Figure 1.
PI3K signaling modulates basal levels of glial Draper expression.
(A) Z-stack confocal images of OR85e+ axons labeled with GFP; repo-Gal4 was used to drive UAS-pdk1RNAi, UAS-pi3k92eRNAi, and UAS-raptorRNAi in a background containing the temperature sensitive Gal4 repressor, Gal80ts; Uninjured, 3 days, and 5 days after maxillary palp ablation are shown. Dotted lines indicate 85e+ glomerulus used for quantification of GFP remaining. (B) Quantification of GFP intensities in 85e+ glomeruli from (A), p-values were calculated using one-way ANOVA and Fisher's LSD post-hoc analysis. Error bars represent standard error of the mean (SEM). (C) Single slice confocal images of adult brains; Draper antibody stains in control (OR85e-GFP,Gal80ts/+; repo-gal4/+) and RNAi backgrounds (OR85e-GFP,Gal80ts/+; repo-gal4/UAS-pdk1RNAi; UAS-pi3k92eRNAi, OR85e-GFP,Gal80ts/+; repo-gal4/+; and OR85e-GFP,Gal80ts/UAS-raptorRNAi; repo-gal4/+). Uninjured and one day after antennal ablation are shown. (D) Western blot analysis of Draper protein levels in ∼3 adult central brain regions of control (OR85e-GFP,Gal80ts/+; repo-gal4/+), pi3k92eRNAi (UAS-pi3k92eRNAi, OR85e-GFP,Gal80ts/+; repo-gal4/+), and pi3k92eCAAX (UAS-pi3k92eCAAX, OR85e-GFP,Gal80ts/+; repo-gal4/+) genotypes. α-Tubulin was used as a loading control. Full blot located in Figure S1. (E) Quantification of Draper protein levels shown on Western blot after normalization to Tubulin, n = 3, p-values were calculated using one-way ANOVA followed by Tukey's post hoc test.
Figure 2.
The draper locus contains a Stat92E-regulated injury responsive element.
(A) Schematic representation of the draper locus and regions used to generate DEEs 2–10. Blue box highlights dee7 region located in the first intron of the draper gene. Red lines indicate Stat92E binding sites in dee7. Asterisk indicates binding site in dee7MUT containing two point mutations. (B) Single slice confocal images of antennal lobe regions; dee7-Gal4 or dee7MUT-Gal4 driving two copies of UAS-mCD8::GFP (dee7>2XmCD8::GFP, dee7MUT>2XmCD8::GFP). Uninjured, one day after antennal ablation and four days after antennal ablation are shown. Dashed circles outline antennal lobes, the site of injury. Asterisk indicates cortex glia responding to axotomy. (C) Quantification for (B), p-values were calculated using Student's t test, n.s., not significant. Error bars represent SEM. (D) Single slice confocal images of antennal lobe regions stained with α-GFP and α-Repo; dee7-Gal4 was used to drive two copies of UAS-mCD8::GFP. Removal of one maxillary palp, two maxillary palps, one antenna, and two antennae are shown one day after injury.
Figure 3.
Stat92E functions in glia to promote axon clearance and regulate Draper expression.
(A) Z-stack confocal images of OR85e+ axons labeled with GFP in control (OR85e-GFP/+; repo-gal4/+) or stat92eRNAi (stat92eRNAi/OR85e-GFP;repo-Gal4/+) backgrounds. Uninjured and 5 days after maxillary palp ablation are shown. Arrows indicate position of maxillary nerves. (B) Quantification of GFP intensities in 85e+ glomeruli from (A), p-values were calculated using Student's t test. Error bars represent SEM. (C) Quantification of maxillary nerves with GFP+ axon fibers from (A). (D) Single slice confocal images; Draper antibody stains in adult brains of control (repo-gal4/+) and stat92eRNAi (stat92eRNAi/+;repo-gal4/+) backgrounds. Uninjured, one day after maxillary palp injury and one day after antennal ablation are shown. Dotted circles and boxes represent regions used for quantification of Draper immunoreactivity. (E) Quantification of data from (D), p-values were calculated using one-way ANOVA followed by Tukey's post hoc test, n.s., not significant. Error bars represent SEM. (F) Western blot analysis of Draper protein levels in ∼3 adult central brain regions of control (repo-gal4/+), stat92eRNAi (UAS-stat92eRNAi/+; repo-gal4/+), and draperRNAi (draperRNAi/+;repo-gal4/+) genotypes. α-Tubulin was used as a loading control. Full blot located in Figure S3. (G) Quantification of Draper protein levels shown on Western blot after normalization to α-Tubulin, n = 3, p-values were calculated using one-way ANOVA followed by Tukey's post hoc test. (H) Real-time PCR analysis of draper mRNA levels in adult central brain region of control (repo-gal4/+), drprRNAi (draperRNAi/+;repo-gal4/+), pi3k92eCAAX(UAS-pi3k92eCAAX/+;repo-gal4/+), pi3k92eRNAi(UAS-pi3k92eRNAi/+;repo-gal4/+), and stat92eRNAi (stat92eRNAi/+;repo-gal4/+) genotypes. Draper Ct values were normalized to ribosomal protein L32 and results are presented as fold induction relative to control. Values represent mean ± SEM from three independent RNA isolations. p-Values were calculated using one-way ANOVA and Dunnett's post hoc test.
Figure 4.
Stat92E transcriptional reporters are expressed in glia and transiently activated after axotomy.
