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First published online 14 January 2009
doi: 10.1242/dev.027334
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Department of Cell and Developmental Biology, Faculty of Life Sciences, University College London, Gower Street, London WC1E 6BT, UK.
* Author for correspondence (e-mail: r.mayor{at}ucl.ac.uk)
Accepted 4 December 2008
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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and activation of PKC
. We show that PKC inhibits Rac
GTPase and that c-Jun is a target of Rac. These findings might account for
previous reports implicating PKC in neural induction and allow us to propose a
link between FGF and PKC signalling pathways during neural induction.
Key words: Neural induction, Syndecan-4, FGF, PKC, Rac, JNK, AP-1, c-Fos
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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; Weinstein
and Hemmati-Brivanlou, 1997). There is compelling evidence that
BMP signalling and its modulation by endogenous inhibitors are involved in the
specification of neural and non-neural domains in Xenopus
(Iemura et al., 1998;
Piccolo et al., 1996;
Sasai et al., 1994;
Smith et al., 1993). However,
several challenges to the default model have originated from studies in chick
and ascidians, as well as from more recent experiments in Xenopus
(for a review, see Stern,
2005). There is now convincing evidence that in addition to BMP
inhibition, other signals are required for neural induction. One of these is
FGF, which is involved in the induction of neural tissue in chick and
Xenopus, as well as in Ciona
(Hongo et al., 1999;
Launay et al., 1996;
Streit et al., 2000;
Tannahill et al., 1992;
Wilson et al., 2000;
Bertrand et al., 2003). It has
been proposed that FGF regulates neural induction in animal caps and in
Xenopus embryos by activation of MAPK, which in turn phosphorylates
the BMP target Smad1, contributing to the inhibition of BMP signalling
(Fuentealba et al., 2007;
Graves et al., 1994;
Grunz and Tacke, 1989;
Hartley et al., 1994;
Kuroda et al., 2005;
Pera et al., 2003;
Sato and Sargent, 1989).
However, it is not known how the activity of FGF is regulated in the embryo to
account for its role during neural induction. As it is well established that
several proteoglycans (PGs) can regulate the activity of FGF, in some cases
working as co-receptors, we decided to study the role of PGs as potential
modulators of FGF during neural induction.
PGs are extracellular glycoproteins that contain sulphated
glycosaminoglycan (GAG) chains. Biochemical and cell culture assays have
implicated PGs as co-regulators of many growth factors, including FGF, HGF,
Wnt, TGFβ and BMP (Bernfield et al.,
1999; Iozzo,
1998). The GAG chains can be of heparan, chondroitin or dermatan
sulphate (Bernfield et al.,
1999; Iozzo,
1998). Syndecan-4 (Syn4) is a heparan sulphate PG reported to
modulate FGF signalling in vitro (Iwabuchi
and Goetinck, 2006; Tkachenko
et al., 2004; Tkachenko and
Simons, 2002). In addition, Syn4 interacts with chemokines
(Brule et al., 2006;
Charnaux et al., 2005) and with
the planar cell polarity (PCP) pathway
(Matthews et al., 2008;
Muñoz et al., 2006). As
Syn4 also interacts with fibronectin and integrins and is required for the
formation of focal adhesions (Woods and
Couchman, 2001), its main role has been thought to be in cell
migration. However, Syn4 is also able to modulate PKC- and small
GTPase-dependent intracellular signalling
(Bass et al., 2007;
Horowitz et al., 1999;
Horowitz and Simons, 1998;
Keum et al., 2004;
Matthews et al., 2008).
Here, we investigate the role of Syn4 in neural induction in
Xenopus. We report that Syn4 is expressed in ectoderm and
becomes restricted to the neural plate. Loss-of-function experiments show that
Syn4 is required for neural induction, whereas misexpression of Syn4 can
induce the expression of neural markers in animal caps or ventral ectoderm. We
also report that Syn4 activates two parallel pathways: the FGF/ERK pathway,
previously implicated in neural induction, and the PKC/Rac/JNK
pathway.
