articles
© 2002 Nature Publishing Group http://neurosci.nature.com
Progressive induction of caudal
neural character by graded Wnt
signaling
Ulrika Nordström1, Thomas M. Jessell2 and Thomas Edlund1
1 Department of Molecular Biology, Umeå University, S-901 87 Umeå, Sweden
2 Howard Hughes Medical Institute, Department of Biochemistry and Molecular Biophysics, Columbia University, New York, New York 10032, USA
Correspondence should be addressed to T.E. (Thomas.Edlund@molbiol.umu.se)
Published online: 13 May 2002, DOI: 10.1038/nn854
Early in differentiation, all neural cells have a rostral character. Only later do posteriorly
positioned neural cells acquire characteristics of caudal forebrain, midbrain and hindbrain cells.
Caudalization of neural tissue in the chick embryo apparently involves the convergent actions of
(i) fibroblast growth factor (FGF) signaling and (ii) signaling from the caudal paraxial mesoderm,
or ‘PMC activity’, which has not yet been defined molecularly. Here we report evidence that Wnt
signaling underlies PMC activity, and show that Wnt signals act directly and in a graded manner
on anterior neural cells to induce their progressive differentiation into caudal forebrain, midbrain
and hindbrain cells.
The early development of the vertebrate nervous system is accompanied by the specification of regionally restricted progenitor
cells along the rostrocaudal axis of the neural tube1,2. Studies in
various vertebrates have indicated that cells of caudal neural character are generated through the reprogramming of cells with an
initial rostral character2–4. In chick embryos, this happens during
late gastrulation4,5. The induction of cells of midbrain and rostral
hindbrain character requires FGF signaling4,6,7. Retinoic acid
(RA) signaling, derived from the paraxial mesoderm that flanks
the caudal region of the neural plate, suppresses the generation of
cells of midbrain and rostral hindbrain character while inducing
caudal hindbrain and spinal cord character4,8–10. However, FGF
and RA signaling are not sufficient (alone or together) to induce
these caudal characters in neural cells grown in vitro. This process
requires an additional paraxial mesoderm caudalizing signal11–13
that has been termed PMC activity4,5. The molecular basis of
PMC signaling is not known.
Genes of the Wnt family are expressed in the posterior region
of vertebrate embryos during stages of gastrulation when caudal
neural cells are generated14–16, and several lines of evidence have
implicated Wnt signaling in the specification of caudal neural
character7,13,17–27. Indeed, Wnt signaling is required at several
different stages, and in several different germ layers, during the
early development of vertebrate embryos. During gastrulation,
for example, Wnt signaling is needed to generate caudal nonaxial mesoderm21,28–30: the inactivation of Wnt genes that are
expressed at gastrula stages in mouse and zebrafish embryos leads
to defects in trunk and tail structures28,29,31. At an even earlier
stage, Wnt signaling also helps to establish the anteroposterior
body axis and to initiate gastrulation21,22. Consistent with these
findings, mis-expression of Wnts or downstream components of
the Wnt signaling pathway before the onset of gastrulation leads
to axis duplications and/or other malformations of the embrynature neuroscience • volume 5 no 6 • june 2002
onic body plan (http://www.stanford.edu/%7Ernusse/wntwindow.html). The multiple patterning roles of Wnts at early developmental stages has made it difficult to determine whether the
later Wnt signals implicated in rostrocaudal neural patterning
act directly or indirectly7,13,18–21,24.
In chick embryos, prospective neural tissue can be separated
from the adjacent mesoderm at stages when neural cells normally acquire caudal regional characters. This way, the rostrocaudal
specification of neural cells and their direct responses to putative
patterning signals may be examined. Our findings show that Wnt
signaling is required for the specification of cells of caudal neural character both in neural plate explants and in chick embryos
grown in New culture. Through in vitro studies and New culture
assays, we found that this caudalizing action of Wnts results from
a direct action on neural cells. Graded Wnt signaling, in combination with FGFs, specifies cells of caudal forebrain, midbrain
and rostral hindbrain character. In the absence of Wnt signaling,
caudal neural cells grown in vitro revert to a rostral forebrain
character. Thus we conclude that Wnt signals mediate the PMC
activity necessary for the establishment of caudal neural fates.
RESULTS
Regional expression of Wnts in chick gastrula
Paraxial mesodermal tissue that underlies the prospective caudal neural plate of Hamburger and Hamilton (HH) stage 4 and 5
chick embryos can induce cells of midbrain and hindbrain character at gastrula stages4,5. In considering candidate mediators of
PMC activity, we noted that Wnt8c (ref. 14) and Wnt11 (ref. 32)
are expressed in the posterior region of the chick embryo at a
time when neural cells are exposed to factors that direct their
caudal neural character4. Using in situ hybridization, we found
that, beginning in early stage 4, Wnt8c is expressed transiently,
and Wnt11 at increasing levels, in the caudal paraxial mesoderm
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Fig. 1. Wnt11 and Wnt8c expression in the posterior region of the
chick embryo at developmental stages when neural cell precursors are
exposed to signals that induce caudal neural characters. Expression of
Wnt11 and Wnt8c in HH stage 4 and 5 embryos was monitored by
whole-mount (a, b) and section (c–f) in situ hybridization. (a, b) Black
arrow, Hensen’s node; black line, level of sections in c and d, respectively. (a–d) Wnt11 was expressed in paraxial mesoderm posterior to
Hensen’s node. (e) Wnt8c was expressed in the primitive streak and
transiently in the mesoderm at early stage 4. (f) At early stage 5, Wnt8c
expression was found in lateral plate mesoderm and in the prospective
caudal neural plate. Scale bars, 0.5 mm.
that underlies the prospective caudal neural plate (Fig. 1a–f).
