Department of Anatomy and Developmental Biology, University College London, Gower Street, London, WC1E 6BT, UK
* Authors for correspondence (e-mail: m.take-uchi{at}ucl.ac.uk, s.wilson{at}ucl.ac.uk)
Accepted 14 November 2002
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SUMMARY |
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We show that Hh signals acting through Smoothened act downstream of the Nodal pathway to promote Vax gene expression. However, in the absence of both Nodal and Hh signals, Vax genes are expressed revealing that other signals, which we show include Fgfs, contribute to Vax gene regulation. Finally, we show that Pax2.1 and Vax1/Vax2 are likely to act in parallel downstream of Hh activity and that the bel locus (yet to be cloned) mediates the ability of Hh-, and perhaps Fgf-, signals to induce Vax expression in the preoptic area. Taking all these results together, we present a model of the partitioning of the optic vesicle along its proximo-distal axis.
Key words: Zebrafish, Vax, Eye, Optic stalk, Coloboma, fgf, hedgehog
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INTRODUCTION |
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During the formation of the optic vesicles, signals derived from axial
structures promote proximal optic stalk fates while inhibiting distal retinal
fates. Hedgehog signalling proteins (Hh) are among the signals that promote
proximal optic vesicle fates. For instance, overexpression of Hh induces optic
stalk marker genes such as pax2 and vax1, while inhibiting
retinal marker genes such as pax6 and Rx
(Ekker et al., 1995;
Hallonet et al., 1999
;
Macdonald et al., 1995
).
Furthermore, loss of function mutations in the Sonic hedgehog gene in mouse
(Shh) lead to a loss of pax2 expression, absence of optic
stalks, and cyclopia (Chiang et al.,
1996
). Although shh mutations in fish do not exhibit this
phenotype (Schaurte et al., 1998), probably because of redundancy with other
Hh proteins (Nasevicius and Ekker,
2000
), absence of pax2.1/noi expression is observed in
the optic vesicles of fish lacking the activity of Smoothened
(Chen et al., 2001
;
Varga et al., 2001
), a
transmembrane protein essential for the transduction of all Hh signals
(Ingham and McMahon,
2001
).
Nodal signals are also implicated in early proximo-distal patterning of the
optic vesicles, as evident from the cyclopic defects in fish carrying
mutations in a variety of genes functioning in the Nodal signalling pathway
(e.g. Gritsman et al., 1999;
Feldman et al., 1998
;
Rebagliati et al., 1998
;
Sampath et al., 1998
). Some of
the forebrain and eye defects in Nodal pathway mutants are indirectly because
of loss of Hh activity in anterior regions of the mutant embryos
(Macdonald et al., 1995
;
Masai et al., 2000
;
Rohr et al., 2001
;
Mathieu et al., 2002
).
However, the extent of cyclopia in Nodal pathway mutants is often more severe
than in zygotic Hh pathway mutants, suggesting that signals in addition to Hhs
contribute to the proximo-distal patterning of the eyes. Indeed, it is
probable that Fgfs contribute to the patterning of proximal optic vesicle
tissue. Several Fgfs are expressed in and around the optic stalks
(Reifers et al., 1998
;
Reifers et al., 2000
;
Shinya et al., 2001
), and
abrogation of Fgf activity leads to disrupted patterning of midline tissue
between the eyes, including the optic chiasm
(Shanmugalingam et al.,
2000
).
Our understanding of genes that function downstream of the signals that
promote proximal optic vesicle fates is fragmentary. In this context, the
best-studied protein is the transcription factor Pax2, which mediates optic
stalk differentiation and chiasm formation in both mice and fish
(Torres et al., 1996;
Macdonald et al., 1997
).
Pax2.1/Noi-expressing optic stalk cells in fish differentiate as reticular
astrocytes of the optic nerve, and it seems probable that these glial cells
play key roles in the guidance of retinal and other axons across the midline
of the brain (Macdonald et al.,
1997
). A second phenotype observed in humans, mice and fish with
disrupted Pax2 function is coloboma, a failure to close the choroid fissure
(Sanyanusin et al., 1995
;
Favor et al., 1996
;
Torres et al., 1996
;
Macdonald et al., 1997
). The
choroid fissure is the opening from the ventral side of the retina into the
optic stalk that allows exit of axons from, and entrance of blood vessels
into, the eye. During normal development, the nasal and temporal retina on
either side of the choroid fissure fuses around the axons and blood vessels.
The conserved nature of the coloboma phenotype indicates that Pax2 is likely
to regulate as yet unknown genes that directly mediate the fusion event.
A second family of genes implicated in optic stalk development and proximal
retinal patterning are the homeodomain protein-encoding Vax genes. Vax genes
are emx-like homeobox-containing genes which have now been identified
in mice, human, Xenopus, rat, chicken and medaka fish
(Barbieri et al., 1999;
Hallonet et al., 1998
;
Ohsaki et al., 1999
;
Schulte et al., 1999
;
Winkler et al., 2000
). Vax
genes are classified into vax1 and vax2 subfamilies
according to their gene structure and expression pattern. vax1 genes
are expressed in the optic stalk and preoptic area, whereas vax2
genes are expressed in the optic stalk, preoptic area and ventral retina. The
developmental roles of Vax genes have been studied through overexpression
experiments in Xenopus and chick
(Barbieri et al., 1999
;
Schulte et al., 1999
), and
gene targetting in mice (Barbieri et al.,
2002
; Hallonet et al.,
1999
; Bertuzzi et al.,
1999
; Mui et al.,
2002
). These studies have implicated Vax gene activity in
the dorso-ventral patterning of retinal cells (and hence in the retinotopic
projection of retinal ganglion cell axons), and similar to Pax2, in optic
nerve differentiation, the guidance of retinal axons and closure of the
choroid fissure. Although vax1 mutant mice do not exhibit cyclopia,
the expression of at least some retinal markers is maintained in the optic
nerves, long after such genes are normally downregulated
(Bertuzzi et al., 1999
;
Hallonet et al., 1999
). This
suggests that proximal optic vesicle tissue maintains some distal (retinal)
character in the absence of Vax1 function.
Here we describe the isolation and characterization of zebrafish vax1 and vax2. vax1 is expressed in the anterior neural keel and later in the preoptic area and optic stalk. vax2 is expressed in the anterior neural keel and later in the preoptic area, optic stalk and ventral retina. Concurrent abrogation of Vax1 and Vax2 function using morpholino oligonucleotides (MOs) results in a coloboma phenotype in which the choroid fissure fails to fuse. Subsequent to this, a medial expansion of retinal tissue along the optic nerve and into the forebrain occurs in Vax activity-depleted embryos. Thus, Vax1 and Vax2 proteins act synergistically to restrict retinal tissue to the optic cup.
To assess the upstream regulators of Vax gene expression, we examined the involvement of Hh, Nodal and Fgf signaling pathways. Overexpression of Hh induces vax1 and vax2 whereas conversely, expression of vax1 and vax2 is lost in Hh pathway mutants. Thus the expression of Vax genes in the preoptic area, optic stalk and ventral retina is dependent on the Hh pathway. We further investigate the nature of genes that might act downstream of Hh signalling to regulate Vax gene expression. We show that Noi/Pax2.1 is not essential for Vax gene expression, whereas Bel function is required within the preoptic area for Hh signals to induce Vax gene expression.
We also show that induction of Vax gene expression is lost in embryos with reduced Nodal activity and that this loss is at least in part because of reduced or absent Hh signalling in anterior regions of the Nodal mutants. However, complete loss of Nodal signalling activity restores expression of Vax genes, despite the apparent lack of Hh activity in such embryos. We provide evidence suggesting that this induction is mediated by Fgf signals.
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MATERIALS AND METHODS |
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Cloning of vax1 and vax2 cDNAs
To isolate zebrafish homologues of the Vax genes, the following oligo
nucleotide primers were used for degenerate reverse-transcription PCR:
Mixed bases are indicated based on IUB codes and I=deoxyinosine. mRNA was purified from total-RNA extracted from 24 hpf zebrafish embryos. Superscript-II (Gibco) was used for the reverse-transcription and two types of cDNA fragments were obtained by subsequent PCR. 5' and 3' regions of the cDNAs were obtained by 5'RACE and 3'RACE. Full-length coding sequence of vax1 and vax2 are available at GenBank (Accession Numbers AY185348 and AY183363, respectively).
