The vertebrate eyes form from a single eye field, an anterior
ectodermal territory containing a retina anlage and an abutting
lens competence field. Under the influence of midline signals,
this field splits into two bilateral regions each containing a
neuroectodermal retina anlage and an ectodermal lens anlage.
During neurulation, the retina anlage evaginates laterally from
the forebrain, giving rise to the optic vesicles. These in turn
invaginate distally to form the optic cups with neural retina
(NR) and retinal pigmented epithelium (RPE) (Grainger, 1996;
Jean et al., 1998). Several transcription factors that are required
for optic vesicle development have been identified so far
(Chow et al., 1999; Jean et al., 1998; Loosli et al., 1999; Porter
et al., 1997). However, it is yet unknown in what manner these
factors interact to initiate and control optic vesicle formation.
What is the molecular link between the determination of the
retina anlage and the formation of an optic vesicle?
The transcription factor Pax6 is an essential regulator for eye
development conserved in evolution (Quiring et al., 1994;
Walther and Gruss, 1991). Pax6 is expressed in the anterior
neuroectoderm before optic vesicle formation in fish (Krauss
et al., 1991; Loosli et al., 1998), frog (Hirsch and Harris, 1997),
and chick (Li et al., 1994). Pax6−/−Small eye mutant mice
develop small optic vesicles, which degenerate during
subsequent development (Hill et al., 1991; Hogan et al., 1986).
Overexpression of Pax6 in Drosophila and Xenopus can lead
to ectopic eye formation (Chow et al., 1999; Halder et al.,
Six3 plays a role in specifying early retinal identity (Bernier
et al., 2000; Loosli et al., 1999). This evolutionarily conserved
transcription factor contains a homedomain and a Six domain
(Cheyette et al., 1994; Seimiya and Gehring, 2000; Toy et
al., 1998). It is specifically expressed in the anterior
neuroectoderm including the eye field and specific parts of the
abutting surface ectoderm in all vertebrates examined
(Bovolenta et al., 1998; Granadino et al., 1999; Loosli et al.,
1998; Oliver et al., 1995; Seo et al., 1998; Zhou et al., 2000).
Overexpression of Six3 leads to expanded and ectopic retinal
primordia in medaka (Loosli et al., 1999) and Xenopus (Bernier
et al., 2000), and to an enlarged forebrain in zebrafish
(Kobayashi et al., 1998). In a feedback loop, Six3 thereby
induces its own expression, and that of Pax6 (Loosli et al.,
1999). As Pax6 also induces Six3 (Chow et al., 1999), both
factors activate each other (Loosli et al., 1999).
Vertebrate members of the Rx gene family are expressed
in the early eye field, and, with the exception of medaka Rx2,
which is exclusively expressed in the developing retina, also
in medial regions of the prosencephalon (Casarosa et al.,
1997; Chuang et al., 1999; Deschet et al., 1999; Furukawa et
al., 1997; Loosli et al., 1999; Mathers et al., 1997; Ohuchi et
al., 1999). Targeted inactivation of Rx in mouse leads to optic
vesicle and forebrain deletions, indicating a function of Rx in
the development of these structures (Mathers et al., 1997).
Similar results have been reported for Xenopus embryos
injected with a XRx1
dominant repressor construct
(Andreazzoli et al., 1999). Conversely ectopic expression of
XRx1 in Xenopus embryos triggers overproliferation of retinal
Development 128, 4035-4044 (2001)
Printed in Great Britain © The Company of Biologists Limited 2001
The complete absence of eyes in the medaka fish mutation
eyeless is the result of defective optic vesicle evagination.
We show that the eyeless mutation is caused by an intronic
insertion in the Rx3 homeobox gene resulting in a
transcriptional repression of the locus that is rescued by
injection of plasmid DNA containing the wild-type locus.
Functional analysis reveals that Six3- and Pax6- dependent
retina determination does not require Rx3. However, gain-
and loss-of-function phenotypes show that Rx3
indispensable to initiate optic vesicle evagination and to
control vesicle proliferation, by that regulating organ size.
Thus, Rx3 acts at a key position coupling the determination
with subsequent morphogenesis and differentiation of the
Key words: Retina determination, Morphogenesis, Proliferation, Size
Medaka eyeless is the key factor linking retinal determination and eye growth
Felix Loosli1, Sylke Winkler1, Carola Burgtorf1, Elisa Wurmbach2,*, Wilhelm Ansorge2, Thorsten Henrich1,‡,
Clemens Grabher1, Detlev Arendt1, Matthias Carl1, Annette Krone1, Erika Grzebisz1
and Joachim Wittbrodt1,§
1European Molecular Biology Laboratory, Developmental Biology Programme, Meyerhofstr. 1, 69117 Heidelberg, Germany
2European Molecular Biology Laboratory, Biochemical Instrumentation Programme, Meyerhofstr. 1, 69117 Heidelberg, Germany
*Present address: Mount Sinai School of Medicine, New York, NY 10029, USA
‡Present address: Kondoh differentiation signalling project, Kyoto, Japan
§Author for correspondence (e-mail: Jochen.Wittbrodt@EMBL-Heidelberg.de)
Accepted 16 July 2001
tissue (Andreazzoli et al., 1999; Mathers et al., 1997, Zhang
et al., 2000). Thus, the specific involvement of Rx genes in
eye development with regard to the determination of
theretina anlage and subsequent specification and
morphogenesis of the retinal progenitor cells remains to be
In the temperature-sensitive, early larval lethal medaka
mutation eyeless (el) optic vesicles do not evaginate at the
restrictive temperature (Winkler et al., 2000). This defect in
morphogenesis leads to a complete lack of eyes. Temperature
shift experiments indicate that gene activity is required before
optic vesicle evagination.
