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First published online September 15, 2003
doi: 10.1242/10.1242/dev.00665
1 Sezione di Biologia Cellulare e dello Sviluppo, Dipartimento di Fisiologia e
Biochimica, Università degli Studi di Pisa, Via Carducci 13, 56010
Ghezzano (Pisa), Italy
2 Università degli Studi di Pisa, Centro di Eccellenza AmbiSEN, Pisa,
Italy
3 Laboratory of Molecular Genetics, National Institute of Child Health and Human
Development, NIH, Bethesda, MD 20892, USA
4 Scuola Normale Superiore, piazza dei Cavalieri, 7 - 56100, Pisa, Italy
* Author for correspondence (e-mail: andream{at}dfb.unipi.it)
Accepted 12 June 2003
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SUMMARY |
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 TOP SUMMARY IntroductionMaterials and methodsResultsDiscussionREFERENCES |
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Key words: Xrx1, Xhairy2, Zic2, p27Xic1, XBF-1, Xenopus laevis, Proliferation, Neurogenesis, Retinoic acid
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Introduction |
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TOPSUMMARYIntroductionMaterials and methodsResultsDiscussionREFERENCES |
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). The role of proneural genes, which code for transcription
factors of the basic helix-loop-helix (bHLH) class, is to define clusters of
cells competent to originate neuronal precursors
(Chitnis, 1999). Within each
cluster, a single cell is selected to become a neuroblast by the expression of
neurogenic genes. This is accomplished by lateral inhibition, a process
mediated by the membrane-bound ligand Delta and the Notch receptor. One cell
in the cluster becomes committed to a neuronal fate by expressing higher
levels of Delta, which in turn activates the Notch receptor in adjacent cells,
forcing them to remain uncommitted. The search for vertebrate homologues of
proneural and neurogenic genes has led to the discovery that key regulators of
neurogenesis are evolutionary conserved
(Chitnis, 1999). In
particular, many studies on vertebrate neural induction and neurogenesis have
been performed in the amphibian Xenopus laevis because of the
experimental accessibility of its embryos and the small number of early
differentiating primary neurons. In this species, it was shown that the dorsal
mesodermal region called Spemann's organizer
(Spemann, 1938) neuralizes the
dorsal ectoderm by secreting noggin, chordin and follistatin which antagonize
BMP4, an epidermalizing signal (Sasai and
De Robertis, 1997). Following neural induction, the spatial
distribution of neuronal precursors within the posterior neural plate is
controlled by prepattern genes of the Xiro, Gli and Zic families. In
particular, Zicr1, Xzic3 and Gli proteins induce neurogenesis, while Zic2,
Xiro1, Xiro2 and Xiro3, acting as anti-neurogenic transcription factors,
restrict the expression domains of proneural genes
(Nakata et al., 1997;
Mizuseki et al., 1998;
Brewster et al., 1998;
de la Calle-Mustienes et al.,
2002). Regulators of these transcription factors include the
Hedgehog genes, which promote proliferation by repressing Gli3 and
activating Zic2, and retinoic acid (RA), a posteriorizing morphogen
that induces neuronal differentiation by inhibiting the expression of Hedgehog
genes (Franco et al., 1999).
As early as the end of gastrulation, the first sites of neurogenesis within
the neural plate are marked by the expression of X-ngnr-1, which
encodes an atonal type bHLH protein, followed by the activation of
X-Delta-1 and finally of N-tubulin, a marker of
differentiated neurons. The expression of these genes is restricted to three
longitudinal rows on either side of the dorsal midline, where individual cells
are selected for differentiation through the action of Delta/Notch
(Chitnis, 1999).
Although the molecular mechanisms underlying the control of neurogenesis in
the posterior nervous system are beginning to be unraveled, less is known
about factors controlling neuronal differentiation in the anterior neural
plate. Lineage tracing and pulse-labeling experiments
(Hartenstein, 1989;
Eagleson et al., 1995), as
well as analysis of neuronal differentiation markers
(Hartenstein, 1993;
Papalopulu and Kintner, 1996),
have shown that anterior neural plate cells undergo neuronal differentiation
significantly later than cells of the posterior neural plate. An as yet
unanswered question is what are the factors controlling this phenomenon and
how they are related to regulators of posterior vertebrate neurogenesis. So
far, only a small group of transcription factors expressed in the anterior
neural plate, including XBF-1, Xanf-1, Xsix3 and Xoptx2,
have been shown to play a role in delaying neuronal differentiation and/or
promoting proliferation (Bourgouignon et al., 1998;
Ermakova et al., 1999;
Zuber et al., 1999;
Bernier et al., 2000;
Hardcastle and Papalopulu,
2000). However, because the spatiotemporal expression of these
genes does not coincide with the entire proliferative region of the anterior
neural plate, additional genes are likely to be involved. In this work, we
propose that Xrx1, a homeobox gene required for eye and anterior
brain development, is one such factor. We report that Xrx1 is
expressed in the entire proliferative anterior neural plate surrounded by
cells expressing X-ngnr-1, X-Delta-1 and p27Xic1, a cell
cycle inhibitor. Xrx1 microinjection inhibits X-ngnr-1,
X-Delta-1 and N-tubulin expression, and counteracts RA- and
X-ngnr-1-mediated differentiation, while at the same time activating
proliferation. These effects are independent of Notch signaling and are
restricted to the most rostral region of the embryo. Xrx1 exerts its
function by activating Xhairy2 and Zic2, the expression of
which in the anterior neural plate overlaps with that of Xrx1, and by
repressing p27Xic1. Accordingly, loss-of-function experiments show
that Xrx1 is required for the normal proliferation of the anterior
neural plate. These data indicate that Xrx1 possesses the appropriate
activities and spatiotemporal expression pattern to be one of the factors
responsible for the maintenance of anterior neuronal precursors in a
proliferative state.
