1 Unité de Génétique Moléculaire de la
Morphogenèse, Institut Pasteur, URA 2578 du CNRS, 25 rue du Dr Roux,
75724 PARIS Cedex 15, France
2 Unité de Biologie Moléculaire du Développement, Institut
Pasteur, URA 2578 du CNRS, 25 rue du Dr Roux, 75724 PARIS Cedex 15,
France
Author for correspondence (e-mail:
brobert{at}pasteur.fr)
Accepted 15 May 2003
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SUMMARY |
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Key words: Mouse Msx genes, Brain development, Subcommissural organ, Prosomeres, Roof plate, Dorsal midline, Wnt, Bmp, In ovo electroporation
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INTRODUCTION |
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Msx genes encode homeodomain transcription factors that are expressed
dorsally in the neural tube of all species throughout chordate evolution. This
may therefore reflect an ancestral function for these genes in central nervous
system (CNS) patterning. Indeed, the single Msx gene of Amphioxus is
expressed exclusively at this site
(Sharman et al., 1999). In
Drosophila, Msh, the Msx ortholog, is required for the specification
of lateral neuroblasts, which can be considered as the homologs of the dorsal
neurons in vertebrates (Cornell and von
Ohlen, 2000
). In the mouse, the Msx gene family comprises three
members (Msx1, Msx2 and Msx3). All three genes are expressed
in the dorsal aspect of the neural tube from early stages of neurogenesis
(Robert et al., 1989
;
Catron et al., 1996
;
Shimeld et al., 1996
;
Wang et al., 1996
). At the
neural fold stage, Msx1 is expressed in the future dorsal neural
cells at the ectoderm/neuroectoderm boundary. Thereafter, at the neural tube
closure stage, Msx1 expression is restricted, in the CNS, to the
dorsal midline along its entire length
(Liem et al., 1995
;
Furuta et al., 1997
) (A.B.,
Y.L. and B.R., unpublished). In the midline, Msx1 is co-expressed
with Bmp and Wnt genes and might therefore play a role in signaling by these
diffusible molecules. Whereas Msx3 expression is restricted to the
neural tube (Shimeld et al.,
1996
; Wang et al.,
1996
), Msx1 and Msx2 are expressed in a number
of structures that are formed by inductive processes between ectoderm and
mesoderm, such as limb buds, craniofacial processes and tooth buds. At these
sites, functional inductive interactions are required for the expression of
the two genes, and these interactions involve signaling molecules of the Bmp
and Fgf families (reviewed in Davidson,
1995
; Peters and Balling,
1999
).
Msx1 homozygous mutants die at birth. They exhibit cleft palate,
an arrest in tooth development, with defects in the craniofacial skeleton and
inner ear (Satokata and Maas,
1994; Houzelstein et al.,
1997
). Msx2 homozygotes are viable and fertile, but
display pleiotropic defects, including abnormal teeth and loss of fur that can
be related to inductive processes
(Satokata et al., 2000
)
(M.-A.N. et al., unpublished). In addition, the cerebellar lobules are reduced
in number and disorganized. Analysis of Msx1/Msx2 compound
mutants has further revealed functional redundancy between these genes in most
of these processes because the absence of both Msx genes results in earlier
and stronger phenotypes, particularly in skull bones and ectodermal organs
(teeth, hair follicles and mammary glands).
The implication of Msx genes in ecto-mesodermal induction processes has
been extensively studied. However, despite a number of data strongly
indicative of a role for Msx genes in the development of the CNS, little is
known about their role in this structure except for the implication of
Msx2 in cerebellum foliation. In this report, we demonstrate, using
Msx1-null mutants that we had produced previously
(Houzelstein et al., 1997),
that this gene is necessary for the correct development of the dorsal midline
of the diencephalon. In the Msx1 mutant, the expression of genes
normally expressed at the dorsal midline is extinguished specifically in
prosomere 1 (P1), the most caudal subunit of the diencephalon. This leads to
the downregulation of genes expressed laterally to the midline, confirming the
importance of this structure for dorsal CNS patterning. Failure to form a
functional dorsal midline in P1 precludes development of the subcommissural
organ and leads to prenatal hydrocephalus. Wnt1 downregulation is
observed early and takes place before the disappearance of the midline. This
observation, together with ectopic expression experiments in the chick embryo,
indicate that Wnt1 is a target of Msx1. In addition,
analysis of Msx1/Msx2 double homozygous mutants confirms that the Msx
genes are required for expression of Wnt1 at the pro- and
mesencephalon dorsal midline. We propose a model in which Msx genes play a
role in Bmp and Wnt signaling to form a functional dorsal midline. The latter
acts as an organizing center to induce or maintain in neighboring tissues the
expression of genes essential for dorsal brain patterning.
