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First published online 12 November 2008
doi: 10.1242/dev.027367
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1 Department of Neurobiology and Anatomy, University of Utah, 401 MREB, 20 N.
1900 E., Salt Lake City, UT 84132, USA.
2 Program in Neuroscience, University of Utah, 401 MREB, 20 N. 1900 E., Salt
Lake City, UT 84132, USA.
3 Brain Institute, University of Utah, 401 MREB, 20 N. 1900 E., Salt Lake City,
UT 84132, USA.
* Author for correspondence (e-mail: richard.dorsky{at}neuro.utah.edu)
Accepted 22 October 2008
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: Wnt, Bmp, Dorsal retina, RPE, Zebrafish
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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). The
activity of the transcription factors encoded by these and other genes
ultimately leads to the correct DV topographical mapping of RGC axons to the
optic tectum in anamniotes and avians, or to the superior colliculus in
mammals, through the regulated expression of guidance molecules (reviewed by
McLaughlin and O'Leary,
2005).
The sequence of events leading to ventral retinal identity is initiated
when Sonic hedgehog (Shh) from the ventral midline triggers the expression of
ventral retinal transcription factors, including Vax2
(Ekker et al., 1995;
Macdonald et al., 1995;
Take-uchi et al., 2003;
Zhang and Yang, 2001). Vax2
can exclude the expression of dorsal retinal genes from the ventral retina and
also induce the graded expression of Ephb2 and Ephb3
(Schulte et al., 1999). This
process leads to retinal progenitor cells that have been `coded' with ventral
positional identity in the form of EphB receptor tyrosine kinase expression
(Barbieri et al., 2002;
Mui et al., 2002;
Schulte et al., 1999).
The establishment of dorsal retinal identity appears to be controlled by
another family of growth factors. A current model of dorsal retinal patterning
posits that Bmp4, expressed in the dorsal retina, triggers the graded dorsal
expression of tbx5, which in turn leads to the graded expression of
Ephrin B molecules (McLaughlin et al.,
2003). Genetic inactivation of Bmp receptors and Bmp4 demonstrates
the requirement of Bmp signaling for dorsal retinal identity in mouse
(Murali et al., 2005), and
misexpression of Bmp4 can dorsalize the ventral retina in chick and
Xenopus (Koshiba-Takeuchi et al.,
2000; Sasagawa et al.,
2002). In zebrafish, multiple Bmp genes, as well as tbx5,
are expressed in the dorsal retina (Rissi
et al., 1995; Thisse and
Thisse, 2005). Furthermore, at least one Bmp family member, Gdf6a,
has been implicated in controlling the expression of dorsal retinal markers,
including tbx5, in multiple vertebrate organisms
(Asai-Coakwell et al., 2007;
Delot et al., 1999;
French et al., 2007;
Hanel and Hensey, 2006).
However, current models do not address whether Bmps or Tbx genes might act in
distinct steps of dorsal patterning, such as initiation, maintenance or
refinement, and leave open the possibility that other factors may also play
key roles.
We were interested in whether canonical Wnt signaling acts in DV retinal
patterning, based on several previous observations. The canonical Wnt pathway
plays key roles in many important aspects of vertebrate CNS development,
including the patterning of CNS structures
(Bonner et al., 2008;
Dorsky et al., 2003;
Ille et al., 2007;
Kapsimali et al., 2004;
Kudoh et al., 2002;
Muroyama et al., 2002).
Several components of the canonical Wnt signaling pathway have also been shown
to be expressed in developing vertebrate eye structures (reviewed by
Van Raay and Vetter, 2004),
suggesting their involvement in eye formation. Both Wnt reporter transgenes
and Wnts themselves are expressed in the dorsal retinal pigmented epithelium
(RPE) and in surrounding tissues during early eye development
(Burns et al., 2008;
Cho and Cepko, 2006;
Fokina and Frolova, 2006;
Liu et al., 2006). In the
developing brain and spinal cord, both Bmp and Wnt signaling are required for
proper dorsal patterning, and Shh induces ventral identities (for reviews, see
Briscoe and Novitch, 2008;
Ulloa and Briscoe, 2007;
Zhuang and Sockanathan, 2006).
