1 Institute of Clinical Pathology, University Hospital, Schmelzbergstrasse 12,
8091 Zürich, Switzerland
2 Division of Molecular Genetics and Centre of Biomedical Genetics, The
Netherlands Cancer Institute, Plesmanlaan 121, 1066CX, Amsterdam, The
Netherlands
3 Institute of Neurology, Queen Square, London WC1N 3BG, UK
* Author for correspondence (e-mail: silvia.marino{at}usz.ch)
Accepted 16 April 2003
![]() |
SUMMARY |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: Cre-LoxP system, Cerebellar development, Rb, Engrailed-2, p107, Granule cell, Purkinje cell, Mouse
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
E2fs comprise a group of at least six closely related proteins that
regulate the expression of genes involved in cell cycle progression,
differentiation, development and apoptosis. Cellular and biochemical analyses
as well as studies of mutant mouse strains derived from gene targeting
indicate distinct in vivo functions of Rb, p107 and p130. Although Rb and the
upstream components of its pathway are mutated in many human cancers (reviewed
by Weinberg, 1995), mutations
in p107 have not been observed and mutations in p130 have been identified so
far only in small cell lung carcinomas
(Claudio et al., 2000
;
Helin et al., 1997
). However,
in mice only Rb loss has been directly associated with tumorigenesis, whereas
loss of the other pocket proteins contributed only to tumorigenesis in
combination with loss of Rb. In accordance with this notion, mice that lack
one Rb allele in a p107-/- background do not show enhanced
tumorigenesis.
During development, Rb is expressed from E9.5 onwards in both, mitotic and
non-mitotic compartments of the brain folds of the neural tube
(Jiang et al., 1997). In the
adult brain, Rb is ubiquitously expressed, including cerebellar granule and
Purkinje cells (Utomo et al.,
1999
). Rb knockout mice show embryonic lethality by E13-E15
(Clarke et al., 1992
;
Jacks et al., 1992
;
Lee et al., 1992
) owing to
major defects in hematopoiesis and central nervous system development. Major
findings in the Rb-deficient CNS are increased and ectopic proliferation, and
massive apoptosis, whereas Rb-deficient neurons in chimeric mice survive and
differentiate, although with an abnormally high proportion of cells arresting
in the G2 phase of the cell cycle
(Lipinski et al., 2001
). In
contrast to the latter findings, Rb-deficient neuronal precursor cells in
telencephalon-specific Rb mutants
(Ferguson et al., 2002
) showed
ectopic cell divisions but not widespread apoptosis. Although these neuronal
precursors differentiated, their fate in the adult CNS cannot be further
investigated due to early postnatal lethality of the mice.
In contrast to the detrimental effects of Rb inactivation, mice lacking
p107 or p130 develop normally and do not exhibit phenotypic
aberrations (Cobrinik et al.,
1996; Lee et al.,
1996
). Functional overlap within this gene family is suggested by
ossification defects in p107/p130 knockout mice and by retinal
dysplasia in Rb+/-; p107-/- mice.
We studied the roles of Rb and p107 in cerebellar development by
conditional inactivation of Rb either in all precursor cells of the cerebellar
vermis [line En2cre-22 (Zinyk et al.,
1998)] or selectively in Purkinje cells [L7-cre
(Marino et al., 2002
)] and
complemented the experiments by introducing p107 null alleles. We chose the
cerebellum to study the roles of Rb and p107 in development, differentiation
and cell death, as it consists of a limited number of distinct cell types, and
because its development and architectural organization are extremely well
documented. The use of a Cre transgenic lines with an expression limited to
the cerebellar vermis or to Purkinje cells allows to study even severe effects
on growth or cell loss without being lethal.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
PCR analysis of recombination
PCR analysis of Cre-mediated recombination on the cerebellar cell fractions
was performed on genomic DNA using Rb212, Rb18 and Rb19E primers, yielding a
283 bp product for the unrecombined RbLoxP allele and a
260 bp product for the recombined Rb19 allele. For details see Marino
et al. (Marino et al.,
2000
).
Analysis of proliferation and apoptosis
In order to examine the fraction of cells in the cerebellar EGL and IGL
that are actively proliferating, P15 littermates were intraperitoneally
injected with 50 mg/kg body weight with 5'-bromo-2'-deoxyuridine
(BrdU) and killed 4 hours after injection. BrdU was immunohistochemically
detected on sections of formalin fixed and paraffin wax-embedded brains (see
Histological analysis). Apoptotic cells were detected with the TUNEL assay kit
(Roche).
BrdU-positive nuclei and the total number of nuclei were counted in five
high power fields separately in EGL and IGL. We counted corresponding areas
located in the dorsal vermis (lobule VI and VII) where the phenotypic
abnormalities were most prominent owing to the transgenic expression pattern.
As the EGL of a wild-type mouse is thinner than the EGL of the mutant (and
vice versa for the IGL), we decided to determine the labeling index by
counting the number of positive cells and all the cells belonging to the EGL
or IGL in a certain visual field (counting grid). The same approach was used
for the TUNEL-positive nuclei. The statistical significance of the differences
was calculated with the Mann-Whitney (P<0.01) and Kruskal-Wallis
(-square above 20) tests and error bars in
Fig. 5 indicate one s.d.
|
Sections were paraffin wax embedded and processed as described below.
