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First published online October 12, 2007
doi: 10.1242/10.1242/dev.007179
1 Department of Cell Biology, MCLM 360, University of Alabama at Birmingham,
Birmingham, AL 35294-0005, USA.
2 Department of Molecular and Cell Biology, and Center for Integrative Genomics,
University of California, Berkeley, Life Sciences Addition, #3200, Berkeley,
CA 94720-3200, USA.
e-mail: cchang{at}uab.edu; harland{at}berkeley.edu
Accepted 14 August 2007
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Neural induction, BMP, Smad1, Nodal, Smad2, Xenopus
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) (reviewed by
Harland, 2000). Since then,
equivalent organizing centers have been identified in other vertebrates,
including rabbit, mouse, chick and fish (Waddington, 1932;
Oppenheimer, 1936;
Beddington, 1994;
Shih and Fraser, 1996;
Knoetgen et al., 2000;
Saude et al., 2000); all of
these organizers induce ectopic neural tissues when transplanted into more
ventral regions. The hunt for substances that confer neural identity to the
ectoderm began soon after Spemann and Mangold's discovery
(Witkowski, 1985), but
progress in identifying endogenous neural inducers came only about 70 years
later with the application of molecular biological tools.
The first direct neural inducer cloned from a vertebrate was
noggin, which was subsequently shown to be an organizer-specific
soluble bone morphogenetic protein (BMP) inhibitor
(Smith and Harland, 1992;
Lamb et al., 1993;
Zimmerman et al., 1996). Other
organizer-secreted BMP antagonists, including chordin, follistatin, Xnr3 and
Cerberus, were then identified. Manipulations involving overexpression,
dominant-negative mutants and antisense depletion of these molecules as well
as BMP signaling components indicate that the BMP pathway plays an essential
role in neural induction (reviewed by
Harland and Gerhart, 1997;
Weinstein and Hemmati-Brivanlou,
1999; Harland,
2000; De Robertis and Kuroda,
2004; Vonica and Brivanlou,
2006). Taken together, the evidence suggests that ectodermal cells
have an inherent tendency toward the neural identity, but constitutive BMP
signaling in the ectoderm prevents realization of the neural fate. Signals
emanating from the organizer disrupt the BMP pathway, so that ectodermal cells
can follow their intrinsic program to adopt the neural lineage. This view is
known as the default model of neural induction
(Hemmati-Brivanlou and Melton,
1997).
Challenges to the default model emerged recently from studies both in
Xenopus and in other vertebrate species, particularly in chick; and
fibroblast growth factor (FGF) and canonical Wnt signals have both been
implicated in the neural induction process. In Xenopus, blocking FGF
signals by different means has been shown by some groups to prevent neural
induction in explants and in embryos, although others find anterior neural
induction to prevail when FGF/Ras signaling is moderately inhibited
(Ribisi et al., 2000).
Activation of FGF signaling was first shown to cause neural differentiation of
isolated cells by Kengaku and Okamoto
(Kengaku and Okamoto, 1993),
and subsequently the FGF-stimulated mitogen-activated protein kinase (MAPK)
pathway has been shown to cooperate with BMP inhibition to promote neural
induction (Lamb and Harland,
1995; Launay et al.,
1996; Hongo et al.,
1999; Linker and Stern,
2004; Delaune et al.,
2005; Wawersik et al.,
2005). Unlike organizer-expressed BMP antagonists, no FGF ligand
has been shown to be specifically localized to the region of neural induction
in Xenopus, so it appears that low-level FGF signaling may at most be
a permissive signal for general neural induction, whereas localized FGF
signaling is important for posterior patterning. This contrasts with the
probable localized role of FGF8 and FGF3 in the chick
(Streit et al., 2000;
Wilson et al., 2000).
