1 Régionalisation Nerveuse CNRS/ENS UMR 8542, Ecole normale
supérieure, 46 rue d'Ulm, 75005 Paris, France
2 GSF-Research Centre, Institute of Mammalian Genetics, Ingolsträdter
Landstrasse 1, D-85764 Neuherberg and Max-Planck-Institute of Psychiatry,
Kraepelinstrasse 2-10, D-80809 Munich, Germany
Author for correspondence (e-mail:
wassef{at}wotan.ens.fr)
Accepted 21 July 2003
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SUMMARY |
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Key words: Cerebellum, Cerebellar midline fusion, Roof plate, Mid/hindbrain morphogenesis, Isthmus, Velum medullaris, Choroid plexus, Wnt1, Engrailed, Otx2, Swaying, Chick-quail chimera, Mouse
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Introduction |
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In the present study we explored how and when midline fusion of the
cerebellum takes place. To gain insight into this process, we first produced a
new fate map of the MHB in avian embryos by following the fate of calibrated
circular grafts and by labeling small groups of cells with DiI. The fate map
revealed marked variations in growth between the medial and lateral domains of
the caudal midbrain neuroepithelium that could influence morphogenetic
movements. In addition, at stage 10-11 according to Hamburger and Hamilton
(HH10-11) (Hamburger and Hamilton,
1951), we detected two distinct domains that contributed cells to
the cerebellar midline: a restricted isthmic midline domain produced divergent
flows of cells that populated the roof plates of the caudal midbrain and
anterior hindbrain, and an adjacent domain contributed to the velum
medullaris, a structure that links the cerebellar vermis to the inferior
colliculus, and also provided a substratum for midline cerebellar fusion. This
domain was considered distinct from the vermis primordium because it produced
none of the typical cerebellar cell types (granule cells, Purkinje, Golgi or
deep cerebellar nuclei neurons).
We then turned our attention to the mouse, in which not only have many regional and cell-type specific markers of the early and late cerebellum been characterized, but also a large number of cerebellar mutants is available. Examination of cerebellar fusion in wild-type mice suggested that BMP signaling was transiently decreased at the cerebellar midline. In addition, just before the onset of granule cell progenitor (GCP) production at early E14.5, expression of the rhombic lip marker Math1 (Atoh1) was downregulated at the medial roof plate segment separating the two vermis primordia, thus allowing for midline cerebellar fusion. Together, these observations indicated that the isthmus-derived cerebellar midline cells have distinct signaling properties.
Based on the avian fate map and on observations in wild-type mice and in
Wnt1sw/sw [swaying
(Lane, 1967;
Bronson and Higgins, 1991
;
Thomas et al., 1991
)] and
En1+/Otx2lacZ mutant embryos
(Broccoli et al., 1999
), and
in En1hd/hd adult survivors
(Wurst et al., 1994
), we
propose that the caudalward movement of isthmus-derived cells is essential for
cerebellar fusion for two reasons. First, because it provides a favorable cell
substratum; second, because its distinct signaling properties modify the edges
of the two cerebellar primordia at the midline rhombic lip allowing for fusion
to occur and possibly restricting the rostral extension of the presumptive
choroid plexus territory that depends on BMP signaling.
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Materials and methods |
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Mouse strains
Wild-type embryos were of the OF1 strain (IFFA Credo, France) or
littermates of the mutant embryos. Mutant strains were described previously:
(i) Wnt1sw/sw (Lane,
1967; Thomas et al.,
1991
) (purchased from the Jackson Labs); (ii)
En1+/Otx2lacZ [Otx2 lacZ knock-in into the
En1 locus (Broccoli et al.,
1999
)]; (iii) En1hd [hd: homeodomain deletion
(Wurst et al., 1994
)]; (iv)
En1Lki [Lki: lacZ knock-in into the En1
locus (Hanks et al., 1995
)].
