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First published online January 23, 2009
doi: 10.1242/10.1242/dev.016204
1IGBMC (Institut de Génétique et de Biologie Moléculaire et Cellulaire), BP 10142, Illkirch, F-67400 France. 2Inserm, U 964, Illkirch, F-67400 France. 3CNRS, UMR 7104, Illkirch, F-67400 France. 4Université Louis Pasteur, Faculté de Médecine, Strasbourg, F-67000 France.
Author for correspondence (e-mail:
dolle{at}igbmc.fr)
Accepted 24 November 2008
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: Retinoids, Gastrulation, Neurogenesis, Spinal cord, Somites, Sonic hedgehog, Gli, Fibroblast growth factor, Mouse
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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).
Fate maps in chick showed that spinal cord precursors are located in the
epiblast lateral to Hensen's node (Brown
and Storey, 2000; Mathis et
al., 2001). Studies in mouse and chicken have shown that neural
and somitic tissues are also derived from the node and node-streak border
(Beddington, 1994;
Cambray and Wilson, 2002;
Cambray and Wilson, 2007;
Mathis and Nicolas, 2000;
Nicolas et al., 1996). Selleck
and Stern (Selleck and Stern,
1991) demonstrated that cells from the chick node contribute
iteratively to the notochord. Thus, from very early in development, the
formation of the posterior central nervous system and the axial and paraxial
mesoderm are tightly linked.
According to Nieuwkoop's `activation-transformation' model
(Nieuwkoop et al., 1952;
Slack and Tannahill, 1992),
further elaborated by Stern and colleagues
(Foley et al., 2000;
Stern, 2001), neural induction
produces cells with a rostral forebrain character that, upon the action of
`transforming' factors identified as Wnts, fibroblast growth factor (Fgf) and
retinoic acid (RA), acquire caudal neural character. These three signalling
pathways are used repeatedly during caudal patterning in the neuroepithelium
and the mesoderm (Diez del Corral and
Storey, 2004; Maden,
2002; Olivera-Martinez and
Storey, 2007; Stern,
2005), suggesting that they interact differently and use distinct
co-factors according to the time and tissue where they are active.
The development of caudal structures relies on the spatiotemporal
distribution of RA, resulting from regulated expression of both synthesizing
(Raldh1, Raldh2 and Raldh3) and metabolizing enzymes (Cyp26a1, Cyp26b1 and
Cyp26c1). Early RA signalling in mouse embryos relies on Raldh2 function
(Niederreither et al., 1999).
Complementary expression patterns of Raldh2 and Cyp26a1 are
observed; during somitogenesis, Raldh2 is expressed in somites and
rostral presomitic mesoderm (Blentic et
al., 2003; Niederreither et
al., 1997; Vermot et al.,
2005), whereas Cyp26a1 expression is restricted to
caudalmost tissues (Fujii et al.,
1997; Reijntjes et al.,
2004). Analysis of mouse mutants deficient in RA signalling
(Raldh2-/- and
Rara-/-;Rarg-/- mutants) or vitamin
A-deficient (VAD) quail embryos revealed that RA acts as a caudalizing signal
in the developing rhombencephalon. Additionally, the entire posterior region
of these embryos is severely shortened. Neural induction occurs in
Raldh2-/- embryos
(Molotkova et al., 2005;
Niederreither et al., 1999);
however, the neuroepithelium remains abnormally thin. In addition, these
mutants exhibit smaller somites, probably as a consequence of increased
Fgf8 expression in the tail bud
(Molotkova et al., 2005;
Vermot et al., 2005). Although
Raldh2 is already expressed during gastrulation in the newly formed
mesenchyme adjacent to the node and primitive streak
(Niederreither et al., 1997;
Zhao et al., 1996), little is
known about its function at this stage.
As development proceeds, the processes of neurogenesis and mesodermal
segmentation rely, at least in part, on a balance between Fgf and RA
signalling (Diez del Corral et al.,
2003; Dubrulle et al.,
2001). Fgf8 is expressed as a posterior (high) to
anterior (low) gradient in caudal tissues, and a crucial threshold (the
`determination front') determines the location of intersomitic boundaries and
the commitment of cells to a given axial identity
(Dubrulle et al., 2001).
Additionally, Fgf8 maintains an undifferentiated `stem' zone adjacent to the
regressing primitive streak (Akai et al.,
2005; Mathis et al.,
2001) [see Rozsko et al. (Rozsko et al., 2007) for a description
of stem cell-like spinal cord progenitors]. Simultaneously, RA emanating from
the anterior presomitic mesoderm (PSM) and the somites attenuates Fgf
signalling and allows neural progenitors to initiate differentiation. A recent
study implicated canonical Wnt signalling in mediating the transition from Fgf
to retinoid activity (Olivera-Martinez and
Storey, 2007).
