Fig. 5: Cap-d3 insufficiency increases cell death in the third instar larval central brain. | Nature Communications

Fig. 5: Cap-d3 insufficiency increases cell death in the third instar larval central brain.

From: Condensin-mediated restriction of retrotransposable elements facilitates brain development in Drosophila melanogaster

Fig. 5

a Third instar larval brains were dissected from male larvae expressing control UAS-GFP dsRNA or UAS-cap-d3 dsRNA, under the control of eyGAL4, GMRGAL4, immunostained with antibodies to detect stem cell marker, Dpn, and mitotic cell marker, PH3, and imaged using confocal microscopy. The percentage of cells that stained positive for both Dpn and PH3 were quantified. b Immunostaining for Dpn and Pros, a marker of differentiation, was performed in male third instar larval brains. The number of cells that stained positive for both Dpn and Pros were quantified. c The number of Dpn-positive cells per square micrometer of larval central brain tissue in larvae expressing cap-d3 dsRNA or control, gfp dsRNA was quantified. For a–c, results shown include larvae harvested from two independent experiments; each data point represents a single brain. P values were determined by performing two-tailed Mann–Whitney analyses. NS not significant. d Immunostaining to detect Dpn and the cell death marker, Dcp1 was performed in male third instar larval brains expressing control UAS-GFP dsRNA (dark blue) or UAS-cap-d3 dsRNA (light blue), under the control of eyGAL4, GMRGAL4. Images were taken with a confocal microscope. Maximum projections of z-stacks are shown. Nuclei are stained with DAPI (blue). All images were taken with 40x magnification. Scale bar = 50 µm. e The percentage of cells that stained positive for both Dpn and Dcp1 in (d) were quantified. Additionally, larval brains expressing eyGAL4 and GMRGAL4 (gray; no RNAi control) were imaged and quantified. Each data point represents a single brain. f Dcp1+, Dpn− cells located adjacent to Dpn-positive cells in male third instar larval brains (experiments described in d, e) were quantified. Each data point represents a single brain. g Adult female head sizes were measured in female flies expressing different combinations of control UAS-GFP dsRNA or UAS-cap-d3 dsRNA and the Drosophila inhibitor of apoptosis, UAS-diap1, under the control of eyGAL4, GMRGAL4. For e–g, results shown include larvae harvested from two independent experiments; each data point represents a single brain (e, f) or a single fly (g). P values were determined by performing two-tailed Mann–Whitney analyses. For (e), *p = 0.0212 and *#p = 0.0411. For (f), *p = 0.0013 and *#p = 0.0024. For (g), *p = 0.0422, ***p = 0.0004, ****p ≤ 0.0001, ****#p ≤ 0.0001, NS not significant. For all experiments, error bars indicate standard deviations from the mean.

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