1 Sezione di Biologia Cellulare e dello Sviluppo, Dipartimento di Fisiologia, e
Biochimica, Universita' degli Studi di Pisa, Via Carducci 13, 56010 Ghezzano
(Pisa), Italy
2 AMBISEN Center, High Technology Center for the Study of the Environmental
Damage of the Endocrine and Nervous Systems, Universita' degli Studi di Pisa,
Italy
3 Department of Anatomy and Developmental Biology, University College of London,
Gower Street, London WC1E 6BT, UK
* Author for correspondence (e-mail: andream{at}dfb.unipi.it)
Accepted 16 February 2005
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SUMMARY |
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Key words: Six3, Cell proliferation, Cell fate, Bmp4, Xenopus, Zebrafish
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Introduction |
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Specification and patterning of the anterior neural plate is crucially
dependent upon the spatial localisation of BMP signalling activity. At early
stages, BMP genes are expressed throughout the ectoderm, but, before the
beginning of gastrulation, transcription is downregulated dorsally, where the
neural plate is going to form (Wilson and
Edlund, 2001; Stern,
2002
; Kuroda et al.,
2004
). Two subsequent steps appear to regulate BMP expression and
activity in the dorsal ectoderm. First, at blastula stages, Wnt and FGF
signalling is crucial to suppress the transcription of BMP genes in the dorsal
ectoderm (Baker et al., 1999
;
Bally-Cuif and Hammerschmidt,
2003
; Kuroda et al.,
2004
). Second, by the late blastula stages, BMP antagonists, such
as Noggin and Chordin secreted by the organizer, interact with BMPs and
prevent their binding to receptors. Bmp4 transcription is maintained
by an autoregulatory loop, where BMP4 protein bound to its receptor stimulates
transcription of the Bmp4 gene
(Jones et al., 1992
;
Hammerschmidt et al., 1996
;
Piccolo et al., 1997
). The
activity of BMP antagonists can interrupt this positive-feedback loop.
How different signalling pathways such as Wnt, FGF and BMP are integrated
during early ectodermal development is still poorly understood. In anamniotes,
it is likely that the activity of Wnt/ßcatenin induces dorsal activation
of genes that modulate FGF signalling; once activated, FGFs mediate an early
restriction of BMP expression and activate the expression of BMP antagonists
in the organizer (Furthauer et al.,
2004; Tsang et al.,
2004
; Kudoh et al.,
2004
). Despite the early repression of BMP activity in presumptive
neural territories, BMP signalling maintains the ability to inhibit expression
of anterior neural plate genes even during neurulation
(Hartley et al., 2001
). This
is of particular relevance considering that the anterior neural plate is
surrounded by non-neural ectoderm and is underlain by anterior mesendoderm,
both of which are sources of BMPs. Persistent suppression of BMP transcription
in the anterior neural plate may be maintained by specific transcriptional
repressors activated after neural induction
(Hartley et al., 2001
). This
is indeed the case for XBF2 and Xiro1, which act as Bmp4
transcriptional repressors at early neurula stage, thus ensuring proper neural
fate acquisition (Mariani and Harland,
1998
; Gomez-Skarmeta et al.,
2001
).
In addition to suppression of BMP signalling, the rostral neural plate must
be protected from the activity of caudalising signals for it to establish
anterior forebrain character. Among the signals that promote posterior neural
identity are Wnts, and a variety of Wnt antagonists ensure that Wnt activity
is suppressed rostrally. The neural plate is patterned along its
anteroposterior axis by the graded activity of Wnts, Wnt antagonists and other
signals (Wilson and Houart,
2004). This is established by the interplay of Wnt antagonists
secreted by the anteriormost neuroectoderm and the underlying mesendoderm, and
local sources of Wnt signals in the posterior neuroectoderm, midbrain,
diencephalon and mesendodermal tissues
(Heisenberg et al., 2001
;
Kiecker and Niehrs, 2001
;
Houart et al., 2002
). An early
event in neural plate patterning is the generation of an anterior region that
comprises the presumptive telencephalon, diencephalon and retina. The
repression of both Wnt and BMP signalling, together with an enhanced
proliferative activity, are crucial for the formation of the anterior neural
plate (Zuber et al., 2003
;
Wilson and Houart, 2004
). In
fact, embryos with blocked cell proliferation or exaggerated Wnt or BMP
signalling display anterior deficiencies
(Hammerschmidt et al., 1996
;
Kim et al., 2000
;
Houart et al., 2002
;
Andreazzoli et al., 2003
;
Zakin and De Robertis,
2004
).
Downstream of the signals that subdivide the ectoderm, a variety of
transcription factors mediate neural plate patterning. Among these Bf1,
Bf2, Rx, Six3 and Otx2, expressed in the anterior neural plate,
are crucially involved in the formation of anterior CNS structures
(Acampora et al., 1995;
Andreazzoli et al., 1997
;
Mathers et al., 1997
;
Bourguignon et al., 1998
;
Mariani and Harland, 1998
;
Andreazzoli et al., 1999
;
Loosli et al., 2001
;
Carl et al., 2002
;
Lagutin et al., 2003
).
However, although there is some evidence that links the activity of these
transcription factors with those of the Wnt and BMP pathways in the formation
of the CNS (Braun et al.,
2003
; Lagutin et al.,
2003
), their exact interplay is still scarcely understood.
In this study, we show that Six3 displays the characteristics
expected from an effector of neural inducers involved in specifying and
maintaining anterior neural plate properties. Xsix3 overexpression
promotes cell proliferation and inhibits neurogenesis at early neurula stage
by activating Xhairy2, Zic2, Xrx1 and Xbf1 and regulating
the expression of p27Xic1 and cyclinD1. Furthermore,
Six3 represses BMP expression in both Xenopus and zebrafish,
and is able to rescue the anterior neural plate defects of chordino
mutants. The effect of Xsix3 on Bmp4 appears to be direct,
as suppression occurs even in the absence of protein synthesis and
Xsix3 can bind the Bmp4 promoter in vitro. Although
Xsix3 efficiently suppresses Bmp4 expression, we observed
that it is unable to induce neural tissue, requiring Xotx2 for this
activity. Taken together with the recent observation that Six3 is
able to repress Wnt expression (Braun et
al., 2003; Lagutin et al.,
2003
), these data indicate Six3 as a crucial factor for
anterior neural plate specification.
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Materials and methods |
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In situ hybridization
Whole-mount in situ hybridization experiments on Xenopus and
zebrafish embryos, and on animal caps, were carried out essentially as
described previously (Harland,
1991; Barth and Wilson,
1995
). Bleaching of Xenopus pigmented embryos was carried
out following colour reaction as described by Mayor et al.
(Mayor et al., 1995
). The
templates for the production of in situ hybridization probes for Xag
(Bradley et al., 1996
),
Xcg (Sive et al.,
1989
), Xsix3 (Zuber
et al., 2003
), Xrx1
(Casarosa et al., 1997
),
rx3 (Chuang et al.,
1999
), Zic2 (Brewster
et al., 1998
), N-tubulin (Hartenstein et al., 1989),
elrC (Perron et al.,
1999
), Xash3 (Turner
and Weintraub, 1994
), Xbf1 (Bourgouignon et al., 1998),
Xdelta1 and Xngnr1 (Ma
et al., 1996
), Xnotch
(Chitnis and Kintner, 1996
),
Xsix1 (Pandur and Moody,
2000
), p27Xic1
(Hardcastle and Papalopulu,
2000
), Xhairy2
(Koyano-Nakagawa et al.,
2000
), Sox9 (Spokony
et al., 2002
), Xslug
(Mayor et al., 1995
),
Xnrp1 (Knecht et al.,
1995
), Bmp4 (Fainsod
et al., 1994
), bmp4
(Nikaido et al., 1997
),
dlx3 (Akimenko et al.,
1994
), Sox2 (Misuzeki et al., 1998), sox3
(Kudoh et al., 2001
),
Xk81 (Jonas et al.,
1985
), Xwnt8
(Christian et al., 1991
),
Xotx2 (Pannese et al.,
1995
), otx2 (Li et
al., 1994
), hoxb1b
(Eisen and Weston, 1993
) and
cyclinD1 (Vernon et al., 2003) have been described previously.
