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First published online 3 July 2006
doi: 10.1242/dev.02450
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1 Zebrafish Neurogenetics Junior Research Group, Institute of Virology,
Technical University-Munich, Trogerstrasse 4b, D-81675, Munich, Germany and
GSF-National Research Center for Environment and Health, Department Zebrafish
Neurogenetics, Institute of Developmental Genetics, Ingolstaedter Landstrasse
1, D-85764 Neuherberg, Germany.
2 Sars Centre for Marine Molecular Biology, University of Bergen,
Thormoehlensgt.55, N-5008 Bergen, Norway.
3 MRC Centre for Developmental Neurobiology, New Hunt's House, King's College
London, SE1 9RT London, UK.
Author for correspondence (e-mail:
bally{at}gsf.de)
Accepted 22 May 2006
 |
SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Zebrafish, Telencephalon, Eye field, Forebrain, Rx3
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Following the specification of forebrain identity during gastrulation,
local organizers refine and maintain forebrain patterning
(Foley and Stern, 2001
;
Wilson and Houart, 2004). One
organizer, located at the anterior margin of the neural plate (ANB or ANR),
controls development of anterior forebrain identities
(Houart et al., 2002;
Houart et al., 1998;
Shimamura and Rubenstein,
1997). The ANB expresses the secreted factors Fgf3 and Fgf8
(Eagleson and Dempewolf, 2002),
as well as potent Wnt antagonists (Houart
et al., 2002). In zebrafish, one of these antagonists is the
secreted Frizzled Related Protein (sFRP) Tlc. Ectopic expression of Tlc mimics
ectopic ANB activity in telencephalic induction, and abrogation of Tlc
function impairs the formation of telencephalon and eyes
(Houart et al., 2002).
Conversely, increased canonical Wnt activity, for instance by overexpression
of Wnt8b normally produced in the posterior diencephalon
(Kelly et al., 1995), or by
the loss of function of Axin1, leads to an enlargement of the diencephalon at
the expense of the telencephalon and eyes
(Heisenberg et al., 2001;
Houart et al., 2002;
van de Water et al., 2001),
and the lack of telencephalon can be corrected by increased levels of Tlc.
Similarly, abrogation of the Wnt inhibitors Tcf3 or Six3 abolishes anterior
forebrain development at the benefit of more posterior identities in zebrafish
and mouse (Dorsky et al., 2003;
Kim et al., 2000;
Lagutin et al., 2003). These
results suggest a model where the level of canonical Wnt activity, determined
by the antagonism between a posterior local source and anterior local
inhibitors, patterns forebrain development during gastrulation
(Wilson and Houart, 2004).
Manipulations of Wnt or its antagonists at an early stage affect
simultaneously the presumptive telencephalon and eye field, suggesting that
these two domains are initially defined as one in their early response to Wnt
activity. The factors controlling the later separation of the telencephalon
and the eye field within the anterior forebrain are unknown. One candidate
might be olSfrp1, a sFRP expressed in the anteriormost region of the neural
plate in Medaka: abrogation of olSfrp1 function using morpholino antisense
oligonucleotides produces embryos with reduced eyes and a complementarily
enlarged telencephalon, without modifying diencephalic size
(Esteve et al., 2004). How
olSfrp1 acts at the cellular and molecular levels, and whether it indeed
controls cell specification choices, however, remains unknown.
The specification of the eye field is correlated with sustained expression
of Pax6, Six3 and Rx1-Rx3, shown to be crucial for eye development
(Bailey et al., 2004;
Graw, 2003;
Hanson, 2003;
Mathers and Jamrich, 2000).
Rx genes encode paired-like homeodomain proteins. At late
gastrulation, expression of the mouse Rx gene is intense in the eye
field and is non-overlapping with the adjacent telencephalic field
(Bailey et al., 2004;
Chuang et al., 1999). A similar
pattern is observed for zebrafish rx3, the earliest and only
rx gene expressed at the open neural plate stage
(Chuang et al., 1999). Knockout
of the single mouse Rx gene, and inhibition of Xenopus Xrx1
function, abolishes the formation of eye structures
(Casarosa et al., 2003;
Mathers et al., 1997).
Conversely, ectopic expression of rx1 and rx2 by mRNA
injection in zebrafish triggers an expansion of retinal tissue
(Chuang and Raymond, 2001).
