1 Department of Pediatric Oncology, Dana-Farber Cancer Institute and Harvard
Medical School, Boston, MA 02115, USA
2 Department of Molecular Biology, Princeton University, Princeton, NJ 08544,
USA
3 Division of Newborn Medicine, Children's Hospital, Boston MA 02115, USA
* Author for correspondence (e-mail: david_rowitch{at}dfci.harvard.edu)
Accepted 2 October 2002
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SUMMARY |
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Key words: Cerebellum, Sonic hedgehog, Proliferation, Cyclopamine, Medulloblastoma, Nmyc, Neural precursor, TRRAP, Mouse
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INTRODUCTION |
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Cerebellar granule neuron precursors (CGNPs) are generated in the rostral
hindbrain during late embryonic development. They then migrate dorsally, where
a second phase of postnatal proliferation takes place in the external granule
layer of the cerebellum (EGL) (Altman and
Bayer, 1997). Proliferating CGNPs express Math1, a bHLH
transcription factor required for development
(Ben-Arie et al., 1997
;
Helms and Johnson, 1998
).
Mature granule cells express other markers, including NeuN and the zinc finger
transcription factor Zic (Aruga et al.,
1994
; Wechsler-Reya and Scott,
1999
). Postmitotic granule precursors migrate to their final
destination in the internal granule layer (IGL), where they undergo terminal
differentiation (Altman and Bayer,
1997
).
Shh pathway activation is implicated in the etiology of cerebellar tumors
in humans as well as other types of cancer, and patched (PTCH), an
inhibitory component of hedgehog signaling, has been identified as a tumor
suppressor (Ruiz i Altaba,
1999). Individuals with Gorlin's syndrome, resulting from
mutations of PTCH, have high rates of basal cell carcinoma and
medulloblastoma (Hahn et al.,
1996
). Medulloblastoma, which is thought to derive from cerebellar
granule cell precursors, is one of the most common solid tumors in children
(Provias and Becker, 1996
).
Mutations of PTCH have also been found in 10-20% of sporadic
medulloblastomas (Pietsch et al.,
1997
; Xie et al.,
1997
) and mice heterozygous for targeted mutations of
Ptch develop cerebellar tumors
(Goodrich et al., 1997
).
However, the intracellular mechanisms that underlie Shh effects on cell cycle
progression during cerebellar development and tumorigenesis are poorly
understood.
Shh is thought to bind to a receptor complex composed of the transmembrane
proteins Ptch and smoothened (Smo). This in turn relieves Ptch-mediated
inhibition of Smo activity (Ingham and
McMahon, 2001). Smo, a member of the serpentine G-protein-coupled
receptor family (van den Heuvel and
Ingham, 1996
), is thought to activate an inhibitory G protein
(DeCamp et al., 2000
).
Consequences of Shh signaling, in vivo and in vitro, can be inhibited
experimentally by increasing cAMP levels or protein kinase A activity
(Dahmane and Ruiz i Altaba,
1999
; Fan et al.,
1995
; Hammerschmidt et al.,
1996
; Kenney and Rowitch,
2000
; Wallace,
1999
; Wechsler-Reya and Scott,
1999
), or by treatment with the alkaloid, cyclopamine and related
compounds (Berman et al., 2002
;
Dahmane et al., 2001
;
Incardona et al., 1998
).
Smo signaling in Drosophila leads to post-translational activation
of Cubitus interruptus protein, a member of the Gli family of zinc-finger
transcription factors (Ruiz i Altaba,
1999). In vertebrates, Gli family members appear to be general
targets of hedgehog signaling. Gli1 behaves as a transcriptional activator,
whereas Gli3 functions as a repressor of hedgehog signaling
(Dai et al., 1999
;
Sasaki et al., 1999
). Although
Gli2 appears to have distinct activator and repressor properties
(Ruiz i Altaba, 1999
;
Sasaki et al., 1999
),
Gli1 can fully compensate for Gli2 function during CNS
development (Bai and Joyner,
2001
). Gli proteins are implicated in regulation of neural
proliferation (Ruiz i Altaba,
1999
), and GLI1 was originally identified as a locus
occasionally amplified in human gliomas
(Kinzler et al., 1987
).
However, cerebellar proliferation is apparently normal in Gli1
knockout mice (Park et al.,
2000
), indicating that additional factors must mediate
proliferative effects of Shh.
Identification of Shh signaling intermediates that function in cell cycle
regulation is key to understanding the role of this pathway in CNS
proliferation during development and tumorigenesis. Shh treatment of CGNPs
results in rapid upregulation of D-type cyclin genes, via a mechanism
requiring protein synthesis (Kenney and
Rowitch, 2000). Many mitogens signal to the cell cycle machinery
by inducing expression of classical immediate-early genes (IEGs), such as
members of the Fos, Jun and Myc families. Mitogen induction of IEGs occurs as
a direct result of signaling pathway activation and does not require new
protein synthesis (Sheng and Greenberg,
1990
).
We report that Shh signaling in CGNPs induces the proto-oncogene Nmyc in a protein synthesis-independent manner. Of the Myc family members tested, only Nmyc was expressed in the proliferative zone of the cerebellum, suggesting a potential role for Nmyc in CGNP proliferation in vivo. Nmyc upregulation also occurred in proliferating neural precursor cells exposed to ectopic Shh in transgenic mice and in medulloblastomas of Ptch heterozygotes. Nmyc overexpression in vitro promoted CGNP proliferation and cyclin D1 protein upregulation in the presence of the Shh signaling inhibitor cyclopamine. Finally, antagonism of Nmyc activity significantly decreased CGNP proliferation induced by Shh. Together, our data provide compelling evidence that Nmyc functions downstream of Shh to promote cell cycle progression in cerebellar neuronal precursor cells during development and tumorigenesis.
