1 Dipartimento di Farmacologia e Fisiologia Umana, Università di Bari;
Università di Torino, Italy
2 Dipartimento di Anatomia Umana ed Istologia, Università di Bari;
Università di Torino, Italy
3 Università di Bari; Dipartimento di Neuroscienze, Università di
Torino, Italy
4 Institute of Biochemistry, University of Zurich, Switzerland
5 Centre for Developmental Genetics, Department of Biomedical Science,
University of Sheffield, Western Bank, Sheffield S10 2TN, UK
* Present address: Cancer and Developmental Biology Laboratory, National Cancer
Institute, Frederick, MD 21702, USA
Authors for correspondence (e-mail:
G.Gennarini{at}tno.it and A.J.Furley@Sheffield.ac.uk)
Accepted 3 October 2002
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SUMMARY |
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Key words: Cerebellar development, Neurite growth, Axonal glycoproteins, F3/contactin, TAG-1, Gene regulation, Cell proliferation, Mouse
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INTRODUCTION |
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Recent studies have revealed the involvement of a number of diffusible
factors in this process, in particular sonic hedgehog
(Dahmane and Ruiz i Altaba,
1999; Wechsler-Reya and Scott,
1999
; Ruiz i Altaba et al.,
2002
), neurotrophins (Schwartz
et al., 1997
; Morrison and
Mason, 1998
; Hirai and Launey,
2000
) and fibroblast growth factor
(Wechsler-Reya and Scott,
1999
). Surprisingly however, although a number of cell adhesion
molecules (CAMs) have been implicated in GC axon outgrowth and fasciculation
(Walsh et al., 1997
;
Buttiglione et al., 1998
;
Meiri et al., 1998
;
Hillenbrand et al., 1999
;
Sakurai et al., 2001
) and in
their migration (Fishell and Hatten,
1991
; Hatten,
1999
; Adams et al.,
2002
), few studies have attempted to address the role of such
molecules in the cell-cell interactions that control cell differentiation in
the cerebellar cortex.
Notable among the adhesion molecules that could mediate such interactions
are members of the immunoglobulin-like L1 subfamily, comprising L1-like
transmembrane (Kadmon and Altevogdt, 1997;
Kamiguchi and Lemmon, 1997)
and F3/contactin-like GPI-linked molecules
(Ranscht, 1988
;
Gennarini et al., 1989
;
Furley et al., 1990
;
Yoshihara et al., 1994
;
Yoshihara et al., 1995
;
Lee et al., 2000
;
Ogawa et al., 2001
), all of
which are expressed on cerebellar neurons: L1, NrCAM, CHL1, TAG-1,
F3/contactin, NB-2 and BIG-2 on GCs
(Yoshihara et al., 1995
;
Hillebrand et al., 1999; Virgintino et
al., 1999
; Sakurai et al.,
2001
; Ogawa et al.,
2001
) and neurofascin (Zhou et
al., 1998
), NrCAM (Sakurai et
al., 2001
), F3/contactin
(Virgintino et al., 1999
), L1
(Jenkins and Bennet, 2001), BIG-1
(Yoshihara et al., 1994
) and
NB-3 (Lee et al., 2000
) on
PCs. Although gene targeting has revealed a critical role for F3/contactin in
GC development (Berglund et al.,
1999
), the effects of deleting other members of the family have
been more subtle (Dahme et al.,
1997
; Fransen et al.,
1998
; Fukamauchi et al.,
2001
) (L. Y. and A. F., unpublished) and there is evidence for
functional redundancy among these molecules (e.g. L1 and NrCAM)
(Sakurai et al., 2001
).
Nonetheless, specific differences in their function in in vitro assays, and
their distinct spatial and temporal expression profiles, suggest that their
deployment at specific times in cerebellar development may be important.
A good example of this is provided by the two GPI-linked glycoproteins
F3/contactin and TAG-1. These molecules share 50% amino acid identity
(Gennarini et al., 1989
;
Furley et al., 1990
), common
binding to L1, NrCAM and neurofascin
(Olive et al., 1995
;
Malhotra et al., 1998
;
Volkmer et al., 1998
;
Faivre-Sarrailh et al., 1999
;
Fitzli et al., 2000
) and the
ability to stimulate neurite elongation
(Furley et al., 1990
;
Gennarini et al., 1991
;
Stoeckli et al., 1991
) and
direct axonal growth (Berglund et al.,
1999
; Fitzli et al.,
2000
; Fujita et al.,
2000
). However, each also has distinct binding capabilities (e.g.
F3/contactin-tenascin R, TAG-1-neurocan)
(Pesheva et al., 1993
;
Milev et al., 1996
) and, in
some contexts, F3/contactin may inhibit neurite outgrowth
(Buttiglione et al., 1996
;
Buttiglione et al., 1998
). In
the cerebellum, TAG-1 is predominantly expressed on premigratory granule
neurons in the external granular layer (EGL) whereas F3/contactin peaks on
postmitotic migrating neurons
(Faivre-Sarrailh et al., 1992
;
Wolfer et al., 1994
;
Wolfer et al., 1998
; Stottmann
et al., 1998; Virgintino et al.,
1999
). Since the function of these two molecules may be
antagonistic (Buttiglione et al.,
1998
), their differential expression may be critical to the
orderly differentiation and morphogenesis of the cerebellar cortex. To address
this possibility, we have deregulated F3/contactin expression in transgenic
mice by placing its cDNA under the control of the proximal promoter region of
the human TAG-1 gene (TAX1). This results in ectopic expression of
F3/contactin in the outer EGL and in a developmentally regulated cerebellar
phenotype in which neuronal proliferation and differentiation are affected.