These observations suggest that the Rx genes are involved in the
specification or maintenance of retinal progenitors
(Bailey et al., 2004), in
contrast with the proposed later function of zebrafish and Medaka rx3
in retinal evagination; in null mutants for Rx3 [chokh (ckh)
and eyeless (el), respectively], early anterior genes such
as six3 or pax6 are expressed, but the optic vesicle fails
to evaginate (Kennedy et al.,
2004; Loosli et al.,
2003; Loosli et al.,
2001; Winkler et al.,
2000). Because forebrain patterning defects had not been noted,
Rx3 was proposed to be an unusual member of the Rx family controlling retinal
morphogenesis.
We describe here a zebrafish mutant, ne2611, with an expanded telencephalon and a lack of eyes. We report that ne2611 is a null allele of rx3, and that retinal precursors in ne2611 ectopically express tlc at late gastrulation and acquire a telencephalic fate. We reanalyzed the published allele ckhs399 and demonstrate that tlc and telencephalic expansion are also apparent in this mutant. These results identify Rx3 as a key determinant controlling specification choices between eye field and telencephalon during anterior forebrain patterning.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). chokh (chks399)
(Loosli et al., 2003) mutants
were obtained by pairwise mating of heterozygous adult carriers.
chkne2611 fish were recovered in a small scale screen
focusing on CNS defects. The screen setup followed that of Haffter et al.
(Haffter et al., 1996), except
that an incross was conducted in the F1 generation. The specific locus rate
was 1/670 against the golden locus and 442 genomes were screened to
recover ne2611.
Molecular identification of ne2611
The rx3 locus of chkne2611 mutants was
analyzed for putative mutations by direct sequencing of PCR products
(Sequiserve) of each of the three exons comparing homozygous wild-type with
homozygous mutant embryos. Primers were designed to bind intronic sequences
flanking the exons to include putative splice site mutations. An exception was
exon 1, where the forward primer was designed containing the ATG-start
site.
rx3_exon1_forward, 5'-GCACGAGGTTCAATGAGGC-3';
rx3_exon1_ reverse, 5'-AAGTTAGAAGTTAGGATAAAGTTGTCAA-3';
rx3_exon2_forward, 5'-TGCACTTTCTCACATATTTCTCACTG-3';
rx3_exon2_ reverse, 5'-TATTATTGCTGTATTAGTTTGAACAGAA-3';
rx3_exon3_forward, 5'-ATAAGCTCCTCAACTACATCTTTAACTT-3'; and
rx3_exon3_ reverse, 5'-AGACCACTGATTTTGAAGATACAAA-3'.
The only significant alteration was found at nucleotide position 382 of the coding sequence. This T to C transition leads to the introduction of a new SatI endonuclease restriction site.
RNA and BAC injections
chk/rx3 cDNA (IMAGp998G108961Q) was obtained from RZPD (Deutsches
Ressourcenzentrum für Genomforschung GmbH,
www.rzpd.de)
and subsequently subcloned into the pCS2+ Vector.
chkne2611/rx3 was cloned by PCR from reverse-transcribed
RNA from homozygous ne2611 mutant embryos, followed by direct cloning
using the TOPO cloning kit (Invitrogen) and subcloning into pCS2+:
rx3_cDNA_forward, 5'-AAATCGTTCAATGAGGCTTGTT-3'; and rx3_cDNA_reverse, 5'-TCTCATCTACCACGTCTTCCCTATA-3'
chk/rx3 and chkne2611/rx3 capped RNA was synthesized using the Ambion mMessage mMachine Kit, following the recommended procedure. Capped RNA was injected at the concentration of 50 or 100 ng/µl into the embryos at the one-cell stage.
The BAC CHORB736A01233Q containing the rx3 locus was obtained from RZPD, amplified and purified with the Large-Construct Kit (QIAGEN) and injected at a concentration of 35 ng/µl into embryos at the one-cell stage.
Bioinformatic analysis
The JPRED algorithm (Cuff et al.,
1998) was used to find a nearly related secondary structure that
has been analyzed in detail
(http://www.compbio.dundee.ac.uk/~www-jpred/).