Canonical Wnt signaling, mediated by ß-catenin and the TCF/Lef family of
transcription factors, also participates in neural induction, although it may
have opposite activities in promoting and inhibiting neural tissues at
different developmental stages (Baker et
al., 1999; Wessely et al.,
2001; Heeg-Truesdell and
LaBonne, 2006). Both FGF and Wnt pathways are reported to
crosstalk with and inhibit BMP signaling. The Erk members of the MAPK family
function downstream of FGF and insulin-like growth factor receptors to
phosphorylate the linker region of BMP-specific Smad1, resulting in
cytoplasmic retention of Smad1 and suppression of BMP signaling
(Pera et al., 2003). By
contrast, early Wnt signals are active over the entire dorsal domain of the
embryo as a result of cortical rotation, and act both to repress transcription
of BMP4 in the dorsal ectoderm and to stimulate expression of the BMP
antagonists noggin and chordin, thus ensuring the clearance of BMP ligands and
inhibition of BMP signals in the neural field
(Baker et al., 1999;
Wessely et al., 2001).
Although FGF and Wnt may have BMP inhibition-independent functions, the nature
of such actions is unknown.
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),
inhibition of BMP signaling by secreted antagonists, a truncated type I
receptor or the inhibitory Smad6 is not sufficient to induce neural marker
expression in prospective ventral epidermis of frog embryos or in the chick
extra-embryonic epiblast (Linker and
Stern, 2004; Delaune et al.,
2005). Clearly other conditions need to be met in order for neural
specification to occur in competent non-neural ectoderm in vivo. One such
condition may be a low level of FGF/ras signaling, as it induces neural
markers in combination with BMP inhibitors in Xenopus
(Linker and Stern, 2004;
Delaune et al., 2005;
Wawersik et al., 2005).
Indeed, even in the absence of localized BMP inhibitors, FGF8a expression
alone can induce ectopic differentiating neurons in the ectoderm
(Hardcastle et al., 2000;
Fletcher et al., 2006). Here we have addressed other potential mechanisms that may cooperate with BMP inhibition in neural induction. Proceeding from observations on the neural-inducing activities of reagents that block both Smad1 and Smad2, we have tested whether Smad2 inhibition may cooperate with Smad1 inhibition in neural induction. Our data suggest that neural induction in early Xenopus embryos exploits simultaneous suppression of BMP and nodal-like signals, and that combined inhibitory rather than instructive signals may be crucial for neural fate determination in vivo.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The embryos were injected with RNAs at 16- to 64-cell stages into one of
the dorsal or ventral animal blastomeres, as indicated in the Results section.
At gastrula stages, some of the embryos were treated with 2 µM
dexamethasone. The injected embryos were incubated to neurula and tailbud
stages and in situ hybridization was performed as described previously
(Harland, 1991). For
histological analyses, embryos were embedded in paraffin and sectioned at 10
µm.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Delaune et al., 2005). To see
whether this also holds true for nuclear proteins, we examined the ability of
two nuclear BMP inhibitors to induce neural markers in the ventral ectoderm. A
dominant-negative mutant of the transcription factor Msx1, VP16-Msx1, was
reported to interfere with the function of wild-type Msx1, a direct target of
BMP signaling, and block BMP-mediated ventralization of early frog embryos
(Suzuki et al., 1997;
Yamamoto et al., 2000). Ski, a
nuclear oncoprotein, was also shown to inhibit BMP signal transduction. It
binds to the BMP-specific Smad1 as well as the common Smad for transforming
growth factor ß (TGFß) signaling, Smad4, and helps to recruit the
transcription co-repressors N-CoR and histone deacetylase (HDAC) to suppress
BMP-dependent gene expression (Wang et
al., 2000; Luo,
2003; Takeda et al.,
2004). Both inhibitors can induce neural markers in animal caps
(Amaravadi et al., 1997;
Wang et al., 2000;
Ishimura et al., 2000). We
therefore co-injected RNAs encoding these nuclear factors with the nuclear
ß-galactosidase tracer (nßGal) into one ventral animal blastomere of
32- to 64-cell stage embryos. The embryos were allowed to develop to neurula
stages before they were collected, stained with a ßGal substrate (Red
Gal, Research Organics) and assayed by in situ hybridization for marker gene
expression. Interestingly, we observed that although both inhibitors blocked
the expression of the type I epidermal keratin XK70
(Winkles et al., 1985) and
induced the neural crest marker Slug
(Mayor et al., 1995), only Ski
was able to induce the neural markers Sox2 and Sox3 efficiently in the ventral
epidermal region (Fig. 1; 51/51
Sox2 and 45/45 Sox3 positive for Ski, and 6/77 Sox2 and 8/69 Sox3 positive for
VP16-Msx1). The induction of the neural genes was direct, as no mesodermal
markers, including chordin (notochord) and MyoD (paraxial mesoderm), were
expressed (see Fig. S1 in the supplementary material); although at higher
doses, we obtained induction of paraxial mesoderm underlying the neural tissue
(data not shown) (Mariani et al.,
2001). This result was intriguing, as it suggested that inhibition
of BMP signaling by Ski, but not by VP16-Msx1, was sufficient to convert
prospective epidermis into neural tissue in vivo.
