University of Technology, Department of Genetics, c/o Max Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauer Strasse 108, 01307 Dresden, Germany
* Author for correspondence (e-mail: picker{at}mpi-cbg.de)
Accepted 2 September 2005
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SUMMARY |
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Key words: Eye development, Axial patterning, Nasal, Temporal, Fgf, Fgf8, Efn, Eph, Zebrafish, Danio rerio
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Introduction |
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The anatomical stages of vertebrate eye development are well described
(Chow and Lang, 2001;
Chuang and Raymond, 2002
). In
the zebrafish, eye morphogenesis commences with the lateral evagination of the
optic vesicle from the diencephalic neural keel at 5- to 6-somite stage
(Schmitt and Dowling, 1994
).
But it is not well understood how early eye morphogenesis relates to axial
patterning of the prospective neural retina and how this influences topography
of the retinotectal map (Peters,
2002
). DV eye patterning involves bone morphogenetic protein 4
(BMP4), which is expressed in the dorsal eye and leads to upregulation of the
dorsal T-box transcription factor Tbx5 and to downregulation of the ventral
homeodomain transcription factors Vax and Pax2 upon misexpression
(Koshiba-Takeuchi et al.,
2000
; Sasagawa et al.,
2002
). BMP loss/inhibition results in ventralization of the retina
(Sakuta et al., 2001
;
Sasagawa et al., 2002
;
Murali et al., 2005
). Vax
genes are required for ventral retina development and to delineate optic stalk
from retinal territories; the latter through a midline-derived Hedgehog (Hh)
signal (Schulte et al., 1999
;
Barbieri et al., 1999
;
Mui et al., 2002
;
Take-uchi et al., 2003
).
Asymmetric distribution of retinoic acid (RA) has also been implicated in DV
eye patterning (Hyatt et al.,
1992
; Kastner et al.,
1994
; Marsh-Armstrong et al.,
1994
; Hyatt et al.,
1996
). A recent study suggested that RA and Fgfs can ventralize
the eye by collaborating with Hh, but the exact temporal requirement for these
factors remains largely unclear (Lupo et
al., 2005
). In the chick, retinal DV polarity is gradually
determined between the 8- and 14-somite stage
(Uemonsa et al., 2002
).
By contrast, retinal patterning along the NT axis is only poorly
understood. The forkhead transcription factors Foxg1 (BF1) and Foxd1 (BF2)
display restricted and complementary expression along the retinal NT axis and
cause RGC axon misprojections upon ectopic expression
(Hatini et al., 1994;
Yuasa et al., 1996
). Two
transcription factors, sensory organ homeobox (SOHo) and Gallus
gallus homeobox 6 (GH6) are expressed in the nasal half of the eye, and
cause RGC axon misprojections upon ectopic expression and downregulation of
temporal expression of Epha3a (Deitcher et
al., 1994
; Schulte and Cepko,
2000
; Stadler and Solursh,
1994
). Foxg1 can induce ectopic nasal gene expression (SOHo, GH6,
Efna5, Efna2) and suppress temporal gene expression (Epha3, Foxd1) upon
misexpression in the temporal retina
(Takahashi et al., 2003
).
Despite increasing knowledge about late DV and NT axial restriction of gene
expression in the retina, it is largely unknown when and through which factors
these patterns are established (McLaughlin
et al., 2003
). Importantly, the currently known factors that
regulate NT eye patterning are themselves already expressed asymmetrically.
Fgf signals have been implicated in various aspects of eye development,
including the segregation of neural from pigmented retina, lens induction and
differentiation, retinal cell fate specification, photoreceptor survival, RGC
axon outgrowth and axon guidance, and are thus good candidates to play a role
in axial eye patterning (Russell,
2003
; Yang, 2004
).
Furthermore, Fgf-dependent differentiation of the retina is mediated by a
combined Fgf8/Fgf3 signal (McCabe et al.,
1999
; Martinez-Morales et al.,
2005
).