MO antisense injections
MO antisense oligo nucleotides (Gene Tools) were designed against 25 bases
including the AUG of the vax1 and the 5' UTR of the
vax2 mRNAs (sequences available on request). Injections of MOs were
done at concentration 25 µg/µl. To confirm the specificity of the Vax
MOs, four constructs were made. Two of the constructs have the MO target
sequence of vax1 or vax2 cloned 5' to the coding
sequence for green fluorescent protein (vax1GFP and vax2GFP,
respectively). The vaxlGFP is an in-frame fusion of the target
sequence of vax1 gene to GFP coding sequence, and the
vax2GFP is a fusion of 5' UTR of the vax2 gene to
5' upstream of GFP coding sequence. The injected transcripts of these
constructs generated fluorescent proteins in the embryos. Co-injection of the
vax1MO or vax2MO removed the fluorescence following
vax1GFP or vax2GFP injection respectively, indicating that
the MOs can prevent translation of the fusion protein. The other two
constructs have three base changes in the MO target sequence of vax1
or vax2 in front of the GFP-coding sequence. The injected transcripts
of these constructs generated fluorescent protein in the embryo both with and
without injected MO. We conclude that the MOs against vax1 and
vax2 are able to inhibit translation from RNA carrying the target
sequence.
Plastic sectioning and toluidine blue staining
Embryos to be sectioned were dehydrated in methanol and embedded in JB4
resin (Agar Scientific Ltd). The polymerized block was cut at 10-µm
sections on a Jung 20555 Microtome with Leica blade, and the sections were
mounted onto glass slides. Toluidine blue staining was performed on the
sections. The slides were mounted with DPX and analysed with DIC optics on a
Nikon optiphot-2 compound microscope.
Confocal microscopy of Dil-labelled optic nerves
Embryos were fixed with 4% paraformaldehyde (PFA) for eight hours at
4°C. The lipophilic fluorescent dye, DiI, was injected intraocularly and
incubated at 4°C for 20 hours to allow dye to diffuse to the tectum.
Melanin pigment was bleached by hydrogen peroxide. DiI-injected embryos were
mounted in 1% agarose in PBS for confocal microscopy. Confocal analysis was
performed on a Leica TCS SP Confocal Microscope using 20X water immersion
objective. Three-dimensional series of images were acquired at 2 µm
intervals and imported into NIH Image to merge.
RNA injection
Capped mRNAs were synthesized in vitro using the MEGAscript kit (Ambion).
Injections were performed as described
(Barth and Wilson, 1995).
In situ hybridization
Digoxigenin-labelled antisense RNA probes were synthesized from cDNA
clones. Staged embryos were fixed in 4% PFA and stored in methanol at
-20°C. In situ hybridizations were performed using standard procedures
(Macdonald et al., 1994).
Sections of embryos were performed after whole-mount stainings.
Fate mapping of optic stalk
Embryos were injected with caged fluorescein (Molecular Probes) at the
one-cell stage. Photoactivation of fluorescent dye was performed as described
(Rohr and Concha, 2000) at
around the 14-somite stage. Following photoactivation, DIC and fluorescent
images were acquired using Openlab software (Improvision) with a cooled CCD
camera (Hammamatsu) attached to an Axioplan microscope (Zeiss). At later
stages, live embryos were mounted in 1% agarose and fluorescent images were
acquired with Leica TCS SP Confocal Microscope using a 20X water immersion
objective. Three-dimensional series of images were acquired at 2 µm
intervals. As an alternative approach, rhodamine-dextran injection was
performed using iontophoresis. Embryos were treated with pronase and mounted
in 1.2% low-melting agarose. Iontophoric injection of one or two medial optic
vesicle cells was performed using 1 µm tip micropipettes filled with 4%
rhodamine-dextran. Fates of fluorescent cells were followed and DIC and
fluorescent images were acquired using Openlab software.
SU5402 treatment
To block Fgf receptor activity, embryos were treated with 21 µM SU5402
from 70% epiboly to the 24 hpf stage as previously described
(Shanmugalingam et al.,
2000).
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RESULTS |
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Zebrafish vax1 encodes 317 amino acids and is most closely related
to medaka vax1 (Winkler et al.,
2000) with which it shares 70% identical amino acids. Zebrafish
vax2 encodes 307 amino acids and has 46% identity to Xenopus
vax2 (Barbieri et al.,
1999
) over the full sequence and 100% within the homeodomain.
Zebrafish Vax genes are expressed in the optic stalk and the preoptic
area
In situ hybridization analysis showed that similar to previously
described Vax genes, zebrafish vax1 and vax2 are expressed
in the anterior forebrain, including the optic stalks, ventral retina and
preoptic area (Fig. 2),
territories that probably all originate from a similar region of the anterior
medial neural plate (Varga et al.,
1999). A low level of vax1 mRNA is first detected in the
anterior neural keel at 7 somites (Fig.
2A). By 12 somites, expression is more robust in the medial
forebrain and forming eyes. By this stage, optic vesicles have evaginated from
the brain and the optic recess of the third ventricle is visible as a
prominent cleft within the rostral brain and optic vesicles
(Fig. 2B,C). The region of the
brain around the optic recess is termed the pre-optic area and is considered
to be the boundary between telencephalon and diencephalon. Laterally, the
preoptic area is in direct continuity with the optic stalks that in turn
connect to the prospective retinae at the site of future choroid fissure
formation.
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By 16 somites, vax1 expression remains within the optic stalk, but
like pax2.1 (see Macdonald et
al., 1997) is predominantly restricted to tissue ventral to the
optic recess in the preoptic area (Fig.
2C). In addition to the optic stalk and ventral preoptic area,
vax1 is weakly expressed in ventral retina near the choroid fissure
region by 24 hpf (Fig. 2G). At
48 hpf, vax1 expression remains prominent in the ventral preoptic
area but is much reduced in the choroid fissure and optic stalk
(Fig. 2H,I).
The expression domain of vax2 is wider than that of vax1 throughout all developmental stages analysed. By 7 somites, vax2 mRNA is detected in the anterior brain (Fig. 2D) and expression is strong within optic stalks, preoptic area and telencephalon by 12 somites (Fig. 2E). By 16 somites, expression in the telencephalon is reduced but that in the preoptic area, optic stalk and ventral retina remains (Fig. 2F), and this pattern of expression is similar at 24 hpf (Fig. 2J). By 48 hpf, vax2 expression is restricted to the ventral preoptic area, anterior dorsal hypothalamus and ventral retina (Fig. 2K,L).
The expression domain of vax1 is nested within that of vax2 at all stages observed, and because of their highly conserved homeodomain sequences, these two transcription factors probably have many of the same target genes. To address the function of these genes, we therefore considered it probable that removal of activity of both Vax1 and Vax2 might reveal phenotypes not detected following loss of function of either gene alone.
Abrogation of Vax1 and Vax2 function results in ectopic ipsilateral
optic nerve projections and severe coloboma
To assess the consequences of reducing the activity of the Vax genes, we
injected MOs against 5'UTR sequence of the Vax gene mRNAs (see Materials
and Methods for controls). Injection of either vax1MO or
vax2MO led to defects in the ventral eye. Early morphogenesis of the
optic stalks and eye cups preceded apparently normally in the injected
embryos. However, by 30 hpf it was apparent that the choroid fissure failed to
close (Fig. 3E,G), a condition
called coloboma that is also observed following loss of Pax2.1/Noi activity
(Macdonald et al., 1997), and
in mice lacking Vax1 or Vax2 activity
(Bertuzzi et al., 1999
;
Hallonet et al., 1999
;
Barbieri et al., 2002
;
Mui et al., 2002
). In injected
embryos, the retinal pigment epithelium around the choroid fissure appeared
reduced and some cell disorganization was evident around the exit point of the
optic nerve from the back of the retina
(Fig. 3F,H,M-P).
