The first morphological evidence of eye formation in
vertebrates is a bilateral expansion of tissue from the early
forebrain to form the optic vesicles. It has been known for
nearly 70 years, however, that presumptive eye tissue (eye
field) exists prior to optic vesicle formation. In the
salamander, a small piece of anterior neural plate can be
isolated 6 hours prior to optic vesicle formation, and
remarkably, after another 24 hours in vitro this tissue will
transform into a single small but histologically normal eye
(Lopashov and Stroeva, 1964). Modern molecular evidence
shows that the eye anlagen is specified at the neural plate
stage when a group of eye field transcription factors, EFTFs,
are expressed in the anterior neural plate. The EFTFs include
ET, Rx1, Pax6, Six3, Lhx2, tll and Optx2 (also known as Six6).
Genetic evidence clearly demonstrates the importance of
these EFTFs in vertebrate eye formation. Mutations of PAX6,
SIX3 and OPTX2 in human result in malformations affecting
the eyes (Wawersik and Maas, 2000). The targeted, or
spontaneous mutation of Pax6, Rx (Rax – Mouse Genome
Informatics) Lhx2, Tll, Six3 and Six6 in mouse, results in
animals with abnormal or no eyes (Hill et al., 1991; Lagutin
et al., 2003; Li et al., 2002; Mathers et al., 1997; Porter et al.,
1997; Tucker et al., 2001; Yu et al., 2000). Similar phenotypes
have been observed when homologues of Six3, Pax6, tll, Rx1
and Optx2 genes have been functionally inactivated in other
vertebrate species (Carl et al., 2002; Chow et al., 1999;
Hollemann et al., 1998; Isaacs et al., 1999; Loosli et al., 2001;
Zuber et al., 1999). Not only are these EFTFs necessary for
eye formation, but in some contexts they are also sufficient.
Overexpression of Pax6, Six3, Rx and Optx2 homologues can
expand or induce eye tissues in the nervous system of
vertebrates (Andreazzoli et al., 1999; Bernier et al., 2000;
Chow et al., 1999; Chuang and Raymond, 2001; Loosli et al.,
1999; Mathers et al., 1997; Oliver et al., 1996; Zuber et al.,
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
Several eye-field transcription factors (EFTFs) are
expressed in the anterior region of the vertebrate neural
plate and are essential for eye formation. The Xenopus
EFTFs ET, Rx1, Pax6, Six3, Lhx2, tll and Optx2 are
expressed in a dynamic, overlapping pattern in the
presumptive eye field. Expression of an EFTF cocktail with
Otx2 is sufficient to induce ectopic eyes outside the nervous
system at high frequency. Using both cocktail subsets and
functional (inductive) analysis of individual EFTFs, we
have revealed a genetic network regulating vertebrate
eye field specification. Our results support a model of
progressive tissue specification in which neural induction
then Otx2-driven neural patterning primes the anterior
neural plate for eye field formation. Next, the EFTFs form
a self-regulating feedback network that specifies the
vertebrate eye field. We find striking similarities and
differences to the network of homologous Drosophila genes
that specify the eye imaginal disc, a finding that is
consistent with the idea of a partial evolutionary
conservation of eye formation.
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
Specification of the vertebrate eye by a network of eye field
Michael E. Zuber1,†, Gaia Gestri2,*, Andrea S. Viczian1,*,†, Giuseppina Barsacchi2and William A. Harris1,‡
1Department of Anatomy, University of Cambridge, Cambridge CB2 3DY, UK
2Laboratorio di Biologia Cellulare e dello Sviluppo, Università di Pisa, Via Carducci 13, 56010 Ghezzano, Pisa, Italy
*These authors contributed equally to this work
†Present address: Departments of Ophthalmology and Biochemistry & Molecular Biology, SUNY Upstate Medical University, Syracuse, NY 13210, USA
‡Author for correspondence (e-mail: firstname.lastname@example.org)
Accepted 4 July 2003
Development 130, 5155-5167
© 2003 The Company of Biologists Ltd
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.,
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
Materials and methods
Fertilised eggs were obtained from pigmented Xenopus injected with
500 U of human chorionic gonadotropin (Sigma-Aldrich Company,
UK) to induce egg laying. Embryos were dejellied with 3.3 mM DTT
in 200 mM TrisHCL (pH 8.8) and staged according to Nieuwkoop
and Faber (Nieuwkoop and Faber, 1994).