We show that the gene affected by the eyeless mutation is
Rx3, a member of the Rx gene family. We combine mutant
analysis with gain-of-function approaches to place Rx3
function into the genetic network of early eye development.
Rx3 is required to initiate and maintain optic vesicle
morphogenesis and proliferation, but is dispensable for the
formation of the retina anlage. These results provide a link
between retinal determination, and the subsequent control optic
vesicle morphogenesis and differentiation.
MATERIAL AND METHODS
The el mutation was crossed into and kept in two different inbred
genetic backgrounds (Cab and Kaga) and kept as closed stocks at
EMBL (Winkler et al., 2000). Embryos were staged according to
Iwamatsu (Iwamatsu, 1994). Sex averaged linkage analysis showed a
close linkage between el and the pigmentation locus b (1.3 cM).
Cleaved amplified polymorphic sequences (CAPS)
DNA of homozygous el hatchlings (three to five individuals pooled)
or individual wild-type fish derived from brother-sister crosses of
el/Kaga carriers was isolated (QIAamp kit, Qiagen). A 333 bp
fragment was amplified using primers specific for the third exon of
Rx3 (up, ACACTCCCTCATCTTTGCTCTCCTTC; low, TGTTCCT-
TGGCCTTCATCCTCA). PCR conditions were 35 cycles of 94°C for
30 seconds, 61°C for 30seconds, 72°C for 1 minute. PCR products
were HaeIII digested and separated on 3% agarose gels, showing
cleavage of a Cab-specific 190 bp fragment into 100 bp and 90 bp
fragments in Kaga.
Isolation of Rx3 full-length cDNA
A neurula stage λZAP cDNA library was screened with a 440 bp Rx3
PCR fragment (Deschet et al., 1999). Positive clones were plaque
purified, converted to pBluescript and sequenced (EMBL Accession
RT-PCR analysis of mutant and wild-type embryos
Total RNA was isolated from two-somite stage homozygous mutant
embryos and wild-type siblings (Sambrook et al., 1989) and
reverse transcribed using an oligo-dT and a Rx3-specific primer
(ATGTTCCATTCTGGGCGTCTCAG) . A Rx3-specific primer pair
(up, TTAGACAAATGTGGCTCCTGGGATCAGCTTCA; low, TGT-
TCCTTGGCCTTCATCCTCA) was used as follows: 35 cycles of
94°C for 45 seconds, 60°C for 1 minute, 72°C for 2 minutes. PCR
products were analysed by gel electrophoresis.
Isolation of a partialTbx2 and aTbx3 cDNA
A 440 bp medaka Tbx2 fragment and a 600bp fragment of Tbx3 were
RT-PCR amplified from stage 18 RNA using degenerate primers (up,
GAGGTIGAG GAYGAYCCIAARGT; low, CCIRTGTCYCTRAAI-
CCYTTIGCRAA). PCR conditions were 5 cycles of 94°C for 1
minute, 53°C for 2 minutes, 72°C of 4 minutes, followed by 30 cycles
with annealing at 58°C. PCR products were cloned (TopoTA,
Invitrogen) and sequenced (EMBL Accession Numbers: OlTbx2,
AJ298301; OlTbx3, AJ298302).
Southern and northern blot analysis
Genomic DNA of adult homozygous mutant el, heterozygous el
carrier and wild-type fish was prepared. The DNA was digested over
night, separated on a 0.7% agarose gel and transferred to HybondN+
(Amersham) by capillary transfer. Filters were hybridised with 32P-
labelled probes under high stringency conditions (Sambrook et al.,
1989). Total RNA was isolated from wild-type embryos. Poly(A)+
RNA was enriched (dynabeads, Dynal). 10 µg of A+RNA were
separated on a formaldehyde agarose gel, blotted and hybridised
(Sambrook et al., 1989).
Genomic library construction and cloning of the mutant el
A lambda FixII (Stratagene) genomic library was generated from
genomic DNA of homozygous el-mutant fish. A phage covering the
3′ part of the mutation was isolated using the Rx3 cDNA as a probe
under high stringency conditions. The remaining part of the mutant
locus was cloned as PCR fragments, and the 5′ breakpoint of the
insertion was isolated by splinkerette (Devon et al., 1995).
Bac and cosmid clones containing the Rx3 locus were isolated from
arrayed libraries (library 756, 73 and 74, RZPD Berlin) probed with
Rx3 cDNA. BAC and cosmid DNA was prepared following standard
protocols. For injection the DNA was further purified on a Caesium
chloride gradient (Sambrook et al., 1989). For the Rx3 rescue plasmid,
an 11 kb SnaBI fragment of the Rx3 cosmid was cloned into
pBluescript SK+ (Stratagene). The remaining cosmid was re-ligated
resulting in the ∆Rx3 plasmid. A frameshift was introduced into the
second helix of the homeodomain by BspEI digestion of the Rx3
rescue plasmid and re-ligation after filling the ends with Klenow
polymerase (Rx3∆HB plasmid).
Plasmid DNA was injected into the blastomere of one-cell stage
embryos from el carrier crosses. Injected embryos were kept under
restrictive conditions. The following DNA concentrations were used
for injection experiments: Rx3 BAC, 50 ng/µl; control BACs, 50
ng/µl; Rx3 cosmid, 40 ng/µl; Rx3 plasmid, 5 and 20 ng/µl; ∆Rx3
plasmid, 45 ng/µl; Rx3∆HB plasmid, 50 ng/µl and pBluescript, 50
A 1.2 kb BamHI-BglII fragment of the medaka Rx3 cDNA cloned into
pCS2+ was verified by sequencing and TNT transcription/translation
(Promega). Linearized pCSmouseSix3, pCSmedakaSix3 (Loosli et al.,
1999) and pCSmedakaRx3 plasmid DNA were in vitro transcribed and
the purified RNA injected into one blastomere at the two-cell stage
(Loosli et al., 1999). RNA concentrations in the injection solution
were 200 ng/µl pCSmouseSix3, 200 ng/µl pCSmedakaSix3, when
injected into embryos of el carrier crosses; 100 ng/µl, when injected
into wild-type embryos; and 50 ng/µl pCSmedakaRx3. For controls,
the respective concentration of hGFP RNA was injected. Injected
embryos from el carrier crosses were kept at the restrictive
In situ hybridisation and vibratome sectioning
Whole-mount in situ hybridisation using antisense riboprobes and
vibratome sections of embryos were performed as described (Loosli
et al., 1998).