These data indicate a novel role for these adhesion molecules and that precise
regulation of their expression is critical to normal cerebellar
differentiation and morphogenesis.
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MATERIALS AND METHODS |
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TAX1/F3 construct
Using a NcoI-HindIII adaptor oligonucleotide, the same
TAX1 genomic fragment was cloned by three-way ligation into
NotI/XbaI-cut pGL3-Basic, along with a 3.1 kb
HindIII-EcoRI fragment containing the full-length
F3/contactin cDNA except for the triplet encoding for the most C-terminal
amino acid and a TGA opal stop codon
(Gennarini et al., 1989). The
junction at the 3' end was achieved by partial filling of EcoRI
and XbaI 5' overhangs, each with two corresponding nucleotides,
resulting in creation of compatible two-letter sticky ends; upon ligation this
restored the last F3/contactin triplet followed by a TAG stop codon. The
integrity of both constructs was verified by sequencing.
Generation and breeding of transgenic mice
DNA fragments were excised by digestion with SalI, diluted in TE
buffer and injected into fertilised oocytes from CBA x C57 Black10
F1 donor mice (Hogan et al.,
1994). Founders carrying the transgene were identified by southern
blot and gene-specific PCR and used to establish colonies at the breeding
facilities of the Department of Pharmacology and Human Physiology of Bari
University. Animal experimentation conformed to the EU directive 86/609
EEC.
Immunohistochemical procedures
F3/contactin immunostaining was performed on paraffin sections from
developing mice cerebella perfused with 2% paraformaldeyde and 0.1%
glutaraldehyde as described previously
(Virgintino et al., 1999). For
calbindin immunostaining a mouse monoclonal antibody (Sigma, St Louis, USA)
was used on adjacent sections; alternatively, 20 µm cryostat sections from
mice cerebella perfused with 4% paraformaldehyde were stained with a rabbit
anti-rat calbindin serum (Swant, Bellinzona, Switzerland) and revealed by the
Vectastain elite ABC kit (Vector Laboratories, Burlingame, CA, USA)
using diaminobenzidine as a substrate. To account for individual variations,
four different mice from each wild-type and transgenic genotype were processed
at each developmental step for both F3/contactin and calbindin
immunohistochemistry.
Staining for ß-galactosidase was performed on 20 µm cryostat
sections from tissues fixed with 2% paraformaldehyde/0.1% glutaraldehyde
according to described protocols (Hogan et
al., 1994). For double TAG-1/lacZ labelling, sections
stained for ß-galactosidase were extensively washed in phosphate-buffered
saline (PBS), incubated with the TAG-1 rabbit antiserum
(Dodd et al., 1988
) and
developed with goat anti-rabbit secondary antibodies. For double
F3/contactin-TAG-1 staining, 20 µm cryostat sections from brains perfused
with 4% paraformaldehyde were incubated with the F3/contactin fusion protein
antiserum 24III (Gennarini et al.,
1991
) and the mouse TAG-1 monoclonal antibody 4D7
(Yamamoto et al., 1986
).
Biotinylated goat anti-rabbit IgG followed by TRITC Avidin D (Vector
Laboratories) and fluorescein-conjugated goat anti-mouse IgM (Jackson
Laboratories, West Grove, Pennsylvania, USA), respectively were used as
secondary antibodies.
Double labelling was performed with polyclonal anti-TAG-1
(Dodd et al., 1988) and
monoclonal anti-proliferating cells nuclear antigen (PCNA) (Novocastra
Laboratories) antibodies, or monoclonal anti-TAG-1 (4D7) and polyclonal
anti-phosphorylated histones H1 and H3 antibodies (Upstate Biotechnology).
Pups were perfused with 4% paraformaldehyde in PBS and brains were then
further fixed for 2 hours before being processed for cryosectioning
(Dodd et al., 1988
). Sections
on slides were incubated for 12 hours at 54°C before being stained with
antibodies as previously described (Dodd
et al., 1988
) and visualised by standard epifluorescent or
confocal (Leica TCS) microscopy.
Cerebellar granule cell cultures and immunocytochemistry
Cerebellar cultures were generated at postnatal day 7 as described
(Buttiglione et al., 1996;
Buttiglione et al., 1998
).
To visualise neurites, after fixation with 4% paraformaldehyde in PBS, aggregate cultures were stained with the 24III serum and dissociated neurons with a rabbit anti-GAP43 serum (Research Diagnostics INC, NY, USA), using Cy3-conjugated goat anti-rabbit immunoglobulins (Jackson laboratories) as secondary antibodies.
