1 Gladstone Institute of Neurological Disease, University of California, San
Francisco, San Francisco, CA 94141-9100, USA
2 Neuroscience Graduate Program, University of California, San Francisco, San
Francisco, CA 94141-9100, USA
* Author for correspondence (e-mail: fgao{at}gladstone.ucsf.edu)
Accepted 11 August 2003
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SUMMARY |
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Key words: Fragile X syndrome, Dendrites, Drosophila, Rac1
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Introduction |
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One family of proteins that may be important in controlling neuronal growth
is that of mRNA-binding proteins. These proteins control gene expression at
multiple steps of mRNA metabolism, such as splicing, transport, localization,
translation and degradation (reviewed by
Darnell, 2002;
Dreyfuss et al., 2002
). Some
of these proteins are highly or solely expressed in neurons, and their
functions are beginning to be revealed (reviewed by
Musunuru and Darnell, 2001
;
Steward and Schuman, 2001
). In
this study, we focus on the role of the fly homolog of the fragile X
mental retardation 1 (FMR1) gene in dendritic development.
The absence of the FMR1 gene activity causes fragile X syndrome,
the most common form of inherited mental retardation in humans, with an
estimated incidence of 1 in 4000 males and 1 in 8000 females (reviewed by
O'Donnell and Warren, 2002).
FMR1 encodes a putative RNA-binding protein with two
ribonucleoprotein K homology (KH) domains and an arginine- and glycine-rich
domain (RGG box). The FMR1 protein preferentially binds to poly(G), poly(U)
and a subset of brain mRNAs in vitro
(Ashley et al., 1993
;
Siomi et al., 1993
;
Brown et al., 1998
). In
addition, FMR1 is associated with polyribosomes and a large number of mRNAs in
vivo, some of which contain G quartet structures as FMR1-binding motifs
(Feng et al., 1997a
;
Corbin et al., 1997
;
Sung et al., 2000
;
Darnell et al., 2001
;
Brown et al., 2001
;
Zalfa et al., 2003
;
Miyashiro et al., 2003
). It
remains to be determined which proteins encoded by mRNAs in FMR1-mRNP
complexes are primarily responsible for the morphological and functional
deficits caused by the absence of FMR1.
The exact molecular function of FMR1 in vivo remains largely unknown. Some
studies suggest that FMR1 affects mRNA localization and translation
(Darnell et al., 2001;
Brown et al., 2001
;
Zalfa et al., 2003
;
Miyashiro et al., 2003
),
although the underlying mechanism is unclear
(Antar and Bassell, 2003
). The
recent demonstration that Drosophila fragile X-related protein (Fmr1;
previously dFXR) interacts with components of the RNA interference (RNAi)
machinery raises the possibility that Fmr1/FMR1 may also function as part of a
gene-silencing mechanism (Ishizuka et al.,
2002
; Caudy et al.,
2002
). FMR1 is highly expressed in neuronal perikaryon and
dendrites and shuttles between the nucleus and the cytosol
(Devys et al., 1993
;
Fridell et al., 1996
;
Feng et al., 1997b
). Studies
of individuals with fragile X syndrome, Fmr1 knockout mice and
cultured neurons, although not entirely consistent with each other, raise the
possibility that FMR1 is involved in the proper development of spines of
central nervous system (CNS) neurons
(Hinton et al., 1991
;
Comery et al., 1997
;
Braun and Segal, 2000
;
Nimchinsky et al., 2001
).
We study the role of Fmr1 in the peripheral nervous system (PNS) of the
Drosophila larva, which is relatively simple and consists of 44
sensory neurons in each abdominal hemisegment
(Ghysen et al., 1986;
Bodmer et al., 1989
;
Orgogozo et al., 2001
).
Dendritic arborization (DA) neurons, one subtype of PNS sensory neurons,
elaborate extensive dendritic arbors just underneath the epidermis to receive
sensory inputs (Bodmer and Jan,
1987
; Gao et al.,
1999
). The ability to visualize the dendritic arbors in living
Drosophila larvae allows us to quantitatively examine the effects of
Fmr1 on dendritic development of identifiable neurons in vivo.
