PMC

(a) HA-tagged Draper constructs and wild type Corkscrew (Csw^WT) were transfected into insect S2 cells. Anti-Csw immunoprecipitation (IP) samples were analyzed by anti-HA WB to compare Csw association with Draper-I and Draper-II. Anti-HA WB was performed on cell lysates to confirm Draper::HA transfection.
(b) Draper-II::HA was co-transfected with Csw^WT or Csw^CS. Anti-HA WB was performed on anti-Csw IP samples to compare Draper-II::HA association with Csw^WT versus Csw^CS. Blots were reprobed for Csw. Anti-HA WBs on lysates confirmed Draper-II::HA expression.
(c) Cells were transfected with indicated constructs and anti-HA IP was performed on cell lysates. Anti-phosphotyrosine and anti-HA WBs were performed on IP samples. Co-transfection of Csw^WT with Draper-II significantly reduced the tyrosine phosphorylation level of bands corresponding to the sizes of Shark and Draper-II (lane 7 versus 8). Csw^WT did not affect phosphorylation of Draper-I::HA or Shark in the absence of Draper-II::HA (lane 5 versus 6).
(d) Anti-phosphotyrosine, anti-HA, and anti-Myc WBs blots were performed on anti-HA IP samples from cells transfected with indicated constructs. Csw^CS increased the phosphorylation status of Shark and Draper-II when transfected together (lane 6 versus 7) but did not significantly alter Draper-I or Shark when expressed together in S2 cells (lanes 4 versus 5).
(e) Anti-Myc IP was performed on cells transfected with Draper-II::HA, Myc::Shark, and noted versions of Csw. Anti-phosphotyrosine, anti-HA, and anti-Myc WBs on IP samples are shown. Tyrosine phosphorylation of Shark was reduced by Csw^WT (lane 2 versus 3) but increased by Csw^CS (lane 2 versus 4).

(a) repo-Gal4 was used to overexpress UAS-draper transgenes in wild type flies that carried OR85e::mCD8-GFP. Projected confocal Z-stacks of OR85e axons (green) in the antennal lobe region are shown before injury and 3 days after injury. Overexpression of Draper-II or the chimeric receptor Draper-I-II completely blocked glial clearance of GFP-labeled degenerating axons. Co-overexpression of Draper-I and Draper-II in glia also robustly blocked clearance. Overexpression of Draper-I or Draper-III had little to no effect on glial engulfment of severed axons. Draper immunostaining (red) shown in single confocal slice images confirmed glial overexpression of each Draper construct.
(b) Graph of GFP quantification of glial clearance assay shown in (a). Bars represent mean ± S.E.M. **p<0.01, ***p<0.0001 as compared to 3 days after ablation in control animals. D0, Day 0; D3, Day 3.
Genotypes and N values:
control=w;OR85e-mCD8::GFP/+;repo-Gal4/+. (D0 N=40; D3 N=40).
Draper-I overexpression=w;OR85e-mCD8::GFP/UAS-Draper-I;repo-Gal4/+. (D0 N=41; D3 N=47).
Draper-II overexpression=w;OR85e-mCD8::GFP/UAS-Draper-II;repo-Gal4/+. (D0 N=8; D3 N=24).
Draper-I and –II overexpression=w;OR85e-mCD8::GFP,UAS-Draper-I/UAS-Draper-II;repo-Gal4/+. (D0 N=8; D3MP N=8).
Draper-III overexpression=w;OR85e-mCD8::GFP/UAS-Draper-III;repo-Gal4/+. (D0 N=16; D3 N=20).
Draper-I-II overexpression=w;OR85e-mCD8::GFP/UAS-Draper-I-II;repo-Gal4/+. (D0 N=18; D3 N=26).