Cell proliferation and apoptosis assays
To estimate cell proliferation in vivo, mice received three subcutaneous
injections of 5-bromo-2'-deoxyuridine (BrdU; Roche Molecular
Biochemicals, Mannheim, Germany; 30 µg/g body weight) at 0, 8, 12 hours and
were sacrificed 8 hours after the last injection. Brains fixed by immersion in
3% acetic acid in ethanol were embedded in paraffin wax. Three µm sections
were incubated with a nuclease-containing anti-BrdU mouse monoclonal antibody
and revealed by alkaline phosphatase-conjugated sheep anti-mouse IgG
(Roche).
To measure cell proliferation in vitro, high density primary cerebellar cultures (0.3x106 cells/cm2 on polylysine-coated LabTek slides) were incubated overnight with BrdU (10 µM in medium) either immediately after plating or 16 hours later. Cells were then washed and fixed with 70% ethanol in 15 mM glycine buffer pH 2 before detecting BrdU as above.
Cell death was estimated by using the ApopTag kit (Intergen, NY, USA) on 5 µm paraffin sections from developing mice cerebella or on high density primary cerebellar cultures according to the manufacturer's instructions.
Morphometric analysis
Image acquisition, processing and measurement were performed by the image
analyser VIDAS 2.5 (Kontron Elektronik GmbH, Eching, Germany).
Cerebellar cortex
Four cerebella from wild-type and four from transgenic mice were examined
at each developmental step (0, 3, 8, 11, 16 and 30 postnatal days). Fifty
Toluidine Blue-stained sections from each cerebellum (in the region of the
cerebellar vermis, from the nucleus medialis habenulae to the nucleus medialis
cerebelli) were digitised. The total area of the cerebellum and of each
cortical layer was measured in µm2 by a custom written
semiautomatic sequence of commands (macros); this included a segmentation step
by a thresholding method able to automatically identify the cerebellar cortex
layers according to the grey level of the individual pixels; mean
(M)±standard error of mean (s.e.m.) were calculated and values were
expressed as their ratios in transgenic versus wild-type (wt) mice.
Cells density in the IGL and EGL was measured by counting Toluidine Blue-labelled nuclei in random fields of identical size (40x magnification) by using a custom written macro. For BrdU incorporation or cell death the number of labelled cells was estimated using VIDAS 2.5 interactive measurement functions. For measuring the number of PCs and the area of their dendritic tree, calbindin-immunostained sections from postnatal days 8 cerebella were used, acquiring the fields at 5x and 40x magnifications, respectively.
Neuronal cultures
To quantify neurite growth, immunolabelled cultures were photographed under
a Leica DRMB microscope connected to a Polaroid DMC camera. The resulting
microphotographs were acquired with a CCD/RGB video camera (TK-1070E; JVC,
Japan) connected to the VIDAS 2.5 and analysed using a specifically written
macro.
For aggregate cultures absolute values for neurite-occupied surface were referred to a total area of 12.9 mm2. A total of 28 cell aggregates was analysed from both wild-type and transgenic mice. To measure neurite length, photographs of dissociate cultures were acquired at a 40x magnification. A total of 408 and 241 isolated neurons from wild-type and TAG/F3 mice, respectively, were analysed. Values were expressed as M±s.e.m.
The number of BrdU-labelled or TUNEL-stained cells in high density primary cerebellar cultures was measured in random fields of identical size (77440 µm2) by using a custom-written macro.
Statistical significance of the morphometric data was assessed by the Student's t-test.
RNA analysis
Total RNA was prepared using the Trizol reagent (Life Technologies). For
RT-PCR amplification, the Superscript One-Step RT-PCR System (Life
Technologies) was used on 3 µg of total RNA. Amplification products were
analysed on 2% agarose gels.
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RESULTS |
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A promoter element from the human TAG-1 (TAX1) gene
drives expression in premigratory granule cells
To define such a regulatory element, the proximal promoter region of the
TAX1 gene (Kozlov et al.,
1995) including the first exon, the ATG-containing second
exon and the intervening first intron was fused in frame to a
lacZ reporter (Fig.
2A, see Materials and Methods) and used to generate transgenic
mice (designated TAG/lacZ). In TAG/lacZ mice, TAG-1
and lacZ expression largely overlapped in the EGL at postnatal day 8
(P8) (Fig. 2B, a-c); however,
transgene activation was weaker in the anterior lobes (compare lobules V-VI in
f with lobules VIII-IX in i). Similarly to TAG-1 protein
(Furley et al., 1990
),
transgene expression was strongest on premigratory neurons (f,i). However,
particularly in lobules VIII-IX, it also extended towards the outermost region
of the EGL (oEGL) (h-i) and in the IGL, where GCs expressed the transgene with
a similar anteroposterior gradient (b,c). These differences between
TAG-1 and lacZ expression may reflect the difference in the
sensitivity of the assays for TAG-1 and ß-galactosidase, and the fact
that the transgene is present in multicopies (data not shown), as low levels
of TAG-1 mRNA are normally detected in both the oEGL and in the IGL
(Furley et al., 1990
).
Moreover, the transgene maintained normal TAG-1 cell-type specificity
and was not expressed, for example, in Purkinje neurons (h) (Stottmann et al.,
1998). Thus, these regulatory elements seemed appropriate to induce premature
expression of F3/contactin.
|
Transgenic mice were then generated using a construct in which the same
TAX1 gene 5' flanking region was fused to a full length
F3/contactin cDNA (Fig. 2A).
