1 Department of Molecular and Medical Pharmacology, University of California,
Los Angeles, CA 90095, USA
2 Molecular Biology Institute, University of California, Los Angeles, CA 90095,
USA
3 Department of Microbiology, Immunology and Molecular Genetics, University of
California, Los Angeles, CA 90095, USA
4 Department of Pathobiological Sciences and Waisman Center, University of
Wisconsin, Madison, WI 53705, USA
5 Department of Pathology and Laboratory Medicine, University of California, Los
Angeles, CA 90095, USA
* Author for correspondence (e-mail: xliu{at}mednet.ucla.edu)
Accepted 5 May 2005
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SUMMARY |
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Key words: PTEN, Bergmann glia, Cerebellar development, Mouse
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Introduction |
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The cerebellar cortex has long provided a model system for studies on
glial-guided neuronal migration and on glial differentiation
(Ramon y Cajal, 1911;
Rakic, 1971
;
Edmondson and Hatten, 1987
;
Solecki et al., 2004
;
Anthony et al., 2004
). Bergmann
glial cells are first seen in the cerebellar cortex in the late embryonic
period, when they express the markers RC2 and BLBP. By birth, the radial glial
population has disappeared, replaced by the Bergmann glia, which extend
processes from midway through the anlagen to the pial surface. The migration
of postmitotic-granule cell precursors begins after birth, with maximal
periods of migration between P7 and P12. By P15, the external granule layer
(EGL) is no longer evident, as the progenitors have all migrated into the
internal granule cell layer (IGL), a zone just deep to the Purkinje
neurons.
PTEN (for phosphatases and tensin homolog, deleted on chromosome 10) is a
tumor suppressor gene frequently mutated in tumors such as glioblastomas,
endometrial carcinomas and advance prostatic cancers
(Li et al., 1997;
Steck et al., 1997
). Although
PTEN functions as both protein and lipid phosphatases in vitro
(Myers et al., 1998
), its
major in vivo substrate is PIP3 (Maehama
and Dixon, 1998
; Sun et al.,
1999
), a lipid second messenger produced by phosphoinositide
3-kinase (PI3K). PIP3 plays a central role in promoting cell proliferation and
survival by activating downstream effectors such as AKT/PKB and mTOR-S6K
pathways (Stocker et al.,
2002
; Stiles et al.,
2002
; Radimerski et al.,
2002
). Germline PTEN mutations are associated with Cowden
disease, Lhermitte-Duclos disease and Bannayan-Zonana syndrome
(Marsh et al., 1998
;
Eng, 2003
). In addition to the
high risk of cancerous developments, these syndromes share a common feature of
disorganized tissue growth with unknown etiology
(Maehama et al., 2001
).
Our previous study demonstrated that Pten deletion in the
embryonic brain leads to abnormal histoarchitecture with severe layering
defects (Groszer et al.,
2001). However, as Pten deletion happens in the neural
stem/progenitor cells, it is unclear whether the abnormal phenotypes observed
are caused by intrinsic migratory defects of Pten-null neurons,
extrinsic micro-environmental cues provided by Pten null radial glia,
or perhaps both (Groszer et al.,
2001
). Recently, Marino et al.
(Marino et al., 2002
) reported
aberrant Purkinje cell positioning in Pten mutant mice, but the
mechanism leading to this defect is unknown.
In this study, we have investigated the role of PTEN in postnatal cerebellar laminar formation. We show that the deletion of Pten in both neurons and glia leads to severe lamination defects. By specifically deleting Pten in Bergmann glial cells, we further demonstrate that PTEN intrinsically controls Bergmann glia differentiation and scaffold organization. Our data suggest that Bergmann glia provide crucial, indispensable and developmental stage-dependent extrinsic cues for cerebellar granule neuron migration and laminar formation. These findings underscore the importance of PTEN as a negative regulator for Bergmann glia differentiation and the essential role of Bergmann glial scaffold for granule neuron migration.
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Materials and methods |
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Isolation of granule neurons and in vitro migration assay
Cerebellar cells were purified by using a modified procedure described by
Hatten (Hatten, 1985).
