1 MRC Centre for Developmental Neurobiology, King's College London, Guy's
Campus, London SE1 1UL, UK
2 Department of Food Science and Human Nutrition, Michigan State University,
East Lansing, MI 48824, USA
3 TIGEM, Telethon Institute of Genetics and Medicine, Naples 80131, Italy
* Author for correspondence (e-mail: studer{at}tigem.it)
Accepted 24 January 2003
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SUMMARY |
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Key words: Retinoids, VAD embryos, Forebrain, Avian
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INTRODUCTION |
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Like FGF and Wnt proteins, retinoic acid (RA) is mainly considered to be a
posteriorizing factor with a high concentration posteriorly and a decreasing
gradient toward the anterior end of the embryo
(Chen et al., 1994;
Maden et al., 1998
;
Maden, 1999
). Retinoids
comprise various isoforms, including all-trans-RA and 9-cis-RA, which are
generated from vitamin A in a two-step oxidation process involving a group of
enzymes belonging to the family of alcohol dehydrogenases
(Duester, 2000
;
Ross et al., 2000
). Retinoids
act via specific nuclear receptors belonging to the steroid superfamily of
ligand-activated transcription factors, which play fundamental roles in
embryological morphogenetic processes (reviewed by
Zile, 2001
). The effects of
retinoid signalling on the CNS have been analysed in gain- and
loss-of-function experiments, which have generally revealed an involvement of
RA in AP patterning. By adding exogenous RA or constitutively activating
retinoic acid receptors (RAR) at early stages of development, the forebrain
becomes dramatically reduced, whereas the hindbrain is expanded
(Durston et al., 1989
;
Sive et al., 1990
;
Simeone et al., 1995
;
Avantaggiato et al., 1996
;
Zhang et al., 1996
). To
reproduce a loss-of-function of retinoid signalling, various strategies have
been employed: vitamin A has been depleted in pregnant mothers of quails or
rodents (Maden et al., 1996
;
Gale et al., 1999
;
White et al., 2000
), dominant
negative RARs have been injected into Xenopus embryos
(Blumberg et al., 1997
;
Kolm et al., 1997
;
van der Wees et al., 1998
) or,
more recently, antagonists to RARs have been added in a temporally and
spatially controlled fashion (Wendling et
al., 2000
; Dupé et al., 2001). These, and the inactivation
of the retinaldehyde dehydrogenase Raldh2 in mouse and zebrafish
(Niederreither et al., 1999
;
Begemann et al., 2001
;
Grandel et al., 2002
) have
pointed to defects restricted mainly to the hindbrain and spinal cord regions.
Thus, it remains unclear whether retinoids have an endogenous role in
forebrain patterning.
Two sites of localized retinoid activity have been described in the
developing forebrain at later stages. One site is localized in the
rostrolateral head mesenchyme and has been visualized with RA-reporter mice;
at this site, activity overlaps with a transient expression of Raldh2
(Rossant et al., 1991;
LaMantia et al., 1993
;
Wagner et al., 2000
). A second
source of RA synthesis is represented by the expression of Raldh3 in
the surface ectoderm and in a large part of the ventral rostral head at later
stages (Li et al., 2000
;
Mic et al., 2000
;
Smith et al., 2001
). It has
been suggested that the ventral ectoderm RA source, which is also present in
the chick embryo, might be involved in sustaining the outgrowth of the
frontonasal process and forebrain
(Schneider et al., 2001
). This
and other reports have therefore confirmed an essential role for retinoids in
the maintenance of a proper craniofacial structure, mainly through the
survival and proliferation of head mesenchyme
(Smith et al., 2001
). However,
a direct role for RA in the early events of anterior neural patterning remains
unclear.