F. Loosli and others
4037 Rx3 links eye determination and growth
Rx3 expression is affected in el-mutant embryos
The el mutation, initially isolated in a strain from the Southern
population of medaka, was crossed into and kept in the ‘Cab’
inbred strain (Winkler et al., 2000). Crossing of el carrier fish
into the Kaga strain revealed a close linkage of el and the
pigmentation locus b. The Kaga strain is homozygous for the
B allele that results in darkly pigmented melanophores. The
Cab background carries the recessive b allele that leads to
unpigmented melanophores. Analysis of the offspring of
el/Kaga intercrosses resulted in 32 recombinant el embryos
(el/el,b/B) in 2422 meiosis. This corresponds to a genetic
distance of 1.3 cM and thus places el on linkage group 12 (LG
12; Naruse et al., 2000) in close proximity to the b locus.
el is a temperature-sensitive mutation. Optic vesicles of
homozygous mutant embryos do not evaginate at the restrictive
temperature (18°C) and subsequent differentiation is perturbed.
At higher temperatures (28°C) in 52% of the el-mutant
embryos optic vesicles of variable size form (Winkler et al.,
2000). Temperature shift experiments showed that el activity
is required at late gastrula/early neurula stages before optic
vesicle evagination. We therefore examined genes that are
expressed in the retina anlage before optic vesicle evagination.
The expression of the retina determination genes Six3 and Pax6
is not affected in the retinal primordia of el-mutant embryos
(Winkler et al., 2000). These genes have been mapped to
different linkage groups (i.e. Pax6, LG 3; Six3, LG 19 (Naruse
et al., 2000)), excluding Pax6 and Six3, as candidates for el.
In the anterior neuroectoderm, the homeobox gene Rx3 is
expressed from late gastrula stages onwards (Deschet et al.,
1999). The initially narrow, single expression domain widens
and is subsequently split into two domains lateral to the
forming axis (Fig. 1A). During neurulation and early
somitogenesis stages, Rx3 is expressed in the anterior ventral
prosencephalon, the optic vesicles and the optic stalk, which
connects these tissues (Fig. 1C). Expression in the retina and
optic stalk is downregulated at later somitogenesis stages, such
that Rx3 expression finally is restricted to the hypothalamus.
In the differentiating retina, Rx3 is expressed in the inner
nuclear layer (INL). In situ hybridisation showed that in el-
mutant embryos, Rx3 expression in all its wild-type expression
domains is completely absent at all stages at the restrictive
temperature (Fig. 1A-D). This indicates that either Rx3 or an
upstream regulator is affected by the el mutation.
CAPS analysis of el
To examine whether Rx3 itself or a regulator of Rx3 expression
is affected in the el mutation we examined whether Rx3 maps
to linkage group 12 and whether the mutation co-segregates
with an Rx3 specific polymorphism. We took advantage of the
highly polymorphic genetic backgrounds of the inbred Cab and
the Kaga strains. A PCR amplified 333 bp genomic Rx3 DNA
fragment contains a polymorphic HaeIII restriction site (Fig.
2A). This cleaved amplified polymorphic sequence (CAPS)
was used to determine the chromosomal location of the Rx3
gene and to examine a potential linkage between Rx3 and the
Cab/Kaga back-cross analysis locates the Rx3 gene to
linkage group 12 in close proximity to the b-locus (Fig. 2D).
el carrier fish of the Cab strain, in which the mutation has
originally been identified, were crossed to Kaga fish and the
wild-type, and mutant progeny of the resulting el/Kaga carrier
intercrosses were examined for co-segregation by CAPS
analysis. The Cab-specific HaeIII polymorphism always co-
segregated with the el phenotype in all 532 meiosis analysed
(Fig. 2). Thus, the genetic distance of the Rx3 gene and the el
mutation is less than 0.19 cM, strongly suggesting that the
mutation affects the Rx3 gene proper.
Genomic organisation of the wild type and mutant
To further analyse the nature of the el mutation we cloned a
full-length Rx3 cDNA. Genomic Southern blots probed with a
600 bp 5′ fragment of the Rx3 cDNA suggested an insertion in
the Rx3 locus of el-mutant fish (Fig. 2B). Rx3 containing BAC
and Cosmid clones were isolated from respective wild-type
libraries. The genomic organisation of the Rx3 locus was
examined by sequence analysis and Southern blot
Fig. 1. Rx3 expression in wild-type and el-mutant embryos. Dorsal
views of whole-mount in situ hybridisation of late gastrula (stage 16;
A,B) and four-somite stage (stage 20; C,D) wild type (A,C) and el-
mutant embryos kept at restrictive temperature (B,D). Anterior is
towards the left. Neural axis is indicated by a broken yellow line
(A,B). (A) Rx3 expression in anterior neural plate. (C) Rx3
expression in hypothalamus (arrow) and optic cup (arrowhead).
(B,D). Note complete loss of Rx3 expression in mutant embryos kept
under restrictive conditions. (F) Under permissive conditions, Rx3 is
weakly expressed in forebrain of el-mutant embryos at the two-
somite stage (stage 19). Note reduced size of evaginating optic
vesicles (arrowhead) at site of reduced Rx3 expression compared
with wild-type embryo of same stage (arrowhead in E). (G) Primers
spanning entire open reading frame detect the wild-type transcript by
RT-PCR in wild-type (lane 1) and el-mutant embryos (lane 2) under
permissive conditions, while no transcript is detected in mutants
under restrictive conditions (lane 3). M, 100 bp ladder.