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To assess changes in gene expression that might accompany the eye defects
in the vaxMO-injected embryos, we assessed expression of markers of
optic stalk, retina and pre-optic area. Although Vax2 is involved in
dorso-ventral patterning of the retina in mouse, chicken and frog
(Barbieri et al., 2002;
Barbieri et al., 1999
;
Mui et al., 2002
;
Schulte et al., 1999
), we were
unable to detect any significant changes in the expression of region-specific
markers, tbx5.1, ephrinB2a and EphB2 in vax2MO
embryos (not shown). The only change we detected was in vax2 itself,
which showed slightly weaker expression in the ventral retina of
vax2MO embryos. Similarly, the optic stalk or choroid fissure markers
fgf8/ace, pax2.1/noi and netrin2 all appeared largely normal
in vax2 morphants. In vax1-MO-injected embryos, expression
of vax1 was slightly reduced in the preoptic area but no other
significant changes in patterns of gene expression were detected (not
shown).
Co-injection of both vax1MO and vax2MO resulted in severe disruptions to eye development. vax1/vax2 morphants showed a severe coloboma and pigmentation defects in the ventral retina (Fig. 3C,D). Consistent with the very localized expression of vax1 and vax2, all other aspects of development appear normal and vax1/vax2 morphants remain viable at 5 dpf. In the vax1-MO/vax2-MO-injected embryos, expression of vax1 is reduced in the preoptic area and vax2 expression is weaker than in wild type in the ventral retina (Fig. 3U,V), suggesting that Vax1 and Vax2 function contributes to the maintenance of vax1 and vax2 expression at these sites.
Defects in the medial optic stalk tissue or preoptic area frequently lead
to defects in commissural axon guidance (e.g.
Macdonald et al., 1997), and
studies in other species have indicated roles for Vax genes in retinal axon
guidance (Schulte et al.,
1999
; Bertuzzi et al.,
1999
; Hallonet et al.,
1999
; Barbieri et al.,
2002
; Mui et al.,
2002
). We therefore examined optic nerve projections by injecting
the lipophilic fluorescent dye, DiI, into the eye. In vax1/vax2
morphants, a minority of axons showed aberrant trajectories at the optic
chiasm and ectopically projected to the ipsilateral tectum
(Fig. 3Y,Z). No ipsilateral
projections were observed in embryos in which vax1MO or
vax2MO were injected alone.
Abrogation of Vax1 and Vax2 function results in a medial expansion of
retinal tissue into the forebrain
To assess retinal defects in Vax abrogated embryos, sections through the
eyes of the vax1/vax2 morphants were examined. Although the
lamination of the retina was normal, there was a progressive expansion of
retinal tissue to the midline of the brain
(Fig. 3K,L,R-T). At 42 hpf,
disruption to the retinal neuro-epithelium and the pigmented epithelium is
evident at the junction between the eye and optic nerve
(Fig. 3K,S). By 72 hpf, retinal
lamination has occurred and retinal neuron differentiation appears
superficially unaffected in the MO-injected embryos. However, in contrast to
wild type, the differentiating neurons extend out of the back of the eye and
along the optic nerve towards the brain
(Fig. 3L). By four days,
retinal neurons are observed all the way to the optic chiasm and pigment
epithelium appears in direct contact with the forebrain neuroepithelium
(Fig. 3R,T).
The expansion of retina to the brain could either be because of degeneration of optic stalk or nerve tissue coupled with over-proliferation of retinal neurons, or alternatively optic stalk or nerve cells may change fate and differentiate as retina. We performed several sets of experiments to determine which of these possibilities is the more probable. First, in 24 hpf vax1/vax2 morphants, expression of the retinal marker rx1 extended into the optic stalks (Fig. 3W,X), suggesting that optic nerve territory does adopt retinal character when Vax activity is abrogated.
To more directly assess whether optic nerve territory forms retina in Vax morphants, we attempted to follow the fate of these cells over time. To do this, cells in the medial region of the optic vesicles of wild-type and vax1/vax2 morphants were labelled by photoactivating caged fluorescein at the 14 somite stage (Fig. 4A). The position and viability of the fluorescently labelled cells was subsequently assessed at 2, 3 and 4 dpf (n=15 morphants; Fig. 4B-G). In both wild-type and vax1/vax2 morphants, cells labelled at the junction of optic stalk and brain gave descendents in the preoptic area and optic stalks (Fig. 4B,E). In contrast to wild type, however, the labelled territory remained very broad and did not constrict to form a nerve in the vax1/vax2 morphants. There was no obvious disappearance of the fluorescent cells in the vax1/vax2 morphants as might be expected if the optic stalks cells were dying and being replaced by retinal neurons. Indeed, labelling of individual medial optic vesicle cells through iontophoresis of rhodamine dextran confirmed the viability of these cells in vax1/vax2 morphants (data not shown). By four days, the labelled cells in vax1/vax2 morphants occupied a broad domain at the interface of the eye with the brain. Given the faintness of the fluorescence at this stage, it was not possible to definitively show that individual fluorescent cells formed retinal neurons. However, by this stage, plastic sectioning suggests that most or all optic vesicle cells of vax1/vax2 morphants are retinal in character (Fig. 3T). These data therefore favour the possibility that cells that should form optic nerve eventually differentiate as retina in embryos lacking Vax1/Vax2 activity.
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Altogether, our data shows that Vax gene activity is required to limit
retinal development to the optic cup. When Vax function is compromised retinal
tissue differentiates in a territory that should only normally contain
reticular astrocytes of the optic nerve
(Macdonald et al., 1997).
Given the importance of Vax genes in regional patterning of eye structures, it
is crucial that the spatial regulation of their expression is tightly
controlled. We therefore next assessed the pathways involved in regulation of
Vax gene expression.
Hedgehog signals acting through Smoothened induce Vax gene
expression
The Hedgehog (Hh) pathway is an upstream regulator of pax2.1
within the optic stalks (Ekker et al.,
1995; Macdonald et al.,
1995
; Varga et al.,
2001
). To determine whether Hh activity also regulates Vax gene
expression, we initially assessed the consequences of overexpression of
shh. In shh-injected embryos, the Hh target gene
ptc2 is strongly induced throughout the eye
(Fig. 5I,L), suggesting that Hh
activity is no longer spatially localized to medial regions of the optic
vesicle. In such embryos, the optic stalk expands at the expense of retinal
tissue (Ekker et al., 1995
;
Macdonald et al., 1995
), and
vax1 and vax2 expression domains are expanded within the
mispatterned optic vesicle (Fig.
5D,E). vax2 expression expands to the dorsal retina
(Fig. 5E), and complimentary to
this, expression of the dorsal retinal markers tbx5.1 and
msxC are reduced or absent in shh-injected embryos
(Fig. 5C,F) (Macdonald et al., 1995
).
Thus, Hh signals expand not only optic stalk markers but also the ventral
retinal marker vax2. These experiments suggest that Hh signals
promote Vax gene expression and change the fate of eye and optic stalk tissue
from distal to proximal fates. Overexpression of shh does not induce
Vax gene expression elsewhere within the CNS, indicating that a spatially
restricted domain of the CNS is competent to express Vax genes in response to
Hh signals.
|
To study the requirement of Hh signals to induce Vax genes, we analysed
expression in smu mutant embryos that lack zygotic activity of the
Smoothened (Smu) membrane protein (Varga
et al., 2001), an essential mediator of Hh signalling
(Ingham and McMahon, 2001
). In
smu-/- embryos, expression of both Vax genes is severely
reduced or absent (Fig. 5G,H),
revealing an essential requirement for Hh signalling in patterning of the
optic stalks, ventral retina and pre-optic area. The weak Vax expression
retained in some smu-/- embryos may be because of the Hh
signalling through residual maternal Smu protein as has been suggested for
pax2.1 and nk2.2 within the brain
(Varga et al., 2001
;
Chen et al., 2001
).
To assess whether the induction of the Vax genes by overexpression of Shh is mediated through the Smu pathway, shh-injected smu-/- mutants were examined. Vax (Fig. 5J,K) and pax2.1 (data not shown) gene expression did not recover in the shh-injected smu-/- embryos, suggesting that, as expected, exogenous Shh acts entirely through a Smu-dependent pathway.