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Materials and methods |
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TOPSUMMARYIntroductionMaterials and methodsResultsDiscussionREFERENCES |
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;
Newport and Kirschner, 1982).
Whole-mount in situ hybridization on embryos and animal caps was performed
essentially as described by Harland
(Harland, 1991). Histological
examination was performed according to Casarosa et al.
(Casarosa et al., 1997). For
HUA treatment, stage 10 devitellinised embryos were added to a 20 mM
hydroxyurea, 150 µM aphidicolin in 0.1x MMR solution, as described by
Hardcastle and Papalopulu (Hardcastle and
Papalopulu, 2000), and kept in this solution until fixation.
Embryo microinjections, animal cap assay, immunostaining, and BrdU
incorporation
Capped synthetic RNAs encoding for Xrx1 (20-100 pg),
X-chh (1 ng), X-shh (1 ng)
(Ekker et al., 1995),
X-ngnr-1 (40 pg) (Ma et al.,
1996), XRALDH2 (1.5 ng)
(Chen et al., 2001),
Notch-ICD (30 pg-1.8 ng) (Chitnis
et al., 1995), X-Delta-1stu (500 pg)
(Chitnis et al., 1995),
XBF-1 (150 pg) (Bourgouignon et al., 1998) and Xhairy2 (125
pg) (Davis et al., 2001) were
generated by in vitro transcription and co-injected with lacZ
RNA(100-500 pg) into one blastomere at the two-cell stage or into a dorsal
blastomere at the four-cell stage. The optimal concentration of each batch of
RNA was identified through injection of various doses followed by analysis of
either the phenotype or the expression of specific markers. For animal cap
experiments, capped synthetic chordin (150 pg per blastomere)
(Sasai et al., 1995),
X-ngnr-1 (40 pg per blastomere) and Xrx1 (360 pg per
blastomere) RNAs were injected into both blastomeres at the two-cell stage and
animal caps dissected at stage 9. When sibling control embryos reached stage
16 or 17, animal caps were fixed and stored in ethanol at -20°C. For
retinoic acid treatment, injected animal caps were incubated in
2x10-6 M RA in 0.5xMMR where they were cultured until
stage 16. For the experiments shown in Fig.
2I-L, Fig. 3E,
Fig. 5P,Q, the total amount of
RNA injected, either in the experimental or in the respective control samples,
is the same. This was achieved by adjusting the amount of lacZ RNA in
the control samples. The Xrx1 antisense morpholino used was:
5'-TCAGGGAAGGGCTGTGCAGGTGCAT-3' (Gene Tools LLC). A standard
morpholino oligo (Gene Tools LLC) was injected as control. Immunostaining with
anti-phosphorylated H3 antibody was performed as described by Saka and Smith
(Saka and Smith, 2001). BrdU
incorporation was performed essentially as described by Hardcastle and
Papalopulu (Hardcastle and Papalopulu,
2000).
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Results |
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TOPSUMMARYIntroductionMaterials and methodsResultsDiscussionREFERENCES |
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;
Mathers et al., 1997;
Andreazzoli et al., 1999). As
cells of the anterior neural plate are characterized by prolonged
proliferation and delayed neuronal differentiation, we decided to analyze
whether Xrx1 is involved in the control of these activities. To
better define Xrx1 expression in the context of early neurogenesis,
we compared the expression domain of Xrx1 with that of early neuronal
differentiation markers. Double in situ hybridization experiments showed that
at early neurula Xrx1 is expressed in a territory that is precisely
circumscribed by the expression of the neurogenic gene X-Delta-1 and
abuts on the anterior expression domains of the neuronal determination gene
X-ngnr-1 (Fig. 1A,B).
Histological sections showed that within this area Xrx1 is expressed
exclusively in the deep sensorial layer of the neuroectoderm where both
primary and secondary neurons will form
(Hartenstein, 1989)
(Fig. 1C). Regions marked by
X-Delta-1 and X-ngnr-1 expression coincide with prospective
sites of neuronal differentiation. In particular, the most anterior
semicircular stripes of X-Delta-1 and X-ngnr-1 correspond to
the presumptive olfactory placodes, part of the telencephalon and laterally to
the epiphysis, while the more posterior X-Delta-1 medial stripe
coincides with the ventral midbrain
(Eagleson and Harris, 1990;
Bourguignon et al., 1998).
Although expression of several other genes partially overlaps with that of
Xrx1, to our knowledge Xrx1 is the only gene described so
far whose expression completely fills the anterior gap of X-Delta-1
expression, thus corresponding exactly to the proliferative region of the
anterior neural plate.