(A–D) Single slice confocal images; (A) 10XStat92E-GFP reporter expression in adult brain in uninjured control animals, one day after maxillary palp, and one day after antennal ablation. Glial nuclei are labeled with α-Repo (blue), glial membranes with α-Draper (red), and Stat92E transcriptional reporter activity with α-GFP (green). (B) 10XStat92E-dGFP in control (10xStat92E-dGFP/+;repo-gal4/+), and stat92ERNAi (10xStat92E-dGFP/stat92eRNAi;repo-gal4/+) backgrounds, uninjured and one day after antennal ablation. (C) High magnification image showing colocalization of Repo+ glial nuclei (arrows) and 10XStat92E-dGFP reporter activity in cortex glial cells following injury. Asterisks mark antennal lobe region. (D) Colocalization of mCherry driven by dee7-Gal4 with the 10xStat92E-dGFP reporter activity one and three days after antennal ablation.
Figure 5.
Injury-induced activation of Stat92E is downstream of the Draper signaling cascade.
(A–C) Single slice confocal images; (A) Glial-specific knockdown of components of the Draper pathway in a 10XStat92E-dGFP reporter background one day after antennal ORN axotomy; control (10XStat92E-dGFP/+) draperΔ5Δ5 (10XStat92E-dGFP/+, draperΔ5/draperΔ5), sharkRNAi (sharkRNAi/10XStat92E-dGFP; repo-Gal4/+), rac1N17(10XStat92E-dGFP/+; repo-Gal4/rac1N17), src42aRNAi (src42aRNAi/10XStat92E-dGFP; repo-Gal4/+), and dCed6RNAi (dCed6RNAi/10XStat92E-dGFP; repo-Gal4/+) genotypes. (B) dee7-Gal4 activity in control (dee7-Gal4,UAS-mCD8::GFP/+) and draperΔ5/Δ5 (dee7-Gal4,UAS-mCD8::GFP/+, draperΔ5/draperΔ5) backgrounds. Uninjured, one day after antennal injury, and one day after maxillary palp injury are shown. Dotted circles indicate antennal lobes. (C) Glial-specific activation of components of the draper pathway in a 10XStat92E-dGFP reporter background; rac1V12 (10XStat92E-dGFP/+; repo-Gal4/rac1v12), and src42aca (10XStat92E-dGFP/+; repo-Gal4/src42aca).
Figure 6.
The draper gene is a primary target of Stat92E after axonal injury.
(A) Z-stack confocal images; OR85e+ axons were labeled with GFP in control (OR85e-GFP/+; repo-gal4/+), stat92eRNAi (stat92eRNAi/OR85e-GFP;repo-Gal4/+), stat92eRNAi/UAS-drprI (stat92eRNAi/OR85e-GFP;repo-Gal4/UAS-drpr-I), and stat92eRNAi/UAS-mCherry (stat92eRNAi/OR85e-GFP;repo-Gal4/UAS-mCherry) backgrounds. Uninjured and 5 days after maxillary palp ablation are shown. (B) Quantification of GFP intensities in 85e+ glomeruli from (A), p-values were calculated using one-way ANOVA followed by Tukey's post hoc, n.s., not significant. Error bars represent SEM. (C) Z-stack confocal images of OR85e+ axons labeled with GFP; controls (OR85e-GFP,Gal80ts/+; repo-gal4/+; and UAS-pi3k92eCAAX/+;OR85e-GFP,Gal80ts/+; repo-gal4/+); stat92eRNAi (OR85e-GFP,Gal80ts/UAS-stat92eRNAi; repo-gal4/+; and UAS-pi3k92eCAAX/+;OR85e-GFP,Gal80ts/UAS-stat92eRNAi;repo-gal4/+); and drprRNAi (OR85e-GFP,Gal80ts/UAS- drprRNAi; repo-gal4/+; and UAS-pi3k92eCAAX/+, OR85e-GFP,Gal80ts/UAS-drprRNAi;repo-gal4/+). Five days after maxillary palp ablation is shown. (D) Quantification of GFP intensities in 85e+ glomeruli from (C), p-values were calculated using one-way ANOVA and Tukey's post hoc analysis, n.s., not significant. Error bars represent SEM. (E) Western blot analysis of Draper protein levels in ∼3 adult central brain regions of controls (OR85e-GFP,Gal80ts/+; repo-gal4/+; and UAS-pi3k92eCAAX/+;OR85e-GFP,Gal80ts/+; repo-gal4/+), stat92eRNAi, (UAS-pi3k92eCAAX/+; OR85e-GFP,Gal80ts/UAS-stat92eRNAi;repo-gal4/+), and drprRNAi (UAS-pi3k92eCAAX/+;OR85e-GFP,Gal80ts/UAS-drprRNAi;repo-gal4/+) backgrounds. α-Tubulin was used as a loading control. Full blot located in Figure S9. (F) Quantification of Draper protein levels shown on Western blot after normalization to α-Tubulin, n = 4, p-values were calculated using one-way ANOVA and Tukey's post hoc analysis.
Figure 7.
Basal and injury-induced Draper expression are regulated through two distinct pathways.
Basal levels of Draper expression are dependent upon the convergent activities of PI3K and Stat92E signaling. After injury Draper signals through the Src family kinase signaling cascade (Src and Shark), and Rac1, and transcriptionally activates its own expression in a Stat92e-dependent fashion. Increased levels of Draper (and perhaps other engulfment genes) likely enhance the ability of glia to engulf axonal debris.