1 Department of Anatomy and Developmental Biology, University College London,
Gower Street, London WC1E 6BT, UK
2 MRC Centre for Developmental Neurobiology, New Hunt's House, King's College
London, London SE1 9RT, UK
* Author for correspondence (e-mail: s.wilson{at}ucl.ac.uk)
Accepted 17 September 2004
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SUMMARY |
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We find that masterblind zebrafish embryos that carry a mutation in Axin1, an intracellular negative regulator of Wnt pathway activity, show an expansion of prospective floorplate coupled with a reduction of prospective hypothalamic tissue. Complementing this observation, transplantation of cells overexpressing axin1 into the prospective floorplate leads to induction of hypothalamic gene expression and suppression of floorplate marker gene expression. Axin1 is more efficient at inducing hypothalamic markers than several other Wnt pathway antagonists, and we present data suggesting that this may be due to an ability to promote Nodal signalling in addition to suppressing Wnt activity. Indeed, extracellular Wnt antagonists can promote hypothalamic gene expression when co-expressed with a modified form of Madh2 that activates Nodal signalling. These results suggest that Nodal signalling promotes the ability of cells to incorporate into ventral midline tissue, and within this tissue, antagonism of Wnt signalling promotes the acquisition of hypothalamic identity. Wnt signalling also affects patterning within the hypothalamus, suggesting that this pathway is involved in both the initial anteroposterior subdivision of ventral CNS midline fates and in the subsequent regionalisation of the hypothalamus. We suggest that by regulating the response of midline cells to signals that induce ventral fates, Axin1 and other modulators of Wnt pathway activity provide a mechanism by which cells can integrate dorsoventral and anteroposterior patterning information.
Key words: Axin, Floorplate, Hypothalamus, Nodal, Wnt, Zebrafish, mbl
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Introduction |
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The different origin and behaviour of ventral midline cells compared to
neural plate tissue destined to form more dorsal CNS structures raises the
question of whether the same signalling pathways establish AP pattern in these
two CNS domains. Indeed, in fish, at stages when the prospective alar neural
plate is receiving signals that influence its anterior character (see
Erter et al., 2001;
Houart et al., 2002
),
prospective hypothalamic cells are still located caudal to the eye field
(Mathieu et al., 2002
;
Varga et al., 1999
;
Woo and Fraser, 1995
) and have
yet to reach their final destination, the ventral anterior tip of the forming
neural tube. Thus, it is unclear if signals that promote anterior identity in
the lateral neural plate/dorsal neural tube, also establish hypothalamic
(anterior) identity in ventral CNS midline tissue.
The signalling pathways responsible for subdivision of the ventral CNS into
hypothalamic and floorplate domains are uncertain (reviewed by
Wilson and Houart, 2004).
Nodal and Hedgehog (Hh) signals have essential roles in the induction of the
ventral CNS, but both pathways influence the entire axis and there is little
evidence to suggest involvement in AP regional patterning. Nodal and Hh
ligands are expressed in the organiser and subsequently in the axial
mesendoderm and axial neuroectoderm, and these signals act in a parallel or
cooperative manner to induce the entire ventral midline and adjacent CNS
tissue (reviewed by Ruiz i Altaba et al.,
2003
; Strähle et al.,
2004
). For example, in zebrafish, the Nodal-related signal Cyclops
and its downstream effector Madh2 can induce shh expression in the
neuroectoderm and promote floorplate fate
(Muller et al., 2000
;
Tian et al., 2003
). In
addition, Nodal signalling is cell-autonomously required for the establishment
of posterior-ventral (PV) hypothalamus and acts indirectly (through
specification of the prechordal plate and PV hypothalamus) to promote
dorsal-anterior hypothalamic fate (Mathieu
et al., 2002
; Rohr et al.,
2001
). Similarly, mutations affecting Hh signalling disrupt both
hypothalamic and floorplate specification (e.g.
Chen et al., 2001
;
Chiang et al., 1996
;
Rohr et al., 2001
;
Varga et al., 2001
).