The input was the protein sequence of Rx3 (ENSDARP00000022866) from the Sanger
Centre zebrafish assembly version 4 (Zv4) using the ENSEMBL server. The
primary output of the algorithm (1FJL.pdb) was used for further sequence and
structure analysis using the MAGE software package version 6.36
(http://kinemage.biochem.duke.edu/software/mage.php).
Cell transplantations between ne2611 and wild type
Wild-type donor embryos were injected with biotin-dextran (Molecular
Probes) at the one-cell stage. Thirty to 40 cells were transplanted
isotopically and isochronically onto the animal pole of shield-stage wild-type
or ne2611 embryos. Recipient embryos were processed at 30 hpf for
immunochemical detection of the biotin tracer.
CLGY469 transgenic donor embryos were injected with 1.5%
lysine-fixable (fluoro-ruby) Tetramethylrhodamine Dextran (10,000
Mr, Molecular Probes) in water (Ambion) at the one-cell
stage. Cell transplantations were as previously described
(Ho and Kane, 1990), with
recipient and donor embryos maintained in the dark at all stages.
Transplantation of around 10 cells was conducted in a homotypic manner at the
animal pole at dome stage. The appropriate localization of transplanted cells
was checked under fluorescent light, and donor and recipient embryos were
documented and subsequently fixed at around 30 hpf.
Uncaging experiments
A solution of DMNB-Caged Fluorescein dextran and biotin, lysine fixable (5
mg/ml; Molecular Probes), was injected into one-cell embryos, which were
allowed to develop further in the dark. At the early tailbud stage, the dye
was activated in a few cells using a UV-beam (DAPI-channel) focused with a
pinhole. Irradiated embryos were imaged at the 24 hpf stage, using the
FITC-channel on a Zeiss Axioplan2 Microscope with a Zeiss Axiocam Hrm Camera
and the Axiovision Software Package (Zeiss), and subsequently fixed overnight
in 4% paraformaldehyde at 4°C and processed for anti-fluorescein
immunocytochemistry.
In situ hybridization and immunohistochemistry
Probe synthesis, in situ hybridization and immunohistochemistry were
carried out according to standard protocols
(Hammerschmidt et al., 1996).
The anti-Phospho-Histone H3 antibody (Upstate Biotechnology) was used in a
final dilution of 1/200. Purified rabbit anti-GFP antibody (ams biotechnology)
was used in a 1/500 to 1/1000 dilution. They were revealed using
FITC-conjugated goat anti-rabbit secondary antibody (Jackson ImmunoResearch
Laboratories) or Cy3-conjugated goat anti-mouse secondary antibody (Jackson
ImmunoResearch Laboratories) (1/200). Embryos were scored and photographed
under a Zeiss SV 11 stereomicroscope or a Zeiss Axioplan photomicroscope.
Isolation and mapping of the CLGY469 insertion
CLGY469 was recovered in a retrovirus-mediated large-scale
enhancer detection screen for its expression in the retina. The 3'
sequence flanking the insertion was identified by linker-mediated PCR as
described previously (Ellingsen et al.,
2005). This sequence (TAAAAAAAAATTTGGGGT-CAATATTACAAG) maps to
chromosome 10, 37.390 base pairs upstream of the rx3 locus (Sanger
Centre zv5 release).
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Telencephalic expansion in ne2611 was confirmed at the 15-somites stage with molecular markers (emx3 - previously emx1-, emx2, pax6.1) (Fig. 1C,D; data not shown), and occurs along the anteroposterior (AP) and mediolateral axes. To determine whether this phenotype reflected broader AP patterning defects, we compared the relative sizes of the different forebrain and midbrain domains (telencephalon, hypothalamus, prethalamus, thalamus, pretectum and midbrain). We used lhx5 and arx as markers of the prethalamus and posterior hypothalamus at 15 somites, and nkx2.1b to reveal the anterior and posterior hypothalamus (Fig. 1E,F; data not shown; see also scheme in Fig. 1H,I). her5 expression identified the midbrain-hindbrain boundary, and the size of the anterior hindbrain, limited by krox20, served as a reference to correct for variations in embryo length. We confirmed that ne2611 embryos suffer from substantially elongated neural tissue anterior to the prethalamus (Fig. 1G, red bars, P<0.01, n=10 embryos measured for each genotype), and found that this phenotype is local, as the prethalamus itself, as well as structures located posterior to the lhx5/arx domain, is unchanged compared with wild-type siblings (Fig. 1G, blue bars, P=0.75, i.e. no significant change, n=10 embryos measured for each genotype) (size of the arx domain between wild type and ne2611: P=0.15, i.e. no significant change, n=10 embryos measured for each genotype) (schematized in Fig. 1H,I).