Histological analysis
Cytospins were thawed, dried at room temperature and fixed for 10 minutes
in 4% paraformaldehyde in PBS. Whole mouse brains were fixed for at least 12
hours in 4% buffered paraformaldehyde. Coronal or sagittal slices were
dehydrated through graded alcohols and embedded in paraffin wax. Sections of 4
µm nominal thickness were mounted on coated slides and routinely stained
with Haematoxylin and Eosin (H&E). Immunohistochemistry for Gfap
(polyclonal, 1:300, DAKO), calbindin-D 28K (monoclonal, 1:400 Sigma),
parvalbumin (polyclonal, 1:300 Swant, CH), NeuN (Neuna60 - Mouse Genome
Informatics) (monoclonal, 1:4000 Chemicon), p27 (polyclonal, 1:400 SantaCruz),
BrdU (monoclonal, 1:50 DAKO), TuJ-1 (Tubb3 - Mouse Genome Informatics)
(monoclonal 1:20, Abcam) and anti phosphorylated histone H3 (polyclonal,1:200
Upstate Biotechnology) was performed on selected sections. A microwave
pre-treatment with 0.01M citrate buffer (pH 6.0) was used for p27, NeuN,
anti-phosphorylated histone H3 and BrdU; Proteinase K digestion (20 µg/ml,
Roche for 20 minutes at 20°C) was used for calbindin-D 28K. Biotinylated
secondary antibodies (goat anti-rabbit and rabbit anti-mouse, DAKO) were used
at a dilution of 1:200. Visualization was achieved using
biotin/avidin-peroxidase (DAKO) and diaminobenzidine as a chromogen.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Both En2cre; RbLoxP/+ and En2cre; RbLoxP/LoxP mice were healthy and fertile and showed no indications of cerebellar malfunction. We examined the cerebella of these mice at time points pivotal for cerebellar development (P1, P8, P15) and at days P20, P35 and P180 (6 months, Table 1).
No macroscopic or histological differences between wild-type and En2cre; RbLoxP/LoxP mice were observed at postnatal days 1 and 8. Although at P15 the outer appearance of mutant brains was not much different from wild type cerebella (Fig. 1A,B), the thickness of the EGL remained greater in En2cre; RbLoxP/LoxP mice (four or five cells thick, Fig. 1F) when compared with those of wild-type littermates (one or two cells thick, Fig. 1E).
|
|
Lack of Rb in all cerebellar cell types located in the vermis of
En2cre; RbLoxP/LoxP mice
It could be argued that the survival of Purkinje cells was due to
incomplete Cre-mediated recombination and retained Rb expression. We therefore
crossed En2cre transgenic mice with the ROSA26 reporter mice
(Zinyk et al., 1998) in which
Cre-mediated recombination results in removal of a Stop cassette and
transcription of a ß-galactosidase gene. lacZ expression was
found in a region largely corresponding to the vermis
(Fig. 3A-C) and immunostaining
with calbindin and NeuN confirmed that lacZ colocalized in granule
neurons and Purkinje cells, respectively
(Fig. 3D,E). To exclude that a
subpopulation of Purkinje or granule neurons, undetected by
ß-galactosidase staining, remained unrecombined, we additionally
performed cell separation and PCR recombination analysis on cell
fractions.
|
Impaired terminal differentiation and migration of granule cell
precursors lacking Rb and p107
The pocket protein family member p107 is expressed from E10.5 in the
ventricular zone of the neuroepithelium, which contains proliferating neuronal
precursor cells (Jiang et al.,
1997). p107 is rapidly downregulated when cells undergo terminal
differentiation. By contrast, the third pocket protein family member, p130, is
expressed at low levels in precursor and mature neurons. In vitro studies have
shown that Rb-deficient neural precursor cells exhibit little change in p130
levels but show a marked increase in the amount of p107 protein during
neurogenesis (Callaghan et al.,
1999
; Lipinski and Jacks,
1999
), implying that p107 might substitute for Rb in facilitating
differentiation.
To assess a possible overlap between Rb and p107 function in the developing
cerebellum, we examined the effects of En2cre mediated Rb
inactivation in a p107-null background. We crossed En2cre;
RbLoxP/LoxP mice with p107-deficient mice
(Robanus-Maandag et al., 1998)
and obtained En2cre; RbLoxP/LoxP;
p107+/-, En2cre; RbLoxP/+;
p107-/- and En2cre; RbLoxP/LoxP;
p107 -/- compound mutant mice
(Table 1). Mice hemizygous or
nullizygous for p107 do not show CNS abnormalities
(Lee et al., 1996
;
Robanus-Maandag et al., 1998
).
In addition, loss of one Rb allele in the vermis of p107 hemizygous or
nullizgous mice (En2cre; RbLoxP/+;
p107-/-) did not result in any abnormality even at 26
weeks of age.
