Neuron, Vol. 35, 255–265, July 18, 2002, Copyright 2002 by Cell Press
Establishment of the Telencephalon during
Gastrulation by Local Antagonism of Wnt Signaling
Corinne Houart,1,3 Luca Caneparo,1
Carl-Philipp Heisenberg,2,4 K. Anukampa Barth,2
Masaya Take-Uchi,2 and Stephen W. Wilson2,3
1
MRC Centre for Developmental Neurobiology
New Hunt’s House
King’s College London
London SE1 9RT
2
Department of Anatomy and Developmental
Biology
University College London
Gower Street
London WC1E 6BT
United Kingdom
Summary
Cells at the anterior boundary of the neural plate (ANB)
can induce telencephalic gene expression when transplanted to more posterior regions. Here, we identify a
secreted Frizzled-related Wnt antagonist, Tlc, that is
expressed in ANB cells and can cell nonautonomously
promote telencephalic gene expression in a concentration-dependent manner. Moreover, abrogation of
Tlc function compromises telencephalic development.
We also identify Wnt8b as a locally acting modulator
of regional fate in the anterior neural plate and a likely
target for antagonism by Tlc. Finally, we show that tlc
expression is regulated by signals that establish early
antero-posterior and dorso-ventral ectodermal pattern. From these studies, we propose that local antagonism of Wnt activity within the anterior ectoderm is
required to establish the telencephalon.
Introduction
The telencephalon is the most complex region of the
brain, and although significant progress has been made
in elucidating the cellular and molecular interactions that
underlie its regional patterning, little is known of the
signals that induce this structure (Monuki and Walsh,
2001; Wilson and Rubenstein, 2000). Studies in several
species have suggested that a necessary initial step in
the establishment of all anterior neural plate fates (which
include telencephalon, diencephalon, and eyes) is the
inhibition of Bmp activity through the action of Bmp
antagonists emanating from the organizer/node (Wilson
and Edlund, 2001). In support of this idea, mice lacking
activity of the extracellular Bmp antagonists Chordin
and Noggin lack forebrain (Bachiller et al., 2000), while
fish with reduced Bmp activity have expanded anterior
neural fates (Barth et al., 1999; Nguyen et al., 1998).
3
Correspondence: corinne.houart@kcl.ac.uk (C.H.), s.wilson@ucl.
ac.uk (S.W.W.)
4
Current address: MPI for Molecular Cell Biology and Genetics,
Pfotenhauerstrasse 108, 01307 Dresden, Germany.
However, graded Bmp activity influences the establishment of ectodermal fates at all head and trunk levels of
the neural axis (Barth et al., 1999), and so it remains
uncertain if this pathway has any specific role in establishment of anteroposterior (AP) identity of induced neural tissue.
Forebrain size is also increased in fish embryos lacking Nodal activity (Sirotkin et al. 2000; Gritsman et al.,
1999), and while it remains possible that Nodal signals
directly inhibit forebrain development (Thisse et al.,
2000), it is more likely that Nodal activity regulates other
signaling pathways that subsequently influence AP and
dorso-ventral (DV) partitioning of the prospective neuroectoderm (Erter et al., 2001). Among the signals potentially regulated by Nodal activity are Fgfs, retinoic acid,
and Wnts, all of which have all been shown to promote
the development of posterior neural tissue at the expense of anterior neural plate fates (Gamse and Sive,
2000; Niehrs, 1999; Yamaguchi, 2001). Overall, for forebrain development to occur, anterior regions of the gastrula embryo must be shielded from the influence of
various posteriorizing signals emanating from more caudal regions of the embryo (Stern, 2001).
Of the signaling pathways that influence early AP patterning, the Wnt pathway is potentially the most critical
for the establishment of the earliest subdivisions of the
neural plate (Yamaguchi, 2001). For instance, an everincreasing number of studies are implicating interplay
between Wnts and Wnt antagonists as being crucial to
the establishment of the vertebrate head. Among the
most studied proteins in this context are Wnt8 and the
extracellular Wnt antagonists Dickkopf-1 (Dkk1), Frzb1,
and Cerberus. Forebrain is absent both in mice lacking
Dkk1 function (Mukhopadhyay et al., 2001) and in fish
lacking activity of Tcf3/Headless, a transcriptional repressor of Wnt target genes (Kim et al., 2000). Conversely, fish in which Wnt8 activity is abrogated have
enlarged forebrains, while more caudal neural tissue is
reduced or absent (Erter et al., 2001; Lekven et al., 2001).
All of these genes function in mesodermal or endodermal territories from the onset of gastrulation or earlier
and may contribute to the formation of a gradient of Wnt
activity (Kiecker and Niehrs, 2001) that helps establish
initial AP subdivisions of the entire neural plate (Yamaguchi, 2001).
Subsequent to the initial subdivision of the neural
plate, each territory undergoes further regional patterning. For instance, in the hindbrain, a cascade of
genetic interactions leads to the establishment of segmentally organized rhombomeres (Lumsden, 1999), and
at the boundary between hindbrain and midbrain, an
organizing center (MHB) patterns adjacent midbrain and
cerebellar structures through the activity of Wnts, Fgfs,
and probably other signals (Joyner et al., 2000). More
rostrally, the anterior neural plate becomes regionally
subdivided into telencephalon, hypothalamus, diencephalon, and eyes—a process that is poorly understood. Ablation studies in mice and zebrafish have, nevertheless, shown that cells located at the margin of the
neural plate (ANB) play an important role in telencephalic
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induction and patterning (Houart et al., 1998; Shimamura
and Rubenstein, 1997). In vivo ablation of these cells
during gastrulation in zebrafish leads to a failure in induction of telencephalic gene expression and subsequent widespread cell death in the forebrain (Houart et
al., 1998). The localization of fgf8 transcripts within the
ANB and the ability of exogenous Fgf8 to induce telencephalic gene expression in forebrain explants raised the
possibility that Fgf8 is an endogenous inducer of the
telencephalon (Monuki and Walsh, 2001; Shimamura
and Rubenstein, 1997). However, loss-of-function studies suggest roles for Fgf signals in telencephalic patterning rather than in telencephalic induction (Meyers
et al., 1998; Shanmugalingam et al., 2000; Shinya et al.,
2001).