We had previously shown that RGCs from fgf8-mutant eyes form
topographically incorrect axon projections on wild-type tecta, suggesting a
role for Fgf8 in axial patterning of the retina
(Picker et al., 1999). Here,
we analyze the exact temporal and spatial requirement for Fgf signaling during
axial patterning of the retina. Gene expression along the NT and DV axis of
the retina in the fgf8-mutant acerebellar (ace),
upon pharmacological inhibition of Fgf-receptor (Fgfr) signaling and upon
ectopic Fgf8 expression shows that Fgf8 together with other Fgf proteins
determines the NT axis. The temporal requirement for Fgfs is tightly
restricted to the onset of eye morphogenesis between the 5- and 10-somite
stage. Heterotopic eye transplantations show that this early patterning phase
is sufficient to drive subsequent autonomous NT eye patterning. We also show
that the early, Fgf-dependent positional code of cells along the NT axis of
the eye determines later topographic mapping of RGC axons along the
anterior-posterior axis of the midbrain tectum. The developmental timing of
(1) fgf8 expression and its patterning activity, (2) of the
requirement for Fgfr signaling and (3) of activation of the Fgf-target gene
spry4 in the diencephalon and eye suggest that the telencephalon acts
as extrinsic Fgf-source and thus is a novel signaling center for retinal
patterning.
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Materials and methods |
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Whole-mount in situ hybridization, immunocytochemistry and cell death analysis
Whole-mount mRNA in situ hybridization were carried out as described
(Reifers et al., 1998).
Digoxigenin-labeled probes were prepared from linearized templates using an
RNA labeling and detection kit (Roche). Receptor alkaline phosphatase staining
with the chick for Epha3-AP fusion protein was carried out as previously
described (Cheng and Flanagan,
1994
; Brennan et al.,
1997
). Apoptotic cells were detected by an in-situ nick-end
labeling procedure using a commercial kit (Roche). Acridine Orange (AO,
Molecular Probes) was used at a concentration of 2 µg/ml to stain live
embryos (Brand et al.,
1996
).
Gene expression intensity profiling
For intensity profiling of retinal gene expression, sets of 8-bit grayscale
images of dissected, flat mounted eyes where captured after in situ
hybridization as described above. Images where subsequently imported into the
ImageJ 1.32j
(http://rsb.info.nih.gov/ij/)
image analysis software. Using the `Analyze>Plot Profile' option intensity
profiles along radial trajectories were captured and further analyzed with
Microsoft Excel. Comparisons of gene expression in wild-type and mutant where
performed on embryos from the same egg lay and were stained, imaged and
analyzed under identical conditions.
Fgfr-inhibitor treatment
The Fgf-receptor signaling inhibitor SU5402 (Calbiochem)
(Mohammadi et al., 1997) was
diluted into 10 mM stocks in DMSO and added to E3 medium at a working
concentration of 5 µM, followed by repeated washing in excess medium.
Control embryos were treated in 0.05% DMSO
(Reifers et al., 2000
).
Tissue transplantations, bead implantations and RGC axon labeling
Optic vesicle transplantation and anterograde RGC labeling was carried out
as described before on the same side of donor and host embryo
(Picker et al., 1999).
Fgf8-protein beads were prepared by overnight incubation of heparin-coated
acrylic beads (Sigma) or 45 µm polystyrene beads (Polysciences) in a 250
µg/ml solution of recombinant zebrafish Fgf8 protein in PBS. Before
implantation, beads were rinsed three times for 10 minutes in PBS. Control
beads were loaded with 250 µg/ml BSA.
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Results |
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In wild-type embryos at 28 hours, expression of efna5a is confined
to cells in the nasal half and epha4b to cells in the temporal half
of the prospective retina (Fig.
1B,E). Fgf8-bead implantation between the 5- and 7-somite somite
stage close to the retina (Fig.
1A,D) leads to a strong `temporal shift' in retinal gene
expression: temporal expansion of the nasally expressed gene efna5a
(n=7/7) and complementary repression of the temporally expressed
epha4b (n=8/8) (Fig.
1C,F). Similar to Fgf8 beads, transplantation of cells from the
telencephalic fgf8 expression domain at the 5-somite stage (see
Fig. 5A,B) into the optic
vesicle can repress temporal epha4b expression (n=3/4)
showing that they can act as source of the patterning activity
(Fig. 1G-I). Implantation of
Fgf8-soaked beads after the 5- to 7-somite stage does not alter NT patterning
(n=17/17). This Fgf8-misexpression phenotype strikingly resembles the
phenotype of the aussicht (aus) mutant, where Fgf8
expression is generally increased
(Heisenberg et al., 1999).