The Fmr1 gene is the only fly homolog of the human FMR1
gene that also has RNA-binding activity in vitro
(Adams et al., 2000;
Wan et al., 2000
). It has been
reported that Fmr1 mutation impairs the synaptic function at the
neuromuscular junction (Zhang et al.,
2001
). In addition, Fmr1 is required for normal circadian rhythm
of adult flies (Dockendorff et al.,
2002
; Morales et al.,
2002
; Inoue et al.,
2002
). To study the role of Fmr1 in dendritic growth, we
isolated Fmr1 protein-deficient mutant fly lines in which specific point
mutations or small deletions were introduced into the Fmr1 gene. We
report that Fmr1 is expressed in DA sensory neurons and limits dendritic
branching during development. In addition, we show that the mRNA encoding the
small GTPase Rac1 is present in Fmr1-mRNP complexes and that the function of
Fmr1 in dendrite development is partially mediated by Rac1.
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Materials and methods |
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Other flies lines used in this study were UAS-mCD8::GFP
(Bloomington Stock Center), UAS-Rac1 (Bloomington Stock
Center), UAS-Fmr1 (Wan
et al., 2000), Fmr13/TM6
(Dockendorff et al., 2002
),
the MD neuron-specific Gal4 line 109(2)80
(Gao et al., 1999
),
Rac1J11, FRT2A/Cyo
(Ng et al., 2002
) and
UAS-Fmr1-GFP (this study). A fly line that contains a 14 kb
fragment that spans the Fmr1 transcriptional unit
(Dockendorff et al., 2002
) was
also used in this study. To study Fmr1 overexpression phenotypes, we selected
third instar larvae with the genotype of Gal4 109(2)80,
UAS-mCD8-GFP/+; UAS-Fmr1/+ and
visualized mCD8::GFP-labeled DA neurons in which Fmr1 was overexpressed.
Similar genetic crosses were made to overexpress Rac1 in DA neurons.
Western blot analysis
The expression of Fmr1 in Drosophila larvae was analyzed by
western blot according to the standard protocol provided by BioRad. Wild-type
or Fmr1 mutants at the third instar larval stage were used to prepare
protein extracts. Anti-Fmr1 monoclonal antibody
(Wan et al., 2000) was used as
the primary antibody (1:1000 dilution). Horseradish peroxidase-conjugated
donkey anti-mouse IgG antibody (Jackson ImmunoResearch Laboratories) was used
as the secondary antibody (1:200).
In situ analysis and immunohistochemistry
The expression of Fmr1 mRNA was analyzed according to a standard
in situ protocol. For antibody immunostaining of DA neurons in dissected third
instar larvae, monoclonal antibody against Fmr1 (1:100) was used as the
primary antibody. Cy3-conjugated goat anti-mouse IgG (Jackson ImmunoResearch
Laboratories, 1:100) was used as the secondary antibody. The dissected larvae
were mounted in 90% glycerol in PBS, and confocal images were obtained with a
confocal microscope (BioRad Radiance 2000). For this experiment, Fmr1
mutant larvae were used as the negative control.
Quantification of dendritic processes
To measure the total number of terminal dendrites of ventral DA neurons in
segments 5 and 6, images of mCD8::GFP-labeled dendrites were taken with a
confocal microscope and were converted with Photoshop 6.0 into grayscale with
a bright background. The dendritic processes were counted in a specific area
between two contralateral ventral clusters of DA neurons from zoom-in images
and presented as the number of processes per 1000 µm2.
Immunoprecipitation and RT-PCR
Cell lysates were made from Drosophila larvae of the desired
genotypes and used in immunoprecipitation experiments in which a monoclonal
antibody raised against Fmr1 (Wan et al.,
2000) was used to pull down Fmr1-mRNP complexes. Total RNA was
extracted from the precipitated complexes and used to generate cDNAs in
reverse transcription (RT) reactions, which served as the template for
polymerase chain reactions (PCR). PCR was performed with oligonucleotide
primers specific for Rac1 or other control mRNAs, and the resulting DNA
fragments were analyzed by electrophoresis in 2% agarose gels.