Experimental evidence from the chick implicates Bmp signalling in the AP
regionalisation of the ventral neural tube. The Bmp antagonist Chordin
probably generates a permissive environment for Shh-mediated induction of the
floorplate in regions of low Bmp signalling activity
(Patten and Placzek, 2002)
whereas Bmp7 from the prechordal mesendoderm acting together with Shh is
proposed to promote hypothalamic/rostral ventral midline identity
(Dale et al., 1997
;
Dale et al., 1999
). However,
in zebrafish, Bmp signalling influences prospective dorsoventral (DV) rather
than AP patterning of the rostral neural plate
(Barth et al., 1999
;
Hammerschmidt et al., 2003
)
and abrogating Bmp activity has little effect upon the initial specification
and regional subdivision of midline neural tissue into hypothalamic and
floorplate domains (Barth et al.,
1999
). These results do not exclude the possibility that Bmps play
a role in AP patterning of the ventral CNS midline of zebrafish, but do raise
the possibility that other signalling pathways may have more critical roles in
the allocation of hypothalamic versus floorplate fates.
The Wnt/Axin/ß-catenin signalling pathway is a candidate to regulate
AP patterning in the ventral CNS midline given its role in AP regionalisation
of other domains of the neural plate. The activity of various Wnt agonists and
antagonists is thought to generate graded Wnt signalling activity (high
caudally and low rostrally), which contributes to the establishment of early
AP subdivisions of the neural plate
(Kiecker and Niehrs, 2001a;
Yamaguchi, 2001
). For
instance, abrogation of activity of the Wnt pathway transcriptional repressors
Tcf3/Headless and Tcf3b results in the loss of anterior CNS fates
(Dorsky et al., 2003
;
Kim et al., 2000
). Conversely,
abrogation of Wnt8 activity results in enlargement of the forebrain and
reduction or absence of more caudal neural tissue
(Erter et al., 2001
;
Lekven et al., 2001
;
Nordstrom et al., 2002
).
Subsequent to the initial regionalisation of the neural plate,
Wnt/ß-catenin signalling has additional roles in the refinement of AP
patterning within discrete domains of the CNS. For instance, within the dorsal
forebrain, masterblind/axin1 mutant (mbl) zebrafish embryos
show a reduction or absence of telencephalon and eyes and expansion of dorsal
diencephalic fates (Heisenberg et al.,
1996; Masai et al.,
1997
). Axin1 is a cytoskeletal scaffolding protein that
participates in the assembly of kinases responsible for ß-catenin
degradation (Dajani et al.,
2003
) and so it is likely that the forebrain phenotype of
mbl embryos is due to overactivation of Wnt signalling in the
anterior neural plate (Heisenberg et al.,
2001
; Houart et al.,
2002
; Van de Water et al.,
2001
). Supporting this interpretation, activity of the Secreted
Frizzled Related Protein Tlc emanating from the anterior border of the neural
plate (ANB) promotes telencephalic identity through local inhibition of Wnt
signalling (Houart et al.,
2002
). Similar roles for Wnts and Wnt antagonists in the regional
patterning of forebrain derivatives are proposed in other species
(Braun et al., 2003
;
Gunhaga et al., 2003
;
Lagutin et al., 2003
). To
date, however, most analyses have focused upon the role of Wnts and Wnt
antagonists in the AP regionalisation of the dorsal CNS and the possibility
that this signalling pathway influences AP identity of ventral CNS structures
has not been thoroughly investigated.
In this study, we have examined the ability of Wnt/Axin/ß-catenin signalling to influence AP identity of ventral CNS midline tissue in developing zebrafish embryos. We find that the abrogation of Axin1 activity in mbl embryos results in expansion of floorplate at the expense of hypothalamic tissue. Conversely, exogenous Axin1 is able to suppress floorplate and induce hypothalamic markers in ventral CNS midline tissue of the midbrain and hindbrain. These results suggest that hypothalamic identity is promoted by suppression of Wnt signalling. We also explore the possibility that activation of Nodal signalling combined with suppression of Wnt signalling promotes expression of hypothalamic markers. Finally, mutant analyses and mis-expression studies suggest that within the hypothalamus, the levels of Wnt/Axin1/ß-catenin signalling activity contribute to the AP regionalisation of this structure.