In this report, we show for the first time that, in avian embryos, RA is
synthesized in the hypoblast before gastrulation, and in anterior definitive
endoderm and prechordal mesendoderm during gastrulation. As a first attempt to
challenge the role of RA in anterior patterning, we used the well-established
vitamin A-deficient quail model (VAD), which has been considered to be similar
to an `RA knockout' (Zile,
2001). We show that VAD embryos have altered molecular properties
in the anteriormost endoderm, which comprises hypoblast and definitive
endoderm, during the process of axial mesoderm specification. Furthermore,
prechordal mesendodermal and prospective telencephalic markers are expanded
posteriorly, and in early somite embryos increased apoptosis is detected in
ventral neuroectoderm and foregut endoderm. At later stages, VAD quail embryos
show a single undivided telencephalic vesicle, increased cell death in ventral
mesenchymal and neuroectodermal cells and abnormal dorsoventral patterning in
the diencephalon. Thus, we hypothesize that, in the forebrain, retinoids have
a biphasic activity that reflects differential functional contexts of RA
signalling in anterior regions: an early phase at gastrula stages, during
which RA refines the rostrocaudal identity of the anterior neural plate by
modulating signalling involved in axial mesoderm specification (this study);
and a late phase at neural pore closure, during which RA is involved in the
survival of the frontonasal mesenchyme mass and forebrain neuroectoderm (this
study) (Schneider et al.,
2001
). We propose that during the early phase, RA might be one of
the signalling molecules involved in the `stabilization step' of the model
proposed by Stern and colleagues (Foley et
al., 2000
; Stern,
2001
).
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MATERIALS AND METHODS |
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In situ hybridization and histological procedures
Whole-mount in situ hybridization with digoxigenin-labelled riboprobes was
performed as previously described (Grove
et al., 1998). Chick riboprobes were used in quail embryos after
careful comparison with their equivalent published chick expression profiles.
The Gsc riboprobe differs from the original version
(Izpisua-Belmonte et al.,
1993
) by covering most of the 5' part of the cDNA sequence
(50 bp of 5' UTR and 738 bp of coding sequence; see also GenBank
Accession Number X70471). Embryos for sectioning were embedded in 20% gelatine
and fixed in 4% PFA/PBS for at least 2 days. Sections were cut at 30 µm on
a vibratome and mounted in 90% glycerol before being photographed using a
digital camera. For histology, embryos were embedded in Fibrowax and sectioned
at 10 µm on a microtome. After the dewaxing and rehydration procedures,
sections were stained with Haematoxylin and Eosin for 2 minutes.
Immunohistochemistry
Free-floating 30 µm gelatine-embedded sections were washed in PBS
followed by three 1 hour washes in a blocking solution consisting of 5% goat
serum and 1% Triton X-100. Sections were then incubated overnight with
anti-phospho-Histone H3 (Upstate, USA) or anti-HNK1 (Zymed Laboratories, USA)
at a concentration of 1:500. After three 1 hour PBS washes, sections were
incubated overnight with Cy3 (Jackson IRL, USA) at a concentration of 1:100.
For the TUNEL method we used the In Situ Cell Death Detection, POD kit
(Roche), with some modifications. Free-floating sections were first washed in
1% Triton X-100 in PBS and, subsequently, incubated in the TUNEL-reaction mix
provided in the kit for 3 hours at 37°C. Sections were then washed in 1%
Triton X-100/PBS and incubated at 4°C overnight in the converter POD
provided in the kit. All sections were mounted in 90% glycerol and viewed
under a confocal microscope (BioRad, MRC-600).
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RESULTS |
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In summary, we found that two RA-synthesizing enzymes are expressed in anterior structures involved in early forebrain induction and patterning. Whereas Raldh2 expression is located in the hypoblast in pre-gastrula embryos and in prechordal mesendoderm during gastrulation, Raldh3 is transiently expressed in the node and in axial mesodermal precursors emerging from the node.
Altered expression of anterior endodermal markers in early VAD
embryos
The striking expression of Raldh2 in anterior endoderm at a stage
when this structure provides signals necessary for axial mesoderm
specification (Vesque et al.,
2000) suggests an endogenous role of RA signalling in patterning
early endodermal and mesodermal properties. To functionally test this
hypothesis, we took advantage of the VAD quail model, which lacks biologically
active RA throughout development. We first assessed whether TGFß family
members were altered in anterior endoderm, which at stage 4/5 is composed of
hypoblast and anterior definitive endoderm
(Fig.
2A-A'',C-C'')
(Vesque et al., 2000
).
Surprisingly, in VAD embryos Bmp2 and Bmp7 have stronger
levels of expression in hypoblast cells when compared with normal embryos
(n=6; asterisks in Fig.
2B,B',D,D'), which suggests an upregulation of BMP
signalling in the hypoblast. Furthermore, the anterior definitive endoderm
domain of Bmp2, and to a lesser extent of Bmp7, expression
is laterally expanded, although levels of transcripts do not change
(arrowheads in Fig.