(a) The pan-glial driver repo-Gal4 was used to overexpress UAS-Draper-II in a wild type background or in animals that carried one copy of csw^va199, a dominant negative allele of corkscrew (DN csw). Reduced Csw activity in csw^va199 heterozygotes partially rescued the engulfment phenotype resulting from glial overexpression of Draper-II. Images of GFP-labeled OR85e axons before and 3 days after maxillary palp ablation are all shown as confocal Z-stack projections.
(b) Quantification of experiment in (a). Bars represent mean S.E.M. ***p< 0.0001. D0, Day 0; D3, Day 3.
Genotypes and N values in (a,b):
DN csw = csw^va199/+. (D0 N=10; D3 N=10).
Draper-II overexpression = w;OR85e-mCD8::GFP/UAS-Draper-II;repo-Gal4/+. (D0 N=14; D3 N=10).
Draper-II overexpression + DN csw= csw^va199/w;OR85e-mCD8::GFP/UAS-Draper-II;repo-Gal4/+. (D0 N=10; D3 N=16).
(c) repo-Gal4 was used to drive UAS-Draper-II in animals that also carried UAS-csw^RNAi to knock down Csw expression. Clearance of GFP-labeled OR85e axons was examined 3 days after maxillary palp ablation. Confocal Z-stack projections show GFP⁺ axonal material in the antennal lobes in each genotype before and after injury. Overexpression of Draper-II in wild type animals blocks glial engulfment of degenerating axons, but this engulfment phenotype is significantly reduced when Csw expression is knocked down by RNAi.
(d) Quantification of the experiment shown in (a). Error bars represent S.E.M. ***p < 0.001. D0, Day 0; D3, Day 3.
Genotypes and N values in (c,d):
Draper-II overexpression = w;OR85e-mCD8::GFP/UAS-Draper-II;repo-Gal4/+. (D0 N=10; D3 N=14).
Draper-II overexpression + cswRNAi = UAS-cswRNAi/w;OR85e-mCD8::GFP/UAS-Draper-II;repo-Gal4/+. (D0 N=12; D3 N=16).

(a) UAS-Draper-I^RNAi, which targets the unique region in the extracellular region of Draper-I (see ) was driven ubiquitously with tubulin-Gal4. Draper WB was performed on adult head protein lysates to confirm specific knockdown of Draper-I (96.4 ± .3% reduction in Draper-I, p<0.01; 2.1 ± 25% reduction in Draper-II/-III, p=0.76). Full-length blots shown in .
(b) Quantification of OR85e-mCD8::GFP shown in (c). Bars represent mean ± S.E.M. ***p<0.001. D0, Day 0; D3, Day 3.
(c) repo-Gal4 was used to knock down Draper-I by driving UAS-Draper-I^RNAi or to knock down all Draper isoforms with UAS-Draper^RNAi. Projected confocal Z-stacks show that glial clearance of axonal debris (green) was inhibited by knockdown of all Draper isoforms and in draper mutants. Notably, glial clearance of severed axons was equally suppressed by selective knockdown of Draper-I. Draper (red) was barely detectable in the brain following expression of UAS-Draper^RNAi or UAS-Draper-I^RNAi in glia. The mean intensity of cortical Draper immunostaining in control, UAS-Draper^RNAi, and UAS-Draper-I^RNAi animals was 82.5 ± 4.7, 39 ± 1.2, and 36 ± 1.3, respectively.
Genotypes in (a): control=tubulin-Gal4/+, Draper-I RNAi=tubulin-Gal4/+;UAS-DraperI^RNAi/+.
Genotypes and N values for (b) and (c):
control=w;OR85e-mCD8::GFP/+;repo-Gal4/+. (D0 N=22; D3 N=23).
draper =w; OR85e-mCD8::GFP/+;draper^Δ5/draper^Δ5
Draper RNAi=w;OR85e-mCD8::GFP/UAS-Draper^RNAi;repo-Gal4/+. (D0 N=20; D3 N=24).
Draper-I RNAi=w;OR85e-mCD8::GFP/+;repo-Gal4/UAS-Draper-I^RNAi. (D0 N=22; D3 N=19).