The acquisition of a neural cell type is concomitant to the onset of
differentiation, and exposure to both RA and sonic hedgehog (Shh)
simultaneously is an obligatory step in the emergence of correct ventral
patterning (Diez del Corral et al.,
2003; Novitch et al.,
2003). The mechanism by which RA and Shh signals interact is
unknown. RA also acts to regulate Hox genes, many of which contain functional
RA-response elements (RAREs) (e.g.
Bel-Vialar et al., 2002;
Gould et al., 1998;
Huang et al., 1998;
Mainguy et al., 2003;
Oosterveen et al., 2003;
Packer et al., 1998).
Positional identity is encoded by the combination of Hox genes expressed at a
given axial level. Expression of Hoxa1 is downregulated in the caudal
region of Raldh2-/- embryos
(Niederreither et al., 1999),
raising the issue of the anteroposterior identity of neural tube cells in the
absence of RA signalling.
In this study, we investigate the function of RA during caudal patterning of the mouse embryo, using Raldh2-/- mutants as a model system. We show that RA is required as early as E7.5 for correct patterning of the caudal neural plate, and later for the onset of spinal cord neurogenesis. We demonstrate that lack of RA signalling interferes with the response of cells to Shh, a defect which may partly result from abnormal Fgf activity. We also report that an Fgf-independent upregulation of Gli2 may be a key mediator of the early embryonic RA-deficiency phenotypes.
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MATERIALS AND METHODS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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; Rossant et al.,
1991). Culture experiments were performed as described
(Ribes et al., 2008), with
embryos collected at E7.5. Recombinant mouse Shh-N (R&D systems) was
rehydrated in culture medium at 2.5 µM, and SU5402 (Calbiochem) in DMSO at
a stock concentration of 85 mM. All-trans-RA (Biomol) was rehydrated in 95%
ethanol at 20 mM. Control embryos were always cultured in presence of the
drug's vehicle. Drug efficacy was tested by analyzing target genes for each
signalling pathway (see Fig. S1 in the supplementary material;
Fig. 6). At least two
independent experiments were performed for each drug condition. After culture,
the embryos were fixed overnight in 4 % paraformaldehyde and processed for in
situ hybridization.
In situ hybridization, X-gal assays and immunohistochemistry
Whole-mount in situ hybridization was performed using an Intavis InSituPro
robot (for details,
http://empress.har.mrc.ac.uk/,
gene expression section). Template plasmids were generated in our institute,
or kindly provided by Drs B. De Crombrugghe (Sox10), S. Ang
(follistatin), J. Deschamps (Cdx1, Hoxb8), D. Echevarria
(Mkp3), R. Kageyama (Hes5), R. Krumlauf (Hoxb9), P.
Gruss (Pax3), F. Guillemot (Ngn2), B. Herrmann (brachyury),
C. C. Hui (Gli1-3), M. Kmita/D. Duboule (lacZ), J. Lewis
(Delta1), R. Lovell-Badge (Sox2), G. Martin
(Spry2), A. McMahon (Shh, Wnt3a), A. Nieto (Snail),
M. Petkovitch (Cyp26a1), B. Robert (Msx1) and C. Tabin
(Ptch1). Following in situ hybridization, some embryos were embedded
in 1% agarose and vibratome sectioned.
Whole-mount X-gal assays and immunolabelling were performed as described
previously (Ribes et al.,
2006; Rossant et al.,
1991). For immunohistochemistry on sections, embryos were fixed
for 20 minutes, impregnated with 10% sucrose and cryosectioned at 10 µm.
All antibodies were incubated in a solution of PBS, 10% foetal calf serum, 3%
BSA and 0.1% triton. Primary antibodies (anti-Foxa2, anti-neurofilament,
anti-Shh-N, Developmental Studies Hybridoma Bank) and secondary
peroxidase-coupled goat anti-mouse antibody (Jackson Immunoresearch) were
incubated overnight at 4°C, whereas goat anti-mouse AlexA4880 or
AlexA594-conjugated (Molecular probes) were incubated for 45 minutes at room
temperature.
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RESULTS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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).
Raldh2 expression starts at the mid-primitive streak stage within the
posterior embryonic region (E6.75; data not shown). At the neural plate stage
(E7.5), it is expressed in posterior mesoderm
(Fig. 1A,B), in primitive
streak (Fig. 1A,B') and
in most of the node cells (Fig.