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MATERIALS AND METHODS |
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Cells destined for retroviral infection were prepared as above, except that Shh (1.5 µg/ml) was included during the adherence period. For 24 hours before infection, CGNPs were incubated in serum-free medium containing Shh (3 µg/ml) or Shh vehicle (indicated in figure legends). For infection, conditioned medium was removed and saved, and cells were exposed to freshly thawed retroviral supernatants for 2-3 hours, with intermittent rocking. Supernatants were removed and conditioned medium, with treatments added as indicated, was replaced. Alternatively, fresh medium was used with new Shh. Control cultures were treated with non-infectious, conditioned packaging cell medium, or UV-treated retroviral supernatants during the infection period. During analyses, no differences were observed between results obtained from cells cultured under these conditions. Cyclopamine, provided by William Gaffield (USDA, Albany, CA), was used at 1 µg/ml, as recommended.
Sample sizes and quantitation
All proliferation assessment experiments were repeated using pups from at
least two separate litters. For immunochemistry experiments, two coverslips
per treatment/infection were used, for each litter. Five fields of 100 cells
per coverslip were analyzed by an unbiased observer, using DAPI staining to
select homogeneously distributed cell fields. Thus, micrograph data shown are
representative of a total n=20, 100-cell fields. Data are portrayed
as fold change from Shh-treated GFP-infected cultures. GFP infected cultures
did not differ from uninfected Shh-treated cultures in levels of proliferation
(not shown). For flow cytometry (see below), experiments were performed using
pups from three separate litters. With each separate litter, two wells of a
six-well plate were used for each treatment/infection. Data represented in
graphs are from n=6 experiments. Error bars represent standard error
of the mean. Significance (P>0.01 in comparison with Shh-treated
uninfected/GFP-infected CGNPs) was determined using the two-tailed
t-test (Excel software). Sub-G1-DNA quantitation was
performed on two samples (two wells at 36 hours post-infection) for each
experimental treatment. For northern blot and immunoblot analysis, two wells
per treatment were pooled to maximize recovery of RNA and protein. Experiments
for those analyses were repeated with three separate litters.
Retroviral constructs and production
Mouse Nmyc cassette (Wood et
al., 2000), and
MB2 cassette were prepared using
standard methods for site-directed mutagenesis. Mxi-SR was a kind
gift of Ron Depinho (Dana-Farber Cancer Institute) and eGFP was
purchased from Clontech. All cassettes were cloned into the pWZL retroviral
vector (Jay P. Morganstern, Millenium Pharmaceuticals, Cambridge, MA). pWZL
IRES-GFP vector was provided by Steve Lessnick (Dana-Farber Cancer
Institute). 293 EBNA (Invitrogen) packaging cells were co-transfected with
retroviral constructs, gagpol, and vesicular stomatitis virus G glycoprotein
plasmids, using Fugene 6 transfection reagent (Roche). Packaging cells were
re-fed 12 hours after transfection. Retroviral supernatants (4 ml) were
harvested every 12 hours for 72 hours and kept at 4°C until they were
pooled, filtered through 0.45 µm syringe filters, aliquoted and stored at
-80°C until use.
Flow cytometry for cell cycle analysis
Flow cytometric analysis of propidium iodide (PI) staining was used to
determine levels of proliferation and cell death as described
(Darzynkiewicz et al., 1997;
Kenney and Rowitch, 2000
),
using a FACScan (Becton Dickinson) and Cellquest software (Becton Dickinson)
for data acquisition. Modfit software (Verity Software House) was used for
quantifying sub-G1 DNA levels.
Immunocytochemical labeling and analysis
For S-phase assessment, CGNPs plated on coverslips were pulsed with 25
µg/ml 8-bromo-deoxyuridine (BrdU) for 2 hours prior to fixation in 4%
paraformaldehyde. Cell were washed in PBS and treated for 2 minutes with 2N
HCl. Cells were then processed for immunocytochemistry using standard methods.
Primary antibodies included mouse anti-BrdU (Becton-Dickinson), rabbit
anti-GFP (Seedorf et al.,
1999), mouse anti-GFAP (Sigma) and mouse-anti-NeuN (Chemicon).
Fluorochrome-conjugated secondary antibodies were Cy-3 anti-rabbit, Cy-2
anti-mouse or FITC-conjugated anti-mouse (Jackson Immunoresearch
Laboratories). Cells were co-stained with DAPI to label nuclei. Staining was
visualized with a Nikon Eclipse E600 microscope. Images were captured using a
SPOT 1 digital camera (Diagnostic Instruments) and processed using Adobe
Photoshop 5.0 software.
RNA preparation and northern blot analysis
Total RNA was prepared by CsCl gradient centrifugation and ethanol
precipitation. Ten µg of each sample was electrophoresed through a 1%
agarose formaldehyde gel and transferred to a Hybond-N+ membrane
(Amersham-Pharmacia). 32P-labeled probes used for northern blotting
were Gli1 (Hui et al., 1994),
Jun (Tom Curran, St. Jude Children's Research Hospital, Memphis TN), Fos
(Charles Stiles, DFCI, Boston MA), Mxi, Myc, Nmyc and Lmyc (all provided by
Ron DePinho, DFCI, Boston, MA). Math1 cDNA was a gift of Jane Johnson
(University of Texas Southwestern Medical Center, Dallas TX). Mouse GAPDH cDNA
probe was PCR amplified from a mouse cDNA library.