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The absence of expression of eye markers could result from the
non-specification or the non-maintenance of eye precursors, and, to resolve
this issue, we probed ne2611 mutants for the expression of the
earliest eye-field markers. In order of appearance, we selected rx3
(at 70% epiboly), followed by rx1 and rx2 (at the 3-somite
stage) (Chuang et al., 1999;
Chuang and Raymond, 2001). We
found that rx3 and rx1 expression followed a normal
spatiotemporal pattern in ne2611 mutants compared with their
wild-type siblings (Fig. 2C-H),
but that their expression was lost during somitogenesis (not shown). By
contrast, rx2 was never expressed
(Fig. 2I-K). Thus, an
incomplete eye-field identity (rx3+,
rx1+ but rx2-) is specified in
ne2611, but this transient phase is followed by the loss of
expression of all retinal markers.
ne2611 is a null allele of rx3/chokh
Reduced or absent eyes characterize the zebrafish mutants headless
(hdl; tcf7l1a) (Kim et
al., 2000), masterblind (mbl; axin1)
(Heisenberg et al., 2001;
van de Water et al., 2001) and
chokh (ckh; rx3)
(Kennedy et al., 2004;
Loosli et al., 2003;
Rojas-Munoz et al., 2005). In
addition, hdl and mbl embryos display various degrees of
brain posteriorization leading to forebrain truncations, while the existing
ckh alleles ckhs399, ckhw29
and ckhhu499 were described as not affecting telencephalic
development (Kennedy et al.,
2004; Loosli et al.,
2003). We found, however, that ne2611 is allelic to
ckh (23 embryos lacking eyes in 78 embryos from a
ne2611/+xckhs399/+ intercross in two
independent experiments). Sequencing of the rx3 cDNA from
ne2611 embryos revealed a T to C transition within exon 2 in
nucleotide position 382 (Fig.
3A), leading to a Serine to Proline substitution at amino acid
position 128 of the Rx3 protein (T382N,
Fig. 3B,C). Comparison using
the JPRED algorithm with the structure of the Drosophila Paired
homeodomain predicts this substitution to a coiled domain separating helix1
and 2 of the Rx3 homeodomain.
|
;
Loosli et al., 2003;
Rojas-Munoz et al., 2005). The
above T382N mutation segregated with the ne2611 telencephalon and eye
phenotypes in DNA sequenced from eight independent embryos. The mutation
creates a Sat1 restriction site that also segregated with loss of
eyes and expanded telencephalon in more than 50 embryos tested by PCR and
Sat1 digestion (not shown). In addition, we found that all eyeless
embryos from a ne2611/+xckhs399/+
intercross, but no embryos with normal eyes from such crosses, had an enlarged
telencephalon (not shown). We also could rescue both the retinal and
telencephalic defects of ne2611 embryos following injection of BAC
CHORB736A01233Q, which contains the rx3 locus (Sanger centre and
RZPD; 79% of the mutant embryos were at least partially rescued,
n=64; Fig. 3G-K).
Finally, a re-analysis of ckhs399 revealed that mutant
embryos display an enlarged telencephalon identical to ne2611 (100%
of cases, n=23; Fig.
3D-F, see also Fig.
4D). We conclude that ne2611 is an allele of
ckh, henceforth referred to as ckhne2611, and
reveals a previously undiscovered role of Rx3 in telencephalic
development.
As mentioned above, structural considerations predict that the
Rx3ne2611 protein is dysfunctional, and we found the telencephalic
and eye phenotype caused by the ne2611 mutation to be as severe as
those of ckhs399, which truncates the Rx3 homeodomain
(Loosli et al., 2003). This
suggests that ne2611 might represent a null allele of rx3.
To support this interpretation, we overexpressed rx3ne2611
RNA in wild-type embryos. Ectopic expression of wild-type rx3 mRNA
produced embryos with head truncations at 24 hpf in a dose-dependent manner
(10% of cases, n=56, Fig.
3L,N). By contrast, no morphological defects were noted following
injection of rx3ne2611 mRNA
(Fig. 3M,N). These results
suggest that ne2611 is a null allele of rx3.