The similarity in functions of Bmp and Shh in patterning both the neural tube
and the retina raises the possibility that Wnt signaling may also have a
conserved function in patterning the dorsal retina. To date, we know of only
one report suggesting a role for canonical Wnt signaling in DV retinal
patterning. In analyzing Lrp6-/- mice, which lack
expression of the Wnt reporter BAT-gal
(Maretto et al., 2003), the
authors observed that Tbx5 is not expressed in the dorsal retina at
E10.5, but they did not assay additional time points or other DV markers.
Here, we test the hypothesis that canonical Wnt signaling plays a role in the establishment of dorsal retinal identity. Through a combination of precisely timed in situ hybridization analyses and conditional misexpression experiments, we show that dorsal retinal identity in zebrafish is initiated at 12 hpf, very early in eye development, and then enters a maintenance phase between 14-16 hpf. We find that Wnt signaling is required for the maintenance of dorsal-specific retinal genes during this second phase, probably through the activation of Bmp signaling. We show that inhibition of the Wnt pathway leads to the loss of dorsal-specific retinal genes, with the concomitant expansion of ventral retinal genes. The loss of dorsal genes reflects a requirement for Wnt signaling in their maintenance, as they initiate their expression normally before Wnt signaling is active in the eye field. Finally, we show that Bmp signaling can rescue dorsal markers in the absence of Wnt signaling, but that activation of Wnt signaling cannot rescue dorsal markers in the absence of Bmp signaling, demonstrating that Wnts signal through Bmps to maintain the dorsal retinal domain.
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MATERIALS AND METHODS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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). The
Tg(TOP:GFP)w25 stable transgenic line was generated by
Dorsky et al. (Dorsky et al.,
2002); the Tg(hsp70l:dkk1-GFP)w32 line was
generated by Stoick-Cooper et al.
(Stoick-Cooper et al., 2007);
the Tg(hsp70l:Tcf3-GFP)w26 line was generated by Lewis et
al. (Lewis et al., 2004); and
the Tg(hsp70l:nog3)fr14 line was generated by Chocron et
al. (Chocron et al., 2007).
Wild-type fish and background of all transgenic lines were of the AB
strain.
In situ hybridization
Digoxigenin-UTP-labeled riboprobes for tbx5, bmp4, bmp2b, gdf6a, vax2,
pax6a, pax6b, vsx2, egfp, efnb2a and ephb2, and
fluorescein-UTP-labeled riboprobe for rx3 were synthesized by in
vitro transcription. Probes for vsx2 and egfp were
synthesized in our laboratory. References for other probes are as follows:
tbx5 (Ruvinsky et al.,
2000), bmp4 (gift from M. Mullins, University of
Pennsylvania), bmp2b (Nikaido et
al., 1997), gdf6a (Open Biosystems EDR1052-524137;
GenBank BI475848), vax2
(Take-uchi et al., 2003),
pax6a (Puschel et al.,
1992), pax6b (Krauss
et al., 1991), efnb2a
(Durbin et al., 1998),
ephb2 (IMAGE Consortium clone 3714371). Whole-mount in situ
hybridization and double in situ hybridization were performed as previously
described (Jowett and Lettice,
1994). For histological analysis, embryos were stained for 40
hours in BM Purple (Roche Applied Sciences), re-fixed for 4 hours in 4%
paraformaldehyde (PFA) in phosphate buffer, dehydrated, embedded in plastic
and sectioned.
Transgenic heat-shock experiments
Adults heterozygous for the 
Tcf and Dkk1 transgenes were outcrossed
to AB strain fish, and Noggin transgenic fish were outcrossed to TL strain
fish. The resulting clutches were heat shocked at various times for 1 hour at
39°C (2 hours at 39°C for hs:Dkk1 and hs:Noggin), sorted for GFP
expression under a fluorescent dissecting microscope, and fixed in 4% PFA at
the required stages. Because the hs:Noggin transgene is untagged, these
embryos were not sorted for GFP.
