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First published online 5 March 2008
doi: 10.1242/dev.015867

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Drosophila fragile X mental retardation protein developmentally regulates activity-dependent axon pruning

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

View larger version (31K): [in this window] [in a new window]   Fig. 1. dFMRP regulates protein and RNA levels during brain maturation. Total RNA (A) and protein (B) at indicated developmental time points from dfmr1 nulls (dfmr150M) and controls (w1118). Bars show the mean µg per head ± standard deviation. *0.05>P>0.01; **0.01<P<0.001. Stages: P3, 60-70 hours APF; P4, 88-96 hours APF; 0-7h, 0-7 hours AE; 1d, 21-24 hours AE; 4d, 96-112 hours AE; 9d, 216-232 hours AE.

View larger version (54K): [in this window] [in a new window]   Fig. 2. dFMRP protein and mRNA are differentially developmentally regulated. (A) Quantitative RT-PCR of dfmr1 mRNA levels normalized to GAPDH2 and reported as fold changes relative to the first time point. Bars show mean±s.e.m. (B) Western blot analysis of dFMRP protein. Each lane represents a single head at indicated stages (2 heads per stage). -Tubulin is the loading control. (C) Immunocytochemistry of dFMRP in control (w1118) brains. CB, central brain; OL, optic lobe. Scale bar: 100 µm. Stages: P3, 60-70 hours APF; P4, 88-96 hours APF; 0-3h, 0-3 hours AE; 1d, 21-24 hours AE; 4d, 96-112 hours AE; 7d, 168-184 hours AE; 9d, 216-232 hours AE.

View larger version (32K): [in this window] [in a new window]   Fig. 3. The dFMRP target chickadee/profilin is developmentally regulated. (A) Quantitative RT-PCR of chickadee/profilin mRNA normalized to GAPDH2 and reported as fold changes relative to the first time point. Bars show mean±s.e.m. (B) Western blot analysis of Chickadee/Profilin from control and dfmr1 animals (one head per lane) at the developmental time points shown. -Tubulin is the loading control.

View larger version (60K): [in this window] [in a new window]   Fig. 4. Single-cell MARCM clonal analysis of MB gamma neuron development. (A) Representative image of Fasciclin II (FASII, red) labeled Mushroom Body containing a single MARCM gamma neuron clone (green). The white box highlights the area of axonal projection. (B) Developmental profile of axon projections of single-cell MARCM gamma neuron clones. Boxed insets are magnifications of areas of small (<5 µm) presynaptic branches (arrowheads), which are subject to pruning. P3, 60-70 hours APF; P4, 88-96 hours APF; 0-3h, 0-3 hours AE; 4d, 96-112 hours AE. Scale bar: 10 µm.

View larger version (39K): [in this window] [in a new window]   Fig. 5. dFMRP overexpression abrogates normal MB neuronal development. (A) Developmental profile of axon projections of single-cell MARCM gamma neuron clones overexpressing dFMRP. Boxed insets highlight presynaptic branches that are pruned (arrows). Scale bar: 10 µm. (B) Quantitation of total axon branch number of dFMRP-overexpressing MARCM clones. Each point is data from a single-cell MARCM clone. Horizontal lines represent the mean for each data set. *0.01<P<0.05. P3, 60-70 hours APF; P4, 88-96 hours APF; 0-3h, 0-3 hours AE; 4d, 96-112 hours AE.

View larger version (16K): [in this window] [in a new window]   Fig. 6. Sensory input deprivation reduces dFMRP expression. (A) Quantitative RT-PCR of dfmr1 mRNA normalized to GAPDH2 from normally reared and sensory-deprived (SD) animals at 4 days (96-112 hours AE). Bars show the mean±s.e.m. (n=4). *0.05>P>0.01. (B) Western blot for dFMRP of normally reared and SD animals at 4 days (96-112 hours AE), with one head per lane. (C) Quantitation of dFMRP western blot normalized to -Tubulin. Bars show the mean±s.e.m. *0.05>P>0.01.

View larger version (46K): [in this window] [in a new window]   Fig. 7. Olfactory and vision mutants reduce dFMRP expression and function. (A) Western blot analysis of dFMRP and Chickadee/Profilin in control, odorant receptor mutant Or83b, and rhodopsin mutant ninaE animals. Single heads from <16-hour-old animals loaded per lane. -Tubulin is the loading control. (B) Quantitation of dFMRP and Chickadee/Profilin western analysis. Bars show the mean±s.e.m. (n=6). *0.05>P>0.01.

View larger version (40K): [in this window] [in a new window]   Fig. 8. Sensory-deprivation modifies dFMRP-dependent axon pruning. (A) Representative images of single-cell MARCM gamma neuron axon projections at 4 days post-eclosion, from animals raised in standard conditions (top row), sensory-deprived conditions (middle row) and sensory-deprived conditions followed by 3 hours of normal sensory stimulation (bottom row). Boxed insets highlight small (<5 µm) branches (arrows). Scale bar: 10 µm. (B) Diagram of dFMRP-dependent changes in MB axonal projections. dFMRP protein and mRNA are expressed maximally during late pupation and the early-use period after eclosion. Activity-dependent pruning in MB axons occurs during this window, dependent on dFMRP. At maturity, transcriptional and translational regulation of dfmr1 becomes uncoupled, as mRNA levels inversely correlate with protein levels.

View larger version (40K): [in this window] [in a new window]   Fig. 9. CHR2-induced neuronal activation drives dFMRP-dependent pruning. (A) Single-cell MARCM clones expressing channelrhodopsin-2 (CHR2) in control or in dfmr1-null backgrounds, from animals grown on food containing all-trans retinal (ATR) or ethanol (ETOH vehicle). Post-eclosion animals (<12 hours AE) were stimulated with 470 nm light at 1 Hz pulses for 6 hours. Scale bar: 10 µm. (B,C) Quantified total axon branch number (B) and <5 µm branch number (C) of CHR2-expressing MARCM clones after stimulation. Horizontal bars are the mean for each data set. *0.01<P<0.05.

View larger version (25K): [in this window] [in a new window]   Fig. 10. Developmental changes in axon branch length and number in Mushroom Body clones in control and dfmr1-null mutants. Cumulative axon branch length (A) and total axon branch number (B) of gamma neurons from the indicated genotypes and stages. Each point is data from a single-cell MARCM clone. Horizontal lines represent the mean for each data set. P3, 60-70 hours APF; P4, 88-96 hours APF; E, 0-3 hours AE; 4D, 96-112 hours AE; SD, sensory-input deprived (96-112 hours AE); 3hr Stim, 3 hours sensory stimulation (sensory deprived for 4 days followed by 3 hours of sensory input stimulation). *0.01<P<0.05; **0.001<P<0.01; ***P<0.001.