Capped RNA was synthesised in vitro from pCS2.Xnoggin,
pCS2+.XRx1, pCS2R.XLhx2 (3), pCS2+mt.X-tll, pCS2.XOptx2,
pCS2.nucβgal or pCS2GFP template DNA using the Message
Machine kit (Ambion, Austin, TX). X-Gal staining was performed on
embryos injected with 200 ng βgal as previously described (Turner
and Weintraub, 1994). GFP was sometimes used (500 ng per embryo)
in place of βgal to label injected embryos, when there was a concern
that βgal staining would obstruct in situ staining.
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.
For multistage analysis, RNA was isolated from three embryos per
stage and a total of four sets of RNAs from staged embryos were
tested yielding similar results. For animal cap assays, each experiment
was performed between three and five times to ensure reproducibility.
Control experiments (not shown) with cloned templates demonstrated
that the amplification efficiencies did not vary between primer sets
Development 130 (21)Research article
Table 1. Primer sets used for PCR analysis
Hollemann et al., 1998
Target geneUpstream primer (5′ to 3′) Downstream primer (5′ to 3′) of bp
CCT ATC CTT GAC TTG CTA CA
GCA ACC TGG CGA GCG ATA AGC
TTG TCT GTC TGT CTC TTG TT
CCC CAA CAG GAG CAT TTA GAA GAC
ACT TGC CTC TCG TGC TGC TCT ACT G
ACC CTC CTC CCC CAT TAC TCA C
ACA GAG CAG CGG CGG CAA AGA
CAC AGT TCC ACC AAA TGC
GGA TGG ATT TGT TAC ATC CGT C
GAC TGG TGC TGT TCA ACC TTG
CGG GAT AAC ATT CAG GGT ATC ACT
GTT TTG GGG AAG GAG GGT AT
CCT GCC GTC TCT GGT TCC GTA GTT
TTC TGT GTT TGG TTT ATC TC
AGG GCA CTC ATG GCA GAA GGT T
ATC CGG TCG GGT TGC TCA TCT T
AGG GCA TAT CTG GGC ATC TTC A
GAG CGC TCC CTG GTA CTG TGA CTG A
GGA ATC AAG CGG TAC AGA
CAC TCT CCG AGC TCA CTT CCC
CAT TGG GAA ATA ACT GGG ACC
ATC CAT GGC GGT AAC TGT CTT CCT
Primer sets were designed from the indicated GenBank sequence or from the indicated source (see Materials and methods for the details of the PCR reaction
conditions). Xenbase primer sequences can be found at http://www.xenbase.org/
5157Genetic network in vertebrate eye formation
using these conditions. A no reverse transcription control was
included in each reaction to check for the presence of contaminating
genomic and plasmid DNA. Subcloning and sequencing confirmed the
identities of the amplified products.
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 βgal 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.
Vertebrate EFTF expression is coordinated
and suggests a genetic hierarchy
To determine the relative timing of vertebrate EFTF
expression, we used RT-PCR to establish the
developmental stage at which each is first and strongly
expressed. Only Six3 is expressed at detectable levels
in the egg (Fig. 1A). Early Six3 expression is transient
and lost by stage 10.5. ET, Pax6, Rx1, tll, Lhx2 and
Optx2 were first detected at stages 10, 10.5, 11, 11.5,
12 and 12.5, respectively. In contrast to the first
detectable expression, strong expression of Pax6, Six3,
Rx1, tll and Lhx2 is nearly simultaneous and starts
between stages 12 and 12.5, while strong induction of
ET and Optx2 occurs by stages 10.5 and 14/15,
respectively. Some variation in the expression of these
genes was observed from experiment to experiment
(Fig. 1B). However, the relative timing of expression
was consistent in each experiment. These results demonstrate
a tightly coordinated, strong expression of five EFTFs within
a 30 minute time span. In addition, these results suggest that:
(1) ET expression does not require the expression of Pax6,
Six3, Rx1, tll, Lhx2 or Optx2; and (2) Optx2 is not required for
the initial expression of ET, Pax6, Six3, Rx1, tll or Lhx2.