Lithium chloride treatment
For Dkk1 rescue, embryos were transferred to embryo water containing 150 mM
LiCl at 11 hpf and removed to fresh water at 14 hpf. Heat-shock was performed
at 12 hpf and embryos fixed at 24 hpf. For hs:Noggin rescue, embryos were
transferred to embryo water containing 200 mM LiCl at 18 hpf until 24 hpf.
Heat shock was performed at 18-20 hpf and embryos fixed at 24 hpf.
Bmp rescue experiments
The DNA construct pDestTol2pA2;hsp70l:bmp4-IRES-GFP was generated
using the Tol2kit (Kwan et al.,
2007). The construct (25 pg), along with 15 pg tol2
transposase mRNA, was injected into one-cell stage embryos. Embryos were heat
shocked at 12 hpf and fixed at 24 hpf.
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RESULTS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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). To
increase sensitivity, we detected the reporter by using in situ hybridization
for gfp mRNA (Fig.
1A-F).
The eyes in zebrafish develop as a bilateral evagination of the anterior
neural keel beginning at approximately 11 hpf to form the optic vesicles. At
this stage, the optic stalk is located at the anterior of the optic vesicle,
and the future dorsal retina is located posteriorly. At 16 hpf, the optic
vesicle begins to invaginate to form the optic cup, and the lens placode forms
from the surface ectoderm in contact with the presumptive neural retina.
Finally, at about 22 hpf, the entire optic cup rotates approximately 90°,
so that the posterior part of the optic cup becomes dorsal. At 12 hpf [6
somite stage (ss)], during early optic vesicle evagination, the rostral limit
of active Wnt signaling is at the midbrain-hindbrain boundary
(Fig. 1A), several cell
diameters caudal to the eye field. At 14 hpf (10 ss), the gfp signal
has extended rostrally along the neural tube to the presumptive telencephalon,
but still appears to be excluded from the optic vesicles
(Fig. 1B,C). By 16 hpf (14 ss),
Wnt signaling activity is clearly evident in the optic vesicles and is
restricted to the dorsoposterior presumptive RPE
(Fig. 1D,F). In embryos
sectioned coronally through the midbrain, TOP:dGFP expression is absent from
the optic vesicles at 14 hpf, and is present in the presumptive RPE but not in
the neural retina at 16 hpf (Fig.
1C,F). As development proceeds, TOP:dGFP expression becomes
stronger in the developing eye, remaining in the dorsal RPE
(Fig. 1E). By 24 hpf, TOP:dGFP
is expressed throughout the entire RPE and ciliary marginal zone
(Dorsky et al., 2002). This
expression analysis shows that Wnt signaling becomes active in the
dorsoposterior RPE between 14-16 hpf. Thus, any role played by Wnt signaling
in the establishment of DV retinal polarity probably begins at this time.
Furthermore, it suggests that the reception of Wnt signaling is localized to
the presumptive RPE and excluded from the neural retina at optic vesicle
stages.
We next analyzed the expression of Tcf transcription factors and Wnt
ligands by in situ hybridization at 12 hpf (6 ss) and 18 hpf (18 ss). There
are five Tcf transcription factor family members in zebrafish: Tcf7, Lef1,
Tcf3a (Headless; Tcf7l1a), Tcf3b (Tcf7l1b) and Tcf4 (Tcf7l2)
(Dorsky et al., 1999;
Kim et al., 2000;
Veien et al., 2005). At 12
hpf, during optic vesicle evagination, tcf3a, tcf3b and tcf4
are expressed throughout the anterior neural tube and optic vesicle primordia,
while the other family members are not expressed in this region
(Fig. 1G-J; data not shown). By
18 hpf, tcf3a and tcf3b are expressed at high levels
throughout the optic vesicles, and expression of tcf4 is present at
somewhat lower levels in the same region. These expression patterns persist
through 24 hpf (not shown). The expression of tcf7 initiates at 16
hpf, specifically in the dorsal retina, and is maintained in this region
through 36 hpf (Veien et al.,
2005). We found no lef1 expression in the optic vesicles
at any stage examined (not shown). Of the approximately 20 Wnt ligands present
in zebrafish, at least two, Wnt2 and Wnt8b, are expressed in or around
developing eye structures. Expression of wnt8b has been previously
observed in the dorsal RPE as early as 16 hpf
(Kelly et al., 1995). We
observed expression of both wnt2 and wnt8b in the dorsal RPE
at 18 hpf (Fig. 1K,L).