Recent results have suggested that in addition to roles
in promoting posterior neural fates and regulating development of tissue adjacent to the MHB, the Wnt pathway
may also be involved in regional patterning within the
anterior neural plate. Zebrafish masterblind (mbl) mutant
embryos show a transformation of telencephalon and
eyes to more caudal diencephalon (Heisenberg et al.,
1996; Masai et al., 1997). As the mbl mutation alters
activity of Axin1, a negative intracellular regulator of
Wnt signaling (Heisenberg et al., 2001), the forebrain
phenotype is likely due to overactivation of the Wnt
pathway (Heisenberg et al., 2001; van de Water et al.,
2001). Early Wnt signaling appears to be unaffected by
the mbl mutation, and so the forebrain fate transformation may be due to altered Wnt activity within the anterior
neural plate itself. The ability of anterior neural plate
cells to respond to Wnt signals (Heisenberg et al., 2001;
Kiecker and Niehrs, 2001) provides further support for
the possibility that locally acting Wnt signals may pattern
this region of the neural plate.
In order to identify secreted proteins involved in the
establishment of telencephalic identity, we have begun
to characterize molecules responsible for the signaling
properties of the ANB. We show here that a novel secreted Frizzled-related protein (sFRP), Tlc, is a component of ANB signaling activity. sFRPs are proteins that
can bind to, and antagonise the activity of, Wnt molecules, presumably by inhibiting receptor/ligand interactions (Wang et al., 1997). Misexpression experiments
have suggested roles for sFRPs in various Wnt-dependent developmental processes such as axial patterning
(Leyns et al., 1997) and heart formation (Marvin et al.,
2001; Schneider and Mercola, 2001), but in vivo requirements for individual sFRP family members have yet to
be determined. We show here that tlc is expressed in
the ANB and that Tlc can mimic ANB function. Moreover,
loss of Tlc function impairs the induction of the telencephalon. We show that the requirement for inhibition
of Wnt activity in the ANB is at least in part due to
the presence of a source of Wnt molecules within the
diencephalic and mesencephalic regions of the neural
plate. We propose that induction of the telencephalon
and partitioning of the prospective forebrain into telencephalic, eye, and diencephalic domains is accomplished through graded modulation of Wnt signaling
within the anterior neural plate.
Results
Tlc Is a Novel sFRP Expressed at the Anterior
Margin of the Neural Plate
In zebrafish, ablation of cells from the ANB during gastrulation results in a loss of telencephalic gene expression (Houart et al., 1998). To elucidate the molecular
basis of ANB activity, we performed a PCR-based subtractive hybridization screen to isolate genes expressed
in the ANB. Through this screen, we isolated a gene
encoding a member of the sFRP family (Wodarz and
Nusse, 1998), named tlc, related to mammalian sfrp5
and sfrp1 (Figure 1A). tlc is expressed from early gastrulation in the anterior neural plate, and over time, expression refines to a domain around the anterior margin
of the neural plate (Figures 1B and 1C) that includes
prospective telencephalic cells (Varga et al., 1999). By
mid-somite stages, expression is undetectable. tlc is
therefore expressed at the right time and place to be
involved in the establishment of telencephalic fates in
the anterior neural plate.
Tlc Can Restore Telencephalic Gene Expression
in ANB-Ablated Embryos
To determine if Tlc contributes to the activity of the ANB,
we first assessed whether Tlc could rescue telencephalic development in embryos in which ANB cells had
been ablated. Telencephalic emx1 (n ⫽ 47/54; Figure
1F), BF1 (n ⫽ 35/38; Figure 1G), and fgf8 (data not shown)
expression was restored or even expanded in embryos
in which ANB cells were replaced with tlc-expressing
cells. At later stages, rescued embryos did not show
the extensive neural cell death characteristic of ANBablated embryos (data not shown). Transplants of control cells gave no such rescue (data not shown). Similarly, the ability of ANB cells to nonautonomously induce
telencephalic gene expression in more posterior regions
of the neural plate is matched by tlc-expressing cells
(n ⫽ 40/59 for emx1, n ⫽ 26/26 for BF1; Figures 1H–1K).
The ability of both ANB and tlc-expressing cells to
promote ectopic telencephalic gene expression raised
the possibility that they might concomitantly suppress
other neural plate fates. To test this, we assessed the
consequences of ANB and tlc-expressing cell transplants upon expression of pax2.1/noi, a marker of prospective midbrain (Brand et al., 1996), and of fgf8/ace,
a gene expressed both in anterior hindbrain/MHB and
in the ANB (Reifers et al., 1998; Shanmugalingam et al.,
2000). Both ANB cells and tlc-expressing cells nonautonomously inhibited pax2.1 expression (n ⫽ 23/27; Figures 1M and 1O) but had no inhibitory effect on fgf8
(n ⫽ 32/36; Figures 1L and 1N), a marker common to
both prospective telencephalon and anterior hindbrain.
Together, these results indicate that tlc-expressing cells
possess similar telencephalon-promoting and midbrainsuppressing properties as ANB cells.
Tlc and Wnts Have Opposite Effects When
Expressed in the ANB
The affiliation of Tlc to the sFRP gene family suggests
that Tlc-mediated antagonism of Wnt signaling contributes to the induction of the telencephalon. To determine
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257
Figure 1. Tlc Activity Promotes Telencephalic Identity
(A) Similarity tree for tlc compared to other
vertebrate sFRP-encoding genes.
