Signaling by the engulfment receptor draper: a screen in Drosophila melanogaster implicates cytoskeletal regulators, Jun N-terminal Kinase, and Yorkie

Abstract

Draper, the Drosophila melanogaster homolog of the Ced-1 protein of Caenorhabditis elegans, is a cell-surface receptor required for the recognition and engulfment of apoptotic cells, glial clearance of axon fragments and dendritic pruning, and salivary gland autophagy. To further elucidate mechanisms of Draper signaling, we screened chromosomal deficiencies to identify loci that dominantly modify the phenotype of overexpression of Draper isoform II (suppressed differentiation of the posterior crossvein in the wing). We found evidence for 43 genetic modifiers of Draper II. Twenty-four of the 37 suppressor loci and 3 of the 6 enhancer loci were identified. An additional 5 suppressors and 2 enhancers were identified among mutations in functionally related genes. These studies reveal positive contributions to Drpr signaling for the Jun N-terminal Kinase pathway, supported by genetic interactions with hemipterous, basket, jun, and puckered, and for cytoskeleton regulation as indicated by genetic interactions with rac1, rac2, RhoA, myoblast city, Wiskcott-Aldrich syndrome protein, and the formin CG32138, and for yorkie and expanded. These findings indicate that Jun N-terminal Kinase activation and cytoskeletal remodeling collaborate in Draper signaling. Relationships between Draper signaling and Decapentaplegic signaling, insulin signaling, Salvador/Warts/Hippo signaling, apical-basal cell polarity, and cellular responses to mechanical forces are also discussed.

Keywords: Draper; Jun N-terminal kinase; Yorkie gene; actin regulator; engulfment.

Figure 1

Overexpression of DrprII using the engrailed-Gal4 driver results in (B) a wing vein phenotype when compared to (A) controls. Removing one copy of (C) the endogenous draper gene suppresses the phenotype associated with DrprII overexpression, as does coexpression of (D) Draper–RNAi. Removing one copy of known downstream components of the Draper pathway, namely (E) ced-6 and (F) shark, is also sufficient to suppress the phenotype. In all cases, female wings are shown. (G) The crossveinless phenotype due to DrprII overexpression is highly penetrant in the wings of female flies (83%) but less so in males (23%), and removal of a single copy of either ced-6 or shark reduces penetrance of the phenotype in females to 25 and 40%, respectively.

Figure 2

Dominant modification of (A) the enGal4>Draper-II over expression phenotype using mutant or P-element insertion lines of (B) lgl, (C) Mad, (D) bsk, (E) pten (F) vps28 (G) rhoA, (H) rac-1, (I) osm-1, (J) CG32138, (K) l(3)bdr, (L) sec23, (M) plx, (N) fer2, (O) tara, (P) 14-3-3-ε, (Q) CG16791, (R) psr, (S) how, (T) crb and (U) wasp. In all cases, wings from females are shown.

Figure 3

The pcv phenotype associated with Draper II overexpression shows (A) weaker penetrance in the wings of male flies when compared to wings of females (A′). Removal of one copy of lid dominantly enhanced the Draper II overexpression phenotype in both (B and B′) males and females. (C) Coexpression of Lid suppressed the en > Draper II phenotype. (D) The Draper II overexpression phenotype was also dominantly enhanced by removing a single copy of E(pc).

Figure 4

Ectopic expression of scabrous with enGal4 gives rise to a nicked wing margin phenotype (A). Deficiencies or specific genes identified in our Draper II overexpression modifier screen were also assayed for their effect on en > Sca. Results are shown for (B) shark², (C) E(pc)¹, and (D) cul-3^gft.

Figure 5

Testing components of putative pathways identified in our screen for modifiers of Draper function. (C) Removal of puc enhances the crossveinless phenotype in the wings of male flies. Conversely, coexpression of puc along with Draper II suppresses the Draper overexpression phenotype in (D) females, as does removal of (E) jun and (F) hep. (I and J) A mutant allele of the Dpp pathway component shn (shn^1B) also suppresses, as does a wing-specific allele of Dpp (Dpp^dr). (K) yki (yki ^Δ5) dominantly suppresses the en > DrprII phenotype as does (L) Rac2 (rac ^2Δ).

Figure 6

When compared to controls (A–A′′) en > DrprII leads to elevated levels of puc expression as seen by the increased activity of a puc–lacZ enhancer trap line (pucLacz^E69) in the posterior compartment of wing imaginal discs (B–B′′). Increased levels of phosphorylated-JNK are also observed in en > DrprII vs. controls (compare C–C′ to D–D′′). Draper II overexpression also leads to increased levels of cells in the posterior compartment that stain positive for the apoptotic marker, cleaved-caspase 3 (compare E′′ to F′′).

Figure 7

Interactions between Drpr isoforms. (A) Absence of posterior crossveins quantified in flies expressing combinations of Drpr isoforms. All statistically significant differences are indicated (Students t-test: *, P < 0.05; **, P < 0.01). These experiments made use of two UAS–drprI insertions and three UAS–drprIII insertions. Since results were similar with each, the mean and observed standard error of the results with distinct insertions is shown here. The lid/+ genotypes were heterozygous for Df(2L)ED385. (B) Wing from normal male fly (w^11-18). (C) Male en > DrprI wing, also heterozygous for Df(2R)ED2219. Only rare male escapers were seen for this genotype. (D) Male en > DrprI wing, also heterozygous for cul3^gft. (E) Female sibling of the fly in D, also heterozygous for the X-linked UAS-DrprII transgene. (F) Male en > DrprIII wing, also heterozygous for cul3^gft. (G) Female sibling of the fly in F, also heterozygous for the X-linked UAS–DrprII transgene.

Figure 8

A scheme of interactions hypothesized to connect the receptor protein Draper to the execution of the engulfment process. Solid arrows represent connections established by previous studies (see Discussion). Shaded arrows highlight the predominant interactions indicated in this study of genetic modifiers. The arrow connecting Ced-6/Shark to actin is dotted because the results do not distinguish whether the Draper pathway affects actin only through the small GTPases or also independently of them. The most parsimonious explanation of JNK activity in response to Draper is shown, whereby JNK is activated indirectly via changes in the actin cytoskeleton. An additional, more direct connection between Draper and Ced-6 or Shark and JNK cannot be excluded. The contribution of Yki activity to engulfment, if any, remains uncertain at present.