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MATERIALS AND METHODS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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).
Embryos were staged according to Niewkoop and Faber (Niewkoop and Faber,
1967). For normal development, embryos were incubated in 0.1x Marc's
Modified Ringer's Solution (MMR) until they reached the appropriate stage.
Animal caps were dissected at stage 9 and analysed at stage 14. Injected mRNA
was synthesised using the mMessage mMachine Kit (Ambion) following the
manufacturer's instructions. For the RacN17 experiments, we added a poly(A)
sequence that was not included in the original clone
(Tahinci and Symes, 2003).
Grafting of neuroectoderm has been described
(Linker and Stern, 2004). For
32-cell stage injection, the cell lineage was as described
(Moody, 1987).
Morpholino oligonucleotide and whole-mount in situ hybridisation
The Syn4 morpholino oligo (MO) was the same as that described previously
(Muñoz et al., 2006;
Matthews et al., 2008). For
rescue experiments, we used point-mutated Syn4 as described
(Matthews et al., 2008).
For in situ hybridisation, we followed the procedures described by Harland
(Harland, 1991), with the modifications described by Kuriyama et al.
(Kuriyama et al., 2006).
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Confocal microscopy
The mRNA for fluorescent fusion proteins (PKC-EGFP or
PKC-EGFP) was injected at the 2-cell stage in both blastomeres. The
membrane was visualised by co-injection of mRNA for membrane monomeric Cherry
(mCherry) protein. In Fig. 5,
the animal caps were dissected at stage 8, treated with 2 µM phorbol ester
(Sivak et al., 2005) or 10
ng/ml FGF2 (R&D), and fixed in MEMFA for 20 minutes. In
Fig. 6, mCherry mRNA with MO
was injected into 16-cell stage embryos after injection of PKC-EGFP
mRNA at the 2-cell stage. Images were taken with a Leica SP2 confocal
microscope.
Clones and constructs
Full-length cDNA clones (NIBB) were used for the analysis of Syn4
expression; these give a stronger signal than the probe previously published
by Muñoz et al. (Muñoz et
al., 2006). The National Institute for Basic Biology (Japan)
reference number of Syn4.1 is XL201e11, and Syn4.2 is XL457P08ex. The cDNA of
the c-Fos gene was isolated from Xenopus neurula cDNA, and
initially cDNA containing the 3'UTR was amplified by RT-PCR with the
following primers (sequences according to EST clone MGC80305): XbaI-Xl-c-Fos
Fw, 5'-CCGTCTAGAACAGAGCAGGATTTGCATTTATA-3' and Xl-c-Fos Rv,
5'-ACAGAATTCACAACAAGTCCATGCCAGT-3'. Xenopus laevis c-Fos
shares 63-65% identity with c-Fos from other species (data not shown). The
Xenopus laevis c-Fos ORF was amplified using the following primers:
ClaI-c-Fos Fw, 5'-ATATCGATGTATCACGCCTTCTCCAGCA-3' and XhoI-c-Fos
Rv, 5'-GCACTCGAGTGCCAATAGGGTAGGGGAGTT-3'. After checking that
there were no mutations in the sequence, the ORF was subcloned into the
pCS2+-GR vector.
Rac activation assay
Rac activity was analysed using the Rac1 Activation Assay Biochem Kit
(Cytoskeleton). Animal caps were dissected at stage 9 and cultured until stage
10.5 (data not shown) or 11.5. The total amount of protein used to bind the
PAK-RBD beads was adjusted in a pilot experiment using Bradford analysis
(BioRad). Around 100 animal caps were dissected for each condition, cell
lysates were centrifuged to remove the yolk fraction, and the supernatants
were used for the GDP/GTP-binding reaction according to the manufacturer's
instructions. Positive (GTP-bound) and negative (GDP-bound) controls were
performed as described by the manufacturer. For pulldown of active Rac, 10
µg of PAK-RBD beads was applied to each sample, boiled with Laemmli sample
buffer (Invitrogen) and loaded onto the gel.