(a) Csw was knocked down in glia by driving UAS-csw^RNAi with repo-Gal4. A “single injury” entailed ablating the maxillary palps to sever ORN axons and then assessing clearance of GFP⁺ maxillary palp ORNs 5 days later. “Double injury” animals were pre-injured by ablating the third antennal segments 5 days prior to ablating the maxillary palps and then analyzing clearance of degenerating GFP⁺ maxillary palp ORNS after 5 days. Representative confocal Z-stack projections shown.
(b) Quantification of GFP intensity in OR85e-innervated maxillary palp glomeruli from experiment in (a). Bars represent mean ± S.E.M. ***p<0.001.
(c) The number of antennal lobes that contained visible tracts of GFP⁺ maxillary palp axonal debris were scored in control and Csw^RNAi animals at the end of the double injury experiment. GFP⁺ axon tracts were faintly visible in 42% of the control brains. In animals expressing glial Csw^RNAi, the majority of brains (83%) contained visible axonal tracts with persistent GFP⁺ debris.
Genotypes and N values:
Control = w;repo-Gal4/+ (single injury (D5MP) N=10; double injury (D10AA + D5MP) N=12).
Csw^RNAi = UAS-csw^RNAi/w; repo-Gal4/+ (single injury (D5MP) N=20; double injury (D10AA + D5MP) N=18).
(d) Projected confocal Z-stacks of GFP-labeled OR85e ORNs in control flies and in flies in which Csw is knocked down in glia by RNAi. Images were collected from uninjured animals and 10 days after antennal ablation to compare the morphology of OR85e axons.
(e) Quantification of GFP levels in OR85e-innervated glomeruli in experiment depicted in (d). Bars represent mean ± S.E.M

(a) Schematic of chimeric Draper receptors, which consist of each possible combination of Draper extracellular (ex) and intracellular (in) domains. The name of each construct indicates the extracellular-intracellular domain within.
(b) repo-Gal4 was used to express each chimeric Draper receptor in glial cells of draper mutant flies carrying an OR85e-mCD8::GFP transgene. Anti-Draper WB was performed on adult head lysates to confirm expression of each domain swap construct. Full-length blot is presented in .
(c) Quantification of axon clearance experiment shown in (d) Bars represent mean ± S.E.M. ***p<0.0001.
(d) Projected confocal Z-stack images of GFP-labeled OR85e ORNs before and 3 days after injury. Single confocal images of Draper immunostaining (red) show robust glial expression of each chimeric Draper transgene.
Genotypes in (b)–(d) and N values for (d):
control=w;OR85e-mCD8::GFP/+;repo-Gal4/+. (D0 N=10; D3 N=12).
draper^Δ5=w; draper^Δ5. (D0 N=18; D3 N=24).
Draper-I-II rescue=w;OR85e-mCD8::GFP/UAS-Draper-I-II;repo-Gal4,drpr^Δ5/drpr^Δ5. (D0 N=16; D3 N=20).
Draper-I-III rescue=w;OR85e-mCD8::GFP/UAS-Draper-I-III;repo-Gal4,drpr^Δ5/drpr^Δ5. (D0 N=20; D3 N=32).
Draper-II/III-I rescue=w;OR85e-mCD8::GFP/UAS-Draper-II/III-I;repo-Gal4,drpr^Δ5/drpr^Δ5. (D0 N=18; D3 N=26).
(e) The intracellular domain of Draper-I contains a classic ITAM sequence (YXXI-(X)₁₁-YXXL) with two immunoreceptor tyrosine phosphorylation sites (underlined) that are required for recruitment of Shark and subsequent Draper signaling. Draper-II contains a unique 11 amino acid insertion (red text) that interrupts the ITAM and introduces an additional immunoreceptor tyrosine phosphorylation site (YXXI) (box). A frame shift in the Draper-III transcript introduces a truncation in the intracellular domain, which deletes half of the ITAM sequence, leaving only one immunoreceptor tyrosine phosphorylation site (underlined).

