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First published online 25 June 2008
doi: 10.1242/dev.022244

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Temporal requirements of the fragile X mental retardation protein in the regulation of synaptic structure

Department of Biological Sciences, Kennedy Center for Research on Human Development, Vanderbilt University, Nashville, TN 37232 USA.

View larger version (39K): [in this window] [in a new window]   Fig. 1. Gene-Switch system drives targeted dFMRP expression in neurons. (A) Schematic of the Gene-Switch (GS) system. The GAL4 DNA-binding domain is fused to the p65 activation domain (p65AD) and a mutated progesterone receptor ligand-binding domain (PR-LBD). In the absence of RU486, the GS is `off'. In the presence of RU486, the hormone-responsive GAL4 drives dFMRP transcription downstream of the UAS regulatory sequence. This approach allows spatial and temporal control of dFMRP expression in the dfmr1-null background. (B) Western blot of isolated third instar Drosophila larval CNS. Genotypes as indicated: w1118 (control), homozygous dfmr150M null allele (dfmr1) and dfmr150M, elav-GSG-301/dfmr150M, UAS-9557-3 (GS). Treatment as indicated: GS fed ethanol vehicle (GS+E) or RU486 (GS+RX, where X is the RU486 concentration in µg/mL). Blot was probed for dFMRP and -Tubulin, illustrating RU486 dosage responsiveness. (C) Quantification of western blot dFMRP levels. Individual band intensities were normalized to -Tubulin and expressed as a percentage of the control. Bars indicate mean±s.e.m. *P<0.05. (D) dFMRP immunohistochemistry of wandering third instar larvae CNS. Bottom row of panels shows higher magnification views of dFMRP staining in the VNC. Note the RU486 dosage dependence of dFMRP expression.

View larger version (60K): [in this window] [in a new window]   Fig. 2. Targeted presynaptic dFMRP rescues all dfmr1-null NMJ structure defects. (A) Representative images of wandering third instar Drosophila larval NMJs; genotypes and treatments as shown. NMJs co-labeled for HRP (presynaptic marker) and DLG (postsynaptic marker), with three examples of each condition shown. (B-D) Quantification of NMJ synaptic branch number and area, defined for presynaptic area (HRP domain) and postsynaptic area (DLG domain). dfmr1-null terminals display overgrowth and over-elaboration. GS+E phenocopies dfmr1, and complete rescue occurs with RU486 constitutively driving presynaptic dFMRP expression. (E) Representative images of mini/satellite boutons; genotypes and treatments as shown. High-magnification images of boxed regions in A showing mini-boutons (arrowheads) in both dfmr1-null and GS+E genotypes and their absence following RU486 induction. (F,G) Quantification of normal (>2 µm diameter) and mini-bouton (2 µm diameter, attached to a normal bouton) number per NMJ in genotypes and treatments shown. n=12 animals for all conditions. Bars indicate mean±s.e.m. Dashed red lines highlight control level quantifications. **P<0.01, ***P<0.001.

View larger version (31K): [in this window] [in a new window]   Fig. 3. Temporal control of dFMRP expression by acute early RU486 treatment. (A) Depiction of time line employed for early Drosophila larval induction of dFMRP indicating points of dFMRP protein analyses. (B) Representative western blot of first instar larval CNS probed for dFMRP and -Tubulin. Samples were taken immediately after a 12-hour treatment with RU486 as indicated. (C) Quantification of western blot dFMRP levels. Individual band intensities were normalized to -Tubulin and expressed as a percentage of the control. Bars indicate mean±s.e.m. (D) Representative western blots of second and third instar larval CNS. dFMRP levels progressively diminish and are minimally detectable 60 hours post-treatment. (E) Quantification of dFMRP levels as a function of age. Individual band intensities were normalized to -Tubulin and expressed as a percentage of the control. Bars indicate mean±s.e.m.

