1 Center for Developmental Biology and Kent Waldrep Foundation Center for Basic
Research on Nerve Growth and Regeneration, University of Texas Southwestern
Medical Center, Dallas, TX 75390-9133, USA
2 Department of Pathology, University of Texas Southwestern Medical Center,
Dallas, TX 75390-9133, USA
3 Department of Neurology, Washington University School of Medicine, St Louis,
MO 63110, USA
4 Division of Molecular Medicine and Genetics, Departments of Internal Medicine
and Cell and Developmental Biology, University of Michigan Medical School, Ann
Arbor, MI 48109, USA
* Author for correspondence (e-mail: yuanzhu{at}umich.edu)
Accepted 12 October 2005
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SUMMARY |
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Key words: Neurofibromatosis type 1, Optic glioma, Glial progenitor, Astrocyte, Tumor suppressor gene, Mouse
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Introduction |
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Individuals afflicted with a familial cancer syndrome, neurofibromatosis
type 1 (NF1), are predisposed to the development of astrocytomas
(Listernick et al., 1999).
Approximately 15 to 20% of children with NF1 develop pilocytic astrocytomas
predominantly within the optic pathway, hypothalamus and, occasionally, in the
other brain areas (Listernick et al.,
1999
; Listernick et al.,
1997
). Like their sporadic counterparts, most NF1-associated
pilocytic astrocytomas are benign and can remain static for many years.
However, despite histological benign features, a significant number of these
tumors will endure and cause vision impairment and other neurological symptoms
(Listernick et al., 1999
). The
NF1 gene encodes the protein product, neurofibromin, which shares
homology with members of the family of Ras GTPase activating proteins (GAPs)
(Ballester et al., 1990
;
Viskochil, 1999
;
Xu et al., 1990
). Like GAPs,
neurofibromin attenuates the Ras-mediated signaling pathway by accelerating
the conversion of activated Ras-GTP to inactive Ras-GDP. Consistent with the
role of the NF1 gene as a tumor suppressor gene, loss of
heterozygosity at the NF1 locus and loss of neurofibromin expression have been
observed in a variety of NF1-associated tumors, including astrocytomas
(Gutmann et al., 2000
;
Kluwe et al., 2001
). In one
case report, loss of neurofibromin expression in an NF1-associated pilocytic
astrocytoma correlated with the elevated Ras-GTP and activation of Ras
downstream effectors such as mitogen-activated protein kinase (MAPK) and
phosphatidylinositol 3-kinase (PI-3K) (Lau
et al., 2000
).
Functional activation of the Ras pathway through upregulation of receptor
tyrosine kinases such as PDGF and EGF receptors has been well documented in
diffusely infiltrative malignant astrocytomas
(Holland, 2001;
Kleihues and Cavenee, 2000
;
Maher et al., 2001
;
Zhu and Parada, 2002
).
However, similar molecular alterations have not been observed in pilocytic
astrocytomas (Gutmann et al.,
2002
; Li et al.,
2001
). It has been suggested that the NF1 gene might be
involved in regulating the proliferation of mature astrocytes
(Bajenaru et al., 2002
).
Although neurofibromin is expressed below detection levels in normal
astrocytes (Daston and Ratner,
1992
; Daston et al.,
1992
; Huynh et al.,
1994
), it was reported that loss of NF1 confers a growth
advantage to neonatal astrocytes in vitro
(Bajenaru et al., 2002
).
Conventional NF1 knockout mice (Nf1-/-) are embryonic
lethal (Brannan et al., 1994
;
Jacks et al., 1994
) and
although heterozygous mice (Nf1+/-) are cancer prone, they
do not develop astrocytomas (Jacks et al.,
1994
). Conditional mutant mice lacking NF1 specifically in neurons
also fail to develop astrocytomas although increased number of non-neoplastic
GFAP (glial fibrillary acidic protein) expressing reactive astrocytes was
observed (Zhu et al., 2001
).
These results suggest that NF1 can regulate the growth of astrocytes both
intrinsically and also indirectly through neurons.
During embryonic development, multipotent neural stem/progenitor cells
progressively lose developmental potential and become lineage-restricted
neuronal progenitor cells or glial progenitor cells
(Gage, 2000;
Temple, 2001
). Gliogenesis
occurs after neurogenesis and extends into postnatal stages. In the setting of
NF1, the greatest risk for development of optic glioma is the first 6 years of
life (Listernick et al.,
1999
). This observation suggests that the NF1 gene might
play a role in regulating the proliferation of progenitor cells. Furthermore,
recent reports demonstrate that pilocytic astrocytomas express molecular
markers reminiscent of glial progenitor cells
(Gutmann et al., 2002
;
Li et al., 2001
).