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RESULTS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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), we focused on the ectodermal expression pattern. A longer
Syn4 probe (see Materials and methods), which gives stronger staining
than the probe described by Muñoz et al.
(Muñoz et al., 2006),
allowed us to characterise the neuroectodermal expression of Syn4.
During blastula stages, Syn4 is expressed transiently in a wide
region of the ectoderm (Munoz et al.,
2006) and very quickly becomes enriched in the prospective neural
tissues at the early and mid-gastrula stages
(Fig. 1A-D). During early
neurula stages, Syn4 expression was detectable only in the neural
plate (Fig. 1E,G,I), resembling
the expression of the neural plate marker Sox2
(Fig. 1F,H,J).
To determine whether Syn4 is required for neural induction, we performed
loss-of-function experiments using a mixture of two antisense morpholino
oligonucleotides (Syn4 MOs), as previously reported
(Muñoz et al., 2006;
Matthews et al., 2008).
Injection of Syn4 MO into dorsal animal blastomeres at the 8-cell stage
produced a strong inhibition of the neural plate markers Sox2 and
Nrp1 on the injected side, whereas no inhibition was observed when a
control MO was injected (Fig.
2A,B,E,F). A similar inhibition of neural plate markers was
observed when the injected embryos were analysed at later stages, indicating
that the effect of Syn4 MO is not merely a delay in gene expression, but a
true inhibition (see Fig. S1A,B in the supplementary material). As the
injection at the 8-cell stage might target some mesodermal cells and affect
neural induction indirectly, we used two approaches to inhibit Syn4
selectively in the prospective neural plate. First, Syn4 MO was injected at
the 32-cell stage into the A1 blastomere, which is fated to contribute to the
neural plate but not to the mesoderm
(Moody, 1987). Sox2
expression was inhibited in descendants of these Syn4 MO-injected cells
(Fig. 2C). This is specific for
Syn4 because it could be rescued by co-injection of mRNA encoding a mutated
Syn4 that does not bind to the MO (Fig.
2D). As an alternative approach, prospective neural plate taken
from an early neurula embryo injected with Syn4 MO or control MO was grafted
into the early neurula of an uninjected host, creating an embryo in which Syn4
MO is present only in the neural plate. The control graft still showed normal
Sox2 expression (Fig.
2G), whereas grafts of Syn4 MO-injected tissue showed loss of
Sox2 expression (Fig.
2H, asterisk). These results show that Syn4 is required in the
ectoderm for neural plate induction.
|
). When
co-injected with these BMP antagonists, Syn4 MO still blocked Sox2
expression in the embryo and in animal caps
(Fig. 2K,N), suggesting that
Syn4 does not function as a BMP antagonist in neural induction. Furthermore,
if Syn4 is a BMP antagonist, it would be expected to dorsalise mesoderm and to
induce a secondary axis, as do all BMP antagonists
(Harland, 1994). Whereas
chordin mRNA did induce a secondary axis, no such effect was observed after
injection of Syn4 mRNA (see Fig. S2A,B in the supplementary material),
consistent with the notion that Syn4 does not block BMP signalling.
Overexpression of Syn4 neuralises the ectoderm
The above results suggest that Syn4 is required for neural plate formation.
To test whether Syn4 can induce a neural fate, Syn4 mRNA was injected at the
32-cell stage into the A4 blastomere (which does not contribute cells to the
neural plate) (Moody, 1987).
Inhibition of BMP in this blastomere does not induce neural tissue
(Linker and Stern, 2004). By
contrast, injection of Syn4 mRNA into A4 did lead to induction of
Sox2 and Sox3 in the ventral epidermis
(Fig. 3A,B; see Fig. S2C,D in
the supplementary material) and to inhibition of epidermal marker expression
(Fig. 3C,D), without induction
of mesodermal markers (Fig.
