|
|
|
|
|
|
|
||||
| Home Help Feedback Subscriptions Archive Search Table of Contents | ||||
First published online 5 March 2008
doi: 10.1242/dev.015867
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Department of Biological Sciences, Kennedy Center for Research on Human Development, Vanderbilt University, Nashville, TN 37232, USA.
* Author for correspondence (e-mail: kendal.broadie{at}vanderbilt.edu)
Accepted 4 February 2008
 |
SUMMARY |
|---|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
Key words: Fragile X Syndrome, Mental retardation, Autism, Neural development, Translation, Synaptogenesis, Synaptic pruning
|
INTRODUCTION |
|---|
|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
; Boccia and
Roberts, 2000; Freund and
Reiss, 1991; Sabaratnam et
al., 2001). The fragile X mental retardation protein (FMRP) is an
mRNA-binding protein proposed to function in mRNA trafficking, stability and
translational regulation (Ashley, Jr et
al., 1993; De Diego Otero et
al., 2002; Feng et al.,
1997; Laggerbauer et al.,
2001; Weiler et al.,
1997; Weiler et al.,
2004; Xu et al.,
2004; Zalfa et al.,
2007; Zalfa et al.,
2003; Zhang et al.,
2007). Although FMRP potentially binds 4% of brain mRNAs, only a
handful of targets have been validated
(Brown et al., 2001;
Hayashi et al., 2007;
Zhang and Broadie, 2005).
Accounting for this discrepancy may be that FMRP binds mRNA targets in
response to neuronal activation (Bear et
al., 2004). Neural activity from sensory experience and
metabotropic glutamate receptor signaling increases FMRP expression and
function (Gabel et al., 2004;
Hou et al., 2006;
Irwin et al., 2005;
Irwin et al., 2000;
Restivo et al., 2005;
Todd and Mack, 2000;
Todd et al., 2003;
Valentine et al., 2000;
Weiler et al., 1997). FMRP may
repress translation via association with polyribosomes
(Aschrafi et al., 2005;
Khandjian et al., 2004;
Stefani et al., 2004),
transported to sites of local synaptic translation in response to
neurotransmission (Ferrari et al.,
2007; Greenough et al.,
1985; Ostroff et al.,
2002).
Activity plays crucial roles in sculpting neural circuits during
development and later in mediating plasticity
(Desai et al., 2002;
Zito and Svoboda, 2002). FMRP
may perform a common function in regulating activity-dependent protein
synthesis in both settings. In FraX patients, mutant mice and
Drosophila, dendritic arbors are overgrown with immature dendritic
spines, suggesting a failure of synapse maturation
(Comery et al., 1997;
Galvez et al., 2003;
Galvez and Greenough, 2005;
Galvez et al., 2005;
Grossman et al., 2006;
Irwin et al., 2002;
Irwin et al., 2001;
Ivanco and Greenough, 2002;
McKinney et al., 2005;
Nimchinsky et al., 2001;
Pan et al., 2004;
Rudelli et al., 1985).
Dendritic defects are robust during early postnatal development and abrogate
with maturation (Galvez and Greenough,
2005; Nimchinsky et al.,
2001). Similarly, mutant neurons exhibit axonal over-branching in
mice and Drosophila, indicating a similar presynaptic requirement
(Antar et al., 2006;
Pan et al., 2004). FMRP is
also required for plasticity in mature synapses; long-term depression (LTD) is
enhanced (Hou et al., 2006;
Huber et al., 2002;
Koekkoek et al., 2005) and
long-term potentiation (LTP) is reduced
(Larson et al., 2005;
Li et al., 2002;
Zhao et al., 2005). These data
suggest two roles for FMRP: during development to regulate the structuring of
neural circuits and during maturity to regulate maintained plasticity.
Drosophila is well suited for the dissection of developmental processes; however, the well-characterized Drosophila FraX model has yet to be exploited for this purpose. Therefore, we investigated the developmental roles of Drosophila FMRP (dFMRP) in the Drosophila brain, specifically the activity-dependent structural changes driven by sensory input. We found that dFMRP expression and function are maximal during late-stage periods of axon pruning, which requires both dFMRP and sensory input activity. These results reveal a prominent role for dFMRP in activity-dependent neural circuit refinement.
|
MATERIALS AND METHODS |
|---|
|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
|
Western blotting
Single heads were homogenized in 1xNupage LDS Sample Buffer
(Invitrogen, Carlsbad, CA) with 55 mM DTT. Debris was pelleted by
centrifugation at 16,000x g at 25°C and samples
boiled for 10 minutes. Extracts were loaded onto a 4-12% Bis-Tris gel and
electrophoresed at 200 V in 1xMOPS or 1xMES buffer (Invitrogen,
Carlsbad, CA). Protein was transferred to nitrocellulose in 1xNupage
Transfer Buffer (Invitrogen, Carlsbad, CA) plus 10% methanol at 100 V for 1
hour. The membrane was blocked for 1 hour in Odyssey Blocking Buffer (Li-Cor,
Lincoln, NE) and probed for 12-16 hours at 4°C with the following
antibodies: dFMRP, 6A15 (Sigma, St Louis, MO), 1:5000 (for developmental
blots) or 1:500 (for sensory-deprivation blots); Chickadee/Profilin, Chi1J
(Developmental Studies Hybridoma Bank, Iowa City, IA), 1:10; 
-Tubulin
(Sigma, St Louis, MO), 1:400,000. Membranes were washed three times with
buffer (25 mM Tris pH 8.0, 150 mM sodium chloride, 0.05% Ige-PAL-CA630). The
secondary antibody, anti-mouse IgG IR800 (Rockland, Gilbertsville, PA) was
diluted 1:10,000 in Odyssey Blocking Buffer and applied for 1 hour at
25°C. The blot was washed three times with buffer and then scanned on the
Odyssey Infrared Imaging System.
Quantitative RT-PCR
cDNA was made from DNase I Turbo-treated (Ambion, Austin, TX) RNA (2 µg)
using random hexamer primers and Superscript II RNase H-Reverse Transcriptase
(Invitrogen, Carlsbad, CA). Quantitative PCR of cDNA (1 µl) was carried out
using SYBR Green JumpStart Taq Ready Mix (Sigma, St Louis, MO). The following
primers were used at 0.5 µM concentrations each per reaction:
gapdh2, 5'-CCTTGCAAGCAAGCCGATAG-3', 5'-CGACATGGTTAACTTTTTGT-3';
dfmr1, 5'-GTTCGGCTCGACAATGGCGC-3', 5'-GCGACAGCTGTCACCTGGCC-3';
chickadee, 5'-CGCAGTCCAGTGGCTTTGAG-3', 5'CGCTGATCAGTTTGGAGAGC-3'.
Cycling parameters were 95°C for 3 minutes, then 95°C for 10 seconds, 60°C for 30 seconds and 72°C for 30 seconds for 40 cycles (Bio-Rad iQ5 Thermal Cycler). Each experiment consisted of three biological replicates for each time point plated in duplicate.
Immunocytochemistry
Brains were dissected in 1xPBS and fixed in 4% paraformaldehyde + 4%
sucrose in 1xPBS for 40 minutes at 25°C. Brains were washed three
times with buffer (1xPBS, 1% BSA, 0.1% Triton X-100) for 30 minutes and
incubated with the following primary antibodies at 4°C for 12-16 hours:
dFMRP, 6A15 (Sigma, St Louis, MO), 1:250; mouse CD8 (Caltag, Burlingame, CA),
1:50; FasII (Developmental Studies Hybridoma Bank 1D4, Iowa City, IA), 1:10.
Secondary antibodies, anti-mouse-IgG-Cy3 and anti-rat-IgG-Cy5 (Jackson
ImmunoResearch, West Grove, PA), and anti-mouse-IgG AlexaFluor 488 (Molecular
Probes, Eugene, OR), diluted 1:400 were applied for 2-3 hours at 25°C.