Hedgehog signals act downstream of the Nodal signalling pathway to
induce Vax genes
In addition to the Hh pathway, Nodal signalling is crucial for various
aspects of midline patterning, including the establishment of ventral
territories in the brain. In some cases, Nodal signalling influences
patterning of the brain through regulation of the Hh pathway, whereas in
others it functions independent of Hh activity
(Masai et al., 2000;
Mathieu et al., 2002
;
Rohr et al., 2001
).
cyc-/- embryos lack activity of the Nodal ligand Cyclops
(Rebagliati et al., 1998
;
Sampath et al., 1998
) and
oep-/- embryos lack zygotic activity of Oep, an EGF-CFC
family membrane protein essential for the reception of Nodal signals
(Zhang et al., 1998
;
Gritsman et al., 1999
). Vax
gene expression was always either severely reduced or absent in
cyc-/- and zygotic oep-/-
(Zoep) embryos, indicating that as for the Hh pathway, Nodal
signalling is required for patterning the preoptic area, optic stalks and
ventral retina (Fig. 6A-D).
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In Nodal pathway mutants, expression of shh is reduced or lost in
anterior regions of the embryo (e.g. Rohr
et al., 2001), indicating that activity of both Nodal and Hh
signalling pathways is compromised. To study the genetic epistasis of the
Nodal and Hedgehog pathways on the induction of Vax genes, we injected
shh into Zoep-/- embryos. The presence of
exogenous Shh in anterior regions of Zoep-/- embryos
restored Vax gene expression in the rostral forebrain
(Fig. 6E,F). In most cases,
expression was induced in medial tissue at the interface between the ventral
telencephalon and the fused eye, a position one would expect preoptic area and
optic stalks to differentiate in wild-type embryos. Thus tissue capable of
expressing Vax genes is present in Nodal mutants, but appears to fail to
receive the signals necessary for induction of expression. These observations
suggest that Hh signalling acts downstream of the Nodal pathway to induce Vax
genes.
Lack of Nodal signalling activity induces Vax genes
Although Zoep-/- embryos lack zygotic expression of
oep, they retain maternally derived oep mRNA
(Zhang et al., 1998) and hence
possess residual Nodal signalling at early developmental stages. In contrast
to Zoep-/-, embryos lacking both maternal and zygotic
oep mRNA (MZoep-/-) showed vax1 and
vax2 expression in the brain adjacent to the fused eye
(Fig. 7A,B). Expression was
broader than that of the optic stalk-specific marker pax2.1
(Masai et al., 2000
)
(Fig. 7C), suggesting that
forebrain tissue with at least some features of preoptic area identity is
present in MZoep-/- embryos but not in
Zoep-/- embryos. The recovery of Vax gene expression in
MZoep-/- embryos indicates that early Nodal activity
indirectly suppresses the induction of Vax gene expression.
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To complement these studies, we also inhibited Nodal activity through
overexpression of Antivin/Lefty1 (Atv/Lft1), an extracellular inhibitor of
Nodal signalling (Bisgrove et al.,
1999; Meno et al.,
1999
; Thisse et al.,
2000
; Thisse and Thisse,
1999
). Injection of a high concentration of atv inhibits
all Nodal signalling and results in a phenotype similar to
MZoep-/- embryos or cyc/sqt double mutants. As
expected, overexpression of high levels of atv/lft1 led to
restoration of Vax expression in a pattern similar to that in
MZoep-/- embryos (data not shown). Altogether these
experiments reveal a complex requirement for Nodal activity in the regulation
of Vax gene expression in that early Nodal activity inhibits Vax gene
expression (through an unknown mechanism), whereas later Nodal signalling
promotes Vax gene expression (at least in part through promoting Hh
activity).
Induction of Vax and pax2.1 gene expression in the absence
of Nodal activity is Smu-independent
The restoration of Vax gene expression in the absence of Nodal activity
could either be because of a restoration of Hh activity in the vicinity of the
Vax expression, or it could be that the Vax genes are induced by other signals
independent of Hh activity. To assess which of these possibilities is more
probable, we assayed whether Hh activity is present in the rostral brain of
MZoep-/- embryos and sought to simultaneously inhibit both
Nodal and Hh signalling to ask whether this suppressed Vax expression.
Neither twhh nor shh appears to be expressed at early
stages in anterior regions of MZoep embryos and later shh
expression is restricted to posterior forebrain tissue far from the site of
pax2.1 induction (see Rohr et
al., 2001). Indeed, induction of the probable Hh target genes
patched2 and nk2.1a is restricted to the posterior forebrain
in the vicinity of the late shh expression
(Fig. 7D) (Rohr et al., 2001
). These
results suggest that Hh signalling is not occurring in the vicinity of the
pax2.1 and Vax expression domains of MZoep-/-
embryos.
Analysis of Vax and pax2.1 expression in embryos compromised in both Hh and Nodal signalling confirmed that Smu activity is unlikely to be required to induce Vax or pax2.1 expression in Nodal activity compromised embryos. By using Atv to inhibit Nodal signalling in smu-/- embryos, we found that there was no apparent difference in the expression of Vax genes or Pax2.1 among all injected siblings from smu+/- x smu+/- crosses (Fig. 7K-M). Thus, Vax genes are induced both in smu-/- and in wild-type embryos when Nodal activity is inhibited and therefore this induction is probably independent of Smu activity. We cannot completely exclude the possibility that maternal Smu promotes Vax expression following inhibition of Nodal activity, but given that maternal Smu has very little ability to promote Vax expression in otherwise wild-type embryos, we think that this is a remote possibility.
MZoep and atv-injected embryos are sensitized to
induction of Vax/pax2.1 expression by Hh signals
Given that repression of Vax gene expression appears to be relieved in
MZoep-/- embryos as compared to
Zoep-/- embryos, we hypothesized that
MZoep-/- forebrain tissue may be sensitive to factors that
promote Vax gene expression. We therefore assessed the consequences of
overexpressing shh in MZoep-/- embryos. This led
to extensive induction of vax1, vax2 and pax2.1 in the
rostral CNS, to a much greater extent than in Zoep-/-
embryos (Fig. 7F-H).
shh injection alters morphogenesis of the eye in
MZoep-/- embryos such that there is no obvious optic cup
or eye structure, and instead eye tissue (as assayed by rx1
expression, Fig. 7J) remains in
continuity with the rest of the diencephalon. Vax gene expression is induced
throughout this eye or diencephalon territory. Similar results were obtained
in embryos injected with both atv and shh. These results
suggest that a complete lack of Nodal signals renders the diencephalon
sensitive to induction of Vax and pax2.1 genes in response to Hh
signals.
Fgf signalling promotes Vax expression in the absence of Nodal
signalling
Fgf activity is required for development of midline tissue between the
eyes, raising the possibility that this pathway may contribute to the
regulation of Vax gene activity. We therefore attempted to assess whether Fgf
signals contribute to the regulation of Vax expression in wild type and in
Nodal signalling-depleted embryos.
Vax gene expression is not obviously affected in ace mutant
embryos that lack Fgf8 activity (data not shown), but other Fgfs, such as
Fgf3, are still active in the anterior CNS of these embryos (see
Shinya et al., 2001).
Therefore to more comprehensively inhibit Fgf signalling, embryos were treated
with SU5402, a chemical inhibitor of Fgf receptor function
(Mohammadi et al., 1997
).
Embryos treated with SU5402 from 70% epiboly to 24-hour stage lacked
expression of Vax genes in the preoptic area
(Fig. 7N,O), consistent with
previous data implicating Fgfs in midline patterning of the anterior forebrain
(Shanmugalingam et al.,
2000
).
Given the likely requirement of Fgf signals in the regulation of Vax gene expression, we next assessed whether the recovery of Vax expression in Nodal-signalling depleted embryos is dependent upon Fgf receptor activity. In support of this possibility, antivin-injected embryos that were treated with SU5402 lacked Vax gene expression (Fig. 7P,Q).
Altogether, these experiments suggest that Fgf signalling promotes Vax gene expression and Fgf activity may be responsible for the recovery of Vax gene expression following severe abrogation of Nodal activity.
Belladonna regulates Vax gene expression in the preoptic area
Next, we attempted to investigate the nature of the genes that might act
downstream of Hh or Fgf signals to regulate Vax gene expression. As
pax2.1 expression is promoted by Hh and Fgf activity
(Ekker et al., 1995;
Macdonald et al., 1995
;
Shanmugalingam et al., 2000
;
Varga et al., 2001
) and
precedes Vax gene expression in the optic stalks, then Pax2.1 is a candidate
for inducing the Vax genes in this region. However, induction and maintenance
of Vax gene expression proceeds normally within the optic stalks of
noi (pax2.1) mutant embryos
(Fig. 8A,B). Furthermore,
exogenous shh efficiently induces Vax gene expression in
noi-/- mutants as in wild type
(Fig. 8C,D).
|
Defects in patterning of medial optic stalk tissue or preoptic area
frequently lead to defects in commissural axon guidance (e.g.