2B,D,B'',D'').
|
Altogether, these data suggest an endogenous role for retinoids in modulating anterior endodermal signalling, which has been shown to be required in forebrain development. However, whereas BMP genes have altered expression levels mainly in primitive endoderm of VAD embryos, the expression of Hex and Crs is affected more in anterior definitive endoderm, suggesting distinct roles for RA in patterning anterior endodermal properties.
Abnormal specification of axial mesodermal and ventral neural midline
cells in early VAD embryos
Considering the fundamental role attributed to anterior endoderm (hypoblast
and anterior definitive endoderm) in the specification of axial mesoderm
(Vesque et al., 2000), we were
interested in establishing whether prechordal mesoderm and notochordal markers
were perturbed in VAD quail embryos.
As described above, anterior Raldh2 expression at stage 5/6 is
specifically upregulated in prechordal mesendoderm but is absent from
notochord cells (Fig. 1; gap of
expression in Fig. 3A and
inset). Surprisingly, we found that stage 6 VAD embryos have patches of
ectopic Raldh2 expression along the whole anterior axial mesoderm
(n=2; Fig. 3B,
arrowheads in inset). Transverse sections at the level of presumptive rostral
notochord indicate the lack of Raldh2 expression in normal embryos
and ectopic mesodermal expression in VAD embryos
(Fig. 3A',B').
Next, we looked at the expression of the transcription factor gene
goosecoid (Gsc) and at the late expression of Crs,
Bmp2 and Bmp7 in prechordal mesodermal cells
(Fig. 3C,E,G; data not shown).
Transverse sections reveal no Gsc expression in the notochord of
normal embryos (Fig.
3C'), whereas abnormal ectopic expression is detected in VAD
embryos (n=5; Fig.
3D'), and Crs and Bmp2 midline expression
is expanded towards the node when compared with normal embryos (n=7;
arrows in Fig. 3F,H). Next, to
ascertain whether the extension of the anterior notochord was altered in VAD
embryos, we used chordin as a molecular marker for notochord cells in
stage 7 embryos (Fig.
3I,I') (Vesque et al.,
2000). In VAD embryos the chordin-free region is posteriorly
expanded, and at presumptive anterior notochord levels, expression is mostly
undetectable (Fig. 3J, inset,
J'). In summary, these data indicate abnormal patterning of axial
mesoderm, i.e. prechordal mesodermal markers are expressed more posteriorly,
in presumptive anterior notochord.
Furthermore, and surprisingly, transverse sections at all rostrocaudal
levels reveal ectopic Bmp2 and Bmp7 expression in the
ventral neural midline of VAD embryos (n=8;
Fig. 3H'; data not
shown), which suggests that BMP signalling is also altered in the ventral
neural tube. It has been reported that the coordinate action of TGFß
family members and SHH is required for the correct induction of anterior
properties of ventral midline cells at prospective forebrain levels
(Dale et al., 1997;
Dale et al., 1999
;
Ericson et al., 1995
). Thus,
to ascertain whether the abnormal distribution of BMP genes accompanied
altered Shh expression, we looked at Shh distribution at
stage 6 of normal and VAD embryos (Fig.
3K-L'). Fig.
3L shows that, in VAD embryos, overall levels of Shh
expression are slightly decreased along the rostrocaudal axis; however, lower
levels are more evident in anterior regions (n=4; inset in
Fig. 3L).
In summary, our molecular analysis suggests an endogenous role for retinoids in the AP specification of axial mesoderm into prechordal mesoderm and anterior notochord.
In addition, abnormal BMP gene expression in the ventral neural tube and lower levels of Shh expression in prechordal mesoderm suggest an altered balance of signalling molecules involved in anterior ventral neural patterning.
The prospective telencephalic territory posteriorly expanded in early
VAD embryos
Heterotopic grafting and tissue recombination experiments have demonstrated
that prechordal mesendoderm is a potent anterior neural inducer (reviewed by
Kiecker and Niehrs, 2001).
