Institut de Génétique et de Biologie Moléculaire et Cellulaire, INSERM/CNRS/ULP, BP 10142, 67404 Illkirch Cedex, C.U. de Strasbourg, France
Author for correspondence (e-mail:
eb{at}igbmc.u-strasbg.fr)
Accepted 18 June 2003
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SUMMARY |
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Key words: Oligodendrocytes, TK ablation system, Transgenic mice, Cerebellum, Development
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Introduction |
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The participation of glial cells in cerebellum development is also very
important. The Bergmann glia, a specialized type of astrocyte, provides a
scaffold for cerebellar neuron migration and positioning
(Rakic, 1971), as well as for
Purkinje cell dendritic outgrowth and synapse formation
(Yamada et al., 2000
).
Selective ablation of these cells in the early postnatal period strongly
impairs cerebellum development (Delaney et
al., 1996
). Astrocytes have also been shown to induce and
stabilize CNS synapses (Ullian et al.,
2001
). The role of oligodendrocytes, the CNS myelin forming cells,
in cerebellum development has not been clearly addressed. However,
neuron-oligodendrocyte interaction at the axonal level is required for the
maturation of CNS neurons (Brady et al.,
1999
; Colello et al.,
1994
; Colello and Schwab,
1994
; Mathis et al.,
2000
), and for the formation and maintenance of the nodes of
Ranvier and the paranodal regions (Arroyo
et al., 2002
; Mathis et al.,
2001
). Furthermore, knockout of contactin, a protein localized
both in neurons and oligodendrocytes (Koch
et al., 1997
), leads to ataxia consequent to an abnormal
microorganization of the cerebellum
(Berglund et al., 1999
). These
results suggest a possible role for oligodendrocytes in cerebellum
development.
To explore this possibility, we used transgenic mice in which herpes
simplex virus 1 thymidine kinase (HSV1-TK) was targeted to OLs under the
control of the MBP promoter. In these mice, OLs can be ablated in a temporal
inducible manner (Mathis et al.,
2000) by injection of FIAU, a non-harmful nucleoside analog that
is converted to a toxic compound by TK activity
(Borrelli et al., 1988
;
Borrelli et al., 1989
). In this
way, the effects of oligodendrocyte ablation can be studied within the context
of cerebellar development.
Loss of oligodendrocytes during the first postnatal weeks results in a
profound perturbation of the cerebellar cytoarchitecture. In particular,
Purkinje cells do not align properly and fail to form dendritic arborizations.
We suggest that oligodendrocytes may be crucial for the proper maturation of
Purkinje cells due to a loss of oligodendrocytes-myelin/neuron interactions.
As a consequence of this, the terminal differentiation of the complete
cerebellar circuitry is aborted leading to a severe phenotype. Importantly,
the effect of oligodendrocyte ablation on cerebellar development is
time-dependent. Furthermore, loss of these cells was concomitantly associated
with the disappearance of basket and stellate cell interneurons in the ML.
This striking observation, together with the white matter origin of these
cerebellar interneurons (Milosevic and
Goldman, 2001; Zhang and
Goldman, 1996b
), raises the possibility of a key role for
oligodendrocytes and/or myelin in the survival/migration of these
interneurons. These results uncover a previously unknown and important role
for oligodendrocytes during cerebellar development.
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Materials and methods |
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Immunohistochemistry
Immunofluorescence analyses were carried out on cerebellar sections, which
were either 4% paraformaldhyde-fixed floating vibratome sections (100 µm)
or cryosections (10 µm) fixed in formalin (Sigma), or on paraffin wax
embedded tissue fixed in BOUIN (10 µm), depending on the primary antibody
used, following the previously described protocols
(Mathis et al., 2000).
Antibody dilutions were as follows: rabbit anti-PAX2 (Zymed), 1:400; mouse
anti-calbindin D-28K (SWant), 1:3000; rabbit anti-BLBP (a generous gift of Dr
N. Heintz), 1:1500; rabbit anti-HSV1-TK (kindly provided by Dr P. Collins,
Glaxo Wellcome, Beckenham, UK), 1:200; rabbit anti-PH3 (Upstate Biotechnology,
Lake Placid, NY), 1:5000; rabbit anti-synaptophysin (Dako), 1:50; mouse
anti-parvalbumin (Chemicon), 1:1000; mouse anti-MBP (Chemicon), 1:1000; mouse
anti-GALC (Roche), 1:50; mouse anti-PLP (Chemicon), 1:800; and goat
anti-rabbit Alexa 594 and goat anti-mouse Alexa 488 (Molecular Probes), 1:600.