Mitotic inhibition with hydroxyurea and aphidicolin treatment
Xenopus embryos were devitellinized at stage 9 and allowed to
recover for 30 minutes in 2% Ficoll in 0.1xMarc's modified Ringer (MMR).
Stage 10 embryos were then incubated in 0.1xMMR containing 20 mM
hydroxyurea and 150 µM aphidicolin (HUA)
(Harris and Hartenstein, 1991;
Hardcastle and Papalopulu,
2000
) until fixation. The effect of hydroxyurea and aphidicolin
treatment on cell division was examined as described previously
(Andreazzoli et al., 2003
).
Embryo microinjections and BrdU incorporation
Capped VP16-Six3 RNA was generated from a construct containing the
full-length Xsix3 cDNA cloned into Cla1/Xhp1 sites
of CS2-VP16 vectors (Kessler,
1997). To make the GR-Xsix3 expression construct, the
open reading frame of Xsix3 was PCR amplified using the primers
5'-GCAGATATCATGGTGTTCAGGTCCCCTC-3' and
5'-GTCCTCGAGTCATACGTCACATTCAGAGTCAC-3' and inserted
into the EcoRV/Xho1 site of pCS2+/GR kindly provided by
Thomas Sargent (see Kodjabachian and
Lemaire, 2001
). RNAs encoding for Xsix3 (20, 40 pg),
GR-Xsix3 (65 pg), Xchh (1 ng)
(Ekker et al., 1995
),
Xngnr1 (40 pg) (Ma et al.,
1996
), tBR (250 pg) and Bmp4 (300 pg)
(Kazanskaya et al., 2000
) were
generated by in vitro transcription using the message machine kit (Ambion,
Austin, TX) and co-injected with lacZ RNA into one blastomere at the
two-cell stage (Xchh and Xngnr1), or into a dorsal animal
blastomere at the four- to eight-cell stage (Xsix3, tBR, Bmp4).
ß-Galactosidase staining was performed on embryos injected with 200 pg of
lacZ RNA as previously described
(Turner and Weintraub, 1994
);
X-Gal and salmon gal substrates were used for blue and red staining,
respectively (Roche, Biosinth-AG). The optimal concentration of each batch of
RNA was identified through the injection of various doses followed by analysis
of either the phenotype or the expression of specific markers. Three different
Xsix3 antisense morpholinos were used (Gene Tools): MoXsix3
targeted against the two pseudoallelic genes of Xsix3, Xsix3.1 and
Xsix3.2 (5'-ACCTGAACACCATGGGATGGCCGG-3')
(Ghanbari et al., 2001
);
MoXsix3.2 targeted against Xsix3.2, the gene that we used
for overexpression experiments (5'-CAGCAAAACTAGCGACAGCGACAGC-3');
and MoSix3.1 targeted against Six3.1
(5'-TGAAAGAAGCGGCAGCAACACTAGC-3'). Differences in the efficiency
of morpholinos were observed; whereas injection of 0.2 mM of MoSix3.1
at the two-cell stage does not induce abnormal development, injection of 0.2
mM of both MoXsix3 and MoXsix3.2 leads to a Xsix3
loss-of-function phenotype. Given that the frequency of affected embryos in
the case of MoXsix3 (95%) injection was higher than with
MoXsix3.2 (45%), we decided to use MoXsix3 for our
studies.
Morpholino oligonucleotides targeted against zebrafish chordin,
reliably phenocopying the mutant chordino phenotype, were injected
into one-cell stage embryos at concentrations of 0.15 mM
(Nasevicius and Ekker, 2000).
In vitro transcription of zebrafish six3 RNA and injection of 19-74
pg was performed as described (Kobayashi
et al., 1998
).
S-phase cells were labelled with BrdU essentially according to the protocol
of Hardcastle and Papalopulu (Hardcastle
and Papalopulu, 2000). To distinguish the injected from the
control side of the embryos, we combined immunohistochemistry with in situ
hybridization using Zic2 expression, a gene strongly upregulated upon
Xsix3 overexpression (see below). In these experiments the number of
BrdU-positive cells in the injected side was compared with that of the
uninjected control side, taking into account also their anteroposterior
distribution. Whole-mount TUNEL staining was performed at stage 13, as
described by Hensey and Gautier (Hensey
and Gautier, 1998
).
Animal cap assay and cycloheximide treatment
For Xenopus animal cap experiments, capped synthetic
chordin (150 pg per blastomere)
(Sasai et al., 1995),
tBR (600 pg) (Wylie et al.,
1996
), Bmp4 (1 ng), Xotx2 (250 pg)
(Vignali et al., 2000
),
Xotx2-GR (50 pg) (Gammill and
Sive, 1997
), Xsix3 (500 pg-2 ng) and GR-Xsix3
(500 pg-1ng) RNA was injected into one-cell stage embryos and animal caps
dissected at stage 9. When sibling control embryos reached stage 14, animal
caps were fixed and stored in ethanol at 20°C. To inhibit protein
synthesis, animal caps were isolated from GR-Xsix3 or
Xotx2-GR injected embryos at stage 9 and aged in high-salt Modified
Barth's Saline (MBS). Stage 10.5 caps where then incubated in high-salt
containing 10 µM of cycloheximide (CHX). Glucocorticoid receptor (GR)
fusion proteins were activated at stage 11, after a 30-minute CHX
pre-incubation step, by adding DEX 10 µM into the medium containing CHX.
Animal caps were then fixed at stage 15.
Gel mobility-shift assay
GST-Xsix3 protein purification was performed as described
(Tessmar et al., 2002). A
315-bp PCR fragment of Bmp4 promoter was 32P-end-labelled
by T4 kinase (Roche). Binding conditions and electrophoresis were as described
by Gomez-Skarmeta et al. (Gomez-Skarmeta
et al., 2001
).
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Results |
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To gain insight into how Six3 may act on proliferation in the anterior neural plate, we analyzed the expression of putative Six3 target genes known to be involved in cell proliferation control that are expressed rostrally at early neurula stage. We found that Xbf1 and Xrx1 are both ectopically activated in the anterior neural plate (Xbf1: 86%, n=45; Xrx1: 72%, n=90; Fig. 1H,I) by exogenous Xsix3. CyclinD1, a positive regulator of the cell cycle expressed in the eye field, is ectopically activated by Xsix3 (stage 14, 82%, n=34; Fig. 1J), whereas p27Xic1, which encodes a cell cycle inhibitor, is repressed by Xsix3 (100%, n=39; Fig. 1K,L). CyclinD1 and p27Xic1 expression is modulated both in the anterior and posterior neural plate (Fig. 1J-L). Thus, Xrx1, Xbf1 and cyclinD1, genes encoding factors promoting cell proliferation, are activated, and p27Xic1, which inhibits cell proliferation, is repressed by Xsix3 at early neurula stage; this strongly suggests that Xsix3 is able to act on the cell cycle machinery already at this early developmental stage.
