|
|
|
|
|
|
|
||||
| Home Help Feedback Subscriptions Archive Search Table of Contents | ||||
First published online December 20, 2005
doi: 10.1242/10.1242/dev.02204
1 Institut de Génétique et de Biologie Moléculaire et
Cellulaire, CNRS, INSERM, Université Louis Pasteur, BP 10142, 67404
Illkirch Cedex, CU de Strasbourg, France.
2 Departments of Medicine and Molecular and Cellular Biology, Baylor College of
Medicine, One Baylor Plaza, Houston, Texas 77030, USA.
Authors for correspondence (e-mail:
karenn{at}bcm.tmc.edu;
dolle{at}igbmc.u-strasbg.fr)
Accepted 9 November 2005
 |
SUMMARY |
|---|
 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Key words: Retinoids, Forebrain development, Eye development, Neural crest, Telencephalon, Diencephalon, Optic vesicle, Pituitary, Sonic hedgehog, FGF, Mouse, Aldh1a2
|
INTRODUCTION |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
;
Pera and Kessel, 1997;
Graham and Begbie, 2000). At
the end of anterior neuropore closure, the forebrain is subdivided into the
anteriorly positioned telencephalon and eye field, and the more caudal
diencephalon, which will contribute to the prethalamus, thalamus and
pretectum, and ventrally to the hypothalamus (reviewed by
Puelles and Rubenstein, 2003;
Wilson and Houart, 2004). At
this stage, neural progenitors have acquired their spatial identity, defined
by distinct combinations of transcription factors
(Shimamura et al., 1997;
Chow and Lang, 2001;
Crossley et al., 2001;
Kobayashi et al., 2002).
Several secreted molecules, including sonic hedgehog (SHH), fibroblast growth
factors (FGFs), WNTs and bone morphogenetic proteins (BMPs), as well as
retinoic acid (RA), have been implicated in early forebrain regionalization
(reviewed by Rallu et al.,
2002a; Echevarria et al.,
2003; Wilson and Houart,
2004).
RA is a diffusible lipophilic molecule that binds to nuclear receptors, the
RARs (
, ß and 
), which act as RXR heterodimers to regulate
the transcription of target genes (reviewed by
Chambon, 1996). In mice, the
three RARs are expressed in different embryonic head tissues
(Ruberte et al., 1991;
Mollard et al., 2000), and
RAR-RAR double mutants lack some frontonasal process derivatives
and display brain abnormalities, especially of the telencephalic vesicles
(Lohnes et al., 1994). Studies
in avian models have revealed sequential roles for RA signaling during
forebrain development. Analysis of RA-deficient quail embryos has implicated
RA as an early signal acting during gastrulation to regulate anteroposterior
(AP) patterning of the neural plate
(Halilagic et al., 2003).
Later on, RA is required for the survival of anterior head mesenchymal cells
and the development of telencephalic structures
(Schneider et al., 2001;
Halilagic et al., 2003). RA
has also been implicated as a signal specifying an intermediate character
within the telencephalon (Marklund et al.,
2004). These studies have highlighted possible crossregulatory
interactions between RA and two signals emanating from local forebrain
organizers: FGF signaling from the anterior neural ridge (ANR)
(Shimamura and Rubenstein,
1997; Ohkubo et al.,
2002); and SHH signaling from the prechordal plate and ventral
diencephalon (Ericson et al.,
1995; Gunhaga et al.,
2000).
The tissue distribution and levels of RA are controlled by the activities
of synthesizing enzymes, the retinaldehyde dehydrogenases (RALDHs), and of
metabolizing enzymes, the cytochrome P450s CYP26 (reviewed by
Duester, 2001;
Stoilov et al., 2001). The
Raldh2 (Aldh1a2 - Mouse Genome Informatics) and
Raldh3 (Aldh1a3 - Mouse Genome Informatics) genes are
differentially activated during early embryonic head and forebrain
development. Raldh2 is transiently expressed in the anteroventral
neuroepithelium prior to optic vesicle outgrowth
(Wagner et al., 2000;
Smith et al., 2001), whereas
Raldh3 is expressed later, although more persistently, in the surface
ectoderm overlying the anterior forebrain neuroepithelium and optic vesicle
(Li et al., 2000;
Schneider et al., 2001;
Blentic et al., 2003). Although
RALDH3 is often mentioned as the principal RA-synthesizing acting during
patterning of the forebrain (Schneider et
al., 2001; Marklund et al.,
2004), no developmental or molecular forebrain abnormalities have
been reported in Raldh3-/- knockout mice, which die at
birth because of nasal defects
(Dupé et al., 2003).
However, Raldh2-/- mutants, which die between embryonic
days (E) 9.5 and 10.5 owing to defective heart development, exhibit an
externally truncated forebrain and frontonasal region
(Niederreither et al., 1999),
as well as impaired optic vesicle morphogenesis
(Mic et al., 2004a).
We have studied the cellular and molecular defects underlying abnormal forebrain development in these mutants. Using a RA-sensitive reporter transgene, we show that RALDH2 is responsible for all detectable RA activity in the mouse forebrain and craniofacial tissues until the 14- to 15-somite-stages. Lack of RA synthesis, at these stages, drastically affects forebrain morphogenesis. By perturbing efficient FGF and SHH signaling, RA deficiency alters cell proliferation and survival, as well as expression levels of important regulators of ventral forebrain development.
|
|
MATERIALS AND METHODS |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
).
Whole-mount in situ hybridization with digoxigenin-labeled riboprobes was
performed as described
(Chotteau-Lelièvre et al.,
2005), using Intavis InSituPro robots (for a detailed procedure,
see
http://www.eumorphia.org/EMPReSS/servlet/EMPReSS.Frameset,
Gene Expression section). Whole-mount X-gal assays were performed as described
(Rossant et al., 1991).
Whole-mount immunolabeling with the phospho-p44/p42 Map-kinase (p-ERK1/2)
antibody (Cell Signaling) was performed according to the Rossant laboratory
protocol
(http://www.mshri.on.ca/rossant/protocols/DpERK%20Immunohistochem.html).