Negative controls were always performed by omitting the primary antibody.
TUNEL experiments were performed on brain cryosections post-fixed in 1%
paraformaldehyde in PBS, using the In Situ Cell Death Detection Kit (Roche,
Germany) and dUTP-coupled with Alexa Fluor 488 (Molecular Probes).
Immunolabeled sections were examined with a conventional microscope (Axiophot;
Zeiss, Overbroken, Germany), and/or with a confocal microscope (DMRE; Leica,
Nussloch, Germany).
In situ hybridization
In situ hybridization was performed as previously described
(Mathis et al., 2000).
35S-labeled RNA probes encoding MBP, PAX2, PAX6, GAD67 (GAD1 -
Mouse Genome Informatics), zebrin-II (ALDO3 - Mouse Genome Informatics) sense
and antisense riboprobes were synthesized using T3, T7 or SP6 polymerase in
the presence of cytidine 5'-
[35S]thiotriphosphate (10
mCi/ml, Amersham), according to the supplier's directions (Stratagene,
Biolabs). After probe hybridization, slides were coated with Kodak NTB2
emulsion and stored at 4°C. Emulsions were finally developed in Kodak 19
and tissues were counter-stained with Toluidine Blue.
Basket and Stellate cells counts
Cresyl Violet stained sections from wild-type and MBP-TK mice treated with
different protocols of FIAU injections (1-20 d, 1-6 d and 6-20 d) were used to
estimate the proportion of interneurons located in the ML of the cerebellum.
Anatomically matched cerebellum sections of wild-type and MBP-TK siblings
identically treated were taken for this study. The number of fields of view
(FOV=600 µm2) examined varied between 25 and 35. Statistical
significance was assessed by ANOVA (Bartlett's test).
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Results |
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Owing to its postnatal development, the cerebellum represents an ideal
model system to analyze the impact of oligodendrocyte ablation on neighboring
cells. MBP-TK transgenic mice expressing the HSV1-TK gene under the control of
a 1.3 kb fragment of the myelin basic protein (MBP) promoter were used in this
study. The MBP-TK transgene is specifically expressed in oligodendrocytes, as
illustrated by the co-localization of MBP and TK immunostaining in the same
cells (Fig. 1A-D)
(Mathis et al., 2000). The
normal white matter tract location and the density of TK-expressing
oligodendrocytes is shown at lower magnification in cerebellar folia IX of p6
MBP-TK untreated mice (Fig.
1E). Upon nucleoside analog [i.e. FIAU
(McLaren et al., 1985
)]
administration, these cells are selectively killed
(Fig. 1F) (Borrelli et al., 1988
;
Mathis et al., 2001
;
Mathis et al., 2000
). This
property allowed us to induce oligodendrocyte cell death in vivo, upon FIAU
treatment (Mathis et al.,
2000
). We chose a treatment protocol that was able to induce 95%
oligodendrocyte ablation in the brain. MBP-TK and wild-type littermates were
administered FIAU through subcutaneous daily injections (40 mg/Kg), starting
24 hours after birth until postnatal day 20 (1-20d)
(Mathis et al., 2000
).
Wild-type and transgenic animals were killed at day 21 (P21) after birth and
analyzed.
|
Sagittal sections from wild-type treated cerebella (1-20d) showed the
classical morphology (Fig.
1G,I). In wild-type mice cerebellar folia are well formed and are
separated by deep fissures. The classical cortical organization is
identifiable by the three well known layers
(Fig. 1G). The ML containing
fibers and interneurons are underlined by the large soma monolayer of Purkinje
neurons (PCL). Just beneath this layer is the IGL, composed of very densely
packed soma of small neurons, the GCs, and the soma of some scattered larger
neurons, the Golgi cells. Thus, FIAU treatment does not alter the structure
and composition of the cerebellum in treated wild-type mice, as previously
reported in the brain and other organs
(Borrelli et al., 1988;
Borrelli et al., 1989
;
Heyman et al., 1989
;
Mathis et al., 2000
).
Similarly, untreated MBP-TK transgenic mice did not show any sign of
cerebellar abnormality (data not shown).
By contrast, 1-20d FIAU-treated MBP-TK mice present an outstanding
phenotype, associated with the almost complete ablation (95%) of
oligodendrocytes (Mathis et al.,
2000). As shown in Fig.
1J, the fissura intercruralis, which normally separates folia VI
and VII in the wild type (Fig.
1I), is absent in MBP-TK mice
(Fig. 1J, star). At higher
magnification, the laminar distribution of cerebello-cortical neurons is
highly altered (Fig. 1H) when
compared with wild-type mice (Fig.