Brains were washed three times for 1 hour before mounting in Vectashield
(Vector Labs, Burlingame, CA) and imaging on a Zeiss Meta 510 confocal
microscope. Images were collected at identical settings and presented as
maximum z-projections. As previously
(Pan et al., 2004), MARCM
branch parameters were determined with LSM software on 3D confocal
z-stacks of each neuron.
|
RESULTS |
|---|
|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
;
Laggerbauer et al., 2001;
Li et al., 2001;
Qin et al., 2005;
Sung et al., 2003). Research
on Drosophila brain development has focused on the first 48 hours
during pupal metamorphosis (Awasaki and
Ito, 2004; Awasaki et al.,
2006; Brown et al.,
2006; Lee et al.,
1999; Marin et al.,
2005; Watts et al.,
2004; Williams and Truman,
2005; Zheng et al.,
2003), but less is known about the latter half of brain
development. However, presumed roles for activity and FMRP coincide with this
time of use-dependent process refinement
(Boothe et al., 1979;
Desai et al., 2002;
Fox and Wong, 2005;
Hinds and Hinds, 1976;
Huttenlocher, 1979;
Lund et al., 1977;
Pan et al., 2004;
Stern et al., 2001;
Turrigiano and Nelson, 2004).
To assay the temporal requirements for dFMRP during brain development versus
maturity, we analyzed developmental time points beginning 60 hours after
puparium formation (APF) and extending into the mature adult (9 days
post-eclosion). Null dfmr1 mutants (dfmr150M) were compared with controls (w1118) at mid-pupal day 3 (P3; 60-72 hours APF), mid-pupal day 4 (P4; 88-96 hours APF), immediately post-eclosion [0-7 hours after eclosion (AE)], and at 1 day (1d), 4 days (4d) and 9 days (9d) in the adult. Total RNA was quantified as µg per head (Fig. 1A). The total amount of RNA was higher in both wild type (WT) and mutants during stages of pupal brain development than at maturity. In dfmr1 nulls, there was a significant increase in RNA only during a restricted window of late pupal development (Fig. 1A). There was a 38% (P3) and 51% (P4) increase in total RNA in the dfmr1 nulls compared with wild type (P3: WT, 0.58±0.14 µg, dfmr1, 0.79±0.08 µg, P=0.025, n=6; P4: WT, 0.47±0.17 µg, dfmr1, 0.71±0.11 µg, P=0.016, n=6). Conversely, there were no significant differences in RNA levels throughout all adult time points (Fig. 1A). Thus, dFMRP functions to negatively regulate RNA levels during a restricted window of late pupal brain development.
As with total RNA, total protein from wild type and mutants was higher in the developing brain than in the mature brain. There was a 2-fold decrease in protein (µg/head) over the 11-day assay period (Fig. 1B). The dfmr1 nulls had significantly elevated protein during a restricted window of development, with elevated protein levels persisting into the early-use period following eclosion (Fig. 1B). There were significant protein increases of 42% (P3), 31% (P4) and 44% (0-7 hours AE) in dfmr1 nulls compared with wild type (P3: WT, 16.7±2.5 µg, dfmr1, 23.73±5.8 µg, P=0.01, n=6; P4: WT, 16.0±3.4 µg, dfmr1, 20.9±3.0 µg, P=0.04, n=6; 0-7 hours: WT, 10.5±3.1 µg, dfmr1, 15.2±2.1 µg, P=0.001, n=12). There were no significant differences in adult animals for several days following this early requirement (Fig. 1B). However, a new period of requirement in the mature brain occurred at nine days, when the dfmr1 null contained 43% more protein than did wild type (9d: WT, 7.68±0.4 µg, dfmr1, 11.0±2.0 µg, P=0.004, n=6). These results suggest that there is a transient window of dFMRP requirement during late pupal development extending into the early-use period of the young adult, followed by a separable requirement much later in the mature brain.
|
;
Lu et al., 2004;
Singh et al., 2007;
Wang et al., 2004), but there
is no indication of whether developmental regulation occurs at the level of
transcription or translation. FMRP represses its own translation
(Ashley, Jr et al., 1993;
Brown et al., 1998;
Ceman et al., 1999;
Schaeffer et al., 2001;
Sung et al., 2000), adding a
complicating factor to understanding the mechanism of regulation. To address
this issue, dfmr1 mRNA and dFMRP protein were assayed from pupal
development to maturity in wild-type animals. dfmr1 mRNA levels were measured by quantitative RT-PCR during the same time points as above, but narrowing the eclosion time point to 0-3 hours AE and including 7 days AE to better define the late expression profile. mRNA levels were normalized to a housekeeping gene, GAPDH2, and reported as a fold change relative to the first time point (Fig. 2A). The expression of dfmr1 mRNA follows two distinct patterns. First, there is high abundance during pupal brain development, which then falls rapidly upon eclosion and remains low for the first day of adult life (Fig. 2A). This pattern is consistent with the reduction in overall RNA abundance during the development to adult transition, as shown in Fig. 1A. The abundance of dfmr1 mRNA, however, increases 50% between days 1 and 4 (P=0.009). In the adult (>7 days post-eclosion), dfmr1 message remains elevated to levels similar to those at pupation (Fig. 2A). This second period of dfmr1 mRNA elevation is divergent from the total RNA profile, which declines into maturity (Fig. 1A). Thus, there are two distinct phases of dfmr1 transcription, with one peak during late brain maturation and a second comparable plateau in the fully mature brain.
The dFMRP protein level in the brain was measured by both immunoblot and immunocytochemistry. Western blot analyses were performed on heads from single animals, in duplicate, from seven developmental time points (Fig. 2B). As predicted from the dfmr1 mRNA levels, dFMRP protein expression is maximal during late stages of brain development and decreases during the first day post-eclosion (Fig. 2B). At maturity (>1 day), dFMRP protein was difficult to detect on western blots and, surprisingly, no longer correlated with dfmr1 mRNA levels (compare Fig. 2A with 2B). Although dfmr1 mRNA was high 4 days AE, dFMRP protein levels remained minimal. This expression profile was also evident by brain immunocytochemistry (Fig. 2C). During late pupation, dFMRP is expressed at high levels throughout the entire brain, primarily in neuronal soma, whereas in the mature brain dFMRP expression is strongly reduced, except in limited central brain regions. These data show that dfmr1 mRNA and dFMRP protein levels correlate closely during brain development, but that dfmr1 transcription and translation become uncoupled in the mature brain.
To test whether dFMRP function is similarly regulated in the same
developmental pattern, we examined the dFMRP target
chickadee/profilin
(Reeve et al., 2005).
Actin-binding Chickadee is upregulated in the absence of dFMRP, but nothing is
known about its developmental regulation. We performed quantitative RT-PCR for
chickadee mRNA on dfmr1-null and wild-type extracts from
development through maturity (Fig.
3A). In wild type, the amount of chickadee mRNA nearly
doubles during late pupation and then falls precipitously after eclosion, and
remains low in the adult brain (Fig.
3A). In dfmr1 mutants, chickadee mRNA levels
follow a similar profile with two important differences. First, the P4 spike
is approximately three times the level of mRNA at P3 and is 46% increased over
controls. Second, by 9 days AE, chickadee mRNA levels are actually
decreased (43%) compared with controls
(Fig. 3A). Wild-type protein
levels are maximal during pupal day 4 and rapidly fall at eclosion
(Fig. 3B), correlating with the
mRNA levels. By contrast, Chickadee protein in dfmr1 nulls is
maintained at aberrantly high levels following eclosion and through the
early-use period in the young adult (Fig.