To determine the role of NF1 in the development of neural cell types and
understand cellular and molecular basis of NF1-associated astrocytoma
formation, we used a bacteriophage Cre/loxP system to target a Nf1
mutation (Zhu et al., 2001)
into multipotent neural stem/progenitor cells and their derivatives, including
glia and neurons (Zhuo et al.,
2001
). We show that loss of NF1 promotes the proliferation of
glial progenitor cells resulting in increased numbers of GFAP-expressing
astrocytes in both developing and adult brains. Furthermore, NF1 also plays an
indispensable role in the maintenance of the differentiation state of mature
astrocytes. Finally, we describe a new mouse model for NF1-associated optic
pathway glioma.
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Materials and methods |
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Size and weight analysis of mice
Nf1hGFAPKO and control littermates at the age of P0.5,
P8 and 2 months were used to determine mass (g). Age-matched mutant and
control mice were perfused with 4% paraformaldehyde (PFA). Brains were
dissected and post-fixed in 4% PFA overnight, and separated into forebrain and
hindbrain for analysis. Statistical analysis was carried out using Student's
t-test. P<0.05 was considered to be significant.
lacZ staining and double immunofluorescence
E10.5, E12.5 embryos or cryostat sections from postnatal or adult tissues
were prepared and subjected to X-gal analysis as described previously
(Zhu et al., 1998). Adjacent
sections were subjected to double immunofluorescence with anti-lacZ
(rabbit, 1:200, 5' and 3') and anti-NeuN (mouse, 1:200, Chemicon)
or anti-lacZ and anti-GFAP (mouse, 1:100, Pharmingen).
BrdU assay
P8 mutant and control littermates were pulsed with BrdU for 2 hours, and
adult mice (4-6 months) were pulsed with BrdU five times a day at 2-hour
intervals. The dose of BrdU was 50 µg/g (gram, body mass). Mice were
perfused with 4% PFA 2 hours after the last pulse. Brains were dissected and
processed for either paraffin-embedded or cryostat sections. BrdU
immunohistochemistry was performed as described previously
(Zhu et al., 1998). The
dilution of BrdU antibody was 1:50 (Becton Dickinson). The number of
BrdU-positive cells was counted in one out of each ten serially prepared
sections. Statistical analysis was carried out using Student's
t-test. P<0.05 was considered to be significant.
Immunohistochemistry
After post-fixed in 4% PFA overnight, tissues were prepared for
free-floating vibratome sections at 50 µm, cryostat sections at 14 µm or
paraffin wax-embedded sections at 5 µm. Paraffin sections were
deparaffinized and rehydrated. Sections were subjected to immunohistochemical
analysis as described previously (Zhu et
al., 2001). The visualization of primary antibodies was performed
with either a horseradish peroxidase system (Vectastain ABC kit, Vector) or
immunofluorescence by using Cy3-conjugated anti-rabbit/mouse and
Cy2-conjugated anti-mouse/rabbit secondary antibodies at 1:200 dilution
(Jackson Laboratories). The dilution of primary antibodies used in this study
were: GFAP (rabbit, 1:2000, DAKO), nestin (mouse, 1:200, Chemicon), BLBP
(rabbit, 1:1000, a gift from N. Heintz), PAX2 (rabbit, 1:1000, a gift from G.
Dressler), P-erk (rabbit, 1:200, Cell Signaling), Cre (mouse, 1:1000, BABCO),
Ki-67 (Rabbit, 1:1000, Novocastra Labs). Sections were examined under either
light or fluorescence microscope (Olympus). The co-localization of two
antigens was further confirmed by confocal microscopy (Zeiss).
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Results |
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In contrast to the diminished body weight, the Nf1hGFAPKO mutant brain mass was slightly larger than that of control brains, although this did not reach statistical significance (0.54±0.01 versus 0.52±0.02, P=0.43), except in the forebrain where the size of mutant tissues was significantly larger than that of controls (see Fig. S1C in the supplementary material, 1.08±0.02 versus 0.94±0.02, P=0.0003).
|
Increased proliferating glial progenitor cells during development
To examine the status of glial progenitors in
Nf1hGFAPKO mutant brains, we selected postnatal day 8 (P8)
for analysis. At P8, neurogenesis is largely complete but glial development
and proliferation is still active (Bayer
and Altman, 1991; Jacobson,
1991
; Qian et al.,
2000
). P8 also coincides with the first detectable morphological
differences between mutant and wild-type pups (see Fig. S1A in the
supplementary material). The results of BrdU incorporation and
immunohistochemical studies indicated that during development, mutant brains
contain excess proliferating cells (Fig.