Therefore, multiple Wnt ligands and Lef/Tcf factors are expressed in the right
place and at the right time to mediate Wnt activation in the dorsal RPE during
mid-somitogenesis stages.
|
;
Delot et al., 1999;
French et al., 2007;
Hocking and McFarlane, 2007;
Liu et al., 2003;
Lupo et al., 2005;
Murali et al., 2005;
Sakuta et al., 2006;
Sasagawa et al., 2002).
Because Bmp signaling can control DV retinal polarity in other vertebrates
(Behesti et al., 2006;
Liu et al., 2003;
Murali et al., 2005;
Plas et al., 2008), we wanted
to determine which Bmp ligands might play a role in zebrafish dorsal retinal
patterning. We therefore examined the expression patterns of the Bmp ligands
bmp4, gdf6a and bmp2b during the initial stages of zebrafish
eye development. bmp4 does not appear in the optic vesicle until 14
hpf, when it begins to be expressed most strongly in the presumptive dorsal
retina (Fig. 2A-C). At 24 hpf,
bmp4 expression is restricted to the dorsal retina
(Fig. 2D). Although
gdf6a and bmp2b are expressed in the ectoderm overlying the
anterior neural plate at 12 hpf, they are not expressed in the retina until 16
and 14 hpf, respectively (Fig.
2E-G,I-K). These ligands also become restricted to the dorsal
retina by 24 hpf (Fig. 2H,L).
Interestingly, the putative Bmp target tbx5 begins its expression in
the retina earlier than do these Bmp ligands, initially in a lateral ocular
domain at 12 hpf, immediately after optic vesicle evagination
(Fig. 2M,N). As the eye
undergoes morphogenetic changes, the tbx5 domain becomes reoriented,
coming to occupy the presumptive dorsal retina at 14 hpf
(Fig. 2O) and eventually the
dorsal retina at 24 hpf (Fig.
2P). A cross-section through the midbrain and optic vesicles
reveals that bmp4 is expressed in both the presumptive RPE and the
retina at 18 hpf (Fig. 2Q,
arrows). Thus, multiple Bmp ligands and tbx5 are expressed in and
around in the optic vesicle during mid-somitogenesis, including in the
presumptive dorsal retina (Fig.
2R). These data suggest that Bmp factors in either the RPE or the
retina could act to maintain tbx5 expression in the dorsal retina,
and that both bmp and tbx5 expression in the retina precede
Wnt activity.
|
Tcf) fused to GFP under the control of the hsp70
promoter [Tg(hsp70l:Tcf3-GFP)w26]. This transgene has been
shown to reliably repress Wnt target genes in an inducible manner
(Lewis et al., 2004). A
heterozygous outcross of these fish was heat shocked and embryos were sorted
for GFP fluorescence to examine the effect of Tcf expression on retinal
patterning. Activation of the Tcf transgene at any of multiple
timepoints resulted in the abolition of tbx5 expression, with no
effect on tbx5 expression in non-transgenic embryos
(Fig. 3A-H). When the transgene
was activated (HS) at 10 hpf and embryos were fixed (F) at 18 hpf (HS10; F18),
tbx5 expression was strongly downregulated in 100% (n=37) of
embryos. In HS10; F24 and HS18; F24 experiments, 100% (n=40, 49) of
embryos showed a similarly strong reduction of tbx5 expression. At
these later timepoints, tbx5 expression was maintained in non-ocular
areas such as the heart and pectoral fin buds (data not shown). When the
transgene was activated as late as 24 hpf (HS24; F30), tbx5
expression was still strongly reduced in 93% (n=109) of embryos.
Thus, regardless of when the heat shock was performed or when the embryos were
fixed, activation of the Tcf transgene eliminated tbx5
expression in the dorsal retina, suggesting that tbx5 is downstream
of Wnt signaling in this region.
|
Tcf represses a gene that normally represses tbx5 in this
region. Together with maintained tbx5 expression in non-ocular areas,
this result suggests that tbx5 may be an indirect transcriptional
target of Wnt signaling. In the course of examining other dorsal markers, we
observed that gdf6a and bmp4 expression were also reduced
following Tcf expression (Fig.