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MATERIALS AND METHODS |
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In situ hybridization
Whole-mount in situ hybridization was performed as described previously
(Houzelstein et al., 1997).
DNA fragments used to generate RNA probes were the following: Wnt3a,
740 bp (3' untranslated region obtained by PCR; F: GGA ATT CCA ATT TGG
GCC GGA AGT CC; R: CGG GAG ATC TGA GTA TTA AGT GTC AGA GC); Otx2, 408
bp (coding for amino-acids 110 to 243); Lim1, 441 bp (coding for
amino-acids 259 to 406); Pax7, Pax6 and Pax3 (complete cDNA
kindly provided by Dr F. Relaix); Wnt1, 1100 bp
(Parr et al., 1993
) (kindly
provided by Dr A. McMahon); Bmp6, 893 bp (EcoRISacI cDNA fragment
kindly provided by Dr B. Hogan); and Gbx2, 620 bp (untranslated
region; kindly provided by Dr G. Martin). In situ hybridization following
staining for ß-galactosidase activity was performed according to
Tajbakhsh and Houzelstein (Tajbakhsh and
Houzelstein, 1995
).
Histology and histochemical staining
For histology, embryos or newborn animals were fixed overnight in Bouin's
fixative, dehydrated in ethanol, cleared in xylene and embedded in paraffin
wax. Sections were cut at 15 µm and stained with Hematoxylin and Eosin.
ß-Galactosidase staining was performed as described by Houzelstein et al.
(Houzelstein et al., 1997).
Nile Blue staining was performed according to Anderson et al.
(Anderson et al., 2002
).
Expression plasmid construction and electroporation in chick
embryos
The complete coding sequence of a chick Msx1 cDNA
(Robert et al., 1991) was
amplified by PCR (Forward primer: CGC CGG TCG ACT GCA TGG CCC CGG CT; Reverse
primer: CGC CGC TCG AGG CGG CTC GGC CCT ATG TAA). These primers were designed
to add SalI and XhoI (underlined) restriction sites at the
5' and 3' of the Msx1 sequence, respectively. The cDNA
was inserted into the XhoI site of the pCIG plasmid
(Megason and McMahon, 2002
)
(kindly provided by Dr A. McMahon). The resulting construct was electroporated
into the diencephalon of chick embryos by a modification of the procedure
described by Mathis et al. (Mathis et al.,
2001
). Plasmid DNA was prepared using Qiagen Maxiprep and
resuspended in water at 3mg/ml of DNA and 0.01% fast green. Eggs were
incubated at 37°C for 40 hours to reach the 8-16 somite stage. After
removal of 3 ml of albumin, a window was made into the eggshell and Hank's
solution (Sigma) was dropped onto the embryo. DNA solution was introduced in
the neural tube or over the head lateral ectoderm using a pressure injection
apparatus (Picospritzer, General Valve). Then, four square pulses (duration=50
ms) of 25 V were applied using curved platinum electrodes (5 mm separation)
placed on either side of the embryo. Embryos were recovered in PBS 6-24 hours
after electroporation, photographed for GFP expression profile, then fixed for
24 hours in PFA 4%, dehydrated progressively in methanol, and conserved at
-20°C. In situ hybridization was performed with a chick Wnt1
probe (Bally-Cuif and Wassef,
1994
) as described above. Double in situ hybridization was
performed using a modification of the protocol by Dietrich et al.
(Dietrich et al., 1997
). In
brief, a chick Msx1 probe (Robert
et al., 1991
) was labeled with fluorescein-UTP and the chick
Wnt1 probe with DIG-UTP (Roche). After hybridization, the
fluorescein-labeled RNAs were detected using INT/BCIP (Roche) as substrate for
the anti-fluorescein antibody-coupled alkaline phosphatase. Then, the alkaline
phosphatase activity was inhibited by heating at 70°C for 30 minutes in
PBS and the alkaline phosphatase-coupled anti-DIG-antibody was applied.