1B,C) that express a neural stem/progenitor cell marker, nestin
(Fig. 2A,B), and an early glial
progenitor cell marker, brain lipid binding protein (BLBP)
(Fig. 2C,D) (Feng et al., 1994
;
Kurtz et al., 1994
).
Consistent with this, the majority of proliferating cells in both control
(Fig. 2E,G) and mutant brains
at P8 (Fig. 2F,H) do not
express mature astrocyte markers, such as GFAP. We observed no difference in
apoptosis between mutant and control brains that could contribute to the
increased numbers of proliferating glial progenitors at this stage (data not
shown).
To determine whether glial progenitor cells in P8 brains express the Cre
transgene and hence are NF1 deficient, we performed immunohistochemical
analysis. In both P8 control and mutant brains, most GFAP-positive astrocytes
express the Cre transgene (Fig.
3; Fig. 4A), which
is consistent with the previous observations in the adult brain
(Malatesta et al., 2003;
Zhu et al., 2005
;
Zhuo et al., 2001
). However,
not all of the Cre-positive cells expressed GFAP
(Fig. 3A, parts a-d; 3B, parts
a-d). Although these Cre-positive/GFAP-negative cells co-exist with
Cre-positive/GFAP-positive astrocytes throughout the brain, they represent the
major cell type in the thalamus (Fig.
3A) and in the periphery of the cerebellar white matter
(Fig. 3B), where we determined
that most of these GFAP-negative cells express both Cre and BLBP
(Fig. 3A, parts e,f; 3B, parts
e,f). These data indicate that the Cre transgene is expressed in the
BLBP-positive glial progenitor cells in both P8 control and mutant brains.
Thus, we conclude that NF1 deficiency as a consequence of Cre-mediated
recombination leads to increased numbers of proliferating glial progenitor
cells in P8 brains.
Nf1-/- glial progenitor cells differentiate
We also determined an increased number of GFAP-positive astrocytes in P8
mutant brains (Fig. 2F,H;
Fig. 3A, part b;
Fig. 3B, part b;
Fig. 4A, parts b,d) as compared
with controls (Fig. 2E,G;
Fig. 3A, part a;
Fig. 3B, part a;
Fig. 4A, parts a,c). To verify
that the GFAP-expressing astrocytes in mutant brains are NF1 deficient, we
used a Cre antibody to label Nf1-/- cells. As shown in
Fig. 4A, most of the
GFAP-expressing cells in Nf1hGFAPKO brains, including the
cerebral cortex (Fig. 4A, part
b) and the hippocampal dentate gyrus (DG)
(Fig. 4A, parts d,f),
co-express Cre and exhibit similar morphology to those astrocytes in control
cortex (Fig. 4A, part a) and
dentate gyrus (Fig. 4A, parts
c,e). Furthermore, similar to normal counterparts
(Fig. 4B, parts a,c), mutant
astrocytes express GFAP but not nestin in most areas of P8 brains
(Fig. 4B, parts b,d). A small
number of cells expressing both GFAP and nestin were observed in both P8
control and mutant dentate gyrus (arrows in
Fig. 4B, parts e,f) and the
subventricular zone, which are probably neural stem/progenitor cells that
persist into adulthood (Alvarez-Buylla et
al., 2001). Thus, loss of NF1 promotes the proliferation of glial
progenitor cells that retain the capacity to differentiate into
GFAP-expressing astrocytes. We conclude that the tumor suppressor gene, NF1,
is a negative regulator of the proliferation for glial progenitor cells but
not for mature astrocytes.
|
|
Tumorigenic potential: a model for optic glioma
Approximately 15-20% of children with NF1 develop benign gliomas along the
optic pathway with characteristics of pilocytic astrocytoma
(Listernick et al., 1999).
Using the Rosa26-lacZ reporter mouse strain
(Soriano, 1999
), we identified
that hGFAP-cre-mediated recombination occurred in the optic nerve and
the retina (Fig. 6A,B). The
density of lacZ-positive cells appeared highest in the area of the
optic nerve immediately adjacent to the retina
(Fig. 6A-D). Double labeling
with antibodies against lacZ and GFAP revealed that the majority of
lacZ-positive cells in the optic nerve were GFAP-expressing
astrocytes (Fig. 6C,D).
Furthermore, hGFAP-cre-mediated recombination in the optic nerve was
confirmed by PCR analysis (Fig.