3I; not shown). This suggested that the downregulation of
tbx5 could be a result of decreased Bmp signaling. To further
investigate this possibility, we examined the expression of Bmp genes and
tbx5 in more detail, focusing on the timecourse of downregulation of
these genes following Tcf expression. Following heat shock at 16 hpf,
we found that bmp4 expression was present in 12% (n=17) of
optic vesicles at 2 hours post-heat shock (18 hpf) and in 0% (n=44)
at 8 hours post-heat shock (24 hpf). gdf6a was present in 9%
(n=11) at 2 hours post-heat shock and in 11% (n=9) at 8
hours post-heat shock. By contrast, tbx5 expression was present in
88% (n=25) of embryos at 2 hours post-heat shock, and in 9%
(n=67) at 8 hours post-heat shock
(Fig. 3J), indicating that Bmp
genes are downregulated before tbx5. These data are consistent with a
model in which tbx5 is indirectly regulated by Wnt signaling through
Bmp activity.
Wnt signaling is required for the maintenance of dorsal retinal genes
A potential concern with the Tcf transgene is that it might repress
targets that contain Tcf-binding sites but are not controlled by endogenous
Wnt activity. Thus, we used a second transgenic fish line that expresses a
secreted inhibitor of Wnt signaling, Dickkopf 1 (Dkk1), upon heat-shock
stimulation [Tg(hsp70l:dkk1-GFP)w32]
(Stoick-Cooper et al., 2007).
This transgene inhibits Wnt signaling at the receptor level instead of at the
transcriptional level, and thus is expected to block only active Wnt
signaling. Embryos in which Dkk1 is activated early (9 hpf) display an
enlarged head and a truncated tail (not shown), phenotypes associated with the
loss of Wnt signaling, and downregulation of the Wnt reporter TOP:dGFP
(Stoick-Cooper et al., 2007).
When Dkk1 expression was activated at 9 hpf and embryos were fixed at 15 hpf,
the dorsal marker tbx5 was expressed normally
(Fig. 4A,B). This result was
consistent with our findings that active Wnt signaling begins in the eye field
between 14 and 16 hpf, after tbx5 expression has been initiated at 12
hpf. However, Dkk1 misexpression resulted in the strong downregulation of
tbx5 in the dorsal retina at 18 hpf and 24 hpf, which is similar to
the results obtained using Tcf; at later timepoints, tbx5 was
reduced, although not completely absent
(Fig. 4C-J). At the HS10; F18
timepoint, 96% (n=71) of embryos had strongly reduced tbx5
expression. At HS10; F24 and HS18; F24, tbx5 expression was strongly
reduced in 88% (n=65) and 83% (n=71) of embryos,
respectively. By contrast, at the last timepoint (HS24; F30), only 26%
(n=19) of embryos showed reduced tbx5 expression in the
dorsal retina. This may indicate that Wnt signaling is required for the
expression of dorsal retinal genes during a time window of approximately 14-24
hpf, a developmental period in which the eye goes through dramatic
morphological changes (C.-B.C. and K. Kwan, unpublished), and when genes that
are initially expressed in a broad retinal domain refine their expression to
the dorsal retina. The finding that tbx5 expression in
Dkk1-expressing embryos initiates normally and then later disappears suggests
that Wnt signaling is necessary for the maintenance of tbx5, but not
for its initiation.
|
;
Koshiba-Takeuchi et al., 2000;
Murali et al., 2005;
Sasagawa et al., 2002); thus,
we examined the expression of the ventral retinal gene vax2 in
embryos induced to express Dkk1 at 18 hpf and fixed at 48 hpf. The expression
of vax2 expanded significantly into the dorsal retina in 76%
(n=13) of these embryos (Fig.
4J). These results demonstrate that, in the absence of Wnt
signaling, the retina forms correctly but is ventralized.