(B–O) Dorsal views of bud-1 somite stage embryos (except [B] and [C]) with anterior to the
left. In this and other figures, where indicated,
stage is shown bottom left, genes analyzed
by in situ hybridization are shown bottom
right, and the experimental procedure is top
right. (B and C) tlc expression. (D) wnt8b expression. Prechordal plate (asterisk) expresses a low level of wnt8b. (E) tlc (black)
and wnt8b (red) expression. The white line
outlines the neural plate. (F and G) Embryos
in which the ANB (white arrowheads) was ablated and replaced with tlc-expressing cells
and analyzed for emx1 or BF1 expression. In
this and other panels, grafted cells are brown.
(H–O) Embryos in which ANB cells (ANB⫹) or
tlc-expressing cells (tlc⫹) were transplanted
to more posterior regions of the neural plate
and analyzed for expression of various genes
(bottom right). flh expression marks the axis
in (M) and (O). Abbreviations: d, diencephalon; ey, eye field; m, midbrain; t, telencephalon.
if this is the case, we transplanted cells expressing extracellular agonists or antagonists of Wnt activity into
the ANB of wild-type host embryos and assessed the
consequences upon telencephalic gene expression.
Transplantation of wnt1- (or wnt8, data not shown) expressing cells in the ANB of wild-type hosts led to nonautonomous inhibition of emx1 and fgf8 expression (n ⫽
23/26; Figures 2A and 2B). Complementing this, expansion of pax2.1 expression was observed in embryos with
grafts expressing high levels of Wnts (n ⫽ 37/37; Figures
2C and 2D). The expression of Wnt proteins in the ANB
therefore has the opposite effect of Tlc, inhibiting telencephalic and promoting midbrain-specific gene expression. To confirm that Tlc is likely to act as an inhibitor
of Wnt signaling, we transplanted cells expressing dominant-negative forms (Hoppler et al., 1996) of Wnt molecules (DNwnt8 or DNwnt1) into the ANB. As with ANB
cells and tlc-expressing cells, DNwnt8-expressing cells
induced fgf8 and emx1 (n ⫽ 17/22; Figures 2E and 2F).
Together, these results indicate that the telencephaloninducing property of the ANB is mimicked by antagonists of Wnt activity and that Tlc is likely to be an endogenous antagonist involved in this process.
Tlc May Regulate the Relative Sizes
of the Different Forebrain Areas
The ability of extracellular activators or inhibitors of Wnt
signaling to nonautonomously influence telencephalic
gene expression raises the possibility that local modulation of Wnt activity may influence early regional patterning of the anterior plate, for instance, by regulating
the size of the prospective telencephalon. To begin to
address this possibility, we replaced ANB cells with cells
expressing increasing levels of tlc mRNA, or we transplanted increasing numbers of tlc-expressing cells (Figures 2G–2L, and data not shown). In both situations,
presumed higher levels of Tlc activity led to an expansion of fgf8 (n ⫽ 41/41; Figures 2G, 2I, and 2K) and to
a lesser extent emx1 (n ⫽ 36/41; Figures 2H, 2J, and
2L) into more medial neural plate territories that would
normally express eye-field markers (Varga et al., 1999).
Expansion of telencephalic gene expression occurred
only within the confines of the neural plate and not laterally or anteriorly into nonneural ectoderm. The size of
the prospective telencephalon relative to other forebrain
domains can therefore be changed by varying the levels
of Wnt signaling within the anterior neural plate. This
may be a way to generate diversity in vertebrate forebrain organization.
Abrogation of Tlc Function Affects Formation
of the Telencephalon
The results above indicate that Tlc activity is sufficient
to induce telencephalic gene expression, but do not
address the endogenous requirement of the gene. To
assess if abrogation of Tlc activity affects formation of
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Figure 2. Tlc Acts in a Concentration-Dependent Fashion as a Wnt
Antagonist
Dorsal views of bud-1s stage embryos ([C] and [D] are slightly older)
with anterior to the left.
(A–F) Control embryos or embryos in which cells expressing wnt1
(⫹wnt1) or dominant-negative wnt8 (⫹DNwnt8) were transplanted
into the ANB.
(G–L) Embryos in which ANB cells were ablated and replaced by
cells expressing increasing levels of tlc mRNA.
the telencephalon, we injected antisense morpholinos
(MO) (Nasevicius and Ekker, 2000) against the 5⬘ sequence of the tlc mRNA (see Experimental Procedures
and Figure 7 for controls). Injections of increasing levels
of tlcMO led to progressive loss of telencephalic emx1,
BF1, and fgf8 expression in 80%–90% of injected embryos (Figures 3C–3H). By mid-somite stage, there was
some recovery of expression (Figures 3I and 3J), suggesting either that Tlc activity does not underlie the
entire telencephalon inducing capacity of ANB cells
(emx1 expression does not recover following ANB ablation) and/or that the MOs did not completely block Tlc
translation. Transplantation of tlc-expressing cells or
ANB cells into the ANB of tlcMO-injected embryos rescued the early induction of emx1 expression (n ⫽ 14/
17; Figures 3A and 3B). At pharyngula stage, tlcMOinjected embryos (n ⫽ 37/41) possessed a reduced forebrain with poorly differentiated telencephalon (Figure
3L). These experiments suggest that endogenous Tlc
activity is required for telencephalic development.