We then obtained mice hemizygous or nullizygous for p107 in which both Rb alleles were recombined in the vermis (En2cre; RbLoxP/LoxP; p107+/-, En2cre; RbLoxP/LoxP; p107 -/-, Table 1). Both compound mutants developed ataxia between P15 and P20. Macroscopic examination at P15 revealed a severe reduction of the vermis size, which was more evident if both p107 alleles were lost (Fig. 1C,D). In keeping with the severity of neurological signs and prominence of macroscopic findings, all histopathological abnormalities were more pronounced in En2cre; RbLoxP/LoxP mice that were nullizygous for p107 than in those hemizygous for p107 (Fig. 1G,H).
At P20, Rb and p107 double mutant mice showed highly disorganized cerebellar architecture of the median cerebellar region (Fig. 2). The EGL was still six or seven cells thick and the molecular layer appeared clearly reduced in size (Fig. 2K,P). EGL cells albeit negative for NeuN were clearly positive for the early neuronal marker TuJ1 (Fig. 2L,Q). Purkinje cells were terminally differentiated, as estimated by the observation of their dendritic tree reaching the pial surface in the immunostaining for calbindin (Fig. 2M,R). However, the dendritic arborization appeared shrunk, with stunted to misoriented dendrites (Fig. 2M,R) and the PC bodies appeared to be poorly aligned with substantial loss of laminar distribution. These abnormalities were at least partially due to the presence of the thick EGL and the impaired formation of an IGL. Occasionally, binucleated Purkinje neurons were observed. Focally, clusters of small, NeuN-negative but TuJ1-positive granule cells were detected in the rudimentary molecular layer and intermingled with Purkinje cells. GFAP immunostaining revealed dystrophic Bergmann glia cells and a marked astrogliosis in the IGL (Fig. 2O,T).
Two weeks later in postnatal development, at day 35, we found a complete loss of granule cells in the IGL, which now mainly contained reactive astrocytes. Surprisingly, Purkinje cells were still preserved, though there was a substantial degree of disarrangement, ballooning of the nucleus and fragmentation of chromatin.
We conclude that p107 can partially compensate for the lack of Rb in promoting differentiation and migration. However, p107 only partially restores normal cell cycle exit. In the absence of Rb and p107, no other pocket protein family member, i.e. p130 or a yet unknown member, takes over the function in granule cells. Instead, Purkinje cell and astrocyte development and differentiation does not appear to be crucially dependent on Rb and p107 function.
Rb and p107 are not required for Purkinje cell differentiation and
survival
Purkinje cells in En2cre; RbLoxP/Lox and
En2cre; RbLoxP/Lox p107 compound mutant mice
showed enlarged soma size, thickened processes and bizarre nuclear shape. In
addition, they were irregularly arranged, in particular upon loss of p107.
Loss of appropriate environment can cause abnormalities in neuronal morphology
and may even lead to cell death. To dissect whether the above effects were
intrinsic to Purkinje cells or rather a consequence of improper environment,
and whether the phenotype could be rescued in an environment expressing Rb and
p107, we ablated Rb either alone or in combination with p107 selectively in
Purkinje cells. To achieve this, we crossed L7cre transgenic mice
(Marino et al., 2002) with
RbLoxP and with p107-/- mice to assess
the role of Rb and p107 in Purkinje cell development and survival. Neither
deletion of Rb alone or in combination with p107 resulted in neurological
deficits until 6 weeks of age. Histological examination of three
L7cre; RbLoxP/Lox and three L7cre;
RbLoxP/Lox p107-/- at postnatal day 20
revealed regular cerebellar architecture and no apparent loss of Purkinje
cells when compared with age- and genetic background-matched controls.
However, as we did not count Purkinje cells, we cannot exclude a minor cell
loss. Purkinje cells were greatly enlarged, occasionally slightly displaced
towards the molecular layer and exhibited enlarged nuclei with abnormal
shapes, resembling hourglass or kidney
(Fig. 4D,E,G,H). These
experiments suggest that the Purkinje cell phenotype is largely
cell-autonomous and cannot be rescued by adjacent, Rb- and p107-expressing
cells. Additionally no delayed differentiation and migration and no abnormal
proliferation and apoptosis of granule cells were observed. Therefore,
Purkinje cell dysplasia in En2cre; RbLoxP/Lox;
p107-/- mice is a result of Rb and p107 loss of function,
rather than an effect of improper positioning or loss of surrounding granule
cells. Moreover the granule cells defects observed in En2cre;
RbLoxP/Lox; p107-/- mice are not secondary to
Purkinje cell abnormalities.
|
We then examined the status of the CDK inhibitor
p27Kip1, which is part of a cell-intrinsic timer that
arrests the cell cycle and initiates differentiation in various cell types
(Durand et al., 1998), among
them neural cells. p27 expression is inversely correlated to the proliferative
status of cells. At P15, wild-type IGL neurons and the inner portion of the
EGL showed strong nuclear p27 expression, while granule cell precursors in the
outer EGL of the molecular layer were p27 negative
(Fig. 5F). Instead, Rb depleted
EGL cells of both regions expressed almost no p27
(Fig. 5I) and most of the
granule cells that have left the EGL and have migrated towards the IGL were
negative, too. As p27 is expressed only in differentiating postmitotic neural
precursors, the findings here are in keeping with the observation that
Rb-deficient EGL precursors and IGL neurons are still actively and abnormally
cycling.