3B). No protein changes are apparent at 9 days when
chickadee mRNA levels are reduced in the mutant. These findings are
consistent with distinct developmentally controlled roles for dFMRP function
during late-brain development/early-use refinement periods and at
maturity.
dFMRP regulates a late development period of axonal pruning
The Mushroom Body (MB) is a primary learning/memory center in the
Drosophila brain, and is therefore the focus of behavioral,
structural and functional studies
(Margulies et al., 2005;
Zars et al., 2000).
dfmr1-null MB neurons exhibit increased axonal growth and
over-branching (Pan et al.,
2004). However, these analyses were carried out in unstaged brains
and the cause of the defects was not determined. As dFMRP expression and
function is differentially regulated during development and maturity, we
re-examined MB neuron axon morphogenesis throughout development. The Mosaic
Analysis with a Repressible Cell Marker (MARCM) genetic clonal technique
(Lee and Luo, 2001) was used
to label single homozygous mutant MB gamma neurons
(Fig. 4A). This method allows
for the direct visualization of individual neuron structure in the intact
brain, and also permits direct analysis of the cell-autonomous function of
dFMRP in that single neuron.
|
|
Eclosion heralds the onset of use-dependent activity in brain circuits. In control neurons at eclosion, there were decreases in both the overall branch length and the branch number relative to P4, which is consistent with a pruning mechanism (Fig. 4B). The specific branches pruned were short processes (<5 µm; Fig. 4B, inset arrows). By contrast, dfmr1-null neurons had no significant decrease in axon branches during this normal pruning period. As a result, dfmr1-null neurons had 24% more and 30% longer axonal branches than controls immediately following eclosion (P=0.02 and P=0.003, respectively; Fig. 4B). In 4 day AE controls, when dFMRP protein is minimally expressed (Fig. 2B), both the branch number and length were static. Conversely, pruning that was absent in dfmr1-null neurons post-eclosion manifested by 4 days AE (Fig. 4B). Again, primarily branches <5 µm were pruned by this mechanism (Fig. 4B, inset). Interestingly, the excessive branching in dfmr1-null neurons present in young animals was absent in mature adults (number and length: P=0.17, 0.84, respectively). In fact, over-pruning is evidenced by a 35% decrease in small (<5 µm) axon branches in dfmr1-null neurons when compared with controls at maturity (P=0.01; Fig. 4B).
The overexpression of dFMRP causes under-branching of gamma neurons, the
inverse of the dfmr1-null phenotype
(Pan et al., 2004). Therefore,
we examined the developmental profile of single-cell MARCM clones
overexpressing (OE) dFMRP. At P3, underbranching is already apparent with 47%
fewer branches (P=0.002), which were on average 61% shorter
(P<0.001) than controls (Fig.
5A). The axon growth that normally occurs during P4, fails in
dFMRP OE neurons, but branch number and length remain unchanged
[Fig. 5B; number: P3,
5.5±3.6 (n=21); P4, 4.1±1.8 (n=20); length:
P3, 25.5±11.6 µm (n=21); P4, 20.5±8.7 µm
(n=20)]. This undergrowth persists into maturity at 4 days
post-eclosion and is worsened by aberrant excessive pruning immediately after
eclosion. Axon branch number in OE neurons decreases by 
30%
(P=0.03) upon eclosion [Fig.
5B; P4, 4.1±1.8 (n=20); 0-3 hours AE,
2.9±2.4 (n=17)]. The only branches available for pruning are
the short filipodial-like branches persistent throughout late pupation
(Fig. 5A, insets). Thus, dFMRP
overexpression inhibits axonal elongation and accelerates post-eclosion
process refinement.
|
|
30 other animals and were maintained in 12:12 light/dark
conditions until 4 days AE.
FMRP expression has been reported to be elevated in response to sensory and
neuronal stimulation (Gabel et al.,
2004; Hou et al.,
2006; Irwin et al.,
2005; Irwin et al.,
2000; Todd and Mack,
2000; Todd et al.,
2003; Valentine et al.,
2000; Weiler et al.,
1997). Therefore, we first tested whether dFMRP expression was
affected by sensory deprivation. The abundance of dfmr1 mRNA was
assayed by quantitative RT-PCR (Fig.
6A). The dfmr1 mRNA level was reduced by 20% in SD
animals (P=0.02). Western analyses on single heads showed dFMRP
protein levels were also significantly reduced (P=0.04;
Fig. 6B), by 20%, in SD
animals (seven pairs of control and SD heads, three trials;
Fig. 6C). Thus, sensory input
activity positively regulates dFMRP mRNA and protein levels.
To refine these analyses to specific sensory modalities, we next assayed
dFMRP expression in two sensory transduction mutants: Or83b, which
eliminates a widely expressed odorant co-receptor required for olfaction of a
broad spectrum of odors; and ninaE, which eliminates rhodopsin
required for visual phototransduction
(Larsson et al., 2004;
O'Tousa et al., 1985). By
single head western analysis, there was a comparable 20% reduction in
dFMRP expression in both mutants (Fig.
7A,B), demonstrating that vision and olfaction positively regulate
dFMRP. Importantly, there was also a significant, opposing increase in the
dFMRP target chickadee/profilin
(Fig. 7A,B). Together, these
results demonstrate that neural activity driven by sensory input is a positive
regulator of dFMRP expression and function.
We next assayed the effect of sensory input activity on MB axon pruning (Fig. 8). Single MB gamma neuron MARCM clones were analyzed in animals grown until 4 days AE in normal versus SD conditions. SD animals displayed a significant 34% (P=0.03) increase in axon branch number in control neurons, but a highly significant 86% (P=0.001) increase in dfmr1-null neurons (Fig. 8A). Axon branch length did not increase in control neurons, but was significantly (P<0.001) increased by 47% in dfmr1-null neurons. As a consequence, after 4 days of sensory deprivation, dfmr1-null axon branches were 30% longer than controls (P=0.002; Fig. 5A). The failure to prune was most apparent in dfmr1-null neurons in branches less than 5 µm in length (P<0.001), but was also apparent in longer branches binned between 5 and 10 µm (P<0.001).
|
|
Neuronal activation enhances dFMRP-dependent axon pruning
Because sensory-deprivation blocks pruning, inducing neuronal activity may
increase pruning. To test this hypothesis, we generated MARCM clones
expressing Chlamydomonas reinhardtii light-gated channelrhodopsin-2
(CHR2) (Nagel et al., 2003).
In the presence of the cofactor all-trans retinal, CHR2 channels open in
response to blue light (Schroll et al.,
2006). We generated recombinant MARCM CHR2 animals in the control
or in the dfmr1 null mutant. Flies were fed all-trans retinal or an
ethanol vehicle throughout development. Upon eclosion (<12 hours AE), both
genotypes were subjected to 1-Hz blue light (470 nm) pulses for 6 hours.
Brains were dissected and single-cell gamma neuron MARCM clones analyzed
(Fig. 9A).
Neuronal activation of MB neurons in CHR2-expressing clones resulted in a significant 21% reduction (P=0.02) in the total number of axonal branches in animals fed the retinal cofactor compared with vehicle-fed controls (Fig. 9B). Importantly, small axon branches (<5 µm) that were not pruned during sensory deprivation were reduced 27% by neuronal activation (Fig. 9C). Induced pruning was totally dependent on dFMRP, as no effect was observed in dfmr1-null neurons expressing CHR2 (Fig. 9). Thus, pruning of axon branches during the early use-refinement phase requires both neuronal electrical activity and dFMRP function.
Two-phase dFMRP requirement: axonogenesis and activity-dependent pruning
The quantification of neuronal architecture reveals two phases of dFMRP
regulation in the MB: axon growth during late-stage pupation and
activity-dependent axon pruning during the early-use phase following eclosion.