3G-J). The induction of neural markers by Syn4 is not transient,
as they were still expressed at the late neurula stages (see Fig. S2H,I in the
supplementary material). Interestingly, this neuralisation by Syn4 was not
blocked by co-injection of a MO against chordin (see Fig. S2E-G in
the supplementary material)
(Oelgeschläger et al.,
2003), which is consistent with the idea that neural induction by
Syn4 is BMP independent. Furthermore, this induction of Sox2 is
cell-autonomous to the descendants of the injected cell, as revealed by
co-injection of nuclear β-galactosidase as a lineage tracer: all
Sox2-positive, epidermal keratin (EpK)-negative cells
exhibited X-Gal staining in the nucleus
(Fig. 3E,F). Finally,
overexpression of Syn4 induced neural plate markers and inhibited
epidermal markers in isolated animal caps
(Fig. 3K-N; see Fig. S2J,K in
the supplementary material), without expression of mesodermal markers (not
shown). Together, these gain- and loss-of-function experiments support a role
for Syn4 in neural plate development.
The FGF/MAPK signalling pathway is required for neural induction by Syn4
As Syn4 is a proteoglycan that binds growth factors, including FGF, through
its extracellular GAG chains, but can also modulate intercellular signalling
through its intracellular domain
(Couchman, 2003), we tested a
set of deletion constructs of Syn4 for neuralising ability. mRNA for each of
these constructs was injected into the A4 blastomere of a 32-cell stage embryo
and their ability to induce neural tissue was compared with that of
full-length Syn4 mRNA. Deletion of the GAG-binding domain (Syn4
GAG)
caused a modest, but reproducible, loss of neural induction ability (see Fig.
S3 in the supplementary material), whereas deletion of the intracellular
domain (Syn4CytCherry) had a stronger effect (see Fig. S3 in the
supplementary material). Together, these experiments implicate both the
extracellular and intracellular domains of Syn4 in neural induction (see Fig.
S3 in the supplementary material).
|
|
) and
FGF is involved in neural induction
(Fuentealba et al., 2007;
Kuroda et al., 2005;
Streit et al., 2000;
Wilson et al., 2000). This
raises the possibility that the effects of Syn4 gain- and loss-of-function are
due to interference with FGF signalling. Neural induction by Syn4 in animal
caps (Fig. 4A,B) was inhibited
by co-injection of a dominant-negative FGF receptor (XFD-1)
(Fig. 4C), as well as by the
presence of the FGF receptor inhibitor SU5402
(Fig. 4D) or the MEK inhibitor
U0126 (Fig. 4E), but not by the
inactive analogue U0124 (Fig.
4F). Moreover, Syn4 induced phosphorylation of MAPK in animal caps
cultured to stage 12.5 (the stage at which neural induction was analysed)
(Fig. 4G, lanes 1, 2). Note
that although FGF promotes MAPK phosphorylation at early gastrula stages
(Sivak et al., 2005), this
effect is not maintained when the animal caps are cultured until stage 12.5
(Fig. 4G, compare lanes 1 and
3). However, a high level of MAPK phosphorylation was observed at these stages
in animal caps treated with FGF and Syn4
(Fig. 4G, lane 4). Together,
these results indicate that Syn4 cooperates with FGF to activate the FGF/MAPK
signalling pathway and that this activation is required for neural
induction.