1G), and the size of the folia is greatly reduced. This phenotype
is accompanied by a misalignment of the Purkinje cell soma, which appears
dispersed among a reduced population of GCs. In addition, the ML is not
clearly defined in these animals. These results indicate that the postnatal
ablation of oligodendrocytes and consequent dysmyelination results in a major
impairment of cerebellar development.
Cellular abnormalities in the cerebellum of treated MBP-TK mice
The white matter tract of the cerebellum is composed of Purkinje cell axons
and the two afferent pathways of the cerebellum, the climbing and the mossy
fibers. Mossy fibers are connected to GC and Golgi neurons. Climbing fibers
have synaptic contacts with Purkinje cells and their primary dendrites in the
ML. Myelinated fibers are visualized (Fig.
1K,L) by immunofluorescence with an antibody raised against MBP.
At P21, all of these fibers are fully myelinated in treated wild-type mice
(Fig. 1K). Oligodendrocyte
ablation, in treated MBP-TK mice, results in a drastic reduction of myelinated
fibers in the white matter tract, where only a few spared MBP-positive fibers
can still be observed (Fig. 1L,
arrow). These fibers are myelinated by residual oligodendrocytes that escaped
ablation; they were probably postmitotic at the time the treatment was started
(Mathis et al., 2000).
Quantification by mRNA analysis of myelin markers shows a very strong
reduction of these proteins. Indeed, the MBP mRNA level, analyzed at P21, was
reduced by 90±10% (n=15) with respect to treated wild-type
mice.
Reactive astrocytes and disorganized Bergmann glia network are
observed in treated MBP-TK mouse cerebellum
Next, we analyzed a second type of glial cell contained in the cerebellum,
the Bergmann glia. These cells are a specialized type of astrocyte that
express two well characterized astrocytic markers: BLBP (brain lipid binding
protein), during development, and GFAP (glial fibrillary acidic protein) in
adult mature cells (Feng et al.,
1994). Projections from these cells extend radially through the ML
toward the cerebellar pial surface. These glial extensions are known to
interact with migrating GCs and mature Purkinje neurons. Several studies have
suggested that Bergmann glia are involved in the guidance of GC migration from
the EGL to the IGL throughout the ML
(Hatten, 1990
;
Hatten and Heintz, 1995
;
Rakic, 1971
). In addition, the
formation of the Bergmann glia network accompanies the formation of the
Purkinje cell dendritic tree during development and synaptogenesis
(Yamada et al., 2000
). Using
BLBP and GFAP, we assessed how loss of oligodendrocytes would affect Bergmann
glia development. BLBP was analyzed at P6, as this marker has been reported to
decrease in the second week after birth. As shown in
Fig. 2A and C, these two
astrocytic markers in treated wild-type mice show the classical palisade of
Bergmann glia extensions in the ML, with glial cell bodies closely associated
with Purkinje cell bodies. BLBP-positive cells are also present in the IGL,
corresponding to astrocytes. In treated MBP-TK mice
(Fig. 2B,D), BLBP-positive
Bergmann glia cells were detected but their radial extensions into the ML were
completely misaligned (Fig. 2D)
with respect to those of wild-type siblings
(Fig. 2C). As in the wild-type
cerebella, BLBP-positive cells were also present in the IGL. GFAP
immunostaining performed at P21 on cerebella from wild-type and MBP-TK mice
confirmed the results obtained by BLBP staining in younger animals
(Fig. 2C,D). We also quantified
Gfap mRNA expression levels in the cerebella of treated wild-type
versus treated MBP-TK mice. As previously reported, a 20±5%
(n=15) increase of Gfap mRNA expression was noticed in
treated MBP-TK mice when compared with wild-type littermates, which is in
agreement with a normal astrocytic response to brain injuries.
|
|
Histological analyses of the cerebellar phenotype of treated MBP-TK mice
(1-20d) showed an abnormal pattern of foliation (loss of fissura
intercruralis) and a reduced IGL compared with wild-type littermates
(Fig. 1G,H). A more in depth
analysis of GCs in wild-type and MBP-TK 1-20d treated mice cerebella was
performed using two markers, PAX6 and RU49. PAX6 is an early marker of these
cells, which starts to be expressed when GCs are still proliferating in the
EGL, and expression is maintained following migration into the IGL
(Engelkamp et al., 1999). In
situ hybridizations were performed on sagittal cerebellar sections from
wild-type and MBP-TK treated mice at P6, using a PAX6 antisense riboprobe
(Fig. 4A,B). These experiments
showed that the EGL thickness in treated MBP-TK animals is reduced in the
depth of the fissure as well as in the extremities of the folia
(Fig. 4B; arrowheads) in
comparison to that of treated wild-type siblings
(Fig. 4A; arrowheads). In
particular, a greater decrease of EGL depth is observed in the future position
of the fissura intercruralis (Fig.
