1 Department of Anatomy, University of Cambridge, Cambridge CB2 3DY, UK
2 Laboratorio di Biologia Cellulare e dello Sviluppo, Università di Pisa,
Via Carducci 13, 56010 Ghezzano, Pisa, Italy
Author for correspondence (e-mail:
harris{at}mole.bio.cam.ac.uk)
Accepted 4 July 2003
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SUMMARY |
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Key words: Neural patterning, Eye field specification, Ectopic eye formation, Genetic network, Noggin, Otx2, ET, Rx1, Pax6, Six3, Lhx2, Tll, Optx2, Xenopus laevis, Transcription factor cocktails
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Introduction |
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Many of these EFTFs were originally identified as homologs of genes
required for eye formation in Drosophila melanogaster. For example,
Pax6 is a homologue of Drosophila eyeless and twin of
eyeless (Quiring et al.,
1994), and Six3 and Optx2 are homologues of
Drosophila sine oculis (Oliver et
al., 1995
). The Drosophila genes, twin of
eyeless (toy), eyeless (ey), eyes
absent (eya), sine oculis (so),
dachshund (dac), eye gone (eyg) and
optix either induce ectopic eyes or are required for normal eye
formation (Hanson, 2001
;
Heberlein and Treisman, 2000
;
Kumar, 2001
;
Wawersik and Maas, 2000
). The
expression patterns of toy, ey, so, eya, dac and eyg overlap
in the Drosophila eye field during its specification
(Kumar and Moses, 2001a
). It
has been proposed that the overlapping expression patterns of these genes
drives eye specification and is regulated by the Notch and EGFR signaling
systems (Kumar and Moses,
2001a
). Dominant-negative Notch receptor blocks compound eye
formation, while constitutively activate Notch induces ey and
toy expression and ectopic fly eyes
(Kurata et al., 2000
). A role
for Notch signaling in vertebrate eye formation is suggested by similar
experiments. Mice homozygous for a hypomorphic Notch2 mutation have bilateral
microphthalmia (McCright et al.,
2001
), while activation of Notch signaling induces the expression
of Pax6, Six3 and Rx and causes eye duplications and ectopic
eye tissue formation (Onuma et al.,
2002
).
In Drosophila, it has been possible using genetics to show that
these genes act as a network with hierarchical components and multiple steps
of feedback regulation including functional protein interactions
(Chen et al., 1997;
Pignoni et al., 1997
). More
recently, overexpression and inactivation studies have begun to shed light on
the transcriptional network of EFTFs involved in vertebrate eye formation.
Overexpression of Pax6, Six3, Optx2 and Rx upregulate each
other's expression, while inactivation of each can reduce the expression of
the others (Andreazzoli et al.,
1999
; Bernier et al.,
2000
; Chow et al.,
1999
; Chuang and Raymond,
2001
; Goudreau et al.,
2002
; Lagutin et al.,
2001
; Lagutin et al.,
2003
; Loosli et al.,
1999
; Zuber et al.,
1999
). For example, Pax6 and Six3 crossregulate
each other's expression in both medakafish and mouse
(Carl et al., 2002
;
Goudreau et al., 2002
). As in
Drosophila, functional interactions among the vertebrate EFTFs
involve protein-protein complexes and multiple levels of regulation
(Li et al., 2002
;
Mikkola et al., 2001
;
Stenman et al., 2003
),
implying that a complex network must exist.
As in the salamander, Xenopus laevis neural plate explants form
eye tissue in vitro. When Xenopus anterior neural plate explants are
isolated with underlying prechordal mesoderm at stage 12.5, two retinas form,
demonstrating that the eye field is specified as early as stage 12.5
(Li et al., 1997). The frog
EFTFs, ET, Pax6, Six3, Rx1, Lhx2, tll and Optx2, are
expressed together in the Xenopus anterior neural plate prior to
stage 15 (Bachy et al., 2001
;
Casarosa et al., 1997
;
Hirsch and Harris, 1997
;
Hollemann et al., 1998
;
Li et al., 1997
;
Mathers et al., 1997
;
Zhou et al., 2000
;
Zuber et al., 1999
). In this
paper, we test the idea proposed by Kumar and Moses for Drosophila
eye field specification, in order to determine if the coordinated expression
of EFTFs can also specify the vertebrate eye field. We find that EFTF
cocktails not only induce ectopic eye fields in Xenopus, but generate
ectopic eyes at high frequency outside the nervous system. In addition we
provide an initial characterisation of the functional interactions among the
EFTFs involved in vertebrate eye field specification.