Genotyping of Wnt1sw/sw embryos was as described
previously (Bally-Cuif et al.,
1995
). Adult cerebella were fixed in 4% paraformaldehyde and
stained in toto with India ink to reveal foliation details.
Transplantation experiments
The different types of homotopic and isochronic transplantation experiments
between quail donor and chick host embryos are schematically represented in
Figs 1 and
2 (left). For microsurgery, we
used hand-made microscalpels and drawn-out glass micropipettes (Clark
Electromedical Instruments, cat. no. GC100T-15), with tips of known diameter
(50 or 100 µm), measured with a micrometer under a dissecting microscope.
Briefly, host chick embryos at the 9- to 13-somite stage (ss) (HH9-11) were
visualized by a sub-blastodermal injection of India ink. Through a cut in the
vitelline membrane, a small plug (100 µm in type 1-3 and 50 µm in type 5
grafts) or a thin strip (type 4 grafts) was removed from the dorsal
neuroepithelium. The corresponding size-matched portion of neuroepithelium was
isolated from a donor quail embryo using the same micropipette without
enzymatic dissociation and transported to replace the ablated region on the
chick host. The eggshell was sealed with tape and returned to the incubator.
Most embryos were collected at HH19-21 (E4) and fixed by immersion in sodium
phosphate buffered 4% paraformaldehyde prior to immunohistochemistry. Type 4
chimeras (see below) that were allowed to survive until E16, E18 or E19 were
fixed by transcardiac perfusion of the same fixative.
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|
Type 2: lateral grafts (Fig.
1, left; lower panel)
Circular lateral grafts (mostly on the right side of the embryo, but some
were placed on the left) that excluded the dorsal midline, subdivided into
three overlapping groups. Type 2a grafts were centered on the isthmus. Type 2b
grafts were placed caudal to the isthmus, with their rostral end positioned at
the level of the constriction. Type 2c grafts were placed rostral to the
isthmus, with their caudal end positioned at the level of the
constriction.
Type 3: paramedial grafts (Fig.
2, left; upper panel)
Circular grafts, intermediate in position between type 1 and type 2 grafts,
extending medially to the dorsal midline (which they barely touched). Type 3a
grafts were centered on the isthmus. Type 3b and 3c grafts partially
overlapped and were placed rostral to the isthmus, the former close to the
midline and the latter slightly more laterally.
Type 4: strip grafts (Fig.
2, left; middle panel)
Bilateral stripped grafts positioned at the caudal end of the midbrain.
Type 5: midline grafts (Fig.
2, left; lower panel)
Small sized grafts (about 50 µm), encompassing the dorsal midline at the
isthmus.
Immunohistochemistry
Embryos were fixed by immersion in 4% paraformaldehyde overnight at 4°C
and the neural tubes were dissected free of surrounding mesenchyme, dehydrated
in methanol and then rehydrated prior to incubation in 0.1%
H2O2 in PGT (PBS, 0.2% gelatin, 1% Triton X-100).
Immunohistochemistry on embryos or cryosections was performed as described
previously (Louvi and Wassef,
2000). Nissl staining was performed by standard procedures.
The QCPN (DSHB, diluted 1:10), anti-BrdU (Becton Dickinson, diluted 1:400), anti-calbindin D28K (Swant; diluted 1:10000), anti-parvalbumin (Sigma; diluted 1:10000), anti-QH1 (a gift from A. Eichmann; diluted 1:5) and anti-vimentin (Amersham; diluted 1:5) monoclonal antibodies, and a rabbit anti-GFAP (Dako, diluted 1:1000) were used to detect quail cells, BrdU, Purkinje cells, interneurons, quail-derived vessels, radial glia and astrocytes, respectively.
Analysis of cell movements
Following immunostaining, camera lucida drawings of individual embryos with
integrated grafts were produced. The embryos were photographed as whole
mounts. Embryos within each graft subtype were divided into four age groups,
based on the number of somites (s; 8/9s, 10s, 11s and 12/13s) and movements of
quail cells with respect to the initial position of the graft were recorded.