The cumulative length of axon branches was significantly increased in
dfmr1-null neurons during P3/P4 [WT: P3, 66.1±30.2 µm
(n=17), P4, 86.5±20.6 µm (n=18), P=0.03;
dfmr1: P3, 82.8±38.3 µm (n=19), P4,
108.1±30.7 µm (n=19), P=0.02;
Fig. 10A]. By contrast, the
number of axon branches was not changed during this development period [WT:
P3, 10.5±4.5 (n=17), P4, 11.4±2.8 (n=18),
P=0.5; dmfr1: P3, 12.2±6.3 (n=19), P4,
10.5±3.2 (n=19), P=0.4;
Fig. 10B]. Thus, dFMRP
regulates axon growth but not branching during late-stage brain
development.
|
|
Sensory deprivation revealed that dFMRP function is activity dependent. In control SD neurons, there was a significant increase in axon branch number [4d control: 9.0±3.3 (n=24); 4d SD: 12.1±3.8 (n=20), P=0.028; Fig. 10B], with a non-significant tendency towards greater length [4d control: 68.9±27.8 µm (n=24); 4d SD: 82.7±21.2 µm (n=20), P=0.17]. In dfmr1-null SD neurons, there was a more pronounced increase in branch number [4d: 7.23±3.1 (n=22); 4d SD: 13.4±3.5 (n=24), P<0.001; Fig. 10B], and branch length was also increased [4d: 72.9±29.7 µm (n=22); 4d SD: 107.3±27.8 µm (n=24), P<0.001; Fig. 10A]. Acute stimulation following SD rearing was unable to induce pruning in either dfmr1-null or control neurons (Fig. 10). Thus, sensory input activity strongly influences both the timing and extent of the dFMRP-dependent pruning of axonal processes.
|
DISCUSSION |
|---|
|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
; Lu et al.,
2004; Singh et al.,
2007; Wang et al.,
2004), very little work has characterized the temporal
requirements of FMRP. We therefore employed the Drosophila FraX model
to analyze dFMRP-dependent processes in neuronal development. Our initial
findings highlighted roles for dFMRP in (1) late stages of brain development
and (2) very early-use circuit refinement, and we therefore focused on these
mechanisms.
In the absence of dFMRP, elevated levels of total RNA/protein are evident
during a restricted period of late pupal brain development, with the protein
increase persisting into an early-use refinement period
(Fig. 1). These increases are
transient and disappear in the mature brain thereby defining a limited
developmental window of dFMRP function. The increase in protein is predicted
as FMRP/dFMRP negatively regulates translation
(Khandjian et al., 2004;
Laggerbauer et al., 2001;
Li et al., 2001;
Qin et al., 2005;
Sung et al., 2003). The
elevated RNA is more surprising. dFMRP/FMRP can both negatively and positively
regulate mRNA stability (Xu et al.,
2004; Zalfa et al.,
2007; Zhang et al.,
2007), and, therefore, dFMRP may have a developmentally-restricted
role primarily as a negative regulator of mRNA stability. Alternatively, the
RNA increase may be caused by elevated transcription, via an uncharacterized
direct or indirect transcriptional inhibition function of dFMRP. Because the
increase in total protein/RNA is not biased towards selected dFMRP targets,
these results suggest globally upregulated transcription/translation in the
dfmr1 mutant brain during a restricted window of late maturation and
early-use refinement.
During brain development, dfmr1 mRNA and dFMRP protein levels
tightly correlate with the above changes, but, surprisingly, dfmr1
mRNA levels inversely correlate with dFMRP protein levels in the mature brain
(Fig. 2). By 4 days AE,
dfmr1 mRNA levels rise to levels nearly as high as those present
during development, but dFMRP protein is maintained at a basal level in the
mature brain. This change strongly suggests a distinct switch in dFMRP
regulation, in which transcription and translation become uncoupled. Because
dFMRP/FMRP represses the translation of its own mRNA
(Ashley, Jr et al., 1993;
Brown et al., 1998;
Ceman et al., 1999;
Schaeffer et al., 2001;
Sung et al., 2000), it is
tempting to speculate that this negative-feedback mechanism specifically
regulates dFMRP in the mature brain. FMRP modulates synaptic plasticity at
maturity, as evidenced by decreased LTP and enhanced LTD in fmr1
knock-out (KO) mice (Hou et al.,
2006; Huber et al.,
2002; Koekkoek et al.,
2005; Larson et al.,
2005; Li et al.,
2002; Wilson and Cox,
2007; Zhao et al.,
2005). Consistent with such a mature function, elevated total
protein levels are once again evident in the fully mature dfmr1-null
brain (Fig. 1). A similar
increase in cerebral protein synthesis occurs in adult fmr1-KO mice
(Qin et al., 2005). Together,
these data suggest that a switch in dFMRP/FMRP regulation defines separate
windows of function in development versus maturity.
It was crucial to determine whether dFMRP function correlated with its
developmental expression profile. A known dFMRP target is
chickadee/profilin; dFMRP binds chickadee mRNA and
negatively regulates its translation
(Reeve et al., 2005).
Importantly, the dynamics of chickadee misregulation in the
dfmr1-null brain indicate that the dFMRP functional requirement
mirrors its developmental expression profile
(Fig. 3). Chickadee expression
normally peaks during late-stage brain development (P4), and it is during this
development window, and shortly following, that overexpression is manifested
in the dfmr1-null brain. Generally, the increase in
chickadee transcripts parallels the increase in protein, suggesting
that dFMRP regulation may be at the level of the mRNA, for example, by
affecting mRNA stability. dFMRP reportedly interacts with miRNA machinery to
control mRNA levels of the sodium channel Pickpocket1
(Xu et al., 2004). A similar
mechanism for chickadee regulation would be consistent with our
results. Interestingly, the increase in Chickadee protein levels coincides
with the period of use-dependent neural circuit refinement at eclosion. At
least one dFMRP/FMRP target mRNA, futsch (MAP1B), is
regulated specifically at postnatal day 10 in fmr1-KO mice
(Lu et al., 2004). These new
insights suggest it will be vital to ascertain the developmental expression of
all putative FMRP targets in the context of these distinct windows of
regulation in order to validate in vivo functions.
During the peak period of dFMRP expression, there are two phases of
dFMRP-dependent axon maturation. During late pupal development, dFMRP inhibits
axon elongation, with dfmr1-null neurons exhibiting branches 25%
longer than controls (Fig. 4).
This function is restricted to very late stages (P4), with no differences in
branch length or number being observed earlier (P3). Immediately upon
eclosion, dFMRP is required for use-dependent pruning, causing a decrease in
both axon branch length and number (Fig.
4). Pruning is most evident in the smallest presynaptic branches
(<5 µm) and occurs quickly (hours) following the onset of adult
activity. Targeted overexpression of dFMRP causes inverse defects in both
phases of dFMRP requirement (Fig.
5). Axon undergrowth is apparent early (P3) and axons fail to grow
later (P4). Axon branches present in these neurons are short, filipodial-like
structures, and, at eclosion, there is excessive pruning to result in 30%
fewer branches than in P4 and 3 times fewer branches than in controls
(Fig. 5). Thus, both
axonogenesis and axon branch pruning are bidirectionally modified by inverse
changes in dFMRP expression.
Blocking sensory input activity maintains dFMRP in its early development
regulative state, with a correlative reduction in both dfmr1 mRNA and
dFMRP protein (Fig. 6). Both
olfactory (Or83b) and phototransduction (ninaE) mutants
(Larsson et al., 2004;
O'Tousa et al., 1985)
similarly suppress dFMRP levels, indicating that these two primary modes of
brain sensory input positively drive dFMRP expression
(Fig. 7). Similarly, mammalian
FMRP expression is elevated following activity stimulation by both
environmental enrichment and mGluR signaling activation
(Gabel et al., 2004;
Hou et al., 2006;
Irwin et al., 2005;
Todd and Mack, 2000;
Todd et al., 2003;
Valentine et al., 2000;
Weiler et al., 1997). Blocking
mGluR activity in Drosophila and mice can rescue some dfmr1
defects, including impaired learning and memory
(McBride et al., 2005;
Yan et al., 2005;
Pan et al., 2008). From these
similar findings, it is tempting to suggest that dFMRP/FMRP may function
downstream of mGluR signaling activity, perhaps differentially in development
versus maturity. Importantly, both Or83b and ninaE sensory
mutants cause elevation of chickadee/profilin at the same
time dFMRP is suppressed (Fig.