Rx3 controls patterning of the telencephalon and eye field at gastrulation
Zebrafish rx3 expression is initiated at late gastrulation and is
first restricted to the presumptive eye field and hypothalamus
(Chuang et al., 1999), which
abut the telencephalic primordium (Wilson
and Houart, 2004). To determine whether the telencephalic
phenotype of ckh reflects an early role of Rx3 in anterior neural
plate development, we examined expression of telencephalic markers during
these stages in both ckhne2611 and
ckhs399.
tlc is one of the earliest markers of the presumptive anterior
forebrain at late gastrulation, and is excluded from the hypothalamus and eye
field (Houart et al., 2002) to
become adjacent to the rx3 domain at tail-bud stage
(Fig. 4E,F). The earliest
phenotype in both ckhne2611 and
ckhs399 mutants was the posterior expansion of
tlc staining at the tail-bud stage (100% of
ckhne2611 mutant embryos, confirmed by genotyping, have
expanded tlc expression, n>50; 24% of embryos from a
cross between ckhs399 heterozygotes have a similar
phenotype, n=82; Fig.
4A-D). Ectopic tlc expression was prominent from bud
stage onwards (Fig. 4E-G).
emx3 and foxg1 (bf1) also label the presumptive
telencephalon, and we compared their expression with that of tlc
expression in a time-course analysis. foxg1 expression appeared
identical in wild type and ckhne2611 mutants at bud stage
(Fig. 4H), but was expanded
posteriorly from the one-somite stage onwards; emx3 expression was
unaffected in ckhne2611 embryos until the 3-somite stage
(Fig. 4K-M), only becoming
visibly ectopic a few hours afterwards (not shown). Double stainings further
demonstrated that tlc and foxg1 expression largely overlaps
rx3 expression at the stages when they become ectopic in ckh
(Fig. 4G,J), suggesting that
these posterior expansions result from a failure to be repressed within the
rx3 domain. Accordingly, overexpression of rx3, but not
rx3ne2611, by mRNA injections into wild-type embryos,
reduced expression of early telencephalic markers such as tlc and
hesx1 (anf) at the tail-bud stage (58% of cases,
n=18; see Fig. S2 in the supplementary material; data not shown).
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Rx3 function accounts for the higher proliferation of eye-field cells when compared with telencephalic precursors
We next addressed the processes underlying this early function of Rx3.
Several non-exclusive mechanisms affecting early telencephalic precursors
might account for telencephalic expansion in ckh: their reduced cell
death, their increased proliferation, or an ectopic specification of such
precursors within the anterior neural plate. We analyzed cell death profiles
in ckhne2611 embryos between 90% epiboly and 3 somites
using acridine orange and immunostaining of cleaved caspase 3. We did not
detect any significant difference between mutant and wild-type siblings at
these stages (n=19), although we did observe apoptosis in the
telencephalon of ckhne2611 mutants at 28 hpf (not
shown).
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|
). We show
that Rx3 may exert this role already within the anterior neural plate.
Rx3 attributes eye versus telencephalic identity to anterior forebrain precursors
Decreased proliferation of retinal precursors might partially account for
the lack of eyes of ckh mutants, but cannot be the direct cause of
telencephalic expansion. By contrast, the co-expression of tlc or
foxg1 and mutant rx3 RNAs in the presumptive eye-field
territory of early ckhne2611 embryos
(Fig. 4G,J) suggests that
telencephalic specification extends posteriorly at the expense of eye identity
in the mutants.
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10) of
eye-field precursor cells at the early tail-bud stage
(Fig. 6A,B); the location of
the progeny of these labeled cells was then determined at 24 hpf by
morphological inspection under fluorescence and Nomarski optics
(Fig. 6C-I). At the early
tail-bud stage, the eye and telencephalic fields are intermingled
(Woo and Fraser, 1995);
however, we found that uncaging at the position indicated in
Fig. 6A mostly revealed retinal
precursors in wild-type embryos (Table
1): pure retinal clones were recovered in 45% of cases
(Fig. 6C), and clones
contributing to the retina and other forebrain derivatives except the
telencephalon in 45+21=66% of cases (Fig.