Staining was performed with BM Purple AP substrate (Roche). The INT/BCIP red
precipitate was washed out during processing of the second antibody.
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RESULTS |
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The Msx1nlacZ expression domain disappears
specifically from prosomere 1 in the Msx1-/- homozygous
mutants
The fate of midline Msx1-expressing cells was analyzed at earlier
stages in heterozygous and homozygous mutant embryos using the nlacZ
reporter gene that we had targeted to the Msx1 locus
(Houzelstein et al., 1997). We
first verified that the ß-galactosidase activity in the CNS of mutant
embryos matched Msx1 transcript distribution. Similar results were
obtained with X-gal staining and in situ hybridization at different stages of
development, confirming that Msx1 is expressed at the midline of the
CNS from, rostrally, the optic stalk to, caudally, the tip of the tail (data
not shown). Analysis of sections further confirmed that, at E12.5,
Msx1nlacZ is expressed in the ependymal layer at the
dorsal midline of the brain over its entire length, particularly in the SCO
cells underneath the posterior commissure (data not shown).
At E14.5, all homozygous mutant embryos tested (15/15) displayed a clear interruption of the ß-galactosidase domain in the midline of the pretectum, between the pineal anlage and the di-mesencephalon boundary (Fig. 2A,B). These anatomical landmarks, together with gene expression data (see below) confirm that all defects are restricted to P1. In a few cases, a short remnant of the nlacZ-expressing domain could be detected just posterior to the pineal anlage, but the main region of the midline that includes the SCO anlage was not labeled.
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This hypothesis is further substantiated by comparing the expression
profiles of several genes normally expressed in the diencephalon dorsal
midline, between Msx1-/- and wild-type or heterozygous
embryos. In normal embryos, Wnt1, Wnt3a and Bmp6 are
expressed in the diencephalon dorsal midline at E11.5
(Parr et al., 1993;
Furuta et al., 1997
). In the
Msx1 homozygous mutants, the expression domains of these three
markers were interrupted in the pretectum midline
(Fig. 3A-D and data not shown).
At E12.5, Wnt3a transcripts were no longer detectable at the
pretectum midline (not shown), whereas expression of several genes that are
excluded from the midline was observed at this site in the Msx1
mutant. This is the case for Pax6 which is normally expressed in two
lateral domains adjacent to the midline
(Fig. 3E; see also
Fig. 5C). We further confirmed
by in situ hybridization after staining for ß-galactosidase in
heterozygous embryos that there is no coexpression of Pax6 and
Msx1nlacZ before E14.5 (data not shown), when
Pax6 begins to be expressed in the SCO layer
(Estivill-Torrus et al.,
2001
). In the mutant, at E12.5, cells of the dorsal midline were
observed to express Pax6, in such a way that the two lateral domains
are no longer separated medially (Fig.
3F). At E14.5, similar results were obtained with Pax7
and Lim1 as well as Pax6. Expression of these genes in the
mutant, in contrast to the normal situation, extends across the midline
(arrows in Fig. 5B,D,F). These
results suggest that in the absence of Msx1, dorsal midline cells are
not maintained in P1.
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The disruption of the dorsal midline affects the expression of
dorsolateral markers specifically in prosomere 1
We further studied the expression pattern of several genes normally
expressed in dorsolateral neural tissues of P1 such as Pax6, Pax7 and
Lim1 (Fujii et al.,
1994; Stoykova and Gruss,
1994
). At E14.5, Pax6 expression is downregulated in most
mutant embryos and even undetectable in some, at the midline and in the
dorsolateral domain (Fig. 5C,D
and data not shown). By contrast, Pax6 expression is maintained in
the epiphysis and in the telencephalon of Msx1 mutants, in accordance
with our observation that defects are limited to P1 (not shown). As
Msx1 and Pax6 are not co-expressed in the diencephalon of
Msx1 mutants, downregulation of Pax6 must be an indirect
effect of the absence of Msx1 in P1. Lim1 is less strongly
expressed laterally in the rostral part of P1 in the mutant at E14.5
(Fig. 5E,F). Similarly,
Pax7 expression is affected in the anterior part of P1 only, with a
clear rostral boundary (Fig.
5A,B). This indicates that there are two functional domains in P1
seen here for Pax7 and Lim1 expression, one that is
dependent on MSX1 activity and the other not. Analysis of the expression
pattern of other genes expressed in the dorsolateral part of P1, such as
Otx2 and Pax3, confirms that a functional dorsal midline is
necessary to specify neural tissues adjacent to the roof plate (not
shown).