7). This finding is consistent with activity-dependent regulation
of dFMRP to regulate chickadee/profilin expression.
This study shows for the first time that Drosophila neurons
undergo activity-dependent pruning coincident with the onset of use. In the
absence of dFMRP, pruning does not occur during the normal developmental
window (Fig. 4). Indeed,
blocking sensory input activity leads to further increases in the axon branch
number and length in dfmr1-null neurons. Moreover, at maturity,
sensory stimulation following sensory deprivation does not induce pruning,
probably because the dFMRP level has fallen too low. We hypothesize that there
is a threshold of dFMRP required for efficient activity-dependent pruning
during the early-use period, which is normally defined by the window of high
dFMRP expression. Reinstated sensory stimulation following sensory deprivation
does cause a significant dFMRP-dependent increase in the number of long axon
branches (>10 µm; Fig.
8). These data are consistent with the need for high dFMRP
expression to both limit axonal growth and mediate the early-use refinement of
circuits. Importantly, we confirmed, by using targeted expression of the
exogenous light-gated channelrhodopsin-2 channel
(Schroll et al., 2006), that
neuronal activation bidirectionally drives the pruning process. Light-driven
activation of CHR2 channels induces pruning of the same small (<5 µm)
axonal processes that aberrantly persist in the dfmr1-null brain
(Fig. 9). As predicted, the
induced pruning process fails to occur in the absence of dFMRP.
Delayed pruning eventually occurs in dfmr1-null neurons to
ultimately rescue the overbranching defect present in younger animals. A
similar transient elongation of dendritic spines occurs in young postnatal
Fmr1-KO mice, although a secondary overgrowth phenotype may appear
months later in adult animals (Galvez and
Greenough, 2005; Nimchinsky et
al., 2001). In Drosophila, the delayed axon pruning in
dfmr1-null neurons actually goes too far, resulting in reduced
neuronal complexity in mature adult animals
(Fig. 10). The small
presynaptic branches (<5 µm) are reduced 35% in dfmr1-null
neurons compared with controls at 4 days. Because pruning normally occurs very
rapidly (<3 hours after eclosion), coincident with initial use, it is
likely that the pruning process is strictly controlled for that developmental
time. By delaying pruning in the absence of dFMRP, it appears that other
factors that buffer the extent of process elimination fail to provide adequate
regulation of the mechanism. Indeed, this mitigation may be a function of
dFMRP itself, as dFMRP levels drop drastically immediately following the
normal pruning window. FMRP potentially regulates many proteins involved in a
diverse set of functions (Brown et al.,
2001; Miyashiro et al.,
2003; Zhang et al.,
2005). Understanding the developmental regulation of proteins that
associate with FMRP and FMRP target mRNAs will be crucial to unraveling the
underlying pruning mechanisms of activity-dependent neural circuit
refinement.
|
ACKNOWLEDGMENTS |
|---|
|
REFERENCES |
|---|
|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
Antar, L. N., Li, C., Zhang, H., Carroll, R. C. and Bassell, G.
J. (2006). Local functions for FMRP in axon growth cone
motility and activity-dependent regulation of filopodia and spine synapses.
Mol. Cell. Neurosci. 32,37
-48.[CrossRef][Medline]
Aschrafi, A., Cunningham, B. A., Edelman, G. M. and Vanderklish,
P. W. (2005). The fragile X mental retardation protein and
group I metabotropic glutamate receptors regulate levels of mRNA granules in
brain. Proc. Natl. Acad. Sci. USA
102,2180
-2185.
Ashley, C. T., Jr, Wilkinson, K. D., Reines, D. and Warren, S.
T. (1993). FMR1 protein: conserved RNP family domains and
selective RNA binding. Science
262,563
-566.
Awasaki, T. and Ito, K. (2004). Engulfing
action of glial cells is required for programmed axon pruning during
Drosophila metamorphosis. Curr. Biol.
14,668
-677.[CrossRef][Medline]
Awasaki, T., Tatsumi, R., Takahashi, K., Arai, K., Nakanishi,
Y., Ueda, R. and Ito, K. (2006). Essential role of the
apoptotic cell engulfment genes draper and ced-6 in programmed axon pruning
during Drosophila metamorphosis. Neuron
50,855
-867.[CrossRef][Medline]
Bear, M. F., Huber, K. M. and Warren, S. T.
(2004). The mGluR theory of fragile X mental retardation.
Trends Neurosci. 27,370
-377.[CrossRef][Medline]
Belmonte, M. K. and Bourgeron, T. (2006).
Fragile X syndrome and autism at the intersection of genetic and neural
networks. Nat. Neurosci.
9,1221
-1225.[CrossRef][Medline]
Boccia, M. L. and Roberts, J. E. (2000).
Behavior and autonomic nervous system function assessed via heart period
measures: the case of hyperarousal in boys with fragile X syndrome.
Behav. Res. Methods Instrum. Comput.
32, 5-10.[Medline]
Boothe, R. G., Greenough, W. T., Lund, J. S. and Wrege, K.
(1979). A quantitative investigation of spine and dendrite
development of neurons in visual cortex (area 17) of Macaca nemestrina
monkeys. J. Comp. Neurol.
186,473
-489.[CrossRef][Medline]
Brown, H. L., Cherbas, L., Cherbas, P. and Truman, J. W.
(2006). Use of time-lapse imaging and dominant negative receptors
to dissect the steroid receptor control of neuronal remodeling in Drosophila.
Development 133,275
-285.
Brown, V., Small, K., Lakkis, L., Feng, Y., Gunter, C.,
Wilkinson, K. D. and Warren, S. T. (1998). Purified
recombinant Fmrp exhibits selective RNA binding as an intrinsic property of
the fragile X mental retardation protein. J. Biol.
Chem. 273,15521
-15527.
Brown, V., Jin, P., Ceman, S., Darnell, J. C., O'Donnell, W. T.,
Tenenbaum, S. A., Jin, X., Feng, Y., Wilkinson, K. D., Keene, J. D. et al.
(2001). Microarray identification of FMRP-associated brain mRNAs
and altered mRNA translational profiles in fragile X syndrome.
Cell 107,477
-487.[CrossRef][Medline]
Ceman, S., Brown, V. and Warren, S. T. (1999).
Isolation of an FMRP-associated messenger ribonucleoprotein particle and
identification of nucleolin and the fragile X-related proteins as components
of the complex. Mol. Cell. Biol.
19,7925
-7932.
Comery, T. A., Harris, J. B., Willems, P. J., Oostra, B. A.,
Irwin, S. A., Weiler, I. J. and Greenough, W. T. (1997).
Abnormal dendritic spines in fragile X knockout mice: maturation and pruning
deficits. Proc. Natl. Acad. Sci. USA
94,5401
-5404.
De Diego Otero, Y., Severijnen, L. A., van Cappellen, G.,
Schrier, M., Oostra, B. and Willemsen, R. (2002). Transport
of fragile X mental retardation protein via granules in neurites of PC12
cells. Mol. Cell. Biol.
22,8332
-8341.
Desai, N. S., Cudmore, R. H., Nelson, S. B. and Turrigiano, G.
G. (2002). Critical periods for experience-dependent synaptic
scaling in visual cortex. Nat. Neurosci.
5, 783-789.[Medline]
Feng, Y., Gutekunst, C. A., Eberhart, D. E., Yi, H., Warren, S.