Ectopic expression of Xsix3 enlarges the neural plate at the expense of adjacent non-neural tissue
Xsix3 is expressed in the anterior neural plate at early neurula
stage, raising the possibility that, besides controlling cell proliferation,
it might participate in neuroectoderm specification
(Zuber et al., 2003). To test
this hypothesis, we analysed the expression of neural plate markers in
Xsix3-injected embryos at early neurula stage. We observed that the
general neural markers Sox2 and Xnrp1
(Knecht et al., 1995
;
Kishi et al., 2000
) are
expanded on the injected side of the embryos (Sox2: 77%,
n=135; Xnrp1: 80%, n=23;
Fig. 2A,B). Notably, ectopic
expression of neural markers in the lateral ectoderm was always contiguous to
the neural plate.
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To test whether the ability of Xsix3 to expand the anterior neural plate at the expense of non-neural tissue might be a consequence of its proliferative activity, we overexpressed Xsix3 in embryos in which cell division was blocked by HUA treatment. HUA treatment severely reduced anti-phosphorylated histone H3 (H3P) staining, a marker of cells in mitosis (Fig. 2I,J). Even under these conditions, Xsix3 is still able to expand Sox2 expression and to repress the expression of Xslug, Sox9 and Xk81 (Sox2: 69%, n=87; Xslug: 73%, n=29; Sox9: 90%, n=19; Xk81: 76%, n=34; Fig. 2K-N). Taken together, these results suggest that the effects of Xsix3 on markers of neural patterning are at least in part independent of proliferation.
To rule out the possibility that the observed effects on neural plate
specification might be a secondary consequence of an early repression of Wnt
gene expression by Xsix3 (Lagutin
et al., 2003), we injected a Dexamethasone (DEX)-inducible form of
the Xsix3 construct. In these experimental conditions, activation of
GR-Xsix3 at mid-gastrula stages is able to promote Sox2 and to
repress Xk81 expression (Sox2: 100%, n=91;
Xk81: 90%, n=90; Fig.
2P,R). In the absence of DEX, the fusion protein GR-Xsix3 is
inactive or only slightly active, and embryos do not display significant
Sox2 and Xk81 expression alterations (Sox2: 89%
normal expression, 11% slightly expanded, n=80; Xk81: 85%
normal expression, 15% slightly reduced, n=59;
Fig. 2O,Q). These results
suggest that Xsix3 has a role in the initial specification of the
neural plate.
To further analyse the requirement for Xsix3 function in anterior neural plate specification, we used two different loss-of-function strategies. In VP16-Xsix3-injected embryos, we observed repression of Sox2 and expansion of Xk81 in the prospective anterior neural plate (Sox2: 72%, n=42; Xk81: 81%, n=32; Fig. 2S,V). The neural crest markers analysed are still expressed, although they are localized closer to the midline (Xslug: 77%, n=27; Sox9: 83%, n=31; Fig. 2T,U). Thus, conversion of presumptive neural plate cells towards an epidermal fate is likely to contribute to the Six3 loss-of-function phenotype.
As an independent loss-of-function approach, we injected an antisense morpholino (MoXsix3). Virtually all the MoXsix3-injected embryos show eye and anterior head defects similar to those observed in VP16-Xsix3-injection experiments (95%, n=130, data not shown). However, the penetrance of MoXsix3 phenotypic alterations is milder than that observed with VP16-Xsix3, as we never observed a complete loss of eye structures in MoXsix3-injected embryos. The phenotype is efficiently rescued by co-injection of Xsix3 RNA (data not shown and Fig. 5Q). As for VP16-Xsix3, overexpression of MoXsix3 reduces Sox2 and expanded Xk81 expression at early neurula stages (Sox2: 100%, n=25; Xk81: 80%; n=75; Fig. 2W,Z). Similarly, the expression of the neural crest markers Sox9 and Xslug is delocalised in a more dorsal position, even though in this case a reduced expression is also observed (Xslug: 100, n=35; Sox9: 100%, n=28; Fig. 2X,Y). Altogether, these results indicate that Xsix3 acts as a transcriptional repressor in the control of anterior neural plate specification.
|
First, we investigated the effects of positive and negative regulators of
cell differentiation on Xsix3 expression. Cephalic hedgehog
(chh) induces proliferation and delays differentiation in
Xenopus anterior neural plate
(Franco et al., 1999).
Injection of Xchh strongly expands the Xsix3 expression
domain at the early neurula stage (75%, n=45;
Fig. 3A). Conversely,
Xngnr1, which encodes a factor promoting neurogenesis in the anterior
neural plate (Ma et al., 1996
)
as shown by its ability to induce ectopic N-tubulin
expression (100%, n=29; Fig.
3B) strongly repressed Xsix3 (100%,
n=24; Fig. 3C).
|
To determine the requirement for Xsix3 function in the control of neurogenesis, we investigated the expression of the same markers in VP16-Xsix3-injected embryos. In the case of Xash3, Zic2 and Xhairy2, we observed a repression of their expression domains (100%; Xash3: n=25; Zic2: n=69; Xhairy2: n=35; Fig. 3M,O,P). Despite this, Xngnr1 expression in VP16-Xsix3-injected embryos was not significantly increased (Fig. 3N). These results suggest that the effects observed in Xsix3 loss of function are mainly independent of precocious neurogenesis.
Xsix3 inhibits epidermal fate but requires Xotx2 to induce neural markers in animal caps
As Xsix3 can expand the neural plate, we tested whether
Xsix3 is able to induce neural tissue independently of mesoderm by
performing animal cap assays. Control uninjected caps showed no expression of
Sox2, Xnrp1, Xash3, Xhairy2 and Zic2 (0% in all caps;
Sox2: n=79; Xnrp1: n=25; Xash3:
n=27; Zic2: n=55; Xhairy2: n=60;
Fig. 4), whereas they did
express Xk81 (100%, n=61;
Fig. 4). None of these markers
was induced in Xsix3-injected caps (Sox2: n=129;
Xnrp1: n=30; Xash3: n=35; Zic2:
n=90; Xhairy2: n=90;
Fig. 4). By contrast,
Xsix3 is able to strongly suppress expression of the epidermal marker
Xk81 (83%, n=78; Fig.
4). These results indicate that Xsix3 negatively
regulates the expression of the epidermal marker Xk81 and that it
requires additional factors, absent in animal caps, to activate neural markers
in the ectoderm. Among these, Xotx2, encoding a factor crucial for
anterior neural plate formation, is co-expressed with Xsix3 in the
presumptive anterior neuroectoderm at early neurula stage (stage 12.5)
(Zuber et al., 2003). Similar
to Xsix3, overexpression of Xotx2 in animal caps strongly
suppresses the expression of the epidermal marker Xk81, but it does
not activate (or only weakly activates) expression of general neural markers
(100%; Sox2: n=20; Xnrp1: n=35;
Fig. 4)
(Vignali et al., 2000
).
However, co-injection of Xsix3 and Xotx2 mRNA strongly
activates the expression of both Sox2 and Xnrp1
(Sox2: 88%, n=60; Xnrp1: 82%, n=60;
Fig. 4). These results suggest
that Xotx2 and Xsix3 may function in a synergistic way in
anterior neural plate specification.
|
Overexpression of Xsix3 resulted in repression of Bmp4 expression both in the embryo (71%, n=49; Fig. 5A) and in animal caps (71%, n=138; Fig. 5C). Control uninjected caps displayed normal Bmp4 expression (100%, n=39; Fig. 5B).