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Materials and methods |
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RNA microinjection
Capped RNA was synthesised in vitro from pCS2.Xnoggin, pCS2.XOtx2,
pCS2R.XET, pCS2R.XPax6, pCS2R.XSix3, pCS2+.XRx1, pCS2R.XLhx2 (3),
pCS2+mt.X-tll, pCS2.XOptx2, pCS2.nucßgal or pCS2GFP template DNA using
the Message Machine kit (Ambion, Austin, TX). X-Gal staining was performed on
embryos injected with 200 ng ßgal as previously described
(Turner and Weintraub, 1994).
GFP was sometimes used (500 ng per embryo) in place of ßgal to
label injected embryos, when there was a concern that ßgal staining would
obstruct in situ staining.
RT-PCR analysis
For animal cap assays, embryos were injected at the two-cell stage with the
indicated RNA(s). Ectodermal explants (animal caps) were isolated from stage
8.5 embryos using the Gastromaster (XENOTEK Engineering, Belleville, IL).
Total RNA was isolated from embryos or pools of ten stage 21 animal caps by
extraction with RNAzol B reagent (Tel-Test, Friendswood, TX, USA). After
treatment with RQ-1 DNAse (Promega, Poole, UK) to remove contaminating genomic
DNA, first-strand cDNA synthesis was performed by reverse transcription with
random hexamers in a volume of 20 µl. Histone H4 PCR was performed using 1
µl of template in a final reaction volume of 12.5 µl to determine the
relative amount of cDNA in each sample. Subsequent PCR was performed using
normalised amounts of template. Cycling conditions were: 92°C, 2 minutes
then 92°C, 45 seconds; 56 or 65°C, 45 seconds; 72°C, 45 seconds,
for 24-30 cycles and ended with a single extension step of 72°C for 10
minutes. An annealing temperature of 65°C was used for the Optx2 primer
set; all other primer sets were annealed at 56°C. The primers used are
shown in Table 1. Radiolabelled
PCR products were separated on 7% polyacrylamide gels, expression levels were
determined using a Storm 860 Phosphoimager with ImageQuant ver. 4.1 software
(Molecular Dynamics, Sunnyvale, CA) and normalised to H4 as a loading
control.
|
cDNA identification and sequence analysis
XSix3 was isolated by screening a stage 42 head cDNA library (a
gift from P. A. Krieg, University of Texas, Austin, TX) with an XSix3
PCR-amplified fragment that was obtained as previously described
(Andreazzoli et al., 1999).
Plating, hybridisation and washing conditions have been described previously
(Franco et al., 1991
). The
XSix3 predicted amino acid sequence is identical to that described by
Zhou and colleagues (Zhou et al.,
2000
). A full-length cDNA was cloned into the
EcoRI/XhoI site of pBS(SK-) vector. A complete description
of the cloning and sequence of the Xenopus Lhx2 will be given
elsewhere (M.E.Z., unpublished). Xenopus Lhx2 sequence has been
submitted to GenBank under Accession Number AY141037.
In situ hybridisation
Whole-mount single and double in situ hybridisation on Xenopus
embryos was performed as previously described
(Andreazzoli et al., 1999;
Harland, 1991
). Bleaching of
pigmented embryos was carried out following color reaction as described by
Mayor et al. (Mayor et al.,
1995
). To determine the change in eye field diameter, the injected
side of embryos was first determined by staining for ßgal expression or
using a fluorescent dissecting microscope to detect GFP. The diameter of the
Rx1 expression domain in the rostrocaudal dimension on the injected
side was then compared with that of the uninjected side.