1A'). RARE-lacZ activity was similarly detected in
the node (Fig. 1C') and
primitive streak (Fig. 1C).
Although Raldh2 is not expressed in the epiblast
(Fig. 1B), the
RARE-lacZ transgene is strongly active in this layer
(Fig. 1D,D'). At headfold
stage (E7.75), RA-responsive cells match the distribution of RA-producing
cells (Fig. 1E,F). However,
close examination showed that Raldh2 expression has been
downregulated in node and primitive streak cells
(Fig. 1E'), whereas some
primitive streak and node cells are still positive for the reporter transgene
(Fig. 1F').
RARE-lacZ activity extinguishes progressively from these cells,
especially within the node where it is detected at least until the four-somite
stage (data not shown) (Sirbu and Duester,
2006). At later stages, RA signalling is restricted to somites,
rostral presomitic mesoderm and neural tube
(Vermot et al., 2005).
Expression of Cyp26a1 appears by E6.5 in the anterior epiblast and
mesendoderm (Fujii et al.,
1997; MacLean et al.,
2001). At headfold stage, Cyp26a1-expressing cells are
adjacent to the rostralmost RARE-lacZ-positive cells
(Fig. 1F,G)
(Sirbu et al., 2005).
Interestingly, Cyp26a1 expression appears in scattered cells of the
primitive streak whereas the RA-responsive transgene becomes downregulated
(Fig. 1F',G'). In
addition, at this stage, Cyp26a1 expression appears in posterior
endodermal cells (Fig.
1G', inset). Eventually, its expression expands anteriorly
and laterally within the caudal neural plate (CNP) and primitive streak to
reach the node cells (data not shown)
(Fujii et al., 1997;
MacLean et al., 2001).
Thus, RA activity is highly dynamic during gastrulation. Interestingly,
primitive streak cells are transiently RA responsive, RA activity being
gradually excluded from the streak and the CNP at the late headfold stage.
Node cells transiently express Raldh2, leading to RA activity both in
the node ectoderm and mesendoderm until late headfold stage, after which
activity is selectively maintained in the node ectoderm (see also
Sirbu and Duester, 2006).
Raldh2-/- embryos display caudal patterning defects
We addressed whether RA activity plays a role in early development of
caudal tissues by analyzing Raldh2-/- mutants, using
marker genes of the posterior epiblast and mesoderm. Follistatin is expressed
from E6.5 in the ectodermal and mesodermal layers near the primitive streak
(Fig. 2A-A''). Its
expression in mesoderm becomes restricted to the primitive streak cells at the
four-somite-stage (Fig.
2C,C'). At E7.75 (Fig.
2B-B'', n=5/5) and in three-somite stage
(Fig. 2D-D'',
n=3/3) Raldh2-/- embryos, follistatin expression
within the epiblast and posterior mesoderm expands more laterally and
anteriorly compared to wild-type embryos (brackets in
Fig. 2C,D). Brachyury
expression in the node and notochord is not significantly altered in mutants
(Fig. 2G,H). However, similarly
to follistatin, brachyury expression within the mutant epiblast and mesoderm
expands more laterally and anteriorly than in wild-type embryos
(Fig. 2E-F'', E7.75;
Fig. 2G-H'', early
somite-stage; n=4/5 and 8/9). The general level of brachyury
expression also appears increased in mutants. We confirmed this patterning
defect by analyzing Wnt3a, a gene required for brachyury expression
(Yamaguchi et al., 1999),
which exhibited a similar expansion in pre- and early somite stage
Raldh2-/- embryos (Fig.
2I-J''; n=5/6 and 4/4, respectively).
|
).
Eventually, it expands posteriorly and is detected in most of the CNP of
three- to four-somite stage wild-type embryos
(Fig. 3A)
(Wood and Episkopou, 1999;
Delfino-Machin et al., 2005).
In stage-matched Raldh2-/- embryos, Sox2
transcripts are undetectable in the CNP, but are present more rostrally in the
neural tube (Fig. 3B;
n=7/7). This defect is transient, as eight-somite stage wild-type and
mutant embryos show comparable Sox2 patterns in the CNP (data not
shown; n=6/6) (Molotkova et al.,
2005). Wnt8a is expressed in mitotic progenitors in the
neural plate, and upon neural tube differentiation is maintained only in
pre-rhombomere 4 (Fig. 3C).
Wnt8a expression was shown to be maintained along the whole neural
tube of Raldh2-/- embryos
(Niederreither et al., 2000).