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;
Luo, 2004). It is therefore
possible that neural induction occurs in Ski-expressing embryos via
simultaneous inhibition of both BMP and TGFß/nodal-like signals in the
ectoderm. To test this hypothesis, we assayed for neural-inducing activities
of various other inhibitors of TGFß signaling. We used two mutant
membrane receptors, a truncated type IA BMP receptor tBMPRIA (also known as
tBR or tALK3), which preferentially blocks the BMP pathway, and a truncated
type IIB activin receptor tActRIIB, which inhibits both BMP and
activin/nodal-like signals. We found that tBMPRIA induced Sox2 and Sox3 weakly
in most embryos (40/65 and 53/67 embryos positive for Sox2 and Sox3,
respectively), but tActRIIB induced Sox2 and Sox3 more strongly, so that
darker staining was observed in the epidermal region (76/83 and 70/72 embryos
positive for Sox2 and Sox3, respectively;
Fig. 1). We also analyzed two
cytoplasmic inhibitors of TGFß signaling. Smad6 preferentially interferes
with BMP/Smad1 signal through direct binding to Smad1 and preventing it from
association with Smad4 (Hata et al.,
1998); whereas Smad7 inhibits both Smad1 and Smad2 signaling
through interaction with different type I receptors and the Smurf E3-ubiquitin
ligases to direct the receptors to the ubiquitin-mediated degradation pathway
(Hayashi et al., 1997;
Kavsak et al., 2000;
Ebisawa et al., 2001;
Murakami et al., 2003). When
expressed in ventral ectodermal cells of early frog embryos, Smad6 was unable
to induce neural markers efficiently (4/69 Sox2-positive), although it induced
Slug expression (39/50 Slug-positive) and could induce secondary axes
effectively when expressed in the ventral marginal region
(Fig. 1 and data not shown). In
comparison, Smad7 was a robust neural inducer in vivo (43/50 and 42/46
positive for Sox2 and Sox3, respectively;
Fig. 1). None of the TGFß
inhibitors induced mesodermal markers at the doses we used, indicating that
the observed neural induction was direct (see Fig. S1 in the supplementary
material). To further confirm that the neural markers were induced in the absence of the mesoderm, we performed double in situ hybridizations. As shown in Fig. 2, tActRIIB, Smad7 and Ski all induced Sox2 and Sox3 in the absence of chordin and MyoD, verifying that neural induction occurred without prior induction of dorsal mesoderm. Taken together, our data demonstrate that inhibitors of both BMP/Smad1 and nodal/Smad2 signals induce neural markers directly in the ventral ectoderm of early frog embryos, whereas specific inhibitors of BMP/Smad1 signals are not efficient neural inducers. This suggests that neural induction in this context may require co-inhibition of both branches of TGFß signals.