MARCM analysis of Rac1 function in DA neuron dendrite
development
Single-cell analysis of Rac1 function in DA neuron dendrite
development was performed as described
(Sweeney et al., 2002).
Briefly, Rac1J11, FRT2A/TM6B male
flies were crossed with GAL4C155, UAS-mCD8::GFP,
hs-FLP1/FM7 virgin female flies. Then,
GAL4C155, UAS-mCD8::GFP, hs-FLP1/+;
Rac1J11, FRT2A/+ male flies were
crossed with GAL4C155, UAS-mCD8::GFP,
hs-FLP/+; tubP-GAL80, FRT2A virgin
female flies. Embryos from this cross were collected and incubated at 25°C
for 3 hours. At 3-6 hours after egg laying (AEL), embryos were heat-shocked in
a 37°C water bath for 40 minutes to induce mitotic recombination. Vials
were then kept at 25°C for 3-4 days. Third instar larvae were collected
and examined for the presence of a single mCD8::GFP-labeled dorsal cluster PNS
neuron, and images of dendritic morphology were obtained as described
above.
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Results |
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Mutant flies with the genotypes of
Fmr14/Fmr14,
Fmr11/Fmr14 or
Fmr12/Fmr14 are viable and develop
into adulthood at the predicted Mendelian ratios, consistent with some of the
previous reports that loss-of-function mutations in Fmr1 are not
lethal events (reviewed by Gao,
2002). No obvious morphological defects were found in adult
Fmr1 mutant flies. In this study, we mainly analyzed developmental
abnormalities of Fmr1 mutant larvae.
Fmr1 is expressed in dendritic arborization (DA) neurons
We have been studying dendritic growth in the Drosophila embryonic
and larval PNS, in which each abdominal hemisegment contains 44 sensory
neurons that can be grouped into dorsal, lateral and ventral clusters. In the
dorsal cluster, eight MD neurons, but not the four external sensory (ES)
neurons, can be labeled by green fluorescent protein (GFP) driven by Gal4
109(2)80 (Gao et al., 1999)
(Fig. 2A). DA neurons, a
subclass of MD neurons, develop complex dendritic branching patterns
(Bodmer and Jan, 1987
;
Gao et al., 1999
;
Gao et al., 2000
).
|
The development of DA neuron terminal dendrites is affected by
Fmr1 mutations
Because Fmr1 mutants were viable, we were able to directly examine
the effects of Fmr1 mutations on dendritic development of specific
neurons in a large number of live flies. To label all dendritic processes, we
expressed UAS-mCD8::GFP, which targets to the cell membrane, in all DA
neurons. We selected third instar larvae 4-5 days after egg laying (AEL) and
recorded the images of dendrites of ventral DA neurons from segments 5 and 6
in live animals. We found that the Fmr1 mutant larvae exhibited more
dendritic processes than wild-type larvae
(Fig. 3A,B). To quantify the
difference, we counted the number of ends of all dendritic terminal processes.
To reduce variation between larvae of the same genotype, we calculated the
number of ends per 1000 µm2 between two ventral cluster DA
neurons, to reflect the density of dendritic processes in a particular area.
On average, Fmr1 mutations increased the number of terminal dendritic
processes of ventral DA neurons by 25% (n=30, P<0.001)
(Fig. 3C). To demonstrate that
the increased number of terminal dendritic processes in Fmr1 mutants
was indeed due to the absence of Fmr1 activity, we introduced one
copy of the wild-type Fmr1 gene into the Fmr14
mutant background and found that the transgene could rescue the dendritic
defects in Fmr1 mutants (Fig.
3C). This Fmr1 transgene construct was previously shown
to be able to rescue the circadian defects in Fmr1 mutants
(Dockendorff et al., 2002). As
shown in Fig. 3D, a large
number of segments in wild-type and Fmr1 mutant larvae exhibit a
similar number of terminal dendritic processes, indicating that there is a
large variation among individual larvae of a given genotype and that
Fmr1 mutations cause subtle changes in neuronal morphology.
|
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Rac1 mRNA is associated with Fmr1-mRNP complex in vivo
The KH domains of Fmr1 share more than 70% identity with the mammalian FMR1
proteins. Indeed, Fmr1 and human FMR1 have similar RNA-binding properties in
vitro (Wan et al., 2000). A
number of recent studies have identified a large number of mRNAs that are
associated with FMR1 in mammalian systems
(Darnell et al., 2001
;
Brown et al., 2001
;
Miyashiro et al., 2003
).