To determine the exact timing of the requirement for Fgf8 in NT eye
patterning, we applied the pharmacological Fgfr inhibitor SU5402 to wild-type
embryos at discrete developmental intervals between the 1- and 15-somite
stage, and studied retinal gene expression at the 28 hour stage
(Mohammadi et al., 1997;
Reifers et al., 2000
;
Raible and Brand, 2001
).
Blocking Fgfr signaling induces a complete `nasal shift' of retinal NT
polarity: in wild-type control embryos, the expression of epha4b is
strictly confined to the temporal half of the retina at the 28 hour stage
(Fig. 2A). After treatment
between the 1- and 5-somite stage, the expression of epha4b partially
expands into the nasal retina (Fig.
2B). Treatment between the 3- and 8-somite stage and the 5- and
10-somite stage leads to an apparently complete expansion of epha4b
throughout the retina (Fig.
2C,D). Complementary to this expansion, the nasal expression
domains of efna5a (Fig.
2G, compare with Fig.
3C for wild-type expression) and foxg1
(Fig. 2F, compare with
Fig. 3A for wild-type
expression) are completely lost. The dorsal expression domain of tbx5
is not affected in its DV extent but shifts position nasally
(Fig. 2H, compare with
Fig. 3G). The ventral domain of
vax2 expression is unaffected
(Fig. 2I, compare to
Fig. 3I). Treatment beyond the
10-somite stage has no influence on epha4b expression
(Fig. 2E) and retinal
patterning in general.
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|
Fgf8 is part of a combinatorial Fgf signal for retinal patterning
To determine if fgf8 is required for retinal patterning, we
analyzed gene expression in fgf8-mutant acerebellar
(ace) embryos at 28 hours of development by in situ hybridization
(Fig. 3A-J).
In wild-type embryos, foxg1 is expressed in the nasal half of the eye, including the complete ventronasal but only the ventral part of the dorsonasal retinal quadrant. In ace mutant embryos, foxg1 expression in the ventronasal quadrant is unaffected but appears reduced in the dorsonasal retina (Fig. 3A,B). Intensity profiling, along a clockwise 180° trajectory through the dorsal eye (mean values, n=8 eyes/gene) (Fig. 3L), reveals that foxg1 is expressed in a nasal-to-temporal decreasing gradient, which is flattened in the dorsonasal retinal segment in the ace mutant, but reaches the same maximum and minimum expression levels (Fig. 3K). Similar to foxg1, efna5a is expressed in the nasal half of the wild-type retina; however, efna5a is also expressed in the complete dorsonasal quadrant. Thus, foxg1 expression is nested within the efna5a expression domain. In ace mutant embryos, efna5a expression is overall strongly reduced (Fig. 3C,D). This leads to a global flattening of the nasal-to-temporal decreasing efna5a expression gradient in the mutant (Fig. 3K). In the wild type, epha4b is expressed throughout the temporal retina, complementary to efna5a. In ace, the epha4b expression domain expands into the dorsonasal retinal quadrant (Fig. 3E,F). As a result, the nasal-to-temporal increasing epha4b gradient is shifted in the ace mutant, indicating a global upregulation of expression (Fig. 3K). In the wild type, tbx5 is expressed in the dorsomedial retina. In ace mutant embryos, the NT extent of tbx5 appears reduced and shifted nasally (Fig. 3G,H). Tb5x is expressed in a bell-shaped gradient, which is centered on the dorsalmost point of the retina and bilaterally fades out into the dorsonasal and dorsotemporal quadrants. In the ace mutant, this bell-shaped gradient is flattened, indicating a global reduction in gene expression (Fig. 3K). In the wild type, vax2 is expressed in the ventral retina. The expression domain covers two spatially distinct sub-domains: one in the ventronasal quadrant and one in the ventrotemporal quadrant, with the latter extending further dorsally than the other. The medial part of the ventral retina only weakly expresses vax2. In the ace mutant, vax2 expression is unaffected (Fig. 3I,J). In sections along the canonical medial DV axis through the retina, we could not detect any differences between wild type and mutant (data not shown).