Strikingly, Pax7 expression profile is not affected in the mesencephalon by the Msx1 mutation (Fig. 5A,B). In situ hybridization with Gbx2, which is expressed dorsolaterally in prosomere 2, showed that the dorsolateral tissues of this prosomere are not affected either (Fig. 5G,H). Noticeably, expression which also takes place in the ventrolateral part of P1 was not affected in the Msx1-/- mutants, indicating a lateral limit to the influence of the midline in this prosomere. These gene expression data further confirm that all defects are restricted to the dorsal part of P1 in the Msx1 mutant, and demonstrate that the loss of Msx1 function does not result in the transformation of P1 into a segment with more rostral or caudal identity.
Wnt1 is downregulated before the dorsal midline disappears in Msx1-/- embryos
Wnt1 has a dynamic pattern of expression with two major phases: at
the neural plate stage, its expression takes place in a broad domain of the
presumptive midbrain; from E9.5, expression is initiated at the dorsal midline
of the neural tube, from the diencephalon through the spinal cord, with the
exception of the r1 region in the metencephalon, and extends rostrally in the
diencephalon up to the level of the P2/P3 boundary
(Parr et al., 1993;
Rowitch et al., 1998
)
(Fig. 8A). In this later phase,
Msx1 and Wnt1 expression domains in the midline overlap in
the spinal cord, the mesencephalon and the posterior diencephalon
(Furuta et al., 1997
) (A.B.,
Y.L. and B.R., unpublished). As mentioned previously, in
Msx1-/- mutant embryos, at E11.5 Wnt1 transcripts
were diminished in the dorsal midline cells of P1, and
Msx1nlacZ expression was also reduced. To determine
whether the downregulation of Wnt1 precedes the disappearance of the
Msx1nlacZ expression domain, we performed in situ
hybridization after staining for ß-galactosidase activity at E10.5 and
E9.5. At E10.5 in heterozygous embryos Wnt1 and
Msx1nlacZ were coexpressed throughout the mesencephalon
and the posterior diencephalon (Fig.
6C). In Msx1-/- embryos, Wnt1 and
Msx1nlacZ were co-expressed in the mesencephalon. In some
Msx1-/- embryos in which Msx1nlacZ
expression was weaker but still continuous in the P1 dorsal midline, the
expression of Wnt1 was reduced and limited to a few patches of cells
(Fig. 6D). At E9.5 in
Msx1 mutant embryos, Msx1nlacZ forms a continuous
domain in P1 in most embryos. At this stage, Wnt1 begins to be
expressed at the dorsal midline of the diencephalon
(Fig. 6A). In some mutant
embryos, Wnt1 expression was limited to a few scattered cells in P1
(Fig. 6B). These results
demonstrate that, in all cases, Wnt1 is downregulated before the
Msx1nlacZ expression domain has disappeared. This
indicates that Msx1 is required for the maintenance of Wnt1
expression in the midline and may also be required for its initiation.
Furthermore, it raises the possibility that the disappearance of a functional
midline in the dorsal domain of P1 may be secondary to the downregulation of
Wnt1.
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To further investigate gene interactions, we performed a kinetics of Wnt1 induction after electroporation of pCIG-Msx1 in the diencephalon (Table 2). Although GFP could be first detected 2 hours after electroporation (hae), no Wnt1 induction was observed before 9 hae and it was still low at 12 hae Maximum induction was reached at 24 hae. Assuming that Wnt1 induction is cell autonomous, this leaves enough time for an indirect, intracellular process of induction, in accordance with the known properties of MSX1 as a transcriptional repressor. However, conspicuous induction requires relatively high levels of MSX1 that might not be reached during the first hours. This leaves open the possibility of a direct activation of the Wnt1 gene by MSX1.
Inactivation of both Msx1 and Msx2 leads to loss of
Wnt1 expression at the dorsal midline of the diencephalon
All three Msx genes in the mouse are expressed at the dorsal midline of the
neural tube. Msx1 and Msx2 are expressed in the anterior
part of the CNS at early stages, while Msx3 expression does not
extend rostrally beyond the isthmus
(Shimeld et al., 1996;
Wang et al., 1996
). We
reasoned that, if Msx genes are required for Wnt1 expression, the
pattern of expression of the latter should be more extensively affected in the
double Msx1/Msx2 mutants than in Msx1 mutants. To test this
hypothesis, we took advantage of an Msx2 mutant strain constructed in
our laboratory by substituting the nlacZ reporter gene to the coding
sequence of the endogenous Msx2 locus, using homologous recombination
(M.-A.N., C.R., T. Paquet, P. J. R. Barton and B.R., unpublished).