Cell movements were consistent across all four age groups, and the presented
results are compilations.
DiI labeling in chick embryos and in
En1+/Otx2lacZ mouse mutants
Fertilized eggs were incubated to the 10ss (somite stage) and windowed. A
small area of the vitelline membrane was removed over the MHB, and a solution
of DiI (Molecular Probes Inc.) at 5 mg/ml in dimethylformamide was injected
onto the dorsal neural tube, from a pulled glass pipette. Embryos were either
fixed 1 hour after DiI application in 4% paraformaldehyde at 4°C, or
allowed to proceed to stages HH19-21, then fixed. Embryos at HH10 were
photographed as whole mounts in fluorescent and polarized light and the two
images were superimposed using Adobe PhotoShop. Embryos at HH19-21 were
dissected and the MHB region was flat-mounted. Pictures were taken using a
Zeiss Axioscope microscope.
Small DiI crystals were inserted into the axonal bundle that links the two hemicerebella in paraformaldehyde fixed En1+/Otx2lacZ mutants at postnatal day (P) 4.
BrdU labeling
Pregnant females were injected intraperitoneally with a solution of BrdU (2
mg/ml in saline) at 20 µg/g of body weight and sacrificed 2 hours later.
Proliferating granule cell precursors at the surface of the cerebellar anlage
were revealed by whole-mount immunohistochemistry.
In situ hybridization
Fixed embryos were hybridized in toto with one or two riboprobes as
described previously (Bally-Cuif et al.,
1995), with minor modifications for cryostat, frozen or vibratome
sections of E16-E20 embryos. Probes used were: Math1 (a gift from F.
Guillemot); Ror
(a gift from B. Hamilton), Ttr (a gift from W. Blaner);
Slit2 (a gift from A. Chédotal and M. Tessier-Lavigne); chick Gdf7 (a
gift from K. Lee); mouse Wnt1 (a gift from J. Kitajewski); Drm (a gift from M.
Marx); rat Gad67 (a gift from A. Tobin); chick GAD67 (a gift from C.
Ragsdale).
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Results |
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Therefore, distinct regions of the developing dorsal midbrain are characterized by differential growth.
Analysis of cell movements in the dorsal mid/hindbrain
I. Dorsomedial (type 1) grafts
To follow the movements of dorsomedially located cells, we analyzed embryos
with grafts encompassing the midline at different rostrocaudal levels
(Fig. 1A-D). Type 1a grafts
(n=27), placed at the isthmus, were mainly (21/27) found in the
hindbrain at HH19-21 (Fig. 1A). A few (3/27) were found elongated and straddling the MHB
(Fig. 1B), while another few
(3/27) ended up anterior to the MHB and were confined to the midbrain. Type 1b
grafts (n=2) remained in the hindbrain
(Fig. 1C), while type 1c grafts
(n=7) were almost invariably (6/7) restricted to the midbrain
(Fig. 1D), with the exception
of one graft which extended thinly into the hindbrain (not shown).
II. Lateral (type 2) grafts
To address whether cells located laterally in the alar plate undergo
movements similar to those observed medially, we analyzed embryos with
dorsolateral grafts (Fig.
1E-G). Type 2a (Fig.
1E) and 2b grafts behaved similarly (n=5+2). Some (3/7)
were displaced rostromedially close to the midbrain midline at HH19-21 and had
increased in size (Fig. 1F);
others (2/7) extended on both sides of the MHB, medially into the midbrain and
laterally into the hindbrain, yet others (2/7), moved caudolaterally and were
confined to the hindbrain (not shown). Type 2c grafts (n=8) moved
rostromedially (5/8), close to (but respecting) the midline of the midbrain
and were significantly enlarged as well
(Fig. 1G), or extended on both
sides of the MHB (3/8), with their rostral part elongated rostromedially and
the caudal part having moved caudolaterally into the hindbrain (not
shown).