Thus, the combined patterns of expression of Wnt8c and Wnt11
in the caudal paraxial mesoderm mimic the known distribution
of tissues that possess PMC activity5. In addition, from late stage
4 onwards, caudal neural plate cells themselves transiently
expressed Wnt8c (Fig. 1f and ref. 14).
Wnt3A induces Wnt expression
As both Wnt8c and Wnt11 were expressed in the caudal paraxial
mesoderm underlying prospective caudal neural plate (Fig. 1a–e),
and Wnt8c was expressed in prospective caudal neural plate cells
(Fig. 1f), we reasoned that Wnt signals derived from the paraxial mesoderm may induce Wnt expression in neural cells. To test
this idea, we examined whether Wnt signaling induces Wnt8c
expression in neural explants. We included FGF8 in these assays
because FGF signaling is required for caudalization of stage 4
forebrain cells by paraxial mesoderm4. Wnt3A and FGF8 in combination, but not Wnt3A or FGF8 alone, induced the expression
of Wnt8c in stage 4 rostral forebrain (RFB) cells (Fig. 2b). This
result supports the view that Wnts derived from the primitive
streak and caudal paraxial mesoderm are involved in inducing
the expression of Wnt8c in neural plate cells.
Wnt signaling specifies caudal neural character
We next examined whether Wnt signaling participates in the
induction of midbrain and hindbrain character by caudal paraxial mesoderm. The positional character of neural cells was
Fig. 2. Wnt3A and FGF8 in combination induces the expression of
Wnt8c in prospective rostral forebrain cells. (a) Schematic drawing of a
late gastrula, HH stage 4, chick embryo. Dotted line, presumptive neural
plate; boxed region, explant of the prospective neural plate used for
in vitro cultivation and RT-PCR. (b) RT-PCR expression analysis of Wnt8c
and the ribosome protein gene S17 in stage 4 RFB explants exposed to
FGF8 (10 ng/ml), Wnt3A (3×, 75 µl of Wnt3A conditioned medium per
ml of culture medium, ∼75 ng/ml) or a combination of Wnt3A (3×) and
FGF8 (10 ng/ml). (c) Schematic representation of the regional neural
markers used in this study. In a 12 somite stage chick embryo, cells in the
rostral forebrain (RFB, red) expressed Otx2; cells in the caudal forebrain
(CFB, yellow) co-expressed Otx and Pax6; cells in the midbrain (MB,
green) co-expressed En1 and Otx2. Gbx2 was widely expressed in the
rostral hindbrain (RHB, blue) and was co-expressed with Krox20 and
Pax6 in cells in rhombomere 3 (dark blue).
526
assayed by monitoring the profile of expression of cell-specific
transcription factors. The expression of Sox2 and Sox3 (Sox2/3)
was used to define neural cells, regardless of their rostrocaudal
position33. Otx2 is expressed in the rostral neural tube with a
caudal limit at the isthmus34, and its expression in the absence
of Pax6, En1, Krox20 or Gbx2 was used as an indicator of neural cells characteristic of rostral forebrain (RFB) levels (prospective telencephalon)35 (Fig. 2c). Co-expression of Otx2 and Pax6
in the absence of En1, Krox20 or Gbx2 was used to define cells
in the caudal forebrain (CFB) (prospective diencephalon)35,36
(Fig. 2c). Co-expression of Otx2 and En1 was used to define
cells of midbrain (MB) character 37 (Fig. 2c). In 12 somite
embryos, Gbx2 (ref. 38) was expressed in rhombomeres (r) 1–4,
and Krox20 (ref. 39) was co-expressed with Pax6 in r3 of the
hindbrain (data not shown). Thus, expression of Gbx2 and the
co-expression of Krox20 and Pax6 defined cells of rostral hindbrain (RHB) character (Fig. 2c), whereas expression of Krox20
in the absence of Pax6 and Gbx2 defined cells of caudal hindbrain (r5-like) character.