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Results |
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We first compared the expression domains of these genes with Otx2,
which is required for the establishment of presumptive forebrain and midbrain
territories (Kablar et al.,
1996; Pannese et al.,
1995
). Because the eye field originates within the forebrain, mice
deficient in Otx2 lack eyes
(Acampora et al., 1995
;
Matsuo et al., 1995
). At
gastrula stages, Otx2 is expressed in the entire presumptive anterior
neuroectoderm (Fig. 2A), but
between the end of gastrulation and the beginning of neurulation (stage
12.5/13) it is downregulated in the medial region of its expression domain
(Fig. 2B). This `hole' in the
Otx2 expression domain, is the approximate location of the eye
field.
|
By midneurula stages (stage 14/15), tll and Optx2
expression can be detected by WISH. tll is first observed in a narrow
stripe of cells in the prechordal region of the neural plate. As described by
Holleman et al. (Hollemann et al.,
1998), the expression domain of tll overlaps the
posterior and lateral Pax6 expression domain
(Fig. 2M), distinct from the
eye field. By contrast, Six3 expression overlaps tll
expression medially (Fig. 2N).
The expression domains of Rx1, ET and Optx2 closely border,
but do not significantly overlap the expression domain of tll
(Fig. 2O-Q). These results
suggest that tll is unlikely to be required for eye field
specification as it is expressed after the eye field forms and only partially
overlaps the eye field region. Optx2 transcripts are detected within
the Pax6, Six3, Rx1 and Lhx2 expression domains
(Fig. 2R-T and not shown).
Clearly, some of the EFTFs are expressed outside the definitive eye field,
consistent with the roles of genes like Pax6 and Six3 in the
development of other nearby structures, such as the olfactory epithelium and
the hypothalamus (Lagutin et al.,
2003; Oliver et al.,
1995
; Van Heyningen and
Williamson, 2002
). Within the eye field - the expression patterns
of the EFTFs are dynamic and follow the morphogenesis of the neural plate,
including the lateral migration of the eye field as it begins to separate.
This is illustrated by comparing their expression patterns at stage 12.5/13
and stage 15 only 3 hours later (Fig.
2U,V). These results demonstrate that the anterior neural plate is
subdivided into molecularly distinct domains that express specific subsets of
the EFTFs.
The coordinated overexpression of EFTFs is sufficient to generate
secondary eye fields and ectopic eyes outside the nervous system
To determine if the coordinated expression of EFTF genes is sufficient to
generate eye fields and eyes in vertebrates, we expressed a cocktail of seven
of the EFTFs in developing Xenopus embryos. We injected Otx2, ET,
Pax6, Six3, Rx1, tll and Optx2 RNAs simultaneously into one
blastomere at the two-cell stage with ßgal to identify the injected side
of the embryo. Lhx2 was intentionally left out of the cocktail, as we
needed an early marker to identify the presence of ectopic eye field.
Preliminary experiments demonstrated that the absence of Lhx2 from
the cocktail had little effect on the observed phenotypes. Coordinated
expression of the EFTF cocktail induced ectopic expression of Lhx2 in
100% of injected embryos. Ectopic Lhx2 was detected both within and
outside the nervous system (Fig.
3B-D), whereas its normal expression domain is limited to the
anterior neural plate (Fig.
3A). When the injected embryos were grown to stage 45, we found
90% of these embryos expressed ectopic retinal pigment epithelium (RPE)
on the injected side. Sections taken through this ectopic tissue and
immunostained for opsin, revealed that photoreceptors were often associated
with the ectopic pigment. Approximately 20% of injected embryos clearly
developed quite large ectopic eyes, the most striking aspect of which was
their location. Ectopic eyes were detected near the CNS, but were also often
found at locations far from the CNS, e.g. in the belly region and even at the
anus (Fig. 3E-H). These tissues
expressed markers for differentiated retinal ganglion, rod and cone
photoreceptor cells, RPE and lens (Fig.
3I-J and not shown), indicating that they were indeed eyes as
defined by the cell types detected as well as their morphology.
|
EFTFs are induced by the combined action of noggin and
Otx2
The above results suggest that eye field formation might result from a
series of progressive inductions. Extending this hypothesis prior to eye field
specification, the ectoderm is converted into the neural plate in response to
neural inducers. Next, presumptive forebrain is specified by the regulated
expression of Otx2. Finally, the eye field forms within the
presumptive forebrain. If this model were correct, one would expect that both
noggin and Otx2 are upstream of the EFTF genes, and may
activate them either directly or indirectly. We therefore used the animal cap
assay to test the effect of noggin and Otx2 on the
expression of the EFTFs.