Compound heterozygous animals were crossed to analyze the phenotype of double homozygous mutant embryos at E10.5 and E11.5. At these stages, a number of such embryos were resorbed. The others were all smaller than normal and most had a very abnormal morphology. In some embryos, an exencephaly encompassing the posterior mesencephalon and the rhombencephalon and a spina bifida were observed (data not shown). In all cases, the brain was small and abnormal. However, in some embryos the different brain regions were clearly visible and these were analyzed for Wnt1 expression (Fig. 8B). In both E10.5 and E11.5 embryos, Wnt1 expression was totally absent from the entire diencephalon and severely reduced in the mesencephalon, where only a small caudal domain remained (Fig. 8A,B and not shown). This remnant of expression may derive from the initial expression domain of Wnt1 in the midbrain, which suggests that in the absence of functional Msx1 and Msx2, Wnt1 is not induced in the dorsal midline of the brain. In the rest of the central nervous system, where Msx3 is also expressed, Wnt1 expression looked normal.
In Msx2nlacZ heterozygous animals, at E10.5, the Msx2nlacZ expression domain extends over the mesencephalon and the diencephalon where it overlaps with that of Msx1nlacZ, but the intensity of the signal was much weaker for Msx2 than for Msx1, especially in P1 (not shown). At E12.5, Msx2nlacZ was further downregulated and almost completely undetectable in P1, while Msx1 was strongly expressed at this site (Fig. 8C,D). On the contrary, the pineal gland displayed a strong staining for the two genes (Fig. 8C,D arrowheads). The transient expression of Msx2 in P1 may explain the variation in the timing and extent of Wnt1 and Msx1nlacz downregulation. Our results suggest that Msx1 and Msx2 have similar functions in dorsal CNS patterning and furthermore that Msx3 may be sufficient to compensate for the loss of Msx1 and Msx2 in the spinal cord and part of the rhombencephalon.
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DISCUSSION |
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All defects are restricted to prosomere 1
According to the neuromeric model, the diencephalon is subdivided into
three anatomical subunits called the prosomeres. Prosomere 1 (P1) constitutes
the most caudal subunit of the diencephalon and is located between the pineal
gland and the mesencephalon. Prosomeres 2 and 3 form the dorsal and ventral
thalamus, respectively. Gene expression studies have shown that prosomeres
have distinct genetic identities provided by the expression of combinatory
sets of genes (Puelles and Rubenstein,
1993; Rubenstein et al.,
1994
). Furthermore, in the Pax6sey/sey mutant,
P1 is partially transformed into a mesencephalic structure
(Mastick et al., 1997
). This
provides functional evidence in favor of the prosomeric model.
In the Msx1 mutant, abnormalities are restricted to the territory between the pineal gland and the mesencephalon, which are landmarks for the P1 territory. Interruption of the Msx1nlacZ midline expression domain also coincides exactly with P1. Therefore, both morphological and genetic evidence indicates that the Msx1-/- mutation affects only P1, confirming that this prosomere constitutes a genetic entity. Msx2 is expressed in the diencephalon at E10.5, but more weakly than Msx1, particularly in P1, and is further downregulated at E12.5. Therefore, Msx2 may partially compensate for the absence of Msx1 in P1 at early stages. The low residual expression of Msx2 is probably responsible for the variability in the time of onset of midline defects that we observed.
In a slightly different neuromeric model, Figdor and Stern
(Figdor and Stern, 1993) have
proposed that P1 is subdivided into two neuromeres: a caudal and a rostral
one. In the Msx1 mutant, Pax7 and Lim1 expression
in P1 is downregulated only in the more rostral region of P1. This indicates
that there are, relative to the expression of these genes, two functional
domains in P1, one that requires Msx1, and the other that is
independent of it.