The EFTFs are expressed in overlapping patterns
during vertebrate eye field formation
Interactions suggested by the synchronised timing of EFTF
expression could only operate if these factors were colocalised.
Therefore, we used double whole-mount in situ hybridisation
to determine the relative expression patterns of the eye field
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
Fig. 1. Relative timing of EFTF expression. RT-PCR was
used to detect the expression of ET, Pax6, Six3, Rx1, tll,
Lhx2 and Optx2 in the unfertilised embryo (E) and until
stage 18 of development. The transient expression of Six3
and tll prior to stage 10.5 was detected in four independent
experiments. PCR amplification of Histone H4 demonstrates
that approximately equivalent amounts of cDNA templates
were used. A duplicate set of reactions from stage 18
embryo RNA were run without reverse transcriptase to test
for contaminating plasmid and genomic DNA (18 –RT). The
PCR products were subcloned and sequenced to confirm
their identities. (B) Schematic showing the results of
multiple experiments. Each dot represents the developmental
stage at which strong induction was observed.
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.
ET, Pax6, Six3, Rx1 and Lhx2 are all first detectable in the
presumptive eye field before the completion of gastrulation and
the beginning of neurulation (stage 12) (not shown). Rx1 is
expressed neatly within the inner limits of the ‘hole’ in the
Otx2 expression domain (Fig. 2C), and within the Rx1 domain
are the even smaller expression domains of Lhx2 and ET (Fig.
2D,E). Both Pax6 and Six3 expression domains are slightly
larger that the Rx1 domain and overlap that of Otx2 (Fig. 2F-
I). To define the anterior and lateral expression boundaries of
these genes more clearly, we used the homeodomain-
containing transcription factor Emx1 as a positional marker.
Emx1 is expressed in the rostral neural plate in the
telencephalic primordium at early neural stages
(Pannese et al., 1998). The expression domains of Rx1
and Emx1 do not overlap, although both Pax6 and Six3
overlie Emx1 expression confirming that the lateral
expression of Rx1 (and therefore Lhx2 and ET) lies
within both the Pax6 and Six3 expression domains (Fig.
2J-L). Although the Six3 expression domain clearly
extends beyond the anterior limit of Emx1 (Fig. 2L), the
most anterior limit of Pax6 expression is coincident with
Emx1 (Fig. 2K). ET, Pax6, Six3, Rx1 and Lhx2 thus have
overlapping, but not identical, expression domains in the
eye field region. The ET expression domain is the most
restricted of these genes within the presumptive eye
field and the Six3 domain is the broadest. One can think
of concentric rings of expression in domains of
decreasing size – Six3 > Pax6 > Rx1 > Lhx2 > ET (Fig.
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.
Development 130 (21)Research article
Fig. 2. Comparison of EFTF expression patterns by double
whole-mount in situ hybridisation. Otx2 expression at stage
12 (A) and 13 (B). In C-I and K-T, the dark blue stain is the
expression pattern of the gene named on the left, while the
magenta stain is the expression pattern of the gene named on
the right, at the stages shown. For example, in C, Otx2 is
dark blue and Rx1 is magenta. (J) Both Emx1 and Rx1 stain
dark blue. (J-L) The Rx1 (J), Pax6 (K) and Six3 (L)
expression borders are indicated by a broken line. A
schematic summary of the overlapping expression patterns of
the eye field transcription factors at stage 12.5/13 (U) and 15
(V) is shown. Scale bars: in A, 300 µm for A-L; in M, 300
µm for M-T.