Telencephalic Fates Can Be Restored in mbl
Mutant Embryos by Abrogation of Wnt
Signaling in the Prospective Diencephalon
To further investigate the role that modulation of Wnt
signaling plays in formation of the telencephalon, we
Figure 3. Tlc Is Required for Telencephalic Development
(A–J) Dorsal views of bud (A–H) and five somite stage (I and J)
embryos. (A) Embryo injected with a 25 pg of tlcMO into which
cells expressing tlc were transplanted to the ANB. emx1 is widely
expressed. (B) Embryo injected with 25 pg of tlcMO into which wildtype ANB cells were transplanted. emx1 expression is similar to
controls (see Figure 1). (C–L) Embryos injected with increasing concentrations of tlcMO. (C–J) A high level of MO leads to the absence
of emx1, BF1, and telencephalic fgf8 expression at early stages and
reduced expression at later stages. (K and L) At 1 day, forebrain
size (lateral views, double headed arrow) and telencephalic neural
differentiation (arrowhead) remain considerably reduced compared
to controls.
made use of embryos that carry a mutation in the Wnt
pathway scaffolding protein, Mbl/Axin1 (Heisenberg et
al., 2001). mbl⫺/⫺ embryos show a reduction or absence
of prospective telencephalon and eyes and an expansion of posterior diencephalic fates (Heisenberg et al.,
2001). Given the role of Axin as an intracellular negative
regulator of Wnt signaling (Ikeda et al., 1998), the fate
changes in mbl⫺/⫺ embryos have been interpreted as
being a consequence of increased Wnt activity (Heisenberg et al., 2001; van de Water et al., 2001). In support
of the possibility that this increase in Wnt activity occurs
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Figure 4. Suppression of Wnt Signals Emanating from the Diencephalon Promotes Telencephalic Development
(A–C) Lateral views of forebrains of a wildtype embryo with control transplanted cells
(A), a mbl⫺/⫺ embryo (B), and a wild-type embryo in which wnt1-expressing cells were
transplanted into the prospective diencephalon during gastrulation (C).
(D) Dorsal view of bud stage wild-type embryo in which ANB cells were replaced with
ANB cells from a mbl⫺/⫺ embryo. emx1 expression, which is normally absent, is restored.
(E and F) Dorsal views of wnt8b expression
in a bud stage mbl⫺/⫺ embryo (E) and in a
mbl⫺/⫺ embryo in which tlc-expressing cells
were transplanted into the ANB (F).
(G and H) Lateral views of bud stage control
embryo (G) and embryo injected with
wnt8bMO, showing expansion of emx1 expression (H).
(I) Dorsal view of a pharyngula stage mbl⫺/⫺
embryo in which wild-type cells were transplanted into the prospective diencephalon
during gastrulation, restoring telencephalic
emx1 expression. Asterisk shows rescued
eye (which is primarily composed of wild-type
cells).
(J and K) Lateral views of a bud stage mbl⫺/⫺
embryo in which cells in the posterior forebrain were ablated (J) and a mbl⫺/⫺ embryo
injected with wnt8b⫹wnt1MOs (K), both
showing restoration of emx1 expression.
(L) Dorsal view of a pharyngula stage mbl⫺/⫺
embryo in which tlc-expressing cells were transplanted in the prospective diencephalon during gastrulation, restoring emx1 expression.
(M–O) Lateral views of forebrains of a pharyngula stage wild-type embryo (M), a mbl⫺/⫺ embryo (N), and a mbl⫺/⫺ embryo injected with
wnt8b⫹wnt1MOs (O), stained with anti-Hu antibody, which recognizes neuronal cell bodies. Arrowheads show telencephalic neurons (which
are absent in the mbl⫺/⫺ embryo). Abbreviations: e, epiphysis; t, telencephalon.
within the neural plate, transplantation of cells expressing Wnt1 (or Wnt8) into the prospective forebrain of wildtype embryos resulted in a mbl-like phenotype in which
telencephalon and eyes were reduced or absent (Figures 4A–4C). Disrupted ANB signaling itself is, however,
unlikely to significantly contribute to the increase in Wnt
activity (and loss of telencephalic fates) in mbl⫺/⫺ embryos, as tlc is expressed during gastrulation in those
embryos (data not shown), and mbl⫺/⫺ ANB cells can
rescue emx1 expression when transplanted into wildtype ANB-ablated embryos (n ⫽ 19/19; Figure 4D). These
observations suggest that the activity of Wnts in the
anterior neural plate is negatively regulated both by extracellular proteins such as Tlc and intracellular proteins
such as Mbl/Axin1.
A requirement for Tlc and Axin1 to induce telencephalon implies a source of Wnt proteins capable of repressing telencephalic fate in the anterior neural plate. If neural tissue caudal to the ANB is a source of Wnt proteins,
we reasoned that interfering with the production, movement, or reception of these signals by transplanting or
ablating cells posterior to the prospective telencephalon
in mbl⫺/⫺ embryos might suppress Wnt signaling and
alleviate the mbl⫺/⫺ telencephalic phenotype. We therefore either ablated posterior forebrain precursors (Figure
4J) or transplanted wild-type neural plate cells (Figure
4I) or tlc-expressing cells (Figure 4L) into the prospective
forebrain of mid-gastrula stage mbl⫺/⫺ embryos. All pro-
cedures led to at least partial restoration of emx1 and
telencephalic fates in mbl⫺/⫺ embryos. One interpretation of these results is that both the ablation procedure
and the insertion of wild-type cells interferes with the
production of Wnt signals in the prospective posterior
diencephalon or with the movement of Wnt proteins
into the prospective telencephalic region of the anterior
neural plate.
Wnt8b Is a Posteriorizing Signal Expressed in
Prospective Posterior Forebrain and Midbrain
To test the notion that establishment of telencephalic
gene expression requires antagonism of Wnts emanating from more posterior regions of the anterior neural
plate, we abrogated the activity of candidate Wnt proteins. wnt1 (Kelly and Moon, 1995) and wnt8b (Kelly et
al., 1995) are both expressed in the prospective caudal
diencephalon and midbrain (Figures 1D and 1E, and data
not shown) and so are good candidates to be involved in
the repression of telencephalic identity. Indeed, wnt8b
is ectopically expressed in the anterior neural plate of
mbl⫺/⫺ embryos (compare Figure 4E to Figure 1D), suggesting that increased Wnt8b activity may contribute
to the loss of telencephalic fate in these embryos. To
address the roles of Wnt8b and Wnt1, we used MOs
to abrogate their activity in vivo. Although injection of
wnt1MO had no obvious effect on telencephalic development (data not shown), injection of wnt8bMO alone
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or wnt8b plus wnt1MOs in wild-type and mbl⫺/⫺ embryos
expanded or restored emx1 expression, respectively
(Figures 4H and 4K). At later stages, wnt8bMO injections
led to restoration of telencephalic neurogenesis in
mbl⫺/⫺ embryos (Figures 4M–4O). Finally, transplantation
of tlc-expressing cells into mbl⫺/⫺ embryos led to suppression of ectopic wnt8b expression (Figure 4F) and
restoration of telencephalic fates (data not shown), indicating that Tlc and Axin may also indirectly modulate
Wnt activity through the regulation of other genes in the
Wnt pathway. Together, our experiments show that the
consequences of reduction of endogenous Wnt8b activity within the neural plate are similar to those observed
when Tlc activity is increased. This supports the hypothesis that local Wnt agonist/antagonist interactions control the induction and extent of the prospective telencephalon within the neural plate.