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As described above, we found that ectopic Fgf8 protein cannot induce NT patterning shifts beyond the 10-somite stage. Nevertheless, late implanted beads are capable of rescuing the ace mutant eye phenotype: an Epha3-alkaline-phosphatase fusion protein detects Efna-ligand protein in the nasal half of the retina of wild-type embryos (Fig. 4A). The expression of Efna ligands is strongly reduced in ace mutant eyes, and rescued after bead implantation into the forebrain at the 10-somite stage (n=10/10) (Fig. 4C,D). Bead implantations into both wild type and ace mutants at this stage lead to ectopic expression of Efna ligands in the ventralmost region of the nasal retina, but do not induce nasal gene expression in the temporal retina (Fig. 4B,D).
One scenario that could explain the observed NT patterning shifts is the selective loss of the nasal and concomitant expansion of temporal retinal fates in ace and Fgfr-inhibitor-treated embryos. We therefore studied cell death during eye development in these embryos. Although we find a general increase in cell death in the eyes of ace and Fgfr-inhibitor treated embryos, this effect is not significant before the 20-somite stage. Likewise we do not observe a bias of cell death in ace and Fgfr-inhibitor treated embryos to the proximal, future nasal half of the eye (see Fig. S1 in the supplementary material). We therefore exclude the possibility that local cell death is the cause of the alterations in retinal NT patterning upon loss of Fgf signaling.
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Early Fgf8 and Fgf-target expression suggests the telencephalon as signaling center for axial retinal patterning
Fgfs bind to high-affinity tyrosine kinase receptors that activate the
intracellular Ras/Mapk signal transduction pathway, and in response the
Fgf-feedback inhibitors Sprouty 2 (Spry2), Sprouty 4 (Spry4), Spred and Sef,
and the ETS domain factors Erm and Pea3
(Furthauer et al., 2001;
Raible and Brand, 2001
;
Lin et al., 2002
;
Furthauer et al., 2002
;
Raible and Brand, 2004
;
Sivak et al., 2005
). To
provide further evidence for a reception of Fgf signals by optic vesicle
cells, we examined the activation of spry4 as Fgf-target gene in
domains overlapping and surrounding sites of Fgf gene expression. If the
proposed Fgf8 signal from the telencephalon has a direct effect on NT eye
patterning, Fgf-target genes should be expressed around the telencephalon,
including the optic vesicle during the crucial patterning phase between the 5-
and 10-somite stage.
We find that at the 5-somite stage spry4 is co-expressed with fgf8 in the telencephalon of wild-type embryos (Fig. 5A-D). Importantly, we find that spry4 is expressed broader than fgf8 and extends along the DV axis of the forebrain into the dorsal diencephalon and the proximal part of the evaginating optic vesicle. Spry4 is also expressed in nasal placode precursors that surround the telencephalon at this stage (Fig. 5C,D). In ace embryos, the telencephalic and diencephalic expression of spry4, including the proximal optic vesicle, is strongly reduced, whereas the nasal placode precursors express spry4 at normal levels (Fig. 5E,F). Thus, spry4 is expressed and selectively dependent on fgf8 in the diencephalon and proximal optic vesicle in the crucial phase for NT eye patterning, supporting the idea of an Fgf signal emanating from the telencephalon and reaching into the optic vesicle. Low-level spry4 expression in the optic vesicle could explain the partial phenotype of ace mutants and argues for at least one other Fgf being involved in retinal NT patterning.
Fgf-dependent alterations of NT eye patterning result in topographic shifts of retinal axon mapping
To test (1) whether the effect of the Fgf signal on retinal patterning is
irreversible during later development and (2) what the functional consequences
of altered Efna/Eph expression in ace and Fgfr-inhibitor-treated
embryos are, we studied the topographic projections of RGC axons to the
midbrain optic tectum. For this purpose we grafted wild-type optic vesicles
with or without prior Fgfr-inhibitor-treatment and optic vesicles from
ace mutant embryos to wild-type hosts at the 10-somite stage
(Fig. 6A). Anterograde
co-labeling of the dorsotemporal (DT) and dorsonasal (DN) retinal quadrants
with the lipophilic tracers DiI and DiO in wild-type control transplantations
shows that DT and DN RGC axons form two spatially distinct termination zones
(TZs) along the anterior-posterior axis of the ventral tectum, according to
the NT position of their somata in the retina
(Fig. 6B-D). Wild-type larvae
with an optic vesicle graft from an Fgfr-inhibitor-treated embryo show a
pronounced topographic shift in the TZs of RGC axons: although the projection
of DT RGC axons is unaltered and forms an anterior TZ, DN RGC axons display a
completely `temporalized projection behavior', terminating in an ectopic
anterior TZ like the DT RGC axons (Fig.