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), we decided to test if they affect Xrx1 expression.
We found that both sonic hedgehog (X-shh) and cephalic
hedgehog (X-chh) are able to activate Xrx1 at early
neurula stage (X-shh 68%, n=44; X-chh 56%,
n=36; Fig. 1D,E).
Xrx1 is ectopically activated by Hedgehog signaling only in an area
that surrounds the endogenous Xrx1 expression domain, despite of the
broader distribution of the injected RNA. Because of the lack of Xrx1
expression in X-Delta-1 and X-ngnr-1 positive regions, we
looked if proneural gene expression plays a role in restricting Xrx1
expression. Overexpression of X-ngnr-1, which efficiently induces
ectopic N-tubulin expression (100%, n=31;
Fig. 1G), strongly represses
Xrx1 expression (100%, n=32;
Fig. 1F). These data indicate
that Xrx1 expression is not compatible with neuronal differentiation
and that the anterior expression of proneural genes like X-ngnr-1
defines the perimeter of the Xrx1 expression domain.
Xrx1 inhibits neuronal differentiation
The coincidence of Xrx1 expression with the proliferating area of
the anterior neural plate, where neuronal markers are not expressed, led us to
think that Xrx1 might be part of the system preventing precocious
neurogenesis in this area. To test this hypothesis, we analyzed the expression
of neuronal differentiation markers in Xrx1-injected embryos during
early neurulation. We observed that X-ngnr-1, X-Delta-1 and
N-tubulin are all repressed in the anterior region by Xrx1
overexpression (X-ngnr-1, 91%, n=58; X-Delta-1,
90%, n=31; N-tubulin, 97%, n=92;
Fig. 2A-D), while the
repressive effects are weak in the posterior expression domains of these
markers (Fig. 2A-C,E).
Sox2, a general neural marker, was not affected at this stage (0%,
n=90; Fig. 2F),
indicating that Xrx1 acts on neuronal differentiation but not on
neural induction. As a positive control, Xrx1 ectopically activates
XBF-1 in the lateral border of the neural plate (58%, n=24;
Fig. 2G), as previously
described (Andreazzoli et al.,
1999). Xrx1 effects on neurogenesis are distinct from
those observed upon XBF-1 overexpression
(Bourguignon et al., 1998). In
fact, injection of XBF-1 at doses that cause suppression of
endogenous N-tubulin also leads to ectopic activation of
N-tubulin along the boundary of the injected area in the posterior
neural plate (94%, n=36; Fig.
2H). In a complementary approach, we tested if Xrx1 has
the ability to inhibit ectopic neurogenesis induced by X-ngnr-1
overexpression. Injection of X-ngnr-1 induced a massive expression of
N-tubulin, the in situ signal of which covered the ß-gal
staining (100%, n=41; Fig.
2I, also compare the injected versus uninjected side in
Fig. 1G). At variance,
co-injection of X-ngnr-1 and Xrx1 resulted in a considerable
attenuation of N-tubulin activation (95% with reduced ectopic
expression, n=45; Fig.
2K). These data were confirmed by animal cap experiments where
X-ngnr-1 ability of inducing N-tubulin
(Ma et al., 1996) was
inhibited by Xrx1 (X-ngnr-1 + lacZ 100% positive,
n=58; X-ngnr-1 + Xrx1 96% negative, 4% weakly
positive, n=62; Fig.
2J,L).
Xrx1 counteracts RA differentiating signals
Retinoic acid has been shown to control the timing of neuronal
differentiation being able to accelerate neurogenesis in anterior neural cells
(Papalopulu and Kintner, 1996;
Sharpe and Goldstone, 2000).
Although RA is thought to function mainly in the posterior neural plate and
mesoderm during early development (Chen et
al., 1994), it has been shown recently that XRALDH2, one
of enzymes involved in RA synthesis, is expressed also in an anterior site
(Chen et al., 2001). A double
in situ hybridization revealed that Xrx1 expression adjoins, but does
not overlap, the XRALDH2 anterior expression domain
(Fig. 3A). To determine the
causes of this spatial relationship between Xrx1 and
XRALDH2, we looked at the effect that the overexpression of each of
these genes exerts on the other. We found that Xrx1 and
XRALDH2 exhibit mutually repressive activities
(XRALDH2-injected embryos: 75% with reduced Xrx1 expression,
n=24; Fig. 3B;
Xrx1-injected embryos: 83% with reduced XRALDH2 expression,
n=24; Fig. 3C), which
could explain the generation of adjacent, non-overlapping expression domains.
To analyze if Xrx1 could also counteract the effects of RA on
neuronal differentiation, we took advantage of an animal cap system that
recapitulates anterior neurogenesis. Papalopulu and Kintner
(Papalopulu and Kintner, 1996)
showed that noggin-injected animal caps cultured until neurula stage
(stage 16) express NCAM but not N-tubulin. Initiation of
N-tubulin expression is observed only when these explants are
cultured until tailbud stage (stage 27); addition of RA accelerates this
process leading to activation of N-tubulin by stage 16. We used
chordin as a BMP antagonist in animal caps, and found that at stage
16 chordin alone does not induce N-tubulin (0% positive,
n=26), while RA treatment of chordin-injected animal caps
robustly activates a punctate expression of N-tubulin (97% positive,
n=33; Fig. 3D).