Because experimental manipulations of Bmp and Tbx5 levels have been shown
to perturb the expression of the Ephrin B and EphB axon guidance molecules
(Koshiba-Takeuchi et al.,
2000; Murali et al.,
2005), we examined the dorsal gene ephrin B2a
(efnb2a) and the ventral gene ephb2 in Dkk1-expressing
embryos heat shocked at 18 hpf and fixed at 30 hpf. In accord with the
observed reduction in tbx5 expression and expansion of vax2
expression, efnb2a was strongly reduced in 93% (n=29) of
embryos, and ephb2 was modestly expanded dorsally in 84%
(n=19) of embryos (Fig.
4U-X). efnb2a is also expressed in the lens, and this
domain of expression was still present after Dkk1 misexpression, again
demonstrating the specific requirement of Wnt signaling for dorsal retinal
gene expression. An obvious prediction from these results is that the
retinotectal map will be perturbed in a predictable way. However,
Dkk1-expressing embryos did not survive until 5 dpf, when retinotectal
pathfinding could be assayed, thereby precluding such an analysis. In
addition, multiple Wnt pathway components, including Wnt3 and Sfrp5, are
expressed in the tectum and Wnt signaling is also required for axon guidance
in this target tissue (Schmitt et al.,
2006; Tendeng and Houart,
2006). Tissue-specific modulation of Wnt signaling in the eye is
thus required to determine the ultimate role of this pathway in pathfinding.
At this point, our data suggest that Wnt signaling is specifically required
for the maintenance of dorsal retinal genes, the loss of which results in a
dorsal expansion of ventral retinal genes.
|
). LiCl
(150 mM) was applied to embryos at 11 hpf, which were then heat shocked to
induce Dkk1 transgene activation at 12 hpf. LiCl was then removed at 14 hpf
and embryos were fixed at 24 hpf. We found that the expression of both
gdf6a (30%, n=47) and tbx5 (33%, n=108)
were expanded in wild-type embryos treated with LiCl
(Fig. 5B,F). Importantly,
application of LiCl rescued tbx5 and gdf6a expression to
wild-type levels in 26% (n=69) and 51% (n=63) of
Dkk1-expressing embryos, respectively (Fig.
5D,H). Although LiCl application at 11 hpf resulted in a highly
variable phenotype, this rescue was significant because wild-type expression
levels of tbx5 and gdf6a were never seen in untreated
Dkk1-expressing embryos. These results confirm a specific role for canonical
Wnt signaling in the maintenance of dorsal retinal gene expression.
Loss of dorsal identity downstream of Wnt inactivation can be rescued by Bmp signaling
In order to examine the relationship between Wnt and Bmp signaling during
the establishment of DV retinal polarity, we investigated whether the
activation of Bmp signaling could substitute for the loss of Wnt signaling. We
injected one-cell stage embryos with DNA for a Bmp4 construct driven by the
hsp70 promoter (pDestTol2pA2;hsp70l:bmp4-IRES-GFP). When
injected into wild-type embryos heat shocked at 12 hpf, this construct led to
the widespread clonal expression of bmp4 and gfp throughout
the embryos, and gfp-expressing clones were found within the retina
in 85% (n=54) of these embryos, as assayed by in situ hybridization
(Fig. 6A,B). We next injected
this construct into Tg(hsp70l:dkk1-GFP)w32 embryos at the
one-cell stage and heat shocked them at 12 hpf, which simultaneously blocked
Wnt signaling and stimulated the clonal expression of Bmp4. In wild-type
embryos, Bmp4 expression led to an expansion of tbx5 into the ventral
retinal domain in 38% (n=46) of embryos
(Fig. 6D), showing that Bmps
can upregulate tbx5 in the eye. This result is consistent with a
recent study which showed that implantation of Bmp4-soaked beads into the
mouse eye causes a ventral expansion of tbx2, tbx3 and tbx5
(Behesti et al., 2006). In
embryos expressing Dkk1, activation of Bmp4 rescued tbx5 expression
in 44% (n=62) of embryos (Fig.