Simultaneous inhibition of Smad1 and Smad2 signals leads to efficient neural induction
While the experiments above are consistent with the idea that simultaneous
inhibition of Smad1 and Smad2 results in neuralization, we wished to test
whether the simultaneous application of reagents that are known to be Smad1-
or Smad2-specific would have the same effect. To test this idea at the
receptor level, we co-expressed the truncated BMP receptor with a truncated
type IB activin receptor (tActRIB, or tALK4), which has been shown to be
incapable of blocking BMP-mediated epidermal induction in dissociated animal
cells, but was efficient in blocking Xenopus nodal-related ligands
(Xnrs) and activin (Chang et al.,
1997; Reissmann et al.,
2001). When expressed alone, tActRIB did not change epidermal cell
fate, whereas tBMPRIA induced neural markers weakly. Co-expression of tActRIB
and tBMPRIA, however, led to strong neural induction. The neural markers Sox2
and Sox3 were detected at high levels, mimicking the situation in which the
truncated type II receptor tActRIIB was ectopically expressed (48/60 and 52/63
were positive for Sox2 and Sox3, respectively;
Fig. 3A). In addition,
co-expression of tActRIB with either Smad6 or VP16-Msx1 led to induction of
Sox2 and Sox3 in the ventral ectoderm (19/30 Sox2- and 32/40 Sox3-positive for
Smad6, 15/37 Sox2- and 30/33 Sox3-positive for VP16-Msx1;
Fig. 3B). The enhanced neural
induction could not be attributed simply to the increased total amount of RNAs
injected, as the same dose of Smad6 RNA alone was not efficient in inducing
neural markers (1/11 Sox2-positive for 2 ng Smad6 injection). Our results thus
support the idea that blocking both Smad1 and Smad2 signals is sufficient for
neural induction in vivo.
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Anterior, but not posterior, neural tissues are induced in the ventral ectoderm
To examine whether the neural tissues induced by the inhibitors of Smad
signals exhibited anteroposterior patterning characteristics, we assayed for
the expression of Otx2 (fore- and midbrain), Engrailed 2 (En2, midbrain),
Krox20 (hindbrain) and HoxB9 (spinal cord) in the ectopic neural tissues. As
shown in Fig. 5, tActRIIB,
Smad7 and Ski all induced Otx2 (13/22, 14/30 and 23/30 positive) and En2
(3/20, 17/29 and 18/30 positive) at different efficiencies, but they failed to
stimulate the expression of Krox20 (0/22, 1/30 and 1/29 positive) and HoxB9
(0/22, 0/30 and 0/32 positive). The results indicate that inhibition of both
Smad1 and Smad2 signals leads to induction of anterior, but not posterior,
neural tissues.
Activation of Smad2 signaling at gastrula stages inhibits neural induction
If neural induction requires simultaneous inhibition of Smad1 and Smad2
signals, we would expect that activation of either signal in the presumptive
neural tissue can interfere with neural development. Consistent with this, it
has been reported that stimulation of BMP signaling via a constitutively
active type I receptor leads to inhibition of neural markers with concurrent
expression of epidermal genes in the neural plate
(Mariani et al., 2001;
Delaune et al., 2005). The
consequence of activation of Smad2 signaling in the neural tissue, however,
has not been examined; and this was the issue we addressed next.
To avoid the early mesoderm-inducing effects of Smad2 activation, we
constructed a chimeric protein containing the hormone-binding domain of
glucocorticoid receptor linked to the full-length Smad2. The resulting
GR-Smad2 exhibited dexamethasone (DEX)-dependent activity in inducing
mesodermal markers in animal caps. Consistent with the previous studies on
temporal responses to activin (Green et
al., 1990), the ability of GR-Smad2 to induce mesoderm declined
during gastrulation, so that if activated by mid-gastrula stages, it no longer
acted as a mesodermal inducer (Fig.
6A). To see whether activation of Smad2 signaling had any effect
on formation of neural tissues, we injected the RNA encoding GR-Smad2 with the
nßGal tracer into one of the dorsal animal cells of 16- to 32-cell stage
embryos and examined the expression of Sox2 at neurula stages. In the absence
of DEX, GR-Smad2 did not disturb the pattern of Sox2 transcription, so that
the red-Gal labeled cells were found to express Sox2 in the neural plate.
Treatment of the injected embryos with DEX at mid-gastrula stages, however,
severely interfered with the expression of Sox2. The nßGal-labeled cells
turned off Sox2 cell-autonomously at all positions along the anteroposterior
axis, so that Sox2-negative, nßGal-positive regions were observed near
the head, the trunk or the caudal end (Fig.
6B). The data indicate that activation of Smad2 signaling in
neural tissues at gastrula stages impairs neural development.
