1 Program in Neurobiology and Behavior, Department of Biological Structure, Box
357420, University of Washington, Seattle, WA 98195, USA
2 Program in MCB, Department of Biological Structure, Box 357420, University of
Washington, Seattle, WA 98195, USA
* Author for correspondence (e-mail: roelink{at}u.washington.edu)
Accepted 24 June 2003
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SUMMARY |
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Key words: Chick, Forebrain, Embryonic induction, Zona limitans, intrathalamica, Wnt, Dickkopf 1
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Introduction |
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An exception to these findings occurs in the developing forebrain, at the
zona limitans intrathalamica (zli), the interface between the future,
anteriorly located ventral thalamus (vT) and the posteriorly located dorsal
thalamus (dT). Before its overt formation, the location of the prospective zli
is demarcated by the adjacent but nonoverlapping expression patterns of
Six3 and Irx3, which encode Iroquois-type transcription
factors. Six3 is expressed in neural tissue overlying the prechordal
plate and Irx3 is expressed above the anterior-most portion of the
notochord. In HH stage 8 chick embryos, the expression of either Six3
or Irx3 can confer anterior or posterior identity, respectively, on
the developing forebrain by determining the competency of this neural tissue
to differentially respond to Fgf and Shh signals
(Kobayashi et al., 2002). A
question that arises from these studies is how the expression domains of
Six3 and Irx3 are established.
Several lines of investigation demonstrate that Wnt signaling in early
forebrain tissue induces differentiation of the posterior forebrain
(van de Water et al., 2001),
whereas the absence of Wnt signaling allows differentiation of the anterior
forebrain (Mukhopadhyay et al.,
2001
; Houart, 2002). These studies indicate a role for Wnts in the
early anteroposterior patterning of the brain
(Nordstrom et al., 2002
). Our
data extend these observations by demonstrating that Wnt signaling is
sufficient to induce Irx3 expression and suppress Six3
expression in explanted forebrain tissue. The source of this Wnt activity
remains unclear. However, somewhat later in development, Wnts are expressed in
and posterior to the zli.
The zli is the first forebrain subdivision to establish, forming above the
transition between the notochord and prechordal plate
(Figdor and Stern, 1993). The
site of zli formation is characterized by the absence of lunatic
fringe expression (Zeltser et al.,
2001
). The zli, a narrow strip of tissue that both expresses
boundary cell markers and restricts the mixing of cell lineages
(Larsen et al., 2001
), defines
the border between the future dT and vT. Although the role of the zli is
unknown, it serves as either the site or limit of expression of several
molecules with inductive capacities. Wnt3a expression deviates from
its pattern along the dorsal neural tube to form a finger-like projection that
extends ventrally at the zli. Wnt3, a Wnt family member with 91%
identity to Wnt3a, is expressed in the prospective dT, with its
anterior limit of expression abutting the zli
(Roelink and Nusse, 1991
;
Salinas and Nusse, 1992
). In
addition to Wnts, the expression patterns of several transcription factors and
cell adhesion molecules have sharp borders at the zli. Gbx2 is
expressed posterior to the zli in the dT
(Bulfone et al., 1993
) and the
zli marks the posterior limit of expression for the vT markers Dlx2,
R-cadherin and cadherin8 (Larsen et al.,
2001
; Price et al.,
1991
; Redies and Takeichi,
1996
). Based on the timing of Wnt3 and Wnt3a
expression, zli-restricted Wnts cannot account for the initial restriction of
Six3 and Irx3 expression.
Here, we demonstrate that activation of the canonical Wnt signaling pathway is sufficient and required to induce dT-specific gene expression, and that the absence of Wnt signaling allows vT-specific differentiation. Blocking the Wnt response resulted in vT-specific gene expression in dT explants, and exposure of vT explants to Wnt3 resulted in the induction of both early (Irx3) and late (Gbx2) dT-specific gene expression. Furthermore, misexpression of either Six3 or the Wnt inhibitor Dkk1 in the presumptive dT initiated differentiation appropriate for the vT. These results indicate that, by determining the domains of Irx3 and Six3 expression, Wnt signaling is crucially important for the initial anteroposterior organization of the forebrain. Our observation that Wnts induced Irx3, which, in turn, allowed the dT-specific response, indicates that Wnt signaling is required at multiple stages of development of the posterior forebrain.
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Materials and methods |
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In headfold experiments, the explants were oriented in a rosette with the anterior end of the explant facing out. This orientation allowed both the anterior and posterior portion of the explants to be scored for the expression of markers following culture.