Five different founder mice were obtained and used to establish lines. Reverse
transcription polymerase chain reaction (RT/PCR) amplification (see
Fig. 3A for details) revealed
that the transgene mRNA was expressed in the cerebellum of several lines
(Fig. 3B and data not shown),
beginning at birth and continuing as late as postnatal day 30 (P30),
reflecting normal TAG-1 expression (Wolfer
et al., 1994; Wolfer et al.,
1998
). Immunohistochemical staining at postnatal day 4 (P4;
Fig. 3C) confirmed that the
transgene directs ectopic expression of F3/contactin protein throughout most
of the EGL, as expected from the TAG/lacZ results
(Fig. 2B, h,i), whereas
wild-type controls were F3/contactin-negative in this region. Having confirmed
the success of this approach, further detailed analysis was performed and
representative results from one of these lines (designated TAG/F3)
are presented below.
|
Deregulation of F3/contactin expression results in a developmentally
regulated reduction in cerebellar size
Cerebellar development was followed using Toluidine Blue-stained sections
(Fig. 4). In newborn
TAG/F3 mice (Fig. 4C,
see also Fig. 6A,B), cerebellar
size and foliation pattern were not significantly different from wild type.
However, by postnatal day 3 (P3) the relative size of the transgenic
cerebellum was slightly reduced (Fig. 4A,
a,b), and by P8 the size of the transgenic cerebellum was markedly
smaller than controls (Fig. 4A,
c,d). Prominent effects were observed in lobules I to V and
VIII-X, while lobules VI-VII displayed comparatively minor changes. The
reduction in cerebellar size persisted at P11; again, lobules I to V and
VIII-X were mostly affected (Fig. 4A,
e,f). However, by P16, only lobules VIII-IX were still slightly
affected (Fig. 4A, g,h), and by
P30 the TAG/F3 cerebellum was indistinguishable from wild-type
controls (not shown).
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Cerebellar cortical layers are differentially affected by
deregulation of F3/contactin as development proceeds
A morphometric analysis was then performed of the overall surface area of
the cerebellar sections and of the different cortical layers (see Methods for
details). Sample sections are presented in
Fig. 4B, and in
Fig. 4C the ratios of the
absolute values of these parameters in TAG/F3 versus wild-type mice
are plotted. Differences in the surface area of the cerebellar sections,
already apparent at P3, become most significant at around P8, followed by a
recovery after P16. The areas of both the IGL and the molecular layer (ML)
were similarly reduced at P8 to almost half their normal values. By contrast,
the EGL, most affected at P3, was already showing signs of recovery by P8, and
by P11 had almost completely recovered normal size, even though the IGL and ML
were still significantly smaller than normal. This suggested that the primary
effect of the transgene was on the number of GCs in the EGL with consequent
effects on cell numbers in the IGL. To rule out the alternative possibility
that the reduction in the IGL size was due to a reduced development of the
surrounding neuropil, we determined the GC density in the IGL at P8. No
differences were found between wild-type (198.15±3.47 s.e.m.
cells/field) and TAG/F3 mice (194.78±3.70 s.e.m.,
P=0.59); similarly, in the EGL, comparable values were obtained for
wild-type and TAG/F3 mice (138.4±4.56 versus
140.04±3.85 cells/field, P=0.8) confirming that changes in the
size of the EGL and IGL were due primarily to a reduction in the absolute
number of cells. Reduction in the size of the ML will be addressed further
below.
F3/contactin overexpression results in changes in granule cell
proliferation
The reduction in the number of GCs in the TAG/F3 cerebellum could
depend upon their reduced production in the EGL, their increased death or a
combination of both. To test the first possibility, cell proliferation was
followed by bromodeoxyuridine (BrdU) incorporation. Significant differences
were not detected at P0 (Fig. 5A,
a,b; 73.51±1.96 s.e.m. labelled cells in TAG/F3
versus 70.96±2.21 in wild-type mice/18667 µm2;
P=0.36). However, at P3 (c,d), a lower density of BrdU-labelled cells
was apparent in the EGL of the TAG/F3 mice cerebellum
(84.54±2.18 s.e.m. labelled cells/field) versus wild-type mice
(103.28±1.58 s.e.m.; P=0.001). Although this difference had
disappeared by postnatal day 6 (e,f) (94.68±1.23 for TAG/F3
versus 96.13±1.19 for wild-type mice, P=0.4), by P8 the
situation was reversed with a significantly higher level of BrdU labelling in
TAG/F3 mice (g,h) (89.38±1.15 versus 82.28±1.21
cells/field, P=0.001). This latter effect was also developmentally
regulated since by P11 it was no longer evident (i-j) (46.04±1.03
labelled cells/field for TAG/F3 versus 46.68±1.0 for wild-type
mice; P=0.6). Thus, overexpression of F3/contactin under the TAG-1
promoter resulted in developmentally regulated changes in GC proliferation,
with a significant decrease in early development followed by an apparently
compensatory increase at the end of the first postnatal week.