Immunolabeling with rabbit polyclonal anti-PAX6 (Babco) and anti-RALDH2
(Berggren et al., 1999) was
performed as described (Scardigli et al.,
2003), using a peroxidase-conjugated goat anti-rabbit (Jackson
Immunoresearch) secondary antibody. Immunohistochemistry with
anti-phospho-Histone H3 (Upstate) were performed on 10 µm cryosections.
Procedures for conventional histology and TUNEL assays can be found at
http://www.eumorphia.org/EMPReSS/servlet/EMPReSS.Frameset
(Necropsy exam, Pathology and Histology section). Three to eight
Raldh2-/- embryos were examined for all assays and at each
developmental stage described hereafter. |
RESULTS |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
To assess the contribution of RALDH2 in mediating active RA signaling in
the embryonic head, we analyzed the activity of the RARE-hsp68-lacZ
transgene (Rossant et al.,
1991), a sensitive reporter for endogenous RA signaling (e.g.
Wagner et al., 2000;
Niederreither et al., 2002a;
Mic et al., 2004b), in
wild-type and Raldh2-/- embryos. In eight- to 12-somite
stage wild-type embryos, the transgene was expressed in a region of the
forebrain encompassing the optic vesicles
(Fig. 1B,B') and
eventually the outgrowing telencephalon
(Fig. 1F,F',J,J'),
but excluding the ANR and the diencephalon
(Fig.
1B',F',J'). The RA-reporter transgene was also
activated between the 10- and 14-somite stages in the anterior head mesenchyme
and surface ectoderm (Fig.
1B',F',J'). In Raldh2-/-
embryos, no transgene activity was detected prior to the 14- to 15-somite
stages, when expression first appeared in scattered ectodermal cells in the
optic region (Fig. 1C,G).
Raldh3 gene expression was induced at a slightly earlier stage (10-12
somites) in both wild-type and Raldh2-/- embryos
(Fig. 1D), and was then
expressed at comparable levels in the head ectoderm of wild-type and mutants
(Fig. 1H,L). Thus, the altered
induction of the RA-reporter transgene in Raldh2-/-
embryos cannot be attributed to a downregulation of Raldh3 gene
expression. At later stages, transgene activity expanded in the facial region
of Raldh2-/- embryos
(Fig. 1K). Section analysis
showed activity in both the surface ectoderm and underlying mesenchyme
(Fig. 1K'); however, the
forebrain and optic vesicle neuroepithelium were almost completely devoid of
transgene activity (compare Fig. 1J'
with 1K').
Together, these data show that RALDH2 activity accounts for the region-specific patterns of RA-response observed in the early forebrain neuroepithelium and the adjacent mesenchyme. Upon RALDH2 deficiency, RALDH3 activity cannot significantly induce the RA-reporter transgene within the forebrain neuroepithelium, although it can eventually activate it in surface ectoderm and mesenchyme, with a delay of about half a day when compared with wild-type embryos.
Raldh2 deficient embryos display defective forebrain and optic development
Raldh2-/- knockout embryos have an externally truncated
craniofacial region (Niederreither et al.,
1999). Detailed histological analysis was performed to
characterize the underlying defects. No abnormality was observed in mutants at
presomitic stages (data not shown). At the 10- to 12-somite stages, before
anterior neuropore closure, the optic sulci did not evaginate properly in
Raldh2-/- embryos (Fig.
2A-B') and the cellular architecture of the forebrain
neuroepithelium was regionally disorganized
(Fig. 2B,B', brackets).
At the 15- to 16-somite stages, the neuroepithelium was disorganized and
misfolded along the optic vesicle-diencephalic junction
(Fig. 2C,D), and cell debris
were seen in the ventricular lumen (Fig.
2D, arrowheads). No evagination of the prospective telencephalic
vesicles was seen in mutant embryos (Fig.
2E,F). Throughout these stages, an abnormal accumulation of loose
mesenchymal cells was observed in the frontonasal region of
Raldh2-/- embryos (Fig.
2B',F).
|
Eye development was compromised in Raldh2-/- mutants,
whose optic vesicles did not closely contact the surface ectoderm
(Fig. 2I,J, insets), an event
that is indispensable for lens placode induction and optic pit formation. The
development of Rathke's pouch was also affected. This structure appears by
invagination of an ectodermal placode in the stomodeum (primitive mouth
cavity), that will contact the forebrain floor plate (infundibulum) to form
the pituitary anlage (Fig. 2K).
Rathke's pouch was absent in most of the Raldh2-/- embryos
(Fig. 2L; n=9/12). A
thin cord of cells was seen ventrally to the infundibulum, which was
molecularly identified as a prechordal plate remnant
(Fig. 2L; see
Fig. 8D). In some mutants
(n=3/12), a rudimentary pouch was present
(Fig. 2M). Raldh2-/- embryos were not analyzed after E9.5, as they
die about 1 day later because of cardiovascular defects
(Niederreither et al.,
2001).
Abnormal patterns of cell death and cell proliferation contribute to the Raldh2-/- forebrain defects
TUNEL analysis was performed to assess whether abnormal apoptosis may
contribute to the forebrain abnormalities in Raldh2-/-
mutants. No abnormal cell death was detected in mutants prior to the 14-somite
stage (data not shown). From this stage, abnormal apoptosis was detected in
the optic vesicle and infundibulum neuroepithelium
(Fig. 3A,B), in the area that
was found to be disorganized upon histological analysis
(Fig. 2D). Between E8.5 and
E9.5, apoptosis extended within the forebrain neuroepithelium to reach the
intermediate telencephalic region, and was also seen among pre-optic
mesenchymal cells (data not shown). Furthermore, whole-mount TUNEL analysis
showed an abnormal distribution of apoptotic cells in the rostral surface
ectoderm of E9.5 mutants, contrasting with the restricted apoptosis along the
ventral midline and placodal ectoderm of wild-type embryos
(Fig. 3C,D).
|
) was
detected in the ventral forebrain neuroepithelium of mutants
(Fig. 3I), whereas in wild-type
littermates it was only observed in the maxillary ectoderm
(Fig. 3H). We also analyzed the
expression of D-cyclin genes, components of the cell cycle machinery that are
dynamically expressed during embryogenesis
(Wianny et al., 1998). At the
14-somite stage, cyclin D1 expression in head mesenchymal cells was similar in
Raldh2-/- and wild-type embryos (data not shown). However,
cyclin D2 expression was reduced in the diencephalic neuroepithelium, and
almost undetectable in the prospective telencephalon of mutant embryos, while
expression at mesencephalic levels was comparable with wild-type embryos
(Fig. 3J,K). Cyclin D3
expression was also downregulated in the forebrain neuroepithelium of
Raldh2-/- embryos, except in the ventralmost telencephalon
(Fig. 3L,M).