III. Paramedial (type 3) grafts
To examine cell movements adjacent to the dorsal midline, we analyzed
embryos with grafts placed between the midline and the lateral edge of the
dorsal midbrain at different rostrocaudal positions
(Fig. 2A-D). Type 3a isthmic
grafts (n=3), became elongated very close to the midline, and moved
along the anterior-posterior axis, either rostrally
(Fig. 2A), or caudally, or even
in both directions, split in half (Fig.
2B). Type 3b grafts (n=7) retained their medial position.
They, too, became elongated close to the dorsal midline, but were larger in
comparison with type 3a grafts (Fig.
2C). Type 3c grafts (n=8) moved rostromedially (7/8)
towards the midline and, compared to type 3a and 3b grafts, increased
significantly in size (Fig.
2D).
IV. Strip (type 4) and midline (type 5) grafts
In order to directly assess the convergent movements of the
neuroepithelium, revealed by type 1-3 grafts, we performed thin bilateral
strip grafts that encompassed the entire dorsal part of the caudal midbrain
(Fig. 2E-H). In agreement with
our previous observations, descendants of these strip grafts (n=17)
were confined to the medial-most MHB structures (13/17); in the hindbrain they
formed two bilateral and distinct protrusions (see for example
Fig. 2F,H). In the remaining
cases (4/17), QCPN-positive cells were exclusively encountered in the
midbrain, rostral to the MHB. Analysis of long survival strip grafts
(n=4) at E16 allowed us to assess their contribution to hindbrain
structures (see below).
Finally, we performed a series of small grafts at the level of the isthmus,
as restricted to the dorsal midline as possible
(Fig. 2I,J). These grafts
became extremely thin and elongated, and remained strictly confined within the
midline. Consistently, only a few quail cells were detected 2 days after
grafting, in the majority of cases (11/19) along the midline of the cerebellar
plate, in agreement with the results obtained with type 1 grafts
(Fig. 2I). Extensive cell death
affecting the MHB midline region at the 14-16ss
(Lumsden et al., 1991) and,
possibly, a lower proliferation rate could explain why the small transplants
only contributed scattered cells to the cerebellar midline. In some embryos
(4/19), quail cells were detected rostrally within the midline of the caudal
midbrain, while in the remaining cases (4/19), they were present on both sides
of the MHB, yet were always confined to the midline
(Fig. 2J).
Tracing the cells at the MHB with DiI
To corroborate the results of our grafting experiments we labeled cells
located at and rostral to the isthmus with DiI at the 10ss, and monitored
their distribution 2 days later. Observation of embryos fixed 1 hour after DiI
application, confirmed that only a small cluster of cells at the desired
position was labeled (Fig.
3A-D). When DiI was applied dorsomedially at the level of the
constriction (Fig. 3A,B),
labeled cells were found either exclusively along the midline of the
cerebellar plate (Fig. 3E), or
in the midline of the caudal midbrain as well
(Fig. 3F), reflecting the
movements of type 1a and 5 grafts. Midline cells located rostral to the
constriction at the time of DiI application
(Fig. 3C), were encountered in
the caudal midbrain at HH19-21 (Fig.
3G), much as type 1c grafts. Finally, when DiI was applied next to
the midline at the constriction (Fig.
3D), labeled cells were found paramedially, in both the caudal
midbrain and the rostral hindbrain (Fig.
3H). Thus, the DiI labeling experiments faithfully reproduced the
results of the transplantation experiments. Cell movements at the MHB are
summarized in Fig. 3I-L.