Although granule cells are mitotically active during postnatal cerebellar
development, Purkinje cells are among the first neurons to leave the
ventricular zone, become postmitotic and migrate to form a temporary
plate-structure during cerebellar development between E11 and E13. It has been
demonstrated that inactivation of Rb family members in Purkinje cells through
a cell-specific expression of a modified T-antigen
(Feddersen et al., 1995),
which was modified not to interact with p53, leads to ectopic Purkinje cell
proliferation and apoptosis. We therefore assessed whether Purkinje cells
lacking Rb or Rb and p107 showed abnormal proliferation and apoptosis.
Both, En2cre- and L7cre-mediated inactivation of Rb alone or in a p107-null background, resulted in terminal differentiation of Purkinje cells, as estimated by calbindin and parvalbumin expression (Fig. 2H,M,R; Fig. 4E,H), and in normal migration from the ventricular zone of the neuroepithelium and formation of a well-defined monolayer. From P15 onwards, a considerable number of Purkinje cells showed enlarged soma size, bizarre nuclei and occasional fragmentation of chromatin. After BrdU incorporation, single Purkinje cells lacking Rb or Rb/p107 displayed nuclear BrdU staining, whereas BrdU labeling was never observed in Purkinje cells of control littermates. These results were confirmed by staining for phosphorylated histone H3 (data not shown). However, while bizarre nuclei and even occasional binucleated Purkinje cells were observed, there were no unambiguous cell divisions found. Interestingly, a large fraction of Purkinje cells deficient for Rb or Rb/p107 showed a strong nuclear p27 accumulation, suggesting a mechanism counteracting cell division (Fig. 4F,I).
Cerebella of wild-type and Rb/p107 double mutant mice were indistinguishable in size, morphology and proliferation rate at P1, but reduction in size and architectural abnormalities became obvious at P15 (Fig. 1), indicating that the cell reduction takes place before complete development of the cerebellum. To assess granule cell death, we performed the TUNEL assay on cerebellar sections at P1, P8, P15 and P20. At P1 and P8, few apoptotic cells were seen in the still actively proliferating EGL and in the postmitotic IGL of wild-type mice. No difference in apoptotic rate was detected in double mutant cerebella mice at P1 but increased by P8. To quantify and compare the results, positive nuclei in EGL and IGL layers were separately counted and labeling indexes were calculated. A trend towards a slightly higher apoptotic index was observed at P8 but did not reach statistical significance. At P15, however, a dramatic increase of the apoptotic rate occurred in the vermis deficient for Rb or Rb and p107 (Fig. 5B,D,G,J; Fig. 6B). Most positive cells were found in the inner half of the EGL and in the IGL (Fig. 5J; Fig. 6B). Similar apoptotic rates were found up to P30 when the granule cell loss was almost complete. Although differentiation still occurred in granule cells lacking Rb, the majority of these cells failed to exit cell cycle and underwent apoptosis within in the inner region of the EGL or after migration towards the IGL. The additional lack of p107 accelerated and aggravated the process.
|
These results indicate that Purkinje neurons achieve terminal differentiation in the absence of Rb or Rb and p107. Like granule cells, Purkinje neurons incorporated BrdU and expressed markers that suggest cell cycle activity well beyond the time they should have become postmitotic, though this did not elicited programmed cell death, as was the case in granule cells.
Apoptosis of ectopically proliferating granule cells lacking Rb is
not p53 mediated
Cell death in Rb mutant embryos has been shown to be p53 dependent in the
developing central nervous system (Macleod
et al., 1996) and in the lens
(Liu and Zacksenhaus, 2000
;
Morgenbesser et al., 1994
). To
determine whether apoptosis of abnormally proliferating, terminally
differentiated granule neurons lacking Rb is dependent on p53, we generated
animals lacking Rb in cerebellar precursor cells and hemizygous for p107 in a
p53-/- background (En2cre; RbLoxP/LoxP;
p107+/-; p53-/-) and examined the
cerebella at postnatal day 15, a time point characterized by a brisk apoptosis
in En2cre; RbLoxP/LoxP; p107+/-. The percentage
of TUNEL-stained nuclei as well as the percentage of BrdU-labeled cells in the
EGL and IGL of compound mutant mice was similar to that in En2cre;
RbLoxP/LoxP; p107+/- mice
(Fig. 6). The structural and
morphological abnormalities observed in double mutants were comparable with
single mutant mice. Similar results were obtained when comparing proliferation
and apoptotic labeling indexes in postnatal day 15 cerebella of En2cre;
RbLoxP/LoxP and En2cre; RbLoxP/LoxP;
p53-/- mice (Fig.