Macdonald et al., 1997;
Shanmugalingam et al., 2000
;
Varga et al., 2001
). We
therefore screened other lines of fish known to have defects in commissure
formation for alterations to Vax gene expression.
belladonna (bel) is a mutation that leads to ipsilateral
projection of the optic nerves and consequent reversal of visual behavioural
responses (Karlstrom et al.,
1996; Neuhauss et al.,
1999
; Rick et al.,
2000
). In bel-/- embryos there is a reduction
of Vax gene expression in the preoptic area, whereas expression in the optic
stalks and ventral retina appears normal
(Fig. 8E,F).
bel has not previously been suggested to be a Hh pathway mutation and normal expression of hh pathway genes in bel-/- embryos suggests that Bel is not an upstream regulator of Hh signalling. To test whether Bel might act downstream or in parallel to Hh signalling, we injected shh into bel-/- embryos. In these embryos, Vax gene expression remained absent from the midline preoptic area, even though expression is expanded in the optic stalks and retina (Fig. 8G,H). Thus Bel function is required within the preoptic area downstream of, or parallel to, Hh signals to induce Vax gene expression.
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DISCUSSION |
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The expression domain of zebrafish vax1 is nested within
that of vax2
The presence of vax1 and vax2 subfamilies of genes in
various vertebrates supports the idea that an ancestral Vax gene duplicated
prior to the divergence of the different vertebrate classes. Despite the
probable ancient nature of this duplication, vax1 and vax2
genes have retained conserved domains of expression between species. Thus,
vax1 genes tend to be expressed strongly in medial regions of the
anterior brain, whereas vax2 genes are expressed prominently in the
ventral retina. This is presumably because of changes in the regulatory
elements that control the spatial extent of expression of the genes (see
Lynch and Force, 2000). The
conservation of vax1 and vax2 expression domains between
vertebrate Classes suggests that the changes in regulation of the two genes
may predate the vertebrate lineage. It will therefore be of great interest to
assess whether non-vertebrate chordates possess both Vax genes and if so, what
their patterns of expression are.
Despite the ancient timing of the duplication that has probably established Vax1 and Vax2, the two proteins remain structurally highly related, and so probably have similar functional activity. Indeed, as Vax1 and Vax2 have highly conserved homeodomains and are expressed in overlapping regions (particularly in zebrafish in which vax1 expression is nested entirely within the vax2 expression domain), then it is probable that these transcription factors regulate many of the same target genes. It is possible that other factors may modulate the transcriptional activity of the Vax proteins by interacting with the two conserved domains, VCDa and VCDb, which lie outside of the Vax homeodomain. As Vax2 has shorter conserved stretches within these domains than Vax1 (data not shown), it is possible that this may impart at least some differences in functional activity between Vax1 and Vax2 proteins.
Given that Vax1 and Vax2 probably regulate the same genes in the same
territory, there must be evolutionary pressure to maintain the activity of the
two genes in the same cells. In other cases of functionally similar
transcriptional regulators, we, and others, have found that presumed sites of
expression of an ancestral gene are often split between the pair of genes that
result from a duplication event, thereby minimizing co-expression (e.g.
Force et al., 1999;
Rohr et al., 2001
). In the
case of the Vax genes, we speculate that the maintenance of the vax1
expression domain within the vax2 expression domain will contribute
to a gradient in the concentration of Vax proteins from medial regions of the
brain to distal regions of the optic vesicle. As we discuss below, there is
now considerable evidence of graded patterning of the optic vesicle along its
proximo-distal axis, and we suggest that variations in the levels of Vax
proteins may contribute to this pattern.
Vax transcription factors regulate closure of the choroid fissure,
limit retinal development to the optic cup and mediate commissural axon
pathfinding
Loss of function studies in mouse
(Barbieri et al., 2002;
Bertuzzi et al., 1999
;
Hallonet et al., 1999
;
Mui et al., 2002
) and now in
zebrafish have revealed requirements for Vax proteins in several different
aspects of eye and forebrain midline development. The most conserved phenotype
following abrogation of Vax activity is a failure in fusion of the choroid
fissure. This phenotype is found in both mouse and fish lacking, or with
reduced, Vax1 or Vax2 activity (with genetic background-dependent penetrance
in mouse Vax2 mutants). The severity of this coloboma phenotype is
increased in fish embryos compromised in both Vax1 and Vax2 activity, strongly
suggesting that both Vax1 and Vax2 co-operate to regulate fusion of choroid
fissure. The proteins that mediate fusion of the retina at the choroid fissure
are unknown, but possible candidates include Eph receptors and their Ephrin
ligands. Members of this family of signalling proteins have been implicated in
fusion events in other epithelia (Holmberg
and Frisen, 2002
), and several family members are expressed in the
ventral retina (Holash and Pasquale,
1995
; Connor et al.,
1998
; Eph Nomenclature
Committee, 1997
). Indeed, in mouse, changes in ephrinB1, ephrinB2
and EphB2 expression occur in the ventral retina of vax2 mutants, and
although these changes have primarily been considered in terms of retinal axon
pathfinding (Barbieri et al.,
2002
; Mui et al.,
2002
), they could potentially contribute to the choroid fissure
defects.
A second conserved phenotype in Vax mutants is disruption to commissural
axon guidance and targeting of retinal axons. In Vax1 mouse mutants,
there is a severe disruption to midline development
(Bertuzzi et al., 1999;
Hallonet et al., 1999
) and
consequently, severe disruption in the pathfinding of axons as they approach
the midline. In vax2 mutants there are also variable defects in the
development of ipsilateral and contralateral retinal projections
(Mui et al., 2002
;
Barbieri et al., 2002
). These
may arise from incorrect assignment of identity to retinal neurons in the
Vax2 mutants (Mui et al.,
2002
; Barbieri et al.,
2002
), but the possibility that midline tissue is also disrupted
has not been excluded. Indeed, in fish, commissural axon pathfinding defects
are much more obvious in Vax1/Vax2 double morphants than in either single
morphant. This suggests, that at least in fish, Vax2 does co-operate with Vax1
to pattern midline tissue.
The third conserved phenotype in animals compromised in Vax protein
activity is a failure to limit retinal development to the optic cup. The
initial indications of this phenotype came from analysis of mouse
Vax1 mutants in which expression of genes normally restricted to
retinal tissue (rx and pax6) was observed to encroach into
the optic nerve (Bertuzzi et al.,
1999; Hallonet et al.,
1999
). Our study provides dramatic confirmation of the requirement
for Vax protein activity to limit retinal development. In Vax1/Vax2 double
morphants, there is a progressive expansion of retinal tissue along the optic
nerve until by four days, neural and pigmented layers of the retina are in
direct continuity with diencephalic cells of the optic chiasm. Early
morphogenesis of the optic stalk and optic cup occurs relatively normally and
indeed, retinal axons navigate out of the eye and along the stalk or nerve to
the midline in the Vax1/Vax2 morphants. It is relatively late in development
that differentiating retinal tissue expands to the midline. Our favoured
hypothesis (given the spread of retina-specific gene expression into the
nerve, and fate tracing experiments) is that this phenotype reflects a change
in the fate of optic stalk or nerve cells to retinal tissue. We have not
however, ruled out the possibility that overproliferation and evagination of
retinal cells from the back of the eye may also contribute to the
phenotype.
Although there are clear similarities in the phenotypes following abrogation of Vax function in mice and in fish, there are also differences. For instance, midline defects are more severe in vax1 mutant mice than in vax1/vax2 morphant fish. This of course could reflect true differences in Vax protein function but there are other possibilities. Perhaps the simplest would be that the vax1 MO does not remove all Vax1 function in fish. Although we cannot discount this possibility, the severity of the retinal expansion phenotype in the Vax1/Vax2 morphants (which is much more severe than either vax1 or vax2 mutant mice) and the penetrance of the coloboma phenotype suggest that the MOs probably severely abrogate Vax function. A further possibility is the presence of other Vax genes in the fish genome. Indeed, it appears that expression of the known Vax genes is initiated a little later in fish than in other vertebrates, raising the question of whether another Vax gene, perhaps with early expression and a stronger role in midline development, might exist. The ongoing sequencing of the fish genome should soon allow us to resolve this issue.