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In animal caps, neural markers can be induced by inhibition of BMP signaling. Activation of Smad2 signaling in naive caps, by contrast, stimulates mesodermal development, and the induced mesoderm can induce neural markers secondarily. To test whether activation of Smad2 signaling in neuralized animal caps may also boost both mesodermal and neural gene expression, we co-injected the RNAs encoding the soluble BMP antagonist noggin and GR-Smad2 into the animal poles of two-cell stage embryos. Assays for gene expression in animal caps by RT-PCR showed that GR-Smad2 did not prevent neural marker induction by noggin in the absence of DEX. However, treating the caps with DEX from blastula to early gastrula stages onward led to a strong inhibition of the neural markers NCAM and NRP-1 with concurrent induction of the mesodermal markers muscle actin and type II collagen. In addition, the neural crest markers Slug and Twist were also induced in these caps (Fig. 9A and data not shown). The ability of GR-Smad2 to inhibit neural markers and induce mesoderm declined progressively when it was activated at mid- to late gastrula stages (Fig. 9A). Our in vitro explant assays were thus consistent with our in vivo results to suggest that activation of Smad2 signaling can inhibit neural development and promote mesoderm formation during gastrulation.
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Activation of Smad2 signaling during gastrulation leads to defective neural development
If aberrant activation of Smad2 signaling interferes with neural
development, we would expect that resulting embryos would display defects in
the nervous system. We therefore examined the embryos injected with GR-Smad2
and treated with DEX at gastrula stages. Tadpoles expressing GR-Smad2 without
exposure to DEX developed normally; but if DEX was added at mid-gastrula
stages, the resulting tadpoles showed severe head defects with malformed or
missing eyes (Fig. 10A). It
has been suggested that Smad2 signaling is important in anteroposterior
patterning of vertebrate embryos (Piccolo
et al., 1999; Sun et al.,
1999; Feldman et al.,
2000; Sirotkin et al.,
2000; Thisse et al.,
2000; Lu et al.,
2001; Andersson et al.,
2006). However, it has been difficult to document any direct
effect of Smad2 signaling on neural patterning against a background of Smad2
induction of mesoderm, which would also secrete caudalizing signals
(Eimon and Harland, 2002). We
therefore addressed whether the head defects might reflect a direct effect on
anteroposterior patterning by Smad2 signaling. We thus examined these embryos
both histologically and by in situ hybridization. Transverse sections of the
embryos revealed that the neural tube was disrupted at both the head and the
trunk levels (Fig. 10B and
data not shown). Ectopic notochord and mesenchyme were often observed to split
the neural tube or compress the size and change the position of the neural
tube (Fig. 10B). The formation
of endodermal and mesodermal derivatives, including the gut, the notochord and
the somites, seemed to be normal. Gene expression studies indicated that Sox2
was greatly reduced along the entire anteroposterior axis, and separate
stripes of weak Sox2 could be seen in some embryos
(Fig. 10C and data not shown),
consistent with the neural tube defects. Otx2 was still expressed, although at
lower levels. In contrast to the neural markers, the neural crest marker Twist
was expressed normally, and the muscle marker MyoD was equally unaffected
(Fig. 10C). The data indicate
that activation of Smad2 signaling during gastrulation in the neural plate
interferes with neural development, resulting in neural tube defects. However,
these results are consistent with the diversion of neural tissues to a
non-neural mesodermal fate by Smad2 signaling, and there remains no evidence
for a direct posteriorizing role of Smad2 signals.