However, systematic identification of Fmr1-binding targets in flies has not
been carried out. To gain mechanistic insights into Fmr1 function in
controlling dendritic growth in flies, we carried out co-immunoprecipitation
experiments to identify mRNAs that are associated with the Fmr1-mRNP complex
in vivo. In this study, using primers specific for genes encoding small GTPase
Rac1,
-tubulin, and the voltage-gated K+ channel molecule
Hyperkinetic, we performed RT-PCR analyses on either total RNAs or the RNAs
that were immunoprecipitated by an anti-Fmr1 monoclonal antibody, from lysates
derived from third instar larvae. All three mRNAs could be readily detected
from total RNAs, while only the Rac1 mRNA was associated with Fmr1 in
lysates derived from wild-type larvae as shown by coimmunoprecipitation
experiments (Fig. 5). The
lysate derived from Fmr1 mutant larvae was used as a negative
control, in which no Rac1 mRNA was detected from identical
co-immunoprecipitation experiments (Fig.
5). These studies demonstrate that Rac1 mRNA is
associated with Fmr1-mRNP complexes in vivo.
|
We were, however, able to test the hypothesis genetically. We first
examined the function of Rac1 in dendritic growth and branching of DA neurons
in Drosophila embryos. We used Rac1J11, a null
allele previously characterized based on biochemical and genetic criteria
(Ng et al., 2002;
Hakeda-Suzuki et al., 2002
).
We used Gal4 109(2)80 (Gao et al.,
1999
) to drive the expression of GFP in DA neurons in
Rac1J11 mutant embryos and did not observe gross defects
in dendritic branching patterns in later embryogenesis stages (data not
shown). DA neuron dendrites develop in discrete phases from the embryonic to
larval stages (Gao et al.,
1999
; Gao et al.,
2000
). In embryos, dorsal dendrites of DA neurons extend from cell
bodies first, and stop elongation 16-17 hours AEL, falling short of the dorsal
midline The lateral dendrites start to extend toward adjacent segment
boundaries and cover the hemisegment before hatching (22-23 hours AEL). Our
findings in Rac1J11 mutant embryos suggest that Rac1 is
not required for the initial growth of dorsal dendrites during
embryogenesis.
During larval stages, the dendritic fields of DA neurons expand many-fold
in accordance with the increase of larval body size. Higher-order dendritic
branches further develop to cover the whole epidermal surface of each
hemisegment (Gao et al., 2000;
Sugimura et al., 2002). Here, we used the MARCM technique
(Lee and Luo, 1999
) to examine
the role of endogenous Rac1 in dendritic growth in the third instar larval
stage. We generated single GFP-labeled wild-type or Rac1 mutant DA
neurons in abdominal segments and counted the number of terminal dendritic
branches (Fig. 6). We found
that Rac1 mutant ddaC neurons developed fewer dendritic branches than
wild-type neurons (Fig. 6A-F),
a phenotype similar to that caused by Fmr1 overexpression. Different
Rac1J11 mutant ddaC neurons exhibited varying severities
of dendritic defects. On average, there was a 23% reduction in the number of
dendritic branches due to the Rac1 mutation
(Fig. 6F). Similar dendritic
defects were also found in other DA neurons (data not shown). These findings
demonstrate that Rac1 is required for normal dendritic branching of DA neurons
in vivo, consistent with several previous studies that rely on the ectopic
expression of dominant mutant forms of Rac1
(Threadgill et al., 1997
;
Gao et al., 1999
;
Ruchhoeft et al., 1999
;
Li et al., 2000
).