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Materials and methods |
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Microinjection and transplantation experiments
Synthetic mRNAs were transcribed in vitro using the mMessage mMachineTM
transcription kit (Ambion). For transplantation experiments, donor embryos
injected at the 2-4-cell stage with gfp or nuclear gfp (100
pg) RNA as lineage tracer and test RNA: zebrafish axin1 (200-300 pg)
(Heisenberg et al., 2001),
dkk1 (150 pg) (Shinya et al.,
2000
), wnt8b (100-200 pg)
(Kelly et al., 1995
), human
lrp6
c (400 pg)
(Tamai et al., 2000
),
zebrafish hdl/tcf3a/tcf7/1a (100-120 pg)
(Dorsky et al., 2003
;
Kim et al., 2000
),
tlc (200-400 pg) (Houart et al.,
2002
), sfrp3/frzb1 (500-700 pg)
(Agathon et al., 2003
). All
reagents were tested for activity by assessing phenotypes following widespread
overexpression. As expected, overexpression of wnt8b posteriorised
embryos whereas injection of axin1 at the 2-4-cell stage gave bigger
eyes (80%) or ventralised (20%) phenotypes (n=510) consistent with
suppressed Wnt activity. Other Wnt antagonists gave similar overexpression
phenotypes.
RNA encoding constitutively active Madh2 (Madh2CA/Smad2CA)
(Muller et al., 2000) was
injected at 10-100 pg in donor embryos. Cells from donors embryos injected
with 20-40pg madh2CA RNA integrated in the floorplate/PV hypothalamus
and/or the notochord (50%), whereas those with higher amounts of
madh2CA (40-80 pg) formed aggregates in the mesendoderm of the host
embryos (80% of the transplanted embryos) or died. As a result, 25 pg of
madh2CA RNA was co-injected with other RNAs in order to facilitate
integration of the transplanted cells in the ventral neural tube.
All test RNA-expressing cells were taken from the animal pole epiblast of
late blastula to 40% epiboly embryos and transplanted to ectoderm adjacent to
the shield of mid-gastrula (50-65% epiboly)-stage hosts. At this stage, donor
epiblast cells are not committed to specific fates and readily incorporate
into most tissue/cell types in recipient hosts. For transplantation, donor and
host embryos were mounted in 3% or 0.5% methyl-cellulose in embryo medium, and
viewed with a fixed stage Nikon Optiphot or a Leica fluo microscope. Cells
were removed from donor embryos by suction using a mineral oil-filled glass
micropipette attached to a 50 µl Hamilton syringe
(Houart et al., 1998) and
gently aspirated into recipient hosts.
In situ hybridization, immunohistochemistry and histology
In situ hybridization was carried out as described previously
(Hauptmann and Gerster, 1994).
Probes used were: dlx3 (Akimenko
et al., 1994
), emx2
(Morita et al., 1995
),
foxa1/fkd7 and foxb1.2/fkd3
(Odenthal and Nusslein-Volhard,
1998
), foxa2/axial
(Strahle et al., 1993
),
hgg1 (Thisse et al.,
1994
), nk2.1a (Rohr
et al., 2001
), ntl
(Schulte-Merker et al., 1994
),
pax2.1/pax2a (Krauss et al.,
1991
), rx3 (Chuang et
al., 1999
), shh
(Krauss et al., 1993
). GFP was
revealed with a rabbit polyclonal antibody (AMS Biotechnology) at 1:1000
dilution and diaminobenzidine (DAB) staining. Some stained embryos were
embedded in JB4 resin (Polyscience) and sectioned with a Leica microtome
(10-14 µm).
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Results |
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nk2.1a is the earliest specific hypothalamic marker in zebrafish
with expression first detected as a stripe of cells in the medial anterior
neural plate of wild-type embryos by the end of epiboly
(Rohr et al., 2001)
(Fig. 1A). Strikingly, in the
neural plate of mbl embryos, nk2.1a expression is reduced to
about half its normal AP length, suggesting that presumptive hypothalamic
tissue is reduced compared to wild-type siblings
(Fig. 1A,B,E,F). As neural
plate size is not obviously affected in mutant embryos
(Heisenberg et al., 2001
)
(Fig. 1I,J and data not shown),
this suggests that neural plate midline tissue may alter its AP regional
character in response to enhanced Wnt signalling. Supporting the idea that
enhanced Wnt signalling antagonises hypothalamic development, local expression
of exogenous Wnt8b suppressed expression of nk2.1a
(Fig. 1K,L; n=13/18,
and data not shown).
|
|
The development of axial neural tissue is dependent upon signals from
underlying mesendoderm (Camus et al.,
2000; Dale et al.,
1997
; Kiecker and Niehrs,
2001b
) and so changes in the AP regionalisation of the neural
plate could be due to altered Axin1 activity in the neural plate itself or in
the underlying axial tissues. We did not observe any major differences in
expression of axial mesendodermal markers in mbl embryos (see
Heisenberg et al., 2001
) and
the regionalisation of this tissue into prechordal plate and prospective
notochord domains appears to be unaffected
(Fig. 1I,J and data not shown).
