 1978;
Wada et al., 1998). Mazet et
al. (Mazet et al., 2005) now
show that a panel of markers homologous to genes expressed in the vertebrate
inner ear, including foxIc, as well as members of the Pax, Eya and
Six families, mark two ectodermal regions on either side of the posterior
brain of embryonic and larval Ciona. These regions go through a
thickened phase resembling placodes before invaginating to form the atrial
openings. It will be of interest to see whether these structures, which appear
to represent the first evolutionary step towards a vertebrate inner ear, show
any signs of AP polarity.
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ACKNOWLEDGMENTS |
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|
Footnotes |
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Supplementary material for this article is available at http://dev.biologists.org/cgi/content/full/133/7/1347/DC1
|
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