Stage 4 and 5 caudal paraxial mesoderm tissue was cultured
together with stage 3 prospective caudal (stage 3C) neural plate
explants (Fig. 3a,c and f) in the absence or presence of a soluble
fragment of the mouse Frizzled receptor 8 protein (mFrz8CRDIgG), a selective antagonist of Wnt signals40,41. Stage 3C explants
grown alone generated Sox2/3+ and Otx2+ neural cells, a molecular profile characteristic of the rostral forebrain4. Similarly, in
the presence of mFrz8CRD-IgG, stage 3C explants generated
Sox2/3+ and Otx2+ cells, indicative of their maintained rostral
forebrain character (Fig. 3b). Early stage 4 caudal paraxial mesoderm induced Otx2+/En1+ cells of midbrain character in stage
a
b
c
nature neuroscience • volume 5 no 6 • june 2002
© 2002 Nature Publishing Group http://neurosci.nature.com
articles
Fig. 3. The induction of midbrain and a
hindbrain cells by caudal paraxial mesob
derm requires Wnt signaling. (a, c, f)
Schematic drawings of HH stages 3, 4 and
5 embryos, respectively. (b, d, e, g, h)
Quail mesoderm was identified by expresd
sion of QCPN, and chick neural tissue by c
expression of Sox2/3 (95 ± 5% cells/section). (a) Dashed line, presumptive neural
plate; red box, prospective stage 3C
neural plate tissue used for in vitro explant
e
studies. (b) Stage 3C explants (n = 14) cultured for 24 h in the presence of
mFrz8CRD-IgG expressed Sox2/3 (95 ±
5% cells/section, n = 5 sections; mean ±
s.e.m.) and Otx2 (97 ± 3% cells/section, n
g
f
= 8 sections) but not Pax6, En1, Gbx2 or
Krox20. (c, f) Caudal paraxial mesoderm
explants used in recombination experiments are marked by gray boxes.
(d) Chick stage 3C explants recombined
h
with early HH stage 4 quail mesoderm
+
+
(n = 12) generated Otx2 /En1 cells (80 ±
20% cells/section, n = 10 sections) and a
few Gbx2+ cells (15 ± 10% cells/section, n
= 10 sections) and Otx2+/Pax6+cells (10 ±
10% cells/section, n = 8 sections), but no
Krox20+ cells. (e) Stage 3C explants recombined with early HH stage 4 mesoderm cultivated in mFrz8CRD-IgG conditioned medium (120 µl/ml culture medium; n = 14) generated Otx2+ cells (97 ± 2% cells/section, n = 9 sections) but no Pax6+, En1+, Gbx2+ or Krox20+ cells. (g) Stage 3C explants
recombined with HH stage 5 mesoderm (n = 12) generated only a few Otx2+/En1+ cells (10 ± 10% cells/section, n = 10 sections) and Otx2+/Pax6+cells
(5 ± 5%, n = 5 sections) but generated Gbx2+ cells (80 ± 20%, n = 8 sections) and Krox20+/Pax6+cells (60 ± 35%, n = 10 sections). (h) Stage 3C
explants recombined with stage 5 mesoderm in the presence of mFrz8CRD-IgG (120 µl/ml; n = 9) generated Otx2+ cells (95 ± 5% cells/section, n = 12
sections) and Pax6+ cells (50 ± 40% cells/section, n = 12 sections) but no En1+, Gbx2+ or Krox20+ cells. Scale bar, 100 µm.
3C explants (Fig. 3d). Exposing these conjugates to mFrz8CRDIgG blocked the generation of En1+ cells but not that of Otx2+
cells (Fig. 3e). Stage 5 paraxial mesoderm induced Krox20+,
Gbx2+ and Pax6+ cells in stage 3C explants, a marker profile
indicative of rostral hindbrain character (Fig. 3g). Exposing these
conjugates to mFrz8CRD-IgG blocked the generation of Krox20+
and Gbx2+cells but not that of Otx2+ cells (Fig. 3h). Thus, Wnt
signaling is required for PMC activity to induce cells of caudal
regional neural character.
Direct caudalizing action of Wnts
We next addressed whether the induction of caudal neural character requires Wnt action on neural cells themselves. By stage 4,
tissue isolated from different regions along the rostrocaudal axis
Fig. 4. Ongoing Wnt signaling in neural plate cells is required for the acquisition of caudal forebrain but not rostral forebrain character.
(a) Schematic drawing of an HH stage 4 chick embryo. Dotted line, presumptive neural plate. Boxed regions, explants of the prospective neural plate
used for in vitro studies: prospective rostral forebrain (RFB, red) and prospective caudal forebrain (CFB, yellow). (b–e) Sox2/3 was used as a general
neural marker (95 ± 5% cells/section).
(b) RFB explants cultured alone for 24 h
a
(n = 12) generated Otx2+cells (95 ± 5%,
cells/section, n = 8 sections) but did not
b
generate Pax6-, En1-, Gbx2- or Krox20expressing cells. (c) RFB explants cultured in mFrz8CRD-IgG conditioned
medium (100 µl/ml culture medium;
c
n = 16) generated Otx2+cells (95 ± 5%
cells/section, n = 5 sections) but did not
generate Pax6, En1, Gbx2 or Krox20
cells. (d) CFB explants cultured alone
(n = 25) generated Otx2+/Pax6+cells
d
(95 ± 5% cells/section, n = 5 sections)
but did not generate En1-, Gbx2- or
Krox20-expressing cells. (e) CFB explants cultured in the presence of
mFrz8CRD-IgG (100 µl/ml; n = 27) gene
erated Otx2+cells (95 ± 5% cells/section, n = 6 sections) but did not generate
Pax6, En1, Gbx2 or Krox20 cells. Scale
bar, 100 µm.