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Discussion |
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Dendritic defects caused by the loss of Fmr1 activity
During the past couple of years, several labs have independently generated
Fmr1 mutant fly lines (Zhang et
al., 2001; Dockendorff et al.,
2002
; Inoue et al.,
2002
) (this study). All of these groups used fly lines provided by
fly stock centers that contain a P-element inserted in the first intron of the
Fmr1 gene. Some labs used the `imprecise hop-out' approach to excise
the P-element and generated deletions in the range of several thousand base
pairs in the Fmr1 locus (Zhang et
al., 2001
; Dockendorff et al.,
2002
; Inoue et al.,
2002
). In this study, we took advantage of the presence of
Gal4-binding sites in the P-element
(Rørth, 1996
) and
designed a different approach to create Fmr1 loss-of-function alleles
(Fig. 1). Consistent with some
of the previous reports, our Fmr1 mutant flies, which contain either
point mutations or small deletions, are fully viable and develop into
adulthood at the expected Mendelian ratios without gross morphological
defects.
The viability of Fmr1 mutant flies allowed us to directly examine
the role of Fmr1 in dendritic development in live larvae. We focused
on DA neurons because these neurons elaborate their dendrites in a
two-dimensional manner just beneath the epidermis
(Gao et al., 1999;
Gao et al., 2000
); therefore,
it was easy to visualize and quantify the number of GFP-labeled dendritic
branches. Secondly, there are only six DA neurons in the dorsal cluster and
four in the ventral cluster in each abdominal hemisegment
(Ghysen et al., 1986
;
Bodmer et al., 1989
;
Orgogozo et al., 2001
). We can
examine the dendrites of these same DA neurons in an area free of other
neurons in a large number of live animals. Our studies indicate that loss of
Fmr1 activity slightly increased the number of terminal dendritic
processes, which can be rescued by expressing wild-type Fmr1 in the mutant
background (Fig. 3). In
addition, specific overexpression of Fmr1 solely in DA neurons in wild-type
larvae dramatically decreased the number of terminal dendritic branches
(Fig. 4), further supporting a
role for Fmr1 in controlling dendritic development.
The dendritic phenotype of DA neurons in Fmr1 mutant larvae is
relatively subtle compared with that of Fmr1 overexpression. The average
number of terminal dendritic processes of ventral DA neurons in Fmr1
mutant larvae showed a statistically significant difference from that of
wild-type larvae; however, the numbers of dendrites in a large number of
segments in Fmr1 mutants show a similar number of terminal dendritic
processes as that seen in wild-type larvae
(Fig. 3D). This finding
suggests that the loss of Fmr1 activity has low expressivity in terms
of the dendritic phenotype, consistent with the wide range of physical and
mental abnormalities found in fragile X patients
(Hagerman, 2002).
mRNA targets regulated by Fmr1
Recent studies suggest that a large number of mRNAs can be found in
association with FMR1-mRNA complexes in mouse brain, and many of them contain
an FMR1-binding motif, the G quartet structure
(Darnell et al., 2001;
Brown et al., 2001
;
Zalfa et al., 2003
;
Miyashiro et al., 2003
). As
Fmr1 shares a high degree of amino acid sequence homology with human and mouse
FMR1, and these proteins behave in several similar ways
(Wan et al., 2000
;
Gao, 2002
), it seems highly
likely that Fmr1 also regulates multiple mRNAs during neural development.
Systematic identification of Fmr1 binding targets in Drosophila has
not been carried out. It was reported before that mRNA encoding the
microtubule-binding protein Futsch could be found in Fmr1-mRNP complexes
(Zhang et al., 2001
). We found
that Rac1 mRNA is also present in Fmr1-mRNP complexes, as shown by
co-immunoprecipitation and RT-PCR analyses
(Fig. 5). Interestingly, part
of the Rac1-coding region is highly conserved at the nucleotide level
from Drosophila to humans. This region contains G-rich nucleotide
sequences, which are not identical but are similar to the FMR1-binding
sequences identified from the in vitro RNA selection experiments
(Darnell et al., 2001
).