6 and data not shown). We conclude that loss of p53 function does
not suffice to rescue Rb deficient neural precursors from apoptosis. This is
important in light of previous evidence showing that apoptosis is
p53-dependent in the CNS of developing Rb-/- embryos
(Macleod et al., 1996
). Our
results demonstrate the critical contribution of p53-independent pathways in
apoptosis occurring in the developing central nervous system after Rb
loss.
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
To study the role of Rb and p107 in cerebellar development, we inactivated Rb or Rb/p107 either in precursor cells of the vermis region or selectively in Purkinje cells. As our mouse models were not limited by postnatal death, we were able to dissect the role of Rb and p107 in development, differentiation and survival of all cerebellar cell populations.
We show that cerebella lacking one Rb allele and both
p107 alleles were indistinguishable from control littermates: cell
cycle proteins were upregulated in proliferating cells located in the outer
zone of the EGL, whereas cells in the inner zone of the EGL have stopped DNA
synthesis and started differentiation and migration towards the IGL as it is
the case in wild-type mice. This was paralleled by upregulation of cell cycle
inhibitors, such as p27 (Miyazawa et al.,
2000). By contrast, the lack of Rb resulted in several cell cycle
abnormalities in granule cell precursors, which were even more pronounced if
one or both p107 alleles were lost. The cell cycle marker p-histone
H3 was expressed throughout the entire EGL, which was reflected by an
increased BrdU incorporation rate, while the expression of the cell cycle
inhibitor p27 was reduced in these cells, suggesting that Rb and Rb/p107
deficient EGL cells cannot properly exit the cell cycle. The presence of IGL
neurons (as indicated by the continued expression of TuJ1) expressing
proliferation markers and incorporating BrdU can be explained by the inability
of Rb-deficient granule cell precursors to switch to a postmitotic state.
However, BrdU incorporation and expression of cell cycle markers in these
granule cells might as well represent an unscheduled re-entry into cell cycle
before death as described by (Herrup and
Busser, 1995
). Our results clearly show that loss of Rb function
is detrimental for controlled differentiation, cell cycle exit and survival of
cerebellar granule cell precursors, and that a single functional Rb
allele is sufficient to impose a completely normal phenotype to the cerebellum
of the mutant mice even in the complete absence of p107.
An essential involvement of Rb in the terminal differentiation process of
these precursors, as has been described for myoblasts
(Novitch et al., 1999) and
adipocytes (Classon et al.,
2000
), seems unlikely because differentiating granule cells
express mature neuronal markers (our data)
(Ferguson et al., 2002
). The
lack of expression of mature neuronal markers described by Lee et al. in
Rb-/- embryos might have been caused by secondary neural
precursor damage as a result of the severe defects in hematopoiesis
(Lee et al., 1992
). Callaghan
and co-workers have suggested that p107 might substitute for Rb function to
facilitate differentiation in vitro, as they detected enhanced levels of p107
but not of p130 during neurogenesis in studies with Rb-deficient neural
precursor cells (Callaghan et al.,
1999
). In agreement with these findings, we report here that p107
does show activities that overlap with those of Rb in promoting cell
differentiation and migration of granule cell precursors in compound mutant
mice. However, at physiological levels, p107 by itself is incapable of
commanding exit from the cell cycle. Purkinje cells and astrocytes instead are
terminally differentiating even in the absence of both Rb and p107.
Forced proliferation of terminally differentiated neurons by the absence of
Rb has been shown to induce apoptosis in embryos in vivo and in cultured cells
in vitro. Most likely, this is mediated by the de-repression of promoters by
the loss of bound Rb/E2f complexes and increased levels of free E2f, leading
to upregulation of E2f-dependent promoters. Among those are several key
regulators of apoptosis, such as APAF1, caspase 3 and caspase 7
(Muller et al., 2001). We show
here that the function of Rb in protecting cells from apoptosis in the CNS is
cell type specific. Although granule cells are forced into programmed cell
death, Purkinje cells and astrocytes do not undergo apoptosis. Purkinje cells
not only achieve terminal differentiation and properly migrate in the absence
of Rb, but a considerable number of them also survive complete loss of granule
cells. Interestingly, transgenic mice expressing, in Purkinje cells, a
modified T antigen (T147) that inactivated all members of the
pocket protein family but did not interact with p53 not only showed cell cycle
re-entry but also apoptosis of these neurons
(Feddersen et al., 1995
). As
we have specifically inactivated Rb and p107, but not p130, it might be
speculated that p130 can protect Purkinje cells from apoptosis, unless the T
antigen mutant exerts other functions that produce apoptosis.
How can the striking difference between granule neurons and Purkinje cells in response to lack of Rb and p107 be explained? First, Purkinje cells differentiate much earlier during development, while granule cells first migrate from the rhombic lip onto the surface of the cerebellar anlage and form the EGL, where they proliferate and migrate inwards. It is therefore possible that proliferative effects mediated by loss of Rb and p107 are counteracted by other signals during Purkinje cell development, but not during the phase in which EGL precursors should exit cell cycle.