However, Wnt8a expression within the CNP of mutants was consistently
weaker, from E7.75 (data not shown; n=2/5) to at least the
four-somite stage (Fig. 3D,
bracket showing lack of expression in lateral regions; n=9/10).
We next analyzed Cdx1, which encodes a transcription factor
required for caudal patterning and directly regulated by RA signalling
(Houle et al., 2003;
Subramanian et al., 1995;
van den Akker et al., 2002).
Cdx1 is expressed in both the mesoderm and ectoderm of the CNP, and
eventually in the neural tube (Meyer and
Gruss, 1993). In four- to five-somite stage
Raldh2-/- embryos, Cdx1 expression is absent from
the neural tube (Fig. 3F) and
the lateral cells of the caudal region
(Fig. 3E,F, brackets)
(n=4/4). As Cdx proteins regulate Hox gene expression
(Lohnes, 2003), we examined
specification of the caudal neural tube in Raldh2-/-
embryos by analyzing several Hox genes. Hoxb9 expression is decreased
in caudal ectoderm and mesoderm, and virtually undetectable in nascent neural
tube, of five- to six-somite stage Raldh2-/- embryos
(Fig. 3G,H, main panels;
n=7/7), probably reflecting a delay in activation. However,
expression of other Hox genes, including Hoxb8
(Fig. 3G,H, insets;
n=4/4), Hoxb6, Hoxc6 and Hoxc8 (data not shown) is
comparable with control embryos. Thus, RA deficiency affects only some Hox
genes in the caudal region, and, collectively, Hox expression patterns reveal
that the posterior neural tube of Raldh2 mutants acquires a spinal
character.
In conclusion, lack of RA signalling leads to an enlargement of the domains of expression of primitive streak markers at E7.5. At early somite stages, the patterning of the caudal region is altered, ectopic expression of mesodermal markers is observed lateral to the node and in anterior/lateral regions of the CNP, whereas prospective neuroepithelial markers are undetectable or shifted anteriorly (see Fig. 9 for a summary).
Impaired neurogenesis in Raldh2-/- embryos
The delay in Sox2 expression and persistence of Wnt8a
expression in the neural tube (Fig.
3) suggest that neurogenesis may be altered in Raldh2
mutants. Previous studies demonstrated a requirement for RA signalling during
specification of neural progenitors along the spinal cord dorsoventral axis
(Novitch et al., 2003;
Diez del Corral et al., 2003;
Wilson et al., 2004;
Molotkova et al., 2005). Here,
we show that RA is required for the proper molecular specification of spinal
cord neural crest cells (NCCs), as observed by a delay in the appearance of
Wnt3a (Fig. 4A,B,
bracket, and data not shown; n=3/3 at E8.75; 4/4 at E9.5) and
markedly decreased expression of Msx1
(Fig. 4C,D; n=4/4) and
Pax3 (data not shown). Lack of Sox9, Sox10
(Fig. 4E,F and data not shown;
n=4/4 for both genes) and Snail (data not shown) transcripts
further indicate an abnormal specification of NCCs in mutants.
Previous work in chick and quail has demonstrated that RA is required for
the proper induction of genes that control spinal cord neurogenesis, including
Ngn1, Ngn2 and Delta1
(Diez del Corral et al.,
2003). We investigated the timing of induction of these genes in
the murine RA-deficient spinal cord. We found that, in wild-type embryos,
Delta1 is activated in scattered neural tube cells from the four- to
five-somite stages (Fig. 4G).
No Delta1-positive cells are present in Raldh2-/-
neural tube until at least the six- to seven-somite stages
(Fig. 4H; n=6/6).
However, at the 14- to 15-somite stage, Delta1 expression has become
comparable with wild type (Fig.
4I,J; n=4/4). Similarly, the effector of Notch signalling
Hes5 is induced with a delay of about 10 hours in
Raldh2-/- spinal cords
(Fig. 4K-N; n=3/3 and
5/5, respectively). Neurogenin 2 (Ngn2) is normally activated at
early somite stages (Scardigli et al.,
2001; Ribes et al.,
2008), whereas it only appears in Raldh2-/-
spinal cord and hindbrain cells around E9.0
(Fig. 4O,P; n=6/6 and
7/7, insets and main panels, respectively). This is in agreement with the
identification of an enhancer element recapitulating the early expression of
Ngn2, the activity of which is dependent on RA signalling
(Ribes et al., 2008). We also
analyzed spinal neuron differentiation. In E9.5 wild-type embryos,
neurofilaments are present along the dorsal root ganglia and the developing
motoneuron axons (Fig. 4Q). No
neurofilament-positive cells are observed along the
Raldh2-/- spinal cord
(Fig. 4R; n=5/5),
although the hindbrain ganglia express this protein (data not shown)
(Niederreither et al., 2000).