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BrdU incorporation was also measured in high density (0.3x106 cells/cm2) dissociated cultures from P3 cerebellum (Fig. 5B). In these conditions, while comparable values were obtained when the labelling was performed immediately after plating (a,c) (497±30.4 s.e.m. labelled cells/77440 µm2 in wild-type versus 470±26.2 in TAG/F3 mice, P=0.33) a consistent difference was observed in cultures labelled 16 hours after plating (b,d) (174±10.3 in wild-type versus 82±4.9 in TAG/F3 mice, P=0.0001). These data, reported in graphic form in Fig. 5C, indicated that GCs overexpressing F3/contactin drop out of cell cycle more rapidly than normal.
TAG-1 is expressed on mitotically active granule cells
The observation that expression of F3/contactin from the TAG-1
promoter effects cell proliferation was unexpected since TAG-1 has usually
been described on post-mitotic neurons (e.g.
Kuhar et al., 1993). To test
the possibility that some TAG-1-expressing cells may still be proliferating in
the EGL, we double labelled P8 wild-type cerebellum with antibodies to TAG-1
and to markers of mitotic activity, including proliferating cells nuclear
antigen (PCNA) (Ino and Chiba,
2000
) and phosphohistones H1 and H3
(Lu et al., 1994
;
Hendzel and Bazett-Jones,
1997
). As shown in Fig.
5D, TAG-1 expression (b) overlapped significantly with both PCNA
(a,c) and phosphohistones H1 and H3 (d). This indicated that a substantial
proportion of the TAG-1-expressing cells in the iEGL are still mitotically
active (
20% (45/215) of TAG-1+ cells also expressed medium to high levels
of PCNA) and, therefore, that F3/contactin is likely to be expressed on
dividing cells in TAG/F3 mice.
Minor differences in cell death occur in TAG/F3 mice
Cell death was estimated in situ by the TUNEL method (see Materials and
Methods). In Fig. 6A sample
elements undergoing apoptosis are shown at postnatal day 3 in lobule IX of
both wild-type and TAG/F3 mice cerebella and in
Fig. 6B the overall density of
such elements in the whole cerebellar cortex is reported from P0 to P8. No
differences were observed between wild-type and transgenic mice at P0
(78±5.7 versus 74±4.0 cells/mm2) and P6
(113±6.8 versus 105±7.2 cells/mm2). However, at P3
the number of apoptotic cells was significantly higher in TAG/F3
(180±11.9 cells/mm2) versus wild-type (125±8.3) mice
(P=0.001) whereas at P8 the reverse situation was observed
(66±1.9 in TAG/F3 versus 94±3.6 in wild-type mice,
P=0.001). Thus, changes in cell death mirror those in cell
proliferation, with cell death increasing when proliferation drops and vice
versa. However, whereas the changes in cell proliferation were observed in GC
within the EGL, changes in cell death mostly affected postmitotic cells in the
IGL.
TUNEL staining was also performed in high density primary cultures from P3 cerebellum plated on polylysine. Sample elements undergoing apoptosis are shown in Fig. 6C in both wild-type and TAG/F3 mice. When their number was estimated in fields of identical size (77440 µm2), comparable values were obtained in wild-type and TAG/F3 mice (67±4.2 s.e.m. labelled cells/field in wild-type versus 62±3.1 in TAG/F3 mice; P=0.44).
Developmentally regulated changes of F3/contactin expression in the
TAG/F3 mice
The above data indicated that ectopically expressed F3/contactin in the EGL
of the TAG/F3 cerebellum transiently inhibits GC proliferation.
Accordingly, the increase in cell proliferation at P8 might be expected to
correlate with a downregulation of F3/contactin. However, RT-PCR analysis
indicated persistent transgene mRNA expression at this time
(Fig. 3B), suggesting that
overall F3/contactin protein levels might not reflect mRNA profile of the
transgene. To test this possibility we performed F3/contactin
immunohistochemistry throughout the postnatal period in both wild-type and
TAG/F3 cerebella.
As shown in Fig. 7A, a,b, at
P0 consistent F3/contactin overexpression was observed in the TAG/F3
mice which, with the exception of lobule X, occurred in both the anterior and
posterior lobes. This overexpression was evident on migrating GC within the
nascent ML as shown in lobules II-III (Fig.
7B, k,l) and VI-VII (m-n). Comparison of adjacent sections stained
for calbindin (Fig. 8A, d) indicated that some of this staining was on PCs (see below). This
overexpression was still evident throughout the cerebellar cortex at P3
(Fig. 7A, c,d). This was
especially evident for lobules I-IV and X which in wild-type mice express very
low levels of F3/contactin (Virgintino et
al., 1999). At this stage, F3/contactin is on both the perikarya
and forming neurites of migrating GCs and on nascent PC dendrites
(Virgintino et al., 1999
); in
TAG/F3 mice, F3/contactin was observed on these neurons at clearly
elevated levels, as shown for lobules II-III (o,p) and VI-VII (q,r); PCs were
identified by comparison with adjacent sections stained with calbindin
(Fig. 8A, g,h). These
alterations were stronger in lobules VI-VII (q,r), than in II-III (o,p), in
agreement with the higher level of transgene activation in the former (see
Fig. 2B). Thus, at P0, P3 and
P4 (Fig. 3C), levels of
F3/contactin protein were elevated in TAG/F3 cerebellum, as predicted
by the levels of transgene mRNA.