T. and Hersch, S. M. (1997). Fragile X mental retardation
protein: nucleocytoplasmic shuttling and association with somatodendritic
ribosomes. J. Neurosci.
17,1539
-1547.
Ferrari, F., Mercaldo, V., Piccoli, G., Sala, C., Cannata, S.,
Achsel, T. and Bagni, C. (2007). The fragile X mental
retardation protein-RNP granules show an mGluR-dependent localization in the
post-synaptic spines. Mol. Cell. Neurosci.
34,343
-354.[CrossRef][Medline]
Fox, K. and Wong, R. O. (2005). A comparison of
experience-dependent plasticity in the visual and somatosensory systems.
Neuron 48,465
-477.[CrossRef][Medline]
Freund, L. S. and Reiss, A. L. (1991).
Cognitive profiles associated with the fra(X) syndrome in males and females.
Am. J. Med. Genet. 38,542
-547.[CrossRef][Medline]
Gabel, L. A., Won, S., Kawai, H., McKinney, M., Tartakoff, A. M.
and Fallon, J. R. (2004). Visual experience regulates
transient expression and dendritic localization of fragile X mental
retardation protein. J. Neurosci.
24,10579
-10583.
Galvez, R. and Greenough, W. T. (2005).
Sequence of abnormal dendritic spine development in primary somatosensory
cortex of a mouse model of the fragile X mental retardation syndrome.
Am. J. Med. Genet. A
135,155
-160.[Medline]
Galvez, R., Gopal, A. R. and Greenough, W. T.
(2003). Somatosensory cortical barrel dendritic abnormalities in
a mouse model of the fragile X mental retardation syndrome. Brain
Res. 971,83
-89.[CrossRef][Medline]
Galvez, R., Smith, R. L. and Greenough, W. T.
(2005). Olfactory bulb mitral cell dendritic pruning
abnormalities in a mouse model of the Fragile-X mental retardation syndrome:
further support for FMRP's involvement in dendritic development.
Brain Res. Dev. Brain Res.
157,214
-216.[CrossRef][Medline]
Greenough, W. T., Hwang, H. M. and Gorman, C.
(1985). Evidence for active synapse formation or altered
postsynaptic metabolism in visual cortex of rats reared in complex
environments. Proc. Natl. Acad. Sci. USA
82,4549
-4552.
Grossman, A. W., Elisseou, N. M., McKinney, B. C. and Greenough,
W. T. (2006). Hippocampal pyramidal cells in adult Fmr1
knockout mice exhibit an immature-appearing profile of dendritic spines.
Brain Res. 1084,158
-164.[CrossRef][Medline]
Hayashi, M. L., Rao, B. S., Seo, J. S., Choi, H. S., Dolan, B.
M., Choi, S. Y., Chattarji, S. and Tonegawa, S. (2007).
Inhibition of p21-activated kinase rescues symptoms of fragile X syndrome in
mice. Proc. Natl. Acad. Sci. USA
104,11489
-11494.
Hinds, J. W. and Hinds, P. L. (1976). Synapse
formation in the mouse olfactory bulb. I. Quantitative studies. J.
Comp. Neurol. 169,15
-40.[CrossRef][Medline]
Hou, L., Antion, M. D., Hu, D., Spencer, C. M., Paylor, R. and
Klann, E. (2006). Dynamic translational and proteasomal
regulation of fragile X mental retardation protein controls mGluR-dependent
long-term depression. Neuron
51,441
-454.[CrossRef][Medline]
Huber, K. M., Gallagher, S. M., Warren, S. T. and Bear, M.
F. (2002). Altered synaptic plasticity in a mouse model of
fragile X mental retardation. Proc. Natl. Acad. Sci.
USA 99,7746
-7750.
Huttenlocher, P. R. (1979). Synaptic density in
human frontal cortex - developmental changes and effects of aging.
Brain Res. 163,195
-205.[CrossRef][Medline]
Irwin, S. A., Swain, R. A., Christmon, C. A., Chakravarti, A.,
Weiler, I. J. and Greenough, W. T. (2000). Evidence for
altered Fragile-X mental retardation protein expression in response to
behavioral stimulation. Neurobiol. Learn. Mem.
73, 87-93.[CrossRef][Medline]
Irwin, S. A., Patel, B., Idupulapati, M., Harris, J. B.,
Crisostomo, R. A., Larsen, B. P., Kooy, F., Willems, P. J., Cras, P.,
Kozlowski, P. B. et al. (2001). Abnormal dendritic spine
characteristics in the temporal and visual cortices of patients with fragile-X
syndrome: a quantitative examination. Am. J. Med.
Genet. 98,161
-167.[CrossRef][Medline]
Irwin, S. A., Idupulapati, M., Gilbert, M. E., Harris, J. B.,
Chakravarti, A. B., Rogers, E. J., Crisostomo, R. A., Larsen, B. P., Mehta,
A., Alcantara, C. J. et al. (2002). Dendritic spine and
dendritic field characteristics of layer V pyramidal neurons in the visual
cortex of fragile-X knockout mice. Am. J. Med. Genet.
111,140
-146.[CrossRef][Medline]
Irwin, S. A., Christmon, C. A., Grossman, A. W., Galvez, R.,
Kim, S. H., DeGrush, B. J., Weiler, I. J. and Greenough, W. T.
(2005). Fragile X mental retardation protein levels increase
following complex environment exposure in rat brain regions undergoing active
synaptogenesis. Neurobiol Learn. Mem.
83,180
-187.[CrossRef][Medline]
Ivanco, T. L. and Greenough, W. T. (2002).
Altered mossy fiber distributions in adult Fmr1 (FVB) knockout mice.
Hippocampus 12,47
-54.[CrossRef][Medline]
Khandjian, E. W., Fortin, A., Thibodeau, A., Tremblay, S., Cote,
F., Devys, D., Mandel, J. L. and Rousseau, F. (1995). A
heterogeneous set of FMR1 proteins is widely distributed in mouse tissues and
is modulated in cell culture. Hum. Mol. Genet.
4, 783-789.
Khandjian, E. W., Huot, M. E., Tremblay, S., Davidovic, L.,
Mazroui, R. and Bardoni, B. (2004). Biochemical evidence for
the association of fragile X mental retardation protein with brain
polyribosomal ribonucleoparticles. Proc. Natl. Acad. Sci.
USA 101,13357
-13362.
Koekkoek, S. K., Yamaguchi, K., Milojkovic, B. A., Dortland, B.
R., Ruigrok, T. J., Maex, R., De Graaf, W., Smit, A. E., VanderWerf, F.,
Bakker, C. E. et al. (2005). Deletion of FMR1 in Purkinje
cells enhances parallel fiber LTD, enlarges spines, and attenuates cerebellar
eyelid conditioning in Fragile X syndrome. Neuron
47,339
-352.[CrossRef][Medline]
Laggerbauer, B., Ostareck, D., Keidel, E. M., Ostareck-Lederer,
A. and Fischer, U. (2001). Evidence that fragile X mental
retardation protein is a negative regulator of translation. Hum.
Mol. Genet. 10,329
-338.
Larson, J., Jessen, R. E., Kim, D., Fine, A. K. and du Hoffmann,
J. (2005). Age-dependent and selective impairment of
long-term potentiation in the anterior piriform cortex of mice lacking the
fragile X mental retardation protein. J. Neurosci.
25,9460
-9469.
Larsson, M. C., Domingos, A. I., Jones, W. D., Chiappe, M. E.,
Amrein, H. and Vosshall, L. B. (2004). Or83b encodes a
broadly expressed odorant receptor essential for Drosophila olfaction.
Neuron 43,703
-714.[CrossRef][Medline]
Lee, T. and Luo, L. (2001). Mosaic analysis
with a repressible cell marker (MARCM) for Drosophila neural development.