Second, it is possible, that downregulation of p27 by Rb family members is
more effective in granule cell precursors than in differentiated Purkinje
cells. In contrast to wild-type Purkinje cells, which were only occasionally
clearly immunopositive for p27, most Rb- or Rb/p107-deficient Purkinje cells
accumulated nuclear p27. Instead, granule cell precursors in the EGL and IGL
neurons are negative or weakly positive for p27, in keeping with the
observation that Rb upregulates p27 in an osteosarcoma cell line
(Alexander and Hinds, 2001). It
is therefore conceivable that upregulation of p27 (and possibly other
functionally related cell cycle regulators) prevents Purkinje cells from
uncontrolled entry into the cell cycle, while EGL precursor cells continue
cycling and eventually undergo apoptosis. Absence of p27 results in delayed
maturation and prolonged proliferation of EGL precursors
(Miyazawa et al., 2000
). In
line with these findings, we observe that lack of Rb and p107 correlates with
reduction of p27 expression and results in a broadened EGL with increased and
prolonged EGL proliferation (Fig.
5I). However, considering the established pro-apoptotic role of
p27, suppression or lack of p27 alone cannot explain the propensity of EGL
neurons that Rb and p107 to undergo apoptosis.
Purkinje cells have been shown to provide trophic support for granule cells
and to stimulate the proliferation of EGL cells mainly through the sonic
hedgehog/patched/Gli1 signaling pathway
(Wallace, 1999). Indeed,
analysis of mice with cerebellar defects, among them the 'staggerer' mouse
(Herrup and Mullen, 1979a
;
Herrup and Mullen, 1979b
), the
lurcher mouse (Caddy and Herrup,
1991
) and the L7-ADT mouse
(Smeyne et al., 1995
), have
shown that the degeneration of Purkinje cells during embryonic or early
postnatal development leads to secondary loss of granule cells.
However, Purkinje cells differentiate and survive in mice lacking granule
cells such as the weaver mutant or Math1 (Atoh1 - Mouse
Genome Informatics) knockout mice (Ben-Arie
et al., 1997). In accordance with these observations, we show here
that even substantial loss of granule cells does not affect Purkinje cell
viability during postnatal cerebellar development. Moreover our experiments
clearly show that the dramatic granule cell loss observed in En2cre;
RbLoxP/LoxP; p107-/- is cell-autonomous.
Apoptosis occurring during lens
(Morgenbesser et al., 1994)
and early CNS development (Macleod et al.,
1996
) in Rb-/- embryos has been shown to be
p53 dependent (Pomerantz et al.,
1998
). However, programmed cell death occurring outside the CNS,
for example in the dorsal root ganglia of the Rb-/-
embryos, was shown to be p53 independent. Likewise, in our setting apoptosis
in the Rb- and Rb/p107-deficient cerebellum cannot be
rescued by inactivating p53. This finding demonstrates an important
contribution of p53-independent apoptosis pathways in the Rb deficient CNS as
it has been shown in other mouse models for granule cell death such as the
lurcher mouse (Doughty et al.,
2000
).
In summary, our results suggest that Rb and p107 are essential for granule cell development and differentiation, while Purkinje cell differentiation and survival does not rely on Rb/p107 function. Rb/p107-deficient granule cells undergo apoptosis by a p53-independent pathway and their loss during postnatal cerebellar development does not affect Purkinje cell survival.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Alexander, K. and Hinds, P. W. (2001).
Requirement for p27(KIP1) in retinoblastoma protein-mediated senescence.
Mol. Cell. Biol. 21,3616
-3631.
Ben-Arie, N., Bellen, H. J., Armstrong, D. L., McCall, A. E., Gordadze, P. R., Guo, Q., Matzuk, M. M. and Zoghbi, H. Y. (1997). Math1 is essential for genesis of cerebellar granule neurons. Nature 390,169 -172.[CrossRef][Medline]
Caddy, K. W. and Herrup, K. (1991). The fine structure of the Purkinje cell and its afferents in lurcher chimeric mice. J. Comp. Neurol. 305,421 -434.[Medline]
Callaghan, D. A., Dong, L., Callaghan, S. M., Hou, Y. X., Dagnino, L. and Slack, R. S. (1999). Neural precursor cells differentiating in the absence of Rb exhibit delayed terminal mitosis and deregulated E2F 1 and 3 activity. Dev. Biol. 207,257 -270.[CrossRef][Medline]
Clarke, A. R., Maandag, E. R., van Roon, M., van der Lugt, N. M., van der Valk, M., Hooper, M. L., Berns, A. and te Riele, H. (1992). Requirement for a functional Rb-1 gene in murine development. Nature 359,328 -330.[CrossRef][Medline]
Classon, M., Kennedy, B. K., Mulloy, R. and Harlow, E.
(2000). Opposing roles of pRB and p107 in adipocyte
differentiation. Proc. Natl. Acad. Sci. USA
97,10826
-10831.
Claudio, P. P., Howard, C. M., Pacilio, C., Cinti, C., Romano,
G., Minimo, C., Maraldi, N. M., Minna, J. D., Gelbert, L., Leoncini, L. et
al. (2000). Mutations in the retinoblastoma-related gene
RB2/p130 in lung tumors and suppression of tumor growth in vivo by
retrovirus-mediated gene transfer. Cancer Res.