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The isthmic interface contributes to the velum medullaris and the
anterior cerebellum
Our transplantation and DiI tracing experiments indicated that the dorsal
isthmic neuroepithelium contributes to the midline of the cerebellar plate. To
assess the differentiation of strip graft-derived cells, we analyzed
long-survival type 4 chimeras at E16, E18 and E19. In all cases (4/4) the
grafts contributed to the anterior cerebellum and to the velum medullaris, a
sheet of cells that links the cerebellar vermis with the midbrain and
constitutes the roof of the fourth ventricle in this region
(Fig. 4A,B). In agreement with
earlier reports (Hallonet et al.,
1990; Martinez and
Alvarado-Mallart, 1989
), the strip grafts contributed neither
granule cells (GCs) nor deep nuclei neurons to the cerebellum. The strip
grafts contributed only a few large neurons to the Purkinje or granular layers
(putative PC and Golgi cells, respectively)
(Fig. 4C) and these neurons
were even completely absent from 2 out of 4 strip grafts at E16
(Fig. 4D). The distribution of
quail cells in the cerebellum of strip graft chimeras (type 4) was distinct: a
great number of small quail cells accumulated around the Purkinje cell (PC)
somata and in the vicinity of the white matter. This pattern closely resembles
that of the small GAD67-positive cells
(Fig. 4E). In order to better
characterize the cerebellar cell types produced by the strip grafts, several
markers were used. Parvalbumin, a marker of PCs and of the interneurons of the
molecular layer (MLI) (Alvarez Otero et
al., 1993
) was not expressed in the MLI at E16; however, a few
putative MLI in the molecular layer of E16 control chick embryos expressed
GAD67 transcripts (Fig. 4E).
Unfortunately, the hybridization procedure prevented the subsequent detection
of the two available quail cell markers: the QCPN epitope and the
nucleolus-associated chromatin. Comparison of GAD and parvalbumin staining at
E18-E20 indicated that only a modest proportion of the GAD-positive MLI
expressed parvalbumin (Fig.
4F-H). Accordingly, only a small proportion of the quail MLI
expressed parvalbumin in type 4 chimeras at E18
(Fig. 4G). A few vascular
segments in the molecular and granular layers expressed the quail endothelium
marker QH1 (Fig. 4I), and some
GFAP-positive quail-derived astrocytes were detected in axonal tracts
(Fig. 4J).
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Fusion of the mouse cerebellar cortex
The chick fate map pointed to the contribution of a dorsal isthmic domain
to the midline of the cerebellum, providing it with a substratum for fusion.
However, limitations of the avian system, notably the lack of genetic tools,
led us to analyze cerebellar cortex fusion in the mouse where many cerebellar
markers and mutants are available. The two major cell types of the cerebellar
cortex, PCs and GCs, have distinct origin: PCs are produced by the cerebellar
neuroepithelium, whereas GCs derive from proliferative precursors (GCPs)
generated by the anterior rhombic lip
(Alcántara et al., 2000;
Gilthorpe et al., 2002
;
Wingate and Hatten, 1999
). We
examined cerebellar development using two cell type-specific markers
(Fig. 5A): for the rhombic lip
and GCPs, Math1, a transcription factor required for the development of
rhombic lip derivatives (Ben-Arie et al.,
1997
; Ben-Arie et al.,
2000
) and for PCs, Ror
an orphan nuclear receptor
(Hamilton et al., 1996
).
|
In addition to the loss of Math1 expression at E14.5, a distinct pattern of
expression of signaling molecules is observed at the cerebellar midline, in
both mouse and chick. For example, expression of BMP/TGFß-related genes
is delayed in the MHB midline: BMP5 and BMP7 in chick
(Fig. 5K), Gdf7 in chick
(Fig. 5L) and mouse (not
shown), whereas between E9.5 and E11.5 in mouse embryos, the cerebellar roof
plate is precisely outlined by a transient expression of Drm, a potent BMP
antagonist of the Cerberus family (Pearce
et al., 1999); (Fig.
5M). This suggests that BMP signaling is transiently down
regulated at the MHB junction. Previous studies have also shown that the
expression of factors belonging to other families of signaling molecules, such
as Slit1/2 in mouse (Yuan et al.,
1999
) (Fig. 5N) and
Wnt1 in both chick and mouse (Bally-Cuif
and Wassef, 1994
), is also interrupted on the cerebellar roof
plate.