To elucidate whether Bmp4 and Xsix3 might antagonise each
other, we analysed the effects that the overexpression of each of these genes
exert on the other. We observed that Bmp4 overexpression leads to a
strong reduction of Xsix3 expression (88%, n=26;
Fig. 5D). Conversely,
interfering with BMP signalling by injection of either tBR, a
dominant-negative BMP receptor, or chordin mRNA
(Weinstein and Hemmati-Brivanlou,
1999) induced a strong activation of Xsix3 both in animal
caps (control: 0%, n=30; chordin: 100%, n=39;
tBR: 100%, n=45; Fig.
5G,H,I) and in the anterior neural plate of the embryo
(tBR: 92%, n=26; Fig.
5E). Conversely, both VP16-Xsix3 and MoXsix3
injection leads to expansion of Bmp4 expression in the presumptive
anterior neural plate (VP16-Xsix3: 45%, n=50;
MoXsix3: 77%, n=27; Fig.
5J,K). Additionally, TUNEL analysis showed that both
Bmp4- and VP16-Xsix3-injected embryos displayed an anterior
accumulation of apoptotic nuclei (Fig.
5L,M).
To analyse whether the effects of Xsix3 loss of function are a consequence of BMP4 expansion in the anterior neural plate, we tested whether interfering with BMP signalling can counteract the reduction of the anterior neural plate in MoXsix3-injected embryos. To achieve this, we analysed the expression of Zic2 (a gene expressed both in the anterior and posterior neural plate that is strongly modulated by Xsix3), in MoXsix3/tBR co-injected embryos. Injection of MoXsix3 alone repressed anterior Zic2 expression (81%, n=73; Fig. 5N). Conversely, MoXsix3/tBR co-injected embryos showed a complete or partial rescue of the Zic2 expression domain (65%, normal expression; 10% slightly reduced expression, 25% expanded expression; n=124; Fig. 5P). None of the co-injected embryos showed the strong expansion of Zic2 seen for tBR alone (100%, n=60; Fig. 5O). As a control, a similar rescue is observed when MoXsix3 is co-injected with Xsix3 (83%, normal expression; 17% slightly expanded; n=68; Fig. 5Q). Taken together, these results indicate a mutual antagonism between Xsix3 and Bmp4.
six3 rescues the anterior alterations in zebrafish chordino mutants
To analyse whether six3 could counteract the effects of BMP
dependent modulation of the neural plate size in vivo, we took advantage of
zebrafish chordino mutants/morphants. These mutants
(Hammerschmidt et al., 1996)
carry a mutation in the chordin gene, a known antagonist of BMP
signalling. The mutation causes the enlargement of tail structures,
alterations in trunk development and a reduction of anterior central nervous
system structures, including the eye (Fig.
6B) (Schulte-Merker et al.,
1997
). Analysis of chordino mutants/morphants at early
neurula stages (90% epiboly) shows a reduced anterior neural plate (rx3,
otx2, sox3) and expanded epidermal (bmp4, dlx3) markers, a
phenotype similar to VP16-Xsix3-injected frog embryos
(Fig. 6F,J,N,R,V).
|
|
Xsix3 directly represses Bmp4 transcription
Because Xsix3 works as a transcriptional repressor, we
investigated whether Bmp4 might be a direct target of Xsix3
and could therefore be repressed by GR-Xsix3 in the absence of
protein synthesis. To achieve this, the protein synthesis inhibitor
cycloheximide (CHX) was added to GR-Xsix3-injected animal caps before
DEX activation.
Injection of GR-Xsix3 resulted in repression of Bmp4
expression in animal caps treated with DEX (88%, n=62;
Fig. 7A), as well as in animal
caps treated with both CHX and DEX (90%, n=120;
Fig. 7A). By contrast, control
caps injected with GR-Xsix3 in the absence of DEX displayed normal
Bmp4 expression (100%, n=60;
Fig. 7A). Similar results were
observed in GR-Xsix3-injected caps following CHX but not DEX
treatment, suggesting that CHX alone had no effect on Bmp4 expression
(100%, n=65; Fig. 7A).
As a positive control for the efficiency of CHX treatment, we injected
Xotx2-GR and analysed the expression of Xcg and
Xag, which are known to be direct and indirect Xotx2
targets, respectively (Gammill and Sive,
1997). As expected, although DEX treatment of injected embryos
activated both Xcg and Xag, addition of DEX and CHX led to
the activation of Xcg but not of Xag
(Fig. 7B).
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Discussion |
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Xsix3 promotes cell proliferation and delays cell differentiation in the neural plate
The ability of Six3 to induce proliferation and inhibit neuronal
differentiation may involve interactions with different cofactors in the
neural plate. Despite the anterior restricted expression of Xsix3,
both anterior and posterior regions of the neural plate are competent to
respond to Xsix3. This suggests that cofactors required for
Xsix3 proliferative activity are expressed throughout the neural
plate. The recently isolated Six3 cofactors can be grouped into two categories
underscoring different mechanisms of action for Six3. One group includes the
groucho family, NeuroD and ATH3/ATH5, which contribute to the specificity of
Six3 transcriptional activity
(Kobayashi et al., 2001;
Zhu et al., 2002
;
Tessmar et al., 2002
;
Lopez-Rios et al., 2003
). The
second category of Six3 interactors is represented by Geminin. Six3 displaces
the DNA replication-inhibitor Geminin from Cdt1 resulting in activation of
cell proliferation in a transcriptional-independent manner
(Del Bene et al., 2004
). Both
types of interactors are expressed along the entire neural plate thus being
potentially available for cooperation with injected Xsix3. Although
the Six3/Geminin complex may contribute to the enhanced cell proliferation
elicited by Six3, this protein is likely to control cell proliferation and
neurogenesis by also acting as a transcription factor. Indeed, Xsix3
misexpression expands the expression of Zic2, Xhairy2 and
cyclinD1, while repressing that of p27Xic1, N-tubulin and
elrC. These effects are unlikely to be explained as a consequence of
cell proliferation induced by Six3/Geminin interaction. Indeed, Xsix3
is able to regulate the expression of the same genes even when cell
proliferation is blocked (Fig.
3K,L and data not shown). Reduction of cell proliferation in
VP16-Xsix3-injected embryos provides additional evidence that
Xsix3 regulates cell proliferation at the transcriptional level.
Six3 represses Bmp4 expression
A prerequisite for dorsal ectoderm to acquire neural fate is the inhibition
of BMP signalling, and one of the ways in which this is accomplished is
through the transcriptional inhibition of BMP genes in the nascent neural
plate (reviewed by Bally-Cuif and
Hammerschmidt, 2003). Prior to gastrulation, Bmp4 is
expressed throughout the embryo, but begins to be cleared from the prospective
neural plate soon after Xsix3 activation at gastrula stage, which
would be consistent with a role for Xsix3 in repressing Bmp4
at these early stages (Fainsod et al.,
1994
; Hemmati-Brivanlou and
Thomsen, 1995
; Zuber et al.,
2003
). Indeed, our overexpression experiments in both embryos and
animal caps provide compelling evidence for a mutual antagonism between
Six3 and Bmp4. In addition, the reduction of neural plate
size elicited by loss of Xsix3 function is significantly alleviated
by attenuation of BMP signalling. Moreover, our study in zebrafish shows that
six3 is able to compensate for the lack of the BMP antagonist
chordin, to specify the size of the anterior neural plate. Because
chordin is not required for six3 induction (data not shown),
it is likely that these two genes act in parallel to exclude Bmp4
from the anterior neural plate.