Abbreviations: perm., permissive temperature; restr., restrictive
hybridisation. The Rx3 open reading frame of 876 bp is split
into three exons and spans a genomic region of about 2.9 kb
(Fig. 2D). The predicted length of the RX3 protein is 292
amino acids. Sequence comparison with other homeobox genes
of the Rx subclass shows that the medaka RX3 protein shares
highest homology with zebrafish RX3. The RX3-specific
octapeptide and the homeodomain are 100% identical and the
conserved region in the C-terminal region (OAR; Furukawa et
al., 1997) differ in only one amino acid between medaka and
zebrafish RX3. However, the medaka paralogue RX2 differs by
two amino acids in the octapeptide, one in the homeodomain
and one in the OAR, compared with medaka RX3. In contrast
to zebrafish, where three Rx genes have been cloned, we
detected two paralogues in medaka.
The homeodomain is encoded by the 3′ part of exon 2 and
the 5′ part of exon 3 (Fig. 2D,E). The mutant locus harbours
an insertion in the intron intervening exon 2 and 3. The inserted
DNA is middle repetitive as indicated by Southern blot
hybridisation (Fig. 2C). The insertion of at least 13 kb has no
significant homology to any known sequence. Apart from the
insertion in intron 2, no further changes were detectable in the
mutant Rx3 locus. The open reading frame is unaffected and
thus the locus has the potential to express a wild-type RX3
Wild-type Rx3 rescues optic vesicle evagination in
the mutant embryo
The tight linkage of el and the pigmentation locus b was used
to genetically mark the el mutation. The homozygous el-
mutant offspring of an el/+,b/B carrier cross lacks the darkly
F. Loosli and others
Fig. 2. Molecular analysis of the
genomic Rx3 region. (A) CAPS
analysis of mutant and wild-type
genomic DNA. A 190 bp
fragment in HaeIII digested
genomic PCR fragment is
diagnostic for the mutant Cab
allele (el/el and el/+ lanes); the
additional restriction site in wild-
type Kaga results in 100 bp and
90 bp fragments (arrowhead, el/+
and +/+ lanes). (B) Genomic
Southern blot hybridisation using
a 600 bp HindIII cDNA fragment
comprising exon 1 and 2 (BamHI
digest) and a 3 kb genomic DNA
fragment (broken line in D; XbaI
digest) as probes. In DNA of
homozygous mutant embryos
(el/el) a larger fragment is
detected than in the wild type
(+/+), indicating an insertion
larger than 13 kb in the mutant el
locus. In DNA of heterozygotes
(el/+), both fragments are
detected. (C) Genomic Southern
blot hybridisation with a 4 kb insertion fragment reveals multiple copies in the genome, indicative of middle repetitive
DNA. (D) Map of Rx3 region indicating relative distance of B and el on linkage group 12 (LG 12). Red boxes
represent three exons on Rx3 cosmid; position of the insertion in intron 2 is shown. Regions comprised in different
rescue plasmids and position of the frameshift (fs) are indicated. (E) Predicted amino acid sequence of the Rx3 protein.
Splice sites are indicated (arrowheads). Conserved octapeptide (yellow), homeodomain (green) and C-terminal region
(red) are highlighted. Abbreviations: B, BamHI; Be, BspEI; S, SnaBI; X, XbaI.
4039Rx3 links eye determination and growth
pigmented melanophores with the exception of less than 1.3%
recombinants (Fig. 3A,B). The pigmentation becomes first
visible at early somitogenesis stages. At the restrictive
temperature, all mutant embryos lack eyes and do not form
retinal pigmented epithelium (RPE), thus the mutant phenotype
is fully penetrant and the expressivity not variable. Control
injections into fertilised eggs affect neither penetrance nor
expressivity (Fig. 3E). Mutant embryos injected with either
the Rx3 BAC or the Rx3 cosmid form eyes of wild-type
morphology in 25% and 39% of the cases, respectively (Fig.
The Rx3 plasmid contains the Rx3 locus (Fig. 2D) and
rescues the el phenotype in 37% of the cases, while the
remaining 8.2kb of the Rx3 cosmid (∆Rx3 cosmid) have no
rescuing activity (Fig. 3E). Northern blot analysis using the
Rx3 plasmid as a probe detects a single transcript of 1.8kb that
corresponds to the Rx3 mRNA (not shown) indicating that
only the Rx3 gene is expressed in the relevant time window
from the Rx3 plasmid. 50% of the rescued homozygous
mutant embryos (n=8) identified by the lack of darkly
pigmented melanophores show wild type morphology and
behaviour (Fig. 3). This rescue is coupled with restored Rx3
expression in mutant embryos (Fig. 4A,B). The finding that
the rescued Rx3 expression is confined specifically to the wild
type expression domains indicates that all regulatory elements
required for wild type expression are contained within the Rx3
plasmid. To examine whether the rescue activity depends on
a functional Rx3 protein we introduced a frameshift in exon 2
of the Rx3 plasmid. This results in a deletion of helices 2 and
3 of the homeodomain and the entire C-terminal portion
(Rx3∆HB plasmid, Fig. 2D). The frameshift completely
abolishes the rescue activity (Fig. 3E) without affecting its
expression (Fig. 4C,D) demonstrating that the rescue depends
on the expression of a functional RX3 protein. On the other
hand, expression from the rescue construct does not require
Rx3 activity (compare Fig. 4C with 4D), indicating that in
contrast to Pax6 and Six3 functional RX3 protein is not
required for its own expression.