tlc Expression Is Induced between Thresholds
of Bmp Activity and Can Restore Telencephalic
Fates to Bmp-Depleted Embryos
Previous studies have indicated that inhibition of both
Bmp and Wnt signals promotes forebrain fates (Glinka
et al., 1997). Severe abrogation of Bmp activity in vivo
leads to a massive increase in the size of the prospective
forebrain (due to conversion of anterior nonneural ectoderm to neural plate; Figures 5A and 5B), but within the
enlarged forebrain, telencephalic fates are reduced or
absent (Barth et al., 1999) (Figures 5C and 5E). Our interpretation of this result is that, as with other DV domains
of the neural plate, the telencephalon can only be established between certain thresholds of Bmp activity (Barth
et al., 1999).
To explore this hypothesis, we examined the relationship between Bmp signaling and Tlc expression and
activity. As expected, tlc expression was severely reduced in embryos with abrogated Bmp signaling (Figure
5I). Increased Bmp activity in chordino⫺/⫺ mutants leads
to reduction of the neural plate, but within the reduced
neural plate, tlc is still expressed at the anterior margin
(Figure 5H), and telencephalic tissue is present. This
again suggests that tlc expression is induced between
thresholds of Bmp activity that are present at the margin
of the neural plate. To directly test this, we altered the
levels of Bmp signaling at the margin of the neural plate
through transplantation of noggin-expressing cells. As
predicted, tlc expression was inhibited where Noggin
activity was highest, while it was ectopically induced in
cells that would have become nonneural ectoderm at a
distance from the graft, presumably due to lowered Bmp
activity in these cells (n ⫽ 19/23; Figure 5K). Thus, Noggin can induce ectodermal cells to become neural, and
within anterior regions, cells at the interface between
neural and nonneural tissue express tlc and hence acquire the ability to induce telencephalic identity. Transplants of noggin⫹ cells into the neural plate had no effect
on tlc expression, fitting the prediction that Bmp activity
is already below the threshold for induction of tlc in this
region (n ⫽ 12/12; Figure 5L). Together, these results
indicate that tlc expression is induced within the anterior
margin of the neural plate between thresholds of Bmp
activity.
The reduction/loss of tlc expression in embryos with
Figure 5. tlc Expression Is Regulated by Bmp Signaling, and Tlc
Can Restore Telencephalic Fates to Bmp-Depleted Embryos
(A and B) Lateral views of wild-type and swirl (swr⫺/⫺) 70% epiboly
embryos. Dorsal is to the right and anterior is up. Expression of the
prospective forebrain marker anf is expanded to the ventral side of
the ectoderm in the swr⫺/⫺ embryo. These data are modified from
Barth et al. (1999).
(C–F) emx1 and telencephalic fgf8 expression (arrows) in noggininjected embryos (C and E) and in noggin-injected embryos with
tlc-expressing cells transplanted to the animal pole (D and F). Expression of both markers is severely reduced following noggin injection (similar to swr⫺/⫺ embryos) and is restored by Tlc. fgf8 expression is shown in lateral view so that posterior expression does not
mask the telencephalic signal.
(G–I) tlc expression in wild-type, chordino mutant, and noggininjected embryos.
(J–L) tlc expression in embryos with a control transplant (J), with a
transplant of noggin-expressing cells to the margin of the neural
plate (K), and with a transplant of noggin-expressing cells within
the neural plate (L). Arrowheads indicate induction of ectopic tlc
expression in cells close to the margin of the neural plate.
compromised Bmp activity may contribute to the failure
of telencephalic specification in the enlarged forebrains
of these embryos. To assess if this is the case, we
transplanted tlc-expressing cells into embryos with abrogated Bmp activity. This led to robust restoration of
telencephalic emx1 and fgf8 expression in all cases (Figures 5D and 5F), supporting the hypothesis that Tlc
imparts telencephalic identity to anterior neural plate
cells and that the loss of Tlc in Bmp-depleted embryos
contributes to the loss of telencephalic fates.
Early Direct or Indirect Manipulations of Wnt
Activity Alter Expression of tlc and wnt8b
Fish mutants with altered expression of Wnt8 have enlarged or reduced heads, depending on the levels of
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261
Figure 6. Alterations in Expression of tlc and
wnt8b in Embryos with Direct or Indirect Alterations in Early Wnt Activity
Dorsal views of bud stage wild-type embryos
(A and E), MZoep (lacking maternal and zygotic Oep activity) embryos (B and F), or embryos injected with dkkMO (C and G) or
wnt8MO (D and H). Genes analyzed are indicated (bottom right).
Wnt8 activity (e.g., Lekven et al., 2001; Erter et al., 2001).
One possibility is that Wnt8 and its antagonists not only
regulate head formation but also mediate regional patterning and inductive events within the prospective forebrain. However, given the distance between the telencephalic anlage and the source of Wnt8 in the involuting
mesendoderm, a more attractive hypothesis is that alterations to early Wnt activity directly or indirectly lead to
changes in the expression of genes that subsequently
act locally within the neural plate to effect regional patterning. To address if this may be the case, we assessed
expression of tlc and wnt8b in embryos with alterations
in early Wnt activity.