Preparation of protein extracts and immunoblot analysis
Protein lysates were prepared and quantified as described
(Kenney and Rowitch, 2000;
Matsushime et al., 1994
). Each
sample (25 µg) was separated on 8% or 12.5% SDS-polyacrylamide gels, then
transferred to Immobilon PVDF (Millipore) membranes. Primary antibodies used
were anti-cyclin D1 and anti-Nmyc (sc-450 and sc-751, Santa Cruz), and
anti-ß-tubulin (T4026, Sigma). Peroxidase-conjugated secondary antibodies
included donkey anti-mouse (Jackson Immunoresearch Laboratories) and goat
anti-rabbit (Pierce). Blots were developed using enhanced chemiluminescence
(ECL) (Amersham-Pharmacia), according to the manufacturer's instructions.
Chemiluminescent immunoreactivity was detected using Kodak X-OMAT X-ray
film.
Tissue preparation and in situ hybridization
Neonatal cerebella (PN1, PN7 and PN15) from 1% formalin/PBS perfused SWB
mice and Ptch adult heterozygotes
(Goodrich et al., 1997) or
Shh-transgenic 12.5 dpc embryos
(Rowitch et al., 1999
) were
drop-fixed in fresh 4% paraformaldehyde at 4°C. Samples were embedded in
OCT and frozen 15-18 µm parasagittal sections were prepared on Superfrost
plus slides. In situ hybridization on frozen sections was performed according
to standard protocols (detailed protocol available upon request). Antisense
digoxigenin-labeled riboprobes were generated from Myc, Nmyc and Lmyc
constructs (provided by Ron DePinho, DFCI, Boston MA), Gli1 (provided
by Alex Joyner, Skirball Institute of Biomolecular Medicine, NY) or cyclin D1
(Ccnd1) (from Steve Elledge, Baylor College of Medicine, Houston TX)
constructs.
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RESULTS |
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As shown (Fig. 1A), 3 hours
of CHX treatment alone resulted in increased levels of mRNA transcripts for
Myc and Fos in CGNPs regardless of treatment, in keeping
with original observations made in fibroblasts
(Greenberg et al., 1986;
Lau and Nathans, 1987
).
Cycloheximide-mediated accumulation of mRNAs for these genes indicates that
they are transcribed in primary granule cell cultures, but it is evident from
our analysis that their transcription is not specifically affected by Shh. Shh
did not induce expression of Lmyc compared with vehicle-treated CGNPs
(Fig. 1A), and Jun mRNA was
undetectable in CGNP cultures (data not shown). Thus, Myc, Fos, Lmyc
and Jun are unlikely to be direct targets of Shh in proliferating
CGNPs.
|
Gli1 is a known transcriptional target of the Shh pathway in CGNPs
(Weschler-Reya and Scott, 1999) and Gli proteins can be activated by hedgehog
activity (Ruiz i Altaba,
1999). Gli1 was strongly induced after 3 hours of
treatment with Shh alone (Fig.
1A). CHX treatment alone did not induce Gli1 mRNA, and
sharply reduced the degree of Gli1 upregulation in the presence of
Shh. These results indicate that Gli1 does not share the
induction/super-induction response characteristic of IEGs. Newly synthesized
Gli1 may regulate its own transcription
(Dai et al., 1999
). This could
explain the antagonistic effect of CHX on Gli1 mRNA induction by
Shh.
Unlike other genes tested, Nmyc expression was strongly
upregulated by Shh in the presence or absence of CHX
(Fig. 1A). CHX treatment alone
resulted in a low level of Nmyc mRNA elevation, in the manner of
classically defined IEGs (Fig.
1A). These data suggest that Shh signaling pathway activation
induces Nmyc by a mechanism that does not require new protein
synthesis. Proliferation in CGNPs reaches high levels by 24 hours of Shh
treatment (Kenney and Rowitch,
2000; Wechsler-Reya and Scott,
1999
). Shh effects on the cell cycle machinery are first observed
after 3 hours of treatment (Kenney and
Rowitch, 2000
). We did not detect significant upregulation of
Nmyc mRNA until 3 hours of treatment with Shh+CHX
(Fig. 1A,B).
Cerebellar homogenate cultures are an established system to characterize
Shh proliferative effects on CGNPs (Kenney
and Rowitch, 2000; Klein et
al., 2001
; Wechsler-Reya and
Scott, 1999
). These heterogeneous cultures comprise
85%
CGNPs, which express the bHLH transcription factor Math1
(Ben-Arie et al., 1997
;
Helms and Johnson, 1998
;
Kenney and Rowitch, 2000
), as
well as glia and a small percentage of other cell types (collectively defined
as `non-granule cell component'). Previous work indicates that CGNPs are the
only cells in these cultures that proliferate in response to Shh
(Dahmane and Ruiz-i-Altaba,
1999
; Kenney and Rowitch,
2000
; Wechsler-Reya and Scott,
1999
). Nonetheless, to minimize any contribution of non-granule
cells, we enriched for CGNPs in cerebellar homogenates (see Materials and
Methods), then assayed cultures prepared in this manner for Nmyc
upregulation by Shh. As shown in Fig.
1B, these enriched cultures exhibited protein-synthesis
independent upregulation of Nmyc in response to Shh. These results
confirm that Shh-induced Nmyc upregulation in cerebellar homogenate
cultures is attributable to CGNPs, and rules out any significant contribution
of the non-granule cell component.
Other neuronal cell types responding to extracellular stimuli typically
upregulate IEG mRNA within minutes (Morgan
and Curran, 1991; Sheng and
Greenberg, 1990
). By contrast, we observed a delay in
Nmyc upregulation, which is most probably due to our culture
conditions (see Materials and Methods) that include a 1 hour `rest' period in
factor-free medium before Shh treatment
(Kenney and Rowitch, 2000
).