In untreated animal caps, only ET and Six3 were detected (U, Fig. 4A), consistent with their early expression in the embryo (Fig. 1). The neural inducer, noggin, dramatically increased the expression of many of the eye field transcription factors including Pax6, Six3, Rx1, Lhx2, tll and Optx2. Interestingly, noggin strongly repressed ET expression. Similar results were found with another neural inducer, chordin (data not shown). Both noggin and Otx2 induced the neural marker NCAM and the cement gland marker XAG. Unlike noggin, however, Otx2 did not alter the expression of ET, Pax6, Six3, Rx1, Lhx2, tll or Optx2. The inability of Otx2 to induce any EFTFs suggests that their regulation is Otx2 independent.
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ET, Rx1 and Pax6 regulate Otx2
expression in the anterior neural plate and presumptive eye field
The loss of Otx2 expression between stages 12 and 13 is
synchronised with the induction of several EFTFs in the eye field
(Fig. 2), suggesting that they
may repress Otx2 expression in the anterior neural plate during
normal embryonic development. It was, in fact, previously shown that
overexpression of Rx1 represses Otx2 expression in the
neural plate (Andreazzoli et al.,
1999). To test if other EFTFs are also capable of regulating
Otx2 expression, we injected the EFTFs into one cell of two-cell
stage embryos and determined their effect on Otx2 expression using
whole-mount in situ hybridisation.
Otx2 expression normally extends both rostral to the neural plate,
in a region corresponding to the cement gland anlagen, and posterior to the
eye field, in the region fated to be the primordium of the mesencephalon
(Eagleson et al., 1995),
Fig. 2B, Fig. 5A). We found that both
ET and Rx1 repressed Otx2 expression throughout the
entire anterior neural plate (Fig.
5B,C). Otx2 expression was repressed in 100% of embryos
injected with ET RNA and 94% of embryos injected with Rx1
RNA (Fig. 5G). In 74% of
embryos injected with Pax6, there was an expansion of the
Otx2 expression domain (Fig.
5D,G). Interestingly, Otx2 expression was expanded
laterally and caudally but not into the eye field by Pax6
(Fig. 5D). Neither
Six3 nor Lhx2 altered Otx2 expression
(Fig. 5E,F). These results
demonstrate that both ET and Rx1 are able to repress the
expression of Otx2 in the eye field region.
|
To test this pathway in vivo, we injected ET or Rx1 RNA and assayed for changes in Rx1 or ET expression in stage 13 embryos. To target the eye field, we injected ET or Rx1 RNA into dorsal blastomeres at the four-cell stage. Consistent with the animal cap assays, Rx1 had no effect on ET expression at stage 13 (compare Fig. 5J-K). As predicted, ET strongly induced the expression of Rx1 in 93% of injected embryos (n=29). Interestingly, Rx1 induction was only observed in the anterior neural plate in the presumptive eye field region (Fig. 5M). When ET was injected ventrally, Rx1 induction was not observed (not shown). When ET was injected at the two-cell stage (resulting in the expression of ET throughout an entire half of the embryo), Rx1 induction was again only detected in the anterior neural plate, including the presumptive eye field region (not shown).
The downregulation of Otx2 in the eye field can thus be explained by the fact that ET induces the expression of Rx1, which then represses Otx2 (Fig. 5N). However, these results, do not rule out the possibility that ET also represses Otx2 independently of Rx1. In fact, there is some indication that this pathway may also be operative as induction of Rx1 by ET was detected in most but not all embryos while ET repressed Otx2 expression in all embryos tested. In addition, we found that in the presumptive cement gland region, ET represses Otx2 (Fig. 5B) without inducing Rx1 (Fig. 5M), implicating an Rx1-independent mechanism in this tissue.
Both Otx2 and noggin potentiate functional
interactions between the EFTFs.
The inducing effect of ET on Rx1 is limited to the
anterior forebrain, suggesting that Otx2, although not an inducer of
EFTFs itself, may provide an environment that primes the anterior
neuroectoderm for eye field formation (Fig.