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a
cells of diencephalic, midbrain and
rostral hindbrain character.
We used New culture methods42 to
examine whether the attenuation of
Wnt signaling imposes a more rostral
character in neural cells in intact
c
embryos. Control beads, or beads containing mFrz8CRD-IgG, were
implanted beneath the neural plate
adjacent to the prospective midd
brain/caudal forebrain regions of
stage 4 embryos (Fig. 6a), and these
embryos were permitted to develop to
the 12–14 somite stage (Fig. 6c and d).
e
Beads containing mFrz8CRD-IgG
induced morphological changes
indicative of an expansion of the caudal forebrain region, which was consistently more pronounced on the side
Fig. 5. Ongoing Wnt signaling in neural plate cells is required for the acquisition of midbrain and ros- of bead implantation (n = 5; Fig. 6d).
tral hindbrain character. (a) Schematic drawing of a HH stage 4 chick embryo. Dotted line, presumptive Analysis of the profile of transcription
neural plate; boxed regions, explants of the prospective neural plate used for in vitro studies: prospec- factor expression in these embryos
tive midbrain (MB, green) and prospective hindbrain (HB, blue). (b–e) Sox2/3 was used as a general
showed that cells normally located in
neural marker (95 ± 5% cells/section). (b) MB explants cultured alone (n = 22) generated
+
+
Otx2 /En1 cells (90 ± 10% cells/section, n = 10 sections) but not Pax6, En1-, Gbx2- or Krox20- the anterior region of the caudal foreexpressing cells. (c) MB explants cultured in the presence of mFrz8CRD-IgG (120 µl/ml culture brain expressed Otx2 but not Pax6,
medium; n = 24) generated Otx2+cells (95 ± 5% cells/section, n = 10 sections), a few Pax6+ cells (5 ± indicating that caudal forebrain cells
5% cells/section, n = 10 sections), but no En1, Gbx2 or Krox20 cells. (d) RHB explants cultured alone had acquired rostral forebrain charac(n = 19) did not generate any Otx2+ or En1+cells but did generate Gbx2+ cells (90 ± 10% cells/section, ter (Fig. 6c and d). The domain in
n = 11) and Krox20+/Pax6+ cells (60 ± 20% cells/section, n = 14). (e) RHB explants cultured in the which Otx2 +/Pax6+ caudal forebrain
presence of mFrz8CRD-IgG (120 µl/ml; n = 24) generated Otx2+cells (90 ± 10% cells/section, n = 12 cells were present extended caudally
sections) that co-expressed Pax6 in 50 ± 30% of the cells/section (n = 11 sections). No En1-, Gbx2- or into the region normally occupied by
Krox20- expressing cells were generated. Scale bar, 100 µm.
En1+/Otx2+ midbrain cells. Consistent
with this, the number of En1+/Otx2+
midbrain cells was reduced and En1 was
expressed at a much lower level by the remaining cells. In addiof the prospective neural plate generates cells of rostral forebrain
tion, the number of En1+/Gbx2+ cells characteristic of rhom(RFB), caudal forebrain (CFB), midbrain (MB) and rostral hindbrain (RHB) character in a position-dependent manner
bomeres 1 and 2 of the rostral hindbrain was reduced (Fig. 6c
(Figs. 4a and 5a) and in the absence of mesodermal signals4. To
and d). Collectively, these results provide evidence of a rostralto-caudal shift in the positional character of neural cells in
examine whether Wnt signaling is required in neural tissue for
embryos exposed to mFrz8CRD-IgG.
the acquisition of caudal neural character, we cultured stage 4
explants isolated from different rostrocaudal levels of the prospective neural plate in the absence or presence of mFrz8CRD-IgG
Distinct caudal fates imposed by graded Wnt signaling
(see Methods).
The requirement for Wnt signaling in the generation of neural
Prospective RFB explants grown with or without mFrz8CRDcells of three different rostrocaudal characters in explant assays,
IgG generated rostral forebrain–like cells that expressed Otx2,
combined with the rostral-to-caudal shift in the positional charbut not Pax6, En1, Krox20 or Gbx2 (Fig. 4b and c). When grown
acter of in the New culture assays, led us to examine whether
alone, prospective CFB explants generated Otx2+ cells and Pax6+
Wnts induce different positional identities through actions at
different concentration thresholds. Wnt3A, Wnt8c and Wnt11
cells, whereas in the presence of mFrz8CRD-IgG, no Pax6+ cells
show similar activities in several different assays
were generated (Fig. 4d and e). In the absence of mFrz8CRD(http://www.stanford.edu/%7Ernusse/wntwindow.html), and
IgG, prospective MB explants generated Otx2+ cells and En1+
the ability of mFrz8CRD-IgG to block Wnt3A signaling was
cells, whereas in the presence of mFrz8CRD-IgG, Otx2+ cells perdemonstrated by assaying the block in induction of epidermal
sisted and no En1+ cells were generated (Fig. 5b and c). When
character in blastula-stage chick epiblast cells in response to
grown alone, prospective RHB explants generated Krox20 +,
Wnt3A (S. Wilson and T.E., unpublished data). Thus, we examGbx2+ and Pax6+ cells, whereas in the presence of mFrz8CRDined the actions of Wnts on stage 4 RFB explants using Wnt3A
IgG, Otx2+ cells were generated, and a subset of these expressed
conditioned medium43 (Fig. 7 and Table 1).