Abnormal craniofacial neural crest distribution
Neural crest cells (NCCs) that colonize the frontonasal and perioptic
regions originate from prosencephalic and mesencephalic levels
(Serbedzija et al., 1992;
Osumi-Yamashita et al., 1994).
To investigate whether the excess mesenchyme observed in the frontonasal area
of Raldh2-/- embryos (see
Fig. 2) corresponds to NCCs, we
used Crabp1 and AP2 as markers of migrating NCCs.
This analysis confirmed that mesenchymal cells in
Raldh2-/- mutants mostly correspond to NCCs
(Fig. 4A,B, insets).
Furthermore, whole-mount analysis indicated a deficit of NCCs posteriorly to
the optic vesicle (Fig. 4A,B,
brackets), while numerous NCCs were found in the pre-optic mesenchyme of 13-14
somite-stage Raldh2-/- embryos
(Fig. 4D, arrow). At E9.5, NCCs
were mainly located along the rostroventral edge of the frontonasal process in
wild-type embryos, whereas these were distributed throughout the frontonasal
region in mutants (Fig. 4C,D,
insets).
We also analyzed NCC molecular determinants that are induced before the
lethality of Raldh2-/- embryos. Sox9 is required
for chondrogenic determination of cranial NCCs
(Mori-Akiyama et al., 2003),
whereas Sox10 is thought to maintain the pluripotency of NCCs, and at
later stages to direct a non-neuronal fate
(Kim et al., 2003). Both of
these determinants were induced in the cranial NCCs of
Raldh2-/- embryos (Fig.
4E-H). The abnormal Sox10 pattern, which was massively
expressed within the frontonasal area (Fig.
4F, arrow), may reflect the abnormal distribution of NCCs and/or
indicate that these cells are arrested at a multipotent state.
Maintenance of telencephalic gene expression requires Raldh2 activity
We then investigated whether regionalization of the forebrain is affected
in Raldh2-/- embryos. The winged helix transcription
factor Foxg1 (Bf1) is expressed in the telencephalic field
from the five-somite stage (Shimamura et
al., 1995) (Fig.
5A,C) and is required for the development of telencephalic
structures (Xuan et al.,
1995). Foxg1 expression was normally induced in
Raldh2-/- embryos until the 13-somite stage (data not
shown), but became abnormally weak by the 15-somite stage
(Fig. 5A,B), especially in the
intermediate telencephalon. By E9.5, its expression was almost extinguished,
with residual expression towards the ventral edge of the
Raldh2-/- forebrain
(Fig. 5C,D).
|
|
).
Emx2 was correctly induced in the telencephalon of 10-somite stage
Raldh2-/- embryos (Fig.
5E,F). However, at E9.5, Emx2 transcripts were expressed
at relatively low and homogeneous levels along the neuroepithelium that failed
to develop telencephalic vesicles, rather than according to a
dorsal-to-ventral gradient as in wild-type embryos
(Fig. 5G,H). Such a pattern was
reminiscent of that observed in wild-type and mutant embryos at an earlier
stage, before telencephalic vesicle outgrowth
(Fig. 5E,F, insets). We then analyzed how ventral cell fates are determined in the Raldh2-/- forebrain. The Nkx2.1 homeobox gene is expressed in ventral regions of the prospective diencephalon and telencephalon (Fig. 5I) The Nkx2.1 expression domain was reduced in Raldh2-/- embryos already at the 11-somite stage (Fig. 5I,J). Diencephalic expression was the most severely affected and Nkx2.1 transcripts were detected, albeit at lower levels, in the ventral telencephalon of Raldh2-/- embryos (Fig. 5I,J, insets). Nkx2.1 diencephalic expression remained markedly downregulated in mutants at E9.5, although the ventralmost diencephalic cells showed higher transcript levels (Fig. 5K,L). At this stage, Nkx2.1 expression in the wild-type telencephalon demarcates the future medial ganglionic eminence (Fig. 5K). The corresponding Nkx2.1-positive domain was both spatially and quantitatively reduced in the mutant embryos (Fig. 5L).
We also assessed whether the onset of neuronal differentiation is affected
in the RA-deficient forebrain by analyzing Delta1
(Bettenhausen et al., 1995) and
Hes5 (Hatakeyama et al.,
2004), which respectively encode a ligand and a downstream
effector of the Notch pathway. Although the first Delta1-expressing
cells were detected in the forebrain of both wild-type and
Raldh2-/- embryos at the 10-somite stage (data not shown),
at the 14-somite stage when Delta1 is highly expressed in the
wild-type telencephalic neuroepithelium, its expression was markedly reduced
in the corresponding region of Raldh2-/- embryos
(Fig. 5M,N). Eventually,
Delta1 expression was seen at E9.5 in the
Raldh2-/- rudimentary forebrain neuroepithelium
(Fig. 5M,N, insets). Similarly,
expression of Hes5 was defective in the E8.5
Raldh2-/- forebrain, although it was present in midbrain
and hindbrain cells (Fig.