Bmp4 transcriptional repression is not sufficient to elicit neural induction
Although Six3 is able to expand the expression domains of the
neural markers Xsox2, Xnrp1, otx2 and sox3, we never
observed ectopic expression of these genes in the ventral ectoderm, where
Six3 represses the epidermal marker Xk81. Furthermore, we
show that in animal caps Xsix3 does not activate the expression of
neural markers, despite being able to repress epidermal markers. This suggests
that Xsix3 activity alone is not sufficient to respecify non-neural
ectoderm to a neural fate. Indeed, we found that neural markers are induced
only if Xotx2 is co-injected with Xsix3, indicating that
these two genes cooperate in neural fate determination. These data suggest
that an additional step besides Bmp4 transcriptional inhibition might
be required to drive unspecified ectodermal cells towards a neural fate. A
number of genes expressed in the presumptive neuroectoderm and involved in
promoting neural fate (e.g. Xanf1, Opl and Sox2) have been
described (Kuo et al., 1998;
Mizuseki et al., 1998
;
Ermakova et al., 1999
). These
genes share the inability to induce neural tissue in animal caps. Because for
most of these genes their effect on Bmp4 expression has not been
tested, at the moment it is not clear how common is the ability of repressing
Bmp4 without inducing neural fate in competent ectoderm. However, it
still remains to be clarified whether the ability of factors like Xiro2,
Soxd and Zic3 to neuralise competent ectoderm is simply the
result of the suppression of Bmp4 expression, as has been previously
reported for Geminin and Xiro1
(Nakata et al., 1997
;
Gomez-Skarmeta et al., 1998
;
Gomez-Skarmeta et al., 2001
;
Mizuseki et al., 1998
;
Kroll et al., 1998
). Moreover,
even though Xiro1 can directly repress Bmp4, Gomez-Skarmeta
and co-workers suggested that this protein may repress additional
uncharacterized factors to neuralise the ectoderm
(Gomez-Skarmeta et al.,
2001
).
Xsix3 controls anterior neural fate, at least in part, independently from Geminin
The recently demonstrated interaction between Six3 and Geminin opens the
possibility that Xsix3 may play a role in neural fate determination
in a Geminin-dependent way. However, Six3 and Geminin activities in the neural
plate appear to be distinct. Geminin is a coiled-coil protein, with two
separable functional domains, one of which neuralises ectoderm, whereas the
other is involved in the inhibition of DNA replication
(Kroll et al., 1998;
McGarry and Kirschner, 1998
).
Thus, Geminin and Six3 have antagonistic functions in the control of cell
proliferation; by contrast, they share the ability to repress Bmp4
and to promote neural plate expansion in Xenopus. However, unlike
Xsix3, Geminin has the ability to induce neural tissue in animal
caps. Moreover, differently from the anterior restricted effects of Xsix3,
Geminin induces neural tissue of posterior but not anterior character in
animal caps. Finally, although the DNA-binding activity of Six3 is dispensable
for the interaction with Geminin, mutagenesis of Six3/Groucho binding sites,
which impairs the transcriptional repressor activity of Six3, completely
abolishes its in vivo function (Kobayashi
et al., 2001
; Zhu et al.,
2002
). Altogether, these data indicate that Six3 may
function in different pathways either acting through the interaction with
Geminin, or controlling the transcription of key regulators of proliferation
and anterior neural plate specification.
A crucial role for Six3 in anterior neural plate specification
Six3 plays a dual role in the anterior neural plate controlling
proliferation and neurogenesis, and protecting the anterior neuroectoderm from
the ventralizing activity of BMPs. The fate of amphibian presumptive
neuroectoderm is reversible during early gastrula stages and can be changed to
epidermis by transplantation to the ventral side
(Spemann, 1938). Although by
the end of gastrulation, the neuroectoderm has little competence left to form
epidermis in transplants, epidermalising factors surrounding the neural plate
maintain their ability to inhibit neural plate genes
(Hartley et al., 2001
).
Six3 is activated by the neural inducers Noggin, Chordin and
ß-catenin, and begins to be expressed at a high level at mid-gastrula
stage, when it is likely to modulate the responsiveness of neuroectodermal
cells rather than the initial fate decisions of uncommitted ectodermal cells
(Bernier et al., 2000
;
Zuber et al., 2003
;
Kuroda et al., 2004
) (this
work). Thus, by repressing Bmp4 expression, Xsix3 might
maintain the competence of neuroectodermal cells to form the anterior neural
plate.
These data, together with the observation that Six3 also
counteracts Wnt signalling (Lagutin et
al., 2003) and promotes cell proliferation, suggests that
Six3 links cell specification and proliferation to maintain and
refine anterior identity.
![]() |
ACKNOWLEDGMENTS |
---|
We thank W. A. Harris, T. D. Sargent, S. Moody, I. Dawid, J. L. Gomez-Skarmeta, J. Wittbrodt and M. Perron for sharing plasmids, and T. Kudoh for chordino mutants. We are indebted to M. Fabbri, G. De Matienzo for technical assistance, and S. Di Maria and C. Wilson and her team for animal care. We gratefully acknowledge S. Banfi and his group for Xsix3 cDNA, E. Landi for help with GST protein purification and L. Pitto for instruction on EMSA. This work was supported by grants from M.I.U.R., Telethon (grant no. GGP04268) and from E.C. Quality of life and Management of Living Resources Program, Contract no. QLG3-CT-2001-01460. Work in S.W.W.'s group is additionally supported by the BBSRC and Wellcome Trust. M.C. is a EMBO Fellow and S.W.W. was a Wellcome Trust Senior Research Fellow.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Acampora, D., Mazan, S., Lallemand, Y., Avantaggiato, V., Maury,
M., Simeone, A. and Brulet, P. (1995). Forebrain and midbrain
regions are deleted in Otx2/ mutants due to a defective anterior
neuroectoderm specification during gastrulation.
Development 121,3279
-3290.
Akimenko, M. A., Ekker, M., Wegner, J., Lin, W. and Westerfield, M. (1994). Combinatorial expression of three zebrafish genes related to distal-less: part of a homeobox gene code for the head. J. Neurosci. 14,3475 -3486.[Abstract]
Andreazzoli, M., Pannese, M. and Boncinelli, E.
(1997). Activating and repressing signals in head development:
the role of Xotx1 and Xotx2. Development
124,1733
-1743.
Andreazzoli, M., Gestri, G., Angeloni, D., Menna, E. and
Barsacchi, G. (1999). Role of Xrx1 in Xenopus eye and
anterior brain development. Development
126,2451
-2460.
Andreazzoli, M., Gestri, G., Cremisi, F., Casarosa, S., Dawid,
I. B. and Barsacchi, G. (2003). Xrx1 controls proliferation
and neurogenesis in Xenopus anterior neural plate.
Development 130,5143
-5154.
Aybar, M. J. and Mayor, R. (2002). Early induction of neural crest cells: lessons learned from frog, fish and chick. Curr. Opin. Genet. Dev. 12,452 -458.[CrossRef][Medline]
Baker, J. C., Beddington, R. S. and Harland, R. M.
(1999). Wnt signaling in Xenopus embryos inhibits bmp4 expression
and activates neural development. Genes Dev.
13,3149
-3159.