Pax6 (Fig. 5d and e). Thus, under these conditions, prospective
hindbrain cells acquired either rostral or caudal forebrain charStage 4 RFB explants (Fig. 7a and b) were exposed to differacter. Stage 4 CFB, MB and RHB explants exposed to mFrz8CRDent concentrations of Wnt3A and to a constant concentration of
IgG were consistently smaller than explants grown alone (data
FGF8. Consistent with previous studies4, Wnt3A alone did not
not shown), suggesting that Wnts exert a proliferative as well as
induce caudal neural cells in stage 4 RFB explants at any concena patterning effect during the early differentiation of neural cells.
tration tested (data not shown). In the presence of Wnt3A (1×)
These results provide evidence that ongoing Wnt signaling in
and FGF8 (10 ng/ml), Otx2+ cells and Otx2+/Pax6+ cells of rosprospective neural cells in vitro is required for the generation of
tral and caudal forebrain character were generated, but no En1+
© 2002 Nature Publishing Group http://neurosci.nature.com
b
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Fig. 6. Wnt signaling imposes rostrocaudal pattern on neural cells in intact chick
embryos. (a, b) Schematic drawings of
late HH stage 4 chick embryos. Dotted
line indicates presumptive neural plate.
(a) Blue dot indicates the site at which
beads soaked in either mFrz8CRD-IgG
or control conditioned medium (CM)
were grafted beneath the neural plate
adjacent to the prospective midbrain
(MB) region. (b) Blue dot indicates the
site at which beads soaked in either
Wnt3A or CM were grafted beneath the
prospective forebrain (FB) region.
(c, d) Dorsal view of 12 somite (12 som)
chick embryos derived from stage 4
embryos grafted with control (c) or
mFrz8CRD-IgG (d) beads adjacent to
the prospective midbrain region, and
maintained in New culture. The numbered horizontal bars indicate the positions of the sections analyzed for marker
expression. (e, f) Dorsal view of 14
somite chick embryos generated in New
culture from stage 4 embryos grafted
with control (e) or Wnt3A (f) beads
beneath the prospective forebrain.
Numbered horizontal bars indicate the
positions of the sections analyzed for
marker expression. (c–f) Sections taken
at an equal distance from the anterior tip
of the respective embryos.
a
c
b
d
midbrain cells or Krox20 Gbx2+ hindbrain cells were detected (Fig. 7c). In
these explants, Otx2+/Pax6+ cells were
located at the periphery and
Otx2+/Pax6– cells at the core. This indicates that cells in the
peripheral regions of the explants are exposed to higher concentrations of Wnt3A than are cells at the core. Wnt3A (2×) and FGF8
(10 ng/ml) induced Otx2 +/Pax6 + caudal forebrain cells and
Otx2+/En1+ midbrain cells, but no Krox20+ Gbx2+ hindbrain cells
(Fig. 7d). Under these conditions, Otx2+/Pax6+ cells were located
at the core and Otx2+/En1+ cells at the periphery of the explants,
again a likely reflection of the exposure of peripheral cells to higher Wnt levels. Wnt3A (4× or 10×) and FGF8 (10 ng/ml) induced
Otx2+/En1+ midbrain cells and Krox20+, Pax6+ and Gbx2+ rostral hindbrain cells (Fig. 7e). Under these conditions, Otx2+/En1+
cells were located at the core and Krox20+ and Gbx2+ cells at the
periphery of the explants. Similar results were obtained with Xenopus laevis Wnt8 conditioned medium (data not shown). The concentration of mFrz8CRD-IgG required to block the generation
of cells of rostral hindbrain character in RHB explants was fourfold higher than that required to block the generation of caudal forebrain character in CFB explants (data not shown). Stage
4 RFB explants exposed to Wnt3A were consistently larger than
explants grown alone (data not shown), supporting the view
that Wnts enhance the proliferation of neural progenitor cells,
in addition to their role in specifying rostro–caudal positional
identity. Taken together, these results provide in vitro evidence
that the caudalizing action of Wnts results from a direct action
on neural cells and that graded Wnt signaling, in combination
with FGFs, specifies cells of caudal forebrain, midbrain and rostral hindbrain character.