5O,P). At E9.5, Hes5 expression was seen in the mutant
forebrain neuroepithelium, albeit at lower levels than in wild type (data not
shown). Thus, RA deficiency may affect the progression of neuronal
differentiation in the embryonic forebrain by controlling genes of the lateral
inhibition pathway.
|
) that is also involved in diencephalon and
telencephalon patterning (Stoykova et al.,
1996; Stoykova et al.,
2000). Its induction was not affected by RA deficiency, as normal
PAX6 protein levels were detected in the forebrain neuroepithelium of six- to
eight-somite stage Raldh2-/- embryos (data not shown).
From the 10-somite stage in wild-type embryos, PAX6 levels are upregulated in
the dorsal optic vesicle neuroepithelium, whereas the dorsal diencephalon and
telencephalon display intermediate protein levels
(Fig. 6A,C,D). PAX6 was not
upregulated in the optic vesicle of Raldh2-/- embryos to
the same extent as in wild-type embryos
(Fig. 6B,D). Thus, its levels
in the mutant optic vesicle were comparable with those seen in the dorsal
diencephalon and telencephalon (Fig.
6D,D').
Six3, a homologue of the Drosophila sine oculis gene, has
been shown to promote anterior fate within the prospective forebrain
(Lagutin et al., 2003).
Six3 expression in the ventral forebrain and optic vesicles was
comparable in Raldh2-/- and wild-type embryos until the
10-somite stage (data not shown), but became abnormally low in the optic
vesicles of mutants by the 12- to 14-somite stages
(Fig. 6E,F). Expression of its
homologue Six6 (Optx2) was similarly reduced in the
Raldh2-/- optic vesicles
(Fig. 6E,F, insets).
Altogether, these results indicate that RA signaling is required for the
proper upregulation of the Pax6 and Six genes within the
optic vesicle. This regulation is gene specific as Pax2, which also
controls eye development (Nornes et al.,
1990), was expressed at normal levels in the optic vesicles of
Raldh2-/- mutants, even at E9.5
(Fig. 6G,H).
To assess hypothalamic and infundibulum patterning, we analyzed
Hesx1 expression. This gene is normally expressed in the rostral
neural folds, which resolve to the hypothalamus
(Hermesz et al., 1996)
(Fig. 6G), and its disruption
leads to septo-optic dysplasia (Dattani et
al., 1998). Hesx1 expression was abnormally low in
Raldh2-/- embryos from the 12-somite stage, especially
within the infundibulum (Fig.
6I,J). Interestingly, expression of Six3 and
Six6, which are involved in neurohypophysis and hypothalamus
development (Lagutin et al.,
2003), was absent in the infundibulum of mutants
(Fig. 6E,F, brackets; data not
shown). Expression of Bmp4, another determinant of pituitary
formation (Takuma et al.,
1998; Ericson et al.,
1998), was maintained in the infundibulum of
Raldh2-/- embryos (Fig.
6K,L). These data suggest that molecular alterations intrinsic to
the hypothalamus and infundibulum would affect development of Rathke's pouch
in Raldh2-/- mutants.
RA deficiency affects FGF signaling in the craniofacial region
FGF8 is one of the key signals produced by the ANR, a secondary forebrain
`organizer' that controls both the outgrowth and specification of
telencephalic territories (Crossley et
al., 2001; Ohkubo et al.,
2002; Storm et al.,
2003; Echevarria et al.,
2003). Fgf8 was expressed at comparable levels in the ANR
of wild-type and Raldh2-/- embryos at the 10- to 14-somite
stages (Fig. 7A,B). Its
expression was maintained in the ANR of mutants at E9.5, although section
analysis showed weaker labeling than in its wild-type counterpart
(Fig. 7C,D, compare insets,
brackets). However, Fgf8 induction was deficient in the facial
surface ectoderm: only few Fgf8-positive cells were detected outside
of the ANR in Raldh2-/- embryos
(Fig. 7C,D).
As an indicator of effective intracellular FGF signaling, we assessed the
levels of phosphorylated ERK1 and 2 (p-ERK1/2)
(Corson et al., 2003) by
whole-mount immunostaining. Between the 8 and 12 somite stages, p-ERK1/2
levels were comparable between wild-type and Raldh2-/-
embryos. Slight reduction in immunostaining first appeared at the 14-somite
stage (data not shown), whereas p-ERK1/2 levels were clearly decreased along
the ANR and anterior head ectoderm of E9.5 mutants
(Fig. 7E,F). Expression of
Mkp3, a negative feedback modulator of FGF signaling whose induction
is FGF dependent (Kawakami et al.,
2003; Echevarria et al.,
2005), was almost undetectable in the anterior head mesenchyme of
Raldh2-/- embryos (Fig.
7E,F, insets). Expression of sprouty 1 (Spry1), another
FGF-dependent intracellular inhibitor
(Hanafusa et al., 2002;
Storm et al., 2003), was
similarly decreased in rostral head tissues of mutant embryos, except in the
ANR, where its expression was higher than in wild-type embryos from the
12-somite stage onwards (Fig.
7G,H).
|
;
Chiang et al., 1996;
Gunhaga et al., 2000), we
assessed whether SHH signaling may be altered in Raldh2-/-
embryos. Shh is initially expressed in the prechordal plate
underlying the neural tube (Fig.
8A). This activity controls the specification of ventral
telencephalic structures (Ericson et al.,
1995; Gunhaga et al.,
2000) and downregulation of Shh expression has been
observed in the prechordal plate of RA-deficient quail embryos
(Halilagic et al., 2003).
However, Shh expression was not altered in the prechordal plate of
Raldh2-/- embryos at presomitic stages
(Fig. 8B). Later on,
Shh is induced along the neural tube floor plate up to the
diencephalic level (Ericson et al.,
1995) (Fig. 8C). No
alteration of Shh expression was detected in the
Raldh2-/- forebrain until the 16-somite stage (data not
shown). From this stage, an abnormal downregulation was selectively observed
in the rostral diencephalon, anterior to the presumptive zona limitans
intrathalamica (ZLI) (Fig. 8D, bracket). In wild-type embryos, Shh expression is downregulated by
E9.5 in the infundibulum region (Shimamura
et al., 1995) (inset in Fig.