Interestingly, Xrx1 expression, which is strongly induced in
chordin-injected animal caps (96% positive, n=25), is
completely abolished by RA treatment (0% positive, n=26;
Fig. 3D). This observation
again inversely correlates Xrx1 expression and neurogenesis. To test
whether the repression of Xrx1 was required to activate neurogenesis
we treated with RA animal caps that had been co-injected with chordin
and Xrx1 RNA. Co-injection of Xrx1, but not of
lacZ, effectively inhibited N-tubulin expression in
RA-treated caps (Chordin + lacZ 100% positive,
n=25; Chordin + Xrx1 80% negative, 20% weakly
positive, n=30; Fig.
3E). Thus, Xrx1 appears to counteract RA-mediated
neuronal differentiation through a dual action: upstream of RA production by
repressing the expression of XRALDH2, and downstream of RA or acting
on a parallel pathway, as shown by its ability to impede RA-mediated
differentiation in chordin-injected caps.
Xrx1 controls proliferation at early neurula stage in a
region-specific manner
Cells of the anterior neural plate are not only characterized by delayed
differentiation but also display a protracted proliferating state. Therefore,
we asked whether Xrx1 plays a role in the control of proliferation at
early neurula stage. To achieve this, embryos injected with either
Xrx1 or Xrx1-EnR, a dominant-negative construct
(Andreazzoli et al., 1999) were
tested for BrdU incorporation. In these experiments the number of
BrdU-positive cells in the injected side was compared with that of the
uninjected control side taking also into account their anteroposterior
distribution. We found that the anterior neural plate of
Xrx1-injected embryos displayed a 44% increase of BrdU-positive cells
in the injected side compared with the control side (an average of 42.2
positive cells per section in the injected side, n=1099, versus 29.2
cells per section in the control side, n=760; P<0.001;
Fig. 4A,C). On the contrary, no
significant difference was detected in the posterior neural plate of
Xrx1-injected embryos (an average of 19.6 positive cells per section
in the injected side, n=413, versus 19.3 cells per section in the
control side, n=406; Fig.
4B,C). However, the anterior neural plate of
Xrx1-EnR-injected embryos shows a 33% decrease of BrdU-positive cells
in the injected side compared with the control side (an average of 18 positive
cells per section in the injected side, n=519, versus 26.8 cells per
section in the control side, n=773; P<0.001;
Fig. 4D,F). In addition, no
significant difference was observed in the posterior neural plate (an average
of 14.3 positive cells per section in the injected side, n=253,
versus 13.9 cells per section in the control side, n=235;
Fig. 4E,F). Altogether, these
results suggest that Xrx1 is involved in controlling cell
proliferation specifically in the anterior region of the neural plate.
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).
Besides being expressed in the posterior neural plate in stripes that
alternate with the primary neurons, Zic2 is also expressed in the
anterior neural plate. Comparing the expression profiles of Xrx1 and
Zic2, we found that they mostly overlap in the presumptive forebrain,
although Zic2 RNA extends slightly more to the anterior and
Xrx1 expression more to the posterior
(Fig. 5A,B). Overexpression of
Xrx1 leads to an expansion of Zic2 expression, which extends
along the mediolateral axis (95%, n=62;
Fig. 5D,E), while no ectopic
expression is observed in the majority of cases in the posterior neural plate.
Xhairy1 and Xhairy2 are homologues of the Drosophila
hairy gene that are known to act as transcriptional repressors and to
inhibit neuronal differentiation (Dawson
et al., 1995; Koyano-Nakagawa
et al., 2000). Both genes display diffuse expression in the
anterior neural plate that overlaps with Xrx1 expression
(Fig. 5C,G). Moreover,
Xhairy2, but not Xhairy1, shows a stronger stripe of
expression that coincides with the anterior and lateral borders of
Xrx1 expression. Injection of Xrx1 induces ectopic
expression of Xhairy2, which, as in the case of Zic2, does
not extend posteriorly (72%, n=50;
Fig. 5F). In addition,
overexpression of Xrx1 also leads to a repression of the anterior
domain of Xhairy1 (90%, n=40;
Fig. 5J). As Xhairy1
and Xhairy2 are known to repress each other, and because there is
evidence that Xrx1 might work as a transcriptional activator
(Andreazzoli et al., 1999), it
is possible that Xhairy2 is first activated by Xrx1 and that
this excess of Xhairy2 could be responsible for the downregulation of
Xhairy1. Xoptx2, a homeobox gene of the Six homeobox family, has been
shown to play a role in controlling the proliferation of the retina
(Zuber et al., 1999). Analysis
of Xoptx2 expression in Xrx1-injected embryos showed that,
although at stage 14 the expression of this gene does not appear to be
affected (89% normal expression, 11% slightly reduced expression,
n=67; not shown), a remarkable expansion is observed at stage 18
(75%, n=56; Fig. 5L).