Thus the AP patterning phenotype is predominantly restricted to the ectoderm,
supporting the idea that the primary requirement for Axin1 in CNS patterning
is within the neural plate.
Abrogation of wild-type Axin1 function leads to expansion of floorplate at the expense of hypothalamic tissue
In order to assess whether the early changes in neural plate expression
domains are reflected in altered patterning of the ventral CNS at later
stages, we analysed the ventral neural tube of mbl embryos at one day
of development. Reduction of nk2.1a expression and rostral expansion
of foxa2 and foxa1 expression is preserved, confirming that
hypothalamic tissue is reduced and floorplate tissue is expanded in the
ventral neural tube of mbl embryos
(Fig. 2A-F). The extent of
reduction of hypothalamic tissue was variable from relatively mild phenotypes
(Fig. 2F), to an almost
complete loss of tissue (not shown). Together these results show that the
reduced activity of Axin1 in mbl embryos results in expanded
floorplate development in the anterior ventral midline of the neuroectoderm at
the expense of hypothalamic fates.
Axin1 is sufficient to promote hypothalamic and suppress floorplate markers in the ventral neural tube
To further investigate the hypothesis that Axin1 activity within ventral
CNS cells promotes hypothalamic identity, we transplanted wild-type epiblast
cells or epiblast cells overexpressing axin1 in the ectoderm adjacent
to the organiser of mbl embryos at 50-65% epiboly. From within this
domain, precursors of both anterior floorplate and hypothalamus extend
rostrally along the AP axis of the ventral CNS midline during gastrulation
(Mathieu et al., 2002;
Woo and Fraser, 1995
). Both
transplanted wild-type and axin1 overexpressing cells integrated into
the hypothalamus and floorplate of mbl embryos
(Fig. 2G-H and data not shown;
n=23/23 and n=29/29 mbl embryos receiving
transplants of gfp and axin1+gfp overexpressing cells
respectively). Furthermore, when wild-type or axin1 overexpressing
transplanted cells incorporated into the rostral ventral neural tube, they
expressed nk2.1a (n=9/11 and 17/18 respectively) and these
mbl embryos often had expanded hypothalamic gene expression compared
to the mbl mutants that did not receive transplants (n=6/11
and n=14/18; compare Fig.
2G,H with 2B). These results show that restoration of Axin1
activity in the anterior ventral neural tube of mbl embryos is
sufficient to restore hypothalamic marker gene expression.
Given the reduction of hypothalamic tissue and the restoration of hypothalamic gene expression by exogenous Axin1 in mbl embryos, we next asked if Axin1 is sufficient to promote anterior/hypothalamic identity at the expense of floorplate fate in the ventral CNS midline. To address this question, we transplanted epiblast cells overexpressing axin1 in the prospective floorplate of 60-65% epiboly stage wild-type embryos. Control transplants of GFP-expressing cells integrated into the ventral neuroepithelium but did not affect the expression of floorplate or hypothalamic markers (Fig. 3A,B and data not shown). In contrast, in the majority of cases (n=58/83 embryos), many transplanted axin1+ cells ectopically expressed nk2.1a in the posterior ventral neural tube where floorplate, motor neurons or ventral interneurons should form (Fig. 3C,D). The ectopic nk2.1a expression in axin1 overexpressing transplants was observed in grafts that incorporated in the ventral midbrain and hindbrain but not in the ventral spinal cord nor in lateral or dorsal regions of the neural tube (Fig. 3D). Ectopic nk2.1a expression was also observed in transplanted cells overexpressing axin1 in the midbrain floorplate of a minority of mbl embryos (n=4/11, data not shown). Unlike the hypothalamic marker nk2.1a, expression of the dorsal anterior forebrain marker foxg1 was unaffected in axin1-expressing transplants in the ventral CNS even when the grafts extended laterally to the floorplate (n=12/12, Fig. 3J,K).