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The PLC/PKC pathway is involved in both FGF and Syn4 signalling
(Simons and Horowitz, 2001;
Sivak et al., 2005). Moreover,
PKC has been implicated in neural induction
(Otte et al., 1988;
Otte et al., 1989;
Otte et al., 1990;
Otte et al., 1991;
Otte and Moon, 1992), although
this has never been clearly connected with FGF or BMP signalling. Activation
of the PLC/PKC pathway by FGF leads to translocation of PKC to the
membrane (Kinoshita et al.,
2003; Sivak et al.,
2005). We tested whether this change in localisation is Syn4
dependent. PKC-GFP distribution was diffuse in untreated animal caps
(Fig. 5A-C), but on addition of
the PKC activator, phorbol ester (PMA), or of FGF2, the fusion construct
translocated to the cell membrane and colocalised with membrane Cherry
(Fig. 5G-L). Strikingly,
overexpression of Syn4 not only failed to promote PKC-GFP membrane
translocation (Fig. 5D-F), but
also inhibited translocation triggered by FGF2
(Fig. 5M-O). These results
suggest that Syn4 modulates FGF signalling by inhibiting PKC
activity.
PKC and PKC as downstream effectors of Syn4 during neural induction
As Syn4 inhibits PKC activity and induces neural tissue, we asked
whether direct inhibition of PKC is sufficient to neuralise ventral
ectoderm. Injection of a dominant-negative PKC RNA (DN-PKC)
(Kinoshita et al., 2003) into
the A4 blastomere induced Sox2
(Fig. 6A), whereas injection of
wild-type PKC mRNA into the endogenous neural plate region led to
inhibition of neural plate marker expression (see Fig. S1C,E in the
supplementary material). Furthermore, neural induction by Syn4 mRNA was
inhibited by co-injection of PKC mRNA
(Fig. 6B). These results
support the conclusion that Syn4 inhibits PKC
expression and that this inhibition is required for the neuralising activity
of Syn4.
To understand more about the mechanism of neural induction by Syn4, we
analysed some candidate downstream effectors of this PKC inhibition. It
has been shown in many systems that PKC and PKC activities
repress each other (Kinoshita et al.,
2003; Choi and Han,
2002) and that PKC is implicated in neural induction
(Otte et al., 1988).
Consistent with these findings, we found that injection of PKC mRNA
into the A4 blastomere induces Sox2
(Fig. 6C) and inhibits
EpK (see Fig. S1H in the supplementary material), whereas
co-injection of PKC mRNA (Fig.
6D) blocks this process.
In conclusion, our data support the hypothesis that activation of
PKC and inhibition of PKC promote neural induction, and that
these two kinases antagonise each other. The inhibition of PKC
expression by Syn4 mRNA and the inhibition of PKC expression
by PKC mRNA prompted us to analyse the relationship between Syn4 and
PKC in neural induction. We found that the induction of Sox2
by PKC mRNA (Fig. 6E) is
inhibited by co-injection of Syn4 MO (Fig.
6F), whereas dominant-negative PKC RNA blocks neural
induction by Syn4 (Fig. 6G).
Observations in cultured cells indicate that Syn4 recruits
phosphatidylinositol 4,5-biphosphate (PIP2) and translocates
PKC to the membrane (Keum et al.,
2004). We analysed the localisation of a PKC-EGFP fusion
protein that retains its neuralising activity when injected into ventral
ectoderm (Fig. 6H).
PKC-EGFP expressed in animal caps showed a spontaneous membrane
localisation that was not affected by co-injection with control MO
(Fig. 6I-K). However, mosaic
expression of Syn4 MO (cells labelled with an asterisk in
Fig. 6L-N) led to a complete
absence of PKC-EGFP from the membrane, indicating that Syn4 is required
for the activation of PKC. We therefore propose that neural induction
by Syn4 is mediated by activation of PKC and that this
activation requires Syn4.
Syn4 induces neural tissue in a MAPK- and PKC-dependent
manner. What is the link between the MAPK and PKC activities?
PKC is known to activate MAPKs, including p38-MAPK, ERK and JNK
(Mauro et al., 2002;
Rucci et al., 2005;
Seo et al., 2004;
Skaletzrorowski et al., 2005;
Wensheng, 2006). However, we
found no evidence that neural induction by PKC depends on MAPK. First,
induction of Sox2 in animal caps by PKC
(Fig. 7A, lane 3) was not
inhibited by the MEK inhibitor U0126 (Fig.