This response is similar to the one previously described for the related gene
Xsix3 (Andreazzoli et al.,
1999). p27Xic1, an inhibitor of cyclin/cdk required for
primary neurogenesis (Su et al.,
1995; Carruthers et al.,
2003; Vernon et al.,
2003), is expressed anteriorly in two semicircles very similar to
those characterized by X-Delta-1 expression. We observed that
Xrx1 expression is complementary to that of p27Xic1, being
delimited dorsally and ventrally by the two p27Xic1 expression
domains (Fig. 5H).
Overexpression of Xrx1 suppresses p27Xic1 expression,
particularly in its most anterior domains (96%, n=83;
Fig. 5K). Finally, we looked at
cyclin D1, which has a strong expression site in the eye field
(http://www.xenbase.org/)
(Vernon and Philpott, 2003)
and has been implicated in mouse eye development
(Fantl et al., 1995). Although
Xrx1-injected embryos did not show any difference in cyclin
D1 expression at early neurula stage (stage 13, 100% normal expression,
n=46; not shown), a notable expansion was observed at late neurula
stage (stage 18, 83%, n=24; Fig.
5I). Thus, Zic2 and Xhairy2 are activated and
p27Xic1 is repressed by Xrx1 at early neurula stage, while
Xoptx2 and cyclin D1 are activated only at a later stage
under the influence of Xrx1.
Xrx1 function is not mediated by lateral inhibition during
early neurulation
X-Notch-1 is expressed in the anterior neural plate in the region
occupied by Xrx1 (Fig.
5M), and its activity in preventing differentiation of neurons has
been described (Chitnis et al.,
1995). Moreover, the mouse Xrx1 homologue (Rx1,
also called Rax) has been shown to activate Notch
transcription during retinogenesis
(Furukawa et al., 2000). For
these reasons, we tested whether, during early neurulation, Xrx1 and
X-Notch-1 affect one another's expression. Analysis of
Xrx1-injected embryos at various stages during early neurulation
failed to show any transcriptional activation of X-Notch-1 (stage 13,
0%, n=54; stage 15, 0%, n=33; stage 18, 0%, n=38;
Fig. 5N). Furthermore,
injection of several doses of a constitutively active form of
X-Notch-1 (Notch-ICD)
(Chitnis et al., 1995) did not
show activation of Xrx1 at early neurula (30 pg 86% normal, 10%
slightly reduced expression, 4% slightly expanded expression, n=30;
500 pg 84% normal, 8% slightly reduced expression, 8% slightly expanded
expression, n=37; 1.8 ng 85% normal, 11% slightly reduced expression,
4% slightly expanded expression, n=27;
Fig. 5O). These results suggest
that during early neurulation, Xrx1 is not affected directly by Notch
signaling, and that Xrx1 does not affect Notch expression.
Finally, to test if the effects of Xrx1 on neurogenesis are mediated
by lateral inhibition, we co-injected Xrx1 with
X-Delta-1stu, an antimorphic version of X-Delta-1
(Chitnis et al., 1995). Even
though under these conditions, lateral inhibition was blocked, as shown by an
excess of N-tubulin-expressing cells in the posterior neural plate
(Fig. 5P,Q, arrow),
Xrx1 was still able to repress the trigeminal ganglia expression of
N-tubulin (75% absent expression, 25% reduced expression,
n=40; Fig. 5Q).
Xrx1 controls the expression of Xhairy2,
Zic2, X-ngnr-1 and p27Xic1 in the absence of cell
division
The ectopic expression of Xhairy2 and Zic2 as well as the
repression of X-ngnr-1 and p27Xic1 in Xrx1-injected
embryos could be triggered independently of cell proliferation or,
alternatively, could result from an expansion of the proliferating
neuroectoderm. To distinguish between these two possibilities, we asked
whether Xrx1 can affect Xhairy2, Zic2, X-ngnr-1 and
p27Xic1 expression in embryos where cell division has been blocked by
treatment with hydroxyurea and aphidicolin (HUA)
(Harris and Hartenstein,
1991). HUA treatment severely affected anti-phoshoH3 staining, a
marker of cells in mitotic prophase, as well as Xoptx2 ability of
expanding Xrx1 (Zuber et al.,
1999) and resulted in smaller embryos with reduced optic vesicles
(Fig. 6E-I). Under these
conditions, Xrx1 is still able to expand Zic2 (96%,
n=78) and Xhairy2 (71%, n=74) expression and to
repress the expression of X-ngnr-1 (97%, n=36) and
p27Xic1 (84%, n=44), although not to the same extent as in
untreated embryos (Fig. 6A-D).
These data suggest that the regulation of Xhairy2, Zic2, X-ngnr-1 and
p27Xic1 observed in Xrx1-injected embryos does not depend
exclusively on proliferation. This observation may be consistent with the
finding that Xrx1 is able to convert competent ectoderm to an
anterior neural fate (Kenyon et al.,
2001).
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). Injection of 10 ng of a morpholino oligo directed against
Xrx1 (MoXrx1) into dorsoanimal blastomeres at the eight-cell
stage generates embryos displaying severe reduction of eyes and anterior head
(97%, n=102; Fig. 8A).