8C). In Raldh2-/- embryos, its expression was
maintained within this region (Fig.
8D, inset), correlating with a rostral expansion of Shh
expression along the ventralmost floor plate cells in younger mutants
(Fig. 8D, arrow).
|
; Marigo and Tabin,
1996). Patched (Ptch1) is expressed at high levels in the
diencephalic neuroepithelium and surrounding mesenchyme of 12- to 14-somite
stage wild-type embryos (Fig.
8E). Ptch1 expression was abnormally low in the
diencephalic neuroepithelium, and undetectable in the mesenchyme, of
Raldh2-/- littermates
(Fig. 8F; data not shown). At
E9.5, Ptch1 was no longer detected in the mutant diencephalon, except
in its rostroventral area close to the optic vesicles (data not shown).
Another direct target of SHH, Gli1
(Lee et al., 1997), is
normally expressed in the ventral diencephalic neuroepithelium and surrounding
mesenchyme, adjacent to the Shh expression domain
(Fig. 8G). From the 10-somite
stage, Gli1 expression was reduced in the diencephalon and abolished
in the mesenchyme of mutant embryos (Fig.
8H). At E9.5, Gli1 transcripts were barely detectable in
the mutant diencephalon, except in its rostroventral area
(Fig. 8G,H, insets).
GLI3 is a peculiar component of the SHH pathway, which primarily functions
as a repressor. Among various murine Gli mutants, only
Gli3-/- embryos exhibit patterning defects in the
forebrain, including downregulation of Emx genes and Pax6
(Theil et al., 1999;
Tole et al., 2000). In
14-somite stage wild-type embryos, Gli3 is expressed in the dorsal
diencephalon, the dorsal optic vesicle neuroepithelium and the telencephalon
(Fig. 8I). From the 10- to
12-somite stages, Gli3 expression was reduced in mutants, especially
in the optic vesicle (Fig. 8J).
Expression was comparatively less affected along the rostral
telencephalon.
SHH has been implicated in the specification of forebrain oligodendrocyte
precursors (OLPs) through the regulation of the Olig1/2 genes
(Nery et al., 2001;
Tekki-Kessaris et al., 2001).
OLPs first appear in the embryonic mouse diencephalon by E9
(Timsit et al., 1995), and
region-specific Olig2 expression is observed 1 day earlier in the
wild-type diencephalon (Fig.
8K). The Olig2-expressing domain was diminished in E8.5
Raldh2-/- embryos and expression was lacking along the
ventral diencephalon (Fig. 8L).
A smaller Olig2 domain was also seen at E9.5 in mutants
(Fig. 8K,L, insets). This
result, along with the defective induction of other SHH downstream genes such
as Nkx2.1 (Pabst et al.,
2000) (see Fig. 5), strengthens the idea that SHH signaling activity is deficient in the
diencephalon of RA-deficient embryos.
|
DISCUSSION |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
;
Appel and Eisen, 2003;
Diez del Corral and Storey,
2004). Owing to the sequential expression of the Raldh2
and Raldh3 enzymes (Wagner et
al., 2000; Li et al.,
2000; Smith et al.,
2001), RA production and signaling emerge in specific regions of
the forebrain neuroepithelium and adjacent tissues while these regions undergo
a dramatic burst of growth and remodeling. Here, we show that Raldh2
is responsible for early RA production within the mouse embryonic head between
the eight- and 14- to 15-somite stages. During this time window, no activity
of the RARE-lacZ reporter transgene is detected in
Raldh2-/- embryos, despite a normal onset of
Raldh3 gene expression in the head surface ectoderm. RALDH3 activity
is likely to account for the subsequent activation of the RA-reporter
transgene in the facial mesenchyme and surface ectoderm of the mutant embryos.
However, it is unable to significantly activate the reporter transgene within
the forebrain and optic neuroepithelium, even at E9.5.
Raldh2-/- embryos exhibit defective morphogenesis of
several forebrain derivatives, including the telencephalic vesicles, the optic
vesicles (see Mic et al.,
2004a) and the diencephalon. Their forebrain-derived neural crest
cells massively migrate towards the preoptic region, leading to an excess of
mesenchyme that may contribute to the phenotypic abnormalities, e.g. by
interfering with optic vesicle morphogenesis and lens placode induction.
Complex molecular alterations underlie these defects. Although the early
induction of several regulators of forebrain development (e.g. Foxg1
or Pax6) is unaltered, we show that RALDH2-mediated RA synthesis is
required for the maintenance of their normal levels of expression. The
earliest molecular alterations (e.g. Nkx2.1) precede the
morphological defects and are found in the ventral diencephalon. Expression of
specific neural determinants of the optic vesicle and
hypothalamic/infundibulum region (e.g. Pax6, Six3, Six6) is also
affected and may contribute, respectively, to the eye and pituitary defects in
mutants.
RA deficiency affects FGF signaling in the forebrain
RA deficiency caused by Raldh2 loss of function interferes with
two major signaling pathways that regulate forebrain development: the SHH and
FGF pathways. Fgf8 induction proceeds normally in the ANR of
Raldh2-/- embryos; at E9.5, its expression is only
marginally decreased. However, Fgf8 almost completely fails to be
activated in the facial ectoderm of mutants. Reduced levels of phosphorylated
ERK1/2, and of Mkp3 and Spry1 transcripts (which are
normally induced in response to FGF) in the anterior head mesenchyme and
surface ectoderm of mutants, collectively indicate impaired FGF signaling in
these tissues. Unexpectedly, however, Spry1 expression was found to
be upregulated within the ANR of mutant embryos. We did not detect abnormal
levels of other Fgf genes (Fgf3, Fgf17 or Fgf18) in the
craniofacial region of mutants, suggesting that this upregulation might be FGF
independent. As Spry1 encodes an intracellular inhibitor of FGF
signaling (Hanafusa et al.,
2002), its upregulation could participate to the defective FGF
signaling within the ANR.