The SCO is missing and this leads to hydrocephalus
The SCO is a specialized part of the ependyma at the diencephalon dorsal
midline, which develops in close association with the posterior commissure
from E11. In the Msx1 mutant, the SCO is absent and the posterior
commissure is affected at late stages. As Msx1 is expressed by the
SCO-forming cells throughout development (P. Fernandez-Llebrez and B.R.,
unpublished), further investigation is needed to determine whether
Msx1 has a direct effect on the development of the SCO or whether
abnormalities in the developing SCO are the consequence of more general dorsal
midline defects.
Msx1 mutant mice display prenatal hydrocephalus with an incomplete
penetrance (data not shown). Although the function of the SCO has not been
completely characterized, prenatal hydrocephalus is correlated with failure of
SCO differentiation in mouse and rat spontaneous mutants
(Takeuchi et al., 1988;
Perez-Figares et al., 1998
).
In the swaying (Wnt1sw/sw) and small eye
(Pax6sey/sey) mutants also, the SCO fails to form and
hydrocephalus is observed (Louvi and
Wassef, 2000
; Estivill-Torrus
et al., 2001
). The absence of the SCO is therefore the most likely
cause of hydrocephalus in the Msx1 mutant. Furthermore, in this
mutant, Wnt1 and Pax6 are downregulated and therefore
crosstalk between these genes may be involved in the proper development of the
SCO.
Relation between Msx1 and Wnt1 expression in the
diencephalon
Wnt1-/- mutant mice present a deletion of the
mesencephalon and rhombomere 1 territories, leading to the apposition of
prosomere 1 and rhombomere 2, and defects in ventral prosomere 2
(McMahon and Bradley, 1990;
Thomas and Capecchi, 1990
;
Mastick et al., 1996
). The
ventral part of P1 is not affected in the Wnt1-/- mutant.
However, the dorsal midline seems to depend on Wnt1 for its
patterning, as E-cadherin, which is normally excluded from the diencephalon
midline and expressed in two bands lateral to it, forms a single medial band
in the Wnt1-/- mutant
(Shimamura et al., 1994
).
Furthermore, as previously mentioned, the subcommissural organ, a P1 midline
derivative, is abnormal in the Wnt1sw/sw mutant
(Louvi and Wassef, 2000
). We
have shown that in the Msx1 mutant, there is a downregulation of
Wnt1 and Wnt3a at E11.5. Therefore the absence of
Wnt signaling may explain the disappearance of the dorsal
midline.
Our data show that in Msx1 mutants, Wnt1 may be
downregulated as early as E9.5, before any morphological alteration is
detectable. This is the stage when the second Wnt1 expression domain,
at the dorsal midline, starts to form
(Parr et al., 1993;
Echelard et al., 1994
).
Furthermore, Wnt1 downregulation just precedes the disappearance of
Msx1nlacZ-expressing cells. These observations suggest
that MSX1 is required for the maintenance of Wnt1 expression in the
diencephalon dorsal midline, and may be required for the activation of the
second phase of Wnt1 expression. This is further supported by the
analysis of the Msx1/Msx2 double mutants. At E10.5 in doubly
homozygous embryos, Wnt1 expression is absent from the diencephalon
and reduced in the mesencephalon. A remnant of expression caudally in the
mesencephalon suggests that the early phase of Wnt1 expression has
been initiated, but the second phase does not seem to take place in the
absence of any functional Msx.
Msx1 has been proposed to be directly implicated in the regulation
of Wnt1 expression (Echelard et
al., 1994; Shang et al.,
1994
; Iler et al.,
1995
; Rowitch et al.,
1998
). Indeed, forced ectopic expression of Msx1 in the
chick brain demonstrates that Msx1 is an inducer of Wnt1 in
the neural tissue. It has been shown that a 110 bp element, which is conserved
between the pufferfish Fugu rubripes and the mouse, within a 1.1 kb
enhancer, is sufficient for the activation of Wnt1 expression in the
mid- and forebrain (Rowitch et al.,
1998
). This element contains at least two homeobox-binding sites
that are capable of binding MSX1 in vitro and in vivo with a high affinity,
suggesting that MSX1 may interact directly with the Wnt1 promoter.