For co-culture experiments, zli explants and explants of prospective dT and vT were incubated either alone or together in collagen. To distinguish zli tissue from prospective dT or vT explant tissue, CellTracker Blue CMAC dye (Molecular Probes) was used to fluorescently label the prospective dT/vT explants according to the manufacturer's instructions. During fixation, the co-cultured explants were visualized and photographed by both fluorescent and light microscopy. These images were merged in Photoshop (Adobe) to create a map of prospective dT/vT versus zli explant tissue for each co-culture condition, thus allowing us to discriminate between zli and prospective diencephalic explant tissue (data not shown).
Wnt was supplied to headfold explants via a soluble Wnt3a-containing
supernatant, generated by growing mouse fibroblast L cells stably transfected
with a Wnt3a-expression construct
(Shibamoto et al., 1998) in
Optimem (Gibco BRL) for 4 days. Control supernatant was obtained from
mock-transfected L cells. The supernatants were added to explants at a 1:1
ratio with complete neurobasal media.
In prospective dT/vT explant experiments, Wnt was provided by one of two
means, with similar results obtained using either Wnt source. First,
prospective dT/vT explants were grown on a monolayer of either RatB1A cells or
RatB1A cells expressing Wnt3 (Shimizu et
al., 1997) in supplemented neurobasal media (see above). Following
culture, the explants were placed in collagen, fixed, and processed by in situ
hybridization. Alternatively, explants were exposed to the Wnt3a supernatants
described above.
To inhibit Wnt signaling in vitro, casein kinase inhibitor 7 (cki7) (Seikasaku America) dissolved in DMSO was added at a final concentration of 50 µM at the start of culture. A similar dilution of DMSO was added to control wells. Alternatively, a Dkk1 supernatant was used to block Wnt signaling in headfold culture experiments. Dkk1 supernatant was generated by growing mouse 293T cells transfected with pRK5-Dkk1 in Optimem (Gibco BRL) for 48 hours. Control supernatant was obtained from mock-transfected 293T cells transfected with pRK5 alone. The supernatants were concentrated 10-fold by filtration through 10KNMWL exclusion membranes (Amicon Ultra-15), and added to explants at a 1:1 ratio with complete neurobasal media.
Explants and embryos to be processed by in situ hybridization were fixed overnight in 4% paraformaldehyde in either PBS or MEM, pH 7.4 at 4°C. After fixation, embryos to be sectioned were rinsed in DEPC PBS, followed by 30% sucrose in DEPC PBS solution, embedded in OCT (Sakura Finetechnical) and then cryosectioned.
In situ hybridization
Whole-mount and slide in situ hybridizations were performed following
established procedures (Jasoni et al.,
1999; Schaeren-Wiemers and
Gerfin-Moser, 1993
). In situ hybridizations were carried out using
antisense Irx3, Six3, Gbx2, Dlx2 and Wnt3 digoxigeninlabeled
(Roche) riboprobes. Antisense riboprobes were prepared from plasmids
containing chicken cDNA sequences for Irx3, a gift from Dr Jessell;
Six3, a gift from Dr Shimamura; Gbx2
(Kowenz-Leutz et al., 1997
), a
gift from Dr Leutz; and Dlx2
(Puelles et al., 2000
), a gift
from Dr Rubenstein. Chicken Wnt3 cDNA was cloned (C.P.R., M.M.B. and
H.R., unpublished) and used to generate an antisense Wnt3
digoxigenin-labeled riboprobe.
Headfold explants exposed to either Wnt3a, Dkk1 or cki7 were processed by in situ hybridization for Irx3 and Six3. The expression domains of these markers were scored as normal, expanded and reduced/absent compared to headfold explants cultured under control conditions.
Prospective dT an vT explants were assayed in one of two ways post in situ hybridization. In both co-culture and Wnt3a-mediated Gbx2 and Dlx2 induction experiments, explants were scored as either positive or negative, as compared to the staining of control tissue. Prospective dT and vT explants cultured in supplemented neurobasal media served as the negative control. Tissue dissected from older embryos was used for both negative and positive controls.
In ovo manipulations
Electroporations were carried out following established protocols
(Watanabe and Nakamura,
2000). xDkk1 (Glinka
et al., 1998
), a gift from Dr Niehrs, was excised from
pCS2+ and cloned into pMiwII
(Watanabe and Nakamura, 2000
),
a gift from Dr Nakamura. eGFP (Clontech) was cloned into
pcDNA3.1/Zeo (Invitrogen). A mixture of 3 µg
µl1 pMiwII-xDKK1 and 3 µg
µl1 pcDNA3.1-GFP in L-I5 (Gibco BRL)
supplemented with 10 mM HEPES was injected into the neuropore of HH stage 9-10
embryos. Full-length chicken Six3
(Kobayashi et al., 2002
) was
subcloned into pMES-IRES-GFP
(Swartz et al., 2001
).
pMES-Six3-IRES-GFP resuspended in L-I5 (Gibco BRL) supplemented with
10 mM HEPES was injected into the neuropore of HH stage 9-10 embryos. The
embryos were electroporated with two 25 msec pulses of 62.5 Volts
cm1. After 48 hours, embryos were fixed and processed by in
situ hybridization as described above.