60,372
-382.
Cobrinik, D., Lee, M. H., Hannon, G., Mulligan, G., Bronson, R. T., Dyson, N., Harlow, E., Beach, D., Weinberg, R. A. and Jacks, T. (1996). Shared role of the pRB-related p130 and p107 proteins in limb development. Genes Dev. 10,1633 -1644.[Abstract]
Doughty, M. L., de Jager, P. L., Korsmeyer, S. J. and Heintz,
N. (2000). Neurodegeneration in Lurcher mice occurs via
multiple cell death pathways. J. Neurosci.
20,3687
-3694.
Durand, B., Fero, M. L., Roberts, J. M. and Raff, M. C. (1998). p27Kip1 alters the response of cells to mitogen and is part of a cell-intrinsic timer that arrests the cell cycle and initiates differentiation. Curr. Biol. 8, 431-440.[Medline]
Feddersen, R. M., Clark, H. B., Yunis, W. S. and Orr, H. T. (1995). In vivo viability of postmitotic Purkinje neurons requires pRb family member function. Mol. Cell. Neurosci. 6,153 -167.[CrossRef][Medline]
Ferguson, K. L., Vanderluit, J. L., Hebert, J. M., McIntosh, W.
C., Tibbo, E., MacLaurin, J. G., Park, D. S., Wallace, V. A., Vooijs, M.,
McConnell, S. K. et al. (2002). Telencephalon-specific Rb
knockouts reveal enhanced neurogenesis, survival and abnormal cortical
development. EMBO J. 21,3337
-3346.
Harbour, J. W. and Dean, D. C. (2000). The
Rb/E2F pathway: expanding roles and emerging paradigms. Genes
Dev. 14,2393
-2409.
Hatten, M. E. (1985). Neuronal regulation of astroglial morphology and proliferation in vitro. J. Cell Biol. 100,384 -396.[Abstract]
Helin, K., Holm, K., Niebuhr, A., Eiberg, H., Tommerup, N.,
Hougaard, S., Poulsen, H. S., Spang-Thomsen, M. and Norgaard, P.
(1997). Loss of the retinoblastoma protein-related p130 protein
in small cell lung carcinoma. Proc. Natl. Acad. Sci.
USA 94,6933
-6938.
Herrup, K. and Busser, J. C. (1995). The
induction of multiple cell cycle events precedes target-related neuronal
death. Development 121,2385
-2395.
Herrup, K. and Mullen, R. J. (1979a). Regional variation and absence of large neurons in the cerebellum of the staggerer mouse. Brain Res. 172,1 -12.[CrossRef][Medline]
Herrup, K. and Mullen, R. J. (1979b). Staggerer chimeras: intrinsic nature of Purkinje cell defects and implications for normal cerebellar development. Brain Res. 178,443 -457.[CrossRef][Medline]
Jacks, T., Fazeli, A., Schmitt, E. M., Bronson, R. T., Goodell, M. A. and Weinberg, R. A. (1992). Effects of an Rb mutation in the mouse. Nature 359,295 -300.[CrossRef][Medline]
Jiang, Z., Zacksenhaus, E., Gallie, B. L. and Phillips, R. A. (1997). The retinoblastoma gene family is differentially expressed during embryogenesis. Oncogene 14,1789 -1797.[CrossRef][Medline]
Juan, G., Traganos, F., James, W. M., Ray, J. M., Roberge, M., Sauve, D. M., Anderson, H. and Darzynkiewicz, Z. (1998). Histone H3 phosphorylation and expression of cyclins A and B1 measured in individual cells during their progression through G2 and mitosis. Cytometry 32,71 -77.[CrossRef][Medline]
Lee, E. Y., Chang, C. Y., Hu, N., Wang, Y. C., Lai, C. C., Herrup, K., Lee, W. H. and Bradley, A. (1992). Mice deficient for Rb are nonviable and show defects in neurogenesis and haematopoiesis. Nature 359,288 -294.[CrossRef][Medline]
Lee, E. Y., Hu, N., Yuan, S. S., Cox, L. A., Bradley, A., Lee, W. H. and Herrup, K. (1994). Dual roles of the retinoblastoma protein in cell cycle regulation and neuron differentiation. Genes Dev. 8,2008 -2021.[Abstract]
Lee, M. H., Williams, B. O., Mulligan, G., Mukai, S., Bronson, R. T., Dyson, N., Harlow, E. and Jacks, T. (1996). Targeted disruption of p107: functional overlap between p107 and Rb. Genes Dev. 10,1621 -1632.[Abstract]
Lipinski, M. M. and Jacks, T. (1999). The retinoblastoma gene family in differentiation and development. Oncogene 18,7873 -7882.[CrossRef][Medline]
Lipinski, M. M., Macleod, K. F., Williams, B. O., Mullaney, T.
L., Crowley, D. and Jacks, T. (2001). Cell-autonomous and
non-cell-autonomous functions of the Rb tumor suppressor in developing central
nervous system. EMBO J.