Expression of Tbx and Vsx genes in the developing
retina require Rx3
The T-box-containing transcription factors Tbx2 and Tbx3 are
expressed in the developing optic vesicles, ventral forebrain
and the otic vesicles starting at early somitogenesis stages in
wild-type medaka (Fig. 4H,K). In el-mutant embryos, however,
cells of the retinal primordia do not express Tbx2 and Tbx3,
whereas the other expression domains are not affected (Fig.
4I,L). Rescued presumptive retinae of mutant embryos express
both Tbx2 and Tbx3 (Fig. 4G,J). This indicates that retinal Tbx2
and Tbx3 expression depend on Rx3 activity and that the Rx3
rescue plasmid can provide also this aspect of Rx3 function.
The paired like homeobox genes Vsx1 and Vsx2 are expressed
in the differentiating retina (Winkler et al., 2000). In the retinae
of rescued mutant embryos, expression of Vsx1 and Vsx2 that
is lost in el-mutant embryos, is specifically restored (Fig. 4M-
Thus, the molecular and morphological analysis of rescued
embryos shows that the Rx3 plasmid fully rescues evagination
of the optic vesicle and subsequent differentiation of the retina,
substantiating that the el-mutant phenotype is caused by the
intronic insertion in the Rx3 gene.
Temperature sensitivity of el correlates with variable
At the restrictive temperature (18°C), Rx3 is not expressed in
el-mutant embryos (Fig. 1B,D). At the permissive temperature
(28°C), however, we found weak Rx3 expression at late neurula
to early somitogenesis stages in 56% of the mutant embryos
(Fig. 1F). Under these conditions, the mutant phenotype is
variable, such that 52% of el-mutant embryos raised at the
permissive temperature form small eyes. Thus, Rx3 expression
in el-mutant embryos kept at the permissive temperature
closely correlates with the variable phenotype. Furthermore,
RT-PCR analysis shows that at the permissive temperature a
correctly spliced transcript is present in el-mutant embryos
(Fig. 1G). Thus, the mutation affects the transcription of the el
locus in a temperature-sensitive way, such that the el mutation
is amorphic at the restrictive temperature and hypomorphic at
the permissive temperature.
Taken together, three lines of evidence indicate that the locus
Fig. 3. Phenotypic rescue of el-mutant embryos. Uninjected
(A,B) and injected (C,D) wild-type (B) and mutant embryos
(A,C,D) raised for 8 days at restrictive temperature. Dorsal views,
anterior is towards the top. Note dark pigmented melanophores in
wild-type embryo (arrowhead in B), lacking in mutants (A,C,D).
(A) No eyes form in mutants. (C,D) Rescued eye formation in
mutant embryos injected with Rx3 plasmid. (C) Note complete
rescue of both eyes. (E) Table displaying results of different DNA
and control injections. N, number of injected embryos; el
(b/b) homozygous mutant embryos as judged by absence of dark
melanophores. No rescue is observed in control injected embryos.
affected in the el mutation is the Rx3 gene: (1) genetic mapping
and the detection of an intronic insertion in the Rx3 gene
correlate; (2) the expressivity of the phenotype correlates with
Rx3 expression levels; and 3) the wild-type Rx3
gene has rescuing activity.
The regulatory interaction of Six3 and Pax6
is independent of Rx3 activity
We had shown that overexpression of medaka or
mouse Six3 leads to retinal hyperplasia and the
formation of ectopic retinal primordia in the mid-
and hindbrain (Loosli et al., 1999). Ectopic retina
formation in response
overexpression in Xenopus and medaka fish is
preceded by a cross-regulatory interaction of these
genes (Bernier et al., 2000; Chow et al., 1999;
Loosli et al., 1999). It has been suggested that
this interaction is a prerequisite for retinal
determination. We examined whether Rx3 functions
in this regulatory interaction of Six3 and Pax6.
To address this, we first investigated whether
ectopic Six3 expression in el-mutant embryos can
relieve the transcriptional repression of the mutant
locus at the restrictive temperature. At the two-
somite stage, no Rx3 expression was detected in the
el-mutant embryos injected with Six3 RNA (n=17,
Fig. 5A,B). On the other hand, in all injected wild
type siblings (n=67) Rx3 expression was detected
(not shown). Therefore, Six3 overexpression does
not relieve the transcriptional repression of the
mutant Rx3 locus under restrictive conditions. This
allows examining the function of Six3 in the
absence of Rx3. The finding that optic vesicle
evagination is not rescued in the Six3 injected
mutant embryos suggests a role of Six3 upstream to
or in parallel of Rx3 (Fig. 5A,B).
Using the el mutation, we analysed whether Rx3
activity is required for the crossregulatory
interaction of Six3 and Pax6. Ectopic medaka Six3
expression in embryos that lack Rx3 results in an
enlarged Pax6 expression domain (n=21/53, 39%)
and in ectopic Pax6 expression in the presumptive
midbrain (n=9/53, 17%) at early somitogenesis
stages (Fig. 5C,D). Similarly, in injected wild-type
siblings used as internal control, expanded as well
as ectopic Pax6 expression was observed. Thus,
ectopic Pax6 expression in response to Six3
overexpression does not require Rx3 activity.
Mouse Six3 overexpression results in the ectopic
activation of the endogenous Six3 gene, indicating
that Six3 functions in a regulatory feedback loop
(Bernier et al., 2000; Loosli et al., 1999). To address
whether this regulatory feedback loop of Six3 will
also function in the absence of Rx3, we injected
mouse Six3 mRNA at the restrictive temperature.
We observed ectopic endogenous Six3 expression at
the early somitogenesis stages in 22% of injected
el-mutant embryos (n=6/27, Fig. 5E,F). Consistent
with expanded Pax6 expression, endogenous Six3
expression was also expanded in 40% (n=11) of the
injected mutant embryos (not shown).
