1 Molecular and Cellular Biology Program, University of Washington School of
Medicine, Seattle, WA 98195, USA
2 Department of Neurobiology and Anatomy, University of Utah, Salt Lake City, UT
84132, USA
3 Department of Biological Structure, University of Washington School of
Medicine, Seattle, WA 98195, USA
4 Howard Hughes Medical Institute and Department of Pharmacology, University of
Washington School of Medicine, Seattle, WA 98195, USA
5 Center for Developmental Biology, University of Washington School of Medicine,
Seattle, WA 98195, USA
Author for correspondence (e-mail:
draible{at}u.washington.edu)
Accepted 25 November 2003
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SUMMARY |
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Key words: Zebrafish, Neural crest, Wnt
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Introduction |
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The Wnt family of secreted signaling molecules, which has been shown to
modulate cell proliferation, fate, and behavior in both vertebrates and
invertebrates (reviewed in Cadigan and
Nusse, 1997), has multiple roles in neural crest development
(reviewed by Dorsky et al.,
2000a
; Yanfeng et al.,
2003
). Several studies have implicated Wnts in neural crest
formation. Overexpression of various Wnts induces ectopic expression of neural
crest markers in Xenopus neuralized animal caps and embryos, while
expression of dominant-negative Wnts represses neural crest markers
(Saint-Jeannet et al., 1997
;
Chang et al., 1998; Labonne and
Bronner-Fraser, 1998
, Bang et
al., 1999
; Tan et al.,
2001
; Villanueva et al.,
2002
). In addition, it was recently shown that inhibition of Wnt
signaling in vivo blocks expression of avian neural crest markers, and that
addition of soluble Wnt is sufficient to induce neural crest from neural tube
explants (García-Castro et al.,
2002
). It should be noted that none of these studies identified
which specific Wnt molecules are required at particular stages of development
for neural crest induction or maintenance. Mice null for Wnt1 and Wnt3A show a
loss of neural crest derived cell types, including peripheral sensory neurons
and pigment cells (Ikeya et al.,
1997
). However, these ligands are expressed after the initial
appearance of neural crest, and expression of markers for pre-migratory neural
crest are not changed in mutant mice. These results suggest that if a Wnt
signal is needed for initial neural crest induction, another Wnt ligand must
be involved. Wnt signals are involved in early patterning of the embryo,
imparting posterior character on neuroectoderm
(McGrew et al., 1995
;
McGrew et al., 1997
;
Bang et al., 1997
;
Lekven et al., 2001
;
Erter et al., 2001
;
Kiecker and Niehrs, 2001
) and
ventral character on mesoderm (Christian et al., 1993;
Hoppler et al., 1996
;
Marom et al., 1999
). It is
thus not clear whether Wnt-mediated effects on neural crest induction are
direct or indirect.
There are several lines of evidence suggesting that Wnt/ß-catenin
signaling is also involved later in neural crest cell fate specification after
crest cells have been formed. Activation of Wnt/ß-catenin signaling in
pre-migratory zebrafish neural crest cells promotes pigment cell determination
at the expense of neurons and glia, while inhibition of the Wnt/ß-catenin
signaling pathway promotes neuronal and glial cell fates
(Dorsky et al., 1998). These
data support a model in which Wnt signaling is required for the pigment cell
lineage. However, they do not identify which Wnts are involved in this fate
decision. As noted, Wnt1/Wnt3A knockout mice are deficient in several neural
crest derivatives, including pigment cells
(Ikeya et al., 1997
), but this
is probably due to lack of early expansion rather than effects on later cell
fate decisions. In addition, it has recently been shown that Wnts influence
the development of crest-derived pigment cells in chicks
(Jin et al., 2001
), and that
Wnt/ß-catenin signaling is necessary for mouse pigment cell
differentiation (Hari et al.,
2002
). Wnts may also promote the proliferative expansion of
melanocyte precursors as well as promote their differentiation
(Dunn et al., 2000
;
Yasumoto et al., 2002
).
Together these studies suggest that Wnts are used at sequential stages of
neural crest development, both initially in crest induction and subsequently
in cell fate determination.
In this study we address how reiterated Wnt signaling influences neural crest development in zebrafish. By inducing expression of a Wnt/ß-catenin signaling pathway inhibitor, we identify specific stages of development during which Wnt/ß-catenin mediated signaling is required cell-autonomously for neural crest induction. We also identify a specific Wnt, Wnt8, and demonstrate that it is crucial for initial neural crest induction. What emerges from our data is the concept of a reiterated Wnt/ß-catenin signaling mechanism. The Wnt/ß-catenin signaling pathway, probably in conjunction with other secreted factors such as BMPs and FGFs, functions early to induce the pre-migratory neural crest cells. Later, secreted Wnt signals provide environmental cues during crest cell migration to specify which cells will adopt the pigment cell fate.