(a) Real-time PCR analysis of normalized expression levels of draper-I and draper-II mRNA in adult central brains before and after antennal ablation (AA). Draper threshold cycle (Ct) values were normalized to ribosomal protein L32 and results are presented as fold-induction relative to uninjured levels. Values represent mean ± S.E.M. from at least 3 independent RNA isolations. draper-I transcript levels were significantly upregulated 1.5, 3.0, and 4.5 hours after injury. draper-II mRNA levels were significantly increased at 4.5 hours after injury (*p<0.05 ANOVA analysis, Dunnett’s post hoc test).
(b) Csw was knocked down in glia by driving UAS-csw^RNAi with repo-Gal4. ORN axons were severed by ablating the third antennal segments. Brains were immunostained for Ced-6 0, 1, 5, 7, or 10 days after injury to compare activation of glia following axon injury in control and Csw^RNAi animals. Representative single confocal slices shown for days 0, 1, and 10.
(c) Quantification of experiment in (b). Bars represent mean ± S.E.M. **p<0.01.
(d) Quantification of experiment in (e). Bars represent mean ± S.E.M. **p<0.01.
(e) Brains were immunostained with anti-Draper to assess the time course of glial responses to AA in the presence and absence of Csw signaling. Representative single confocal slices shown for days 0, 1, and 10.
Genotypes and N values:
Control = w;repo-Gal4/+. (D0 N=20; D1AA N=20; D5AA N=24; D7AA N=20; D10AA N=20).
Csw^RNAi = UAS-csw^RNAi/w; repo-Gal4/+. (D0 N=20; D1AA N=20; D5AA N=24; D7AA N=20; D10AA N=20).

(a) Draper isoform schematic. Ovals represent extracellular EGF-like motifs. NPXY is the predicted Ced-6 binding site. Draper-I contains an ITAM (YXXI-X₁₁-YXXL), which binds Shark. Draper-II contains a unique insertion (asterisk) within the ITAM. Draper-III lacks an ITAM due to a frame shift and premature stop codon.
(b) Draper Western blot (WB) on control yw adult heads. At least two bands were detected: Draper-I (113 kD) and at least one smaller band corresponding to Draper-II (65 kD) and Draper-III (59 kD). Full-length blot is presented in .
(c) repo-Gal4 was used to express UAS-driven transgenes of each Draper isoform in draper^Δ5 mutants that also carried OR85e-mCD8::GFP to label maxillary palp 85e ORNs. Projected confocal Z-stacks show GFP⁺ axons (green) in the antennal lobe before and after axotomy. Confocal slices of Draper immunostaining (red) show robust glial expression of Draper transgenes.
(d) Quantification of GFP⁺ axon material in Draper rescue experiments shown in (c). Bars depict mean ± S.E.M. ***p<0.001.
(e) Draper WB on head lysates of control yw, draper^Δ5, and rescue flies to confirm expression of UAS-Draper transgenes. Blot was reprobed for tubulin. Less protein lysate was loaded in rescue lanes because GAL4/UAS drives robust expression. D0, Day 0; D3, Day 3. Full-length blots shown in .
Genotypes and N values in (c,d): control=w;OR85e-mCD8::GFP/+;repo-Gal4/+. (D0 N=12, D3 N=12).
draper^Δ5=w; draper^Δ5. (D0 N=20; D3 N=14).
Draper-I rescue=w;OR85e-mCD8::GFP/UAS-Draper-I;repo-Gal4,drpr^Δ5/drpr^Δ5. (D0 N=31; D3=39).
Draper-II rescue=w;OR85e-mCD8::GFP/UAS-Draper-II;repo-Gal4,drpr^Δ5/drpr^Δ5. (D0 N=16; D3 N=16).
Draper-III rescue=w;OR85e-mCD8::GFP/UAS-Draper-III;repo-Gal4,drpr^Δ5/drpr^Δ5. (D0 N=23; D3 N=26).