6E) (Zhu et al.,
2002
; Zhu et al.,
2005
). We next examined the optic nerves from
Nf1hGFAPKO mutant mice along with control littermates. Of
optic nerves from twenty nine mutant mice analyzed, 18 were significantly
enlarged in diameter (Fig.
6F-H; Fig. 7A,B) and reminiscent of similarly prepared optic nerves from individuals with NF1
with optic gliomas (Kleihues and Cavenee,
2000
). Histological analysis revealed evidence of disorganization
and increased cellularity throughout the optic nerves from all of the 29
mutant mice (Fig. 7C,D). The
dysplastic nature of the cells in the mutant optic nerves is illustrated by
double labeling with nestin and GFAP (Fig.
7E-H). We found that mature optic nerve astrocytes retain low
levels of nestin expression (Frisen et
al., 1995
). Furthermore, consistent with the pattern of
hGFAP-cre-mediated recombination in the optic pathway, the most
pronounced changes were found in the area immediately adjacent to the retina
(Fig. 7I,J), which contained
dense clusters of randomly oriented glial nuclei. Detailed pathological
analysis revealed that six out of 29 mutant mice (6/29) developed conspicuous
neoplastic lesions (Fig. 8A-C).
In contrast to normal nerves, which are comprised of well-organized astrocytes
expressing both nestin and GFAP (Fig.
8D), neoplastic cells are completely disorganized and downregulate
nestin expression (Fig. 8E,F).
Although the optic nerve lesions observed in this tumor model lack some common
features of pilocytic astrocytomas, such as Rosenthal fibers and granular
bodies, these lesions display pathological features similar to human tumors,
which include the location in the anterior optic nerve, coarse fibrillary
appearance and nuclear pleiomorphism with clustered atypical tumor nuclei
(also see Fig. 9K,M).
|
We confirmed that the pathology observed in the optic nerves results from loss of NF1 by immunohistochemical analysis with a Cre antibody (data not shown). Consistently, mutant glial cells have high MAPK activity (Fig. 10A-D) with a subset of mutant glial cells showing activated MAPK but lacking GFAP. These results suggest that both glial progenitor cells (Fig. 10F, arrowheads) and astrocytes (Fig. 10F, arrows) in mutant optic nerves had activated MAPK. These observations suggest that activation of Ras/MAPK pathway as a consequence of NF1 inactivation may underlie hyperplasia and glioma formation in the mutant optic nerve.
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Discussion |
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NF1 and proliferation of glial progenitor cells
Consistent with the role of NF1 as a tumor suppressor gene, recent studies
have demonstrated that loss of NF1 function confers a growth advantage to a
variety of cell types in vitro, including neural stem/progenitor cells in the
embryonic forebrain (Dasgupta and Gutmann,
2005), oligodendrocyte precursor cells from the embryonic spinal
cord (Bennett et al., 2003
),
Schwann cells (Kim et al.,
1997
) and fibroblasts
(Rosenbaum et al., 1995
), etc.
However, because of early embryonic lethality of NF1 homozygous mutants,
insights into the function of NF1 during late developmental stages and
adulthood remain limited (Bajenaru et al.,
2003
; Bajenaru et al.,
2002
; Zhu et al.,
2002
; Zhu et al.,
2001
). For example, despite the fact that several lines of
evidence point to the Nf1 gene as a negative regulator of astrocyte
proliferation (Bajenaru et al.,
2002
), the in vivo role of the Nf1 gene in mature
astrocytes has not been fully addressed. Our studies indicate that
NF1-deficient mature astrocytes in the adult brain do not show a significant
increase in proliferation. Instead, we show significantly increased
proliferation during postnatal CNS development. These proliferating cells
express progenitor cell markers found in both multipotent neural
stem/progenitor cells and glial progenitor cells
(Anthony et al., 2004
;
Feng et al., 1994
;
Frisen et al., 1995
;
Kurtz et al., 1994
;
Lendahl et al., 1990
). We
conclude that these proliferative cells most probably represent glial
progenitor cells, as neurogenesis is largely complete and gliogenesis persists
in the P8 brain (Bayer and Altman,
1991
; Jacobson,
1991
; Qian et al.,
2000
). Thus, our studies demonstrate that the Nf1 gene is
a negative regulator of proliferation in glial progenitor cells and provide
evidence that the NF1-associated pathology observed in the adult brain could
result from developmental defects.