6E-G). Wild-type larvae that received an optic vesicle graft from
an ace embryo show an `intermediate' RGC axon projection phenotype:
the projections of DT RGC axons are unaltered and form an anterior TZ, but DN
RGC axons form an ectopic anterior TZ in addition to the topographically
correct posterior TZ (Fig.
6H-J).
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The NT axis of the prospective retina is established prior to the 7-somite stage
Finally, we asked whether any other or later signal derived from the
forebrain or head of the embryo, apart from the Fgf signal, is required for
retinal NT patterning. To achieve this, we grafted optic vesicles between the
5- and 7-somite stage ectopically onto the yolk cell, by slipping the graft
into a pocket between the host epidermis and yolk cell membrane
(Fig. 7A-C). Earlier
transplants were not possible, because optic vesicle morphogenesis has just
started at this stage. The transplantation spatially isolates the optic
vesicle from the embryonic body proper and the above-described Fgf sources in
the head region. Observation of live embryos shows that these heterotopic
grafts integrate, grow and develop autonomously and ultimately form an
ectopic, morphologically normal eye (Fig.
7D,E). At the 28 hour stage (16 hours post-transplantation) we
tested NT gene expression, and find that both nasal expression of
efna5a (n=5/7) and temporal epha4b expression
(n=7/8) are correctly established and maintained in such grafts
(Fig. 6F-I). This shows that
the retina can undergo correct NT patterning in the absence of all signals
from the forebrain or head after the 5- to 7-somite stage. Together with the
results of the Fgfr inhibition experiments, this argues for an early Fgf
signaling phase around the 5-somite stage that is sufficient to trigger
autonomous NT eye patterning. The missing retinal pigment epithelium in the
grafts at later stages of development (inset in
Fig. 7E) is probably due to a
lack of induction from the head mesenchyme in the ectopic situs
(Fuhrmann et al., 2000).
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Discussion |
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Combined Fgf signals including Fgf8 determine NT patterning of the prospective retina
We show that NT gene expression patterns in retinae compromised for Fgf
signaling display a `nasal shift' compared with the wild type
(Fig. 8A-C). The complete
`nasal shift' in retinal polarity upon blocking of all Fgfr signaling,
compared with the partial effect in the ace mutant and remnant
spry4 expression in ace mutant early optic vesicles,
demonstrates the combinatorial nature of the Fgf signal
(Fig. 8C). fgf3 and
fgf24 have been previously described to function combinatorially with
fgf8 and are thus good candidates to be part of such a combinatorial
signal (Leger and Brand, 2002;
Walshe and Mason, 2003
;
Draper et al., 2003
;
Martinez-Morales et al.,
2005
). fgf3 is strictly co-expressed with fgf8
in the telencephalon at the 5-somite stage and there it regulates
spry4 expression together with fgf8
(Fürthauer et al., 2001
;
Raible and Brand, 2001
;
Herzog et al., 2004
). It thus
could signal from the same source as fgf8 during retinal patterning.
fgf24 is not expressed in the telencephalon, but in the mesenchymal
precursor cells of the nasal placode, which are surrounding the anterior
neural keel at the 5-somite stage, where spry4 is also expressed but
not dependent on Fgf8 (Whitlock and
Westerfield, 2000
). Previous studies have suggested the
extraocular mesenchyme could act as source of extrinsic factors for optic
vesicle differentiation (Fuhrmann et al.,
2000
). It is therefore possible that the fgf24-expressing
precursors cells of the nasal placode act as an alternative source of Fgf
during retinal NT patterning. Another candidate is the recently published
fgf17b, which may be co-expressed with fgf8 and
fgf3 in the telencephalon (Cao et
al., 2004
). Preliminary results with Fgf3 morpholinos
(Leger and Brand, 2002
)
injected into ace mutant embryos suggest that Fgf8+Fgf3 cannot
account for the signal (A.P. and M.B., unpublished). Further loss-of-function
experiments will clarify the molecular nature of the Fgfs that function with
Fgf8 in retinal NT patterning.