Bally-Cuif, L. and Hammerschmidt, M. (2003). Induction and patterning of neuronal development, and its connection to cell cycle control. Curr. Opin. Neurobiol. 13, 16-25.[CrossRef][Medline]
Barth, K. A. and Wilson, S. W. (1995).
Expression of zebrafish nk2.2 is influenced by sonic hedgehog/vertebrate
hedgehog-1 and demarcates a zone of neuronal differentiation in the embryonic
forebrain. Development
121,755
-768.
Barth, K. A., Kishimoto, Y., Rohr, K. B., Seydler, C.,
Schulte-Merker, S. and Wilson, S. W. (1999). Bmp activity
establishes a gradient of positional information throughout the entire neural
plate. Development 126,4977
-4987.
Bernier, G., Panitz, F., Zhou, X., Hollemann, T., Gruss, P. and Pieler, T. (2000). Expanded retina territory by midbrain transformation upon overexpression of Six6 (Optx2) in Xenopus embryos. Mech. Dev. 93,59 -69.[CrossRef][Medline]
Bourguignon, C., Li, J. and Papalopulu, N.
(1998). XBF-1, a winged helix transcription factor with dual
activity, has a role in positioning neurogenesis in Xenopus competent
ectoderm. Development
125,4889
-4900.
Bradley, L., Wainstock, D. and Sive, H. (1996).
Positive and negative signals modulate formation of the Xenopus cement gland.
Development 122,2739
-2750.
Braun, M. M., Etheridge, A., Bernard, A., Robertson, C. P. and
Roelink, H. (2003). Wnt signaling is required at distinct
stages of development for the induction of the posterior forebrain.
Development 130,5579
-5587.
Brewster, R., Lee, J. and Ruiz i Altaba, A. (1998). Gli/Zic factors pattern the neural plate by defining domains of cell differentiation. Nature 393,579 -583.[CrossRef][Medline]
Carl, M., Loosli, F. and Wittbrodt, J. (2002).
Six3 inactivation reveals its essential role for the formation and patterning
of the vertebrate eye. Development
129,4057
-4163.
Casarosa, S., Andreazzoli, M., Simeone, A. and Barsacchi, G. (1997). Xrx1, a novel Xenopus homeobox gene expressed during eye and pineal gland development. Mech. Dev. 61,187 -198.[CrossRef][Medline]
Chitnis, A. B. (1999). Control of neurogenesis lessons from frogs, fish and flies. Curr. Opin. Neurobiol. 9,18 -25.[CrossRef][Medline]
Chitnis, A. and Kintner, C. (1996). Sensitivity
of proneural genes to lateral inhibition affects the pattern of primary
neurons in Xenopus embryos. Development
122,2295
-2301.
Christian, J. L., McMahon, J. A., McMahon, A. P. and Moon, R. T. (1991). Xwnt-8, a Xenopus Wnt-1/int-1-related gene responsive to mesoderm-inducing growth factors, may play a role in ventral mesodermal patterning during embryogenesis. Development 111,1045 -1055.[Abstract]
Chuang, J. C., Mathers, P. H. and Raymond, P. A. (1999). Expression of three Rx homeobox genes in embryonic and adult zebrafish. Mech. Dev. 84,195 -198.[CrossRef][Medline]
Dawson, S. R., Turner, D. L., Weintraub, H. and Parkhurst, S. M. (1995). Specificity for the hairy/enhancer of split basic helix-loop-helix (bHLH) proteins maps outside the bHLH domain and suggests two separable modes of transcriptional repression. Mol. Cell. Biol. 15,6923 -6931.[Abstract]
Del Bene, F., Tessmar-Raible, K. and Wittbrodt, J. (2004). Direct interaction of geminin and Six3 in eye development. Nature 427,745 -749.[CrossRef][Medline]
Eisen, J. S. and Weston, J. A. (1993). Development of the neural crest in the zebrafish. Dev. Biol. 159,50 -59.[CrossRef][Medline]
Ermakova, G. V., Alexandrova, E. M., Kazanskaya, O. V.,
Vasiliev, O. L., Smith, M. W. and Zaraisky, A. G. (1999). The
homeobox gene, Xanf-1, can control both neural differentiation and patterning
in the presumptive anterior neurectoderm of the Xenopus laevis embryo.
Development 126,4513
-4523.
Ekker, S. C., McGrew, L. L., Lai, C. J., Lee, J. J., von
Kessler, D. P., Moon, R. T. and Beachy, P. A. (1995).
Distinct expression and shared activities of members of the hedgehog gene
family of Xenopus laevis. Development
121,2337
-2347.
Fainsod, A., Steinbeisser, H. and de Robertis, E. M. (1994). On the function of BMP-4 in patterning the marginal zone of the Xenopus embryo. EMBO J. 13,5015 -5125.[Abstract]
Franco, P. G., Paganelli, A. R., Lopez, S. L. and Carrasco, A.
E. (1999). Functional association of retinoic acid and
hedgehog signaling in Xenopus primary neurogenesis.
Development 126,4257
-4265.
Furthauer, M., van Celst, J., Thisse, C. and Thisse, B.
(2004). Fgf signalling controls the dorsoventral patterning of
the zebrafish embryo. Development
131,2853
-2864.
Gammill, L. S. and Sive, H. (1997).
Identification of otx2 target genes and restrictions in ectodermal competence
during Xenopus cement gland formation. Development
124,471
-481.
Ghanbari, H., Seo, H. C., Fjose, A. and Brändli, A. W. (2001). Molecular cloning and embryonic expression of Xenopus Six homeobox genes. Mech. Dev. 101,271 -277.[CrossRef][Medline]
Gomez-Skarmeta, J. L., Glavic, A., de la Calle-Mustienes, E., Modolell, J. and Mayor, R. (1998). Xiro, a Xenopus homolog of the Drosophila Iroquois complex genes, controls development at the neural plate. EMBO J. 2,181 -190.[CrossRef]
Gomez-Skarmeta, J., de la Calle-Mustienes, E. and Modolell,
J. (2001). The Wnt-activated Xiro1 gene encodes a repressor
that is essential for neural development and downregulates Bmp4.
Development 128,551
-560.
Hammerschmidt, M., Serbedzija, G. N. and McMahon, A. P. (1996). Genetic analysis of dorsoventral pattern formation in the zebrafish: requirement of a BMP-like ventralizing activity and its dorsal repressor. Genes Dev. 10,2452 -2461.[Abstract]
Hardcastle, Z. and Papalopulu, N. (2000).
Distinct effects of XBF-1 in regulating the cell cycle inhibitor p27(XIC1) and
imparting a neural fate. Development
127,1303
-1314.
Harland, R. M. (1991). In situ hybridization: an improved whole-mount method for Xenopus embryos. Methods Cell Biol. 36,685 -695.[Medline]
Harris, W. A. and Hartenstein, V. (1991). Neuronal determination without cell division in Xenopus embryos. Neuron 6,499 -515.[CrossRef][Medline]
Hartenstein, V. (1989). Early neurogenesis in Xenopus: the spatio-temporal pattern of proliferation and cell lineages in the embryonic spinal cord. Neuron 3, 399-411.[CrossRef][Medline]
Hartley, K. O., Hardcastle, Z., Friday, R. V., Amaya, E. and Papalopulu, N. (2001). Transgenic Xenopus embryos reveal that anterior neural development requires continued suppression of BMP signaling after gastrulation. Dev. Biol. 238,168 -184.[CrossRef][Medline]
Heisenberg, C. P., Houart, C., Take-Uchi, M., Rauch, G. J.,
Young, N., Coutinho, P., Masai, I., Caneparo, L., Concha, M. L., Geisler, R.
et al. (2001). A mutation in the Gsk3-binding domain of
zebrafish Masterblind/Axin1 leads to a fate transformation of telencephalon
and eyes to diencephalon. Genes Dev.