Thus, the crossregulatory interactions of Six3 and Pax6, as
well as the regulatory feedback loop of Six3 are independent
of Rx3. The finding that Rx3 is not required for these regulatory
F. Loosli and others
Fig. 4. Restored gene expression in rescued mutant embryos. (A-F) Rx3
expression at the four-somite stage in mutant (A-C,E) and wild-type
(D,F) embryos, dorsal views (A-D,F), injected with Rx3 plasmid
(A,B), Rx3∆HB plasmid (C,D) and controls (E,F); lateral view (E). Anterior is
towards the left. (A,B) Optic vesicle evagination correlates with level of
restored Rx3 expression (compare arrowheads in A,B). (C) Restored expression
of nonfunctional Rx3 protein does not rescue. (A-D) Note specific Rx3
expression in mutant (A-C) and wild-type (D) embryos. Compare the
homogenous wild-type Rx3 expression in control injected (F) and clonal
expression in Rx3 plasmid injected mutant (A-C) and wild type (D) embryos.
(E) Control injected mutant embryo lacks Rx3 expression. (G-L) Rx3 plasmid
(G,J) and control injected (H,I,K,L) 12-somite mutant (G,I,J,L) and wild-type
(H,K) embryos. Tbx2 and Tbx3 expression in the retina is rescued in Rx3
plasmid-injected embryos (compare arrowheads in G-I for Tbx2, and J-L for
Tbx3). Note that expression of Tbx2 and Tbx3 in the hypothalamus (arrows in
G-I and J-L, respectively) and Tbx2 expression in the otic vesicle (compare G-I)
is not affected by the el mutation. (M-P) Transversal section of 22-somite
(M,N) and 35-somite (O,P) uninjected wild-type (M,O) and Rx3 plasmid-
injected mutant embryos (N,P). Dorsal is towards the top. (M,N) Vsx2
expression in wild-type (M) and rescued mutant embryo (N) in the ventral
retina. (O,P) Vsx1 expression in inner nuclear layer of the wild type retina
(O) is rescued in injected mutant embryo (P). Abbreviation: ov, otic vesicle.
4041 Rx3 links eye determination and growth
interactions preceding the formation of ectopic retinal
primordia indicates that Rx3 is not essential for retina
Six3 induces ectopic retinal primordia in an Rx3
To investigate whether Rx3 is required for Six3 mediated
ectopic retina formation, we further analysed the effect of Six3
overexpression in the el-mutant background. As a specific
retina marker we used Rx2 that in wild type embryos is
exclusively expressed in presumptive neural retina.
In el-mutant embryos at early somitogenesis stages Rx2 is
expressed indicating that retinal determination is not affected
by loss of Rx3 activity (Fig. 5H). Furthermore, in the Six3
injected mutant embryos we found strongly enlarged Rx2
expression domains (n=12/43, 28%) at early somitogenesis
stages (Fig. 5G). Importantly, in injected mutant embryos
(n=4/45, 9%) ectopic Rx2 expression was detectable in the
presumptive midbrain, as in the wild type siblings (n=10/104,
10%; Fig. 5G arrowhead). This shows that also in the absence
of Rx3 activity, Six3 can mediate respecification of presumptive
midbrain to a retinal fate. This is in good agreement with a role
of Six3 in the determination of retinal primordia independent
which acts subsequently in optic vesicle
morphogenesis and proliferation.
Rx3 acts downstream of Six3 and controls
proliferation in the optic vesicle
At late gastrula stages (stage 16), Rx3 is expressed in
a narrow domain across the anterior neuroectoderm. Using the
forming axis as a morphological landmark, we found that Rx3
expression overlaps with both the Six3 and Pax6 expression
domains at this stage (Fig. 6A-C). The crossregulatory
interactions of Six3 and Pax6 in early retina determination are
not affected by the absence of Rx3 activity in el-mutant
embryos at the restrictive temperature, suggesting that Six3 and
Pax6 expression do not depend on Rx3. To address whether Rx3
acts downstream or in parallel to Six3, we examined the effects
of Six3 overexpression on Rx3 expression and vice versa.
Fig. 5. Rx3 is not required for retina determination. Dorsal views of
two-somite stage (A-F) and six-somite stage el-mutant embryos
(G,H). Anterior is towards the left. (A,E,G) Mouse Six3 RNA
injected, (C) medaka Six3 RNA injected, (B,D,F,H) control injected
el-mutant embryos raised at restrictive temperature. (A,B) Six3
overexpression does not result in Rx3 expression in el-mutant
embryos. (B,C) Ectopic Pax6 expression (arrowhead) in the midbrain
in response to Six3 overexpression. (E) Six3 overexpression results in
ectopic Six3 expression in the presumptive midbrain (arrowhead)
(G) Overexpression of Six3 results in expanded and ectopic Rx2
expression (arrowhead) in forebrain and presumptive midbrain,
respectively. Abbreviations: fb, forebrain; hb, hindbrain; mb,
Fig. 6. Regulatory interactions of Six3 and Rx3. Dorsal
views of late gastrula (stage 16, A-D), two-somite stage
(stage 19, E and F) and six-somite stage embryos (stage 21,
G-L). Anterior is towards the left. (A-C) The expression of
Six3 (A), Rx3 (B) and Pax6 (C) partially overlap in
presumptive forebrain. Anterior end of neural axis indicated
by a broken line. (D,E) Overexpression of Six3 results in
expanded Rx3 expression (arrowhead) and ectopic
expression in the presumptive midbrain (arrow) at early
neurula stage (D) and two-somite stage (E). (E) Note
expanded expression in enlarged optic vesicle (arrow).
(F) Control injected embryo showing wild-type expression.
(G-I) Rx3-injected embryos with enlarged optic vesicles
(arrowhead) show expanded Six3 (G), Pax6 (H) and Rx2
(I) expression. (J-L) Control injected embryos at same stage.