Cerebellar homogenates were initially maintained in serum-containing media to
allow them to adhere and recover from harvest (Hatten, 1998). Although serum
is not mitogenic for CGNPs, it is likely to contain factors that affect
signaling pathways, which could antagonize or synergize with Shh activity. The
resting period was thus used to minimize any potential contributions of
serum-regulated pathways and reveal effects specific to Shh signaling. The
timecourse we show for induction of Nmyc coincides with that of
D-type cyclins in the absence of CHX
(Kenney and Rowitch, 2000
;
Wechsler-Reya, 1999). Similarly, Gli1 upregulation was first
detectable after 3 hours of treatment in the absence of CHX
(Fig. 1C), and lasted for the
duration of this time course analysis. In summary, 3 hours is the earliest
time point at which we have observed concerted activation of the hedgehog
pathway and effects on cell cycle.
Proliferation in CGNPs reaches high levels by 24 hours of Shh treatment
(Kenney and Rowitch, 2000;
Wechsler-Reya and Scott,
1999
). Cultures treated with Shh for 24 hours showed maintenance
of Math1 (Atoh1 Mouse Genome Informatics) expression
(Fig. 1C), a CGNP-specific
marker in the cerebellum required for granule cell development
(Ben-Arie et al., 1997
).
Expression of Math1 was used to confirm the presence of immature
CGNPs in heterogeneous Shh-treated homogenate cultures
(Kenney and Rowitch, 2000
;
Wechsler-Reya and Scott,
1999
). Like Gli1, a classical Shh pathway target
(Ruiz i Altaba, 1999
),
Nmyc was upregulated in CGNPs treated with Shh for at least 24 hours
(Fig. 1C), indicating that the
induction of Nmyc is not a transient response to Shh.
Nmyc is expressed in proliferating granule cell precursors
in vivo
In mice, granule neuron precursor expansion in the EGL occurs primarily
during the first 2 weeks of life (PN1-PN14)
(Altman and Bayer, 1997).
However, a role for Nmyc in this process has not been described, nor has
Nmyc expression been characterized during expansion of granule
neurons in vivo. We used in situ hybridization to determine expression of
Nmyc, cyclin D1 and Gli1, an established Shh transcriptional
target, in the PN7 cerebellum. As shown in
Fig. 2A-C, we observed
co-expression of Nmyc, cyclin D1 and Gli1 in the EGL. To
assess whether Nmyc is expressed in an appropriate developmental time
course in vivo, we subjected cerebella from PN1-15 mice to analysis.
Nmyc expression was readily detectable at PN1 and PN7
(Fig. 2D,E). At PN7,
Nmyc expression was restricted to the proliferating outer zone of the
EGL (EGLa) and was not detected in the inner granule layer
(Fig. 2E,G), where
differentiated granule cells lie. Furthermore, at PN15, when CGNP
proliferation has ceased, Nmyc mRNA transcripts were no longer
detected (Fig. 2F). Other myc
family members tested (Myc, Lmyc) were not specifically expressed in
the EGL (Fig. 2H,I) at any
stage, consistent with results in Fig.
1. Thus, the temporal-spatial expression pattern of Nmyc,
but not other myc family members, precisely coincides with regions of hedgehog
proliferative activity in the developing cerebellum
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Nmyc overexpression promotes cell-autonomous CGNP
proliferation without smoothened activation
Gene over-expression has been employed to study hedgehog-stimulated
proliferation in CNS precursors in cerebellar slice preparations
(Solecki et al., 2001) and
transgenic mice (Epstein et al.,
1996
; Rowitch et al.,
1999
). These methods, however, cannot distinguish primary versus
secondary proliferative effects on surrounding cells. By contrast, monolayer
cultures of CGNPs are suitable for the study of the cell-autonomous functions
expected of transcription factors. Moreover, they minimize formation of
aggregates, which can stimulate granule cell proliferation
(Gao et al., 1991
). Retroviral
infection of monolayer cultures was the method we chose to investigate the
role of Nmyc in Shh-regulated CGNP proliferation. Retroviruses exclusively
infect proliferating cells (Roe et al.,
1993
), so we optimized conditions to enhance proliferation of
CGNPs in vitro and minimize proliferation of non-CGNP cell types, such as glia
and fibroblasts (see Materials and Methods). In particular, we observed that
cultures grown in the presence of Shh for 24 hours before infection
demonstrated high rates of infection with jellyfish green fluorescent protein
(GFP)-carrying retroviruses (Fig.
3B) relative to cultures pre-treated with vehicle alone
(Fig. 3A). To confirm selective
infection of CGNPs under our conditions, primary CGNP cultures were infected
with GFP-carrying retroviruses, then incubated in serum-free medium
without Shh for 48 hours to ensure that infected cells exited the cell cycle
and commenced differentiation. As shown in
Fig. 3C,D, fluorescence
immunocytochemistry indicated co-localization of GFP with NeuN, a marker of
post-mitotic granule cells (Wechsler-Reya
and Scott, 1999
), but not cells expressing the astroglial marker,
glial fibrillary acidic protein (GFAP).
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For proliferation studies, CGNP cultures were infected with retroviruses
expressing either GFP, wild-type mouse Nmyc or mutant Nmyc
lacking the Myc box 2 domain (MB2). The MB2 domain is required
for myc interaction with TRRAP (McMahon et
al., 1998
), a co-factor essential for recruitment of histone
acetyltransferase to target DNA. Cyclopamine, an alkaloid derived from the
veratrum lily (Keeler, 1969
),
antagonizes hedgehog signaling during development in vivo
(Incardona et al., 1998
)
because it prevents activation of Smo
(Taipale et al., 2000
).