However, mutation of the main binding site for MSX1 does not prevent transgene
expression in the diencephalon (Iler et
al., 1995
). Furthermore, MSX1 has been characterized as a
transcriptional repressor, and as such is unlikely to activate Wnt1
directly (reviewed by Bendall and
Abate-Shen, 2000
). Msx1 might be involved indirectly in
the regulation of Wnt1 expression, either by inducing a paracrine
signaling loop or, intracellularly, by repressing a transcriptional inhibitor
of Wnt1. The former hypothesis is unlikely, as expression of
Wnt1 in electroporated embryos is always contained within
Msx1 expression domain and strictly limited to
Msx1-expressing cells. The kinetics of induction is compatible with
repression of a Wnt1 repressor, as no activation of Wnt1 is
observed before 9 hae. As a point of comparison, expression of Pax6,
which by several criteria is a likely direct target of En2, is
affected as early as 3 hae of an En2-expressing vector
(Araki and Nakamura,1999
).
Msx1 and signaling in induction processes
Expression of Msx1 may be induced by several Bmps, such as Bmp2,
Bmp4 and Bmp7, in different organs during embryogenesis (reviewed by
Davidson, 1995). Reciprocally,
Msx gene products may regulate Bmp expression. The most compelling
evidence for interplay between Bmp and Msx genes is provided by the tooth
germ, where Bmp4 induces Msx1 expression in the mesenchyme, which in
turn is required for induction and maintenance of Bmp4 expression at
this site (Chen et al., 1996
).
Both Bmp4 and Bmp7 have the capacity to induce roof plate cells in spinal
neural plate explants, which also leads to Msx1 expression, while
Bmp4 can induce ectopic Msx1 expression in lateral explants of the
telencephalon (Liem et al.,
1995
; Furuta et al.,
1997
; Lee and Jessell,
1999
). Accordingly, application of noggin, a Bmp2-Bmp4 and Bmp7
inhibitor, to the dorsal neural plate downregulates Msx1
(Muroyama et al., 2002
). At
the neural plate stage, Msx1 gene expression is restricted to the
lateral, prospective dorsal, region of the neural folds
(Robert et al., 1989
) (A.B.,
Y.L. and B.R., unpublished) (Liem et al.,
1995
), in a manner consistent with a lateral induction coming from
the ectoderm which expresses Bmp4 and Bmp7. After neural
tube closure, Bmp7 expression is maintained at a high level in the
epidermal ectoderm of the forebrain while Bmp4 is expressed in the
roof plate (Liem et al., 1995
;
Furuta et al., 1997
). A
hierarchical relationship between Bmp and Wnt genes in the dorsal neural tube
has been suggested, but not definitely established. Application of Bmp4 on
forebrain explants only led to a slight extension in the Wnt1
expression domain (Golden et al.,
1999
). On the contrary, ectopic expression of a constitutively
active Bmp receptor (caBmpr1a) under the control of nestin regulatory
sequences led to an expansion of the Wnt1 expression domain, to
include the entire alar domain of the neural tube
(Panchision et al., 2001
).
This suggests that Bmp signaling may induce Wnt1 expression.
On the other hand, roof plate ablation has demonstrated that the dorsal
midline is an essential patterning center for the dorsal neural tube
(Lee et al., 2000). Roof plate
cells produce signaling molecules such as Wnts or Bmps that are essential for
the specification and proliferation of dorsolateral neural cells
(Liem et al., 1995
;
Liem et al., 1997
;
Furuta et al., 1997
;
Lee and Jessell, 1999
;
Megason and McMahon, 2002
).
Mutations in the genes for these signaling factors lead to patterning defects
of the dorsal CNS (Ikeya et al.,
1997
; Lee et al.,
1998
; Kim et al.,
2001
; Muroyama et al.,
2002
). In Msx1-/- mice, several of these genes
are downregulated in the dorsal midline of the diencephalon, and this is the
most likely explanation for the downregulation of genes normally expressed in
the dorsolateral domains.
Based on these data, a model for dorsal midline formation is proposed, in which a Bmp signal coming from the lateral/dorsal ectoderm or mesoderm induces midline expression of Msx1 at early stages (Fig. 9). This in turn is essential to maintain a functional midline that produces the diffusible signals Bmp6, Wnt1 and Wnt3a, leading to patterning of adjacent dorsolateral neural tissues. We propose that, in the case of the dorsal midline in the diencephalon, Msx genes are intermediaries between Bmp and Wnt and that this is essential for the maintenance of a functional dorsal midline.
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Footnotes |
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Present address: Departamento de Biologia Celular, Facultad de Biologia,
Avenue Diagonal, 645, 08071 Barcelona, Spain
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