This phenotype, which is very similar to the one obtained after
Xrx1-EnR injection and to Rx1-/- mice
(Mathers et al., 1997;
Andreazzoli et al.,1999), is
completely rescued by co-injection of 80 pg of Xrx1 RNA (99%,
n=86; Fig. 8B) and is
not observed upon injection of control morpholino (96% normal embryos,
n=75; Fig. 8C).
Analysis of X-ngnr-1 expression in both MoXrx1- and
Xrx1-EnR-injected embryos showed an anterior expansion of the
expression domains of this gene, which tend to fuse medially. This phenotype
is first observed at stage 14 (Xrx1-EnR: 82% n=34;
MoXrx1: 83%, n=79; Fig.
8D-F) and is maintained at stage 18 (Xrx1-EnR: 75%,
n=28; MoXrx1: 78%, n=38;
Fig. 8H-J). The expanded
expression domains of X-ngnr-1 appeared to correspond to the
telencephalon rather than the trigeminal ganglia. This was confirmed by the
observation that MoXrx1 did not affect the expression of
FoxD3, a marker of neural crest cells that contribute also to the
trigeminal ganglia (100%, n=19;
Fig. 8G,K). To analyze
simultaneously the effects of Xrx1 knockdown on neurogenesis and
eye-field specification we co-hybridized MoXrx1-injected embryos with
X-ngnr-1 and Xrx1 (Fig.
8L-N). We found that the medial expansion of
X-ngnr-1correlates with a smaller Xrx1 expressing area,
which in extreme cases was totally abolished, with no overlap of the two
markers (85% reduced Xrx1 expression,
Fig. 8M; 15% absent
Xrx1 expression, Fig.
8N, n=33). Analogous results were also obtained using
Xsix3 as eye field marker (not shown). A very similar phenotype was
observed in HUA-treated embryos (100% reduced Xrx1 expression,
n=25; Fig. 8O),
suggesting that the effects observed in Xrx1 loss of function are
mainly due to a reduced proliferation of the anterior neural plate. As
gain-of-function experiments indicated a potential role of Xhairy2
downstream of Xrx1, we tested whether Xhairy2 could rescue
MoXrx1 effects. Bilateral injection of MoXrx1 resulted in
reduction, but not complete abolishment, of the expression of Xhairy2
(98%, n=50; Fig.
8P,Q), whereas Xhairy2 overexpression led to a severe
repression of X-ngnr-1 expression (67%, n=36;
Fig. 8R). Co-injection of
MoXrx1 and Xhairy2 resulted in embryos displaying the
typical anterior expansion of X-ngnr-1 observed in embryos injected
with MoXrx1 alone, thus indicating that Xhairy2 is not able
to rescue the MoXrx1 phenotype (84%, n=94;
Fig. 8S). Among the genes
activated by Xrx1 overexpression and repressed by Xrx1
inactivation (Andreazzoli et al.,
1999), XBF-1 is of particular interest, because, like
Xrx1, it acts by controlling p27Xic1 expression
(Hardcastle and Papalopulu,
2000). Therefore, we tested whether XBF-1 could rescue
the effects of MoXrx1 injection. Overexpression of XBF-1
alone repressed anterior X-ngnr-1 expression while inducing ectopic
X-ngnr-1 in the posterior neural plate (90%, n=30;
Fig. 8T,U). When co-injected
with MoXrx1, XBF-1 was able to restore a wide X-ngnr-1-free
area in the anterior neural plate (82%, n=70;
Fig. 8V). These results suggest
that XBF-1 might work downstream of Xrx1 and/or that these
genes function by controlling common mechanisms.
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Discussion |
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TOPSUMMARYIntroductionMaterials and methodsResultsDiscussionREFERENCES |
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; Zuber et al.,
1999). XBF-1 is expressed only in the most anterior
border of the neural plate, adjacent to the rostral expression of
X-Delta-1 and p27Xic1 (Bourgouignon et al., 1998;
Hardcastle and Papalopulu,
2000), where it plays a crucial role in defining the border
between proliferative and non-proliferative areas. Xoptx2 is
expressed in the anterior neural plate later than Xrx1
(Zuber et al., 1999) and
Xanf-1 is expressed in a domain broader than the region occupied by
Xrx1 (data not shown). Thus, the early Xrx1 expression
appears to be restricted, spatially and temporally, to the proliferative area
of the anterior neural plate more specifically than that of other
transcription factors. How is Xrx1 expression localized to this area
and how are its expression boundaries defined? We found that both positive and
negative signals contribute to this positioning, as Xrx1 is activated
by chordin and Hedgehog and is repressed by retinoic acid and
X-ngnr-1. As BMP antagonists are known to act as general forebrain
inducers (Sasai and De Robertis,
1997), chordin may be responsible for the initial
activation of Xrx1 in the anterior neuroectoderm. Hedgehog signaling
has been shown to delay differentiation and promote proliferation at early
neurula stage (Franco et al.,
1999); thus, the observation that Hedgehog signaling can activate
Xrx1 correlates with the expression of Xrx1 in anterior
proliferating cells. Different Hedgehog genes share the same activity, with
regional specificity being achieved by differential expression of the family
members. Thus, it is expected that X-shh and X-chh were
equally effective in Xrx1 activation, although it is likely that
X-chh, which displays anterior expression
(Ekker et al., 1995), assumes
this function in normal development. At later stages, when the proximodistal
axis of the diencephalon/eye-tract develops, the response of Xrx1 to
the Hedgehog gradient may become more complex. In fact, Xrx1 is
expressed in the retina and retinal pigmented epithelium, where the level of
Hedgehog is low, is absent in the optic stalk, and is expressed again in the
diencephalon floor, a region exposed to the highest level of Hedgehog signal.