Trends Neurosci. 24,251
-254.[CrossRef][Medline]
Lee, T., Lee, A. and Luo, L. (1999).
Development of the Drosophila mushroom bodies: sequential generation of three
distinct types of neurons from a neuroblast.
Development 126,4065
-4076.[Abstract]
Li, J., Pelletier, M. R., Perez Velazquez, J. L. and Carlen, P.
L. (2002). Reduced cortical synaptic plasticity and GluR1
expression associated with fragile X mental retardation protein deficiency.
Mol. Cell. Neurosci. 19,138
-151.[CrossRef][Medline]
Li, Z., Zhang, Y., Ku, L., Wilkinson, K. D., Warren, S. T. and
Feng, Y. (2001). The fragile X mental retardation protein
inhibits translation via interacting with mRNA. Nucleic Acids
Res. 29,2276
-2283.
Lu, R., Wang, H., Liang, Z., Ku, L., O'Donnell, W., T., Li, W.,
Warren, S. T. and Feng, Y. (2004). The fragile X protein
controls microtubule-associated protein 1B translation and microtubule
stability in brain neuron development. Proc. Natl. Acad. Sci.
USA 101,15201
-15206.
Lund, J. S., Boothe, R. G. and Lund, R. D.
(1977). Development of neurons in the visual cortex (area 17) of
the monkey (Macaca nemestrina): a Golgi study from fetal day 127 to postnatal
maturity. J. Comp. Neurol.
176,149
-188.[CrossRef][Medline]
Margulies, C., Tully, T. and Dubnau, J. (2005).
Deconstructing memory in Drosophila. Curr. Biol.
15,R700
-R713.[CrossRef][Medline]
Marin, E. C., Watts, R. J., Tanaka, N. K., Ito, K. and Luo,
L. (2005). Developmentally programmed remodeling of the
Drosophila olfactory circuit. Development
132,725
-737.
McBride, S. M., Choi, C. H., Wang, Y., Liebelt, D., Braunstein,
E., Ferreiro, D., Sehgal, A., Siwicki, K. K., Dockendorff, T. C., Nguyen, H.
T. et al. (2005). Pharmacological rescue of synaptic
plasticity, courtship behavior, and mushroom body defects in a Drosophila
model of fragile X syndrome. Neuron
45,753
-764.[CrossRef][Medline]
McKinney, B. C., Grossman, A. W., Elisseou, N. M. and Greenough,
W. T. (2005). Dendritic spine abnormalities in the occipital
cortex of C57BL/6 Fmr1 knockout mice. Am. J. Med. Genet. B
Neuropsychiatr. Genet. 136,98
-102.[Medline]
Miyashiro, K. Y., Beckel-Mitchener, A., Purk, T. P., Becker, K.
G., Barret, T., Liu, L., Carbonetto, S., Weiler, I. J., Greenough, W. T. and
Eberwine, J. (2003). RNA cargoes associating with FMRP reveal
deficits in cellular functioning in Fmr1 null mice.
Neuron 37,417
-431.[CrossRef][Medline]
Nagel, G., Szellas, T., Huhn, W., Kateriya, S., Adeishvili, N.,
Berthold, P., Ollig, D., Hegemann, P. and Bamberg, E. (2003).
Channelrhodopsin-2, a directly light-gated cation-selective membrane channel.
Proc. Natl. Acad. Sci. USA
100,13940
-13945.
Nimchinsky, E. A., Oberlander, A. M. and Svoboda, K.
(2001). Abnormal development of dendritic spines in FMR1
knock-out mice. J. Neurosci.
21,5139
-5146.
Ostroff, L. E., Fiala, J. C., Allwardt, B. and Harris, K. M.
(2002). Polyribosomes redistribute from dendritic shafts into
spines with enlarged synapses during LTP in developing rat hippocampal slices.
Neuron 35,535
-545.[CrossRef][Medline]
O'Tousa, J. E., Baehr, W., Martin, R. L., Hirsh, J., Pak, W. L.
and Applebury, M. L. (1985). The Drosophila ninaE gene
encodes an opsin. Cell
40,839
-850.[CrossRef][Medline]
Pan, L., Zhang, Y. Q., Woodruff, E. and Broadie, K.
(2004). The Drosophila fragile X gene negatively regulates
neuronal elaboration and synaptic differentiation. Curr.
Biol. 14,1863
-1870.[CrossRef][Medline]
Pan, L., Woodruff, E., 3rd, Liang, P. and Broadie, K.
(2008). Mechanistic relationships between Drosophila fragile X
mental retardation protein and metabotropic glutamate receptor A signaling.
Mol. Cell. Neurosci. doi:10.1016/j.mcn.2008.1.003
Qin, M., Kang, J., Burlin, T. V., Jiang, C. and Smith, C. B.
(2005). Postadolescent changes in regional cerebral protein
synthesis: an in vivo study in the FMR1 null mouse. J.
Neurosci. 25,5087
-5095.
Reeve, S. P., Bassetto, L., Genova, G. K., Kleyner, Y., Leyssen,
M., Jackson, F. R. and Hassan, B. A. (2005). The Drosophila
fragile X mental retardation protein controls actin dynamics by directly
regulating profilin in the brain. Curr. Biol.
15,1156
-1163.[CrossRef][Medline]
Restivo, L., Ferrari, F., Passino, E., Sgobio, C., Bock, J.,
Oostra, B. A., Bagni, C. and Ammassari-Teule, M. (2005).
Enriched environment promotes behavioral and morphological recovery in a mouse
model for the fragile X syndrome. Proc. Natl. Acad. Sci.
USA 102,11557
-11562.
Rudelli, R. D., Brown, W. T., Wisniewski, K., Jenkins, E. C.,
Laure- Kamionowska, M., Connell, F. and Wisniewski, H. M.
(1985). Adult fragile X syndrome. Clinico-neuropathologic
findings. Acta Neuropathol.
67,289
-295.[CrossRef][Medline]
Sabaratnam, M., Vroegop, P. G. and Gangadharan, S. K.
(2001). Epilepsy and EEG findings in 18 males with fragile X
syndrome. Seizure 10,60
-63.[CrossRef][Medline]
Schaeffer, C., Bardoni, B., Mandel, J. L., Ehresmann, B.,
Ehresmann, C. and Moine, H. (2001). The fragile X mental
retardation protein binds specifically to its mRNA via a purine quartet motif.
EMBO J. 20,4803
-4813.[CrossRef][Medline]
Schroll, C., Riemensperger, T., Bucher, D., Ehmer, J., Voller,
T., Erbguth, K., Gerber, B., Hendel, T., Nagel, G., Buchner, E. et al.
(2006). Light-induced activation of distinct modulatory neurons
triggers appetitive or aversive learning in Drosophila larvae.
Curr. Biol. 16,1741
-1747.[CrossRef][Medline]
Singh, K., Gaur, P. and Prasad, S. (2007).
Fragile x mental retardation (Fmr-1) gene expression is down regulated in
brain of mice during aging. Mol. Biol. Rep.
34,173
-181.[CrossRef][Medline]
Stefani, G., Fraser, C. E., Darnell, J. C. and Darnell, R.
B. (2004). Fragile X mental retardation protein is associated
with translating polyribosomes in neuronal cells. J.
Neurosci. 24,7272
-7276.
Stern, E. A., Maravall, M. and Svoboda, K.
(2001). Rapid development and plasticity of layer 2/3 maps in rat
barrel cortex in vivo. Neuron
31,305
-315.[CrossRef][Medline]
Sung, Y. J., Conti, J., Currie, J. R., Brown, W. T. and Denman,
R. B. (2000). RNAs that interact with the fragile X syndrome
RNA binding protein FMRP. Biochem. Biophys. Res.