Zebrafish embryos lacking Nodal signaling have enlarged heads as a consequence of reduced Wnt8 activity
(Erter et al., 2001). To assess whether this Nodal pathway-dependent reduction in Wnt8 signaling is likely to
affect the activity of locally acting Wnt pathway genes
in the anterior neural plate, we assessed expression of
tlc and wnt8b in Nodal-depleted embryos. We observed
dramatic alterations in expression of both genes, with
tlc expressed in a broad anterior domain of the neural
plate and wnt8b reduced (Figures 6B and 6F). Thus,
although early Wnt8 activity is altered in Nodal-depleted
embryos (Erter et al., 2001), it is likely that the consequent alterations in Tlc and Wnt8b activity contribute to
the expansion of telencephalic fates. More direct interference with early acting Wnt pathway genes through
the use of Dkk or Wnt8 MOs also led, respectively, to a
reduction or expansion of tlc expression and a concomitant rostral or caudal shift in wnt8b expression (Figures
6C, 6D, 6G, and 6H). All together, these observations
indicate that although manipulations or mutations that
alter the activity of early acting Wnt pathway genes can
influence forebrain size (including telencephalon), aspects of this phenotype may be an indirect effect of
altering the activity of later acting genes functioning
locally within the anterior neural plate.
Discussion
Previous studies have shown that cells at the anterior
margin of the neural plate produce signals both necessary and sufficient to induce telencephalic gene expression. Here, we identify the novel Wnt antagonist Tlc as
a component of the activity of the ANB. Tlc is expressed
within the ANB, can mimic its inductive abilities, and is
required for telencephalic development. We show that
local antagonism of Wnt signaling within the anterior
neural plate can result in ectopic induction of telencephalon in wild-type embryos and restoration of telencephalic identity both in ANB-ablated embryos and in
mbl⫺/⫺ embryos. Furthermore, we provide evidence that
the posterior diencephalon is a likely source of Wnt
ligands, including Wnt8b, which antagonize telencephalic specification. All together, these data provide
compelling evidence that telencephalic identity is established through local suppression of Wnt signaling in the
anterior neural plate during late gastrulation.
Tlc Is a Novel sFRP Required for Establishment
of the Telencephalon
Sequence analysis shows Tlc to have its greatest homology to mammalian sFRP1 and sFRP5. However, there
is currently insufficient data to assess whether Tlc is a
true ortholog of either of these genes. There has been
relatively little expression analysis of sFRP-encoding
genes, and of those analyzed in other species, none
shows patterns similar to tlc. Tlc can function as a bona
fide Wnt antagonist, as can several other sFRP family
members (e.g., Wang et al. 1997; Leyns et al., 1997).
This is evident from assays in which we have shown
that other extracellular antagonists of Wnt signaling
mimic the activity of Tlc and from the ability of Tlc to
produce embryonic phenotypes (enlarged heads) characteristic of reduced Wnt activity when ubiquitously
overexpressed.
In gain-of-function assays, Tlc is able to reproduce
the ability of ANB cells to nonautonomously induce telencephalic and inhibit mesencephalic gene expression.
This suggests that endogenous Tlc activity is likely to
contribute significantly to the organizer activity of the
ANB cells. Although we have yet to identify a genetic
loss-of-function mutation in the tlc gene, the tlc morphant phenotype provides evidence in support of an
endogenous requirement for Tlc in the establishment of
telencephalic identity. Unlike in embryos in which ANB
cells are ablated, there is some later recovery of telencephalic gene expression in tlc morphants, perhaps most
likely because other genes also contribute to the induction of telencephalic identity.
The Prospective Diencephalon Is a Source of Wnt
Proteins that Inhibit Telencephalic Development
The requirement of an sFRP within the anterior margin
of the neural plate implies a source of Wnt proteins
Neuron
262
Figure 7. tlc and wnt8b Morpholinos Abrogate Tlc and Wnt8b Activity
(A and B) Control 60% epiboly and prim5 stage embryos.
(C–R) Columns 1 and 3 show lateral views of gastrula stage embryos injected with RNA encoding GFP-tagged Tlc and increasing levels of
tlcMO (column 1), or with RNA encoding GFP-tagged Wnt8b and increasing levels of wnt8bMO (column 3). Increasing levels of the MOs inhibit
translation of the tagged proteins, resulting in decreasing levels of fluorescence. Columns 2 and 4 show lateral views of the heads of 24 hr
embryos resulting from the experiments shown in columns 1 and 3. High levels of exogenous Tlc activity enlarge brain and eyes, while high
levels of tlcMO lead to reduced eyes (and telencephalon). High levels of exogenous Wnt8b result in loss of eyes, while higher levels of
wnt8bMO lead to increased eye size.
capable of antagonizing telencephalic development. We
have presented several lines of evidence implicating
the anlage of the posterior diencephalon as a site of
production of Wnt ligands that influence anterior neural
plate patterning. First, we have suggested that the loss
of telencephalon in mbl⫺/⫺ embryos is due to local overactivation of Wnt signaling within the anterior neural
plate. Telencephalic fates can be restored in mbl⫺/⫺ embryos by ablation of posterior diencephalic cells or
transplantation of tlc-expressing cells into the posterior
diencephalon. We predict that both of these procedures
would limit the amount of Wnt protein moving from posterior diencephalon into the anterior neural plate. Second, we show that wnt8b is expressed in the prospective
posterior diencephalon from mid/late stages of gastrulation. Abrogation of Wnt8b activity in mbl⫺/⫺ embryos
restores telencephalic fates, implying that excessive
Wnt8b activity contributes to the loss of telencephalic
fates in mutant embryos. Similarly, abrogation of Wnt8b
activity in wild-type embryos leads to expansion of the
telencephalon, implying that endogenous Wnt8b activity
may limit the extent of telencephalic tissue. All together,
these results imply that Wnt8b (and perhaps other Wnts)
are produced in the posterior diencephalon and move
into the anterior neural plate where their activity is antag-
onized or modulated by other components of the Wnt
pathway, such as Tlc. This suggests an organizing role
for the diencephalic anlage during gastrulation, and so,
early brain patterning may involve complex interactions
between three organizing centers (the ANB, MHB, and
a domain within the diencephalon).