Midline signals induce Vax genes in the preoptic area, optic stalk
and ventral retina
The Hh pathway is a key regulator of Vax gene expression in the preoptic
area, optic stalk and ventral retina. In smu-/- embryos,
Vax gene expression is lost in all these sites and so Hh signals emanating
from midline tissues are required to induce Vax genes. Overexpression of Hh
signals can expand Vax expression in wild-type embryos and restore Vax
expression in Nodal pathway mutants, indicating that Hh signals are both
necessary and sufficient to induce Vax expression within the rostral forebrain
and eyes. Within the optic vesicles, Hh signalling may act in a graded way
with the highest levels of activity promoting the most medial fates (preoptic
area and optic stalks), with lower levels promoting choroid fissure and
ventral retina. Although definitive proof of such a model is still lacking,
gain of function experiments consistently show graded effects of Hh activity
in proximodistal patterning of the optic vesicles (e.g.
Macdonald et al., 1995;
Ekker et al., 1995
; Ungar et
al., 1996; this study; Hallonet et al.,
1999
).
The loss of Vax gene expression in Nodal pathway mutants is probably
because of loss of hh expression in anterior regions of the mutant
embryos. Indeed, activation of the Nodal pathway can lead to direct induction
of Hh gene transcription and can consequently expand optic stalk
pax2.1 expression (Muller et al.,
2000). Furthermore, restoration of Hh signals in Nodal mutants
leads to induction of Vax genes, implying that tissue capable of Vax
expression is present in the Nodal mutants but does not receive the signals
necessary for induction. The loss of Hh activity in Nodal mutants may also
underlie defects in patterning of ventral telencephalon
(Rohr et al., 2001
) and in
aspects of hypothalamic development
(Mathieu et al., 2002
).
However, Hh signals are not sufficient to induce the hypothalamus in the
absence of Nodal activity (Rohr et al.,
2001
), and there is an absolute requirement for direct reception
of Nodal signals by cells in this part of the brain
(Mathieu et al., 2002
).
Indeed, given that the severity of optic vesicle cyclopia is greater in Nodal
pathway mutants than in zygotic smu-/- mutants, we suggest
that it is unlikely that the regulation of Hh gene transcription is the only
route by which Nodal signals pattern the optic vesicles.
Vax genes can be induced in the absence of Hh and Nodal signals
Although Vax expression is absent in smu-/-,
cyc-/- and embryos that lack zygotic activity of Oep, it
is present in embryos that lack both maternal and zygotic Oep activity, and in
both wild-type or smu-/- embryos that overexpress the
Nodal antagonist Lefty1/Atv. As both Hh and Nodal signals are severely
depleted in anterior regions of these embryos, then we can conclude that under
certain circumstances, Vax gene induction (and pax2.1)
(Masai et al., 2000) can occur
in the absence of both Nodal and Hh signals. Our interpretation of this result
is that removal of all Nodal signals increases the level of activity of other
inducers of Vax gene activity, decreases the levels of inhibitors of Vax gene
expression, or may do both.
Despite the absence of ventral forebrain derivatives such as the
hypothalamus (Rohr et al.,
2001), the forebrain is enlarged in MZoep embryos
(Gritsman et al., 2000
). This
may be because of reduced activity of Wnt proteins emanating from the margin
that posteriorize the developing neural tissue
(Erter et al., 2001
). One
might therefore speculate that Wnt signals emanating from the marginal zone of
the embryo normally inhibit Vax gene expression and the failure to induce such
signals in MZoep embryos relieves this inhibition. However, we argue
elsewhere (Houart et al.,
2002
) that it is unlikely that marginally derived Wnt signals
directly influence pattern within the forebrain, and instead do so by
regulating the expression of other genes that act more locally within the
neural plate.
The Vax-expressing cells in MZoep-/- embryos and the
cells competent to express Vax genes in Zoep-/- are
located at the interface between the eye and the telencephalon. Indeed, within
the neural plate, optic stalks and probably preoptic tissues, derive from
cells at the interface between axial midline tissues and the anterior border
of the neural plate (Varga et al.,
1999). As the anterior neural plate border is a known source of
signalling proteins (Houart et al.,
1998
; Shimamura and
Rubenstein, 1997
;
Shanmugalingam et al., 2000
;
Houart et al., 2002
), then
such signals are very good candidates for being inducers of Vax gene
expression. Furthermore, the activity of signals emanating from the anterior
border of the neural plate is probably enhanced in MZoep embryos
(Houart et al., 2002
),
providing a possible explanation for the restoration of Vax gene expression in
these embryos.
Our data suggests that Fgfs are good candidates for being inducers of Vax
gene expression in the absence of Nodal activity. fgf8 is expressed
in the anterior border of the neural plate adjacent to the eye field, and the
midline patterning defects and loss of medial pax2.1 expression in
acelfgf8 mutants suggest that Fgf8 has an important role in the
development of the medial optic stalk
(Shanmugalingam et al., 2000).
In this study, we show that Fgf receptor activity is necessary for expression
of Vax genes in the preoptic area and for the restoration of Vax expression in
Nodal-depleted embryos. We therefore suggest that medial cells in the anterior
neural plate express Vax genes in response to Hh signals arising from axial
tissues and Fgf signals arising from the anterior margin of the neural plate
(and perhaps elsewhere).
One further issue to consider is that in other regions of the CNS, Shh
contributes to the establishment of pattern by relieving Gli3-mediated
inhibition of Hh target genes. Thus removal of Gli3 function restores ventral
spinal cord and telencephalic fates that are normally absent in the absence of
Shh activity (Litingtung and Chiang,
2000; Rallu et al.,
2002
). It is not known if Gli3 functions within Vax-expressing
territories, but this is an issue that deserves further investigation.
Belladonna and Fgf signalling regulate Vax gene expression in the
preoptic area
There are striking similarities between the expression, upstream regulation
and function of Vax genes and Pax2 genes within the forming optic vesicles and
midline tissues of the brain (Fig.
9). For instance, the noi/pax2.1 gene is expressed in the
optic stalks prior to the expression of Vax genes
(Macdonald et al., 1997;
Pfeffer et al., 1998
; this
study), is regulated by Nodal and Hedgehog activity
(Ekker et al., 1995
;
Macdonald et al., 1995
;
Masai et al., 2000
;
Varga et al., 2001
), regulates
choroid fissure closure and guidance of axons at the midline
(Macdonald et al., 1997
;
Torres et al., 1996
) and
suppresses pax6 expression in the optic stalk (Schwartz et al.,
2000). Given these similarities, we wondered whether there might be regulatory
interactions between the Pax2 and Vax genes. However as yet, no such
interactions have been clearly demonstrated in either fish or mice in which
Pax2 or Vax gene function is disrupted. Thus pax2 is still expressed
in embryos with reduced Vax activity (this study,
Bertuzzi et al., 1999
;
Hallonet et al., 1999
;
Mui et al., 2002
,
Barbieri et al., 2002
), whereas
Vax gene expression is retained in noi fish mutants that lack
activity of Pax2.1. These observations suggest that although pax2 and
Vax genes may be regulated in a similar way and function in the same
patterning events, they may do so in parallel pathways. One caveat is that
pax2 and Vax gene expression has not been examined in embryos in
which it is certain that all Pax2 activity or all Vax activity is completely
absent (for instance, in noi mutants, a second pax2-related
gene may still have activity in the optic stalks)
(Pfeffer et al., 1998
).
|
Bel is required to specify Vax gene expression in the preoptic tissue
epistatic to Hh signals. It is probable that the activity of Bel in mediating
Hh activity is very localized within the CNS as bel-/-
embryos do not exhibit the U-shaped somites characteristic of many other Hh
pathway mutants (van Eeden et al.,
1996; Karlstrom et al.,
1999
; Schauerte et al.,
1998
). Conversely, not all mutants affecting Hh-dependent
patterning exhibit alterations in Vax gene expression. For instance,
ubo-/- embryos show the U-shaped somite phenotype
characteristic of defects in Hh signalling
(van Eeden et al., 1996
), but
have no defects in retinal axon pathfinding
(Karlstrom et al., 1996
) and
no alterations to Vax gene expression (data not shown). As Vax morphants and
bel-/- embryos share some phenotypes, this raises the
possibility that the bel locus may encode one or other of the Vax
genes. However, given the importance of Fgf activity for induction of Vax
expression in the preoptic area, it is perhaps more probable that Bel
functions in the Fgf signalling pathway.