6D). This compared with 34% of cases contributing to the
telencephalon (Fig. 6E;
n=29). By contrast, all labeled clones contributed to the
telencephalon in ckhne2611 embryos (100% of cases,
n=10; Table 1,
Fig. 6F-I). Because extensive
labeled clones were recovered in all activated ckhne2611
mutants (Table 1), we conclude
that retinal precursors lacking Rx3 tend to join the telencephalon. We never
detected expression of retinal markers in the expanded telencephalon of
ckhne2611 (see otx2 on
Fig. 2B, or atoh7 and
vax2, not shown), supporting the interpretation that presumptive
eye-field cells may acquire a telencephalic fate in the absence of Rx3
function.
|
)
proposed that Medaka Eyeless, the ortholog of zebrafish Rx3, is responsible
for retinal evagination in a cell-autonomous manner. The same is true for
zebrafish Rx3: when large numbers of wild-type cells (25-40) are homotopically
transplanted to the animal pole of a shield-stage
ckhne2611 embryo, they often give rise to a normally
evaginated retina (36% of cases, n=11), which is always composed
exclusively of wild-type cells (Fig.
7A). By contrast, ckhne2611 cells transplanted
into a wild-type host never contribute to the retina (not shown). Our results support an earlier role of Rx3 in specification choices of eye field versus telencephalic identity. To determine whether this earlier function of Rx3 was also cell autonomous, we uncoupled it from morphogenesis control by transplanting a small number of cells (4-5). Indeed, we observed that when a low number of wild-type cells are transplanted into the presumptive eye field of a ckhne2611 host, these integrate into the anterior forebrain and no evagination takes place (0% of cases, n=14; Fig. 7B). This finding suggests either that the wild-type cells are topologically misplaced in a mutant environment but keep their eye-field identity, or that when in a minority inside a Rx3-depleted environment, the wild-type cells are subjected to a non-autonomous cell fate change and adopt telencephalic identity.
To discriminate between these two possibilities and to assess the identity
of the progeny of these transplanted cells, we made use of a sensitive
transgenic retinal marker. In an enhancer detection screen following the
technology described by Ellingsen et al.
(Ellingsen et al., 2005), we
recovered a transgenic line, CLGY469, which expresses YFP in the
retina but not in the telencephalon (Fig.
7D). As mapping indicates, this line is likely to detect an
rx3 enhancer (see Materials and methods). YFP expression is observed
in CLGY469 transgenic embryos from the tail-bud stage onwards (see
Fig. 7C for a 5-somite embryo),
and thus precedes retinal evagination and is an early marker of retinal
specification. We therefore used CLGY469 expression as a selective
and sensitive marker for retinal specification in our transplantation
experiments with a low number of cells. When a few wild-type cells taken from
the animal pole of a CLGY469 transgenic donor were homotopically and
isochronically transplanted into a ckhne2611
non-transgenic host at the sphere stage, we repeatedly observed that some of
these cells turn on expression of the transgene (80% of cases, n=5;
Fig. 7F), in conditions where
no morphological retina is visible. We can rule out that the transplanted
cells, although YFP-negative at the time of transplantation, were already
determined to express the transgene: similar grafts into non-transgenic
wild-type hosts usually gave rise to YFP expression in cells that populated
the retina, but not in cells that contributed to other structures (usually the
telencephalon; Fig. 7G; 86% of
cases, n=7). We conclude that the maintenance of eye-field identity,
and repression of telencephalon fate, is cellautonomously encoded by Rx3
expression.
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|
DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Loosli et al.,
2003; Loosli et al.,
2001; Winkler et al.,
2000) and formation of the pigmented retinal epithelium
(Rojas-Munoz et al., 2005),
they also suggest that the link between several crucial steps of retinal
development, namely the maintenance of retinal precursors, the morphogenetic
shaping of the retina into an optic cup and the specification of the pigmented
retinal epithelium, are accomplished at least in part by the use of the same
molecule, Rx3.