Cyclopamine also inhibits Shh-induced expansion of chick mid-brain precursor
populations (Britto et al.,
2002
). Effects of cyclopamine on cycling CGNPs have not been
previously investigated.
We used cyclopamine to assess whether (1) Smo inhibition blocks Shh-induced
CGNP proliferation, (2) Nmyc lies downstream of Smo and (3) Nmyc can
functionally rescue CGNP proliferation when Smo is inactivated. Proliferation
was assessed 48 hours post-infection (80 hours post-plating). Fresh
medium with new Shh was provided after the infection period. It is known that
GCNPs will proliferate at high levels with Shh treatment for up to 2 weeks and
that proliferation is maximal at 3-4 days post-plating
(Wechsler-Reya and Scott,
1999
). Thus, our assay was performed within the time window during
which Shh proliferative effects have been observed. Further, we confirmed
upregulation of cyclin D1 proteins and ongoing effects of Shh on the molecular
apparatus controlling cell cycle progression at these times
(Fig. 4A,B). As shown in
Fig. 4A, S-phase levels in
cyclopamine/Shh-treated cells were significantly reduced in comparison with
Shh-treated CGNPs. Western blot analysis confirmed that Shh-treated CGNPs
expressed higher levels of Nmyc protein than vehicle-treated cells
(Fig. 4A), and that cyclopamine
treatment reduced levels of Nmyc protein in Shh-treated control/GFP-infected
CGNPs.
|
Using flow cytometry, we measured levels of proliferation in CGNP cultures
that were treated with cyclopamine/Shh and infected with Nmyc- or
MB2-carrying retroviruses. Retroviral protein expression was
verified by western blotting (Fig.
4A).
MB2-infected, cyclopamine/Shh-treated CGNPs
showed similar levels of cells in S-phase as GFP-infected
Shh/cyclopamine-treated CGNPs (Fig.
4A). By contrast, Nmyc-infected cyclopamine/Shh treated
cells maintained levels of proliferation similar to CGNPs exposed to Shh
alone. These findings indicate that Nmyc expression is sufficient for
CGNP proliferation in the absence of Smo-mediated hedgehog pathway activation.
To confirm results of flow cytometry by an independent means, we measured
proliferation in CGNP-enriched cultures by BrdU immunochemistry. As shown in
Fig. 4B, this analysis yielded
similar results. Cyclopamine alone had no effect on the ability of Nmyc to
promote proliferation in CGNPs (Fig.
4B). CGNPs infected with Nmyc+GFP and subsequently
treated with Shh vehicle alone also showed levels of proliferation similar to
cells infected with GFP and treated with Shh (data not shown). Infection with
Nmyc
MB2 and treatment with cyclopamine or Shh vehicle resulted
in proliferation and cyclin D1 protein levels and similar to GFP-infected
cells treated with Shh+cyclopamine (Fig.
4B and data not shown).
Nmyc might promote synthesis and release of secondary mitogens from infected cells. To determine whether proliferative effects of Nmyc on cell division were cell-autonomous, we used a retrovirus expressing both Nmyc and GFP (see Materials and Methods) to follow infected cells in Shh/cyclopamine-treated cultures. As shown in Fig. 4C, when Shh/cyclopamine-treated cultures were infected with Nmyc+GFP virus, we observed immunolabeling for BrdU exclusively in cells that co-expressed GFP. No BrdU-positive, GFP-negative cells were observed, out of twenty 100-cell fields examined. These results suggest that Nmyc acts within individual CGNPs to promote cell cycle progression.
Although our culture conditions were optimized for preferential infection of CGNPs, it was formally possible that the few glial cells present may have been infected with the Nmyc retrovirus. However, in Nmyc+GFP retrovirus-infected cultures treated with Shh/cyclopamine, we observed that both BrdU and GFP labeling consistently segregated away from cells expressing the astroglial marker, GFAP (Fig. 4C). Furthermore, the similar proliferative effects of Nmyc virus (Fig. 4B) despite the lack of glial cells in pre-plated, CGNP-enriched cultures indicates that these cells are not required to support CGNP proliferation in vitro. Together, these data rule out that CGNP proliferation in Nmyc-infected cultures reflects any significant contribution from glial cells.
A hallmark of the CGNP response to Shh is upregulation of D-type cyclin
mRNA and protein (Kenney and Rowitch,
2000). Immunoblot analysis showed increased levels of cyclin D1 in
Shh-treated cells, compared with vehicle treated controls
(Fig. 4A,B). Cyclopamine
co-treatment eliminated cyclin D1 protein expression in Shh-treated control
cells and those infected with GFP or
MB2
retroviruses. By contrast, Nmyc-infected CGNPs maintained cyclin D1
protein expression despite treatment with cyclopamine. These results suggest
that a downstream consequence of Nmyc activity is upregulation of cyclin D1
protein expression.
Evidence that Nmyc activity is necessary for the full CGNP
proliferative response to Shh
To further investigate potential roles for Nmyc, we asked whether Nmyc is
required for Shh-mediated CGNP proliferation. A well-established means of
blocking Myc function in vitro is by overexpression of Mad family members
(Ayer and Eisenman, 1993;
Lahoz et al., 1994
;
Lee and Ziff, 1999
;
Wu et al., 1996
). Mad
proteins, including Mad1, 3, 4 and Mxi, repress Myc target genes
(Baudino and Cleveland, 2001
)
by binding to E-box sequences and recruiting histone deactelylase. Mxi
overexpression blocks Myc- and Nmyc-mediated, but not E1a-mediated
transformation of fibroblasts (Lahoz et
al., 1994
).