7A, lane 4), in spite of the strong inhibition of phosphorylated
MAPK (p-MAPK in Fig. 7A, lane
4). Second, PKC did not affect the phosphorylation of MAPK, as analysed
by western blot (Fig. 7A,B).
Therefore, our results do not support a direct link between MAPK activity and
PKC during neural induction, a discovery that prompted us to look for
downstream effectors of PKC in neural induction.
Rac/AP-1 as downstream effectors of PKC during neural induction
We have recently shown that Syn4 is a repressor of the small GTPase Rac
during neural crest migration in vivo
(Matthews et al., 2008),
whereas the Syn4/PKC/Rac/RhoA signalling complex appears to be a key regulator
of cell migration in vitro (Couchman,
2003). Could a similar pathway be involved in neural induction?
Our results showed that the normal levels of Rac activity found in a control
animal cap (Fig. 7C, AC lane 2)
are strongly inhibited by expression of Syn4
(Fig. 7C, Syn4 lane 4).
Furthermore, our data suggest that the inhibition of Rac activity by
PKC is a requirement for neural induction. Neural induction by
PKC misexpression (Fig.
7D) was inhibited by co-injection of a constitutively active form
of Rac (Fig. 7E,F). In
addition, expression of active Rac in the neural plate led to inhibition of
the endogenous neural plate (see Fig. S1D,F in the supplementary material). By
contrast, injection of a dominant-negative form of Rac into the A4 blastomere
strongly induced Sox2 (Fig.
7G), supporting the hypothesis that inhibition of Rac activity by
Syn4/PKC can induce neural tissue.
|
). Previous reports suggest that the AP-1 complex is required
for neural induction and that it binds directly to the promoter of the neural
plate gene Zic3 during this inductive process
(Leclerc et al., 1999;
Lee et al., 2004). One
hypothesis is that the PKC/Rac pathway facilitates formation of the
heterodimeric AP-1 complex (c-Fos/c-Jun) during neural induction. To test this
hypothesis, we constructed a hormone-inducible derivative of Xenopus
c-Fos that cannot homodimerise
(Halazonetis et al., 1988;
Nakabeppu et al., 1988); it
will only form the heterodimer c-Fos/c-Jun when c-Fos is
overexpressed, and is therefore expected to increase formation of the AP-1
complex. A similar approach has been described using c-Fos-ER to activate AP-1
in fibroblasts (Reichmann et al.,
1992). We made a construct containing the Xenopus c-Fos
gene fused to the human glucocorticoid receptor (GR; see Materials and
methods). Ectopic expression of c-Fos-GR was achieved by injecting mRNA into
the A4 blastomere and adding dexamethasone at stage 10.5, which induced
Sox2 (Fig. 7H),
whereas embryos that were not treated with dexamethasone did not upregulate
Sox2 (Fig. 7I). In
addition, western blot analysis revealed that PKC increases c-Fos
protein levels in animal caps (Fig.
7B).
Overexpression of c-Fos-GR was also used to rescue neural induction
inhibited by activation of Rac. Animal caps injected with chordin mRNA
expressed Sox2 (Fig.
7J,K), and, as expected, this induction was inhibited by
expression of activated Rac (Fig.
7L). However, this inhibition of neural induction could be
reversed by activation of the c-Fos-GR construct with dexamethasone
(Fig. 7M). It should be noted
that activation of c-Fos-GR is sufficient to neuralise the animal caps
(Fig. 7N,O). In conclusion, our
data are consistent with the idea that PKC promotes the formation of
AP-1 complexes that are required for neural induction through the inhibition
of Rac.