The complete ne2611 phenotype results from the loss of Rx3 function
Previous analyses of ckh mutant alleles did not report
telencephalic defects (Kennedy et al.,
2004; Loosli et al.,
2003; Rojas-Munoz et al.,
2005), making it crucial to ascertain that the ne2611
phenotype results solely from the loss of Rx3 function. Our observations that
telencephalic and eye phenotypes in ckhne2611 always
co-segregate and are concomitantly rescued by injection of an
rx3-containing BAC support the interpretation that Rx3, directly or
indirectly, controls the early development of both telencephalon and eyes. A
further decisive argument towards this interpretation is provided by our
re-analysis of ckhs399, and the finding that
ckhs399 mutants display expanded tlc and
emx3 expression from the tail-bud and somite stages onwards,
respectively, in a manner indistinguishable from ckhne2611
(Fig. 3F,
Fig. 4D). These observations
lead us to propose an early role for Rx3 in limiting extension of the
telencephalic field at late gastrulation. This finding is in keeping with the
onset of zebrafish rx3 expression during gastrulation, earlier than
rx1 and rx2, and in a domain adjacent to but non-overlapping
with the presumptive telencephalon (Chuang
et al., 1999). The phenotype of Rx mutant mice is complex
and includes both lack of eyes and anterior forebrain truncations
(Mathers et al., 1997). Given
that the mouse harbors a single Rx gene, successive functions for Rx
might be difficult to appreciate experimentally; however, the precocious
downregulation of retinal markers in these mutants is compatible with an early
role of Rx in controlling retinal specification. Medaka, where loss of Rx3/el
seems to be only associated with morphogenesis defects, is a more puzzling
case (Loosli et al., 2001;
Winkler et al., 2000). Unlike
zebrafish Rx3 (Chuang and Raymond,
2001) (this paper), overexpression of Rx3/el in Medaka does not
cause head truncations (Loosli et al.,
2001). Rx3/el can, however, rescue the ckh phenotype
(Loosli et al., 2003). Thus,
zebrafish Rx3 and Medaka Rx3/el might have similar regulatory capacities, but
not all can be revealed in the Medaka context. This observation might relate
to the recent demonstration of a genetic backgrounddependent effect of Rx3 in
zebrafish (Rojas-Munoz et al.,
2005). However, the evolutionary conservation of an early
expression of Rx genes during brain development in vertebrates and
invertebrates (Bailey et al.,
2004) lends support to a primary ancestral role of Rx in the
specification of early anterior progenitors.
Rx3 function maintains retinal versus telencephalic identity
The expanded telencephalon and lack of eyes of ckh mutants might
have been subsequent but unrelated phenomena reflecting two independent roles
of Rx3. Contrary to this hypothesis, we found that retinal precursors,
although at least partially specified (e.g. expressing rx3 and
rx1 RNAs, Fig.
2C-D,H), are affected already at late gastrulation in
ckhne2611 mutants. This is noticeable in two ways: first,
their proliferation is reduced (Fig.
5B,E); and second, their identity is altered, as revealed by the
co-expression of tlc or foxg1 and rx3 within the
eye field in ckhne2611 at the tail-bud or one-somite stage
(Fig. 4G,J), combinations never
observed in wild-type embryos. Direct tracing of eye-field cells from the
tail-bud stage further demonstrates that these cells, in mutants, populate the
telencephalon instead of the retina and probably acquire a telencephalic fate
(Fig. 6). Together, these
results are best interpreted by postulating an early role of Rx3 in permitting
the maintenance of retinal versus telencephalic specification during
gastrulation.
Based on overexpression studies, zebrafish Rx1 and Rx2 were postulated to
promote retinal versus telencephalic identity
(Chuang and Raymond, 2001).
Notably however, the telencephalic to retinal fate switch imposed by injection
of rx1 or rx2 mRNA was only observed at a late stage (neural
keel), while no alteration in emx3 or foxg1 expression could
be detected at the neural plate stage. It will be important to support these
data by loss-of-function analyses to determine whether the distinction between
the eye and telencephalic identities is a general property of Rx factors, and,
if so, whether these factors act in a timely regulated cascade.
An additional phenotype of ckhne2611 mutants is the
decreased proliferation of eye-field precursors at the tail-bud stage
(Fig. 5), suggesting that Rx3,
in addition to imparting cell identity, also promotes proliferation of its
expressing cells at late gastrulation. This finding is in line with previous
analyses of Xenopus Xrx1, which has been shown to increase the clonal
proliferation of retinal progenitors in ectopic expression experiments
(Casarosa et al., 2003).