Decreased FGF signaling can explain several abnormalities in the
RA-deficient embryos that have been similarly observed in other models where
the FGF pathway was experimentally altered. Reduction of Foxg1 and
Emx2 expression, as well as increased apoptosis, are seen following
Spry1 overexpression in mouse telencephalic explants
(Storm et al., 2003).
Inhibition of pERK signaling (Shinya et
al., 2001) or Fgf8 mutation
(Shanmugalingam et al., 2000)
in zebrafish lead to a reduction of the prospective medial ganglionic
eminence, which is also seen in the Raldh2-/- embryos by
Nkx2.1 expression. Interestingly, correct expression of Fgf8
in the chick ANR and head ectoderm has been shown to rely on facial NCCs
(Creuzet et al., 2004). NCCs
show an abnormal distribution in the frontonasal region of
Raldh2-/- embryos and maintain high levels of
Ap2a and Sox10 expression, genes that are normally turned
off when cells undergo differentiation
(Kim et al., 2003; Mitchell et
al., 1991). Impaired NCC-facial ectoderm interactions (and/or abnormal
specification of NCCs) could therefore play a role in the defective
Fgf8 induction in the RA-deficient embryos.
Altered FGF signaling could also account for the cell proliferation defect
observed in the forebrain neuroepithelium of mutants. FGF8 produced by the ANR
induces Foxg1 (Shimamura and
Rubenstein, 1997), which then regulates regionalization and growth
of the telencephalon by promoting the proliferation of neuroectodermal cells
(Xuan et al., 1995;
Martynoga et al., 2005). In
Xenopus, Foxg1 regulates expression of the cyclin-dependent kinase
inhibitor p27 (Hardcastle and Papalopulu,
2000). In the Raldh2-/- mutants,
downregulation of Foxg1 is accompanied by regionally reduced
expression of cyclin D2 and cyclin D3. D-cyclins are induced by mitogens and
act as sensors of the extracellular environment that link mitogenic pathways
to the core cell cycle machinery (reviewed by
Sherr and Roberts, 2004).
Their effect can be inhibited by the cyclin-dependent kinase inhibitors p21
and p27 (Sherr and Roberts,
1999). Increased p21 expression, which was observed in
the ventral forebrain neuroepithelium of E9.5 Raldh2-/-
embryos, may further contribute to the cell proliferation defect.
RA is required for proper Sonic hedgehog signaling in head tissues
Analysis of the expression of two SHH direct target genes, Gli1
and Ptch1, revealed decreased levels of SHH signaling in post-optic
ectomesenchymal cells and diencephalic neuroepithelium of
Raldh2-/- mutants, from the 10- to 12-somite stage
onwards. This decrease is not linked to a downregulation of Shh
itself, whose expression is not affected prior to the 16-somite stage in
mutants. Altered SHH signaling is likely to be responsible for the early
patterning defects observed in the Raldh2-/- ventral
diencephalon. Studies in various models have demonstrated that ventral genes,
such as NKx2.1, are downregulated or not induced when SHH signaling
is impaired (Ericson et al.,
1995; Gunhaga et al.,
2000; Rallu et al.,
2002b) and that pituitary development requires SHH signaling
(Treier et al., 2001;
Sbrogna et al., 2003).
Accordingly, we find that key genes involved in infundibulum specification
(such as Six3, Six6 or Hesx1) are affected in the
Raldh2-/- mutants. Whether our observations reflect a
possible role of RA in regulating the processing of SHH to an active form or
its transport (Okada et al.,
2004), or the convergence of RA and SHH pathways for proper
regulation of common target genes (Tsukui
et al., 1999; Novitch et al.,
2003; Schafer et al.,
2005), remains to be clarified.
Abnormal SHH signaling might in turn affect the FGF pathway in the
RA-deficient embryos. Several studies have shown that both in the forebrain
and the limb bud a decrease in SHH signaling leads to a downregulation of Fgf
gene expression (Sun et al.,
2000; Kraus et al.,
2001; Ohkubo et al.,
2002). Thus, RA activity in the forebrain, as postulated for the
limb (Helms et al., 1994;
Schneider et al., 2001;
Niederreither et al., 2002b;
Mic et al., 2004b), would be
required for the establishment of a functional FGF-SHH signaling loop.
Schneider et al. (Schneider et al.,
2001) have inhibited RAR/RXR signaling in the chick embryonic
head, and have correlated the resulting forebrain and facial deficiencies to a
loss of Fgf8 and Shh expression. Our model of endogenous RA
deficiency refines the relationship between these pathways, by showing that:
(1) response to the SHH signal is affected before any detectable change in
Shh expression; (2) Fgf8 expression in the head ectoderm
(rather than the ANR) is critically RA dependent; and (3) abnormal levels of
Spry1 might participate to the decreased FGF signaling in the
ANR.
A model for RA action in the embryonic forebrain
The brain develops from multipotent, self-renewing stem cells that are
defined by their ability, in vitro, to generate floating aggregates
(neurospheres) capable of differentiating into neurons, astrocytes and
oligodendrocytes (Reynolds and Weiss,
1992; Gritti et al.,
1996). Among adult forebrain precursors there are stem-like cells
that can be cultured in the presence of mitogens such as FGFs
(Gritti et al., 1996;
Johe et al., 1996). Embryonic
dorsal neurospheres acquire Olig2 expression (and oligodendrocyte
identity) only after exposure to FGF, which promotes the ventralization of the
stem cell population (Gabay et al.,
2003). The mouse model of RA deficiency described herein offers
insight into how RA regulates neural stem cell proliferation in a temporally
regulated manner. A localized burst of ventrally enriched RA production occurs
in the mouse forebrain at the 8- to 10-somite stages. We hypothesize this
serves to `prime' the ventralization of forebrain neural stem cell
populations. In the absence of RA several events occur: (1) reduction in
ventral forebrain gene expression; (2) impaired response to the SHH signal;
and (3) at a later stage, reduced FGF signaling. The initial patterning
alterations affect ventral neuronal determinants (such as Nkx2.1), as
well as the oligodendrocyte determinant Olig2. Several hours later
(after the 12- to 14-somite stages), RA acts in a broader manner, affecting
both dorsal and ventral cell populations, as evidenced by the defective
telencephalic vesicle outgrowth and overall thinning of the forebrain
neuroepithelium in the Raldh2-/- embryos. Throughout these
stages, a reduction in the expansion of neuronal progenitors, accompanied by
cell survival changes, could account for the abnormal forebrain phenotype. The
challenge will be to understand how crosstalk between RA, FGF and SHH
signaling lead to their sequential actions in regulating stem cell cycle exit
and induction of molecular determinants of patterning and/or cell-type
specification.