Cerebella were dissected from P6 animals. After removal of meninges,
cerebellar tissue was treated with Trypsin-EDTA (Gibco-BRL) and triturated
into single cell suspension using fire polished glass pipettes. The cell
suspension was applied to a Percoll gradient and separated by centrifugation.
Enriched granule neurons were applied to glass bottom microwell dishes (MatTek
Corporation) containing shattered Whatman glass fiber filters that are
pre-coated with polylysine (Sigma) and laminin (BD Bioscience). Granule neuron
migration along the glass fiber was imaged by a Hamamatsu video camera mounted
on a Leica inverted microscope and analyzed by computer software
(Fishman and Hatten, 1993
).
Competitive migration assay was performed as described previously
(Hirotsune et al., 1998).
Fresh dissociated granule neurons were stained by CellTracker Green dye
(Molecular probes) at 20 µM for 45 minutes. Equal numbers of labeled
wild-type neurons were mixed with unlabeled mutant neurons (and vice versa)
and allowed to form reaggregates overnight before plating on laminin-coated
culture chambers. After 24 hours, the reaggregates were fixed with 4%
paraformaldehyde and assayed for migration.
For glia-mediated granule neuron migration
(Hatten et al., 1986),
enriched glia isolated from P6 cerebella were cultured at low density. After 7
days in vitro, cultured glia were free from neuron contamination. Then fresh
dissociated granule neurons were applied to glia to yield four different
co-culture combinations: (1) wild-type granule neurons/wild-type glia; (2)
wild-type granule neurons/mutant glia; (3) mutant granule neurons/wild-type
glia; (4) mutant granule neurons/mutant glia. After additional 36 hour
incubation, the cells were fixed with 4% paraformaldehyde and subject to
immunohistochemical staining with antibodies against Tuj1 (COVANCE) and GFAP
(Dako).
Primary granule neuron culture
Granule neurons were obtained from dissociated P6 cerebella as described
(Dudek et al., 1997). Cells
were grown in DMEM/F12 medium (Gibco) supplemented with 10% calf serum, 25 mM
KCl, 30 mM glucose, 5 mM HEPES buffer, 2 mM glutamine and
penicillin-streptomycin. Cytosine arabinoside (10 µM) was added 24 hours
after plating to prevent proliferation of non-neuronal cells. After culturing
7 days in vitro cells were washed and switched to serum-free medium with or
without 25 mM KCl for 24 hours, chromatin condensation was visualized by DAPI
staining.
Immunohistochemistry
Immunohistochemical analysis was performed on either 4%
paraformaldehyde-fixed paraffin sections (4 µm) or cryosections (10 µm).
Immunostaining of BLBP (kindly provided by Dr Heintz at Rockefeller
University), BrdU (G3G4, DSHB), Calbindin D-28K (Chemicon, Sigma), DsRed (BD
Bioscience), GFAP (Biomeda, DAKO), TAG1 (DSHB), phospho-histone H3 (Upstate),
GABAA receptor alpha6 (Santa Cruz), Tuj1 (COVANCE) and p27 (Santa
Cruz) were detected by either Vector MOM Kit or Vector ABC Kit (Vector
Laboratories). Whole-mount X-gal staining was performed according to standard
protocol.
Western blot analysis
For surveying of PTEN, P-AKT and AKT expression in cerebella, whole-cell
extract was prepared by lysing fresh isolated P6 cerebellar granule neurons
and glia in RIPA buffer. Cell lysates were subjected to SDS-PAGE
electrophoresis and western blot analysis, using antibodies specific for PTEN,
P-AKT, AKT (Cell Signaling Technologies), Vinculin or Actin (Sigma)
antibodies.
Retrovirus production
Recombinant pantropic replication-incompetent retroviruses were produced
according to manufacturer's suggestions (BD biosciences). For generation of
retroviral construct, a SalI/DraI fragment containing
IRES2-DsRed was isolated from pIRES2-DsRed2 vector and inserted into
SalI/StuI sites of the pLNCX2 vector. Next, the
XhoI/NotI fragment containing a nuclear targeting CRE/GFP
fusion protein was inserted into the pLNCX2-IRES-DsRed vector to generate the
pLNCX2-CRE/GFP-IRES-DsRed retroviral construct.