4). If so, co-injection of Otx2 might potentiate the
effects of ET on the activation of downstream EFTFs. ET is
an obvious candidate for co-injection experiments as it is expressed earlier
than all other EFTFs and has the most restricted expression domain.
As predicted by the above model, Otx2 strongly potentiates the induction of Rx1 by ET in the animal cap assay (Fig. 6A). This is in spite of the fact that Otx2 alone does not induce Rx1 expression in vitro. The effect is dose dependent as co-injection of 50 and 100 pg of Otx2 with ET induced a three- and eleven-fold increase in Rx1 expression. Noggin also potentiates the Rx1 induction by ET. Weak Rx1 expression was detected in animal caps from embryos injected with suboptimal amounts of ET or noggin. However, co-injection of noggin and ET RNAs at these same concentrations induced a greater than five-fold increase in Rx1 expression (Fig. 6B).
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The circuitry of the EFTF network revealed by systematic
overexpresssion studies in animal caps
The relative timing, spatial expression patterns, cocktail subset
experiments and directed overexpression studies suggest that early genes such
as ET may be required for the expression of later expressed genes in
the eye field and rule out the possibility that later genes such as
Optx2 are required for the initial induction of earlier EFTFs.
Nevertheless, dominant-negative Optx2 and tll constructs and
Optx2 knockouts retard eye field growth and thus lead to reduced
levels of other EFTFs, including ET and Pax6
(Hollemann et al., 1998;
Li et al., 2002
;
Zhu et al., 2002
;
Zuber et al., 1999
). Because
of the possibility of such indirect feedback effects on EFTF expression, it is
difficult to unravel the EFTF network using a loss-of-function approach alone,
especially when the initial expression of these genes in the eye field is so
closely synchronised. To overcome this problem, we used a systematic approach,
injecting embryos with one EFTF at a time, and screening the injected caps
using RT-PCR to detect changes in the expression of the remaining EFTFs.
A representative experiment and a summary of our results are shown in Fig. 7A,B. This data was then assembled into a circuit using Occam's razor (Fig. 7C) that shows the most parsimonious set of necessary interactions needed to explain the results. These results confirm many of the predictions made from the expression studies (Figs 1, 2) and incomplete cocktail experiments (Fig. 3). ET, positioned at the front of the circuit induces the expression of Rx1, Lhx2 and tll, which in turn induce the expression of Pax6:Lhx2:tll:Optx2, Pax6 and Pax6:Six3:Lhx2, respectively. However, ET is unique in that none of the EFTFs studied here can induce its expression in the animal cap assay. Conversely, Optx2, the last of these genes to be expressed during eye formation, is induced by both Pax6 and Rx1, yet is unable to induce any of the earlier expressed EFTFs. The four EFTFs expressed earliest and deemed most crucial by the incomplete cocktail method are not only situated towards the front end of the circuit, but are also factors like Pax6, Six3 and Otx2 that previous studies have demonstrated are central to eye formation. Pax6 and Six3 induce each other's expression as well as that of Lhx2 and tll. Pax6 also induces Optx2. Lhx2 and tll were induced by five and four of the six EFTFs, respectively, confirming the hypothesis that a sufficient amount of these genes could be induced by the remaining EFTFs to compensate for their removal from the eye inducing cocktails. In summary, ET at the front of the circuit induces Rx1, which activates a crossregulatory network, including Pax6, Six3, Lhx2 and tll, followed by Optx2 induced by Pax6.
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Discussion |
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In the fly, toy, ey, so, eya, dac and eyg are
co-expressed in the second larval stage and the elimination of any of them
reduces the probability of eye formation
(Kumar and Moses, 2001a). In
Xenopus, ET, Rx1, Pax6 and Six3 are co-expressed in the
anterior neural plate and the elimination of any of them from a cocktail of
EFTFs injected into the Xenopus embryo reduces the frequency of
ectopic eye tissue formation.