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DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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The role of Syn4 during Xenopus development has recently
been analysed, revealing its key role as a new element of the PCP pathways
during convergent extension and neural crest migration
(Muñoz et al., 2006;
Matthews et al., 2008). The
apparent lack of any effect on neural plate or neural crest induction in these
previous reports is likely to be due to the targeting of different regions of
the embryo. In order to see the effect of Syn4 MO on neural induction, the
injection has to be targeted to the prospective neuroectoderm, whereas
injections into prospective mesoderm, as published by Muñoz et al.
(Muñoz et al., 2006),
lead to convergent extension defects. In addition, Syn4 is expressed
in neural crest cells just before their migration starts, once they are
already specified (Matthews et al.,
2008) (and this work), which explains why the MO does not affect
neural crest induction. Taken together, these previous publications and the
data presented here indicate that the same signalling molecule can be involved
in induction and cell migration at different times during development.
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;
Alvarez et al., 1998;
Hongo et al., 1999) were
controversial, the evidence is now strong that FGF is indeed involved in the
induction of neural tissue (Sasai et al.,
1994; Smith et al.,
1993; Launay et al.,
1996; Linker and Stern,
2004; Pera et al.,
2003; Streit et al.,
1998; Streit et al.,
2000; Wilson et al.,
2000). Moreover, FGF contributes to the inhibition of BMP
signalling, at least in part by phosphorylation of Smad1 during neural
induction (Fuentealba et al.,
2007; Kuroda et al.,
2005).
Syn4 modulates FGF signalling through its extracellular domain (containing
the GAG-binding region, which will present heparin sulphates to which FGF is
expected to bind) and by an effect on the transduction of intracellular
signals (Hou et al., 2007;
Iwabuchi and Goetinck, 2006;
Horowitz et al., 2002). Our
data support the idea that FGF is required for neural induction and that Syn4
is a likely modulator, by showing that the inhibition of FGF receptor and of
MAPK activity impair neural induction by Syn4. Syn4 could act as a co-receptor
of the FGF receptor (Hou et al.,
2007) or as a presenter of the FGF ligand, through binding of FGF
to the GAG side-chains, to facilitate the activation of FGF receptor
(Fig. 8A).
However, Syn4 also plays a separate role in neural induction involving PKC
(Fig. 8B). We propose that this
involves inhibition of PKC and activation of PKC, and that
PKC is an inhibitor of the small GTPase Rac. Since the BMP-inhibiting
effects of FGF act through MAPK (Kuroda et
al., 2005), this pathway could account for the
BMP-inhibition-independent role of FGF signalling in neural induction
(Linker and Stern, 2004;
Delaune et al., 2005;
de Almeida et al., 2008). Rac
is a well-known regulator of cell migration that acts by controlling actin
polymerisation, but has not previously been implicated in neural induction.
Evidence that Rac can control JNK activity
(Chen et al., 2006;
Habas et al., 2003) suggested
the hypothesis that Syn4/PKC might inhibit Rac activity by an increase
in AP-1 (c-Fos/c-Jun) activity that is mediated through inhibition of JNK.
|
). The
relative abundance of AP-1 subunits seems to play a key role in gene
regulation (Eferl and Wagner,
2003). c-Fos cannot dimerise and requires c-Jun to be active
(Eferl and Wagner, 2003). When
c-Jun is phosphorylated by JNK it becomes latent and unable to bind to DNA
(Boyle et al., 1991).
Activation of JNK inhibits heterodimeric AP-1.
PKC activated by TPA (12-O-tetradecanoylphorbol-13-acetate)
dephosphorylates c-Jun and simultaneously increases AP-1 DNA-binding activity
(Boyle et al., 1991). This is
consistent with our results suggesting that Syn4/PKC promotes the
formation of the c-Fos/c-Jun complex (Fig.
8B). Additional support comes from the finding that overexpression
of PKC increases c-Fos levels in animal caps. Studies of the preneural
gene Zic3 revealed that AP-1 binds directly to the Zic3
promoter rather than to the c-Jun homodimer
(Lee et al., 2004). Taken
together, these data suggest that during neural induction, Syn4/PKC
might inhibit Rac to minimise JNK activity, facilitating formation of the
c-Fos/c-Jun (AP-1) complex.