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Results |
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To confirm the in vitro requirement for Wnt signaling on Irx3
induction in posterior forebrain tissue, medium conditioned with
Xenopus Dickkopf1 (Dkk1) was added to cultured headfolds. In
Xenopus, Dkk1 antagonizes Wnt action
(Glinka et al., 1998) by
binding to LRP and Kremen (Mao et al.,
2002
; Nusse,
2001
), members of a receptor complex for the Wnt ligand. Treatment
with Dkk1 recapitulated the cki7 results, with 90% of the headfolds tested
showing a downregulation of Irx3 expression
(Fig. 2H versus 2E). Again, the
Six3 expression pattern was unaffected by inhibition of the
Wnt-signaling pathway (Fig.
2D).
These headfold explant experiments indicated that an early, endogenous Wnt signal was required for the maintenance of the posterior forebrain determinant Irx3. Moreover, if not inhibited, Wnt signaling can preclude the proper specification of the anterior forebrain by preventing the maintenance of Six3 expression and the expansion of Irx3 expression. To determine if this Wnt activity occurs in combination with other mesoderm-derived signals, we tested if neural plate explants exhibited a similar response.
Using the axial mesoderm transition as a guide, explants containing the prospective dT and vT were dissected from HH stage 8 chick embryos (Fig. 3A), cultured for 48 hours under serum-free conditions, fixed and then assayed for the induction of Irx3, Gbx2 and Dlx2 by in situ hybridization. Prospective dT explants, and not prospective vT explants, were Irx3-positive at the time of dissection (data not shown), consistent with the expression of Irx3 in the posterior forebrain at HH stage 8.
Exposure to Wnt3a-conditioned medium increased the level of Irx3 expression (Fig. 3F) and induced the expression of Gbx2 (Fig. 3J), but only in dT explants. No significant induction of either Irx3 or Gbx2 was observed in vT explants (Fig. 3B). The anterior forebrain marker Dlx2 was not expressed in dT and vT explants in the presence or absence of Wnts (Fig. 3K-N). These data demonstrate that Wnt signaling was sufficient to maintain posterior forebrain identity in explants. The Wnt-mediated induction of Gbx2 indicates that a continuous Wnt signal is necessary for the development of the dorsal thalamus. Furthermore, the absence of Wnt does not cause expression of Dlx2, indicating that additional signals that are not provided in these in vitro cultures are required are required for its expression.
Blocking the response to forebrain-derived Wnt causes specific
differentiation of the anterior forebrain
To test if zli tissue is capable of inducing vT- and dT-specific gene
expression, prospective dT and vT explants from stage 8 embryos were cultured
adjacent to zli explants from HH stage 17-18 embryos
(Fig. 4A). Following 2 days in
culture, induction of Gbx2 and Dlx2 was assayed by in situ
hybridization. Gbx2 was induced in 42% of the prospective dT explants
cultured in contact with zli tissue, but in none of the prospective vT
explants co-cultured with zli tissue (Fig.
4B,G,C). Co-culture of zli tissue with prospective vT explants
induced Dlx2 in 44% of these explants, whereas no significant
Dlx2 induction was observed in prospective dT explants cultured under
these conditions (Fig. 4B,D,H). Because the zli explants are taken from HH stage 17-18 embryos, Gbx2
and Dlx2 expression are sometimes observed in zli tissue (e.g.
Fig. 4C). These co-culture
results confirm the observation that neural tissue anterior and posterior to
the prospective zli has different competencies, which is consistent with the
differential Wnt response in explants from these regions.
|
Together, these results demonstrate that, in vitro, a Wnt signal is
required for both the induction of Gbx2 and the repression of
Dlx2 in prospective dT explants. Apparently, blocking Wnt-mediated
signaling causes an anterior to posterior change in the response to other
signals derived from the zli. To address if blocking Wnt activity in the
posterior forebrain allowed an anterior forebrain-specific response in ovo, we
misexpressed Xenopus Dickkopf1 (xDkk1). pRK5-xDkk1
and pCDNA3.1-GFP were co-injected into the neuropore of HH stage 9-10
embryos and diencephalic misexpression of these plasmids was achieved
following electroporation. Induction of Dlx2 and Gbx2 was
visualized by in situ hybridization in serial sections in which GFP was
present. Misexpression of xDkk1 resulted in down regulation of
Gbx2 in dT tissue and a concomitant expression of Dlx2 in
the same region (Fig. 5C,D).