These remarkable similarities in general developmental design are perhaps
logically predicated based on the functional and structural homologies between
the Drosophila eye genes and the vertebrate EFTFs
(Hanson, 2001;
Wawersik and Maas, 2000
).
orthodenticle (otd) the Drosophila homolog of Otx
genes is required for development of the eye, antenna and anterior brain, and
is normally expressed in a wide domain that spans the dorsal midline and
encompasses the entire dorsal head ectoderm
(Finkelstein and Boncinelli,
1994
). Its expression is turned off in the head midline during
development and in the part of the visual primordium that forms the posterior
optic lobe and the larval eye (Royet and
Finkelstein, 1996
). This is strikingly similar to the changes we
see in the Xenopus Otx2 expression pattern. The
optomotor-blind (omb) gene is a member of the Tbx2
T-box subfamily. ET shares more sequence homology with omb
than any other gene in the fly genome (not shown). omb expression is
first detected in the optic lob anlagen, later expanding to a larger part of
the developing larval brain (Poeck et al.,
1993
). In the eye imaginal disc, omb is detected in glial
precursors, posterior to the morphogenetic furrow and in the optic stalk. Null
omb mutants die in pupal stage and show severe optic lobe defects
(Pflugfelder et al., 1992
).
The Drosophila Rx homolog is not expressed in the larval eye imaginal
discs nor the embryonic eye primordia
(Eggert et al., 1998
;
Mathers et al., 1997
).
However, it is expressed prior to ey in the procephalic region from
which the eye primordia originates, suggesting a role for Drosophila
Rx prior to ey during eye formation in the fly
(Eggert et al., 1998
;
Mathers et al., 1997
). It has
therefore been suggested that Drosophila Rx may only be required for
early brain development (Eggert et al.,
1998
). Finally, our results showing Pax6 as the most
critical component of the Xenopus EFTF cocktail with respect to the
induction of ectopic eyes meshes well with the general prominence given to
Pax6 and its Drosophila homologues ey and
toy as transcription factors centrally involved in early eye
development (Wawersik and Maas,
2000
).
The functional interactions among the genes required for
Drosophila eye formation have been extensively investigated
(Heberlein and Treisman, 2000;
Kumar and Moses, 2001b
). Using
the ectodermal explant assay, we identified functional epistatic interactions
among the vertebrate EFTFs. There are some striking similarities with the
functional interactions among the fly EFTFs. For example, we see induction of
Six3 and Optx2 by Pax6 and induction of
Pax6 by Six3 in ectodermal explants
(Fig. 7B). In Drosophila,
ey can induce ectopic so and optix expression and
ectopic eye formation induced by co-expression of so with
eya results in the activation of the ey gene
(Halder et al., 1998
;
Niimi et al., 1999
;
Pignoni et al., 1997
;
Seimiya and Gehring,
2000
).
Some differences between fly and vertebrate eye formation are also evident.
We found that tll was able to induce the expression of Pax6,
Six3 and Lhx2, and that Pax6 and Six3 induce
tll expression. Drosophila tll does not require ey
or so in the embryonic visual system
(Daniel et al., 1999;
Rudolph et al., 1997
). We
found Lhx2 to be induced by all the EFTFs investigated in this report
with the exception of Optx2 (Fig.
7A,B). The gene apterous (ap) is the most
homologous Drosophila gene to Lhx2; however,
apterous loss-of-function mutants have no reported defect in eye
formation (Bourgouin et al.,
1992
; Cohen et al.,
1992
; Lundgren et al.,
1995
).
Vertebrate EFTFs and their functions
It is interesting to examine the results of this paper in light of studies,
particularly knockout studies, on specific EFTFs in other vertebrates.
Otx2-/- mice lack forebrain and midbrain
(Acampora et al., 1995;
Matsuo et al., 1995
). In
Xenopus, anterior structures are also lost when Otx2 fused
to the engrailed transcriptional repressor is expressed in embryos
(Isaacs et al., 1999
). An
early requirement for Otx2 in vertebrate eye formation is implied
from studies in which Pax6, Six3, Optx2 or Rx overexpression
result in the formation of ectopic eye tissues, because as Chuang and Raymond
observed, ectopic eye tissue is only generated in the head region defined by
Otx2 expression (Chuang and
Raymond, 2002
). Using EFTF cocktails, we were able to generate
ectopic eyes outside of the nervous system
(Fig. 3). Otx2 clearly
potentiates the functional interaction among the EFTFs and is a crucial
component of the mix (Figs 3,
6). However, a more detailed
analysis will be required to determine if ectopic eye formation outside the
nervous system is a result of including Otx2 in the cocktail.