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Delaune et al.,
2005; Wawersik et al.,
2005). To date, FGF signaling has been emphasized as the `missing
ingredient' in this. However, in our current study, we find that inhibition of
both BMP/Smad1 and nodal-like/Smad2 signaling is sufficient for efficient
neural induction in ventral ectoderm of early frog embryos. Single reagents,
such as Ski or Smad7, which inhibit both signals, are efficient neuralizing
agents (Figs 1 and
2); and separate reagents, such
as the truncated nodal receptor and Smad6, which are well-characterized
inhibitors of individual pathways, work together to neuralize ectoderm (Figs
3 and
4). To some extent, there is an
expectation that both pathways would need to be inhibited, as each will induce
a non-neural tissue, epidermis or mesoderm, on its own. It has always been
assumed that the epidermis has not experienced significant Smad2 signaling,
and thus it was unexpected that inhibition of Smad2 signaling potentiates the
neural-inducing activity of Smad1 inhibitors so strongly in the context of
ventral epidermis. However, evidence that the ectoderm does experience some
Smad2 activation comes from experiments in which animal caps have been cut
large or late, where mesoderm is induced in the presence of the competence
modifying signals from Wnts or noggin
(Christian et al., 1992;
Lamb et al., 1993;
Sokol, 1993). In any case, our
results emphasize the requirement in neural induction that all TGFß
signals need to be inhibited for neural specification to occur.
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; Branford and Yost,
2002; Tanegashima et al.,
2004; Cha et al.,
2006). In ectoderm, a Dan/Cerberus family member, Coco, moderates
both BMP and nodal/activin signaling to regulate cell fate specification and
competence (Bell et al., 2003).
Both BMP and nodal-like signals are also inhibited by an ectodermally
expressed Smad4 ubiquitin ligase, Ectodermin
(Dupont et al., 2005).
Furthermore, a transcription factor belonging to the foxi-class of
winged-helix proteins, Xema/FoxI1e, is localized exclusively in the ectoderm
and represses mesoderm induction by activin/nodal-like growth factors
(Suri et al., 2005;
Mir et al., 2007). Depletion
of Lefty/Antivin, Ectodermin and Xema all leads to expansion of mesodermal
genes toward the animal region, and in the case of Ectodermin and FoxI1e, the
neural tissue is reduced (neural development in Lefty morphant embryos has not
been studied in detail). Thus the available evidence is consistent with the
view that ectodermal cells adopt a neural fate when both nodal-like and BMP
signals are inhibited simultaneously to prevent mesodermal and epidermal
development, respectively. In line with this idea, it has been reported that
both phosphorylated Smad2 and Smad1 are completely cleared from the neural
plate by early neurula stages in frog embryos
(Schohl and Fagotto,
2002).
Despite the presence of multiple stage- and tissue-specific endogenous
modifiers of Smad2 activity, the ventral ectoderm must perceive significant
Smad2 signaling, as its response to BMP inhibitors is radically altered by the
simultaneous blocking of Smad2 signaling. Activation of Smad2 has been studied
in the relevant stages from blastula to gastrula
(Faure et al., 2000;
Lee et al., 2001;
Schohl and Fagotto, 2002).
Numerous ligands, such as Xnr1 and Derriere
(Sun et al., 1999;
Lee et al., 2001;
Eimon and Harland, 2002), are
deployed close to the ventral ectoderm, so it is not surprising that the
ventral epidermis would have experienced some Smad2 signaling at the relevant
stages. Consistent with this, phosphorylated Smad2 has been detected in the
ventral ectoderm of frog gastrulae and in the epidermis of neurula and tailbud
embryos (Schohl and Fagotto,
2002). It is not clear whether this low level of signaling has an
effect on epidermis; and perhaps a more detailed examination of different
epidermal markers might reveal an instructive role for Smad2 in the diversion
of the atypical epidermis that is induced in animal caps compared to the
epidermis that develops in whole embryos.
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; Basch and Bronner-Fraser,
2006), this raises the possibility that Smad2 activity may also
contribute to neural crest specification.
FGF/MAPK signaling in neural induction: inhibition of Smad1 and Smad2?
In Xenopus neural induction, several models have been proposed
that emphasize the involvement of different signals. The default model states
that BMP-free ectoderm assumes neural fate autonomously, whereas other views
stress the importance of additional signaling, such as the IGF/FGF pathways.