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|
In contrast, by P8 F3/contactin protein expression in TAG/F3 mice
(f) appeared, if anything, slightly reduced compared to the controls (e). At
higher magnification (s,t) this correlated with markedly reduced width and
labelling of the ML, and less elaborate dendrites of PCs whose perikarya were
notably smaller than in controls. At P11 (g,h), the level of F3/contactin
expression was partially recovered in TAG/F3 mice. This reflected
increased expression on PCs, which by this time had partially recovered their
normal morphology, as well as on GC axonal extensions in the ML (u,v). By P16
(i,j,w,y) no differences could be observed among the different cortical
layers. F3/contactin immunostaining was restricted to the ML and, as
previously reported (Virgintino et al.,
1999), no expression could be observed on PCs.
Together, these data suggested that, in contrast to the situation at P0-P3, from P8-P16 there was no evidence of F3/contactin protein overexpression in TAG/F3 mice, even though the levels of transgene mRNA remained relatively constant throughout this period (Fig. 3B). Indeed, at P8 we detected lower levels of F3/contactin than in wild type which, together with decreased ML width and PC dendrite arborisation, suggested delayed differentiation of granule and Purkinje neurons.
Biphasic effects on Purkinje cells differentiation in TAG/F3
transgenic mice
The effects of the transgene on PC development were further studied by
calbindin immunostaining. Similar to the elevated F3/contactin expression seen
at birth (Fig. 7), calbindin
expression in TAG/F3 mice (Fig.
8A, b,d) was also higher than in controls (a,c). This premature
expression was restricted to lobules I-V and IX but at P3 was also apparent on
lobule X (e,f). Thus, by this criterion, PCs begin to differentiate
prematurely in TAG/F3 mice.
However, there was a reduction in the arborisation of Purkinje cell dendrites at P3 (g,h) in transgenic mice, as suggested by F3/contactin immunostaining (Fig. 7). By P8, this reduction, together with a reduction in PC cell body size, became striking (Fig. 8A, i,j). Similar, though reduced differences were apparent at P11 (k,l), but by P16 (m,n) transgenic PCs took on an essentially normal appearance. Morphometric analysis at P8 indicated an approximately threefold reduction of the overall extension of the PC dendritic three in TAG/F3 mice (801.37 µm2±27.29 s.e.m. versus 273.54 µm2±8.39/field; P=0.007). When studied at higher power in thicker sections (20 µm) from postnatal day 6 cerebellar cortex (Fig. 8B) Purkinje cells dendrites clearly displayed a simplified branch network, suggestive of a delayed growth of the spiny branchlet compartment.
These results are consistent with an apparent delay in PC maturation. However, their overall number per section was not significantly different from wild type (411±44.05 s.e.m. in TAG/F3 versus 527±58.2; P=0.14), their cell bodies were aligned in a single row and their dendrites were properly oriented along parasagittal planes, indicating that other aspects of their development were normal.
Thus, the data above confirmed that the terminal differentiation of PCs is delayed in TAG/F3 mice. Paradoxically, however, premature elevation of calbindin expression suggested that aspects of their early differentiation may occur prematurely.
F3/contactin misexpression in granule cells enhances axonal
fasciculation and inhibits axonal growth in vitro
The reduced width of the ML and delayed PC dendritic arborisation seen in
TAG/F3 mice from P3-P11 suggested that, in addition to effects on
granule cell proliferation, F3/contactin misexpression may affect granule cell
axon growth. We tested this in granule cell reaggregate cultures in vitro. In
Fig. 9A-D, F3/contactin
immunostaining of 2 day-old reaggregate cultures from P7 wild-type (A,C) and
TAG/F3 (B,D) mice cerebella are shown. In TAG/F3 mice,
neurites appeared shorter and the surface area they covered was significantly
lower than in controls (1.82 mm2±0.10 s.e.m. versus
2.74±0.12; P=0.0001). Consistent with this, neurites displayed
a strong tendency to fasciculate, noticeably more so than in cultures from
wild-type mice (C,D). To discriminate between the effects on neurite
fasciculation and growth, dissociated cultures of cerebellar neurons were
generated and grown on CHO cells monolayers. In
Fig. 9E-F, representative
cultures from wild-type (E) and transgenic (F) cerebellum at P7 are shown,
stained with GAP-43 antibodies and in G neurite growth is quantified by
cumulative neurite length histogram. Cultures from transgenic mice developed
shorter neurites than the controls (60.43±2.9 s.e.m. versus
106.02±3.7 µm; P=0.0014), indicating that, as well as
enhancing axonal fasciculation, F3/contactin misexpression also exerted a
specific inhibitory effect on neurite growth from cerebellar neurons.
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DISCUSSION |
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TAG/F3 transgenic mice display a developmentally regulated
decrease of the cerebellar size
The most striking effect we observe in TAG/F3 mice was a decrease
in the cerebellar size, predominant at the end of the first postnatal week.