Note that anterior and posterior boundaries of respective
expression domains are not shifted in response to Rx3
overexpression (compare G with J,H with K and I with L).
Insets in I and L show horizontal sections at comparable
level. Note the increased cell number in the Rx3-injected
Ectopic expression of mouse Six3 results in an expansion of
the Rx3 expression domain (n=19/29, 65%) and in ectopic Rx3
expression (n=8/29, 27%) already at late gastrula/early neurula
stages (Fig. 6D). At late neurula/early somitogenesis stages an
expansion of the Rx3 expression domain (n=33/49, 67%) and
ectopic Rx3 expression in the presumptive midbrain of medaka
Six3 injected embryos (n=8/49, 16%) is observed (Fig. 6E).
This strongly suggests that Six3 acts as an upstream regulator
of Rx3 expression at these stages.
To test whether, in reverse, Rx3 can activate Six3 we
overexpressed Rx3. Ectopic Rx3 expression does not affect Six3
expression at the time point when they are first co-expressed
in wild-type embryos (late gastrula/early neurula stage) and at
midneurula stages (not shown). Neither size nor morphology
of the forebrain or optic vesicles is altered by Rx3
overexpression at these stages (not shown). After optic vesicle
evagination, however, the optic vesicles of injected embryos
are often enlarged as visualised by Six3 (n=13/36, 36%) and
Pax6 (n=15/41, 37%) expression (Fig. 6G-I), without affecting
their expression domains in the forebrain. The finding that Rx3
overexpression at somitogenesis specifically results in enlarged
optic vesicles containing significantly more cells was
confirmed by the expanded expression domain of Rx2
(n=34/101, 34%; Fig. 6I) that in wild-type embryos is
expressed exclusively in the presumptive neural retina
Thus, Six3 overexpression results in ectopic Rx3 expression.
Rx3 overexpression, however, does not alter Six3 expression at
stages before optic vesicle evagination, consistent with a role
of Rx3 downstream of Six3. The enlargement of optic vesicles
caused by higher numbers of retinal cell in response to Rx3
overexpression indicates a role of this gene in the control of
proliferation in the optic vesicle.
In this study, we show that the eyeless mutant phenotype in
medaka fish is caused by an intronic insertion in the homeobox
gene Rx3, resulting in a transcriptional repression of the locus.
We show that determination of the retina anlage occurs
normally in the absence of Rx3 function, whereas subsequent
morphogenesis and differentiation of the retinal primordia do
not occur. Our studies indicate a role of Rx3 downstream of the
retina determination gene Six3 in morphogenesis and growth
control of the optic vesicle. Thus, Rx3 plays a pivotal role in
the manifestation of a fate that has been laid down in the organ
anlage by upstream players.
Furthermore, we show that the regulatory interactions of
Six3 and Pax6 that precede the formation of ectopic retinal
primordia do not depend on Rx3 activity. Our results provide
novel insights into the regulatory interactions of key players
involved in retina determination and subsequent steps of organ
Temperature-sensitive repression of the mutant el
The detailed phenotypic analysis of genetically marked el-
mutant embryos shows that the penetrance is complete and the
expressivity not variable at the restrictive temperature. At the
permissive temperature, however, the expressivity is variable,
while the penetrance is not affected. This temperature-sensitive
expressivity of the mutant phenotype is tightly correlated with
the expression levels of Rx3 in the presumptive retina (Fig. 1).
Furthermore, in our rescue experiments the degree of rescue
correlates well with restored expression in the evaginating
optic vesicles of el-mutant embryos (Fig. 4A,B). Thus, in both
situations, optic vesicle evagination and Rx3 expression
correlate. In line with this, at the permissive temperature a
correctly spliced transcript is detectable by RT-PCR. This
strongly suggests that under permissive conditions a functional
protein is made, which accounts for the formation of small eyes
in the mutant embryos.
The nature of the transcriptional repression remains unclear.
Database searches did not reveal any significant homology of
the insertion to known sequences. It is conceivable that the
insertion influences intronic enhancer elements near the
insertion site, thereby resulting in a repression of the locus.
However, preliminary data do not suggest the presence of
regulatory elements in the second intron (not shown). This
does not rule out the possibility that the insertion represses
the Rx3 locus by affecting its general accessibility for the
transcriptional machinery. Temperature-sensitive splicing
appears unlikely, as we cannot detect 5′ parts of the Rx3
transcript using RT-PCR or whole-mount in situ analysis at the
restrictive temperature. Further analysis of the regulatory
elements of the locus will address the nature of the repression
and its temperature sensitivity.
The Rx3 plasmid rescues all aspects of the mutant
F. Loosli and others
Fig. 7. Genetic hierarchy underlying early retina development. X, Y
and Z represent factors that pattern the anterior neuroectoderm,
leading to the overlapping expression of Six3 and Pax6 in the retina
anlage, which results in the determination of retinal fate. The
crossregulatory interaction and the feedback loops of Six3 and Pax6
(arrows) result in the maintenance of retinal fate. Rx3 expression is
regulated by these genes, but may also receive input of upstream
factors X, Y and Z. Rx3 activity is required for morphogenesis (optic
vesicle evagination) and organ size regulation (proliferation in optic
vesicle). The retina specific expression of Rx2 does not depend on
Rx3 activity. Genes involved in subsequent differentiation steps
require preceding Rx3 activity (Tbx2/3, Vsx1/2).
4043 Rx3 links eye determination and growth
phenotype, namely optic vesicle evagination and retinal
differentiation in embryos raised under restrictive conditions
(Figs 3 and 4). The regulatory elements required for correct
expression are contained in 11 kb of genomic DNA present in
the Rx3 plasmid as evidenced by specifically restored Rx3
expression in the wild-type expression domains. A modified
Rx3 plasmid encoding a non-functional protein also results in
specific Rx3 expression in mutant and wild-type embryos.