Graded Wnt Signaling May Mediate the Regional
Subdivision of the Anterior Neural Plate into
Telencephalon, Eye Field, and Diencephalon
In addition to affecting the specification of the telencephalon, local levels of Wnt activity affect eye specification. In mbl⫺/⫺ embryos, eyes are lost, presumably due
to overactivation of Wnt signaling within the prospective
eye field (Heisenberg et al., 2001). However, here we
have also shown that enhanced Tlc activity in the anterior margin of the neural plate can expand telencephalic
gene expression into the prospective eye field, in this
case presumably by lowering the level of Wnt activity.
Together, these results raise the possibility that eye-field
fates are specified by a level of Wnt signaling intermediate
between that which promotes posterior diencephalon
and that found in the prospective telencephalon. Although this simple gradient model of Wnt activity in the
anterior neural plate is attractive, it may be an oversim-
Telencephalon Formation by Local Wnt Antagonism
263
plification, as some observations still lack explanation.
For instance, abrogation of Wnt8b activity restores telencephalon but not eyes to mbl⫺/⫺ embryos, and indeed,
there appears to be a cell autonomous requirement for
wild-type Axin1/Mbl in the eye fields (Heisenberg et al.,
1996) but not elsewhere in the forebrain. Finally, recent
studies in Xenopus have suggested that Frizzled3-mediated Wnt signaling promotes, rather than inhibits, eye
development (Rasmussen et al., 2001). All together,
these results suggest a complex, and as yet poorly understood, role for the Wnt pathway in eye development.
Forebrain Fates and Bmp Signaling
Our data suggest that tlc is expressed between thresholds of Bmp activity found at the rostral margin of the
neural plate. Severe abrogation of Bmp activity leads to
a loss of tlc expression, as has been observed for other
marginal neural plate markers at all head and trunk levels
(Barth et al., 1999; Nguyen et al., 1998). This loss of
tlc expression may explain why telencephalic fates are
reduced in Bmp-depleted embryos despite an overall
increase in the extent of forebrain tissue (Barth et al.,
1999). The robust restoration of telencephalic gene expression in Bmp-depleted embryos by Tlc provides support for this hypothesis. Given these and other results,
it is probably an oversimplification to consider Bmp antagonists to be inducers of the anterior brain. As we
discuss further below, signals that establish initial AP
and DV pattern in the embryo are likely to influence
the regional character of anterior neural plate cells by
regulating the expression of genes, such as tlc, that
locally mediate regional fate determination.
Repeated Use of the Wnt Pathway in AP Patterning
The canonical Wnt signaling pathway is used repeatedly
during establishment of AP and DV pattern in the embryo. There is an initial requirement for ␤-catenin-dependent activation of target genes on the dorsal side of the
embryo. In its absence, organizer formation fails and
embryos develop without heads and other dorsal/anterior structures (Joubin and Stern, 2001). Subsequently,
Wnt8 (and possibly other Wnt proteins) is expressed in
prospective mesendoderm cells. It is thought that this
phase of Wnt activity promotes the development of posterior and ventral tissues and inhibits dorsal and anterior
fates (Sokol, 1999). Indeed, recent studies have suggested that a gradient of Wnt activity may contribute to
the establishment of initial positional values along the
entire AP axis of the neural plate (Kiecker and Niehrs,
2001). Finally, Wnts are implicated in the development
of dorsal neural fates such as neural crest and dorsal
telencephalon (Deardorff et al., 2001; Lee et al., 2000)
and establishment or maintenance of midbrain and cerebellar tissue (Brault et al., 2001). To this list, we now
add evidence that suppression of Wnt signaling is necessary for induction of the telencephalon. Resolving how
these Wnt-dependent patterning events are integrated
both spatially and temporally in the embryo is a major
challenge.
One possibility is that the telencephalon is a default
neural fate, specified at the low point on an early acting
gradient of Wnt activity that assigns AP positional values
to the forming neural ectoderm. There are many argu-
ments against this idea. Alterations in the activities of
the genes (such as dkk, frzb1, wnt8, and cerberus) that
may influence the postulated Wnt gradient all lead to
increases or decreases in the size of the whole forebrain
and not to alterations in induction of specific regional
fates within the forebrain. Indeed, fish embryos possibly
lacking all early posteriorizing signals still possess patterned forebrain tissue consisting of telencephalon, diencephalon, and eyes (Thisse et al., 2000). Second, although it appears that Wnts can have activities at a
distance, it seems unlikely that Wnts expressed in prospective mesendoderm could travel far enough through
the ectoderm to mediate regional patterning within the
anterior neural plate. Third, all of the manipulations that
we describe in this study lead to dramatic alterations in
regional patterning of the anterior neural plate without
affecting early Wnt-dependent patterning events.
Fourth, there is no evidence that telencephalic identity
is established at the time at which early acting Wnt
pathway genes are initially active; rather, the first indications of telencephalon-specific gene expression appear
around the stage that Tlc, Wnt8b, and other locally acting genes are likely to function in the anterior neural
plate. Finally, it is probably incorrect to consider the
telencephalon to be the anteriormost subdivision of the
CNS. The most widely accepted model of CNS regionalization suggests the telencephalon and eyes both to be
derivatives of the alar region of the most anterior CNS
(Redies and Puelles, 2001). Thus, fate selection between
telencephalon and eye field should probably not be regarded as an AP subdivision of the neural plate.
If telencephalic induction is not a direct consequence
of signaling by early acting Wnts and their antagonists,
then an attractive hypothesis is that early Wnt activity
regulates expression of genes that function later and
locally mediate regional patterning of the neural plate.