These results corroborate data obtained in avian systems
(Novitch et al., 2003;
Diez del Corral et al., 2003)
by showing that RA synthesized by Raldh2 is required, in the mouse embryo, for
the onset of neurogenesis and neural differentiation at the trunk level. The
delayed induction of various genes in the spinal cord of
Raldh2-/- mutants may reflect the presence of other
sources of RA in this tissue (Mic et al.,
2002).
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;
Helms et al., 1994;
Helms et al., 1997). We
investigated whether crosstalk between these two signalling pathways occurs
during caudal development. We analyzed Shh transcripts and protein
distribution in Raldh2-/- mutants, and found that
Shh is correctly transcribed and translated in the prechordal plate,
notochord and node until the 12- to 14-somite stages
(Fig. 5A-B';
n=6/6; and data not shown). However, at the 14- to 15-somite stages
(Fig. 5C) and at E9.5
(Fig. 5D), Shh protein is
almost undetectable in Raldh2-/- floor-plate cells
(Fig. 5E,F, and data not shown;
n=5/5 at each stage). This is not due to a lack of floor plate, as
both Shh transcripts and Foxa2 protein are similarly detected in
ventral midline cells in controls and mutants
(Fig. 5I,J and data not shown;
n=9/9 for Shh in mutants). Shh transcript and protein
expression in notochord is unaffected by RA deficiency, at least until E9.0
(Fig. 5C-F,G,I; notice the
enlarged notochord with low Shh expression at E9.5 in 5J).
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; Ingham and McMahon,
2001; Lee et al.,
1997; Ruiz i Altaba et al.,
2003; Sasaki et al.,
1997; Stamataki et al.,
2005). Gli1 expression is markedly downregulated in
Raldh2-/- embryos as early as E7.75
(Fig. 5K-L';
n=4/4). In wild-type embryos, Gli1 is expressed in axial
mesoderm, medial neural plate, newly formed mesoderm
(Fig. 5K, bracket), lateral
epiblast (Fig. 5K';
bracket) and node (asterisk). In Raldh2-/- embryos,
Gli1 expression is present in the anterior neural plate
(Fig. 5L), but is downregulated
in mesoderm (Fig. 5L, bracket),
lateral epiblast (Fig.
5L', bracket) and node (asterisk). At E8.5, Gli1 is
severely downregulated in most Raldh2-/- tissues
(Fig. 5M-N'';
n=9/9). Noteworthy, its distribution in the posterior hindbrain of
mutants (Fig. 5N') is not
ventrally restricted as in controls (Fig.
5M'). Patched 1, another Shh target gene
(Goodrich et al., 1996;
Marigo et al., 1996), showed
the same spatial and temporal downregulation in Raldh2-/-
embryos (data not shown). These results suggest that in the absence of RA
activity, Shh signalling cannot proceed efficiently in the prospective spinal
cord and adjacent tissues.
RA activity is required for proper response to Shh signalling
The downregulation of Shh target genes in Raldh2-/-
mutants suggests that cells require RA during late gastrulation and early
neurulation to respond properly to the Shh signal. Should it be the case,
administration of active Shh protein (Shh-N) may not rescue Shh signalling,
and therefore Gli1 expression, in Raldh2-/-
embryos. We tested this hypothesis using whole-embryo culture. Headfold stage
(E7.5) embryos were cultured for 14 hours in presence or absence of Shh-N. An
increase in Gli1 expression is observed in wild-type embryos (5/6
embryos) in both mesodermal and neural tissues after addition of Shh-N,
already at a physiological concentration of 5 nM
(Fig.
6A,A',C,C',E,E',G,G'). By contrast, the
addition of 5 nM, 50 nM or 150 nM Shh-N does not augment significantly
Gli1 expression in Raldh2-/- embryos (0/2; 0/9;
2/15 embryos showing elevated Gli1 levels, respectively;
Fig.
6B,B',D,D',F,F'). Gli1 upregulation in
mutants is only seen by increasing Shh-N to 200 nM (5/9;
Fig. 6H,H'). Higher Shh-N
doses (300, 400 nM) drastically affect both wild-type and
Raldh2-/- embryos, preventing further analysis (data not
shown). These experiments demonstrate that tissues of
Raldh2-/- embryos are not able to respond to exogenous Shh
at physiological or even supra-physiological concentrations, although they do
respond to Shh-N if administered at a concentration about 40-fold over the
physiological range.