|
ACKNOWLEDGMENTS |
|---|
), C. Sherr (Cyclins) and A. Simeone
(Emx2) for template plasmids. This work was supported by grants from
the American Heart Association (0330265N) and the National Institute of Health
(R01 HL070733) (to K.N.); and by funds from the CNRS, INSERM, Hôpitaux
Universitaires de Strasbourg, Ministère Français de la Recherche
(ACI 03-2-490), Institut Universitaire de France and European Union
(EVI-GENORET: LSHG-CT-2005-512036) (to P.D.). |
Footnotes |
|---|
|
REFERENCES |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Appel, B. and Eisen, J. S. (2003). Retinoids run rampant: multiple roles during spinal cord and motor neuron development. Neuron 40,461 -464.[CrossRef][Medline]
Ashery-Padan, R. and Gruss, P. (2001). Pax6 lights-up the way for eye development. Curr. Opin. Cell Biol. 13,706 -714.[CrossRef][Medline]
Berggren, K., McCaffery, P., Drager, U. and Forehand, C. J. (1999). Differential distribution of retinoic acid synthesis in the chicken embryo as determined by immunolocalization of the retinoic acid synthetic enzyme, RALDH-2. Dev. Biol. 210,288 -304.[CrossRef][Medline]
Bettenhausen, B., Hrabe de Angelis, M., Simon, D., Guenet, J. L. and Gossler, A. (1995). Transient and restricted expression during mouse embryogenesis of Dll1, a murine gene closely related to Drosophila Delta. Development 121,2407 -2418.[Abstract]
Blentic, A., Gale, E. and Maden, M. (2003). Retinoic acid signalling centres in the avian embryo identified by sites of expression of synthesising and catabolising enzymes. Dev. Dyn. 227,114 -127.[CrossRef][Medline]
Chambon, P. (1996). A decade of molecular biology of retinoic acid receptors. FASEB J. 10,940 -954.[Abstract]
Chiang, C., Litingtung, Y., Lee, E., Young, K. E., Corden, J. L., Westphal, H. and Beachy, P. A. (1996). Cyclopia and defective axial patterning in mice lacking Sonic hedgehog gene function. Nature 383,407 -413.[CrossRef][Medline]
Chotteau-Lelièvre, A., Dollé, P. and Gofflot, F. (2005). Expression analysis of murine genes using in situ hybridization with radioactive and non-radioactively labeled probes. In Methods in Molecular Biology: In Situ Hybridization Protocols, 3rd edn (ed. I. A. Darby and T. D. Hewitson). Totowa (NJ): Humana Press.
Chow, R. L. and Lang, R. A. (2001). Early eye development in vertebrates. Annu. Rev. Cell Dev. Biol. 17,255 -296.[CrossRef][Medline]
Corson, L. B., Yamanaka, Y., Lai, K. M. and Rossant, J.
(2003). Spatial and temporal patterns of ERK signaling during
mouse embryogenesis. Development
130,4527
-4537.
Creuzet, S., Schuler, B., Couly, G. and Le Douarin, N. M.
(2004). Reciprocal relationships between Fgf8 and neural
crest cells in facial and forebrain development. Proc. Natl. Acad.
Sci. USA 101,4843
-4847.
Crossley, P. H., Martinez, S., Ohkubo, Y. and Rubenstein, J. L. (2001). Coordinate expression of Fgf8, Otx2, Bmp4, and Shh in the rostral prosencephalon during development of the telencephalic and optic vesicles. Neuroscience 108,183 -206.[CrossRef][Medline]
Dattani, M. T., Martinez-Barbera, J. P., Thomas, P. Q., Brickman, J. M., Gupta, R., Martensson, I. L., Toresson, H., Fox, M., Wales, J. K., Hindmarsh, P. C. et al. (1998). Mutations in the homeobox gene HESX1/Hesx1 associated with septo-optic dysplasia in human and mouse. Nat. Genet. 19,125 -133.[CrossRef][Medline]
Diez del Corral, R. and Storey, K. G. (2004). Opposing FGF and retinoid pathways: a signalling switch that controls differentiation and patterning onset in the extending vertebrate body axis. BioEssays 26,857 -869.[CrossRef][Medline]
Duester, G. (2001). Genetic dissection of retinoid dehydrogenases. Chem. Biol. Interact. 30,469 -480.
Dupé, V., Matt, N., Garnier, J. M., Chambon, P., Mark, M.
and Ghyselinck, N. B. (2003). A newborn lethal defect due to
inactivation of retinaldehyde dehydrogenase type 3 is prevented by maternal
retinoic acid treatment. Proc. Natl. Acad. Sci. USA
100,14036
-14041.