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Materials and methods |
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Morpholino injections
Anti-sense Morpholino oligonucleotides (GeneTools) were dissolved in 1X
Danieau's buffer (Nasevicius and Ekker,
2000) for a stock concentration of 20 ng/nl. Morpholino
oligonucleotides (MOs) used in this study have the same sequences as wnt8 MO1
and MO2 (Lekven et al., 2001
).
Each MO was injected into embryos at the 1-2 cell stage using an ASI pressure
injector (ASI Systems).
In-situ hybridization
Whole-mount in-situ hybridization was performed as described
(Thisse et al., 1993).
Digoxigenin-labeled RNA antisense probes were prepared from templates encoding
gsc (Stachel et al.,
1993
), wnt8.1 and wnt8.2
(Lekven et al., 2001
),
pax3 (Seo et al.,
1998
), foxd3
(Odenthal and Nüsslein-Volhard,
1998
; Kelsh et al.,
2000
), sox10 (Dutton
et al., 2001
), mitfa
(Lister et al., 1999
),
crestin (Rubenstein et al.,
2000
; Luo et al.,
2001
), dlx2 (Akimenko
et al., 1994
), and huC
(Kim et al., 1996
). Following
in-situ hybridization, embryos were cleared in 70% glycerol. Embryos were
photographed using an Olympus SZX12 dissecting microscope and DP12 camera.
Antibody staining
To detect Foxd3 expression, fixed embryos were stained with anti-Foxd3
rabbit antisera (1:1000) with 20% goat serum, and then incubated with
anti-rabbit Alexa568-conjugated secondary antibodies. To detect Hu expression,
fixed embryos were stained with mouse monoclonal anti-Hu antibody (Molecular
Probes; 1:1000) with 10% goat serum, followed by incubation with anti-mouse
Alexa568-conjugated secondary antibody. All incubations were performed either
at room temperature for 4 hours or at 4°C for 12-16 hours. Following
antibody staining, serial washes in PBS+0.1% TritonX-100 were performed to
reduce background. Staining was visualized with a Zeiss LSM 510 Pascal
confocal microscope.
Transplantation assays
Donor embryos were labeled at the 1-4 cell stage with rhodamine-dextran.
Cells from blastula stage donor embryos were transplanted to mediolateral
regions of shield-stage, unlabeled host embryos
(Moens and Fritz, 1999). After
transplantation, embryos were placed in embryos medium
(Westerfield, 1994
) containing
50 units penicillin and 5 µg streptomycin at 28.5°C. To activate
expression of the hs
Tcf-GFP, embryo incubation temperature was shifted
to 37°C for 1 hour. Transplanted cells were visualized using a Zeiss
compound microscope.
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Results |
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Canonical Wnt signaling is required for neural crest induction
If Wnt signals are indeed necessary for zebrafish neural crest induction,
then inhibition of Wnt/ß-catenin mediated signaling should result in loss
of neural crest. We globally blocked Wnt signaling at various stages of early
neural development by activating expression of Tcf in transgenic fish
(Fig. 2). Embryos were
incubated at 37°C for 45 minutes, and then returned to 28.5°C before
fixing and staining with an antibody that recognizes zebrafish Foxd3 (fkd6)
(Odenthal and Nusslein-Volhard,
1998
), revealing neural crest cells flanking the neural plate. At
this stage, foxd3 appears to be expressed in virtually all
pre-migratory neural crest cells, completely overlapping with
snail2/slug (Kelsh et al.,
2000
). Wnt/ß-catenin signaling is crucial for neural crest
induction from the end of gastrulation (bud stage) through the 3-somite stage
since
Tcf inhibits expression of Foxd3 at these stages
(Fig. 2D,E, compared with
control Fig. 2A,B). In
contrast, Foxd3 expression is comparable to that of wild-type siblings upon
expression of the inhibitor 1 hour later at the 6-somite stage
(Fig. 2C, compared with
control, Fig. 2F). Expression
of the neural crest marker sox10 as assayed by in-situ hybridization
is similarly affected (data not shown). We conclude that there is a temporal
limit to the requirement for Wnt/ß-catenin mediated signaling in neural
crest induction, and that cells lose sensitivity to transgene activation
between the 3- and 6-somite stages.