The overlapping expression of these two markers was never observed in control
embryos (Fig. 5A,B). Together,
these results demonstrate that Wnt can be the sole determinant that allows
posterior differentiation but that the induction of Dlx2 requires
other, unknown signals present in the zli or other parts of the forebrain.
Because Wnt signaling efficiently represses Six3 expression, and Six3
and Irx3 are mutually inhibitory, we tested if expression of Six3 in
the posterior forebrain resulted in differentiation appropriate for the
anterior forebrain, as would be predicted based on a previous study
(Kobayashi et al., 2002).
Six3 predicates vT-specific differentiation
Full-length chicken Six3
(Kobayashi et al., 2002),
cloned into pMES-IRES-GFP, was injected into the neuropore of HH
stage 9-10 embryos, and diencephalic misexpression of this plasmid was
achieved via electroporation. In serial sections where GFP was present
posterior to the zli, Dlx2 and Gbx2 expression was
visualized by in situ hybridization. Misexpression of Six3 resulted
in repression of Gbx2 in dT tissue
(Fig. 6A,B). In 25% of these
embryos, a concomitant induction of Dlx2 was observed in the dT
(Fig. 6C). Independent
electroporations demonstrated that misexpression of Six3 in the
posterior forebrain alters the normal levels of Wnt3 in the dT; in
regions of the dT where GFP was present, Wnt3 was repressed
(Fig. 6D,E,F).
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Discussion |
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Wnt family members have been implicated as posteriorizing agents during
neural development. In the Nieuwkoop model, neural induction occurs via a
two-step activation-transformation process
(Nieuwkoop, 1952). Following
the initial induction of neural tissue, all of which is anterior in nature,
subsequent events underlie the induction of more caudally-fated tissue. Wnts
appear capable of mediating these secondary inductive events, thereby
initiating posterior neural fates (McGrew
et al., 1995
). In Xenopus, misexpression of
xWnt8 results in loss of anterior structures, including the forebrain
(Fredieu et al., 1997
).
Treatment with lithium, which activates the transforming Wnt pathway, has a
similar effect.
In addition, the zebrafish mutant mbl/,
which has an overactive Wnt response caused by a nonfunctional axin
gene, demonstrated a role for Wnt signaling in conferring posterior identity
in the developing forebrain (van de Water
et al., 2001). In mbl/ mutants,
there is a loss of telencephalon and vT with a concomitant expansion of the
region that gives rise to dT. Because the telencephalon and vT develop from
structures that are initially localized anterior to the dT, the fate shift in
mbl/ represents a gain of
posterior-forebrain fates at the expense of anterior-forebrain fates. Although
the exact identity of the Wnt ligands that mediate this posteriorization are
unknown, several candidate molecules are present in and around the developing
forebrain during early and later stages of development
(Nordstrom et al., 2002
;
Roelink and Nusse, 1991
).
How are anterior and posterior forebrain competency differences
established?
Although Gbx2 and Dlx2, the markers of dT and vT
forebrain fates, are not yet induced by HH stage 8, Six3 and
Irx3 are expressed in distinct anterior- and posterior-forebrain
domains at this time. The interface of Six3 and Irx3
expression corresponds with the boundary used to obtain prospective dT and vT
explants: Six3 is expressed above the prechordal plate and
Irx3 is expressed above the notochord. A study
(Kobayashi et al., 2002)
showed that the expression of either Six3 anteriorly or Irx3
posteriorly differentially primes anterior and posterior forebrain tissue to
respond to Shh and Fgf signals, and results in the induction of either
anterior forebrain or posterior forebrain-specific genes, respectively.
The Wnt inhibitor Dkk1 is produced in the prechordal plate and is present
at the right time and place to block a tonic Wnt signal that confers posterior
identity on the forebrain at neural-plate stages
(Glinka et al., 1998).
Moreover, Wnt family members are expressed in the posterior neural plate by HH
stage 4-5 (Nordstrom et al.,
2002
), which indicates that Wnt signaling could be involved in
early regionalization events of the developing forebrain. Our headfold culture
experiments implicate differential Wnt signaling as the mechanism by which the
forebrain determinants Six3 and Irx3 are induced in the
neural plate. Ectopically supplied Wnt3a was sufficient to inhibit the
expression of the anterior forebrain determinant Six3, whereas
inhibition of the Wnt pathway eliminated the expression of the posterior
forebrain determinant Irx3. These experiments support a model whereby
Dkk1 from the prechordal plate inhibits a Wnt signal in the most anterior
neural tissue, thus causing a switch from a Wnt-induced,
Irx3-positive posterior forebrain fate to a Six3-positive,
anterior forebrain fate (Fig.