15,1427
-1434.
Hemmati-Brivanlou, A. and Thomsen, G. H. (1995). Ventral mesodermal patterning in Xenopus embryos: expression patterns and activities of BMP-2 and BMP-4. Dev. Genet. 17,78 -89.[Medline]
Hensey, C. and Gautier, J. (1998). Programmed cell death during Xenopus development: a spatio-temporal analysis. Dev. Biol. 203,36 -48.[CrossRef][Medline]
Houart, C., Caneparo, L., Heisenberg, C., Barth, K., Take-Uchi, M. and Wilson, S. (2002). Establishment of the telencephalon during gastrulation by local antagonism of Wnt signaling. Neuron 35,255 -265.[CrossRef][Medline]
Jonas, E., Sargent, T. D. and Dawid, I. B. (1985). Epidermal keratin gene expressed in embryos of Xenopus laevis. Proc. Natl. Acad. Sci. USA 82,5413 -5417.[Abstract]
Jonas, E. A., Snape, A. M. and Sargent, T. D. (1989). Transcriptional regulation of a Xenopus embryonic epidermal keratin gene. Development 106,399 -405.[Abstract]
Jones, C. M., Lyons, K. M., Lapan, P. M., Wright, C. V. and
Hogan, B. L. (1992). DVR-4 (bone morphogenetic protein-4) as
a posterior-ventralizing factor in Xenopus mesoderm induction.
Development 115,639
-647.
Kazanskaya, O., Glinka, A. and Niehrs, C. (2000). The role of Xenopus dickkopf1 in prechordal plate specification and neural patterning. Development 122,4981 -4992.
Kessler, D. S. (1997). Siamois is required for
formation of Spemann's organizer. Proc. Natl. Acad. Sci.
USA 94,13017
-13022.
Kiecker, C. and Niehrs, C. (2001). A morphogen
gradient of Wnt/beta-catenin signalling regulates anteroposterior neural
patterning in Xenopus. Development
128,4189
-4201.
Kim, C. H., Oda, T., Itoh, M., Jiang, D., Artinger, K. B., Chandrasekharappa, S. C., Driever, W. and Chitnis, A. B. (2000). Repressor activity of Headless/Tcf3 is essential for vertebrate head formation. Nature 407,913 -916.[CrossRef][Medline]
Kishi, M., Mizuseki, K., Sasai, N., Yamazaki, H., Shiota, K.,
Nakanishi, S. and Sasai, Y. (2000). Requirement of
Sox2-mediated signaling for differentiation of early Xenopus neuroectoderm.
Development 127,791
-800.
Knecht, A. K., Good, P. J., Dawid, I. B. and Harland, R. M.
(1995). Dorsal-ventral patterning and differentiation of
noggin-induced neural tissue in the absence of mesoderm.
Development 121,1927
-1935.
Kobayashi, M., Toyama, R., Takeda, H., Dawid, I. B. and
Kawakami, K. (1998). Overexpression of the forebrain-specific
homeobox gene six3 induces rostral forebrain enlargement in zebrafish.
Development 125,2973
-2982.
Kobayashi, M., Nishikawa, K., Suzuki, T. and Yamamoto, M. (2001). The homeobox protein Six3 interacts with the Groucho corepressor and acts as a transcriptional repressor in eye and forebrain formation. Dev. Biol. 232,315 -326.[CrossRef][Medline]
Kodjabachian, L. and Lemaire, P. (2001). Siamois functions in the early blastula to induce Spemann's organiser. Mech. Dev. 108,71 -79.[CrossRef][Medline]
Koyano-Nakagawa, N., Kim, J., Anderson, D. and Kintner, C.
(2000). Hes6 acts in a positive feedback loop with the
neurogenins to promote neuronal differentiation.
Development 127,4203
-4216.
Kroll, K. L., Salic, A. N., Evans, L. M. and Kirschner, M.
W. (1998). Geminin, a neuralizing molecule that demarcates
the future neural plate at the onset of gastrulation.
Development 125,3247
-3258.
Kudoh, T., Tsang, M., Hukriede, N. A., Chen, X., Dedekian, M., Clarke, C. J., Kiang, A., Schultz, S., Epstein, J. A., Toyama, R. and Dawid, I. B. (2001). A gene expression screen in zebrafish embryogenesis. Genome Res. 11,979 -987.
Kudoh, T., Concha, M. L., Houart, C., Dawid, I. B. and Wilson,
S. W. (2004). Combinatorial Fgf and Bmp signalling patterns
the gastrula ectoderm into prospective neural and epidermal domains.
Development 131,3581
-3592.
Kuo, J. S., Patel, M., Gamse, J., Merzdorf, C., Liu, X., Apekin,
V. and Sive, H. (1998). Opl: a zinc finger protein that
regulates neural determination and patterning in Xenopus.
Development 125,2867
-2882.
Kuroda, H., Wessely, O. and de Robertis, E. M. (2004). Neural induction in Xenopus: requirement for ectodermal and endodermal signals via Chordin, Noggin, ß-Catenin and Cerberus. PLoS Biol. 2,623 -634.[CrossRef]
Lagutin, O. V., Zhu, C. C., Kobayashi, D., Topczewski, J.,
Shimamura, K., Puelles, L., Russell, H. R., McKinnon, P. J., Solnica-Krezel,
L. and Oliver, G. (2003). Six3 repression of Wnt signaling in
the anterior neuroectoderm is essential for vertebrate forebrain development.
Genes Dev. 17,368
-379.
Li, Y., Allende, M. L., Finkelstein, R. and Weinberg, E. S. (1994). Expression of two zebrafish orthodenticle-related genes in the embryonic brain. Mech. Dev. 48,229 -244.[CrossRef][Medline]
Loosli, F., Winkler, S. and Wittbrodt, J.
(1999). Six3 overexpression initiates the formation of ectopic
retina. Genes Dev. 13,649
-654.
Loosli, F., Winkler, S., Burgtorf, C., Wurmbach, E., Ansorge, W., Henrich, T., Grabher, C., Arendt, D., Carl, M., Krone, A. et al. (2001). Medaka eyeless is the key factor linking retinal determination and eye growth. Development 128,4035 -4044.[Medline]
Lopez-Rios, J., Tessmar, K., Loosli, F., Wittbrodt, J. and
Bovolenta, P. (2003). Six3 and Six6 activity is modulated by
members of the groucho family. Development
130,185
-195.
Ma, Q., Kintner, C. and Anderson, D. J. (1996). Identification of neurogenin, a vertebrate neuronal determination gene. Cell 87,43 -52.[CrossRef][Medline]
Mariani, F. V. and Harland, R. M. (1998).
XBF-2 is a transcriptional repressor that converts ectoderm into
neural tissue. Development
125,5019
-5031.
Mayor, R., Morgan, R. and Sargent, M. G.
(1995). Induction of the prospective neural crest of Xenopus.
Development 121,767
-777.