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Impaired Fgf signalling in Raldh2-/- embryos accounts in part for the defects in Shh signalling
Studies in chick and mouse have shown that RA activity controls the extent
of Fgf8 expression in caudal tissues
(Diez del Corral et al., 2003;
Vermot et al., 2005;
Molotkova et al., 2005;
Sirbu and Duester, 2006). Fgf
and Shh signalling pathways interact in several developing systems (see
Introduction), and exposure of chicken caudal neural tissue to Fgf inhibits
onset of Shh and Ptc2 expression
(Diez del Corral et al.,
2003). To investigate whether alterations in Fgf signalling could
be responsible for the decrease in Shh response in
Raldh2-/- embryos, we analyzed two targets of this
pathway: sprouty 2 (Minowada et al.,
1999) and Mkp3
[(Dickinson et al., 2002); for
studies in chick (Eblaghie et al.,
2003; Kawakami et al.,
2003)]. Sprouty 2 is expressed at E7.75 in the CNP nearby the
primitive streak of wild-type embryos (Fig.
7A). Its domain of expression expands laterally in
Raldh2-/- embryos (Fig.
7B; n=5/5), reminiscent of the expansion of brachyury and
follistatin at the same stage (Fig.
2). Then, at early somite stages, sprouty 2 expression in the
caudal region of Raldh2-/- embryos is comparable with that
seen in controls (Fig. 7C,D;
n=5/5). At E9.5, sprouty 2 is markedly downregulated in caudal
tissues of mutants (Fig. 7E,F,
n=4/4).
In early somite-stage wild-type embryos, Mkp3 is expressed in
caudal tissues, with higher levels around the node
(Fig. 7G). Mkp3 caudal
expression is comparable in Raldh2-/- mutants, although
higher expression levels are seen in a domain posterior to the node
(Fig. 7H; n=9/9).
Whereas expression is observed in the somites and caudal tissues of wild-type
embryos from the 12- to 13-somite stages, Mkp3 expression is
abnormally low in the caudalmost region of the mutants
(Fig. 7I,J, brackets;
n=12/12). In summary, Fgf signalling is laterally expanded within the
CNP of Raldh2-/- embryos at the late gastrulation stage.
However, at early somite stages, no ectopic anterior expression of Fgf target
genes is seen in the Raldh2-/- neural plate and presomitic
mesoderm, and by the 15- to 16-somite stage Fgf signalling is downregulated
both in caudal tissues and somites. These results indicate that Fgf signalling
is only transiently expanded in caudal tissues of RA-deficient embryos prior
to E8. Consistent with previous observations in the VAD quail model, we found
no evidence of ectopic expression of target genes of Fgf signalling within the
developing neural tube (Diez del Corral et
al., 2003).
To test whether the ectopic Fgf activity in gastrulating
Raldh2-/- embryos contributes to the decrease in Shh
signalling efficiency, embryos were cultured for 6 or 14 hours in the presence
of 50 µM SU5402, an inhibitor of Fgf receptor signalling
(Mohammadi et al., 1997).
Blockade of Fgf signalling for 14 hours dramatically increases Gli1
expression in both controls (9/9) and mutants (3/3)
(Fig. 7J-Q). An increase of
Gli1 expression is observed in few controls (3/13), but not in
mutants (0/5), after 6 hours of SU5402 administration
(Fig. 7N-Q), suggesting that
its effect on Gli1 is indirect, similar to the effect of AT-RA. These
findings suggest that the abnormal Fgf signalling observed in
Raldh2-/- mutants contributes to the decrease of Shh
signalling efficiency. As SU5402 increases Gli1 levels even in
tissues lying at a distance from the ventral source of Shh, they might also
suggest that Gli1 can be stimulated independently of Shh.
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To test this hypothesis, wild-type and mutant embryos were cultured for 6
hours in the presence of 200 nM AT-RA. Gli2 levels are comparable and
relatively low in vehicle and RA-treated control embryos
(Fig. 8E,F). However, in the
presence of AT-RA, Gli2 is downregulated in most heterozygous and
null mutants, especially at the trunk level, when compared with
vehicle-treated controls (n=12/15 and 7/16)
(Fig. 8G,H; data not shown).
Similar results were obtained upon longer (14 hour) cultures with 200 nM AT-RA
(data not shown). Gli2 was also found to be downregulated following a
6-hour culture of wild-type embryos in the presence of BMS493 (5 µM), a
pan-RAR antagonist that stabilizes the association of RAR/RXR heterodimers
with transcriptional co-repressors
(Germain et al., 2002)
(n=31/40; data not shown), suggesting that it may be directly
regulated by RARs. This effect of BMS493 seems paradoxical, but probably
reflects a forced conformation of RAR/RXRs in a repressive state, also called
`inverse agonist' activity. Paradoxical effects observed upon treatment of
mouse embryos with BMS493 have already been discussed
(Niederreither et al., 2001).