Preparation and infection of organotypic cerebellar slices
Organotypic cerebellar slices were prepared according to Tomoda et al.
(Tomoda et al., 1999). To
visualize migrating granule neurons within slices, cerebella from P6 mice were
cut coronally into 300 µm slices by a chopper (Stoelting). The slices were
transferred onto culture inserts (Millicell; Millipore) and submerged in
culture media. Two hours after dissection, retroviruses were applied to
medium. Forty-eight to 72 hours after infection, slices were fixed and
processed for immunostaining.
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Results |
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Ptenlox/lox;hGfap-cre+ mice were viable but showed significant macrocephaly. Brain mass increased progressively until death occurred around postnatal day 21 (P21, M.G., H.W. and X.L., unpublished). Dissection of Pten mutant brains revealed a twofold increase in brain mass at P21 (Fig. 1A). To determine the efficiency of Pten deletion, we performed western blot analysis on fresh isolated P6 cerebellar granule neurons (95% purity) and glia (70% purity). Pten deletion is near complete in granule neurons but to a lesser degree in glial cells of mutant cerebella (Fig. 1B). We also detected prominent CRE immunoreactivity in mutant Bergmann glia throughout postnatal development (data not shown).
AKT is a downstream effector that is negatively regulated by PTEN; increased AKT phosphorylation (AKT-P) is a hallmark of PTEN loss. We examined AKT-P level in both granule neurons and glia. As predicted, AKT-P was significantly increased in both granule neurons and glia from Pten mutant cerebella. Interestingly, Pten expression level was notably higher in control glia compared with wild-type neurons, whereas AKT-P level was higher in mutant glia than in mutant neurons (Fig. 1B), implying cerebellar glia have stronger PI3K activity than do granule neurons.
Chronological analysis of histological samples revealed a bi-phasic developmental defect in PTEN mutant cerebella: before P3, PTEN mutant cerebella were significantly larger than controls but had relatively normal EGL and cerebellar lobules (Fig. 1C, parts a-b'). Dramatic impairment occurred after P6 when mutants showed a marked cerebellar enlargement and lack of IGL and folia, the hallmarks of postnatal cerebella (Fig. 1C, parts c-d'). We also observed a time-dependent Purkinje layer defect in mutant mice, demonstrated by immunohistochemical staining with a Purkinje cell specific marker Calbindin (Fig. 1D). Before P3, the positioning of mutant Purkinje cells was largely normal (Fig. 1D, parts a-b'), indicating normal Purkinje cell migration during embryonic development. The first sign of Purkinje layer defect was seen in mutant mice around P4-P6 (Fig. 1D, parts c,c'). By P9, numerous Purkinje neurons, which had previously adopted normal positioning, now were randomly scattered (Fig. 1D, parts d,d'). As Pten is not deleted in Purkinje neurons (Zhuo et al., 2002) (data not shown), the aforementioned layering defect could be secondary to the abnormality of granule neuron migration or Bergmann scaffold.
|
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Previous studies have indicated that deletion of Pten in Purkinje
neurons leads to vacuolization, degeneration and progressive loss of Purkinje
cells (Marino et al., 2002).
To exclude the possibility that there was a loss of granule neurons either in
the IGL or during cell migration, we tested the effect of PTEN on cell
survival by culturing isolated granule neurons in the presence or absence of a
depolarizing level of potassium. Pten-null granule neurons were
highly resistant to non-depolarizing level of potassium (5 mM)-induced
apoptosis, and the neuron size was significantly enlarged
(Fig. 4A, and quantified in
Fig. 4B). Similarly, no
significant cell death could be detected in mutant brains as measured by TUNEL
assay (data not shown).