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ACKNOWLEDGMENTS |
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![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Barbieri, A. M., Broccoli, V., Bovolenta, P., Alfano, G.,
Marchitiello, A., Mocchetti, C., Crippa, L., Bulfone, A., Marigo, V.,
Ballabio, A. et al. (2002). Vax2 inactivation in
mouse determines alteration of the eye dorsal-ventral axis, misrouting of the
optic fibres and eye coloboma. Development
129,805
-813.
Barbieri, A. M., Lupo, G., Bulfone, A., Andreazzoli, M.,
Mariani, M., Fougerousse, F., Consalez, G. G., Borsani, G., Beckmann, J. S.,
Barsacchi, G. et al. (1999). A homeobox gene, vax2,
controls the patterning of the eye dorsoventral axis. Proc. Natl.
Acad. Sci. USA 96,10729
-10734.
Barth, K. A. and Wilson, S. W. (1995).
Expression of zebrafish nk2.2 is influenced by sonic
hedgehog/vertebrate hedgehog-1 and demarcates a zone of neuronal
differentiation in the embryonic forebrain.
Development 121,1755
-1768.
Bertuzzi, S., Hindges, R., Mui, S. H., O'Leary, D. D. and Lemke,
G. (1999). The homeodomain protein Vax1 is required for axon
guidance and major tract formation in the developing forebrain.
Genes Dev. 13,3092
-3105.
Bisgrove, B. W., Essner, J. J. and Yost, H. J.
(1999). Regulation of midline development by antagonism of lefty
and nodal signaling. Development
126,3253
-3262.
Chen, W., Burgess, S. and Hopkins, N. (2001).
Analysis of the zebrafish smoothened mutant reveals conserved and divergent
functions of hedgehog activity. Development
128,2385
-2396.
Chiang, C., Litingtung, Y., Lee, E., Young, K. E., Corden, J. L., Westphal, H. and Beachy, P. A. (1996). Cyclopia and defective axial patterning in mice lacking Sonic hedgehog gene function. Nature 383,407 -413.[CrossRef][Medline]
Connor, R. J., Menzel, P. and Pasquale, E. B. (1998). Expression and tyrosine phosphorylation of Eph receptors suggest multiple mechanisms in patterning of the visual system. Dev. Biol. 193,21 -35.[CrossRef][Medline]
Ekker, S. C., Ungar, A. R., Greenstein, P., von Kessler, D. P., Porter, J. A., Moon, R. T. and Beachy, P. A. (1995). Patterning activities of vertebrate hedgehog proteins in the developing eye and brain. Curr. Biol. 5, 944-955.[Medline]
Eph Nomenclature Committee (1997). Unified nomenclature for Eph family receptors and their ligands, the Ephrins. Cell 90,403 -404.[Medline]
Erter, C. E., Wilm, T. P., Basler, N., Wright, C. V. and
Solnica-Krezel, L. (2001). Wnt8 is required in lateral
mesendodermal precursors for neural posteriorization in vivo.
Development 128,3571
-3583.
Favor, J., Sandulache, R., Neuhauser-Klaus, A., Pretsch, W.,
Chatterjee, B., Senft, E., Wurst, W., Blanquet, V., Grimes, P., Sporle, R. et
al. (1996). The mouse Pax2(1Neu) mutation is identical to a
human PAX2 mutation in a family with renal-coloboma syndrome and results in
developmental defects of the brain, ear, eye, and kidney. Proc.
Natl. Acad. Sci. USA 93,13870
-13875.
Feldman, B., Gates, M. A., Egan, E. S., Dougan, S. T., Rennebeck, G., Sirotkin, H. I., Schier, A. F. and Talbot, W. S. (1998). Zebrafish organizer development and germ-layer formation require nodal-related signals. Nature 395,181 -185.[CrossRef][Medline]
Force, A., Lynch, M., Pickett, F. B., Amores, A., Yan, Y.-I. and
Postlethwait, J. (1999). Preservation of duplicate genes by
complementary, degenerative mutations. Genetics
151,1531
-1545.
Gritsman, K., Zhang, J., Cheng, S., Heckscher, E., Talbot, W. S. and Schier, A. F. (1999). The EGF-CFC protein one-eyed pinhead is essential for nodal signaling. Cell 97,121 -132.[Medline]
Gritsman, K., Talbot, W. S. and Schier, A.
(2000). Nodal signaling patterns the organizer.
Development 127,921
-932.
Hallonet, M., Hollemann, T., Pieler, T. and Gruss, P.
(1999). Vax1, a novel homeobox-containing gene, directs
development of the basal forebrain and visual system. Genes
Dev. 13,3106
-3114.
Hallonet, M., Hollemann, T., Wehr, R., Jenkins, N. A., Copeland,
N. G., Pieler, T. and Gruss, P. (1998). Vax1 is a
novel homeobox-containing gene expressed in the developing anterior ventral
forebrain. Development
125,2599
-2610.
Holash, J. A. and Pasquale, E. B. (1995). Polarized expression of the receptor protein tyrosine kinase Cek5 in the developing avian visual system. Dev. Biol. 172,683 -693.[CrossRef][Medline]
Holmberg, J. and Frisen, J. (2002). Ephrins are not only unattractive. Trends Neurosci. 25,239 -243.[CrossRef][Medline]
Houart, C., Westerfield, M. and Wilson, S. W. (1998). A small population of anterior cells patterns the forebrain during zebrafish gastrulation. Nature 391,788 -792.[CrossRef][Medline]
Houart, C., Caneparo, L., Heisenberg, C.-P., Barth, K. A., Take-uchi, M. and Wilson, S. W. (2002). Establishment of the telencephalon during gastrulation by local antagonism of Wnt signalling. Neuron 35,255 -265.[Medline]
Ingham, P. W. and McMahon, A. P. (2001).
Hedgehog signaling in animal development: paradigms and principles.
Genes Dev. 15,3059
-3087.
Karlstrom, R. O., Talbot, W. S. and Schier, A. F.
(1999). Comparative synteny cloning of zebrafish
you-too: mutations in the Hedgehog target gli2 affect
ventral forebrain patterning. Genes Dev.
13,388
-393.
Karlstrom, R. O., Trowe, T., Klostermann, S., Baier, H., Brand,
M., Crawford, A. D., Grunewald, B., Haffter, P., Hoffmann, H., Meyer, S. U. et
al. (1996). Zebrafish mutations affecting retinotectal axon
pathfinding. Development
123,427
-438.
Litingtung, Y. and Chiang, C. (2000). Specification of ventral neuron types is mediated by an antagonistic interaction between Shh and Gli3. Nat. Neurosci. 3, 979-985.[CrossRef][Medline]
Lynch, M. and Force, A. (2000). The probability
of duplicate gene preservation by subfunctionalization.
Genetics 154,459
-473.
Macdonald, R., Xu, Q., Barth, K. A., Mikkola, I., Holder, N., Fjose, A., Krauss, S. and Wilson, S. W. (1994). Regulatory gene expression boundaries demarcate sites of neuronal differentiation in the embryonic zebrafish forebrain. Neuron 13,1039 -1053.[Medline]
Macdonald, R., Barth, K. A., Xu, Q., Holder, N., Mikkola, I. and
Wilson, S. W. (1995). Midline signalling is required for Pax
gene regulation and patterning of the eyes.
Development 121,3267
-3278.
Macdonald, R., Scholes, J., Strahle, U., Brennan, C., Holder,
N., Brand, M. and Wilson, S. W. (1997). The Pax protein Noi
is required for commissural axon pathway formation in the rostral forebrain.
Development 124,2397
-2408.
Masai, I., Stemple, D. L., Okamoto, H. and Wilson, S. W. (2000). Midline signals regulate retinal neurogenesis in zebrafish. Neuron 27,251 -263.[Medline]
Mathieu, J., Barth, A., Rosa, F. M., Wilson, S. W. and
Peyriéras, N. (2002). Distinct and cooperative roles
for Nodal and Hedgehog signals during hypothalamic development.
Development 129,3055
-3065.