nature neuroscience • volume 5 no 6 • june 2002
e
f
We used New culture methods42 to examine whether Wnt3A
induced caudal character in anterior neural tissue in intact chick
embryos. Control beads or beads containing Wnt3A were
implanted beneath the prospective forebrain of stage 4 embryos
(Fig. 6b), and embryos were permitted to develop to the 12–14
somite stage (Fig. 6e and f). Embryos with grafted Wnt3A beads
typically showed a reduction in rostral forebrain tissue. The
domain of Otx2+ rostral forebrain cells was reduced and the
domains of Otx2 + /Pax6 + caudal forebrain cells and of
En1+/Otx2+ midbrain cells were shifted rostrally (Fig. 6e and
f). These findings support the idea that elevated Wnt signaling
in the anterior region of embryos leads to a loss of anterior
neural tissue and/or head structures and to a rostral-to-caudal
shift in neural pattern7,16,18,25,26,44.
Permissive action of FGF signaling
To examine whether a variation in the level of FGF signaling
might also influence caudal regional character, we added FGF8
(10 or 40 ng/ml) to stage 4 RFB explants exposed to Wnt3A (1×)
or to Wnt3A (4×). Varying the concentration of FGF did not
change the proportion or distribution of caudal neural cells
induced by Wnt signals (data not shown), suggesting that FGFs
act solely in a permissive manner during the establishment of
caudal neural character. Taken together, these findings indicate
that Wnts act directly, and in a concentration-dependent manner, to induce cells of caudal forebrain, midbrain and rostral
hindbrain character in RFB explants.
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b
© 2002 Nature Publishing Group http://neurosci.nature.com
c
d
e
Fig. 7. Graded Wnt3A activity, in combination with FGF8, induces caudal regional character in prospective rostral forebrain cells. (a) Schematic
drawing of an HH stage 4 embryo. Dotted line, presumptive neural plate; red box, prospective RFB epiblast explant used for in vitro studies. (b) RFB
explant cultured alone expressed Sox2/3 (95 ± 3% cells/section, n = 8 sections) and Otx2+ cells (95 ± 5% Otx2+ cells/section, n = 8 sections). (c) RFB
explants cultured in the presence of FGF8 (10 ng/ml) and Wnt3A (1×, 25 µl of Wnt3A conditioned medium per milliliter of culture medium,
∼25 ng/ml) had a central domain of Otx2+/Pax6– cells (95 ± 5% Otx2+ cells/section, n = 9 sections), whereas peripheral cells (typically 1–2 cell diameters) co-expressed Otx2 and Pax6 (96 ± 4% Otx2+/Pax6+ cells/section, n = 7 sections). (d) RFB explants cultured in the presence of FGF8
(10 ng/ml) and Wnt3A (2×) had a central domain of Otx2+/Pax6+ cells (95 ± 5% Otx2+/Pax6+ cells/section, n = 8 sections) and a peripheral domain
(typically 2–3 cell diameters) of En1+/Otx2+ cells (98 ± 2% Otx2+/En1+ cells/section, n = 8 sections). (e) RFB explants cultured in the in the presence
of FGF8 (10 ng/ml) and Wnt3A conditioned medium (4×) had a central domain of Otx2+/En1+ cells (95 ± 5% Otx2+/En1+ cells/section, n = 12 sections) and a peripheral domain (typically 3–5 cell diameters) of Gbx2+ (90 ± 8% Gbx2+cells/section, n = 12 sections), and Krox20+/Pax6+ cells
(60 ± 25% Krox20+/Pax6+ cells/section, n = 12 sections). In all conditions, >95% of the cells expressed Sox2/3. Scale bar, 100 µm.
DISCUSSION
This study reports three main findings: (i) Wnt signaling is
required to reprogram neural cells of initial rostral forebrain character during the acquisition of caudal regional neural characters;
(ii) Wnts, in combination with FGFs, can induce cells of caudal
forebrain, midbrain and rostral hindbrain characters; and (iii)
Wnts act in a graded manner directly on neural plate cells. These
findings support Wnt involvement in the signaling pathway by
which prospective neural plate cells acquire diencephalic, midbrain and rostral hindbrain identity during the early phases of
chick neural tube development.
The developmental patterns of expression of Wnts and Fgfs
are consistent with the notion that that combined Wnt and FGF
signaling in neural plate cells induces caudal regional neural characters. At early gastrula stages, when prospective caudal neural
cells possess a rostral forebrain character, Wnts are preferentially expressed in the posterior region of the primitive streak, and
thus are located at a considerable distance from caudal neural
cells (ref. 14 and data not shown). At these developmental stages,
secreted Wnt antagonists such as crescent and caronte are
expressed in prospective neural plate cells or in tissues that underlie the entire prospective neural plate45, and thus may help to
decrease the exposure of anterior neural cells to Wnt signals. Over
this early period, Fgf8 is expressed along the entire length of the
developing primitive streak4. At late gastrula stages, when neural cells are exposed to signals that direct their caudal character,
both FGFs and Wnts are expressed in the posterior regions of the
chick embryo, whereas Wnt inhibitors are expressed in rostral
530
neural plate cells and in the head mesendoderm that underlies
the prospective rostral forebrain 16,44. This latter domain of
expression may explain the finding that gastrula-stage head
mesoendodermal tissue possesses rostralizing activity4,5,46. Thus,
the exclusion of Wnt signaling from the anterior region of the
early embryo is probably involved in maintaining the rostral
(Pax6−) forebrain character of neural progenitor cells.