Pten null granule neurons do not have cell-autonomous migration defects
The granule neuron migration defect seen in Pten mutant mice
warranted a more detailed examination of their migration properties. Previous
studies demonstrated that granule neurons isolated from cerebella could
readily migrate on either laminin-coated glass fibers or cerebellar glial
fibers, but poorly on fibronectin coated glass fibers
(Hatten, 1985;
Fishman and Hatten, 1993
). We
first tested the intrinsic migration properties of Pten-null granule
neuron migration on laminin coated glass fibers. The migration distance of an
individual neuron was recorded with a time-lapse microscope and
Fig. 5A showed an example of
such a study. Notably the mutant neuron retained a typical migration profile
consisting of cycles of extension and contraction on the laminin-coated glass
fiber substratum. Comparing 50 migrating neurons from mutants and controls, we
found the overall migration velocity was similar (speed of mutant
neuron=6.1±4.6 µm/hour; speed of control neuron=6.9±6.3
µm/hour).
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We next studied granule neuron migration in cerebellar organ cultures. P6
Ptenlox/lox cerebellar slices were infected by a
replication incompetent retrovirus that was engineered to express both a
CRE-GFP fusion protein and a DsRed protein bicistronically (see Materials and
methods). Because granule neurons are the only cell type that undergoes rapid
proliferation in the cerebellar cortex at P6, this retrovirus vector will
preferentially infect and delete Pten in granule neurons
(Tomoda et al., 1999).
Forty-eight to 72 hours post-infection, Pten-deficient granule
neurons (indicated by nuclear GFP expression) exhibited the typical mode of
migration, as judged by the morphology of the leading dendrite and trailing
axon, indicated by DsRed (Fig.
5C), suggesting that migrating polarity could be maintained in
Pten-deficient granule neurons in vivo. Thus, the intrinsic migratory
properties of Pten-null granule neuron does not differ significantly
from its wild-type counterparts, based on the individual migration assay, the
competition measurement and semi in vivo labeling.
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Severe Bergmann glia defects in Ptenlox/lox; Gfap-cre+ mice
We then turned our attention to cerebellar glial cells in which
Pten was efficiently deleted. Bergmann glia are the major glial cell
type in the cerebellar cortex. During postnatal development, Bergmann glia
undergo significant morphological changes to accommodate granule neuron
differentiation and migration. At P3, Bergmann glia are BLBP immunoreactive
and occupy the outer region of the cerebellar cortex
(Fig. 6A). Around P7, Bergmann
glia show strong BLBP immunostaining and their cell bodies align roughly in a
single layer next to Purkinje cells (Fig.
6B). Subsequently, Bergmann glia extend their fibers that link the
EGL and IGL (Fig. 6C), thereby
providing a substratum or cue for granule neuron migration
(Rakic, 1971). Detailed
immunohistochemical analysis revealed a time-dependent Bergmann glial defect
in Pten mutant cerebella. Pten-null Bergmann fibers were
significantly enlarged when compared with the controls at P3
(Fig. 6A'). At P7,
Pten-null Bergmann glial cell bodies were randomly distributed, some
had completely lost their contacts to the pial surface and positioned deep
within IGL region (Fig.
6B', white arrowheads). These ectopic Bergmann glia were
negative for Ki67 labeling (data not shown), suggesting that they were not
generated in situ but, instead, disintegrated from the Bergmann glial layer
during the rapid expansion of cerebellar cortex. At P14,
Pten-deficient Bergmann glia branched out numerous processes and
acquired an astrocyte-like cell morphology
(Fig. 6C'). Although some
Bergmann glia still maintained their endfeet at pial surface, others had
withdrawn theirs completely. Accompanying premature Bergmann glia to astrocyte
differentiation, numerous granule neurons were evidently retained within the
same region (Fig. 6C',
granule neuron nucleus shown in blue). Together, these results suggest that
Bergmann glia premature differentiation may affect granule neuron migration
and IGL layering formation.