4B). This might explain the absence of this fissure in treated
MBP-TK mice observed at P21. By P10, almost all of the EGL has disappeared in
treated MBP-TK mice cerebella, with the exception of the fissura secunda and
parafloccularis (data not shown). The decreased thickness of the EGL in MBP-TK
mice is suggestive of an altered rate of proliferation of these cells in these
mice compared with in wild-type animals. This hypothesis was confirmed by
immunofluorescence studies using the anti-phosphorylated histone 3 (PH3)
antibody that specifically labels mitotic cells
(Fig. 4C,D). Moreover, TUNEL
experiments performed on sections from these mice revealed a larger number of
GC cells undergoing apoptosis in treated MBP-TK animals
(Fig. 4F) in comparison to in
treated wild-type mice (Fig.
4E). These data indicate that the reduced EGL size is the result
of both a decrease of GC progenitor proliferation and increased apoptosis.
These findings were confirmed at P21 by in situ hybridization using RU49
(Yang et al., 1996
) as a probe
(Fig. 4G,H), which labels GCs
in the IGL. To exclude the possibility that the decrease of GCs could be due
to an ectopic expression of the transgene in GC precursors, we performed
double immunostaining using anti-PAX6 (Fig.
4I,L) and HSV1-TK (Fig.
4J,L) antibodies. This analysis showed the absence of TK
expression in PAX6-positive cells in MBP-TK mice
(Fig. 4I-L). These analyses
were repeated at different time points and always gave negative results (data
not shown).
|
|
FIAU-induced cerebellar abnormalities in MBP-TK mice are
time-dependent
The biggest advantage of the TK-ablation system resides in the ability to
kill cells in an inducible manner. We therefore investigated the status of the
cerebellar cortex and of interneuron migration into this area following
different FIAU treatment protocols. We thus compared the structure of the
cerebellar cortex of transgenic animals treated from day 1 to 20, from day 1
to 6 and finally from day 6 to 20 (Fig.
6). In agreement with previous data, we found that ablation of
oligodendrocytes during the first postnatal week resulted in a great
impairment of the development of the cerebellar cortex. Indeed, Cresyl Violet
staining of the cerebella of 1-6d treated MBP-TK mice
(Fig. 6C,G) showed a phenotype
very similar to that of 1-20d chronically treated mice
(Fig. 6B,F). In wild-type
animals, no difference (Fig.
6A,E) was observed in the organization of the cerebella cortex of
animals that had followed the 6-20d FIAU treatment protocol
(Fig. 6D,H).
|
This observation prompted us to analyze the presence of these cells at early stages of postnatal cerebellar development. To do this, we examined cerebella of P3 and P6 treated MBP-TK and wild-type mice receiving FIAU from 24 hours after birth (Fig. 5A-D). At early stages of development, interneurons can be traced using the PAX2 marker. Cerebella of treated P3 MBP-TK mice already showed a reduction in interneurons when compared with wild-type siblings (Fig. 5A,B, arrowheads). Interneuron reduction became even more evident at day 6, where few PAX2-positive interneurons localized in the white matter tract were detectable, despite the presence of IGL localized Golgi cells (Fig. 5C,D, arrowheads).
Absence of interneurons could be related to their migratory pathway through the white matter tract in the wild type (Fig. 5E, arrowheads). Indeed, ablation of oligodendrocytes leads to a reduced development of this structure and only rare PAX2-positive interneurons in migration are found in the treated MBP-TK animals (Fig. 5F, arrowhead). This suggests that oligodendrocytes could be required for a trophic and/or migratory support of interneurons during the first postnatal week of cerebellum development.
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Discussion |
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We have analyzed the impact of oligodendrocyte loss on the development of the cerebellum, which occurs mainly in the postnatal period. Strikingly, ablation of oligodendrocytes following daily treatment with FIAU during the first three weeks after birth results in a dramatic phenotype characterized by an unstructured cerebellar cortex.
The cerebellum is composed of different cell types that are closely and
orderly interconnected. Several studies have demonstrated the importance of
cell-cell interactions in the normal development of this structure
(Baptista et al., 1994;
Changeux and Mikoshiba,
1978
).