Development of the velum medullaris
In the late mouse embryo the medial cerebellum (delimited rostrally by the
extension of the external granular layer; EGL) almost abuts the caudal
midbrain boundary (Fig. 6A).
The ependymal domain (Fig. 6A,
arrow) corresponding to the isthmus at this stage is narrow. During the next
few days, a wide thin sheet of cells devoid of neurons, the velum medullaris,
characterized by En1 expression, develops in this region, linking the anterior
vermis to the inferior colliculus of the midbrain
(Fig. 6B,C). Two processes
contribute to the growth of the velum medullaris in the mouse. On the one
hand, active proliferation, as the proportion of BrdU-labeled ependymal cells
is higher in the velum than in the cerebellum in perinatal rodents
(Fig. 6D). On the other hand,
we observed a shortening of the midline cerebellar ependyme in P4 pups, in
comparison to late gestation embryos (not shown). A posterior retraction of
the anterior end of the vermis causes the velum to delaminate form the
anterior cerebellum and expand. This process may involve a set of radial glia,
labeled by vimentin, which link the anterior and posterior edges of the vermis
(Fig. 6E,F) and express high
levels of En1 (Fig. 6G). These
glia progressively detach from the ependymal surface
(Fig. 6G and data not shown).
Thus, our observations in avian and rodent embryos indicate that the
cerebellum fuses over an isthmic-derived territory, which, in rodent, is
subsequently relinquished to the velum medullaris.
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Discussion |
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Formation of the hindbrain roof plate
The roof plate is a signaling center involved in dorsoventral patterning.
In the spinal cord, the roof plate arises at the site of neural fold fusion
under the influence of TGFß family signals derived from the adjacent
surface ectoderm (Liem et al.,
1995). In the hindbrain, the dorsal part of the neural tube loses
its epithelial organization and forms a sheet of cells on which the choroid
plexus organizes. Roof plate structures form at the boundary between the
choroid plexus and the dorsal edges of the neural epithelium, possibly under
the influence of choroid plexus-derived BMP signals
(Alder et al., 1999
). At the
12ss, the presumptive choroid plexus territory extends in rhombomere 1
(Gilthorpe et al., 2002
) up to
the isthmic constriction (this study). Cerebellar midline cells migrating from
the isthmic region could prevent the rostral extension of the choroid plexus
either by being refractory to a choroid plexus fate, or by propagating
inhibitory signals. BMP signaling is essential for choroid plexus development,
at least in the forebrain (Furuta et al.,
1997
; Hébert et al.,
2002
; Panchision et al.,
2001
), while BMP expression is downregulated on the cerebellar
midline where Drm, a potent BMP antagonist, is specifically expressed at the
onset of choroid plexus development
(Pearce et al., 1999
).
Transient expression of Math1 on the cerebellar midline
At early stages the two cerebellar primordia are delineated by a
Math1-expressing rhombic lip. Consistent with earlier findings
(Hallonet et al., 1990;
Martinez and Alvarado-Mallart,
1989
), we observed that the caudal midbrain strip grafts populate
the cerebellar midline but do not produce GCs. The requirements for GC
production are not met on the cerebellar midline: first, the rhombic lip to
GCP switch requires BMP signals (Alder et
al., 1999
), which we find markedly decreased on the cerebellar
midline and second, Math1, whose function is essential for GCP development
(Ben-Arie et al., 1997
) is
also downregulated on the cerebellar midline. Slit2 has been shown to repel
GCP (Gilthorpe et al., 2002
).