Therefore, a functional Rx3 protein is not required for wild-
type Rx3 expression, in contrast to Pax6 and Six3, which act in
regulatory feedback loops (Chow et al., 1999; Loosli et al.,
Rx3 functions downstream of the determination of
the retina anlage
Our study shows that the el mutation genetically separates the
determination of the vertebrate retina anlage from its
subsequent morphogenesis, organ size control and neuronal
Rx3 is not involved in the determination of the retina anlage,
as the expression of the retina determination genes Six3 and
Pax6 are not affected by either gain or loss of Rx3 activity.
Furthermore, Rx2 expression indicative of retinal fate is still
present in el-mutant embryos. However, also in absence of Rx3
activity, overexpression of Six3 results in the formation of
ectopic retinal primordia.
The targeted inactivation of a mouse Rx homologue results
in anophthalmic embryos (Mathers et al., 1997). Similar to the
situation in the medaka eyeless mutant, at the neural plate
stage, early expression of Six3, Pax6 and Otx2 is not affected
in Rx mutant mice (Zhang et al., 2000). This indicates that in
Rx−/−mouse embryos, analogous to medaka, patterning of the
anterior neural plate and thus the determination of the retina
anlage is not affected.
However, at later stages (E9.0-10.5), upregulation of retina
specific expression of Six3 and Pax6 is not detected in Rx−/−
embryos, indicating that retinal progenitor cells are not present
(Zhang et al., 2000). At the corresponding stage, the expression
of the respective homologues is still detectable in the medaka
mutant embryo. The difference is most probably due to a split
of function of the medaka Rx3 and Rx2 genes, respectively (see
Rx2 and Rx3 may have non-overlapping functions
The spatial and temporal expression patterns of the two
medaka paralogues Rx2 and Rx3 differ significantly. Medaka
Rx3 is expressed already at late gastrula stages in the anterior
neuroectoderm, and its expression domain comprises the
ventral forebrain as well as the optic vesicle. Rx2, on the other
hand, is first expressed several hours later at the late neurula
stage in the evaginated optic vesicle, and is thus exclusively
expressed in retinal progenitor cells. Thus, Rx2 and Rx3 in
medaka are not co-expressed except for a short period in the
retinal progenitor cells of the evaginated optic vesicle,
suggesting mainly non-overlapping functions of these genes,
while Rx3 function is required in the evaginating optic vesicle
Rx2 is expressed independently and may play a role in later
aspects of retinogenesis.
In zebrafish, three Rx homologues have been isolated. Rx1
and Rx2, which are most similar to medaka Rx2, share the early
expression domain with zebrafish Rx3, but show a slightly later
onset of expression (Chuang et al., 1999). In the differentiating
retina at late organogenesis stages, medaka and zebrafish Rx3
are expressed in the inner nuclear layer overlapping with Six3
expression. Expression of medaka Rx2 is confined to the outer
nuclear layer (photoreceptor layer) and the ciliary margin as is
Rx1 and Rx2 in zebrafish (not shown).
Genetic hierarchy underlying early retina
Based on our study, we propose the following model for early
vertebrate retina development (Fig. 7). Patterning of the
anterior neural plate culminates in defined expression patterns
of Six3 and Pax6. This anterior neural plate patterning relies
on the repression of wnt and BMP signalling (Niehrs, 1999),
and requires the activity of the Otx transcription factors
(Simeone, 1998). In the region where Six3 and Pax6 expression
overlap, retinal fate is specified. An Rx3-independent
regulatory feedback loop of these genes then ensures the
maintenance of the retinal fate.
Six3 overexpression in el-mutant embryos results in
dramatically enlarged retinal primordia (Fig. 5G). This
expansion does not occur on the expense of forebrain tissue,
suggesting that Six3
also affects cell proliferation
independently of Rx3 and thereby regulates the size of the
retina anlage. Consistent with the suggested role of Six3 in cell
proliferation, the closely related Xenopus Optx2 gene controls
the size of the optic vesicles by regulating proliferation (Zhou
et al., 2000; Zuber et al., 1999).
Under the influence of midline signalling, the retinal anlage
is split into two retinal primordia (Varga et al., 1999).
Mutations in Six3 cause holoprosencephaly in humans,
indicating a requirement for Six3 in this process (Wallis and
Muenke, 1999). The two retinal primordia then become
localised to the lateral wall of the prosencephalon during
Subsequent evagination of the primordia results in the
formation of the optic vesicles. For this process, Rx3 function
is essential. Functional studies consistently argue for a
regulatory role of vertebrate Rx genes in proliferation of
retinal progenitor cells in the optic vesicle (Andreazzoli et
al., 1999; Mathers and Jamrich, 2000), thus regulating its
growth. In the absence of Rx3 function, there is no sign of
morphogenesis and the specified retinal precursors do not
proliferate and eventually die. Rx3 acts downstream of Six3
and Pax6 that determine the retina anlage. However, it is
possible that Rx3 initially also receives input from neural plate
Subsequent development divides the optic vesicle into
specific regions that then give rise to neural retina (NR), retinal
pigmented epithelium (RPE) and optic stalk. Several genes that
are expressed during these later steps of retinal development
require Rx3 function directly or indirectly. Interestingly, the
expression of Tbx2 and Tbx3 is specifically affected in the
retinal primordium, but not in the hypothalamus, where they
are also co-expressed. This indicates a differential regulation
of Tbx2 and Tbx3 in these tissues.
The authors thank Milan Jamrich and Peter Gruss for discussion of
unpublished results, J.-S. Joly for materials, and S. Cohen and M.
Treier for comments on the manuscript. This work was supported by
an EC-Biotech grant (BIO4-CT98-0399) to J. W.
4044 Download full-text
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