Whether the impaired proliferation of the eye field in
ckhne2611 mutants is a consequence of the altered identity
of these cells or reflects an independent role of Rx3 in proliferation control
remains to be resolved. Similarly, Xrx1 promotes both proliferation and
retinal identity.
|
), and
identifying the upstream regulators of the Rx3-mediated retinal specification
cascade will be an important step in our understanding of anterior forebrain
patterning. Known targets of Rx3 include rx2 and mab21l2,
which are absent or downregulated in ckh
(Kennedy et al., 2004).
six3 or pax6.1 expression appeared unaffected in
ckhne2611 at the 3-somite stage (not shown); however, both
genes also label the telencephalic primordium at that stage, preventing a
straightforward interpretation of these results. We identify here, however,
two new and possibly important targets of Rx3: tlc and
foxg1. Their expression is modified in ckhne2611
prior to the normal timing of optic vesicle evagination, strongly suggesting
that these genes respond to the early function of Rx3. Whether Rx factors
display transcriptional repressor or activator functions is unresolved
(Chuang and Raymond, 2001), and
our results do not permit us to determine whether the regulation of expression
of tlc and foxg1 by Rx3 is direct. Finally, we observed that ckhne2611 cells transplanted into the animal pole of a wild-type host preferentially populate the telencephalon (not shown). Thus, in addition to attributing an eye-field identity to its expressing cells, Rx3 may also endow these cells with specific cell surface recognition properties that distinguish them from telencephalic precursors. This phenomenon might be an integral part of the Rx3-encoded specification maintenance process.
A model for the subdivision of the anterior forebrain into telencephalon and eye field
A major finding of our study is that, at late gastrulation, Rx3 controls
cell identity choices between the presumptive telencephalic and retinal fields
but spares most other diencephalic domains, with the possible exception of the
anterior hypothalamus (see Fig.
1E-I). These results permit for the first time the proposal of a
model for the segregation of the telencephalon and eye field
(Fig. 8). Following global AP
forebrain patterning during gastrulation, a boundary of the patterning field
is established between the diencephalon and eye field
(Fig. 8, asterisk), demarcating
the posterior limit of a patterning process specific to the anterior forebrain
and segregating eye versus telencephalic identities. Without Rx3 function,
retinal precursors adopt a telencephalic fate, demonstrating that Rx3 biases
cell fate choices in bipotential precursors. Whether telencephalic precursors
at that stage would also be capable of acquiring a retinal fate remains to be
demonstrated. It is also unknown whether these precursors are defined by
default by the absence of Rx3 expression, or whether they also necessitate
instructive information.
Whether the specific Rx3-mediated anterior forebrain patterning process
involves graded positional information, in a manner reminiscent of global
forebrain patterning at an earlier stage
(Wilson and Houart, 2004),
remains unknown. Several findings suggest a possible involvement of Wnt
signaling in this early Rx3-dependent process. First, the earliest alteration
in gene expression in ckhne2611 is the ectopic maintenance
of tlc expression (Fig.
4A-G; Fig. 8, blue
gradient). Second, overexpression of rx3 by mRNA injection leads to
head truncations (Fig. 3), and
a similar phenotype is caused by exaggerated Wnt signaling
(Kim et al., 2000). Thus, the
Rx3 overexpression phenotype might result from a (premature) positive
interaction of Rx3 with Wnt activity. Finally, olSfrp1, which was recently
proposed to also participate in anterior forebrain patterning, belongs, like
Tlc, to the sFRP family of Wnt-binding factors
(Esteve et al., 2004). Thus,
whether Tlc activity is instrumental in the anterior forebrain
cell-specification defects of ckhne2611, and whether an
appropriate source of Wnt is positioned at the eye field/diencephalon border
at late gastrulation, remain important issues to address. Another,
non-exclusive, interesting candidate that might account for the
ckhne2611 phenotype is Foxg1
(Fig. 8, blue bars). However,
the early expression domain of foxg1, in wild type, is not completely
restricted to the telencephalic primordium but also includes a small portion
of the ventral retinal field (Lisa Winstanley and C.H., unpublished). Thus,
Foxg1 involvement in telencephalic versus eye specification would be complex
and is likely to imply context-dependent activities, which might be related to
the presence or absence of Rx3.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/133/15/2925/DC1
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ACKNOWLEDGMENTS |
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|
Footnotes |
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Present address: Mischterlich and Partners, Postfach 330609, 80066 Munich,
Germany
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