Slit1 and 2, expressed elsewhere in the rhombic lip, are also downregulated on
the cerebellar midline (this study and unpublished observations), an event
that could trigger cerebellar fusion, by first allowing GCP to populate the
midline and then attracting PCs, known to migrate beneath ectopic patches of
GCP in Unc5h3 mutants (Ackerman et
al., 1997
; Leonardo et al.,
1997
; Przyborski et al.,
1998
). Finally, although the cerebellar midline is not favorable
for the acquisition of roof plate or rhombic lip identities, several
observations indicate that choroid plexus-derived signals, in particular BMPs,
are capable of inducing or maintaining rhombic lip markers
(Alder et al., 1999
;
Bally-Cuif et al., 1995
;
Furuta et al., 1997
). The
transient presence of a putative source of GCs in the midvermis could explain
the previously puzzling observation that, whereas in situ the vermis
primordium does not produce GCs (Hallonet
et al., 1990
; Martinez and
Alvarado-Mallart, 1989
) (present study), the ectopic structures
with anterior cerebellum identity induced by FGF8 bead implantation have a
normal complement of GCs (Martinez et al.,
1999
).
Involvement of the dorsal isthmus in cerebellar morphogenesis
The observations reported here indicate that migration from the isthmic
region provides the cerebellar midline with both a substratum and signals
essential for cerebellar fusion. In rodent, an En1-dependent caudal retraction
of the fused rostral cerebellar cortex is subsequently observed, partially
reversing the cell movements leading to cerebellar fusion (see
Fig. 7). Isthmus-derived
cerebellar midline cells could be specialized in the production of GABAergic
interneurons both in chick and mouse. However, the isthmus cannot be the sole
source of this cerebellar cell type, as GABAergic interneurons are still
present in the cerebellum of Wnt1sw/sw mutant mice that
lack isthmic structures (unpublished observation).
The cerebellar phenotype of adult Wnt1sw/sw mutants,
fusion of the cerebellum with the midbrain, deletion of intervening structures
and anterior displacement of the choroid plexus, is consistent with our
previous reports on swaying embryos
(Bally-Cuif et al., 1995;
Louvi and Wassef, 2000
). In
contrast, the increase in isthmic structures (generally considered as
intermediate between rhombomere 1 and the midbrain) that we observed in the
En1+/Otx2lacZ mutants was unexpected. Indeed, the shift in
the caudal limit of Otx2 expression in En1+/Otx2lacZ
mutants has been interpreted as an expansion of the midbrain territory at the
expense of the anterior vermis (Broccoli
et al., 1999
). According to this scenario, any intervening isthmic
territory should have been at best reduced, if not deleted. The present
finding suggests that the isthmic territory is really an intermediate domain
depending on both Otx2 and Gbx2. Delineation of the isthmic domain could occur
at an early stage where the Otx2 and Gbx2 domains overlap
(Garda et al., 2001
).
Alternatively, the isthmic domain could be composed of cells that cross the
Otx2 caudal limit and switch off Otx2 expression. Although the caudal limit of
Otx2 expression has been considered as fixed
(Millet et al., 1996
), several
observations challenge this notion. Cells have been shown to cross the Otx2
boundary (Jungbluth et al.,
2001
), no restriction to cell coupling, a hallmark of rhombomere
boundaries, has been found at the MHB junction
(Martinez et al., 1992
), and
extensive DiI tracing experiments never detected any restriction to cell
movements at the MHB junction except on the midline
(Alexandre and Wassef, 2003
).
Finally the mode of progressive differentiation and growth of both the mid-
and hindbrain centered at the MHB allows for much regulation when gene
expression is turned off and on (Millet et
al., 1999
). We propose that maintenance of a moderate level of
Otx2 expression in the hindbrain predisposes boundary cells towards an isthmic
identity.
The observations reported here indicate that the isthmic region is crucial for the formation of the cerebellar midline, first, by controlling divergent midline cell movements and by contributing cells to both the cerebellar roof plate and the adjacent domain, second, by endowing the isthmus-derived cells in the cerebellar plate with specific properties and allowing them to restrict the influence of the choroid plexus, a hindbrain-specific midline cell population.
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ACKNOWLEDGMENTS |
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Footnotes |
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* Present address: Department of Neurobiology, Pharmacology and Physiology,
University of Chicago, Chicago, IL 60637, USA
Present address: INSERM U106, Hôpital de la Salpêtrière,
47 bd de l'Hôpital, 75013 Paris, France
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