Gene expression along the retinal DV axis is not affected upon
Fgf-dependent alterations in NT patterning. By experimentally interfering with
Fgfs through gastrulation stages, two studies had suggested Fgfs to signal in
parallel with Nodal, Hedgehog (Hh) and retinoic acid (RA) in DV eye patterning
(Take-uchi et al., 2003;
Lupo et al., 2005
). In
addition, interactions between the DV and NT patterning mechanisms have been
proposed (Huh et al., 1999
;
Mui et al., 2002
;
Sakuta et al., 2001
;
Koshiba-Takeuchi et al.,
2000
). Our results show that the crucial phase for Fgf-dependent
retinal NT patterning is after gastrulation; interference with Fgf signaling
during this time does not change DV patterning. The reduction in the dorsal
expression domain of tbx5, that we observe in the ace mutant
is restricted along the NT axis and does not affect the DV extent of the
domain. This argues for the independence of the NT and DV eye patterning
mechanisms.
The critical phase for NT patterning is at the onset of eye morphogenesis
The exact developmental timing of the events that lead to axial patterning
of the retina has not been clearly resolved. Grafting studies in chick embryos
suggest that the retinal NT axis is determined before HH stage 10-11 (10-13
somite stage) (Dutting and Meyer,
1995). Through combining loss- and gain-of-function analysis, we
show that the crucial phase for Fgf-dependent NT patterning is between the 5-
and 10-somite stage, when fgf8 expression is clearly confined to the
telencephalon and the optic vesicle has just started to evaginate from the
diencephalon and is still connected along its full AP extent to the forebrain
(Schmitt and Dowling, 1994
).
Fgfr inhibitor application during discrete developmental intervals was crucial
for this observation, as a complete nasal shift in gene expression along the
NT axis of the eye can only be induced by treatment between the 5- and
10-somite stage. Partial NT shifts induced by treatments directly before that
stage, are most readily explained by persistence of the inhibitor. The
importance of the phase between the 5- and 10-somite stage for eye patterning
is independently supported by the reduced expression of the Fgf target
spry4 in the diencephalon and optic vesicle in ace mutant by
the 5-somite stage, which indicates a non-autonomous requirement for Fgf8 in
eye development at this early stage.
Interestingly, the ace mutant eye shows an extended responsiveness
to Fgf8, beyond the early NT patterning phase, in rescue experiments. This is
reminiscent of the observed plasticity in the midbrain-hindbrain region
(Jaszai et al., 2003). But the
observed, retained competence to respond to Fgf8 is limited as Fgfr-inhibitor
treated and ace mutant eyes do not restore normal NT patterning, when
transplanted into wild-type host brains beyond the 10-somite stage. Instead,
patterning is terminally altered in the grafts, which is reflected by
topographic RGC axon misprojections 5 days after transplantation. Thus, beyond
the 10-somite stage, the endogenous patterning activity of Fgfs is not
effective.
Previous studies have taken advantage of culturing optic vesicles or
retinal explants to determine the influence of extrinsic signals on eye
development (Pittack et al.,
1997; Fuhrmann et al.,
2000
). Unexpectedly, our heterotopic transplantations of optic
vesicles to the yolk cell at the 5- to 7-somite stage demonstrate that the
inductive events prior to this stage are sufficient to drive correct NT
patterning, autonomously in the absence of extrinsic cues from the
forebrain/head. Taken together with our findings on the requirement for Fgfs,
in particular Fgf8, during that period, we propose that a short pulse of Fgfs
around the 5-somite stage triggers NT eye patterning, and the eye becomes
independent of exogenous signal supply shortly thereafter.
Early Fgf-dependent eye patterning is required for topographic mapping of RGC axons
Because epha4b expression is confined to the temporal retina in
normal zebrafish, its expansion could directly explain the misprojection of
RGCs from the nasal retina of ace mutant and Fgfr-inhibitor treated
eyes to an ectopic, anterior termination zone (TZ) by increasing the
sensitivity of axons for repellent Efna-ligands expressed in gradients on the
tectum (Brennan et al., 1997;
Picker et al., 1999
).
Remarkably, nasal RGC axons from an ace mutant eye form an ectopic
anterior TZ in addition to a normal posterior TZ, indicating a partial
temporalization, in contrast to a full temporalization upon blocking of all
Fgfr signaling. These duplicated TZs are similar to the observations made in
mice with targeted deletions of Efna5, Efna2 and Efna5/Efna2
(Frisen et al., 1998
;
Feldheim et al., 2000
).