Our experiments offer an alternative explanation for several unresolved
issues. For example, inhibition of BMP signaling in animal caps is sufficient
for neural induction, but is not efficient in neural specification in ventral
ectoderm. We now recognize the involvement of Smad2 signals in the suppression
of neural induction in the whole embryo context. Another puzzle is the role of
the Ras/MAPK pathway in neural induction. IGF and FGF have been shown to
activate Ras/MAPK to inhibit Smad1 through phosphorylation of the linker
region of Smad1, and FGF may also regulate BMP expression
(Pera et al., 2003;
Delaune et al., 2005;
Kuroda et al., 2005). The
Ras/MAPK signaling may thus converge with the BMP pathway during neural
induction. However, it is also reported that FGF may have BMP-independent
effects in neural specification, although the mechanism is unknown
(Delaune et al., 2005). In
light of our current finding and previous studies in mammalian cell culture
(Kretzschmar et al., 1999), we
propose that in addition to blocking Smad1, FGF/MAPK signaling may also
inhibit Smad2 through linker phosphorylation, and this may contribute to the
synergistic effect on neural induction by BMP inhibitors and low FGF/ras/MAPK
signaling (Linker and Stern,
2004; Delaune et al.,
2005; Wawersik et al.,
2005). Indeed, inactivation of Smad2 by linker phosphorylation has
been correlated with loss of competence of gastrula ectoderm to respond to
activin-mediated mesodermal induction
(Grimm and Gurdon, 2002). In
this case, the relative levels of Smad2 and FGF signals may be important, as
high levels of both signals are required for mesoderm induction, while low
levels of both signals may lead to Smad2 inhibition and neural
development.
Conserved and divergent mechanisms of neural induction during animal evolution
When compared with other animals, we find that both similar and divergent
mechanisms may be utilized during neural induction. One common, although
under-emphasized, theme for cells to adopt a neural fate in all species is
that cells choose not just between epidermal and neural fates, but also neural
and mesendodermal fates (Harland,
2000). This is demonstrated, for example, by a common precursor
for spinal cord and notochord in the non-vertebrate chordate ascidian
(Lemaire et al., 2002) and by
conversion of dorsal mesoderm to neural ectoderm in nodal-signaling-deficient
zebrafish embryos (Feldman et al.,
2000; Schier and Talbot,
2001). In the chick, the transcription factor Churchill acts
through upregulation of Smad-interacting-protein-1 (Sip1) to block mesoderm
induction and permit neural development in competent epiblast
(Sheng et al., 2003). In the
mouse, the lack of the mesendodermal specification signaling factor nodal
leads to precocious neural differentiation
(Camus et al., 2006). All
these results indicate that obstruction or loss of response to
mesoderm-inducing factors may be an essential first step for cells to adopt a
neural fate. A less conserved mechanism among different chordates concerns
which signaling pathways are involved in neural specification. In ascidians,
the Ras/MEK/Erk pathway downstream of FGFs has been shown to regulate neural
development directly by modulating the promoter activities and therefore the
expression of neural-specific genes
(Hudson and Lemaire, 2001;
Bertrand et al., 2003;
Hudson et al., 2003). BMP
inhibition does not appear to be important for early neural induction in
ascidian (Lemaire et al.,
2002), and may not be an ancestral mechanism in the chordates, as
the hemichordate outgroup also shows no correlation of BMP signaling in the
neural versus epidermal choice (Lowe et
al., 2006). FGF signaling has been strongly implicated in neural
induction in the chick (Alvarez et al.,
1998; Streit et al.,
2000; Wilson et al.,
2000), and inhibition of Wnt signaling is also required for early
neural induction in the chick (Wilson et
al., 2001). In these cases, FGF and Wnt signals may crosstalk with
the BMP pathway to affect neural induction. Indeed, it has been shown that in
both chick and zebrafish, FGF signaling regulates expression of BMP ligands
and/or BMP antagonists (Wilson et al.,
2000; Furthauer et al.,
2004; Londin et al.,
2005). Nodal signaling may also have a direct effect on neural
induction in mammals, as in human embryonic stem cells nodal inhibits neural
differentiation while promoting cell maintenance in a pluripotent state
(Vallier et al., 2004); and in
mice deficient in nodal, anterior neural tissues form precociously
(Camus et al., 2006). Although
it may be surprising that different chordates have exploited different
pathways as precursors to neural induction, the ultimate loss of BMP signaling
and an absence of mesoderm-inducing signals in the neural precursors remain a
common theme in the vertebrates.
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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