|
Intracellular inhibition of Wnt/ß-catenin signalling promotes hypothalamic identity
Axin1 inhibits canonical Wnt signalling and so our favoured hypothesis to
explain the results described above is that suppression of Wnt signalling
promotes hypothalamic identity. To test this idea, we assessed whether other
Wnt antagonists could promote hypothalamic identity. Indeed, cells
overexpressing hdl/tcf3a, a potent intracellular repressor
of Wnt canonical/ß-catenin activity
(Kim et al., 2000) often
expressed ectopic nk2.1a expression when they incorporated into the
ventral CNS (Fig. 4A,B,
n=7/12). Surprisingly, however, when cells overexpressing the
secreted Wnt/Axin/ß-catenin antagonists Dkk1
(Glinka et al., 1998
;
Niehrs et al., 2001
), Tlc
(Houart et al., 2002
) or Frzb1
(Agathon et al., 2003
) were
transplanted in the prospective floorplate of 60-65% epiboly stage hosts,
nk2.1a was not ectopically expressed (n=21/21,
n=9/9, n=25/25; Fig.
4D and data not shown).
|
Activation of Nodal signalling promotes incorporation of cells into the CNS midline
Although we always transplanted cells to the same region (the prospective
hypothalamus/anterior floorplate) of host embryos, we noticed a difference in
the distribution of the cells expressing different reagents one day later.
axin1+ cells had a high incidence of integrating into medial regions
of the floorplate rather than the adjacent neuroepithelium (n=28/83
predominantly in the medial floorplate (e.g.
Fig. 4C); n=39/83 in
the medial floorplate and adjacent cells (e.g.
Fig. 3C lower inset and
Fig. 3F,G,H) and
n=16/83 dispersed more widely in the ventral CNS (e.g.
Fig. 3C upper inset and
Fig. 3D,K). In contrast,
gfp+, dkk1+ and sfrp+ cells tended to integrate in the
neuroepithelium of the ventral neural tube lateral to the most medial domain
of the floorplate of the host embryos (Fig.
3B, Fig. 4D and
data not shown, n=31/31 for dkk1 and n=34/34 for
sfrp genes, respectively). As Nodal signalling is essential for cells
to form the medial floorplate and posterior/ventral hypothalamus
(Schier, 2003), we
hypothesised that the exogenous Axin1 in the transplanted cells may facilitate
Nodal activity. Indeed, in certain assays, Axin1 can function as an adapter
for Smad/Madh proteins to facilitate Nodal signalling
(Furuhashi et al., 2001
).
To explore the possibility that exogenous Axin1 influences Nodal
signalling, we analysed the behaviour of ventral CNS cells with activated
Nodal signalling. To do this we transplanted cells expressing a constitutively
active form of the Nodal pathway effector Madh2 (Madh2CA)
(Muller et al., 2000) into the
prospective hypothalamus/anterior floorplate. From this position, cells
expressing a high level of madh2CA RNA (40-80 pg) moved out of the
ectoderm and predominantly incorporated into axial mesendoderm (data not
shown). However, cells expressing less madh2CA (20-40 pg), behaved in
a similar way to Axin1-expressing cells, preferentially incorporating into the
most medial floorplate (Fig. 4E
and compare to 4C).
Furthermore, expression of low levels of madh2CA + axin1 led
to many cells entering the mesendoderm (not shown) suggesting that the
addition of exogenous Axin1 facilitates Madh2 activity. Axin1 and Madh2CA also
both promote the lateral expansion of medial floorplate markers
(Muller et al., 2000
)
(Fig. 3L,M, n=12/20).
Thus, both Nodal signalling and Axin1 promote the acquisition of midline
identity and enhance the ability of donor cells to incorporate into the
ventral CNS midline.