5159Genetic network in vertebrate eye formation
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 βgal 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.
Cocktail subsets reveal crucial circuit components
The high efficiency with which cocktails of EFTFs generate
ectopic eye tissue enabled us to determine those most crucial
for eye formation. To do this, we systematically injected
cocktail subsets lacking one of the EFTFs and determined their
efficiency at inducing ectopic eye tissues. The most dramatic
reductions in ectopic eye tissue were observed when Pax6 was
Fig. 3. Coordinated expression of EFTFs induces ectopic Lhx2
expression and ectopic eye-like structures outside the nervous system.
(A-D) In situ hybridisation for Lhx2 expression (violet) in stage 20
embryos. (A) Uninjected embryo shows the normal expression pattern
of Lhx2. (B-D) Otx2, ET, Pax6, Six3, Rx1, tll, Optx2 and β-gal RNAs
were injected into one cell of two-cell stage embryos. β-gal staining
(light blue) shows the injected side. Arrow indicates to ectopic Lhx2
expression (violet). (E-H) Embryos injected with Otx2, ET, Pax6, Six3,
Rx1, tll and Optx2 RNAs, and grown to stage 45. Arrows indicate
ectopic eyes and arrowheads point to lens. (I,J) Sections through
ectopic eyes reveal the layering of ganglion (GCL), inner nuclear
(INL) and outer nuclear (ONL) cell layers. (I) The retinal ganglion
cells are detected using the marker, hermes (violet). Rod
photoreceptors are identified in the outer nuclear layer, by the
detection of opsin (green, J). Opsin also stains a rosette of cells
between the GCL and the lens. Lens was detected using anti-
crystalline antibodies and stains red in J. (K) Cocktail subsets reveal
the relative importance of EFTFs for eye tissue induction. Animals
were scored according to severity of phenotype – from ectopic pigment/eye tissue (most severe) to normal animals.
When all the factors were present, most embryos developed ectopic pigment or eye tissue (Ect. Pig./Eye Tissue).
When Pax6 was left out of the cocktail, for example, the frequency of ectopic pigment or eye tissue was greatly
reduced and 20% of the embryos were unaffected (Normal).
removed (Fig. 3K), followed by Otx2, Six3 and ET. Removal
of Rx1, Optx2 or tll from the EFTF cocktails affected ectopic
eye tissue induction to a lesser extent (Fig. 3K). The strong
effect of removing Pax6, Otx2 and Six3 from the cocktails may
have been predicted, as numerous studies have demonstrated
these genes are required for eye formation. However, a crucial
role for ET in early eye formation has not been reported.
Conversely, the relatively small effects of removing Rx1,
Optx2, tll and Lhx2 from the cocktails is intriguing because
each of these genes has been shown to be required for normal
eye formation. Remembering that this is non-mutant tissue, a
possible explanation is that Rx1, Optx2, tll and Lhx2 can be
induced to sufficient levels by the remaining EFTFs –
compensating for their removal. The strong ectopic expression
of Lhx2 seen in embryos injected with EFTF cocktails (Fig.
3B-D) certainly supports this hypothesis. The genetic hierarchy
Development 130 (21)Research article
Fig. 4. Noggin but not Otx2 regulates eye field
transcription factor expression while Otx2 blocks
the repression of ET by noggin. (A) RT-PCR was
used to detect changes in the expression of the
EFTFs in response to noggin (10 pg) and Otx2
(200 pg). The effect of Otx2 on its own expression
was not determined (ND). The presence of ET and
Six3 in uninjected animal caps (U) was not a result
of DNA contamination as neither transcript was
detected when duplicate samples were amplified
in the absence of reverse transcriptase (–RT).