Commun. 275,973
-980.[CrossRef][Medline]
Sung, Y. J., Dolzhanskaya, N., Nolin, S. L., Brown, T., Currie,
J. R. and Denman, R. B. (2003). The fragile X mental
retardation protein FMRP binds elongation factor 1A mRNA and negatively
regulates its translation in vivo. J. Biol. Chem.
278,15669
-15678.
Todd, P. K. and Mack, K. J. (2000). Sensory
stimulation increases cortical expression of the fragile X mental retardation
protein in vivo. Brain Res. Mol. Brain Res.
80, 17-25.[Medline]
Todd, P. K., Mack, K. J. and Malter, J. S.
(2003). The fragile X mental retardation protein is required for
type-I metabotropic glutamate receptor-dependent translation of PSD-95.
Proc. Natl. Acad. Sci. USA
100,14374
-14378.
Turrigiano, G. G. and Nelson, S. B. (2004).
Homeostatic plasticity in the developing nervous system. Nat. Rev.
Neurosci. 5,97
-107.[CrossRef][Medline]
Valentine, G., Chakravarty, S., Sarvey, J., Bramham, C. and
Herkenham, M. (2000). Fragile X (fmr1) mRNA expression is
differentially regulated in two adult models of activity-dependent gene
expression. Brain Res. Mol. Brain Res.
75,337
-341.[CrossRef][Medline]
Wang, H., Ku, L., Osterhout, D. J., Li, W., Ahmadian, A., Liang,
Z. and Feng, Y. (2004). Developmentally-programmed FMRP
expression in oligodendrocytes: a potential role of FMRP in regulating
translation in oligodendroglia progenitors. Hum. Mol.
Genet. 13,79
-89.
Watts, R. J., Schuldiner, O., Perrino, J., Larsen, C. and Luo,
L. (2004). Glia engulf degenerating axons during
developmental axon pruning. Curr. Biol.
14,678
-684.[CrossRef][Medline]
Weiler, I. J., Irwin, S. A., Klintsova, A. Y., Spencer, C. M.,
Brazelton, A. D., Miyashiro, K., Comery, T. A., Patel, B., Eberwine, J. and
Greenough, W. T. (1997). Fragile X mental retardation protein
is translated near synapses in response to neurotransmitter activation.
Proc. Natl. Acad. Sci. USA
94,5395
-5400.
Weiler, I. J., Spangler, C. C., Klintsova, A. Y., Grossman, A.
W., Kim, S. H., Bertaina-Anglade, V., Khaliq, H., de Vries, F. E., Lambers, F.
A., Hatia, F. et al. (2004). Fragile X mental retardation
protein is necessary for neurotransmitter-activated protein translation at
synapses. Proc. Natl. Acad. Sci. USA
101,17504
-17509.
Williams, D. W. and Truman, J. W. (2005).
Cellular mechanisms of dendrite pruning in Drosophila: insights from in vivo
time-lapse of remodeling dendritic arborizing sensory neurons.
Development 132,3631
-3642.
Wilson, B. M. and Cox, C. L. (2007). Absence of
metabotropic glutamate receptor-mediated plasticity in the neocortex of
fragile X mice. Proc. Natl. Acad. Sci. USA
104,2454
-2459.
Xu, K., Bogert, B. A., Li, W., Su, K., Lee, A. and Gao, F.
B. (2004). The fragile X-related gene affects the crawling
behavior of Drosophila larvae by regulating the mRNA level of the DEG/ENaC
protein pickpocket1. Curr. Biol.
14,1025
-1034.[CrossRef][Medline]
Yan, Q. J., Rammal, M., Tranfaglia, M. and Bauchwitz, R. P.
(2005). Suppression of two major Fragile X Syndrome mouse model
phenotypes by the mGluR5 antagonist MPEP.
Neuropharmacology 49,1053
-1066.[CrossRef][Medline]
Zalfa, F., Giorgi, M., Primerano, B., Moro, A., Di Penta, A.,
Reis, S., Oostra, B. and Bagni, C. (2003). The fragile X
syndrome protein FMRP associates with BC1 RNA and regulates the translation of
specific mRNAs at synapses. Cell
112,317
-327.[CrossRef][Medline]
Zalfa, F., Eleuteri, B., Dickson, K. S., Mercaldo, V., De
Rubeis, S., di Penta, A., Tabolacci, E., Chiurazzi, P., Neri, G., Grant, S. G.
et al. (2007). A new function for the fragile X mental
retardation protein in regulation of PSD-95 mRNA stability. Nat.
Neurosci. 10,578
-587.[CrossRef][Medline]
Zars, T., Fischer, M., Schulz, R. and Heisenberg, M.
(2000). Localization of a short-term memory in Drosophila.
Science 288,672
-675.
Zhang, M., Wang, Q. and Huang, Y. (2007).
Fragile X mental retardation protein FMRP and the RNA export factor NXF2
associate with and destabilize Nxf1 mRNA in neuronal cells. Proc.
Natl. Acad. Sci. USA 104,10057
-10062.
Zhang, Y. Q. and Broadie, K. (2005). Fathoming
fragile X in fruit flies. Trends Genet.
21, 37-45.[CrossRef][Medline]
Zhang, Y. Q., Friedman, D. B., Wang, Z., Woodruff, E., 3rd, Pan,
L., O'Donnell, J. and Broadie, K. (2005). Protein expression
profiling of the drosophila fragile X mutant brain reveals up-regulation of
monoamine synthesis. Mol. Cell. Proteomics
4, 278-290.
Zhao, M. G., Toyoda, H., Ko, S. W., Ding, H. K., Wu, L. J. and
Zhuo, M. (2005). Deficits in trace fear memory and long-term
potentiation in a mouse model for fragile X syndrome. J.
Neurosci. 25,7385
-7392.
Zheng, X., Wang, J., Haerry, T. E., Wu, A. Y., Martin, J.,
O'Connor, M. B., Lee, C. H. and Lee, T. (2003). TGF-beta
signaling activates steroid hormone receptor expression during neuronal
remodeling in the Drosophila brain. Cell
112,303
-315.[CrossRef][Medline]
Zito, K. and Svoboda, K. (2002).
Activity-dependent synaptogenesis in the adult Mammalian cortex.
Neuron 35,1015
-1017.[CrossRef][Medline]
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati 
Twitter What's this?
Related articles in Development:
This article has been cited by other articles:
|
|
 |
|
  Y. Zhang, D. Chen, and Z. Wang Analyses of mental dysfunction-related ACSl4 in Drosophila reveal its requirement for Dpp/BMP production and visual wiring in the brain Hum. Mol. Genet., October 15, 2009; 18(20): 3894 - 3905. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. E. Pfeiffer and K. M. Huber The State of Synapses in Fragile X Syndrome Neuroscientist, October 1, 2009; 15(5): 549 - 567. [Abstract] [PDF]
|
|
|
|
 |
|
 I. Bureau The development of cortical columns: role of Fragile X mental retardation protein J. Physiol., May 1, 2009; 587(9): 1897 - 1901. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Chiang, R. Priya, M. Ramaswami, K. VijayRaghavan, and V. Rodrigues Neuronal activity and Wnt signaling act through Gsk3-{beta} to regulate axonal integrity in mature Drosophila olfactory sensory neurons Development, April 15, 2009; 136(8): 1273 - 1282. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. Bushey, G. Tononi, and C. Cirelli The Drosophila Fragile X Mental Retardation Gene Regulates Sleep Need J. Neurosci., February 18, 2009; 29(7): 1948 - 1961. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Repicky and K. Broadie Metabotropic Glutamate Receptor-Mediated Use-Dependent Down-Regulation of Synaptic Excitability Involves the Fragile X Mental Retardation Protein J Neurophysiol, February 1, 2009; 101(2): 672 - 687. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. R. Tessier and K. Broadie Drosophila fragile x mental retardation protein developmentally regulates activity-dependent axon pruning J. Cell Sci., April 15, 2008; 121(8): e807 - e807. [Full Text]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
