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Figures
- Fig. 1. Sleep and expression levels are altered in adult flies expressing hMeCP2 in astrocytes.
(A) An individual Drosophila astrocyte visualized by CD8:GFP expression in a UAS-CD8:GFP/alrm-Gal4 adult brain. (B) The Gal4/UAS bipartite system drives transgenic hMeCP2 expression in astrocytes via alrm-Gal4. All alrm-Gal4 progeny contained both the alrm-Gal4#3 and alrm-Gal4#2 transgenes. (C,D) The minutes of sleep over a 24 hour period covering a 10 day span from individual control males (alrm-Gal4/+,alrm-Gal4/+, UAS-hMeCP2FL/+, or alrm-Gal4/+;alrm-Gal4/UAS-EGFP) and experimental (alrm-Gal4/+; alrm-Gal4/UAS-hMeCP2FL) males. (C) Eduction graph displaying 12 hour night (black bar) and 12 hour day (white bar) periods in control and experimental males. (D) Males expressing hMeCP2FL in astrocytes exhibited a decrease in sleep during the day-to-night transition and through hours 22–24 as compared to controls (F: 20.122, P<0.001, d = 1.458; one-way ANOVA of bootstrapped residuals; Nrep = 50,000; d = effect size; pairwise comparisons indicate significant differences in all control-experimental group pairs) (arrow in panel C). (E–E″) Astrocytes in control adult brain expressing nuclear dsRed in an alrm-Gal4/UAS-dsRed;alrm-Gal4/+ progeny. (E′) Nuclear-labeled astrocytes from the central brain projected onto a standard Drosophila brain representation. (E″) Quantification of astrocytes located in the central brain region in control brains (alrm-Gal4/UAS-dsRed;alrm-Gal4/+). (F–F″) Astrocytes expressing hMeCP2FL in experimental alrm-Gal4/+;alrm-Gal4/UAS-hMeCP2FL progeny. hMeCP2 is detected by immunofluorescence using a rabbit hMeCP2 antibody (green, Cell Signaling). (F′) Central brain hMeCP2FL-labeled astrocytes projected onto a standard Drosophila brain representation. (F″) Quantification of astrocytes expressing detectable hMeCP2FL in alrm-Gal4/+;alrm-Gal4/UAS-hMeCP2FL experimental progeny. Scale bar represents 50 µm.
- Fig. 2. The alrm-Gal4 population of astrocytes is present in hMeCP2FL-expressing brains.
(A–C) Adult brain co-expressing hMeCP2FL and dsRed in astrocytes (UAS-dsRed/alrm-Gal4;alrm-Gal4/UAS-hMeCP2FL). hMeCP2FL expression is reduced or not detectable in a substantial number of astrocytes (B,D) that co-express dsRed (C,D). (D) Quantification of astrocytes expressing detectable hMeCP2FL in UAS-dsRed/alrm-Gal4;alrm-Gal4/UAS-hMeCP2FL progeny. Scale bars represent 50 µm. (E–G) hMeCP2R106W expression is reduced or not detectable in alrm-Gal4 expressing astrocytes in a large part of the adult brain including the protocerebrum (arrow) at 5 (E) or 15 (F) days post-eclosion. (G) Schematic representation of the single point mutation in the MBD of the RTT allele hMeCP2R106W.
- Fig. 3. hMeCP2 expression is developmentally regulated and does not alter glial differentiation.
(A) At 1 day post-eclosion, hMeCP2 expression is detected by immunofluorescence in the majority of alrm-Gal4 expressing astrocytes. (B) At 5 days post-eclosion, hMeCP2 protein is reduced or not detectable in astrocytes located outside of the SOG region. (C) The transcription factor Repo is co-expressed with β-gal in optic lobe astrocytes (arrow) in alrm-Gal4/UAS-nucLacZ;alrm-Gal4 progeny. (D) hMeCP2-expressing astrocytes maintain glial differentiation as assayed by Repo expression in the optic lobe (arrow) and the SOG (arrowhead) in the alrm-Gal4/+;alrm-Gal4/UAS-hMeCP2FL adult brain. Scale bars represent 50 µm. (E,E′) Confocal image of the large number of astrocytes labeled by a cell-membrane GFP reporter in the brain of a control alrm-Gal4/+;alrm-Gal4/UAS-CD8:GFP adult. (E′) Optical sections of a hs-flp;alrm-Gal4/UAS->stop>CD8:GFP;alrm-Gal4/+ control brain at a high magnification showing a single GFP-expressing astrocyte. (F,F′) hMeCP2FL-expressing astrocytes labeled with the same cell-membrane GFP reporter in an adult alrm-Gal4/UAS-CD8:GFP;alrm-Gal4/UAS-hMeCP2FL brain five days post-eclosion. GFP-expressing astrocytes with or without detectable hMeCP2 (red nuclear expression) cover the entire brain as in control brains. (F′) Optical sections from a hs-flp;alrm-Gal4/UAS->stop>CD8:GFP;alrm-Gal4/UAS-hMeCP2FL brain at high magnification showing a single GFP and hMeCP2FL -expressing astrocyte. The dense, fine processes observed in control astrocytes are present in astrocytes expressing hMeCP2FL. Scale bar represents 50 µm.
- Fig. 4. The hMeCP2Δ166 allele is expressed in all astrocytes.
(A,B) hMeCP2Δ166 expression is detected in alrm-Gal4 expressing astrocytes throughout the brain at 5 (A) or 15 (B) days post-eclosion (alrm-Gal4/UAS- hMeCP2Δ166;alrm-Gal4/+). (C) Schematic representation of the hMeCP2Δ166 allele containing a deletion of the N-terminus including the MBD. hMeCP2Δ166 expression is detected by immunofluorescence with the rabbit hMeCP2 antibody (green, Cell Signaling). (D,D′) Confocal images of two UAS-dsRed;EAAT1-Gal4/UAS-hMeCP2FL adult brains demonstrating hMeCP2FL protein is largely absent or reduced in the Eaat1 glial population. Co-expression of dsRed and hMeCP2FL is maintained in a number of glia in the optic lobe (arrow) or around the antennal lobe. Scale bars represent 50 µm.
- Fig. 5. hMeCP2FL transcript levels are reduced compared to hMeCP2Δ166 levels.
(A) Primer locations depicted in a schematic representation of hMeCP2 locus. (B) For semi-quantitative RT-PCR experiments, RNA from the heads of adults expressing hMeCP2FL in astrocytes (alrm-Gal4/+;alrm-Gal4/UAS-hMeCP2FL, green column), and hMeCP2Δ166 in astrocytes (alrm-Gal4/UAS-hMeCP2Δ166;alrm-Gal4/+, blue column). Adult alrm-Gal4;UAS-hMeCP2FL flies at 5–10 days post-eclosion showed significant decreases in hMeCP2 transcript levels as compared to age-matched alrm-Gal4;UAS-hMeCP2Δ166 adults (Mann–Whitney test, **P<0.05). (C) In comparison, transcript levels were not statistically different in adults expressing hMeCP2FL in octopamine neurons (Tdc2-Gal4;UAS- hMeCP2FL, green column) compared to age matched controls expressing the hMeCP2Δ166 in octopamine neurons (Tdc2-Gal4/UAS-hMeCP2Δ166, blue column) (Mann–Whitney test, P>0.7). Reactions were performed in quadruplicate for each gene. Rpl32 expression was used as the reference control to normalize expression between treatment groups (error bars indicate s.e.m.).
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