In such a model, the postulated gradient of early Wnt
activity would divide the prospective neural plate into
broad domains, perhaps forebrain, midbrain, hindbrain,
and spinal cord. One consequence of this would be the
local activation of genes within these specific territories,
and it would be interactions between these induced
genes that regulate subsequent regional patterning. We
have provided evidence in support of such a model by
examining expression of tlc and wnt8b in embryos in
which early Wnt activity is directly or indirectly manipulated. For instance, it is thought that the expansion of
forebrain fates in Nodal-depleted embryos is a consequence of the loss of posteriorizing signals, including
Wnt8, from the prospective mesendoderm (Erter et al.,
2001). In such embryos, there is an increase in tlc expression, suggesting that early alterations in posteriorizing signals lead to later changes in the activity of patterning genes that act locally in the neural plate.
Although we suggest that the consequences of modulating early and later Wnt activity are distinct, this does
not necessarily imply that they are completely discrete
events. Establishment of regional fates in the neural
plate may involve a continuous refinement of positional
values that are influenced by Wnt signals secreted from
various locations and acting at various different stages.
Although several studies have suggested the presence
of a graded nuclear readout of Wnt activity in the ectoderm (Dorsky et al., 2002; Kiecker and Niehrs, 2001),
Neuron
264
much more study is required to dissect the interplay
between agonists and antagonists responsible for mediating such variations in the output of the Wnt pathway.
Over all, we suggest that changes in regional patterning of the anterior neural plate following early manipulation of posteriorizing signals in the embryo may be
a secondary consequence of alterations in the activity
of genes functioning locally within the neural plate.
Given the complexity of expression of genes that can
modulate Wnt activity, it remains a challenge to unravel
how early and later Wnt-dependent signaling events are
integrated both spatially and temporally during formation of anterior regions of the vertebrate brain.
stage Nikon Optiphot microscope. Cells were moved by suction
using a mineral oil-filled glass micropipette attached to a 50 ␮l
Hamilton syringe (Houart et al., 1998).
Experimental Procedures
Bachiller, D., Klingensmith, J., Kemp, C., Belo, J.A., Anderson, R.M.,
May, S.R., McMahon, J.A., McMahon, A.P., Harland, R.M., Rossant,
J., and DeRobertis, E.M. (2000). The organizer factors Chordin and
Noggin are required for mouse forebrain development. Nature 403,
658–661.
Isolation of tlc
A partial fragment of tlc cDNA was isolated from a subtractive library
made between cDNAs from the ANB and more posterior neural plate
cells using the Clontech PCR-Select kit. Full-length tlc cDNA was
isolated by screening an anterior neural plate cDNA library. All antibody and in situ hybridization protocols used standard procedures
(Shanmugalingam et al., 2000).
MO Experiments
MO antisense oligonucleotides (GeneTools) were designed against
25 bases around the AUG of the tlc, wnt1, dkk and wnt8b mRNAs
(sequences available on request). wnt8MO ORF1 was used as previously described (Lekven et al., 2001). Injections of MOs were done
at concentrations between 0.5 and 5 ␮g/␮l. Routine controls involved injection of four nucleotide-modified versions of the MO
tested. We assessed the specificity of the MO phenotypes by testing
whether the phenotype observed could be rescued by expressing
wild-type wnt8b mRNA devoid of the sequence 5⬘ to the AUG or by
transplanting cells expressing tlc. In addition, we confirmed that
tlcMO and wnt8bMO inhibit in vivo translation and activity of GFPtagged versions of the respective proteins (Figure 7).
Overexpression of tlcGFP alone leads to an increase anterior brain
size when injected in one to two cell stage embryos, mimicking the
activity of early acting Wnt antagonists such as Cerberus, Dkk1,
and Frzb1 (Yamaguchi, 2001). Coinjection with tlcMO decreased
fluorescence and led to concentration-dependent rescue of the
TlcGFP overexpression phenotype (Figures 7E–7H). When tlcGFP
RNA was coinjected with a very high amount of tlcMO, the embryos
began to show a decrease in telencephalon and eye size typical of
tlcMO-injected embryos (Figures 7I–7J), indicating that the tlcMO
was reducing both exogenous and endogenous Tlc. Similarly, injection of wnt8bGFP RNA alone induces a loss of anterior brain structures when injected in one to two cell stage embryos (Figures 7K
and 7L), mimicking the effect of overexpression of early acting Wnts
such as Wnt8 (Kelly et al., 1995). Coinjection with wnt8bMO decreased fluorescence and led to concentration-dependent rescue
of the wnt8bGFP overexpression phenotype (Figures 7M–7P). When
wnt8bGFP was coinjected with a very high amount of wnt8bMO,
the embryos began to show an increase in telencephalon and eye
size typical of wnt8bMO injected embryos (Figures 7Q and 7R).
Injections and Transplantations
Injection of tlc mRNA in one to two cell stage donor embryos was
done at low (10 pg/embryo), moderate (50 pg/embryo), and high (100
pg/embryo) doses, and noggin injections were done as previously
described (Barth et al., 1999). For transplantations, donor embryos
were injected with test RNA, GFP-RNA, and the tracer rhodamine
biotinylated dextran shortly after fertilization. Test RNA-expressing
cells were from taken from various random positions in late blastula
donor embryos and transplanted to mid-gastrula stage hosts. Cells
from the margin of the embryo were avoided, as they are a likely
source of endogenous Wnt-signaling molecules. Control transplants
used cells from donor embryos injected with GFP-RNA alone. Ablations and transplantations were performed on embryos mounted in
4% methyl-cellulose in embryo medium and viewed with a fixed
Acknowledgments
We thank many of our colleagues, especially Randy Moon, for providing reagents used in this study. This study was supported by
grants from the Wellcome Trust, MRC, and BBSRC to S.W.W. and
C.H. S.W.W. is a Wellcome Trust Senior Research Fellow.
Received: March 6, 2002
Revised: April 29, 2002
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Accession Numbers
The Genbank accession number for the tlc sequence reported in
this paper is AF520426.