6F). This rescue was specific to the eye, as no other part of the
embryo displayed ectopic tbx5 staining. We next examined whether the
loss of gdf6a expression in embryos expressing Dkk1 could be rescued
by the activation of Bmp4. No significant rescue was seen in this case
(n=49; data not shown). Together with the rescue of dorsal genes by
Wnt pathway activation described previously, these results show that: (1)
tbx5 is downstream of both Wnt and Bmp signaling; and (2) the
activation of Bmp signaling rescues tbx5 but not gdf6a
expression. This supports a model in which Wnt signaling maintains dorsal
retinal identity through the regulation of Bmp signaling.
Activation of Wnt signaling does not rescue dorsal identity lost from Bmp inhibition
Our results suggest that Wnt signaling maintains dorsal retinal markers by
activating Bmp signaling, but another formal possibility is that Wnts and Bmps
act in parallel. To address this point, we first confirmed that tbx5
expression is lost following Bmp inhibition, then tried to rescue
tbx5 expression by activating Wnt signaling. The implantation of
beads soaked with the Bmp inhibitor Noggin just dorsal to the optic vesicle
was recently shown to abolish tbx5 expression
(Behesti et al., 2006). We used
the transgenic fish line Tg(hsp70l:nog3)fr14, which
expresses Noggin upon heat-shock stimulation
(Chocron et al., 2007). To
achieve robust activation of Wnt signaling, embryos were placed in 200 mM LiCl
from 18-24 hpf and heat-shocked at 18 hpf for 2 hours to activate Noggin
expression. Embryos were fixed at 24 hpf and processed for tbx5
expression by in situ hybridization. To confirm that these embryos had
increased Wnt signaling, we treated the TOP:dGFP reporter line with LiCl from
18-24 hpf, which revealed strongly upregulated reporter expression
(Fig. 6G,H). Because these
embryos were obtained from a heterozygous outcross, we expected 50% of them to
express Noggin. For embryos untreated with LiCl, 55% (n=49) embryos
lost tbx5 expression whereas, for embryos treated with LiCl, 57%
(n=122) embryos lost tbx5 expression
(Fig. 6I-L). No significant
rescue was seen; thus, inhibiting Bmp signaling indeed abolishes dorsal
identity, and this effect is downstream of the dorsal-promoting effect of Wnt
activation. Taken together, these experiments reveal a linear pathway in which
Wnt signaling in the RPE maintains the identity of the dorsal retinal domain
through the activation of Bmp signaling in the RPE and retina.
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|
DISCUSSION |
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|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Our expression analysis revealed that several Wnt pathway components are
expressed in and around the developing eyes from the optic vesicle stage
through 24 hpf. Of the five Tcfs present in zebrafish, only tcf3a,
tcf3b and tcf4 are present in or around the optic vesicles at
the stage when Wnt signaling becomes active in the dorsal RPE (14-16 hpf).
tcf3a and tcf3b are both expressed at high levels in the
evaginating optic vesicles, and tcf4 is expressed in the same domain
but at a slightly lower level (Fig.
1G-J). Although Tcf3 is usually referred to as a `repressor' in
the literature (for a review, see Arce et
al., 2006), it may also function as an activator under conditions
in which β-catenin is stabilized, and therefore Tcf3 and/or Tcf4 are
likely to be the transcription factors through which Wnt signaling maintains
the expression of dorsal retinal genes. A practical difficulty in testing this
idea stems from the fact that Tcf3 loss of function results in embryos lacking
anterior forebrain structures, including eyes
(Kim et al., 2000), whereas
fish mutant for tcf4 do not have a retinal phenotype on their own
(Muncan et al., 2007) (our
unpublished observations). Thus, the unique contribution of Tcf3a, Tcf3b
and/or Tcf4 in mediating Wnt signaling relevant to the expression of dorsal
retinal genes is still unknown. tcf7 is also expressed in the dorsal
retina, but it appears after the initial onset of Wnt signaling in the dorsal
neural retina, a domain slightly different from that of the Wnt reporter
(Veien et al., 2005).
Therefore we believe that tcf7, rather than mediating Wnt function in
the dorsal RPE, may in fact be a downstream target of the pathway, and that it
is possibly regulated through Bmp activity.
|
;
Rossi et al., 2007;
Van Raay and Vetter, 2004).