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Discussion |
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Our studies are the first to identify a specific Wnt necessary for neural
crest induction in zebrafish. However, wnt8 is apparently needed only
during the initial phase of neural crest formation, and perhaps other Wnts are
needed after this period. In mouse embryos, loss of Wnt1 and
Wnt3a does not block the initial induction of neural crest but
affects subsequent neural crest expansion
(Ikeya et al., 1997). Although
Wnts are thought to be involved in regulating proliferation of neural tissue
(McMahon and Bradley, 1990
;
Dickinson et al., 1994
;
Megason and McMahon, 2002
),
proliferation of neural crest precursors is reported to be unaffected in
Wnt1/Wnt3a knockout mice
(Ikeya et al., 1997
). These
results suggest the possibility that there are several stages of neural crest
induction regulated by different Wnts: an early phase involving wnt8
and a later phase utilizing wnt1 and wnt3a. Other Wnts, such
as wnt3, wnt6, wnt7 and wnt10b, may also be involved.
Several studies have implicated Wnts in promoting ventral/posterior
patterning of mesoderm and neuroectoderm. Blocking wnt8 in zebrafish
results in loss of posterior brain and spinal cord
(Erter et al., 2001;
Lekven et al., 2001
). In
addition, there is an anterior expansion of posterior markers when zebrafish
tcf3 genes are inactivated (Kim
et al., 2000
; Dorsky et al.,
2003
), including an anterior shift of neural crest
(Itoh et al., 2002
). Since
relief of Tcf3 repressor activity is a result of Wnt signal transduction, loss
of tcf3 function is thought to behave similarly to Wnt activation
(Brannon et al., 1997
;
Kim et al., 2000
). Together
these observations suggest the possibility that the effects we see on neural
crest are indirect: interfering with Wnt signals might simply convert
posterior neural tissue to anterior, from which no neural crest would normally
be produced. However, our experiments with the
Tcf transgenic line
suggest that this is not the case. We have found that neural crest development
can be blocked at a time when the development of dorsal spinal Rohon-Beard
sensory neurons is unaffected. This result implies that we have not simply
eliminated the domain from which neural crest cells are derived, since neural
crest cell precursors arise intermingled among Rohon-Beard neurons
(Cornell and Eisen, 2000
). In
addition, cell transplantation experiments suggest that neural crest
precursors specifically require activation of the Wnt/ß-catenin signaling
pathway, and that small groups of wild-type cells can form neural crest in the
correct position even when Wnt signaling is blocked in all other cells of the
host embryo. These results suggest that Wnt-mediated induction of zebrafish
neural crest is probably independent of its roles in anterior/posterior
patterning, as has been recently suggested for FGF-mediated induction of
neural crest in Xenopus
(Monsoro-Burq et al.,
2003
).
At first glance, the phenotype of the headless/tcf3 mutant, in
which neural crest markers are expanded anteriorly, appears to contradict our
results with the Tcf transgenic line, in which neural crest markers are
lost. However, the headless mutation results in a loss-of-function of
a repressor while the
Tcf transgenic results in a gain-of-function of a
repressor, which is consistent with the different phenotypes. Furthermore,
describing neural crest cells as `expanded' in headless mutants is
perhaps inaccurate, as the neural crest domain instead is shifted anteriorly.
Finally, the
Tcf transgene is likely to act as a dominant repressor in
the place of all members of the TCF/Lef family, not just Tcf3.
There are several caveats to the interpretations of the data we present
here. Although wnt8 is expressed at the right place and time to be
involved in the induction of neural crest cells from ectoderm and
wnt8 MO injection blocks neural crest formation, we cannot eliminate
the possibility that activation of the Tcf transgene is instead
blocking signaling from some other Wnt. The period we identified as critical
using the transgenic line roughly corresponds to the period of wnt8
expression; however, exactly when Wnts are needed cannot be easily determined
by transgene activation. Although it takes several hours to inactivate the
TOPdGFP reporter, suggesting that the Wnt requirement for neural crest
formation might be later than the period in which we performed the heat shock,
this multicopy reporter transgene may not respond with the same kinetics as
endogenous genes. Currently identified zebrafish Wnts known to be expressed in
the dorsal neural tube, such as wnt1, wnt3a and wnt10b, are
expressed too late to be involved in this initial phase of neural crest
induction, and a deletion eliminating zebrafish wnt1 and
wnt10b has little effect on neural crest
(Lekven at al., 2003
). Future
studies identifying other zebrafish Wnt genes and their functions will be
needed to fully address this point. Another caveat is that the
Tcf
transgene may additionally act to repress genes not regulated by Wnt signals.