We checked whether the regulation of Gli2 is dependent on Fgf
signalling by analyzing embryos cultured in the presence of SU5402. After 6 or
14 hours of culture, Gli2 expression is comparable in vehicle-treated
(n=7 and 10) and SU5402-treated embryos (n=8 and 11)
(Fig. 8I-L). These experiments
indicate that RA signalling represses Gli2 expression independently
of Fgf signalling (Fig.
9B).
|
|
DISCUSSION |
|---|
|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
RA signalling controls the partitioning of the epiblast into neural and mesodermal domains by inhibiting Fgf signalling
The generation of caudal structures relies on the orchestration within the
epiblast of two processes: cell ingression movements through the primitive
streak occurring during gastrulation and neural induction. The segregation of
these processes probably relies on the early subdivision of the epiblast into
mesodermal and neural domains (Delaune et
al., 2005; Sheng et al.,
2003). Our data indicate that the transition between gastrulation
and neural induction occurs in mouse around the late-headfold stage, when
epiblast cells along the anterior primitive streak start expressing early
neural markers such as Sox2 (Fig.
3 and data not shown). This raises the question of how is the
commitment of anterior epiblast cells towards a neural fate temporally and
spatially regulated? Studies in chick led to a model stating that this
commitment relies on opposing actions of RA emanating from the somites and
anterior presomitic mesoderm and Fgf signalling within caudalmost tissues
(Diez del Corral and Storey,
2004; Delphino-Machin et al., 2005). The present results lead us
to refine this model.
First, we found that RA signalling is dynamically regulated prior to the
onset of somitogenesis and that, from mid-gastrulation to headfold-stage, all
caudal progenitors, including primitive streak and node cells respond to the
RA signal. Thus, during gastrulation and early neural induction, RA and Fgfs
act in conjunction within the epiblast. Second, we show that this early RA
activity is required to restrict the expression of mesodermal markers,
including Wnt3a, brachyury and follistatin
(Fig. 2;
Fig. 9A). Although Wnt3a
directly regulates brachyury (Yamaguchi et
al., 1999), RA signalling is another candidate to regulate its
expression. Upon RA treatment, wild-type mouse embryos display within 4 hours
a downregulation of brachyury expression
(Iulianella et al., 1999). In
RA-deficient embryos, the expansion of these mesodermal markers probably
accounts for the abnormal accumulation of mesodermal cells within the tail
bud. Indeed, brachyury is necessary for caudal elongation, and analysis of
mouse chimeras showed that it controls cell movements in the posterior
mesoderm cell autonomously (Wilson et al.,
1995). Therefore, the expansion of brachyury expression in
Raldh2-/- embryos could alter mesodermal cell motility.
Moreover, by limiting the expansion of mesodermal markers and preventing the
ingression of the epiblast, RA signalling may control the timing of anterior
epiblast cells commitment towards a neural fate. This is supported by our
findings in Raldh2-/- embryos, including a delay in
Sox2 induction in the CNP and an absence of Wnt8a expression
postero-laterally to the node (Fig.
9A).
|
|
;
Sirbu and Duester, 2006), our
analysis of target genes of Fgf signalling did not confirm an anterior
expansion of this signal within the newly formed spinal cord or somites.
However, during gastrulation and early neurulation, a transient ectopic Fgf
activity mimics the lateral expansion of primitive streak markers in the
caudal region of Raldh2-/- embryos, arguing that RA
modulates Fgf signalling within epiblast cells. Because, in chick and
Xenopus, high levels of Fgf signalling enhance mesoderm formation and
cell ingression through the primitive streak, whereas lower levels drive the
epiblast towards a neural fate (Delphino-Machin et al., 2005;
Delaune et al., 2005), we
conclude that this abnormal Fgf activity could be one of the cues responsible
for the expansion of mesodermal markers in Raldh2 mutants. Altogether, our
results indicate that within the primitive streak, RA activity is required to
attenuate Fgf signalling, thereby limiting the progression of mesodermal
markers and the cell ingression movements through the primitive streak.
RA signalling sustains the cells response to Shh activity during specification and differentiation of neural and mesodermal derivatives
Previous studies have shown that in several developmental processes, Shh
and RA signalling converge and influence target gene regulation (e.g.