Echevarria, D., Vieira, C., Gimeno, L. and Martinez, S. (2003). Neuroepithelial secondary organizers and cell fate specification in the developing brain. Brain Res. Brain Res. Rev. 43,179 -191.[CrossRef][Medline]
Echevarria, D., Martinez, S., Marques, S., Lucas-Teixeira, V. and Belo, J. A. (2005). Mkp3 is a negative feedback modulator of Fgf8 signaling in the mammalian isthmic organizer. Dev. Biol. 277,114 -128.[CrossRef][Medline]
Ericson, J., Muhr, J., Placzek, M., Lints, T., Jessell, T. M. and Edlund, T. (1995). Sonic hedgehog induces the differentiation of ventral forebrain neurons: a common signal for ventral patterning within the neural tube. Cell 81,747 -756.[CrossRef][Medline]
Ericson, J., Norlin, S., Jessell, T. M. and Edlund, T. (1998). Integrated FGF and BMP signaling controls the progression of progenitor cell differentiation and the emergence of pattern in the embryonic anterior pituitary. Development 125,1005 -1015.[Abstract]
Gabay, L., Lowell, S., Rubin, L. L. and Anderson, D. J. (2003). Deregulation of dorsoventral patterning by FGF confers trilineage differentiation capacity on CNS stem cells in vitro. Neuron 40,485 -499.[CrossRef][Medline]
Gong, Q. and Shipley, M. T. (1995). Evidence that pioneer olfactory axons regulate telencephalon cell cycle kinetics to induce the formation of the olfactory bulb. Neuron 14, 91-101.[CrossRef][Medline]
Goodrich, L. V., Johnson, R. L., Milenkovic, L., McMahon, J. A.
and Scott, M. P. (1996). Conservation of the hedgehog/patched
signaling pathway from flies to mice: induction of a mouse patched gene by
Hedgehog. Genes Dev. 10,301
-312.
Graham, A. and Begbie, J. (2000). Neurogenic placodes: a common front. Trends Neurosci. 23,313 -316.[CrossRef][Medline]
Gritti, A., Parati, E. A., Cova, L., Frolichsthal, P., Galli,
R., Wanke, E., Faravelli, L., Morassutti, D. J., Roisen, F., Nickel, D. D. et
al. (1996). Multipotential stem cells from the adult mouse
brain proliferate and self-renew in response to basic fibroblast growth
factor. J. Neurosci. 16,1091
-1100.
Gunhaga, L., Jessell, T. M. and Edlund, T. (2000). Sonic hedgehog signaling at gastrula stages specifies ventral telencephalic cells in the chick embryo. Development 127,3283 -3293.[Abstract]
Halilagic, A., Zile, M. H. and Studer, M.
(2003). A novel role for retinoids in patterning the avian
forebrain during presomite stages. Development
130,2039
-2050.
Hanafusa, H., Torii, S., Yasunaga, T. and Nishida, E. (2002). Sprouty1 and Sprouty2 provide a control mechanism for the Ras/MAPK signalling pathway. Nat. Cell Biol. 4, 850-858.[CrossRef][Medline]
Hardcastle, Z. and Papalopulu, N. (2000). Distinct effects of XBF-1 in regulating the cell cycle inhibitor p27(XIC1) and imparting a neural fate. Development 127,1303 -1314.[Abstract]
Hatakeyama, J., Bessho, Y., Katoh, K., Ookawara, S., Fujioka,
M., Guillemot, F. and Kageyama, R. (2004). Hes genes regulate
size, shape and histogenesis of the nervous system by control of the timing of
neural stem cell differentiation. Development
131,5539
-5550.
Helms, J., Thaller, C. and Eichele, G. (1994). Relationship between retinoic acid and sonic hedgehog, two polarizing signals in the chick wing bud. Development 120,3267 -3274.[Abstract]
Hermesz, E., Mackem, S. and Mahon, K. A. (1996). Rpx: a novel anterior-restricted homeobox gene progressively activated in the prechordal plate, anterior neural plate and Rathke's pouch of the mouse embryo. Development 122, 41-52.[Abstract]
Johe, K. K., Hazel, T. G., Muller, T., Dugich-Djordjevic, M. M.
and McKay, R. D. (1996). Single factors direct the
differentiation of stem cells from the fetal and adult central nervous system.
Genes Dev. 10,3129
-3140.
Kawakami, Y., Rodriguez-Leon, J., Koth, C. M., Buscher, D., Itoh, T., Raya, A., Ng, J. K., Esteban, C. R., Takahashi, S., Henrique, D. et al. (2003). MKP3 mediates the cellular response to FGF8 signalling in the vertebrate limb. Nat. Cell Biol. 5, 513-519.[CrossRef][Medline]
Kim, J., Lo, L., Dormand, E. and Anderson, D. J. (2003). SOX10 maintains multipotency and inhibits neuronal differentiation of neural crest stem cells. Neuron 38, 17-31.[CrossRef][Medline]
Kobayashi, D., Kobayashi, M., Matsumoto, K., Ogura, T.,
Nakafuku, M. and Shimamura, K. (2002). Early subdivisions in
the neural plate define distinct competence for inductive signals.
Development 129,83
-93.
Kraus, P., Fraidenraich, D. and Loomis, C. A. (2001). Some distal limb structures develop in mice lacking Sonic hedgehog signaling. Mech. Dev. 100, 45-58.[CrossRef][Medline]
Lagutin, O. V., Zhu, C. C., Kobayashi, D., Topczewski, J.,
Shimamura, K., Puelles, L., Russell, H. R., McKinnon, P. J., Solnica-Krezel,
L. and Oliver, G. (2003). Six3 repression of Wnt signaling in
the anterior neuroectoderm is essential for vertebrate forebrain development.
Genes Dev. 17,368
-379.
Lee, J., Platt, K. A., Censullo, P. and Ruiz i Altaba, A. (1997). Gli1 is a target of Sonic hedgehog that induces ventral neural tube development. Development 124,2537 -2552.[Abstract]
Li, H., Wagner, E., McCaffery, P., Smith, D., Andreadis, A. and Dräger, U. C. (2000). A retinoic acid synthesizing enzyme in ventral retina and telencephalon of the embryonic mouse. Mech. Dev. 95,283 -289.[CrossRef][Medline]
Lohnes, D., Mark, M., Mendelsohn, C., Dollé, P., Dierich, A., Gorry, P., Gansmuller, A. and Chambon, P. (1994). Function of the retinoic acid receptors (RARs) during development (I). Craniofacial and skeletal abnormalities in RAR double mutants. Development 120,2723 -2748.[Abstract]
Marigo, V. and Tabin, C. J. (1996). Regulation
of patched by sonic hedgehog in the developing neural tube. Proc.
Natl. Acad. Sci. USA 93,9346
-9351.