As shown in Fig. 5A,
endogenous Mxi is expressed in CGNPs in vitro. We infected
Shh-treated CGNPs with a retrovirus carrying Mxi-SR, the strong
repressor form of Mxi (Schreiber-Agus et
al., 1995), then compared proliferation levels with
control/GFP-infected CGNPs. Expression of Mxi-Sr is shown in
Fig. 5A. Thirty-six hours after
infection, proliferation in CGNPs was assayed by flow cytometry.
Mxi-infected CGNPs treated with Shh showed significantly reduced
proliferation compared with controls (Fig.
5A). To determine whether this decrease in proliferation reflected
cell death, we quantified levels of fragmented `sub-G1' DNA, a
hallmark of cell death that can be quantified by flow cytometry
(Darzynkiewicz et al., 1997
).
The anti-proliferative effects of Mxi were not due to increased levels of cell
death, as sub-G1 DNA levels in Mxi-infected cells (5.3%±0.5,
n=2) were similar to those in GFP-infected CGNPs (5.6%±0.7,
n=2). These findings indicate that Mxi overexpression negatively
regulates Shh-stimulated proliferation in CGNPs. Because Mxi represses Myc
targets, and Nmyc is the only Myc family member expressed in CGNPs,
our observations imply that Nmyc target gene activity is crucial for Shh to
achieve its full mitogenic potential in CGNPs.
|
Although cell growth regulation targets substantially overlap between Myc
and Mad (James and Eisenman,
2002), several targets for activation by Myc are not targets for
Mad-mediated repression, and vice versa
(O'Hagan et al., 2000
). We
wished to confirm our findings using a more specific means of blocking Nmyc
activity. To this end, we infected CGNPs with a virus expressing
MB2+GFP and assessed levels of proliferation in these cells,
compared with cells infected with GFP-carrying retroviruses alone.
Proliferation was measured by quantitation of BrdU incorporation. These data
indicated that
MB2 overexpression results in a significant decrease in
Shh-induced CGNP proliferation (Fig.
5B). This suggests that overexpression of Nmyc
MB2
can compete with endogenous Nmyc in regulating growth-promoting target genes,
resulting in reduced levels of proliferation. Together, the findings suggest
that Nmyc activity is required to achieve the full proliferative response to
Shh within CGNPs.
Hedgehog pathway activation causes Nmyc upregulation at
ectopic locations in the neural tube and in medulloblastoma
Our findings indicate that Nmyc acts a link between hedgehog pathway
activation and cell cycle progression within CGNPs. Shh has proliferative
effects elsewhere in the CNS (Dahmane et
al., 2001; Jensen and Wallace,
1997
; Kalyani et al.,
1998
), and ectopic expression of Shh using the
Wnt1 regulatory element results in increased levels of neural
precursor proliferation in the dorsal spinal cord of transgenic mice
(Rowitch et al., 1999
). We
observed Nmyc upregulation in dorsal spinal cord of such transgenic
embryos (Fig. 6D), suggesting
that Nmyc is similarly upregulated in other CNS regions where Shh has
mitogenic effects. These data further show that, in contrast to Gli1,
Nmyc is not a general Shh transcriptional target. For example,
Nmyc expression is absent in the floorplate, where Shh is
strongly expressed (fp) (compare Fig. 6A,C
with Fig. 6B,D). Second, it is clear that Nmyc can be
activated by mitogens other than Shh. Nmyc expression is observed in
proliferating progenitors in the dorsal half of wild-type spinal cord at 12.5
dpc (at a long distance from a source of Shh;
Fig. 6B) and in the dorsal root
ganglion (drg; Fig. 6B).
Together with results shown in Figs
1,
2, these findings indicate that
Nmyc can be recruited by Shh solely within immature, proliferating
CNS precursor cells.
|
Mutation of the tumor suppressor Ptch is associated with abnormal
activation of hedgehog signaling and generation of medulloblastoma
(Goodrich et al., 1997). We
performed in situ hybridization on sections of medulloblastoma from
Ptch adult heterozygotes. As shown in
Fig. 6E, we observed dramatic
induction of Nmyc in the tumors, in contrast to adjacent normal
cerebellum. Indeed, the borders of the tumor appeared to be well demarcated by
Nmyc expression, which highlighted an area of continuity between the
IGL and tumor that largely occupied the space dorsal and posterior to the
cerebellum (Fig. 6F). These
findings suggest that Nmyc upregulation is a consequence of
pathological hedgehog pathway activation as well as normal Shh signaling
during development.
![]() |
DISCUSSION |
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Of numerous IEGs tested by northern blotting, only Nmyc expression
was upregulated by Shh in the presence of cycloheximide in CGNPs, suggesting
its transcription is directly regulated by the Shh signaling pathway.
Nmyc overexpression was sufficient for CGNP proliferation, despite
treatment with the hedgehog pathway antagonist cyclopamine, which interferes
with Shh signaling by affecting the activation state of Smo
(Taipale et al., 2000).
Cyclopamine has been shown to interfere with the expansion of chick midbrain
precursors, a Shh-mediated process (Britto
et al., 2002
). Interestingly, cyclopamine also reduces
proliferation of certain brain tumor cells
(Dahmane et al., 2001
), which
do not express Shh but do express Shh signaling targets. More recently, use of
cyclopamine to inhibit the hedgehog signaling pathway was shown to block
growth of mouse medulloblastomas in vitro and in vivo
(Berman et al., 2002
). Our data
indicate that cyclopamine blocks Shh-induced proliferation and reduces levels
of Nmyc protein in CGNPs. Nmyc overexpression sustained CGNP
proliferation, despite cyclopamine treatment, indicating that Nmyc function
lies downstream of Smo in the Shh signaling pathway.