This appears to depend primarily upon a reduction in granule cell number,
first obvious in the EGL at P3 and in the IGL at P8, and secondly upon a
parallel reduction in the growth and fasciculation of their axons in the
ML.
F3/contactin expression affects granule cell proliferation
Members of the L1-like family have been proposed to have roles in cell
migration, axon growth, fasciculation and guidance, and in synaptic plasticity
(Schachner, 1997;
Walsh and Doherty, 1997
;
Kamiguchi and Lemmon, 2000
).
Here we show that premature expression of F3/contactin in GCs in the EGL from
P0 to P3 is accompanied by a nearly 20% reduction in the number of
proliferating GCs. The simplest interpretation of our observations is that
F3/contactin expression antagonises proliferation-promoting contacts between
GCs (Gao et al., 1991
),
perhaps by stimulating cell repulsion, as has been observed when F3/contactin
acts as a receptor for tenascin-R in vitro
(Pesheva et al., 1993
;
Xiao et al., 1996
). In fact,
in preliminary studies, no changes of the adhesive behaviour of granule cells
could be directly demonstrated in primary cultures from TAG/F3 mice
(data not shown); however, the possibility cannot be excluded that more subtle
effects may occur in vivo among contacting granule cells, resulting in a
reduction of their adhesive strength. Alternatively, the effects on GC
proliferation could depend upon changes in PC differentiation, which were
evident as early as P0 in TAG/F3 mice, particularly since it is known
that GC proliferation depends on PC-derived signals
(Wetts and Herrup, 1983
;
Smeyne et al., 1995
;
Baader et al., 1998
;
Chomez et al., 2000
;
Dahmane and Ruiz i Altaba,
1999
; Wechsler-Reya and Scott,
1999
). However, since the TAX-1 promoter used for
transgene generation is not expressed in PCs, it is likely that these changes
are anyway initiated through contacts with F3/contactin-overexpressing GCs.
Therefore, we favour the hypothesis that F3/contactin expression in granule
cells directly modulates proliferation. In support of this, our in vitro
experiments show that granule cells from TAG/F3 mice cultured at high
density have substantially reduced proliferative capacity.
An important question is whether this F3/contactin effect has physiological
significance. For this to be the case, F3/contactin would be expected to be
found on the proliferating cells themselves, or on cells with which they
contact. Our data indicate that F3/contactin-expressing GCs normally are
separated from the proliferative oEGL by TAG-1-expressing cells
(Fig. 1), conventionally
thought to be post-mitotic. However, as suggested by in vitro studies
(Wolf et al., 1997), we have
found that a substantial proportion of TAG-1-expressing GCs also express cell
proliferation markers (Fig.
5D). Thus, it seems plausible that in normal development,
expression of F3/contactin on or close to proliferating TAG-1-expressing cells
may contribute to the process of cell cycle exit. Precedent for a similar role
for this class of molecule comes from studies in vitro in which soluble NCAM
was found to inhibit hippocampal neuronal progenitor proliferation and
concurrently induce differentiation
(Amoureux et al., 2000
).
In several mouse mutants reduced cerebellar size has been attributed in
part to increased cell death (Sonmez and
Herrup, 1984; Herrup and
Sunter, 1987
; Smeyne and
Goldowitz, 1989
; Harrison and
Roffler-Tarlov, 1998
; Doughty
et al., 2000
). While we also observed an increase in cell death in
the IGL early on, the changes we saw were small (a maximum increase of 40%)
compared to, for instance, those seen in the rev-erbA
mutant
mouse (up to 400% increase) (Chomez et
al., 2000
) and even in this case the effects on cerebellar size
were small. We believe, therefore, that effects on cell death have only a
minor role in the phenotype observed in TAG/F3 animals. In any case
the effects we observe are likely to be developmental in nature, triggered by
modified interactions among cerebellar neurons as no differences in cell death
could be observed in primary cultures.
Effects on neurite growth and fasciculation
While the smaller number of GCs in the IGL may simply reduce axon input to
the ML our observations of reduced neurite outgrowth from transgenic GCs in
primary culture, accompanied by an increase in axon fasciculation, suggest
that F3/contactin misexpression also directly affects axon growth. In previous
studies, substrata of F3/contactin were found to exert inhibitory effects on
neurite growth and to promote fasciculation in primary cerebellar cultures
(Buttiglione et al., 1996;
Buttiglione et al., 1998
).
Moreover, results from our gain-of-function experiment are exactly
complementary to those from the F3/contactin loss-of-function mouse insofar as
aggregate cultures from those mice exhibited a decrease in neurite
fasciculation and, in vivo, their parallel fibres were less compacted
(Berglund et al., 1999
).
Although we have not checked this directly by electron microscopy, the
reduction in the width of the ML in TAG/F3 mice is consistent with an
increase in the compaction of parallel fibres. Together these results confirm
a direct role for F3/contactin in parallel fibre growth and fasciculation.
However, the occurrence of comparable levels of apoptosis in cultured GC from
TAG/F3 and wild-type mice together with the minor differences
occurring in vivo exclude the possibility that the effects on neurite growth
from GC may reflect differences in their viability.
In other ways, our results were not predicted from previous experiments.