The present studies support the view that Wnts have distinct
roles in the development of the chick nervous system at blastula and gastrula stages. At the blastula stage, epiblast cells acquire
generic neural fates; Wnt signaling at this stage promotes epidermal fate and blocks neural fate, apparently by preventing epiblast cells from responding to the neuralizing actions of FGFs41.
At late gastrula stages, after neural cells have become committed to a neural fate, graded Wnt activity instead induces progressively more caudal neural characters, through actions in
combination with FGF signaling. RA signaling at these stages is
involved in inducing cells of caudal hindbrain and spinal cord
character4,10,47, suggesting that the joint actions of RA, Wnts
and FGFs are required to induce cells in the most caudal regions
of the neural axis.
The functions of Wnts in neural patterning reported here
extend previous findings in vertebrate embryos mutant in components of the Wnt signaling pathway. In the mouse,
Wnt3/Wnt3A double mutant embryos lack all mesoderm, and
Wnt3A mutants generate ectopic neural plate tissue in place of
caudal paraxial mesoderm 21,30,31. Moreover, mutant mouse
embryos that lack the function of the Wnt inhibitor dickkopf-1
nature neuroscience • volume 5 no 6 • june 2002
© 2002 Nature Publishing Group http://neurosci.nature.com
articles
fail to develop head structures rostral to the Table 1. Marker expression in rostral forebrain explants exposed to FGF8 and
midbrain 26 . In zebrafish, Wnt8 mutant Wnt3A.
embryos lack trunk and tail structures and
have ectopic neural tissue29. In addition, in
Otx2 (RFB)
Otx2, En1 (MB)
zebrafish masterblind mutant embryos, an
apparent reduction in Axin1-dependent inhiOtx2, Pax6 (CFB)
Gbx2, Krox20, Pax6 (RHB)
bition of Wnt signaling is accompanied by a
loss of telencephalic structures and an expanTotal no.
sion of more caudal neural tissue25. Thus,
[Wnt3A]
of explants
these genetic studies provide evidence that
Wnt signaling is required for the induction
and patterning of neural tissue. Such genetic






45
38
7
analyses do not, however, address whether
Wnts act directly or indirectly on neural cells


58
19
1
4
12
22
1×
to regulate their caudal regional character.
Wnts have been implicated in caudal

72
2
17
21
26
2
4
2×
neural patterning in X. laevis, but again the
earlier involvement of Wnts in induction of


78
15
2
24
32
4×
5
mesoderm and epidermal ectoderm complicates the task of distinguishing direct from Colored discs represent schematic figures of explants and the typical distribution of cells expressing
indirect Wnt action in rostrocaudal neural different region specific markers, characteristic of rostral forebrain (RFB), caudal forebrain (CFB),
patterning17,44. In blastula-stage ectoderm, midbrain (MB) and rostral hindbrain (RHB). For detailed quantification of marker expression, see
overexpression of the neural inducer Noggin Fig. 7 legend.
together with Wnts or the Wnt effector βcatenin induces the expression of both caudal neural and mesodermal markers. In contrast, in blastula-stage
ing signals in the neural tube by similar or identical signals
ectoderm that has been neuralized by dissociation in Ca2+- and
derived from extrinsic tissues may represent a common strategy for axial neural patterning.
Mg2+-free medium, X. laevis Wnt8 induces caudal regional neural markers in the absence of markers of dorsal mesoderm24. In
gastrula-stage ectoderm, enhanced Wnt signaling leads to the
METHODS
Isolation and growth of tissue explants. Prospective neural plate explants
induction of caudal neural markers in adjacent cells, in the
were isolated from HH stage 3 and 4 chick embryos, and paraxial mesoabsence of induction of mesodermal markers7. Under these conderm explants were isolated from stage 4 and 5 quail embryos. Explants
ditions, cells expressing caudal neural markers are induced by an
were cultured in vitro in serum-free OPTI-MEM containing N2 suppleindirect, non-cell-autonomous mechanism that apparently
ment (Gibco-BRL/ Invitrogen, Paisley, UK) and fibronectin (Sigma). The
involves FGF signaling. Thus, these studies in X. laevis are conuse of chick embryos in this study was approved by the ethical commitsistent with our findings in chick, indicating that the actions of
tee at Umeå University.
FGFs and graded Wnt signaling on neural cells induce cells of
progressively more caudal neural character.