Mathers, P. H., Grinberg, A., Mahon, K. A. and Jamrich, M. (1997). The Rx homeobox gene is essential for vertebrate eye development. Nature 387,603 -607.[CrossRef][Medline]
McGarry, T. J. and Kirschner, M. W. (1998). Geminin, an inhibitor of DNA replication, is degraded during mitosis. Cell 93,1043 -1053.[CrossRef][Medline]
Mizuseki, K., Kishi, M., Matsui, M., Nakanishi, S. and Sasai,
Y. (1998). Xenopus Zic-related-1 and Sox-2, two factors
induced by chordin, have distinct activities in the initiation of neural
induction. Development
125,579
-587.
Morgan, R. and Sargent, M. G. (1997). The role
in neural patterning of translation initiation factor eIF4AII; induction of
neural fold genes. Development
124,2751
-2760.
Munoz-Sanjuan, I. and Brivanlou, A. H. (2002). Neural induction, the default model and embryonic stem cells. Nat. Rev. Neurosci. 3,271 -280.[CrossRef][Medline]
Nasevicius, A. and Ekker, S. C. (2000). Effective targeted gene `knockdown' in zebrafish. Nat. Genet. 26,216 -220.[CrossRef][Medline]
Nakata, K., Nagai, T., Aruga, J. and Mikoshiba, K.
(1997). Xenopus Zic3, a primary regulator both in neural and
neural crest development. Proc. Natl. Acad. Sci. USA
94,11980
-11985.
Nieuwkoop, P. D. and Faber, J. (1967).Normal table of development of Xenopus laevis (Daudin) . North-Holland, Amsterdam: Elsevier.
Newport, J. and Kirschner, M. (1982). A major developmental transition in early Xenopus embryos: I. characterization and timing of cellular changes at the midblastula stage. Cell 30,675 -686.[CrossRef][Medline]
Nguyen, V. H., Trout, J., Connors, S. A., Andermann, P.,
Weinberg, E. and Mullins, M. C. (2000). Dorsal and
intermediate neuronal cell types of the spinal cord are established by a BMP
signaling pathway. Development
127,1209
-1220.
Nikaido, M., Tada, M., Saji, T. and Ueno, N. (1997). Conservation of BMP signaling in zebrafish mesoderm patterning. Mech. Dev. 61, 75-88.[CrossRef][Medline]
Pandur, P. D. and Moody, S. A. (2000). Xenopus Six1 gene is expressed in neurogenic cranial placodes and maintained in the differentiating lateral lines. Mech. Dev. 96,253 -257.[CrossRef][Medline]
Pannese, M., Polo, C., Andreazzoli, M., Vignali, R., Kablar, B.,
Barsacchi, G. and Boncinelli, E. (1995). The Xenopus
homologue of Otx2 is a maternal homeobox gene that demarcates and specifies
anterior body regions. Development
121,707
-720.
Perron, M., Furrer, M. P., Wegnez, M. and Theodore, L. (1999). Xenopus elav-like genes are differentially expressed during neurogenesis. Mech. Dev. 84,139 -142.[CrossRef][Medline]
Piccolo, S., Agius, E., Lu, B., Goodman, S., Dale, L. and de Robertis, E. M. (1997). Cleavage of Chordin by Xolloid metalloprotease suggests a role for proteolytic processing in the regulation of Spemann organizer activity. Cell 91,407 -416.[CrossRef][Medline]
Sasai, N., Mizuseki, K. and Sasai, Y. (2001).
Requirement of FoxD3-class signaling for neural crest determination in
Xenopus. Development
128,2525
-2536.
Sasai, Y., Lu, B., Steinbeisser, H. and de Robertis, E. M. (1995). Regulation of neural induction by the Chd and Bmp-4 antagonistic patterning signals in Xenopus. Nature 377, 757.[CrossRef][Medline]
Schulte-Merker, S., Lee, K. J., McMahon, A. P. and Hammerschmidt, M. (1997). The zebrafish organizer requires chordino. Nature 387,862 -863.[CrossRef][Medline]
Sive, H. and Bradley, L. (1996). A sticky problem: the Xenopus cement gland as a paradigm for anteroposterior patterning. Dev. Dyn. 205,265 -280.[CrossRef][Medline]
Sive, H. L., Hattori, K. and Weintraub, H. (1989). Progressive determination during formation of the anteroposterior axis in Xenopus laevis. Cell 58,171 -180.[CrossRef][Medline]
Spemann, H. (1938). Embryonic Induction and Development. New Haven, CT: Yale University Press.
Spokony, R. F., Aoki, Y., Saint-Germain, N., Magner-Fink, E. and Saint-Jeannet, J. P. (2002). The transcription factor Sox9 is required for cranial neural crest development in Xenopus. Development 129,421 -432.[Medline]
Stern, C. D. (2002). Induction and initial patterning of the nervous systemthe chick embryo enters the scene. Curr. Opin. Genet. Dev. 12,447 -451.[CrossRef][Medline]
Tessmar, K., Loosli, F. and Wittbrodt, J. (2002). A screen for co-factors of Six3. Mech. Dev. 117,103 -113.[CrossRef][Medline]
Tsang, M., Maegawa, S., Kiang, A., Habas, R., Weinberg, E. and
Dawid, I. B. (2004). A role for MKP3 in axial patterning of
the zebrafish embryo. Development
131,2769
-2779.
Turner, D. L. and Weintraub, H. (1994). Expression of achaete-scute homolog 3 in Xenopus embryos converts ectodermal cells to a neural fate. Genes Dev. 8,1434 -1447.[Abstract]
Vernon, A. E. and Philpott, A. (2003). The developmental expression of cell cycle regulators in Xenopus laevis. Gene Expr. Patterns 3,179 -192.[CrossRef][Medline]
Vignali, R., Colombetti, S., Lupo, G., Zhang, W., Stachel, S., Harland, R. M. and Barsacchi, G. (2000). Xotx5b, a new member of the Otx gene family, may be involved in anterior and eye development in Xenopus laevis. Mech. Dev. 96, 3-13.[CrossRef][Medline]
Weinstein, D. C. and Hemmati-Brivanlou, A. (1999). Neural induction. Annu. Rev. Cell Dev. Biol. 15,411 -433.[CrossRef][Medline]
Wilson, P. A. and Hemmati-Brivanlou, A. (1995). Induction of epidermis and inhibition of neural fate by Bmp-4. Nature 376,331 -333.[CrossRef][Medline]
Wilson, S. I. and Edlund, T. (2001). Neural induction: toward a unifying mechanism. Nat. Neurosci. 4,1161 -1168.[Medline]
Wilson, S. W. and Houart, C. (2004). Early steps in the development of the forebrain. Dev. Cell 6, 167-181.[CrossRef][Medline]
Wylie, C., Kofron, M., Payne, C., Anderson, R., Hosobuchi, M.,
Joseph, E. and Heasman, J. (1996). Maternal beta-catenin
establishes a `dorsal signal' in early Xenopus embryos.
Development 122,2987
-2996.
Zakin, L. and de Robertis, E. M. (2004).
Inactivation of mouse Twisted gastrulation reveals its role in promoting Bmp4
activity during forebrain development. Development
131,413
-424.
Zhu, C. C., Dyer, M. A., Uchikawa, M., Kondoh, H., Lagutin, O. V. and Oliver, G. (2002). Six3-mediated auto repression and eye development requires its interaction with members of the Groucho-related family of corepressors. Development 129,2835 -2849.[Medline]
Zuber, M. E., Gestri, G., Viczian, A. S., Barsacchi, G. and
Harris, W. A. (2003). Specification of the vertebrate eye by
a network of eye field transcription factors.
Development 130,5155
-5167.