Activation of Nodal signalling in combination with inhibition of Wnt signalling promotes hypothalamic identity
In light of the possible influence of Axin1 on both Nodal and Wnt pathways,
we next assessed whether activation of Nodal signalling coupled with the
activity of other extracellular Wnt antagonists promotes hypothalamic
identity. To do this, we transplanted cells expressing madh2CA alone
or with dkk1, or with RNA encoding a C-terminal truncated form of the
human Lrp6 (Lrpc) Wnt co-receptor, in which the Axin-binding
domain is deleted, thus preventing the translocation of Axin to the membrane
and consequently enhancing ß-catenin degradation
(He et al., 2004
;
Tamai et al., 2000
). Cells
overexpressing madh2CA alone did not induce any ectopic
nk2.1a expression and had no effect on the regional subdivision of
the ventral neural tube into floorplate and hypothalamic domains
(Fig. 4E). In contrast, embryos
containing cells co-expressing dkk1 + madh2CA or
lrp
c + madh2CA showed ectopic patches of
nk2.1a expression in the posterior ventral neural tube
(Fig. 4F and data not shown;
n=11/18 and n=5/10 respectively). Control transplants
expressing the ligand Wnt8b in combination with Madh2CA showed no ectopic
nk2.1a expression (n=19/19, data not shown). Altogether,
these results suggest that Nodal signalling promotes the incorporation of
cells into ventral midline tissue, and that within this tissue, antagonism of
Wnt signalling promotes the expression of hypothalamic markers.
Compromised Axin1 activity results in loss of rostral hypothalamic tissue and expansion of posterior diencephalic/midbrain floorplate
Although Axin1 activity promotes hypothalamic identity, a small domain of
nk2.1a-expressing hypothalamic tissue is retained in mbl
embryos that are compromised in Axin1 function
(Fig. 2B). To examine the
identity of this remaining hypothalamic tissue, we analysed expression of
markers of regional identity within the hypothalamus. In wild-type embryos,
rx3 is expressed in the anterior hypothalamus from early
somitogenesis (Chuang et al.,
1999) (Fig. 5A) and
shh expression is consolidated in the anterior-dorsal hypothalamus by
mid-somite stages (Fig. 5C). In
contrast, emx2 is expressed in the posterior and ventral hypothalamus
by mid-somite stages (Fig. 5E)
(Mathieu et al., 2002
). In
mbl embryos, emx2 is expressed at the anterior tip of the
ventral neural tube similar to nk2.1a
(Fig. 5F compare with
Fig. 2B) whereas rx3
expression and the most anterior domain of shh expression is lost
(Fig. 5B,D). Wild-type Axin1
activity is therefore required for the establishment of anterior hypothalamic
identity. Consistent with this observation, transplantation of gfp+
or axin1+ cells restored rx3 expression in the hypothalamus
of mbl embryos (n=6/12 and 7/12;
Fig. 5G,H and data not shown).
However, when Axin1-expressing cells incorporated into floorplate domains of
wild-type embryos, they expressed the caudal hypothalamic marker,
emx2 (Fig. 5I,J).
|
Wnt/ß-catenin signalling influences the patterning of the hypothalamus along its AP axis
Enhanced Wnt pathway activity in mbl embryos is correlated with a
loss of anterior hypothalamic fate suggesting that Wnt signalling may play a
role in the regionalisation of the hypothalamus. To further explore this
possibility, we locally manipulated levels of Wnt activity by transplanting
cells expressing wnt8b into the prospective hypothalamus of wild-type
embryos. wnt8b expression is upregulated in mbl embryos and
Wnt8b activity is thought to play a role in the AP patterning of the
alar/dorsal forebrain (Houart et al.,
2002; Kim et al.,
2002
). wnt8b-expressing cells failed to express
rx3 when located in the anterior hypothalamus and suppressed
rx3 expression in adjacent host hypothalamic tissue
(n=22/29, Fig. 6A-C).
Complementing this, expression of the caudal hypothalamic marker emx2
was rostrally expanded in the presence of transplanted wnt8b (or
wnt8b + madh2CA)-expressing cells in the hypothalamus
(n=16/22; n=18/21; Fig.
6D-F and data not shown). Thus, expression of exogenous
wnt8b in the presumptive hypothalamus leads to expansion of posterior
at the expense of anterior hypothalamic marker gene expression.
|
![]() |
Discussion |
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Axin1/Wnt signalling regulates AP regionalisation of the ventral CNS
Our data show that Wnt/Axin/ß-catenin signalling must be suppressed
for ventral CNS cells to adopt hypothalamic rather than floorplate identity.