Our results suggest a role for ET as an initiator of eye field
specification. Originally identified as a T-box family member expressed very
early in the eye field, (Li et al.,
1997), subsequent overexpression studies showed that ET
is involved in the dorsoventral patterning of the eye
(Wong et al., 2002
). The more
than 50 T-box family members identified have been classed into five
subfamilies (Papaioannou and Silver,
1998
; Wilson and Conlon,
2002
). ET is a member of the Tbx2 subfamily that
includes the Tbx2, Tbx3, Tbx4 and Tbx5 genes, and is most
similar to Tbx3. The mouse and chicken orthologues of Tbx2,
Tbx3 and Tbx5 are all expressed in overlapping domains within
the dorsal neural retina of the embryonic optic cup
(Chapman et al., 1996
;
Gibson-Brown et al., 1998
;
Sowden et al., 2001
) - very
similar to the expression pattern of Xenopus ET
(Li et al., 1997
;
Takabatake et al., 2000
).
Evidence of a role for Tbx3 in mammalian eye formation is limited.
Mouse Tbx3 has been detected in preimplantation embryos as early as
3.5 days post coitum (Bollag et al.,
1994
; Chapman et al.,
1996
) and in the retinal primordia
(Takabatake et al., 2000
), but
no Tbx3 null mutants have been reported. Hypomorphic mutations in
human Tbx3 cause Ulnar-mammary syndrome (UMS), an autosomal dominant
disorder affecting limb, apocrine-gland, tooth, hair and genital development
with no apparent effect on the eye (Bamshad
et al., 1997
). However, the mouse Tbx3 is clearly
expressed in some embryonic tissues that are unaffected in the human syndrome
(Bamshad et al., 1997
;
Chapman et al., 1996
). It may
be that the human mutations responsible for UMS do not affect its role in
these other tissues, or that other T-box family members compensate for a
defective form of Tbx3. Interestingly, the putative orthologue of
zebrafish Tbx2, tbxc, is expressed in the single eye field at the end
of gastrulation (
10 hours post fertilisation, hpf), while the putative
zebrafish Tbx3 orthologue is not expressed prior to 24 hpf and is not
reported to be expressed in the retina
(Dheen et al., 1999
;
Ruvinsky et al., 2000
;
Yonei-Tamura et al., 1999
).
Thus, it may be that the zebrafish tbx2, not zebrafish tbx3
is the functional homologue of Xenopus ET.
Overexpresssion studies are very useful in characterising genetic networks, but clearly do not rule out the existence of parallel pathways and additional intermediates. For example, the fact that noggin can induce Rx1 while repressing ET means that a parallel pathway for Rx1 induction must exist and that ET expression is not essential for Rx1 induction. Whether ET is required in vivo for Rx1 induction is not known. However, the question of requirement and the normal pathway of activation are different issues. The observation that ET is expressed prior to Rx1 and induces Rx1 expression, and that this activity is enhanced in neuralised tissue suggests very strongly that this pathway is active in the embryo.
The role of Rx in vertebrate eye formation has been investigated
in more detail than ET. Rx homologues have been identified in humans,
rodents (mouse and rat), chicken, fish (zebrafish, medakafish and cavefish),
as well as frog (Casarosa et al.,
1997; Loosli et al.,
2001
; Mathers et al.,
1997
; Ohuchi et al.,
1999
; Strickler et al.,
2002
; Tucker et al.,
2001
). Mice lacking functional Rx homologues do not
develop eyes (Mathers et al.,
1997
; Tucker et al.,
2001
). In Rx-/- mice, neither Pax6
nor Six3 are upregulated in the presumptive optic area as early as
E9.0 (Zhang et al., 2000
).
These results are consistent with our own, indicating that Rx has an
early role in eye formation and is upstream of Pax6 and
Six3. The medakafish mutant eyeless (el), is the result of
an intronic insertion into the Rx3 locus
(Loosli et al., 2001
).