Uninjected sibling embryos ‘E’ were used as a
positive control for PCR. Histone H4 was used as
a loading control. (B) RT-PCR was used to
determine the relative expression of ET in
ectodermal explants from embryos injected with
Otx2, noggin or both. The percent of ET
expression relative to uninjected controls is shown
above each bar of the graph. (C) Interpretation of
the combined results from A and B.
Fig. 5. ET, Rx1 and Pax6 regulate Otx2 expression. Embryos were injected into one blastomere at the two-cell stage with RNA of the indicated
gene. Whole-mount in situ hybridisation was used to detect Otx2 expression in embryos injected with 100 pg ET (B), 400 pg Rx1 (C), 200 pg
Pax6 (D), 200 pg Six3 (E) or 500 pg Lhx2 (F) RNA. Embryos in A,D-F were co-injected with βgal RNA to identify the injected side. In B and
C, the embryos were not stained for βgal expression so that the repression of Otx2 could be more easily visualised. Scale bar: 300 µm.
(G) Quantitation of the effect of EFTFs on Otx2 expression. Percent of embryos with an increase (↑), decrease (↓) or no change (NC) in Otx2
expression. ET induces Rx1 expression. (H,I) Rx1 injection did not effect ET expression, while ET induced Rx1 expression in Xenopus animal
caps in a dose-dependent manner. Histone H4 was used as a loading control; U, uninjected; E, parallel, uninjected embryo. (J-M) Whole-mount
in situ hybridisation was used to detect ET (J-K) and Rx1 (L-M) expression in stage 13 Xenopus embryos injected with 200 pg Rx1 (K) or ET
(M) RNA. In (J,K), embryos were injected with βgal RNA. In L,M, GFP RNA was used to detect the injected side of the embryo. The right side
is the injected side in J-M. Scale bar: 300 µm. (N) Interpretation of the results of Figs 4, 5.
5161Genetic network in vertebrate eye formation
suggested by the timing of EFTF expression (Fig. 1) is also
consistent with this idea as the four EFTFs that are deemed
most crucial by the cocktail subset method are expressed
earlier than, and may therefore induce the expression of, Rx1,
Optx2, tll or Lhx2.
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.
The strong repression of ET by noggin and chordin,
however, raised the question of how the initial expression of
ET is turned on in the eye field. We therefore considered the
possibility that Otx2 inhibits the repression of ET by noggin.
To test this idea, noggin mRNA was injected with and without
Otx2 mRNA, and the effect on ET expression was determined
in animal caps. Otx2 alone had no effect on ET, while noggin
repressed ET expression to 15% of control levels (Fig. 4B).
However, co-expression of Otx2 with noggin not only rescued
ET expression, but increased ET levels beyond that of controls
in a dose-dependent manner. These results indicate a
potentially crucial role for Otx2 in eye field formation. Neural
induction alone results in a neural plate resistant to ET
expression; however, subsequent expression of Otx2 in the
anterior neural plate permits ET expression and perhaps
subsequent eye field formation (Fig. 4C).
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.
As ET is expressed before Rx1, it is possible that the
repression of Otx2 by ET is indirect – mediated through Rx1.
To test this possibility, we first used the animal cap assay to
determine the effect of ET and Rx1 on each other’s expression.
Rx1 neither induced nor repressed ET expression in animal
caps at concentrations as high as 1000 pg (Fig. 5H). However,
ET strongly induced the expression of Rx1 in a dose-dependent
manner (Fig. 5I).
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).
We next examined the effect of Otx2:ET co-injection on Rx1
expression in vivo. Otx2 (25 pg) only slightly increased Rx1
expression in stage 13 embryos (Fig. 6D,G), while ET (10 pg)
expanded the average domain of Rx1 expression by 15% (Fig.
6E,G). However, co-injection of Otx2 with ET increased the
average eye field diameter by nearly 35% (Fig. 6F,G),
approximately equivalent to the effect of 25 pg of ET alone
(Fig. 6G). These results are consistent with the model of
progressive tissue specification described above.