Therefore, the posterior expansion of prechordal mesendoderm markers observed
in VAD embryos could result in the induction of neural tissue with a more
anterior character. To test this hypothesis, we looked at the expression of
various anterior neural markers in stage 6-11 VAD embryos. The transcription
factor Otx2 is a marker for presumptive fore- and midbrain regions in
all vertebrate species examined, and its expression is reduced in presence of
excess RA (Bally-Cuif et al.,
1995
; Simeone et al.,
1995
; Swindell et al.,
1999
). Concordantly, we observed an AP enlargement of the
Otx2 expression domain in the absence of RA (n=3;
Fig. 4B), which suggests an
expansion of prosencephalic and mesencephalic tissue. To assess the degree of
expansion at the level of the prosencephalon, we used Pax6 as a
presumptive diencephalic marker (Bell et
al., 2001
). At stage 7+, Pax6 is expressed in the caudal
half of the prosencephalon but is excluded from the most anterior region of
the embryo (Fig. 4C). In VAD
embryos the posterior Pax6-positive region is not affected, whereas
the most anterior Pax6-negative region has an increased AP length
(n=3; Fig. 4D). To
verify that the affected region corresponds to the future telencephalon, we
used Nkx2.1 as a ventral regional marker
(Crossley et al., 2001
) and
Bf1 (qin) as a prospective ventral telencephalic marker
(Bell et al., 2001
). At stage
9, the Nkx2.1 expression domain encompasses mainly the hypothalamic
primordium (Fig. 4E; inset). In
VAD embryos the posterior expression boundary is clearly shifted caudally,
whereas the anterior boundary has not changed (n=5;
Fig. 4F; arrowhead in
F'). Furthermore, the caudomedian expression boundary of Bf1
(red line in Fig. 4G) has
shifted towards the prospective diencephalon at stage 11+ (n=4;
Fig. 4H; inset). Thus, our
analysis with regional forebrain-specific markers shows an expansion of
telencephalic tissue in the absence of retinoids, and suggests a role for RA
in maintaining a proper boundary between the future telencephalon and
diencephalon.
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DISCUSSION |
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In the mouse, the AVE is necessary for the development of anterior
structures (Thomas and Beddington,
1996); however, its transplantation does not generate an ectopic
forebrain (Tam and Steiner,
1999
; Kimura et al.,
2000
). In the chick, the hypoblast can only transiently induce
early anterior and neural markers
(Knötgen et al., 1999
;
Foley et al., 2000
).
Therefore, the role of the AVE and the hypoblast remain unclear. One
hypothesis is that the hypoblast might pre-pattern the naïve epiblast,
before it becomes responsive to additional stabilizing signals secreted by the
node. Alternatively, both the AVE and the hypoblast could be involved in
regulating cell movements, as suggested by fate mapping in mouse and
transplantation studies in chick (Foley et
al., 2000
; Kimura et al.,
2000
). Finally, removal of the hypoblast in chick causes the
formation of ectopic primitive streaks, which suggests that the hypoblast
could position the primitive streak by emitting an inhibitor of axis formation
(Bertocchini and Stern, 2002
).
Interestingly, it has been shown that local application of RA can cause a
pronounced curving of the primitive streak, away from the retinoid source
(Knezevic and Mackem, 2001
),
in a manner reminiscent to that observed with hypoblast rotation
(Foley et al., 2000
); however,
no presence of retinoids has been documented in pre-gastrula stage embryos so
far. We found that Raldh2 is highly expressed in the expanding
hypoblast and thus it is conceivable that retinoids participate in the early
events in which the hypoblast is implicated. We propose that one of the roles
of RA in the hypoblast might be to provide a proper developmental environment
for migrating cells, a role that has already been suggested in branchial arch
patterning (Wendling et al.,
2000
; Zile et al.,
2000
).
During early gastrulation the anteriormost endoderm, comprising the
hypoblast and anterior definitive endoderm, is required for proper forebrain
development. In particular, it has been shown that BMP2 and BMP7 can partially
mimic the action of anterior endodermal cells in suppressing notochord and in
inducing prospective prechordal mesoderm characteristics in the axial mesoderm
(Vesque et al., 2000). Our
study shows that during axial mesodermal specification, retinoid synthesis is
present in anterior endoderm, as seen by the expression profile of
Raldh2. Therefore, RA could freely diffuse from the endoderm into the
overlying anterior neural plate and towards the emerging axial mesoderm.
However, the presence of the RA-degrading enzyme CYP26 in anterior
neuroectoderm from stage 3+/4 onwards (data not shown)
(Swindell et al., 1999
)
excludes a vertical action of RA directly into the neural plate at this stage,
and favours instead a direct or indirect role of RA in axial mesoderm
specification (see model in Fig.