NF1 in differentiation of the astrocyte lineage
Increased numbers of NF1-deficient, GFAP-positive mature astrocytes were
observed in both developing and adult Nf1hGFAPKO mutant
brains. Because hGFAP-cre is expressed in a majority of astrocyte
precursors, radial glia in the embryonic brain
(Malatesta et al., 2003) and
glial progenitor cells in the postnatal brain (this study), it is reasonable
to assume that the majority of mutant astrocytes arise from NF1 deficient
progenitor cells. Thus, in vivo, NF1 is dispensable for astrocytic
differentiation. As no increased proliferation or reduced apoptosis was
observed in NF1-deficient mature astrocytes in both P8 and adult brains, the
increased numbers of GFAP-positive astrocytes in adult mutant brains probably
result from excess generation of glial progenitor cells, as observed during
development. In brains of a subset of aged Nf1hGFAPKO
mutant mice, we identified an unusual population of cells that express both
nestin and GFAP. These nestin/GFAP-positive cells morphologically resemble
reactive astrocytes that are hypertrophic with thickened and increased
processes (Ridet et al.,
1997
), which are observed in both human and mouse NF1-deficient
brains (Gutmann et al., 1999
;
Nordlund et al., 1995
;
Rizvi et al., 1999
;
Zhu et al., 2001
). As these
nestin/GFAP-positive cells were not observed in the brains of developing (P8)
or young adult (P30) mutant mice, these observations suggest that NF1 may be
required for the maintenance of the astrocytic differentiation state or
possibly preventing mature astrocytes from undergoing abnormal
differentiation. Previously, we have reported that neuronal loss of NF1
results in activation of MAPK in neurons but not in astrocytes, leading to
reactive astrogliosis in a non-cell autonomous fashion
(Zhu et al., 2001
). Despite
exhibiting morphological similarities, the reactive astrocytes observed in
neuronal-specific NF1 (Nf1SynIKO) mutant brains, do not
express nestin (Zhu et al.,
2001
). Therefore, the aberrant expression of this CNS progenitor
cell marker (nestin) could result in part from intrinsic loss of NF1 within
astrocytes or its progenitor cells. This is consistent with the observation
that astrocytes in Nf1hGFAPKO brains have elevated levels
of activated MAPK, while reactive astrocytes in Nf1SynIKO
brains do not (Zhu et al.,
2001
).
|
A model for optic pathway glioma
Histologically, pilocytic astrocytomas may contain a surprisingly wide
range of patterns (Kleihues and Cavenee,
2000). In addition to some of the more common features, including
recognizable bipolar cytoplasmic processes, brightly eosinophilic Rosenthal
fibers and hyaline granular bodies, pilocytic astrocytomas may also contain
areas of substantial nuclear pleomorphism, oligodendroglioma-like regions,
areas of infiltrative growth and cells similar to those of diffuse WHO grade
II astrocytoma. In the case of lesions lacking Rosenthal fibers and hyaline
granular bodies, the distinction between diffuse and pilocytic astrocytomas
may be extremely difficult (Kleihues and
Cavenee, 2000
). Thus, within the accepted variability of pilocytic
astrocytomas, the lesions found in the mutant mice are consistent with early
stage optic pathway gliomas. This phenotype is further consistent with the
fact that individuals with NF1 develop astrocytic neoplasms predominantly
along the optic pathway (Listernick et
al., 1999
).
While this manuscript was in preparation, a mouse model for NF1-associated
optic pathway glioma was published using the conditional NF1 flox mouse strain
described here together with the GFAP-cre* transgene (see Fig. S3
in the supplementary material) (Bajenaru et
al., 2003). The pathology described by Bajenaru et al. is less
severe, reminiscent of that described as hyperplasia in this study
(Fig. 7). In addition, Bajenaru
et al.'s study describes hyperplastic optic nerves only in the
Nf1flox/- genetic configuration and not in the
Nf1flox/flox configuration. Our
Nf1hGFAPKO mice exhibit fully penetrant hyperplasia in
either the flox/- or the flox/flox configurations and, additionally, 20%
incidence of optic pathway gliomas. Possible explanations for the discrepancy
between these two studies include the timing of Cre transgene activation
(E10.5 for hGFAP-cre versus E14.5 for GFAP-cre*),
contributions of NF1 deficient neighboring cells in the tumor
micro-environment and differences in genetic background. In line with this
study, it is tempting to speculate that the timing of NF1 inactivation in the
glial cell lineage during optic nerve development may account in part for the
heterogeneous nature of the NF1-associated optic gliomas
(Listernick et al., 1999
;
Rubin and Gutmann, 2005
).
Thus, our model may mimic a subset of individuals with NF1 who have severe
tumor phenotypes as a consequence of loss of NF1 in a progenitor cell
population, while the mouse strain developed by Bajenaru et al. may model less
aggressive lesions owing to NF1 inactivation in more differentiated cells.
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ACKNOWLEDGMENTS |
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