Granule neuron migration defect is secondary to abnormal Bergmann glial layering
Detailed analysis of PTEN mutant cerebella suggested that disruption of
Bergmann glial layering might cause the granule neuron migration defect. At
P7, concomitant with the mislocalized Bergmann glia in the deep IGL
(Fig. 7A, part b), many granule
neurons failed to complete their migration and were accumulated within the ML
(Fig. 7A, part b'). To
directly analyze PTEN function in Bergmann glia development, we constructed a
helper-dependent adenovirus (HDA) vector expressing Cre and YFP (HDA.Cre/YFP)
(Dorigo et al., 2004). As
adenovirus preferentially infects glial cells rather than neurons
(Iino et al., 2001
), this
vector allowed us to delete Pten in Bergmann glia. We surgically
injected HDA.Cre/YFP into
Ptenloxp/loxp;Rosa26floxed-Stop-lacZ cerebella
at P3 and examined at 8 days post-injection. Cre-mediated Pten
deletion, as indicated by lacZ-positive staining
(Fig. 7B, part a; area
indicated by paired arrows), correlated well with changes in Bergmann glial
scaffold (Fig. 7B, part b;
paired arrows) and retention of granule neurons in the ML region
(Fig. 7B, part c; compare red
boxed area with blue boxed area, P<0.05), although these granule
neurons were negative for lacZ expression (red boxed area in
Fig. 7B, part a). Thus,
Pten deletion in Bergmann glia can affect granule neuron migration,
even when these granule neurons are wild type for Pten expression. As
a control experiment, HDA.Cre/YFP was also injected into
Rosa26floxed-Stop-lacZ cerebella; no structural changes
were observed resulting from viral infection alone (data not shown).
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Discussion |
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The cerebellum is the target of numerous gene mutations
(Goldowitz and Hamre, 1998).
The creation of laminated structure of postnatal cerebellar cortices is
achieved by directional migration of committed granule neurons from the EGL to
their final destination in the IGL. Many intrinsic and extrinsic factors are
known to influence this differentiation process
(Hatten, 1999
). Therefore, the
cerebellum can serve as an excellent model for studying brain morphogenesis.
Conditional Pten deletion in cerebellum has been reported recently
using different Cre lines. Two groups
(Backman et al., 2001
;
Kwon et al., 2001
) used a
GFAP-CRE line that restricted Pten deletion in postmitotic neurons
and observed a cell-autonomous increase in neuronal soma size. The third group
(Marino et al., 2002
) used
En2-CRE and L7-CRE lines and observed Purkinje cell positioning defect and
degeneration.
In our study, we focus on the role of Pten during early postnatal cerebellar development. Our data further extend previous findings and suggest that (1) PTEN intrinsically controls Bergmann glia differentiation, and (2) Bergmann glia provide critical environment cues for granule neuron migration. We speculate that this model may be relevant for explaining some of the mutant phenotypes observed by the previous studies.
Factors controlling granule neuron migration have been extensively studied
using gene targeted mouse models. Mutations in Cdk5, Pax6 and
Pafah1b1 genes cause cell-autonomous granule neuron migration
aberrations (Ohshima et al.,
1999; Engelkamp et al.,
1999
; Yamasaki et al.,
2001
; Hirotsune et al.,
1998
). Mutations in astrotactin and neuregulin
(Adams et al., 2002
;
Rio et al., 1997
) affect
granule neuron-Bergmann glia interaction and cell migration. The relationship
between Pten deletion and cell migration defects has been studied
recently. PTEN-deficient Dictyostelium cells are poorly chemotactic as result
of polarity defects and an inability to restrict gradient sensing
(Iijima and Devreotes, 2002
;
Funamoto et al., 2002
).
PTEN-deficient mammalian cells, however, are more mobile, although different
mechanisms have been proposed (Tamura et
al., 1998
; Liliental et al.,
2000
; Raftopoulou et al.,
2004
). By contrast, the effect of Pten deletion on
gliophilic migrating granule neurons remains largely unknown. Although
dysplastic granule neurons are present in the adult EGL or ML of PTEN mutants
(Backman et al., 2001
;
Marino et al., 2002
), their
number is relatively small and may not reflect a genuine cell intrinsic
migration defect. As demonstrated in this study, Pten-null neurons
are highly resistant to apoptosis. Thus, the persistent survival of ectopic
granule neurons within the ML, which otherwise should be eliminated through
apoptosis in wild type, may contribute granule neuron dysplasia seen in these
studies (Backman et al., 2001
).