Further work is necessary to identify other members of the Wnt family that are
expressed in the RPE and their individual contributions to DV retinal
patterning.
Both the Tcf and Dkk1-expressing zebrafish lines are powerful tools
with which to study the loss of Wnt signaling in a temporally-controlled
manner, acting through distinct mechanisms. Tcf directly represses
target genes containing Wnt response elements (WREs) within their promoters,
and Dkk1 specifically inhibits Wnts from signaling through the canonical
pathway by competing for Lrp receptor occupancy (reviewed by
Arce et al., 2006). The early
activation of either transgene resulted in the loss of tbx5 and Bmp
genes in the dorsal retina, providing evidence that the observed phenotype is
not a result of ectopic repression of genes that are not normally responsive
to Wnt signaling. The downregulation of multiple Bmp ligands suggests two
possible nonexclusive mechanisms: Wnt signaling may transcriptionally regulate
multiple Bmp genes, or there may be a positive-feedback mechanism through
which one Wnt-dependent Bmp molecule positively regulates the expression of
other Bmp genes. At later heat-shock timepoints, the activation of Tcf
still led to the loss of dorsal markers, but tbx5 expression was seen
ectopically in the dorsal diencephalon. This suggests that a factor(s) present
in the diencephalon normally represses tbx5 in this region, and is
itself repressed by Tcf. In addition, this finding, together with
maintained non-ocular tbx5 expression and the downregulation of Bmp
genes before tbx5 in Tcf embryos, suggests that tbx5
is an indirect target of Wnt signaling and supports our hypothesis in which
Wnt signaling maintains the dorsal retinal domain through the regulation of
Bmp signaling. However, we cannot completely rule out the possibility that
tbx5 is also a direct target of Wnt signaling in the retina, and
direct analysis of tbx5 regulatory elements is required to further
address this issue. The activation of Dkk1 at 24 hpf led to a modest
downregulation of tbx5 in the dorsal retina at 30 hpf, suggesting
that Wnt signaling is required during a specific time window, approximately
14-24 hpf, as the dorsal retinal domain is being established.
Wnt and Bmp signaling are known to co-regulate gene expression in several
parts of the developing vertebrate embryo. For example, in zebrafish, Wnt8 and
Bmp2b have recently been shown to be required for the establishment of
ventrolateral mesoderm through their cooperative regulation of vent,
vox and ved (Ramel et al.,
2005), and Wnt and Bmp signals also function cooperatively in the
formation of posterior structures through their regulation of genes such as
tbx6 (Szeto and Kimelman,
2004). Wnt and Bmp signals coordinately control the specification
of dorsal spinal cord neurons by regulating Olig3 transcription in
mice (Zechner et al., 2007).
Conditional ablation of the Bmp receptor 1a and β-catenin in the mouse
heart revealed that Bmp signaling is required for the expression of
Tbx5 and specification of the first heart field, and that Wnt
signaling is required for the expression of Bmp4 and specification of
the second heart field (Klaus et al.,
2007). Thus, Wnt/Bmp co-regulation of gene expression and pattern
formation is a general mechanism used in multiple places and at multiple times
in developing embryos. Our results show that this mechanism is also used
during maintenance of the dorsal retinal domain, and that Wnt signaling is
itself required for Bmp pathway activity in this region. In addition to the
possibility that Bmp genes may be direct targets of Wnt signaling, another
possible mechanism by which Wnt signaling could regulate the Bmp pathway is
through the Gsk3-dependent phosphorylation of Smad proteins
(Fuentealba et al., 2007). The
presence of differential Smad phosphorylation in the developing optic vesicle,
particularly in the dorsal versus ventral presumptive RPE, would support such
a model. The use of multiple signaling pathways for patterning a complex organ
such as the eye has obvious advantages. If Bmp signaling alone regulated the
expression of dorsal retinal genes, it would be difficult to maintain this
expression in a static domain during morphogenetic tissue movements. Localized
to the dorsal RPE, Wnt signaling could stabilize dorsal retinal identity by
coordinating the development of these two tissues during eye patterning.
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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