7).
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A Wnt signal is necessary and sufficient for specifying dT
identity
Although the influence of Wnt signaling on Irx3 expression
indicates an early role for Wnts in forebrain patterning, the subsequent
expression of Wnt3 and Wnt3a indicate a subsequent role for
Wnts in dT specification. An exogenous Wnt3/3a signal appears to be capable of
acting as a posteriorizing agent that specifies the dT fate, as measured by
Gbx2 induction in our prospective forebrain-explant system. The
observation that not all explants respond to Wnt3a-conditioned medium by
expressing Gbx2 indicates differences in these explants that might be
caused by small variations between dissections and the embryonic stages from
which these explants are derived. In addition, it remains to be determined
whether higher doses of Wnt3a result in a higher percentage of
Gbx2-expressing explants.
The ability of endogenous zli-derived signals to induce either vT or dT markers in prospective vT and dT explants was assessed using a heterochronic co-culture system. Because the expression patterns of inductive molecules at the zli are well-described at HH stage 17, zli tissue from these older embryos was used as a source of inductive molecules. Culture of zli tissue adjacent to prospective vT or dT explants is capable of inducing vT- and dT-specific gene induction. Because zli explants are not homogenous sources of inductive signals, we expect that the relative position of the prospective dT/vT explants to zli explants affects the induction of Gbx2 and Dlx2, which might explain why just over 50% of the explants do not respond under these co-culture conditions.
The induction of Gbx2 in prospective dT explants by zli tissue is mediated through a transforming Wnt signal, which is likely to be Wnt3 or Wnt3a. Inhibiting the Wnt response in co-cultures caused prospective dT tissue to acquire a vT-specific fate. Therefore, a Wnt signal that is either from the zli or present in the prospective dT is instructive in specifying the posterior/dT tissue in two ways. First, a Wnt signal induces the dT markers Irx3 and Gbx2. Second, a Wnt signal inhibits the induction of the vT markers Six3 and Dlx2 (see Fig. 7). In turn, Six3 inhibits the expression of Gbx2 and Wnt3 and promotes the expression of Dlx2.
Our observation that the vT is seemingly insensitive to Wnt signals could
be caused by the lack of a crucial component of the Wnt signaling pathway in
the vT. Interestingly, Tcf4 is expressed in the prospective dT but
not the vT (Galceran et al.,
2000) and might be an important mediator of the differences in
competency to respond to Wnt signaling on either side of the zli.
Alternatively, Wnt inhibitors expressed in the prospective vT could prevent
Wnt-receptor activation. SFRP-2, a known Wnt antagonist
(Ladher et al., 2000
), is
expressed in the developing vT with a sharp posterior boundary of expression
at the zli.
Although the in vitro studies presented in this work indicate a role for Wnt signaling in diencephalic development, they also demonstrate the existence of an undetermined factor or factors that induce the vT fate. Possible candidates for vT inducers include Shh and Fgf8. Both are expressed at the zli and we are currently examining their roles in Dlx2 induction.
Our results agree fundamentally with the Nieuwkoop model of neural induction. However, the loss of anterior forebrain in Dkk1 mutants indicates that active suppression of Wnt signaling is required for the formation of anterior neural tissue, which implies the presence of tonic Wnt signals in the developing forebrain. It appears that the presence of Dkk1 is required for the expression of Six3, but it is unknown if Six3 expression, in turn, requires a distinct inducer. Expression of Irx3 in the posterior forebrain is likely to be induced by a Wnt signal, consistent with our observation that Wnt3a can induce Irx3 in forebrain explants.
The expression of Six3 and Irx3 are crucial for the subsequent distinct differentiation of tissue in the anterior and posterior forebrain. Our observation that misexpression of Six3 in the Irx3 domain causes the repression of dT-specific and the activation of vT-specific gene expression demonstrates the key role of Six3 in the induction of anterior forebrain fates. The normal pathway for vT specification, in which expression of Six3 predicates that of Dlx2, involves continual inhibition of the Wnt response, whereas the inductive steps that allow Gbx2 expression in Irx3-positive cells are mediated by Wnts, presumably Wnt3 and Wnt3a. The mechanism by which the interface of Irx3 and Six3 domains becomes the zli is unclear, but indicates the presence of signaling events at this border.
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ACKNOWLEDGMENTS |
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