Furthermore, within the nascent hypothalamus Wnt signalling promotes posterior
at the expense of anterior identities. Taken together, it appears that Wnt
signalling influences both the initial subdivision of the ventral CNS into
hypothalamic and floorplate domains, and subsequently influences
regionalisation within the hypothalamic subdomain (promoting posterior at the
expense of anterior identity). A similar mechanism is likely to operate in
dorsal regions of the CNS (reviewed by
Wilson and Houart, 2004) where
early-acting Wnt signals are proposed to contribute to the initial regional
subdivision into forebrain, midbrain, hindbrain and spinal cord domains,
whereas later-acting Wnts and Wnt antagonists locally modulate levels of
signalling within individual domains, thereby further refining cell fate
decisions.
In our experiments, exogenous Axin1 only induced hypothalamic markers at the expense of floorplate in the midbrain and hindbrain and did not do so in all expressing cells. Furthermore posterior rather than anterior hypothalamic markers were induced in these experiments. A reasonable interpretation of these findings is that the increased levels of Axin1 activity only partially suppress Wnt signalling, neither sufficiently to induce markers of anterior hypothalamus nor sufficiently to suppress floorplate markers in the caudal CNS. This interpretation is also supported by the observation that exogenous Axin1 was less efficient at inducing nk2.1 in the floorplate of mbl embryos (which have enhanced Wnt signalling) than wild-type embryos.
Axin1 may promote Nodal signalling and integration into midline tissue
Our data show that activation of Nodal signalling promotes the
incorporation of epiblast cells into the MFP and PV hypothalamus. This is
consistent with previous data showing that cells unable to receive Nodal
signals are excluded from the PV hypothalamus
(Mathieu et al., 2002), and
that Nodal signalling is required for formation of MFP
(Strahle et al., 2004
). One
unexpected observation was that exogenous Axin1 promotes incorporation of
cells into midline neural tissue in a manner similar to reagents that activate
Nodal signalling. Indeed, Axin1 also promotes the cell-autonomous lateral
expansion of shh expression as does Madh2CA (this study)
(Müller et al., 2000
).
Furthermore, co-expression of axin1 together with low levels of
madh2CA leads to exclusion of cells from the CNS and incorporation
into mesendoderm, phenocopying the consequences of expression of high levels
of madh2CA alone. Altogether these results suggest that Axin1 may
facilitate Nodal signalling in addition to its well-established role in
antagonising Wnt signalling (Jones and
Bejsovec, 2003
; Tolwinski and
Wieschaus, 2004
). Indeed biochemical studies have suggested a
mechanism by which this could occur as Axin1 can bind and promote the activity
of Madh proteins functioning in the Nodal signalling cascade
(Furuhashi et al., 2001
). It
is also possible that Tcf/Lef proteins could directly modulate the
transcriptional activity of the Nodal pathway Madh proteins
(Labbe et al., 2000
;
Nishita et al., 2000
). Embryos
completely lacking Axin1 function have not been generated in fish but in mice,
such animals show multiple axes (Zeng et
al., 1997
), a phenotype unlikely to occur if Nodal signalling was
severely compromised. Therefore, although Axin1 may facilitate Nodal
signalling, it is unlikely to be an essential signal transduction component of
this pathway.
What is the source of Wnts and their inhibitors that influence ventral CNS development?
Although our studies provide compelling evidence that Wnt/ß-catenin
signalling influences the AP regionalisation of the ventral neural tube, we
have not identified the source of Wnts that promote posterior development nor
do we know the exact timing at which modulation of the pathway influences fate
decisions. One challenge for resolving this issue is that it is not known when
the fate choice between hypothalamic and floorplate identity is made. At early
stages, all axial midline neural cells appear to express similar genes
(Dale et al., 1997) and it is
not until late during gastrulation that hypothalamus-specific markers are
first expressed (Dale et al.,
1999
; Rohr et al.,
2001
). This suggests that fate decisions may only be determined
once the axial cells have extended along the CNS midline. However, there is no
evidence to rule out the possibility that fate decisions are initiated at
earlier stages than this, when the precursor populations are located close to
the organiser, prior to the extension movements of gastrulation. Given that
many other fate decisions are being made in and around the organiser from the
onset of gastrulation, or even earlier, then hypothalamic/floorplate fate
decisions could be initiated around this time. If so, there are both Wnt
ligands, such as Wnt8, and Wnt antagonists, such as Dkk1, that could influence
levels of Wnt activity in and adjacent to the domain of floorplate and
hypothalamic precursors (Erter et al.,
2001
; Foley et al.,
2000
; Kiecker and Niehrs,
2001a
; Pera and De Robertis,
2000
).