Indeed, co-expression of F3/contactin and TAG-1 in CHO cells as a substratum
negated the inhibitory effects on GC neurite outgrowth and fasciculation that
F3/contactin had when present alone
(Buttiglione et al., 1998). In
TAG/F3 mice, however, F3/contactin was overexpressed as a receptor
rather than as a substratum, which may invoke different binding partners
(Pesheva et al., 1993
;
Xiao et al., 1996
); in
addition, evidently higher levels of F3/contactin may have outweighed TAG-1
modulatory effects.
Purkinje neuron terminal differentiation is delayed in
TAG/F3 transgenic mice
Unlike GCs, no significant changes were observed in the number of PCs,
consistent with the time course of their proliferation which may be complete
before the onset of transgene activation
(Altman and Bayer, 1985).
However, increased calbindin and F3/contactin expression at P0 suggested that
early differentiation events occurred prematurely. Therefore as in the case of
peripheral neurons (Gennarini et al.,
1991
), F3/contactin may positively modulate PC differentiation.
However, since the transgene is not activated on Purkinje neurons, these
effects are likely to be triggered by F3/contactin expressed at the surface of
contacting granule cells or released in soluble form in their microenvironment
(see Fig. 7). Paradoxically,
however, by P3, and most significantly at P8, the PC morphological
differentiation was delayed, as judged by reduced extension and arborisation
of their dendrites. Again, these effects seem to be non-autonomous and may
reflect the known interactions between granule and Purkinje cells
(Hatten and Heintz, 1995
;
Altman and Bayer, 1997
). In
particular, they may arise from the delayed development of GC axons since PC
dendritic growth can be regulated by interactions with outgrowing parallel
fibres (Sotelo, 1978
;
Baptista et al., 1994
;
Altman and Bayer, 1997
;
Morrison and Mason, 1998
;
Catania et al., 2001
).
The effects of F3/contactin misexpression are developmentally
regulated
The restoration of normal cerebellar morphology in TAG/F3 mice
after P8 was particularly surprising as, at the mRNA level, the transgene is
expressed at high levels until at least P30, reflecting normal TAG-1
expression (Furley, 1990; Wolfer, 1994; Yoshihara, 1995). At least two factors
may contribute to this recovery. First, while ectopic expression of the
F3/contactin protein is clearly evident at P4
(Fig. 3), by P8 its levels are
lower than in controls (Fig. 6
and data not shown), potentially reflecting delayed neuronal differentiation
in TAG/F3 mice. Second, there is evidence to suggest that
F3/contactin function may change during the course of the first postnatal
week, reflecting changes in the expression profile of some of its binding
partners. For instance, tenascin R, whose interaction with F3/contactin is
inhibitory for axonal growth (Pesheva et
al., 1993), is upregulated on both myelinating cells and neurons
at the end of the first postnatal week, when we observe the strongest
phenotypic alterations in the ML and IGL, and is downregulated thereafter when
we observe recovery (Fuss et al.,
1993
), suggesting that some aspects of TAG/F3 phenotype
could be accounted for by developmentally regulated interactions between
F3/contactin and tenascin R. Indeed, we find that the developmental profile of
expression of tenascin R mRNA (estimated by quantitative RT/PCR) in
TAG/F3 mice is similar to those found in wild-type mice (data not
shown).
An alternative, but not exclusive possibility is that the mechanism of GC
maturation may change with time with respect to F3/contactin function. Indeed,
loss of F3/contactin only appears to affect later-differentiating granule
cells (Berglund et al., 1999),
whereas our gain-of-function experiment seems to affect early-differentiating
GCs. Thus, although granule cell differentiation is often assumed to be a
uniform process operating throughout the first two postnatal weeks (e.g.
Kuhar et al., 1993
), these
observations suggest that, at least with regard to F3/contactin function, it
may be divided into two phases. The first one operates perinatally and
involves the ability of F3/contactin to modulate granule cell precursor
proliferation, the second begins towards the end of the first postnatal week
and mostly relates to axonal growth control. This differential role during
development in turn may depend upon regulated interactions of F3/contactin
with its various receptors that are known to have different profiles of
developmental activation (Pesheva et al.,
1993
; Xiao et al.,
1996
; Buttiglione et al.,
1998
; Volkmer et al.,
1998
; Faivre-Sarrailh et al.,
1999
).
Conclusions
Together, our data strongly support our main hypothesis that the precise
regulation of axonal CAMs expression is critical to normal morphogenesis of
the cerebellum. In particular, the differential deployment of the antagonistic
functions of F3/contactin and TAG-1 may define different stages of cerebellar
neuron differentiation. F3/contactin may be important in promoting cell cycle
exit, stabilising axon growth and inducing early events of Purkinje cell
differentiation. By contrast, the antagonistic activity of TAG-1, suggested by
a previous study (Buttiglione et al.,
1998), may allow granule cells to continue dividing while
extending axons and migrating. Our results also indicate that the emphasis on
these differing activities seems to change with time; in the first postnatal
week, low F3/contactin activity appears to be critical for the cerebellum to
expand appropriately, whereas its upregulation in the second week may be
important to stabilise axonal extensions and to take part in the process of
shutting down granule cell proliferation.
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ACKNOWLEDGMENTS |
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