Rx3 is required for evagination and proliferation of the optic
vesicle. In medaka Rx3 mutants, both Tbx2 and Tbx3
expression in the retina is lost, suggesting that Rx3 is genetically
upstream of these genes or that Tbx2/3 are expressed in tissues lost or
re-patterned in Rx3 mutants
(Loosli et al., 2001
). This is
in contrast to our results, which show Rx1 is downstream of
ET. Two Rx homologues have been reported in Xenopus
(Rx1 and Rx2) and medakafish (Rx2 and
Rx3), while three have been identified in zebrafish (rx1,
rx2 and rx3) (Casarosa et
al., 1997
; Chuang et al.,
1999
; Loosli et al.,
2001
; Mathers et al.,
1997
; Winkler et al.,
2000
). Medakafish Rx3 shares greater sequence homology
with Xenopus XRx2 than XRx1 (not shown). In medaka
Rx3 mutants, Rx2 expression is unaffected and morphogenetic
movements are normal until optic vesicle evagination, Rx2-positive
retinal tissue forms and the separation of the single retinal field into the
two eye primordial is unaffected (Winkler
et al., 2000
). Medakafish Rx2 is exclusively expressed in
presumptive and differentiated retinal tissue during and after gastrulation
(Loosli et al., 2001
;
Mathers et al., 1997
). These
results suggest that medakafish Rx2, or an as yet unidentified medaka
Rx homolog is acting as the Rx1 functional homologue in
Xenopus.
Rx-/-, Pax6-/-,
Lhx2-/- and Six3-/- mice all lack eyes
(Grindley et al., 1995;
Mathers et al., 1997
;
Porter et al., 1997
;
Tucker et al., 2001
), but the
morphological defects seen in these embryos also give clues to the order in
which they are required during eye formation. Rx-/-
embryos do not develop optic sulci, vesicles or cups, which normally form
between stages E8.5 and E9.5 (Zhang et
al., 2000
). Pax6-/- (Sey) and
Lhx2-/- mice, however, do develop optic vesicles, which
form optic stalks and rudimentary optic cups
(Grindley et al., 1995
;
Porter et al., 1997
). In
Pax6-/- animals, Rx1, Six3 and Lhx2
expression is unaffected as late as E10.5
(Bernier et al., 2001
;
Zhang et al., 2000
). Recently,
Lagutin and colleagues demonstrated a requirement for Six3 during
forebrain development (Lagutin et al.,
2003
). Six3-/- mice die at birth, and lack
head structures anterior to the midbrain, including the eyes. Mouse
Six3 expression is first detected at E7.0 to E7.5 in the anterior
neuroectoderm and the first morphological abnormalities in
Six3-/- mice are seen at E8.0. Rx1 expression,
although significantly reduced, is still detected at E8.5 in the anterior
neural plate of Six3-null animals, demonstrating that early
Rx1 expression does not require Six3. By contrast, neither
Rx1 nor Pax6 is detectable at optic vesicle stages, as these
structures do not develop. Interestingly, we also detect Six3
expression prior to eye field formation in the frog. Xenopus Six3 is
detected weakly until stage 9, is lost, and then increases dramatically during
eye field specification (Fig.
1). Perhaps there results point to a twofold role for
Six3 in eye formation - an early neural patterning function then as a
component of the self-regulating network responsible for eye field
specification. Our animal cap analysis indicates that Optx2 does not
regulate the expression of any of the EFTFs, consistent with the observation
that Pax6, Six3 and Rx expression are normal in the small
eyed Six6-/- mouse (Li
et al., 2002
). Dominant-negative tll constructs inhibit
the growth of the optic vesicle in Xenopus
(Hollemann et al., 1998
), and
tll-/- mice show signs of retinal degeneration 3 weeks
after birth that eventually result in visual defects
(Yu et al., 2000
). Our results
similarly argue that tll and Optx2 are not involved in the
earliest steps of eye field formation, but that ET, Rx1, Pax6, Six3
and Lhx2 are part of a self-regulating network of nuclear factors in
vertebrates that helps specify the eye field.
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ACKNOWLEDGMENTS |
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Footnotes |
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* These authors contributed equally to this work
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