Hypothalamic versus floorplate identity different inducing signals or different responses to the same signals?
There are two classes of model to explain how ventral midline CNS tissue
acquires either hypothalamic or floorplate identity
(Wilson and Houart, 2004). The
first proposes that signals from underlying axial mesendoderm vary along the
AP axis and that depending upon the nature of the signals received, the
midline neural tissue differentiates either with floorplate or with
hypothalamic identity (Dale et al.,
1997
; Dale et al.,
1999
; Patten et al.,
2003
; Placzek et al.,
2000
). The second class of models proposes that the signals from
underlying axial mesendoderm are the same along the entire axis and that it is
intrinsic AP differences within the neural ectoderm that determine the
responses to these signals (Ericson et
al., 1995
; Kobayashi et al.,
2002
; Pera and Kessel,
1997
; Shimamura and
Rubenstein, 1997
). As we discuss below, these two models are not
mutually incompatible, and indeed it is likely that elements of both models
are correct.
Analysis of mbl embryos favours the idea that signals from axial
mesendoderm are largely unaffected in the mutants, but the responsiveness of
ectoderm to these signals is perturbed. This study and others have found
neither major changes in the prechordal plate or prospective notochord nor
phenotypes, such as cyclopia or defective induction of floorplate to suggest
that axial mesendodermal signals are perturbed. Conversely, there is ample
evidence to indicate that disrupted Axin1 activity within the neural plate is
responsible for defective regionalisation of this tissue. For instance,
restoration of Axin1 activity within anterior neural plate cells of
mbl embryos is sufficient to restore hypothalamic marker gene
expression. Axin1 only promotes hypothalamic markers in medial (prospective
ventral) neural plate cells, whereas in more lateral (prospective dorsal)
domains, it promotes expression of alar plate markers
(Heisenberg et al., 2001;
Houart et al., 2002
). It
appears therefore that Axin1 activity modulates the response of medially
positioned neural plate cells to axial signals, such as Nodals and Hhs that
induce ventral CNS identity.
There are many mechanisms by which levels of Wnt activity are modulated
within the forming neural plate, including spatially and temporally localised
expression of a variety of intracellular and extracellular agonists and
antagonists of signalling. Although many of the factors modulating levels of
Wnt activity are intrinsic to the neural plate
(Houart et al., 2002;
Dorsky et al., 2003
;
Kim et al., 2000
;
Kim et al., 2002
), there are
several secreted Wnt antagonists, such as Dkk1
(Kiecker and Niehrs, 2001b
;
Mukhopadhyay et al., 2001
),
expressed in mesendodermal tissues underlying the rostral neural plate. This
implies that the level of Wnt pathway activity, and hence the differential
responsiveness of ventral neural tissue to axial mesendodermal signals, may in
part be determined by secreted proteins that themselves originate in the
mesendoderm. Thus both the signals produced by mesendoderm and the
responsiveness of ventral neural tissue varies along the AP axis of the
nascent neural plate, and both are likely to contribute to the regionalisation
of axial CNS in response to signals that induce ventral fate.
One intriguing possibility is that intracellular modulators of
Wnt/ß-catenin signalling, such as Axin1, could influence levels of
pathway activity to some extent independent of extracellular ligands. Within
the prospective forebrain, the transcriptional repressor activity of Tcf
proteins is crucial for regionalisation of the neural plate
(Dorsky et al., 2003;
Kim et al., 2000
). Wnt ligand
signalling leads to alleviation of Tcf-dependent repression but, at least in
theory, transcriptional or translational regulation of intracellular Wnt
pathway proteins independent of Wnt ligand activity could also lead to changes
in levels of nuclear Tcf-dependent repression.
Although our study has focused upon the subdivision of axial neural tissue
into hypothalamic and floorplate domains, there is likely to be further
regionalisation within the floorplate itself. For instance, in fish, various
mutant conditions differentially affect anterior versus posterior floorplate
(Amacher et al., 2002;
Schier et al., 1997
), and this
fact may reflect different origins of anterior and posterior parts of the
floorplate and/or variation in the AP extent of expression of various
floorplate markers (Dale et al.,
1999
; Le Douarin and Halpern,
2000
; Patten et al.,
2003
).
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ACKNOWLEDGMENTS |
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