In support of this assumption, early reports demonstrated an intact ML during
early postnatal development (P10), although PTEN expression is significantly
downregulated in the inner EGL of Pten mutants, suggesting deletion
of PTEN in granule neurons prior to migration may not cause a cell-intrinsic
migration defect (Backman et al.,
2001
). To clarify the effect of Pten deletion on
migrating granule neurons, we performed a systematic investigation on cell
intrinsic migration property. We show that Pten deletion has minimal
effect on intrinsic migratory properties of granule neurons in both in vitro
and in vivo settings, suggesting that migration defect of Pten
mutants is due to changes of extrinsic factors within the local
microenvironment.
The intimate structural relationships between migrating granule neurons and
Bergmann fibers suggest that elongated radial Bergmann glia serve as a
scaffold that is crucial for granule neuron migration
(Rakic, 1971). Using genetic
model systems, continuing efforts have been made towards the establishment of
the indispensable role of Bergmann glial scaffold during granule neuron
migration. One of the examples is the studies of weaver mutants; the close
association between abnormal Bergmann glial scaffold and impaired granule
neuron migration suggested that the morphological change of Bergmann glia was
the major cause of granule neuron migration defect
(Sidman and Rakic, 1973
).
However, other studies have demonstrated that weaver Bergmann glia were
phenotypically normal in the chimeric studies, and weaver granule neurons
failed to form contacts with Bergmann glia
(Hatten et al., 1986
;
Goldowitz, 1989
) and express
differentiation markers Tag1, suggesting that Bergmann glia scaffold defects
in weaver mice might be secondary to mutant granule neurons. The
identification of a mutation in the Girk2 gene as being responsible
for the weaver mutant (Patil et al.,
1995
), which might result in a loss of inwardly rectifying K+
current and cytotoxicity in cerebellar granule cells
(Surmeier et al., 1996
),
further complicated the interpretation of the role of weaver Bergmann glia in
granule neuron migration. Thus, it is still not clear whether the migration
defect in weaver mutants is due to abnormality of Bergmann glial scaffold or
cell-intrinsic ionic changes in granule neurons. Another example is meander
tail mice, which have the abnormal Bergmann glial scaffold, but can be rescued
in the chimeric study, suggesting they might also be secondary to mutant
granule neurons (Hamre and Goldowitz,
1997
).
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Whether PTEN can modulate cell differentiation events remains ambiguous:
PTEN loss is not crucial for cell-fate determination in central nervous system
(Groszer et al., 2001;
Marino et al., 2002
), but
precocious morphogenesis of mammary gland development
(Li et al., 2002
) and hair
follicle changes (Suzuki et al.,
2003
) have been observed in Pten conditional deleted
mammary glands and skin. In the current study, we observed striking
morphological changes in Bergmann glia in
hGfap-cre+/;Ptenloxp/loxp mice during
early postnatal development. The elongated Bergmann glial fibers prematurely
develop numerous lateral processes that are indistinguishable from stellate
astrocytes. This change highly resembles the cortical radial glia to stellate
astrocyte differentiation. By selectively deleting Pten in Bergmann
glia through targeted adenovirus delivery, we show that the Pten-null
Bergmann glia premature differentiation is a cell intrinsic event, suggesting
PI3K/Akt activity is important for radial glia to astrocyte transformation. In
contrast to the dramatic changes in Bergmann glia after Pten
deletion, we did not observe a significant alternation of cerebellar granule
neuron differentiation by examining several differentiation markers. The
discrepancy may arise from the relatively short duration of granule neuron
differentiation, which could obscure the recognizable changes under current
detection means. Alternatively, granule neuron and Bergmann glia may differ in
their response to Pten deletion in the aspect of cell
differentiation.
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ACKNOWLEDGMENTS |
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