Consistent with normal gene expression along the DV axis of the retina, we do
not find DV mapping defects of RGC axons from ace or Fgfr-inhibitor
treated eyes. It has recently been shown that nasally expressed foxg1
(CBF1) represses temporal and induces nasal gene expression, similar
to the results that we obtain for Fgfs
(Takahashi et al., 2003
). It
is thus possible that Fgf signaling acts upstream of the transcriptional
regulator foxg1.
Propagation of the Fgf signal from a novel signaling center in the telencephalon
We propose that the telencephalon is the source of the Fgf8 signal that
influences NT eye patterning. At the onset of optic vesicle evagination,
between the 5- and 10-somite stage, the telencephalon is the only
fgf8 expression domain in the head region. Expression there coincides
with the timing of the observed effects of Fgfs on NT eye patterning.
(1) Fgfr inhibitor treatments show that Fgf signaling is crucial between the 5- and 10-somite stage, and not required for NT patterning beyond the 10-somite stage, when Fgf8 starts to be expressed in the optic stalk and retina.
(2) Ectopic Fgf8 protein expression shows a restricted competence to induce NT patterning shifts between the 5- and 10-somite stage.
(3) Reduction of the graded expression of the Fgf target spry4 in the dorsal diencephalon, including the proximal optic vesicle in the ace mutant at the 5-somite stage, shows active Fgf signaling in the region.
Although our loss- and gain-of-function experiments consistently show that
Fgf8 influences retinal NT patterning, this effect could still be indirect and
reflect a previously reported, autonomous requirement for Fgf8 during
telencephalon development rather than a direct signal
(Shanmugalingam et al., 2000;
Walshe and Mason, 2003
).
However, the fact that (1) the Fgf target gene spry4 is dependent on
fgf8 in the diencephalon and optic vesicle and (2) altered NT
patterning after Fgf8-bead implantation support that Fgf8 itself is the
signaling entity. As the simplest model, we thus favor a
`trans-neuroepithelial' propagation of the Fgf8 signal from the dorsal
telencephalic source along the DV axis of the neural tube into the ventrally
located diencephalon and the contiguous proximal-distal axis of the
evaginating optic vesicle (Fig.
8E). Fate-mapping of cells in the optic vesicle at the 10-somite
stage shows that the proximal-distal and posterior-anterior axis of the
vesicle match the NT and DV axis of the retina at later stages
(Li et al., 2000
). Thus, in
the crucial patterning phase between the 5- and 10-somite stage, the NT axis
of the prospective retina is perpendicular to the AP axis of the
telencephalon, which therefore could function as an asymmetric Fgf source.
Such a `trans-neuroepithelial' Fgf8 distribution could well explain (1) the
graded activation of spyr4 in the diencephalon and optic vesicle, and
(2) the Fgf-dependent induction of proximal/future nasal (foxg1- and
efna5a-expressing) and suppression of distal/future temporal
(epha4b-expressing) retinal cell fates. The similarity between the
ace phenotype and the effects caused by partial Fgfr-inhibition
suggests that the other Fgf(s) act via a similar mechanism, but analysis of
further candidate Fgfs will be needed to determine whether the other molecular
components of the combinatorial signal also emanate from the
telencephalon.
Considering the role of Fgf expression at the midbrain-hindbrain boundary
for tectal AP patterning (Lee et al.,
1997; Picker et al.,
1999
), it is intriguing to find that Fgfs also determine eye
patterning along the NT axis. Because both axes are matched in the topography
of the retinotectal projection, it is tempting to speculate that this dual
requirement reflects a common control mechanism for the positional code of
cells in the source and target area of retinotectal map. If this were true,
graded interference with Fgfs, common to source and target field, would create
parallel shifts in axial patterning of eye and tectum. In evolutionary terms,
this might confer `robustness' to the map, because global fluctuations in Fgf
signaling would result in coordinate changes of axial patterning of eye and
tectum, which are compensated for each other in the retinotectal
topography.
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ACKNOWLEDGMENTS |
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Footnotes |
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Supplementary material for this article is available at http://dev.biologists.org/cgi/content/full/132/22/4951/DC1
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