Bertrand and Dahmane, 2006;
Helms et al., 1994;
Helms et al., 1997). Some key
target genes may actually be regulated by direct combinatorial inputs from
both signalling pathways, as recently demonstrated in the case of the enhancer
responsible for Ngn2 onset of expression, which contains functional
Gli and RAR-RXR-binding sites (Ribes et
al., 2008). By overexpressing a dominant-negative RAR
,
Novitch et al. (Novitch et al.,
2003) reported a lack of response of cells to Shh signalling
within the chicken neural tube. Our present work strongly suggests that a
decrease in Shh signalling efficiency demonstrated in
Raldh2-/- embryos contributes to defects in both neural
and mesodermal patterning and differentiation. Many known Shh target genes are
downregulated in mutant tissues, such as the markers of somite differentiation
Pax1 and Myf5, or genes involved in specification of the
ventral neural tube Olig2, Pax6, Nkx6.2 and Nkx2.2
(Briscoe and Ericson, 2001;
Diez del Corral et al., 2003;
Lee and Pfaff, 2001;
Molotkova et al., 2005) (V.R.,
I.L.R. and P.D., unpublished). We provide further evidence that RA activity
acts at least at two different levels of Shh signalling
(Fig. 9B), by controlling the
cells response to Shh and the levels of Gli2.
As early as E7.5, Raldh2-/- neural and mesodermal tissues exhibit abnormally low levels of expression of Shh target genes (Gli1, Ptch1), despite unaltered levels of Shh mRNA and protein. Using embryo culture experiments, we show that: (1) Raldh2-/- embryos are less efficient that wild-type embryos to respond to exogenous Shh protein; (2) Gli1 and Ptch1 expression can be rescued following exposure for 14 hours, but not 6 hours, to exogenous RA, arguing for an indirect regulation; (3) exogenous RA enhances the response of cells to Shh in both wild-type and RA-deficient conditions. These results support the idea that RA is a necessary signal for cells to efficiently respond to Shh.
Importantly, we found that RA negatively regulates expression of
Gli2 (see Fig. 9B), a
key player of the Shh signalling pathway. An upregulation of Gli2 has
been reported in mice deficient for Shh
(Bai and Joyner, 2001),
although at later stages than in the RA-deficient mutants. This may suggest
that Gli2 upregulation in Raldh2 mutants is a consequence of
decreased Shh signalling. This is, however, unlikely, as we have not observed
any changes in Gli2 levels upon administration of Shh-N for 14 hours
to cultured embryos, either wild-type or Raldh2-/- (data
not shown).
We propose that the upregulation of Gli2 in RA-deficient embryos
could be one of the molecular cues underlying the specification defects both
in the caudal region and the forming somites
(Fig. 9B). Experiments in
Xenopus and mice have demonstrated that Gli2 is involved in
maintenance and AP patterning of the mesoderm, and establishment of ventral
identities within the spinal cord (Ruiz i
Altaba, 1999; Borycki et al.,
1998; Brewster et al.,
1998; Mullor et al.,
2001; Bai et al.,
2002; Bai et al.,
2004). Interestingly, Gli2 ability to induce ectopic expression of
mesodermal markers relies on an activating form of the protein
(Ruiz i Altaba, 1999;
Brewster et al., 1998;
Mullor et al., 2001),
suggesting that, in RA-deficient embryos, this transcription factor is acting
as an activator. Thus, unlike Gli3, which requires Shh signalling in order not
to be processed into a negative form
(Litingtung et al., 2002;
Persson et al., 2002;
Wang et al., 2000),
Gli2-activating function can be maintained independently of Shh signalling.
This indicates that, in mice, as described in chick and Xenopus
(Borycki et al., 1998;
Brewster et al., 1998;
Ruiz i Altaba, 1999), Gli2 may
not only be the major mediator of Shh signalling in neural and mesodermal
tissues, but could also have independent functions. From our results, Gli2
could play a pivotal function by integrating the RA and Shh signals. As these
two signalling pathways crosstalk in various cells types, including cancer
cells (Goyette et al., 2000;
So et al., 2004), our data
might suggest new routes to combat diseases due to alteration in the
transduction of these pathways, such as basal cell carcinoma
(So et al., 2006;
So et al., 2004).
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/136/4/665/DC1
|
Footnotes |
|---|
* These authors contributed equally to this work 
Present address: Department of Developmental Neurobiology, National
Institute of Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK
Present address: Department of Developmental Biology, CNRS URA 2578,
Pasteur Institute, 25 rue du Dr Roux, 75724 Cedex 15, Paris, France
|
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TOP
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MATERIALS AND METHODS
RESULTS
DISCUSSION
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