View larger version (50K): [in this window] [in a new window]   Fig. 4. Early presynaptic dFMRP rescues dfmr1-null NMJ architectural phenotypes. (A) Representative images showing NMJs from Drosophila either vehicle- (GS+E) or experimentally treated (GS+RU486 at 0.25 mg/mL, R0.25) for 12 hours immediately post-hatching and then analyzed as third instar larvae (108 hours AEL), co-labeled for HRP and DLG. (B-E) Quantification of NMJ structure. Statistically significant rescue occurs for synaptic branch number (B), synaptic bouton number (C), presynaptic junctional area (D), and postsynaptic junction area (E). n=12-14 animals for each condition. Bars indicate mean±s.e.m. Dashed red lines highlight dfmr1-null conditions. *P<0.05, **P<0.01, ***P<0.001.

View larger version (46K): [in this window] [in a new window]   Fig. 5. Acute late presynaptic dFMRP partially rescues dfmr1-null NMJ structure. (A) Depiction of time line employed for late larval stage intervention. Drosophila larvae were raised to 96 hours AEL, transferred to RU486-containing food for 12 hours, and then immediately processed. (B) Representative western blot of third instar larval CNS probed for dFMRP and -Tubulin. (C) Quantification of western blot dFMRP levels. Individual band intensities were normalized to -Tubulin and expressed as a percentage of the control. Bars indicate mean±s.e.m. **P<0.01, ***P<0.001. (D) Representative NMJ images from animals treated during the late 12-hour time period, co-labeled for HRP and DLG. Acute dFMRP expression reduces the excess number of boutons observed in the dfmr1-null. (E) Quantification of synaptic bouton number. Late 12-hour treatment with RU486 to induce dFMRP effects partial rescue of NMJ structural alterations. n=10-12 animals for each condition. Bars indicate mean±s.e.m. Dashed red line highlights the dfmr1-null condition. **P<0.01.

View larger version (46K): [in this window] [in a new window]   Fig. 6. Targeted presynaptic dFMRP rescues dfmr1-null NMJ cytoskeletal defects. (A) Representative images of NMJs from wandering third instar Drosophila larvae probed for HRP and Futsch. GS animals were constitutively fed either EtOH vehicle or RU486 at 2 µg/mL. Note the increased number of Futsch loops present throughout the dfmr1-null synaptic terminals. (B) Quantification of Futsch loops. In wandering third instar larvae, constitutive treatment with RU486 partially rescues the cytoskeletal alteration of the null mutant. n=11-13 animals for each condition. (C) Quantification of Futsch loops after either early or late 12-hour treatment with RU486 (taken at 108 hours AEL). Age-matched control and dfmr1-nulls are presented. n=10-15 animals for each condition. Bars indicate mean±s.e.m. Dashed red line highlights control level quantifications. *P<0.05, **P<0.01, ***P<0.001.

View larger version (67K): [in this window] [in a new window]   Fig. 7. Constitutive presynaptic dFMRP does not rescue dfmr1-null FM1-43 function defect. (A) Representative images of wandering third instar Drosophila larval NMJs loaded with FM1-43 and then subsequently unloaded with high [K+] depolarizing saline. Note the elevated relative fluorescence retained in control versus dfmr1-null and GS mutants. Insets show representative synaptic boutons at higher magnification. (B) Quantification of the FM1-43 unload:load fluorescence intensity ratio. Sample sizes: n=8 each control and dfmr1-null; n=13 GS+E; n=6 GS+R0.5; and n=9 GS+R2. (C) Quantification of average fluorescence intensity per bouton in loaded and unloaded conditions. Sample sizes: n=5 for each control, dfmr1-null, GS+E, GS+R0.5 and GS+R2. Bars indicate mean±s.e.m. *P<0.05, **P<0.01.

View larger version (35K): [in this window] [in a new window]   Fig. 8. Constitutive presynaptic dFMRP does not rescue dfmr1-null mEJC function defect. (A) Sample miniature excitatory junctional current (mEJC) traces from wandering third instar Drosophila larval NMJs showing 3 seconds of recording from control, dfmr1-null, GS+E, GS+R0.5 and GS+R2. Note the increased number of events in the dfmr1-null compared with the control, and the further increase upon dFMRP induction. (B) Quantification of mEJC peak amplitude. (C) Quantification of mEJC frequency. Bars indicate mean±s.e.m.; n=10 for each category. *P<0.05, **P<0.01, ***P<0.001.

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