Meno, C., Gritsman, K., Ohishi, S., Ohfuji, Y., Heckscher, E., Mochida, K., Shimono, A., Kondoh, H., Talbot, W. S., Robertson, E. J. et al. (1999). Mouse Lefty2 and zebrafish Antivin are feedback inhibitors of Nodal signaling during vertebrate gastrulation. Mol. Cell 4,287 -298.[Medline]
Mohammadi, M., McMahon, G., Sun, L., Tang, C., Hirth, P., Yeh,
B., Hubbard, S. and Schlessinger, J. (1997). Structures of
the tyrosine kinase domain of fibroblast growth factor receptor in complex
with inhibitors. Science
276,955
-960.
Mui, S. H., Hindges, R., O'Leary, D. D. M., Lemke, G. and
Bertuzzi, S. (2002). The homeodomain protein Vax2 patterns
the dorsoventral and nasotemporal axes of the eye.
Development 129,797
-804.
Muller, F., Albert, S., Blader, P., Fischer, N., Hallonet, M.
and Strahle, U. (2000). Direct action of the Nodal-related
signal Cyclops in induction of sonic hedgehog in the ventral midline of the
CNS. Development 127,3889
-3897.
Nasevicius, A. and Ekker, S. C. (2000). Effective targeted gene `knockdown' in zebrafish. Nat. Genet. 26,216 -220.[CrossRef][Medline]
Neuhauss, S. C., Biehlmaier, O., Seeliger, M. W., Das, T.,
Kohler, K., Harris, W. A. and Baier, H. (1999). Genetic
disorders of vision revealed by a behavioral screen of 400 essential loci in
zebrafish. J. Neurosci.
19,8603
-8615.
Ohsaki, K., Morimitsu, T., Ishida, Y., Kominami, R. and
Takahashi, N. (1999). Expression of the Vax family homeobox
genes suggests multiple roles in eye development. Genes
Cells 4,267
-276.
Pfeffer, P. L., Gerster, T., Lun, K., Brand, M. and Busslinger,
M. (1998). Characterization of three novel members of the
zebrafish Pax2/5/8 family: dependency of Pax5 and Pax8 expression on the
Pax2.1 (noi) function. Development
125,3063
-3074.
Rallu, M., Machold, R., Gaiano, N., Corbin, J. G., McMahon, A.
P. and Fishell, G. (2002). Dorsoventral patterning is
established in the telencephalon of mutants lacking both Gli3 and Hedgehog
signaling. Development
129,4963
-4974.
Rebagliati, M. R., Toyama, R., Haffter, P. and Dawid, I. B.
(1998). cyclops encodes a nodal-related factor involved
in midline signaling. Proc. Natl. Acad. Sci. USA
95,9932
-9937.
Reifers, F., Bohli, H., Walsh, E. C., Crossley, P. H., Stainier,
D. Y. and Brand, M. (1998). Fgf8 is mutated in zebrafish
acerebellar (ace) mutants and is required for maintenance of
midbrain-hindbrain boundary development and somitogenesis.
Development 125,2381
-2395.
Reifers, F., Adams, J., Mason, I., Schulte-Merker, S. and Brand, M. (2000). Overlapping and distinct functions provided by fgf17, a new zebrafish member of Fgf8/17/18 subgroup of Fgfs. Mech. Dev. 99,39 -49.[CrossRef][Medline]
Rick, J. M., Horschke, I. and Neuhauss, S. C. (2000). Optokinetic behavior is reversed in achiasmatic mutant zebrafish larvae. Curr. Biol. 10,595 -598.[CrossRef][Medline]
Rohr, K. B. and Concha, M. L. (2000). Expression of nk2.1a during early development of the thyroid gland in zebrafish. Mech. Dev. 95,267 -270.[CrossRef][Medline]
Rohr, K. B., Barth, K. A., Varga, Z. M. and Wilson, S. W. (2001). The Nodal pathway acts upstream of Hedgehog signaling to specify ventral telencephalic identity. Neuron 29,341 -351.[Medline]
Sampath, K., Rubinstein, A. L., Cheng, A. M., Liang, J. O., Fekany, K., Solnica-Krezel, L., Korzh, V., Halpern, M. E. and Wright, C. V. (1998). Induction of the zebrafish ventral brain and floorplate requires cyclops/nodal signalling. Nature 395,185 -189.[CrossRef][Medline]
Sanyanusin, P., McNoe, L. A., Sullivan, M. J., Weaver, R. G. and Eccles, M. R. (1995). Mutation of PAX2 in two siblings with renal-coloboma syndrome. Hum. Mol. Genet. 4,2183 -2184.[Medline]
Schauerte, H. E., van Eeden, F. J. M., Fricke, C., Odenthal, J.,
Strähle, U. and Haffter, P. (1998). Sonic hedgehog is
not required for the induction of medial floor plate cells in the zebrafish.
Development 125,2983
-2993.
Schulte, D., Furukawa, T., Peters, M. A., Kozak, C. A. and Cepko, C. L. (1999). Misexpression of the Emx-related homeobox genes cVax and mVax2 ventralizes the retina and perturbs the retinotectal map. Neuron 24,541 -553.[Medline]
Schwarz, M., Cecconi, F., Bernier, G., Andrejewski, N.,
Kammandel, B., Wagner, M. and Gruss, P. (2000). Spatial
specification of mammalian eye territories by reciprocal transcriptional
repression of Pax2 and Pax6. Development
127,4325
-4333.
Shanmugalingam, S., Houart, C., Picker, A., Reifers, F.,
Macdonald, R., Barth, A., Griffin, K., Brand, M. and Wilson, S. W.
(2000). Ace/Fgf8 is required for forebrain commissure formation
and patterning of the telencephalon. Development
127,2549
-2561.
Shimamura, K. and Rubenstein, J. L. R. (1997).
Inductive interactions direct early regionalization of the mouse forebrain.
Development 124,2709
-2718.
Shinya, M., Koshida, S., Sawada, A., Kuroiwa, A. and Takeda,
H. (2001). Fgf signalling through MAPK cascade is required
for development of the subpallial telencephalon in zebrafish embryos.
Development 128,4153
-4164.
Thisse, B., Wright, C. V. and Thisse, C. (2000). Activin- and Nodal-related factors control antero-posterior patterning of the zebrafish embryo. Nature 403,425 -428.[CrossRef][Medline]
Thisse, C. and Thisse, B. (1999). Antivin, a
novel and divergent member of the TGFß superfamily, negatively regulates
mesoderm induction. Development
126,229
-240.
Torres, M., Gomez-Pardo, E. and Gruss, P.
(1996). Pax2 contributes to inner ear patterning and optic nerve
trajectory. Development
122,3381
-3391.
Ungar, A. R. and Moon, R. T. (1996). Inhibition of protein kinase A phenocopies ectopic expression of hedgehog in the CNS of wild-type and cyclops mutant embryos. Dev. Biol. 178,186 -191.[CrossRef][Medline]
van Eeden, F. J., Granato, M., Schach, U., Brand, M.,
Furutani-Seiki, M., Haffter, P., Hammerschmidt, M., Heisenberg, C.-P., Jiang,
Y. J., Kane, D. A. et al. (1996). Mutations affecting somite
formation and patterning in the zebrafish, Danio rerio.Development 123,153
-164.
Varga, Z. M., Amores, A., Lewis, K. E., Yan, Y.-L.,
Postlethwait, J. H., Eisen, J. S. and Westerfield, M. (2001).
Zebrafish smoothened functions in ventral neural tube specification and axon
tract formation. Development
128,3497
-3509.
Varga, Z. M., Wegner, J. and Westerfield, M.
(1999). Anterior movement of ventral diencephalic precursors
separates the primordial eye field in the neural plate and requires cyclops.
Development 126,5533
-5546.
Westerfield, M. (1993). The Zebrafish Book. Eugene, OR: The University of Oregon Press.
Winkler, S., Loosli, F., Henrich, T., Wakamatsu, Y. and
Wittbrodt, J. (2000). The conditional medaka mutation eyeless
uncouples patterning and morphogenesis of the eye.
Development 127,1911
-1919.
Zhang, J., Talbot, W. S. and Schier, A. F. (1998). Positional cloning identifies zebrafish one-eyed pinhead as a permissive EGF-related ligand required during gastrulation. Cell 92,241 -251.[Medline]