Xrx1 expression in the anterior neural plate is also controlled by
negative regulators such as RA and X-ngnr-1, which appear to prevent
Xrx1 expansion to surrounding regions. This is reflected in the
spatial relationship between Xrx1 on one side, and XRALDH2,
a RA-producing enzyme (Chen et al.,
2001), and X-ngnr-1 on the other. Xrx1 is not
simply regulated by factors controlling neuronal differentiation, but plays an
active role in this process. In fact, Xrx1 is able to repress
neuronal differentiation markers and to counteract the effects of
X-ngnr-1 and RA on neurogenesis. Both in the case of neurogenin and
RA, Xrx1 action appears to be twofold. Xrx1 acts upstream by
repressing X-ngnr-1 and XRALDH2 expression, and downstream
or in parallel, by blocking the differentiation promoting activity of these
factors. Although at early neurula stage, XRALDH2 and Xrx1
expression domains are adjacent but do not overlap, at later stages
XRALDH2 expression extends into the retina
(Chen et al., 2001). This
indicates that although at later stages RA is required for retina formation,
for the initial specification of the eye field it is important to keep RA in
the anterior neural plate at a very low level. The RA degrading activity of
Cyp26 provides protection from RA coming from the posterior neural plate
(Hollemann et al., 1998).
However, the lack of expression of Cyp26 in the most rostral region of the
embryo leaves this area exposed to RA generated by the anterior domain of
XRALDH2. Xrx1, the expression pattern of which is complementary to
that of Cyp26 (data not shown), may be one of the elements that counteracts
the RA signal in the anterior neural plate.
Besides counteracting neuronal differentiation, Xrx1 promotes
proliferation in the anterior neural plate. In fact, BrdU incorporation in
gain- and loss-of-function experiments provides a direct evidence that
Xrx1 is both necessary and sufficient to regulate proliferation in
the anterior neural plate. In particular, loss of Xrx1 activity
reduces anterior neural plate proliferation to levels similar to those
observed in the posterior neural plate, indicating that Xrx1 is one
of the main factors responsible, directly or indirectly, for the increased
proliferation of the anterior neural plate. So far, among the Rx genes that
promote an enlargement of the retina, a proliferation inducing activity has
been suggested for medaka Rx3
(Loosli et al., 2001) but not
for zebrafish rx1 and rx2
(Chuang and Raymond, 2001).
Although species-specific differences may exist, the orthology relationship
between vertebrate Rx genes has not yet been completely clarified.
Anterior-specific activities of Xrx1
We previously noticed that the phenotypic effects of Xrx1
overexpression are restricted to the eye-anterior brain region, despite of the
wider distribution of the injected RNA
(Andreazzoli et al., 1999). In
the present work, we find that Xrx1 is able to induce proliferation
and repress neuronal differentiation in an anterior-specific manner. As Xrx1
is a transcription factor, it presumably acts by regulating the expression of
target genes. Interestingly, previous experiments have shown that
Xrx1 microinjection activates ectopic expression of XBF-1 in
the lateroanterior border of the neural plate
(Andreazzoli et al., 1999)
(Fig. 2G). As the effects of
Xrx1 on neurogenesis are also observed in regions where
XBF-1 cannot be activated by Xrx1, and the Xrx1
expression domain is larger than that of XBF-1, additional factors
are likely to be regulated by Xrx1 in the anterior neural plate.
Although Xrx1 appears to be a transcriptional activator
(Andreazzoli et al., 1999;
Chuang and Raymond, 2001), its
overexpression repressed X-ngnr-1, X-Delta-1, N-tubulin, XRALDH2 and
p27Xic1. Consistently, Xrx1 activates Zic2 and
Xhairy2, two transcriptional repressors involved in delaying neuronal
differentiation. As these two genes have an anterior expression domain that
partially overlaps with that of Xrx1, they may mediate the repressive
effects of Xrx1 in this system. Interestingly, mouse Rx1 can
activate Hes1, a hairy homologue, during retinogenesis
(Furukawa et al., 2000),
indicating that genes of the Hairy family might be evolutionary conserved
Rx1 targets. Moreover, mutations in human ZIC2 induce
holoprosencephaly, and the mouse knockout of Hes1 affects eye
morphogenesis, phenotypes that are similar to those produced by the loss of
function of the Rx1 gene (Tomita
et al., 1996; Mathers et al.,
1997; Brown et al.,
1998; Andreazzoli et al.,
1999). However, as Zic2 is not induced by Xrx1
in animal caps, additional factors are likely required for Zic2
activation. Both the ectopic activation of Zic2 and Xhairy2,
and the repression of X-ngnr-1, X-Delta-1, N-tubulin, XRALDH2 and
p27Xic1 by Xrx1 overexpression are restricted to the
anterior neural plate, suggesting that only this region is competent to
respond to Xrx1. Xrx1 loss-of-function experiments resulted in
reduction, but not abolishment, of Xhairy2 and Zic2
expression (Fig. 8Q; data not
shown), indicating that Xrx1 is not the only factor responsible for
their anterior activation. Conversely, X-ngnr-1 anterior expression
was expanded medially, probably as a consequence of the reduction of the eye
field. This phenotype, which is essentially reproduced in HUA-treated embryos,
is consistent with a severely reduced anterior proliferation. Accordingly, the
functional inactivation of Xrx1 does not appear to be sufficient to
induce widespread ectopic X-ngnr-1 across the anterior neural plate,
presumably because of the persistence of Zic2 and Xhairy2
expression. Co-injection experiments revealed that XBF-1, but not
Xhairy2, is able to rescue the anterior expansion of
X-ngnr-1 observed in MoXrx1-injected embryos. These data
indicate that Xhairy2 cannot maintain a normal level of proliferation
in the anterior neural plate in the absence of Xrx1 function. The
ability of XBF-1 to re-establish a X-ngnr-1-free region
suggests that Xrx1 might work in part through XBF-1 and/or
that both genes control anterior neural plate proliferation acting on common
regulators, as is the case for p27Xic1.