Whole-embryo culture. HH stage 4 chick embryos were maintained in
Our results also provide evidence that Wnts mediate the
New culture42 to the 12–14 somite stage. Blue-Beads (Bio-Rad, Hercules,
PMC activity described previously in chick assays4,5,12. In prinCalifornia) soaked in mFrz8CRD-IgG, Wnt3A or control conditioned
medium were grafted beneath different regions of the prospective neurciple, the expression of Wnts in prospective caudal neural cells
al plate of host embryos. Embryos in which the bead was still in contact
does not exclude the possibility that a distinct signal derived
with the forebrain or midbrain region at stage 7 (1–3 somite stage) were
from the paraxial mesoderm induces Wnt expression in neural
maintained in culture until the 12–14 somite stage. Eight embryos graftcells, and that neurally derived Wnts impose caudal neural chared with control beads, six grafted with mFrz8CRD-IgG beads and five
acters. However, the expression of Wnt8c and Wnt11 in the caugrafted with Wnt3A beads were cryosectioned (9 µm). Each section was
dal paraxial mesoderm precisely mimics the distribution of
collected and analyzed by immunohistochemistry for marker expression.
tissues that possess PMC activity 5 (Fig. 1a–d and data not
shown). Moreover, Wnt3A, in combination with FGF8, induces
Preparation of inducing factors. Soluble mouse Wnt3A and control conWnt8c in prospective rostral forebrain cells. Although we canditioned media (CM) were obtained from stably transfected, or untransnot exclude the possible involvement of other Wnts, we show
fected, mouse L-cells, respectively43, grown in Dulbecco’s modified Eagle’s
here that Wnt8c and Wnt11 are likely mediators of PMC activmedium (DMEM) with 10% Knockout Replacement Serum (Gibcoity.
BRL). Under these conditions, the CM contain ∼100 µg/ml of Wnt3A
protein (R. Nusse, personal communication). The CM were concentratThe specification of caudal region neural characteristics by
ed using Centriprep 10,000-MWCO filters (Amicon/Millipore, Bedford,
Wnt signaling may, however, be a two-step process in which an
Massachusetts), divided into aliquots and stored at –80°C. Wnt3A was
initial phase of mesodermally derived Wnt signaling is consolused at an estimated concentration of 25–250 ng/ml (1–10×) in explant
idated by the expression of Wnts in prospective caudal neural
assays. The mFrz8CRD-IgG40 and control CM were prepared by transcells. This sequential ‘like-inducing-like’ strategy is reminiscent
fecting HEK-293 cells with the mFrz8CRD-IgG expression construct or
of the mechanisms that establish dorsoventral pattern in the
with a lacZ reporter construct using Gene-PORTER 2 (GTSINC, San
neural tube. Sonic hedgehog (Shh) protein derived from the
Diego, California). Cells were transferred to serum-free OPTI-MEM
axial mesoderm, and bone morphogenetic proteins (BMPs)
(Gibco-BRL), and the CM was harvested after 48 h, concentrated with
derived from the flanking epidermal ectoderm, induce the
Centriprep filters (Amicon), divided into aliquots and stored at –80°C.
expression of Shh and Bmps, respectively, in ventral and dorsal
mFrz8CRD-IgG or lacZ CM were used at 100–160 µl/ml of explant medium. FGF8 (Gibco-BRL) was used at 10–40 ng/ml.
midline neural cells48. Thus, the induction of secreted patternnature neuroscience • volume 5 no 6 • june 2002
531
articles
© 2002 Nature Publishing Group http://neurosci.nature.com
Immunohistochemistry, in situ hybridization and RT-PCR. In situ
hybridization and immunohistochemistry was carried out as
described49,50. Rabbit anti-Gbx2 antibodies were raised against the peptides EEAKGREENFSMDSD and QNRRAKWKRVKAGN. Other antibodies used in this study have been described 5,41. Conditions and
primers used to analyze Wnt8C (35 cycles) and S17 (26 cycles, as semiquantitative control) expression by RT-PCR in pooled explants (n = 9)
have been described41.
Acknowledgments
We thank Y. Renoncourt for experimental contributions, members of the Edlund
lab for discussions and H. Alstermark for technical assistance. We are grateful to
R. Nusse for providing Wnt3A-expressing cells, to J. Nathans for the mFrzCRDIgG plasmid and Xwnt8 cell line and to C. Tabin for Wnt probes. T.E. is
supported by the Swedish Medical Research Council and by the Foundation for
Strategic Research. T.M.J. is supported by grants from US National Institute of
Neurological Disorders and Stroke (NIH-NINDS) and is an Investigator of the
Howard Hughes Medical Institute.
Competing interests statement
The authors declare that they have no competing financial interests.
RECEIVED 25 MARCH; ACCEPTED 19 APRIL 2002
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nature neuroscience • volume 5 no 6 • june 2002
corrigenda
Progressive induction of caudal neural character by graded Wnt signaling
Ulrika Nordström, Thomas M. Jessell and Thomas Edlund
Nat. Neurosci. 5, 525-532 (2002)
The authors wish to correct the phrase “rostral-to-caudal shift” on page 528, which should read “rostrocaudal shift”. The error
occurs twice on this page.
nature neuroscience • volume 5 no 7 • july 2002
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