20,3402
-3413.
Liu, Y. and Zacksenhaus, E. (2000). E2F1 mediates ectopic proliferation and stage-specific p53-dependent apoptosis but not aberrant differentiation in the ocular lens of Rb deficient fetuses. Oncogene 19,6065 -6073.[CrossRef][Medline]
Maandag, E. C., van der Valk, M., Vlaar, M., Feltkamp, C., O'Brien, J., van Roon, M., van der Lugt, N., Berns, A. and te Riele, H. (1994). Developmental rescue of an embryonic-lethal mutation in the retinoblastoma gene in chimeric mice. EMBO J. 13,4260 -4268.[Abstract]
Macleod, K. F., Hu, Y. and Jacks, T. (1996). Loss of Rb activates both p53-dependent and independent cell death pathways in the developing mouse nervous system. EMBO J. 15,6178 -6188.[Abstract]
Marino, S., Vooijs, M., van Der Gulden, H., Jonkers, J. and
Berns, A. (2000). Induction of medulloblastomas in p53-null
mutant mice by somatic inactivation of Rb in the external granular layer cells
of the cerebellum. Genes Dev.
14,994
-1004.
Marino, S., Krimpenfort, P., Leung, C., van der Korput, H. A., Trapman, J., Camenisch, I., Berns, A. and Brandner, S. (2002). PTEN is essential for cell migration but not for fate determination and tumourigenesis in the cerebellum. Development 129,3513 -3522.[Medline]
Miyazawa, K., Himi, T., Garcia, V., Yamagishi, H., Sato, S. and
Ishizaki, Y. (2000). A role for p27/Kip1 in the control of
cerebellar granule cell precursor proliferation. J.
Neurosci. 20,5756
-5763.
Morgenbesser, S. D., Williams, B. O., Jacks, T. and DePinho, R. A. (1994). p53-dependent apoptosis produced by Rb-deficiency in the developing mouse lens. Nature 371, 72-74.[CrossRef][Medline]
Muller, H., Bracken, A. P., Vernell, R., Moroni, M. C.,
Christians, F., Grassilli, E., Prosperini, E., Vigo, E., Oliner, J. D. and
Helin, K. (2001). E2Fs regulate the expression of genes
involved in differentiation, development, proliferation, and apoptosis.
Genes Dev. 15,267
-285.
Novitch, B. G., Spicer, D. B., Kim, P. S., Cheung, W. L. and Lassar, A. B. (1999). pRb is required for MEF2-dependent gene expression as well as cell-cycle arrest during skeletal muscle differentiation. Curr. Biol. 9, 449-459.[CrossRef][Medline]
Pomerantz, J., Schreiber-Agus, N., Liegeois, N. J., Silverman, A., Alland, L., Chin, L., Potes, J., Chen, K., Orlow, I., Lee, H. W. et al. (1998). The Ink4a tumor suppressor gene product, p19Arf, interacts with MDM2 and neutralizes MDM2's inhibition of p53. Cell 92,713 -723.[Medline]
Robanus-Maandag, E., Dekker, M., van der Valk, M., Carrozza, M.
L., Jeanny, J. C., Dannenberg, J. H., Berns, A. and te Riele, H.
(1998). p107 is a suppressor of retinoblastoma development in
pRb-deficient mice. Genes Dev.
12,1599
-1609.
Smeyne, R. J., Chu, T., Lewin, A., Bian, F., S-Crisman, S., Kunsch, C., Lira, S. A. and Oberdick, J. (1995). Local control of granule cell generation by cerebellar Purkinje cells. Mol. Cell. Neurosci. 6,230 -251.[CrossRef][Medline]
Utomo, A. R., Nikitin, A. Y. and Lee, W. H. (1999). Temporal, spatial, and cell type-specific control of Cre-mediated DNA recombination in transgenic mice. Nat. Biotechnol. 17,1091 -1096.[CrossRef][Medline]
Vooijs, M. and Berns, A. (1999). Developmental defects and tumor predisposition in Rb mutant mice. Oncogene 18,5293 -5303.[CrossRef][Medline]
Wallace, V. A. (1999). Purkinje-cell-derived Sonic hedgehog regulates granule neuron precursor cell proliferation in the developing mouse cerebellum. Curr. Biol. 9, 445-448.[CrossRef][Medline]
Weinberg, R. A. (1995). The retinoblastoma protein and cell cycle control. Cell 81,323 -330.[Medline]
Williams, B. O., Schmitt, E. M., Remington, L., Bronson, R. T., Albert, D. M., Weinberg, R. A. and Jacks, T. (1994). Extensive contribution of Rb-deficient cells to adult chimeric mice with limited histopathological consequences. EMBO J. 13,4251 -4259.[Abstract]
Zinyk, D. L., Mercer, E. H., Harris, E., Anderson, D. J. and Joyner, A. L. (1998). Fate mapping of the mouse midbrain-hindbrain constriction using a site-specific recombination system. Curr. Biol. 8,665 -668.[Medline]