Several pieces of evidence indicate that Rac1 is partially responsible for
the effects of Fmr1 on dendritic development of DA neurons. First, Rac1 mRNA
is present in Fmr1-mRNP complexes in vivo
(Fig. 5). Second, MARCM
analysis demonstrates that Rac1 is required for dendritic branching of DA
neurons in a cell-autonomous manner, consistent with a previous report that
three small GTPases together, Rac1, Rac2 and Mtl, are required for dendritic
branching in mushroom body neurons (Ng et
al., 2002). Third, overexpression of Rac1 promotes dendritic
branching of DA neurons, a phenotype partially similar to that caused by
Fmr1 loss-of-function mutations. Fourth, decreased dendritic
branching caused by ectopic expression of Fmr1 can be partially rescued by
co-expression of Rac1. It is worth noting that overexpression of Rac1
increases dendritic branching more dramatically than Fmr1
loss-of-function mutations. We suspect that this is because of the level of
overexpression of Rac1 in the UAS-Gal4 system being much higher than Rac1
expression in Fmr1 mutant DA neurons. Conversely, overexpression of
Fmr1 causes a more dramatic decrease in dendritic branching than Rac1
mutations in MARCM-generated single neurons. This can potentially be accounted
for in two ways. The first is that, in MARCM-generated single mutant neurons,
wild-type Rac1 protein and mRNA inherited from its precursor cell before the
FRT-mediated recombination event may reduce the severity of the dendritic
phenotype (Ng et al., 2002
).
Second, it is highly likely that the expression of more than one protein
encoded by mRNAs in Fmr1-mRNP complexes is affected by the overexpression of
Fmr1. Indeed, our coimmunoprecipitation experiments indicate that other mRNAs
are also associated with Fmr1 in vivo (K.X. and F.-B.G., unpublished).
FMR1/Fmr1 proteins are found in dendrites of mammalian or fly neurons
(Devys et al., 1993;
Feng et al., 1997b
) (this
study). Although difficult to prove in vivo, local regulation of Rac1
expression by FMR1/Fmr1 may play a role in controlling the branching process
of terminal dendrites. The identification of Rac1 mRNA as one of the
targets of Fmr1 may also provide a partial explanation for the reported subtle
axon guidance defects found in Fmr1 mutant flies
(Morales et al., 2002
;
Dockendorff et al., 2002
).
After the submission of this manuscript, Schenck et al.
(Schenck et al., 2003)
reported the biochemical association between Rac1 and CYFIP, and between CYFIP
and Fmr1 in vitro and in transfected S2 cells. Based on biochemical and
genetic analyses in different cell types, the authors proposed that Rac1
regulates Fmr1 activity through CYFIP
(Schenck et al., 2003
). Taken
together, their studies and the findings reported here may suggest that there
is feedback loop between Rac1 and Fmr1 functions in vivo.
Molecular functions of Fmr1
How does Fmr1 function at the molecular level? Recent studies demonstrate
that Fmr1 is associated with some proteins that are known to function in the
RNAi complex (Ishizuka et al.,
2002; Caudy et al.,
2002
). Although Fmr1 can affect the efficiency of the RNAi, Fmr1
is not required for the process, and its exact role in the complex remains
unclear (Ishizuka et al.,
2002
; Caudy et al.,
2002
). RNAi can act at the post-transcriptional level to influence
the stability of mRNAs with sequences complementary to the silence trigger
(Fire et al., 1998
), or they
can inhibit protein synthesis without causing message degradation
(Olsen and Ambros, 1999
). In
our Fmr1 mutants, we did not detect any significant change in
quantity of Rac1 mRNA (data not shown). Fmr1 may inhibit
Rac1 mRNA translation in vivo, although the exact underlying
molecular mechanism remains to be determined. Based on the observation that
Fmr1 can be found in DA neuron dendrites, our results also raise the
possibility that Fmr1 regulates the translation of its target mRNAs in
dendrites, which may play an important role in regulating local protein
synthesis and neuronal function (reviewed by
Steward and Schuman, 2001
). It
would be interesting to dissect whether Fmr1 functions in controlling neuronal
morphology through an RNAi-based mechanism or in some other mRNA
degradation/translation regulatory pathways.
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ACKNOWLEDGMENTS |
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