A role for PKC in neural induction was first suggested almost 20
years ago (Otte et al., 1988;
Otte et al., 1989;
Otte et al., 1990;
Otte et al., 1991;
Otte and Moon, 1992) but had
never been connected with the signalling pathways now known to be involved in
neural induction. It was originally shown that PKC is activated and
translocated to the membrane during neural induction, and it was suggested
that this is required to confer neural competence on the ectoderm
(Otte et al., 1988;
Otte et al., 1989;
Otte et al., 1990;
Otte et al., 1991;
Otte and Moon, 1992). We have
confirmed and extended these observations by showing that expression of
PKC in ventral ectoderm or in animal caps can act as a neuralising
signal and that PKC activity is regulated by interactions with Syn4 and
PKC. PKC appears to work as a repressor of PKC, whereas
Syn4 appears to be required for PKC activity; however, we also show
that PKC is required for the neuralising activity of Syn4. Thus, our
finding allows us to propose a link between the PKC and FGF pathways, both of
which have been identified previously as being involved in neural
induction.
These observations have parallels in studies of migrating cells. Syn4
interacts with PIP2, and this stabilises the oligomeric structure
of Syn4 and promotes the association of PKC and Syn4
(Oh et al., 1997a;
Oh et al., 1997b;
Horowitz and Simons, 1998;
Lim et al., 2003); the
catalytic domain of PKC binds to the cytoplasmic domain of Syn4, and
PKC is `superactivated' (Lim et
al., 2003; Murakami et al.,
2002). This interaction between PKC and Syn4 provides a
satisfactory explanation for our observation that neural induction by
Syn4 requires PKC and vice versa. In addition, during cell
migration, PKC phosphorylates Syn4, decreases its affinity for
PIP2 and abolishes its capacity to activate PKC
(Couchman et al., 2002;
Murakami et al., 2002). We
have found a similar negative regulation between PKC and PKC
during early neural plate development.
Despite several previous reports demonstrating direct phosphorylation of
MAPK by PKC (Mauro et al.,
2002; Seo et al.,
2004), we found no evidence that the PKC and MAPK pathways
interact during neural induction other than indirectly, through Syn4.
Neuralisation by PKC is evidently MAPK-independent and PKC does
not affect MAPK activity. Another possibility is that Rac can affect MAPK
signalling via PAK-MEK interactions, the amino acids T292 and S298 of MEK1
being essential for PAK-dependent ERK activity
(Eblen et al., 2002). However,
T292 is not conserved in Xenopus MEK1 (not shown), which could
explain the absence of this regulatory pathway.
During cell migration, targets of the PKC pathway include small GTPases
that control cytoskeletal organisation and adhesion to the extracellular
matrix (Ridley et al., 2003).
Our results suggest that Syn4/PKC inhibits Rac activity during neural
induction, as it does in migrating cells
(Bass et al., 2007;
Matthews et al., 2008).
Expression of a dominant-negative Rac neuralises ventral ectoderm strongly,
whereas activation of Rac inhibits neural induction by PKC. However,
activation of Rac in ventral ectoderm has no effect on neural plate markers,
but induces neural crest markers (not shown), supporting recent reports of
induction of neural crest by Rac/Rho activities (Broders-Brondon et al., 2007;
Guemar et al., 2007).
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/136/4/575/DC1
|
Footnotes |
|---|
constructs; J.-K. Han for the pCS2+-human
PKC clone; N. Kinoshita for PKC constructs; and Oh and P. Kyung
for the PKC-EGFP construct; and NIBB Xenopus Resources (Japan)
for cDNAs. This work was supported by
MRC and
BBSRC grants to R.M. and by the
Uehara Memorial Foundation to S.K. Deposited in
PMC for release after 6 months. |
REFERENCES |
|---|
|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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