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
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
Development 130 (21) Research article
Fig. 6. Otx2 and noggin potentiate the induction of Rx1 by ET. (A,B) RT-PCR was used to detect changes in Rx1 and XAG expression in
ectodermal explants from Xenopus embryos injected with noggin, Otx2 and ET. ET (100 pg) was injected alone, with 50 or 100 pg of Otx2
(A), or 5 pg noggin (B). (A) Lane 1, uninjected; lane 2, ET (100 pg); lane 3, Otx2 (50 pg); lane 4, Otx2 (100 pg); lane 5, ET (100 pg) + Otx2 (50
pg); lane 6, ET (100 pg) + Otx2 (100 pg); lane 7, embryo, no reverse transcription; lane 8, embryo, XAG induction was used as a positive control
for Otx2 activity. (B) Lane 1, uninjected; lane 2, ET (100 pg); lane 3, noggin (5 pg); lane 4, ET (100 pg) + noggin (5 pg). (C-G) Rx1 expression
was normalised to Histone H4 then set relative to uninjected controls. Otx2 potentiates the ET induced expansion of Rx1 expression in the
anterior neural plate. Whole-mount in situ hybridisation was used to detect Rx1 expression at stage 13 in embryos injected with βgal alone (C), or
in combination with 25 pg Otx2 (D), 10 pg ET (E) or both Otx2 and ET (F). (G) The rostrocaudal diameter of the Rx1 expression domain on the
injected side (βgal-positive) was measured and compared with the uninjected (βgal-negative) side of the embryo (see F for an example).
5163Genetic network in vertebrate eye formation
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.
Additional interactions may also exist and are not ruled out
by the present data set. The working model illustrated in Fig.
Fig. 7. Epigenetic interactions among the
eye field transcription factors define a
genetic network during eye field formation.
(A) ET induces the expression of a subset
of EFTFs. RT-PCR was used to detect
changes in EFTF expression in ectodermal
explants isolated from embryos injected
with 200 pg of ET. The fold induction
represents the relative expression of the
EFTFs when compared with uninjected
controls. (B) Epigenetic interactions
between noggin, Otx2 and the EFTFs. ↑,
induction of target gene; ↓, repression of
target gene; NC, no change in target gene
expression; *some variability in these
inductions was observed. (C) Summary
model of eye field induction in the anterior
neural plate. Light blue indicates the neural
plate, blue shows the area of Otx2
expression and dark blue represents the eye
7C forms a framework that further experiments may build on
to define more precisely the genetic interactions required for
vertebrate eye formation.
Eye specification in flies and vertebrates
Kumar and Moses proposed a two-stage mechanism for
Drosophila eye specification. First the eye-antennal imaginal
disk complex is subdivided into two distinct presumptive organ
fields. Next, specific organ identities (eye versus antenna) are
defined. In the embryo prior to eye specification, toy, ey, so,
eya, dac and eyg have only partially overlapping expression
patterns. However, at the second larval stage their expression
patterns are coordinated and the eye becomes specified (Kumar
and Moses, 2001a). In the vertebrate, we have shown that
inductive and patterning events prepare the anterior neural
plate for formation of the vertebrate eye field. However, it is
the coordinated expression of the EFTFs that is required for
the specification of the eye field. We find this to be a
remarkable example of mechanism conservation, given the
large evolutionary distance between these species and the
differences in the development and morphology of fly and
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
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,
Development 130 (21)Research article
5165 Genetic network in vertebrate eye formation
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.
We thank Yi Rao, Tomas Pieler and Anna Philpott who provided
us with the pCS2R.XET, pCS2MT.Xtll, pCS2.noggin, respectively.
We would also like to thank Susannah Hopper, Dean Pask, Katie Baird
for technical assistance and Giuseppe Lupo for the sharing of
unpublished data. This work was supported by grants from European
Commission and the Wellcome Trust, UK. M.Z. and A.V. were
supported by the Burroughs Wellcome Fund through a Hitchings-
Elion Fellowship and an NEI (NRSA grant EY-07051), respectively.
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