Lateral inhibition is not involved in Xrx1 activities
An important mechanism used during development to prevent neuronal
differentiation is lateral inhibition, a process mediated by transduction of
the Notch signal. We considered the possibility that Xrx1 might work
by increasing lateral inhibition. This hypothesis was supported by the
co-expression of Xrx1 and X-Notch-1 at early neurula and by
data indicating that mouse Rx1 activates Notch transcription
during retinogenesis (Furukawa et al.,
2000). By contrast, we did not find activation of
X-Notch-1 in Xrx1-injected embryos during early-mid
neurulation (stages 13-18). Furthermore, Xrx1 expression could not be
stimulated by expression of a constitutively active form of Notch at
early neurula stage. Another way in which lateral inhibition could be
triggered is by overexpression of Delta, but this possibility could
also be ruled out as Xrx1 represses X-Delta-1 expression,
probably as a consequence of X-ngnr-1 inhibition. Finally, we checked
if Xrx1 repression of neuronal differentiation could be prevented by
blocking lateral inhibition. We observed that co-injection of Xrx1
and an antimorphic form of Delta, known to block lateral inhibition, does not
affect the ability of Xrx1 of repressing neuronal differentiation in
the anterior regions of the embryo.
These data suggest that Xrx1 does not work through lateral
inhibition involving Delta and Notch, but may bypass this system through the
activation of Xhairy2, a target gene of Notch
(Davis et al., 2001). In
general, lateral inhibition is probably not responsible for preventing
precocious neuronal differentiation in the anterior neural plate. In fact, the
inability of noggin-injected animal caps, which display an anterior
neuroectodermal character, to undergo neuronal differentiation at early
neurula stage is not mediated by lateral inhibition
(Papalopulu and Kintner,
1996). Similarly, the inhibition of neuronal differentiation after
injection of high doses of XBF-1 is not due to increased lateral
inhibition (Bourguignon et al.,
1998).
Distinct anterior and posterior gene systems control neuronal
differentiation
In Drosophila, prepattern genes that are expressed before the
onset of neurogenesis control the region-specific activation of proneural
genes. Prepattern genes include hairy and the Iroquois family
homeobox genes (Gomez-Skarmeta et al.,
1996; Fisher and Caudy,
1998). In vertebrates, homologues of the Iroquois genes play a
similar role, functioning during early neurulation in the specification of
neural precursors in the posterior neural plate
(Bellefroid et al., 1998;
Gomez-Skarmeta et al., 1998;
de la Calle-Mustienes et al.,
2002; Itoh et al.,
2002). We notice several similarities between the activities of
Xenopus Iroquois (Xiro) genes and Xrx1, as these
genes: (1) repress neuronal differentiation at early neurula; (2) do not work
through lateral inhibition; (3) are repressed by X-ngnr-1 and
activated by hedgehog signaling; (4) upregulate Xhairy2 and
Zic2; and (5) act after neural induction and before the selection of
neuronal precursor cells.
Moreover, the loss of function of Rx genes in vertebrates as well as of the
Iroquois complex in Drosophila, results in the absence of the
structures where these genes are normally expressed
(Cavodeassi et al., 2001;
Mathers et al., 1997;
Andreazzoli et al., 1999;
Loosli et al., 2001). Beside
these similarities, it is worth noting that while the Iroqouis genes play a
role in positioning domains of proneural gene expression, this function has
not been demonstrated for the Rx genes. However, the complementary expression
of Xrx1 and Xiro genes together with their similar
activities suggest the existence of two gene systems, one acting in the
anterior and the other in the posterior neural plate, the function of which is
to control the timing and delimit the location of neuronal
differentiation.
In conclusion, Xrx1, by counteracting differentiating signals and promoting proliferation in a region-specific manner, plays a crucial role in executing a program that, after neural induction, leads to the correct differentiation and patterning of the anterior neural plate.
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ACKNOWLEDGMENTS |
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