7). Accordingly, our functional data demonstrate that in the
absence of retinoids, Bmp2 and Bmp7 expression is
upregulated in the hypoblast, and axial mesodermal cells acquire altered
prechordal mesoderm properties. Thus, we propose that retinoids cooperate in
refining the extent of prechordal mesododerm characteristics by modulating
adequate levels of BMP signalling in the hypoblast
(Vesque et al., 2000
). The
need to maintain precise levels of both RA and BMP signalling has been already
described in other systems (Schultheiss et
al., 1997
; Yatskievych et al.,
1997
; Andree et al.,
1998
; Ladd et al.,
1998
; Ghatpande et al.,
2000
; Schlange et al.,
2000
). In the head, low levels of BMP proteins and higher levels
of RA are necessary to pattern the frontonasal mesenchyme in chick embryos
(Lee et al., 2001
). Our data
validate that precise levels of RA and BMP proteins are also a requisite for
early anterior patterning, and add further insight into the regulation of BMP
proteins by retinoids.
|
In summary, we propose that among the various signalling molecules involved in endodermal and early anterior CNS patterning, RA is an essential component that might act at the level of primitive and definitive anterior endoderm thereby modulating levels of expression of various target genes. More studies need to be performed in order to characterize the precise role of retinoids in the genetic cascade of endodermal signalling.
Retinoids are required in the specification and regionalization of
the developing prosencephalon
Various reports have shown that prechordal mesoderm is a potent anterior
inducer when transplanted ectopically, and is absolutely necessary for
maintaining and/or reinforcing anterior neural plate specification and, thus,
head structures (reviewed in Kiecker and
Niehrs, 2001). Moreover, in chick, signals provided by axial
mesendoderm refine the rostrocaudal character of the overlying neuroectoderm
(Rowan et al., 1999
).
Therefore, accurate AP specification of prechordal mesoderm is a prerequisite
for correct regionalization of the overlying anterior neuroectoderm. Our data
are in accordance with these findings and show that an extension of prechordal
mesendoderm caused by lack of retinoids induces a reciprocal expansion of the
anterior neural plate. In Xenopus, injection of a dominant-negative
retinoid acid receptor that can inhibit RA signalling leads to an expansion of
anterior structures, as observed by the increased expression of the
forebrain/midbrain marker Otx2
(Blumberg et al., 1997
).
Conversely, in mouse and chick, RA exposure abolishes Otx2 expression
in midline mesendoderm, and induces various degrees of forebrain malformations
in mouse embryos (Bally-Cuif et al.,
1995
; Simeone et al.,
1995
). Our data not only confirm the endogenous requirement of
retinoids in restricting Otx2 expression in avian embryos, but also
identify the ventral telencephalon as the main target subregion within the
developing prosencephalon that is affected by the absence of retinoids.
Therefore, the early endogenous role of RA might be to restrict the AP extent
of the ventral telencephalon. However, our data cannot distinguish between a
posterior to anterior fate conversion or an expansion of the future
telencephalon. The maintenance of a normal diencephalic Pax6 domain
prompts us to opt for the latter possibility; however, studies involving
tissue explants with the use of regionalized molecular markers in the presence
or absence of RA would help solve this issue.
Finally, the expansion of the posterior expression boundary of
Nkx2.1, which normally marks the border between prechordal and
epichordal neural plate (Shimamura et al.,
1995), could imply a specific role of retinoids in maintaining the
correct position of morphological boundaries within the prosencephalon.
Various observations are in agreement with this hypothesis. First, VAD embryos
fail to develop a morphological zli boundary, as seen in histological
preparations and in the absence of Shh expression in the zli (A.H.
and M.S., unpublished). Second, VAD embryos present ectopic constrictions
and/or ridges in the dorsal diencephalon
(Fig. 6B). Finally, in the
hindbrain, local application of RA causes the disappearance of posterior
hindbrain boundaries (Nittenberg et al.,
1997
) and, conversely, treatment with a retinoid receptor
antagonist fails to specify gene expression borders involved in boundary
formation (Dupé et al., 2001). Thus, as in the hindbrain, the
endogenous role of RA in anterior regions might be to restrict the expression
domains of regulatory genes involved in regionalizing the developing
prosencephalon during a crucial time-window.