Future studies using other tools to block Wnt signals, such as identifying and
eliminating Frizzled receptors expressed at the right place and time, or
expressing other reagents that interfere with Wnt signals such as
axin or kinase-dead gsk3, are needed.
Both BMP and Notch signaling have been implicated in neural crest induction
in zebrafish, but these signaling pathways may be required at different times
from Wnt signaling. Mutations in zebrafish bmp2b result in loss of
both neural crest and Rohon-Beard cells
(Barth et al., 1999;
Nguyen et al., 2000
).
Disruption of Notch signaling also results in loss of neural crest but instead
concomitantly increases the number of Rohon-Beard cells
(Cornell and Eisen, 2000
). In
contrast, blocking Wnt signals can interfere with neural crest without
affecting Rohon-Beard cells, suggesting that Wnts maintain a role independent
of Notch regulation.
Previous studies have implicated Wnt signaling in the specification and
differentiation of pigment cells from neural crest
(Dorsky et al., 1998;
Dunn et al., 2000
;
Jin et al., 2001
;
Hari et al., 2002
), and that
mitfa, a gene encoding a bHLH transcription factor necessary and
sufficient for pigment cell formation, is a direct target of the Wnt pathway
(Dorsky et al., 2000b
;
Takeda et al., 2000
;
Widlund et al., 2002
). We
demonstrate here that the Wnt requirement for mitfa expression is to
some degree temporally separable from Wnt regulation of neural crest
induction, suggesting that reiterated Wnt signaling plays sequential roles in
neural crest development. When the
-Tcf transgene is activated at the
18-somite stage, mitfa expression is almost completely eliminated. An
alternative explanation is that transgene activation at this stage
specifically blocked the formation of a subpopulation of melanogenic neural
crest cells, consistent with the observed reduction of sox10-positive
cells. However, our previous results demonstrated that zebrafish melanogenic
neural crest cells have already segregated from the neural tube by the
18-somite stage (Raible and Eisen,
1994
; Raible and Eisen,
1996
), suggesting this possibility is less likely.
Our results also suggest that Wnt signals promote dlx2 expression
in branchial arches. Unfortunately, the poor long-term survival of
heat-shocked transgenic animals has not let us assess the final effects of
transgene activation on cartilage differentiation. Although ectomesenchyme
that gives rise to craniofacial cartilages is traditionally thought of as
derived from neural crest, some studies suggest that these cells are a
distinct population (e.g. Dutton et al.,
2001). Although our results might suggest that Wnt signals are
needed for ectomesenchyme as well as other neural crest derivatives, the
inference that these cells thus have common origins should be reached with
caution since Wnt signals have widespread use during embryogenesis.
One way the same signaling pathway could play different roles at different
times is if different genes are induced in the context of other signaling
pathways. Indeed, neural crest induction has been proposed to occur after a
combination of signals, including both Wnts and BMPs
(LaBonne and Bronner-Fraser,
1998). Moreover, Wnts work with FGF during anterior/posterior
neural patterning (McGrew et al.,
1997
). Another possibility is that genes induced by initial Wnt
exposure modify the response to subsequent Wnt signals. A good candidate for
such a role would be sox10, which we show here requires Wnt signals
for its initial expression. Sox10 is necessary for the development of a subset
of neural crest derivatives, including pigment cells
(Southard-Smith et al., 1998
;
Pingault et al., 1998
;
Dutton et al., 2001
;
Honore et al., 2003
;
Aoki et al., 2003
), and
directly regulates the mitf promoter in vitro and in vivo
(Verastegui et al., 2000
;
Bondurand et al., 2000
;
Lee et al., 2000
;
Potterf et al., 2000
;
Elworthy et al., 2003
).
The use of the same signaling pathways in different places and at different
times is common throughout development, and understanding how specificity is
generated in particular contexts remains a major challenge. It is interesting
to consider that BMPs or Notch may also have sequential roles within the
neural crest, first needed for induction and subsequently in generation of
neuronal or glial sublineages (Shah et
al., 1996; Reissmann et al.,
1996
; Varley and Maxwell,
1996
; Morrison et al.,
2000
; Wakamatsu et al.,
2000
). Wnts, BMPs and Notch also play roles in the survival of
neural crest cells (Graham et al.,
1996
; Maynard et al.,
2000
; Ellies et al.,
2000
; Brault et al.,
2001
; Hasegawa et al.,
2002
). Understanding the specifics of how Wnt signals regulate
distinct steps in neural crest induction and pigment cell specification might
reveal general mechanisms for reiterated signaling during development.
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ACKNOWLEDGMENTS |
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Footnotes |
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