Purkinje cell differentiation depends on local epigenetic factors, provided
partly by granule neurons (Baptista et al.,
1994). Interestingly, we show that oligodendrocyte ablation leads
to the misalignment of Purkinje cell bodies, accompained by poorly developed
dendritic arborizations and loose fasciculation of their axons. This suggests
that the presence of oligodendrocytes, and possibly myelination of Purkinje
axons, during the final maturation of these cells is crucial for the
completion of their differentiation program.
Oligodendrocyte ablation also affects granule neuron proliferation and
migration. Indeed, in treated MBP-TK cerebella, we observed reduced
proliferation of GC progenitors, paralleled by an increased apoptotic rate of
these cells. This results in a smaller cerebellum, abnormal foliation (loss of
fissura intercruralis) and a sparse IGL in transgenic treated mice (1-20d and
1-6d). It is known that proliferation of GC progenitors requires factors
including sonic hedgehog, which is secreted by Purkinje neurons
(Dahmane and Ruiz-i-Altaba,
1999). The defective maturation of Purkinje neurons in treated
MBP-TK mice could thus directly affect the generation of granule neurons.
However, future in depth studies are required to trace the series of molecular
events leading to the reduced numbers of granule cells.
In addition, GCs use the Bergmann glia scaffold to reach their final
location in the IGL (Rakic,
1971). In treated MBP- TK cerebella, the Bergmann glia are
disorganized, which probably has an impact on the migration and maturation of
the GC precursors, leading to their premature death and, consequently, to the
formation of a sparse IGL. Lack of organization of Bergmann fibers is also
very likely to be dependent upon the lack of maturation of Purkinje cells, as
the transformation of Bergmann fibers is synchronized with the dendritic
differentiation of these cells (Yamada et
al., 2000
). These events combine to result in an unstructured
ML.
It should also be taken into account that afferent pre-cerebellar and
pontine climbing and mossy fibers are in contact with, and myelinated by,
oligodendrocytes (Palay and Chan-Palay,
1974). Thus, we cannot exclude the possibility that
oligodendrocyte ablation may affect the connections of these two afferent
pathways with their cerebellar targets, thus contributing to the severe
phenotype observed in treated MBPTK mice.
Importantly, loss of myelin in genetic myelin mutants (shiverer, jimpy,
quaking) was never reported to affect cerebellum development as has been
observed in MBP-TK mice (Mikoshiba et al.,
1979; Nagaike et al.,
1982
). In these mutants, and in contrast with MBP-TK mice, the
oligodendrocytes are present and are able to myelinate axons at early
postnatal stages of development. However, during the second postnatal week,
oligodendrocytes and myelin eventually degenerate in these mutants
(Knapp et al., 1986
;
Nave, 1994
;
Readhead and Hood, 1990
).
Thus, it appears that the presence of oligodendrocytes during the first
postnatal week is an absolute requirement for the normal development of the
cerebellum. In agreement with this, MBP-TK mice treated from day 6 to 20,
despite a myelin reduction close to 50% in the cerebellum, do not present any
structural cerebellar abnormality.
We can exclude the possibility that the cerebellar phenotype might be due either to an ectopic expression of TK in other cell types, or to FIAU non-specific toxic effects as TK expression has never been observed in other cerebellar cells and FIAU treatment does not alter cerebellar development in wild-type controls. We can also exclude a potential toxic cell-autonomous effect of TK expression in oligodendrocytes, as in untreated MBP-TK mice cerebellar development is normal. Similarly, the observed phenotype is not due to a non-specific toxicity on granule cells. Indeed, FIAU treatments started at postnatal day 6 does not have any effect on cerebellar structure, despite the still active proliferation of GCs during the second postnatal week.
This study also shows that the absence of oligodendrocytes impairs basket
and stellate cell proliferation and/or migration into the ML. This finding
suggests that oligodendrocytes might secrete a factor(s) that helps their
survival and migration, and/or that serves as a substrate for the migration of
interneurons to their final location. A recent study on interneuron
progenitors located in the white matter of postnatal cerebellum suggests that
these cells begin to differentiate during migration
(Milosevic and Goldman, 2001).
Thus, another explanation for the disappearance of cortical interneurons might
be that oligodendrocytes are involved in the differentiation process of these
progenitors into basket and stellate cells. This is an important contribution
of oligodendrocytes to cerebellar development that has not been previously
observed, and which warrants further future investigations.
In conclusion, oligodendrocytes appear to have a crucial role in the establishment of the cerebellar cytoarchitecture. Our findings clearly show that the role of glial cells in brain development should be re-evaluated. New tools, represented by genetically engineered mice, are of great importance to analyze the relevance not only of genes, but also of cell populations, in the development, function and organization of tissues in vivo.
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ACKNOWLEDGMENTS |
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Footnotes |
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