A dual role of RA action in patterning the forebrain
Although VAD embryos show an expansion of anterior neural tissue at early
stages, the telencephalic vesicles are not separated and are dramatically
reduced at later stages. How are these events correlated? We propose various
hypotheses to explain this discrepancy. First, we found a reduction of
Shh expression in prechordal mesoderm before the appearance of an
increased rate of cell death in ventral neuroectoderm and foregut endoderm.
SHH has been shown to act as an anti-apoptotic factor and might therefore be
responsible for the increased ventral cell death seen in VAD embryos (see also
Ahlgren and Bronner-Fraser,
1999; Britto et al.,
2002
; Charrier et al.,
2001
). Second, VAD embryos show abnormal expression of
Bmp2 and Bmp7 in ventral neural midline, which might induce,
together with reduced Shh expression, altered rostrocaudal properties
in the overlying neuroectoderm. Therefore, mis-specification of axial identity
in the mesenchyme and neuroepithelium could lead to programmed cell death, a
phenomenon referred to as positional apoptosis and already described in the
hindbrain (Maden et al.,
1997
). Third, decrease of retinoid signalling in the frontonasal
mass, either through inactivation of RAR at the gene level or through RAR
antagonists, generates various degrees of forebrain malformations and
increased death of head mesenchymal cells
(Lohnes et al., 1994
;
Schneider et al., 2001
). The
study by Schneider et al. (Schneider et
al., 2001
) has shown that, in stage 10 chick embryos, retinoids
might be involved in maintaining a proper growth rate of head mesenchymal
cells, and thus an adequate outgrowth of the frontonasal process and forebrain
structures. Our study demonstrates that, at stage 10, VAD embryos have already
increased cell death in ventral neuroectoderm and endoderm but migration of
cranial neural crest cells is normal. Subsequently, from stage 12 onwards, VAD
embryos show a dramatic increase in programmed cell death of mesenchymal and
neuroectodermal cells. Thus, in contrast to the findings of Schneider and
collaborators, we identified apoptotic cells in ventral neuroectoderm before
the appearance of dying cells in neural-crest derived cells, which suggests
that retinoids might have different roles at different stages in patterning
the developing forebrain. We propose, therefore, that the overall increase of
cell death observed in VAD embryos at stage 20, is derived from an additive
effect between an early event restricted mainly to the ventral neuroectoderm
and foregut endoderm, and a later event confined to the head mesenchyme.
Conclusion
In summary, this study has unravelled a novel role for RA signalling during
gastrulation in anterior patterning that has not been reported previously (see
model in Fig. 7). We speculate
that RA synthesized in hypoblast cells might be involved in providing a proper
developmental environment for cell motility and/or regulating primitive streak
formation. Subsequently, during the process of axial mesoderm specification
into prechordal mesoderm and notochord, RA is synthesized from two sources,
one located in the node and another in anterior endoderm, which is composed of
remnants of hypoblast and anterior definitive endoderm. Whereas the node
source might be involved in the differentiation of axial mesodermal
precursors, the anteriormost endodermal source might be required in modulating
regulatory genes and signalling molecules during the specification of axial
mesodermal cells (Fig. 7A). In
particular, RA would restrict the AP extent of prechordal mesoderm, thereby
maintaining and stabilizing anterior neural plate characteristics in
conjunction with other signalling pathways, in accordance with the
`stabilization step' proposed by Stern and collaborators
(Stern, 2001)
(Fig. 7B). Therefore, altered
vertical signals that are due to the absence of retinoids and to abnormal AP
specification of prechordal mesoderm would fail to maintain a proper forebrain
anlage (Fig. 7C). Previous data
obtained from Xenopus and mouse embryos with exogenous doses of RA
had suggested a direct action of RA on axial mesodermal cells
(Ruiz i Altaba and Jessell,
1991
; Bally-Cuif et al.,
1995
; Simeone et al.,
1995
); however, no endogenous sources of RA synthesis had been
described at that time to justify such a hypothesis.
Finally, a late source of retinoids localized in the head mesenchyme and involved in sustaining proper forebrain outgrowth would be responsible for the late phase of cell death observed in VAD and antagonist-treated embryos (this study) (Schneider et al., 2000). Thus, although presumptive telencephalic structures are more widely induced in early VAD embryos, these cannot be maintained at later stages.
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ACKNOWLEDGMENTS |
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