It seems likely that Nmyc is not the only Smo target with a role
in regulating cell cycle progression. Investigation of the proliferative
targets of Shh has often focused on the Gli transcription factors, orthologs
of the Drosophila hedgehog target cubitus interruptus
(ci). Increased levels of Gli1 mRNA transcripts are
characteristic of Shh signaling pathway activation, and can be seen in CGNPs
treated with Shh (Kenney and Rowitch,
2000; Wechsler-Reya and Scott,
1999
). Our studies have not focused on a relationship between Gli
activity and Nmyc regulation per se. Recently, a microarray analysis
by Yoon and colleagues identified cyclin D2 as a Gli1 target in transformation
assays (Yoon et al., 2002
).
Nmyc was not among the Gli1 targets described in that study.
Mutation of the DNA-binding domain of Gli1 is not reported to
result in cerebellar defects (Park et al.,
2000), indicating the existence of Gli1-independent
mechanisms for regulating cerebellar expansion. It is possible that Gli2
activity or antagonism of Gli3-mediated repression promotes Shh-stimulated
Nmyc expression. The regulation of these factors is thought to involve
post-translational processing (Ruiz i
Altaba, 1999
; Wang et al.,
2000
), a process that would enable target gene activation in the
presence of protein synthesis inhibitors. However, this mechanism may not
involve the induction/super-induction property of IEG mRNA regulation. An
intriguing alternative is that Nmyc up-regulation by Shh occurs via a
Gli-independent mechanism.
Shh-mediated regulation of Nmyc during cerebellar
development and tumorigenesis
Nmyc is a member of the myc proto-oncogene family, which also
includes Myc and Lmyc. Activity of Myc proteins is known to
promote proliferation and/or transformation in many cell types
(Henriksson and Luscher,
1996). Complete loss of Myc activity results in severely
compromised cell cycle regulation (Mateyak
et al., 1997
). We found that Nmyc was highly expressed in
the expansion zone of the EGL, where precursor proliferation is known to
depend upon Shh activity. By contrast, we did not detect Myc or
Lmyc expression in the developing EGL, nor were they upregulated by
Shh in CGNP cultures. These findings suggest an important role for Nmyc in
proliferation of CGNPs. We observed Nmyc upregulation in CGNP
cultures and the dorsal embryonic spinal cord exposed to Shh, suggesting that
signaling via Nmyc may be a general feature of Shh-induced
proliferation in the CNS. However, in contrast to known general
transcriptional targets of Shh (e.g. Gli genes), we note that Shh signaling
effects on Nmyc regulation are unique to immature, proliferating
precursor cells. Thus, cellular competence is evidently a critical determinant
of the transcriptional response of Nmyc to Shh.
Increased levels of MYCN mRNA have been reported in certain cases
of human medulloblastoma (Garson et al.,
1989). More recently, MYCN upregulation has been observed
in the desmoplastic form of medulloblastoma, the specific subtype of
medulloblastoma associated with pathological activation of the hedgehog
pathway (Pomeroy et al.,
2002
). By contrast, MYCN expression was not elevated in
the majority of other (non-desmoplastic) medulloblastoma types analyzed.
Consistent with findings in humans, we observed dramatic Nmyc
upregulation in medulloblastomas from Ptch heterozygotes
(Goodrich et al., 1997
).
Together, these findings are strong evidence that similar mechanisms operate
downstream of hedgehog signaling during central nervous system development and
tumorigenesis. Our results further suggest that MYCN expression in
desmoplastic medulloblastoma is a direct consequence of hedgehog pathway
activation.
Evidence that Nmyc activity is required for the full Shh
proliferative response in CGNPs
To establish a role for Nmyc in the Shh proliferative pathway in developing
cerebellar precursors, it is necessary to examine cell cycle progression in
the absence of Nmyc function. However, mice homozygous for
Nmyc null or certain hypomorphic alleles mice die in utero or at
birth (Charron et al., 1992;
Moens et al., 1992
;
Sawai et al., 1991
;
Stanton et al., 1992
),
preventing assessment of Nmyc function during the proliferative
postnatal phase of CGNP development. Recently, conditional targeting of murine
Nmyc in the developing CNS has been reported to result in severe
hypoplasia of cerebellum and defects in granule cell precursor proliferation
(Knoepfler et al., 2002
).
These findings indicate that Nmyc function is necessary for
cerebellar development. Yet, they leave unanswered the question of whether
Nmyc activity is required downstream of Shh signaling during the postnatal
phase of granule cell expansion.
In studies of Myc biology, overexpression of Mad family members is an
established method for inhibiting Myc functions in proliferation,
transformation and apoptosis (Ayer and
Eisenman, 1993; Hurlin et al.,
1995
; Schreiber-Agus et al.,
1995
; Wu et al.,
1996
). These studies indicate that the repressive activity of Mad
family members is specific, and Mad proteins are unlikely to behave as general
transcriptional repressors; indeed, Mxi overexpression cannot inhibit
transformation by E1a, another potent oncogene
(Lahoz et al., 1994
). The
ability of Mxi overexpression to abrogate the mitogenic effects of
Shh in vitro raises the possibility that Mad family members may regulate CGNP
cell cycle exit during cerebellar development in vivo. In keeping with this,
expression profiling studies indicate upregulation of several Mad
family members at PN7, which coincides with the major wave of CGNP
differentiation (Q. Zhao, A. Kho, I. Kohane and D. H. R., unpublished).
Our results imply that Mxi represses Nmyc targets, whose activity is
crucial for Shh signaling to achieve its full proliferative capacity in CGNPs.
To show that Nmyc activity is specifically involved in the proliferative
response of CGNPs to Shh, we overexpressed a mutant Nmyc that lacks
the Myc box 2 domain. This also resulted in a significant reduction in CGNP
proliferation. Analogous mutants of Myc have dominant negative effects on
transformation (Sawyers et al.,
1992), and can attenuate entry into S-phase in fibroblasts
(Conzen et al., 2000
). The
Nmyc
MB2 mutant is incapable of interacting with TRRAP, a co-factor
required for the transformation activity of Myc
(McMahon et al., 1998
).
Although the Nmyc
MB2 mutant lacks transforming activity, it can
partially rescue cell cycle defects in Myc-null fibroblasts
(Nikiforov et al., 2002
). The
activity of the Nmyc
MB2 mutant, as well as requirements for TRRAP in
neuronal precursors, have not been investigated. The inability of
Nmyc
MB2 to rescue proliferation in cyclopamine-treated CGNPs and its
interference in Shh-induced CGNP proliferation raise the interesting
possibility that neuronal precursors possess unique requirements for TRRAP
activity in regulating proliferation-associated gene activation.
Although the levels of proliferation were reduced comparably with Mxi-SR or
NmycMB2, in neither case did we observe complete cessation of
proliferation. This is not surprising, as both of these methods allow for some
residual Nmyc activity. When Myc activity is reduced, but not eliminated,
Myc-dependent processes can still occur
(Bazarov et al., 2001
). Indeed,
cultured fibroblasts that lack any Myc family member remain capable of
proliferation albeit with an extended cell cycle length
(Mateyak et al., 1997
). Thus,
abrogation of Nmyc activity might not necessarily lead to cell cycle exit
within the time frame of our studies. Taken together, our findings of
decreased CGNP proliferation with specific Nmyc antagonists in vitro, coupled
with data that demonstrate essential functions for Nmyc during
cerebellar development in vivo (Knoepfler
et al., 2002
), strongly suggest that Nmyc functions as an
integral component of a Shh regulated pathway during proliferation of granule
neuron precursors.
Mechanisms underlying cell cycle regulation by Nmyc in proliferating
neuronal precursors
Myc proteins are characterized by specific functional domains, including a
C terminus DNA-binding domain and a helix-loop-helix/leucine zipper domain,
which mediates dimerization with the obligate partner of Myc, Max [Myn in the
mouse (Prendergast et al.,
1991] (Henriksson and Luscher,
1996
). Two highly conserved amino acid regions, Myc box 1 and Myc
box 2, reside in the N-terminal transactivation domain. Myc box 2 associates
with TRRAP protein (McMahon et al.,
1998
), which functions to recruit histone acetyltransferase to the
transactivation domain of Myc (Park et
al., 2001
). This interaction is required for the transforming
potential of Myc and Nymc
(McMahon et al., 1998
). The
Myc box 2 domain also appears to be required for the proliferative function of
Nmyc in CGNPs, as overexpression of an Nmyc mutant lacking Myc box 2
was unable to rescue CGNP proliferation in the absence of Shh signal, and
interfered with CGNP proliferation in the presence of Shh.
It is likely that Nmyc activates yet to be identified genes involved in
cell cycle regulation. Recently, Id2 was shown to be an Nmyc target
in neuroblastoma (Lasorella et al.,
2002). Some Myc targets include genes such as cyclin D2
(Bouchard et al., 1999
) and
Cdc25 (Galaktionov et al.,
1996
), which control cell cycle progression. Metabolic regulators
that enhance cell growth may also be targets of Myc
(Dang, 1999
). Finally, Myc has
been implicated in repression of genes that promote cell cycle exit and
differentiation (Claassen and Hann,
1999
). Given the high level of relatedness between Nmyc and Myc
protein structure, and the observation that Nmyc can rescue many of the
defects in Myc null mutant mice (Malynn et
al., 2000
), it is possible that they have similar transcriptional
targets.
We observed increased levels of cyclin D1 in Nmyc-infected cells
despite the presence of cyclopamine. Nmyc is a direct transcriptional
target of Shh signaling, whereas cyclin D1 mRNA upregulation was inhibited by
cycloheximide (Kenney and Rowitch,
2000). Nmyc could be a candidate regulator of cyclin D1
expression. However, our present cyclin D1 protein measurements were taken 48
hours after infection and treatment, too long a length of time to assay for
direct activation of Nmyc target genes. Nmyc may act indirectly, via
activation of genes that otherwise promote cell growth, or by repressing genes
that control the degradation of cyclin D1 message.
In summary, our results indicate that Nmyc is a direct downstream target of the canonical Shh signaling pathway in proliferating CGNPs. Nmyc expression was also found in medulloblastomas of Ptch mutant mice. Nmyc function is a key component of a Shh proliferative pathway essential for normal expansion of CGNP populations. These findings are the first to identify a direct connection between the Shh signaling pathway and a cell-intrinsic regulator of the cell cycle apparatus in primary neuronal cells. Proximal events in the progression from Smo activation to upregulation of Nmyc expression remain to be determined. Identification of Nmyc transcriptional targets will be crucial for understanding Shh-mediated cell cycle progression in CNS development. Moreover, because cerebellar granule cells are postulated to be the cell-of-origin for medulloblastoma, effective means to inhibit Nmyc activity might provide new approaches to control the growth of cerebellar tumors.
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ACKNOWLEDGMENTS |
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