Fig 1.
EGFRAP Knock down enhances RasV12-dependent tissue hyperplasia in Drosophila wing imaginal discs.
(A-D) Maximal projection of confocal views of wing imaginal discs from third-instar larvae expressing GFP (green) and the indicated UAS transgenes under the control of apterous Gal4 (ap-Gal4), stained with anti-GFP (green) and Rhodamine Phalloidin to detect F-actin (RhPh, red) (A’-D’). Scale bars, 50 μm (A-D).
Fig 2.
EGFRAP knockdown increases RasV12-dependent cell shape changes and growth in Drosophila wing imaginal discs.
(A-C) Maximal projection of confocal images of wing imaginal discs from third-instar larvae expressing myristoylated-Tomato (MyrT, red) and the indicated UAS transgenes under the control of ap-Gal4, stained with anti-Tomato (red) and the nuclear marker Hoechst (DNA, blue). (A’-C’) Confocal xz sections along the white dotted lines of wing discs shown in A-C. The apical side of wing discs is at the top. Brackets indicate cell height. (D-F) Apical and (G-I) basal surface views of the indicated genotypes. (J-L) Box plots of the cell height (J), apical cell area (K) and basal cell area (L) of the indicated genotypes. The statistical significance of differences was assessed with a t-test, ****P value<0.0001. Scale bars, 50 μm (A-C), 30 μm (A’-C’) and 10 μm (D-I).
Fig 3.
EGFRAP downregulation increases apoptosis and JNK activity in wild-type cells adjacent to RasV12 expressing cells.
(A-G) Maximal projection of confocal views of wing imaginal discs from third-instar larvae expressing GFP (green) and the indicated UAS transgenes under the control of ap-Gal4, stained with anti-GFP (green), anti-Dcp-1 (red in A-C, white in A’-C’), anti-pJNK (red in E-G) and Hoechst (DNA, blue). (D, H) Box plots of mean fluorescent Dcp-1 (D) and pJNK (H) intensities of wing discs of the designated genotypes. (I-K) Confocal yz sections parallel to the A/P axis of wing imaginal discs from third-instar larvae expressing GFP (green) and the indicated UAS transgenes under the control of ap-Gal4, stained with anti-GFP (green), anti-Dlg (red) and Hoechst (DNA, blue). Apical side of wing discs is to the top. Arrows in I-K point to the border between dorsal experimental and ventral control cells. The statistical significance of differences was assessed with a t-test, **** and *** P values <0.0001 and <0.001, respectively. Scale bars, 50 μm (A-G) and 30 μm (I-K).
Fig 4.
Generation of EGFRAP mutant alleles supports its role as modulator of RasV12-mediated tissue hyperplasia.
(A) Schematic representation of the EGFRAP locus (3rd chromosome), EGFRAP mutants generated, sgRNAs used for generation of mutants (green boxes 1–3) and sequence targeted by EGFRAPRNAi construct (purple box). (B-D) Maximal projection of confocal images of wing imaginal discs from third-instar larvae of the indicated genotypes stained with anti-GFP (green), RhPh (red) and Hoechst (DNA, blue). (B’-B”, C’-C” and D’-D”) Confocal sections of wing discs of the specified genotypes along the white dotted lines shown in B, C and D, respectively, parallel (B’, C’ and D’) or perpendicular (B”, C” and D”) to the A/P border. Apical sides of wing discs are to the left (B’, C’ and D’) or to the top (B”, C” and D”). Scale bars, 60 μm (B-D).
Fig 5.
PVRAP downregulation enhances EGFRAP loss of function phenotypes.
(A-D’) Maximal projection of confocal images of third instar wing imaginal discs expressing the indicated UAS transgenes under the control of ap-Gal4 stained with anti-GFP (green) and RhPh (red). (E-H) Dorsal view of Drosophila noti of the specified genotypes. (I-L) Images of female flies of the indicated genotypes. Scale bars, 50 μm (A-C).
Fig 6.
Expression of EGFRAP in Drosophila wing discs.
(A) In situ hybridization of a third instar wing imaginal disc with a probe for EGFRAP mRNA. (B) Maximal projection of confocal images of a wild-type (wt) third instar wing imaginal disc stained with anti-EGFRAP. (C) Quantification of EGFRAP levels in the region of the white dotted line in (B). (D) Maximal projection of confocal images of an EGFR-sfGFP third instar wing imaginal disc stained with anti-EGFRAP (red) and anti-GFP (green). (D’) Confocal yz section, along the white dotted line in D. (E, F) Maximal projection of confocal views of wing discs expressing the indicated UAS transgenes under the control of ap-Gal4 stained with anti-GFP (green), anti-EGFRAP (red) and Hoechst (DNA, blue). (E’-E” and F’-F”) Confocal yz sections along the white dotted lines shown in E and F, respectively. Apical side of wing discs is to the left. White arrows indicate wing margin cells (wm). (G, H, I, K, L) Maximal projection of confocal images of wing discs expressing GFP and the specified UAS transgenes under the control of hedgehog-Gal4 (hh-Gal4) (G-I) and ap-Gal4 (K, L), stained with anti-EGFRAP (red), anti-GFP (green) and Hoechst (DNA, blue). (J) Box plot of the posterior/anterior EGFRAP intensity ratio of wing discs of the designated genotypes. (I’, L’) Confocal yz sections along the white dotted lines are shown in I and L, respectively. Apical side of wing discs is to the left. The statistical significance of differences was assessed with a t-test, *** P value<0.001. wm (wing margin). Scale bars, 60 μm (A, B) and 50 μm (D, E, F, G, H, I, K, L).
Fig 7.
EGFRAP regulates EGFR activity in Drosophila wing imaginal discs.
(A-C’) Maximal projection of confocal images of third instar wing imaginal discs expressing the indicated UAS transgenes under the control of en-Gal4, stained with anti-RFP (red) and anti-pERK (green). (D) Box plot of the posterior/anterior pERK intensity ratio of wing discs of the designated genotypes. (E, F) Adult flies expressing the indicated transgenes under the control of ap-Gal4. Insets in E and F show confocal images of third instar wing imaginal discs expressing GFP and EGFRAP-GFP, respectively, under the control of ap-Gal4, stained with anti-GFP (green) and anti-EGFRAP (red). (G) Maximal projection of confocal images of an EGFR-sfGFP third instar wing imaginal disc expressing EGFRAP under the control of Hh-Gal4 stained with anti-GFP (green), anti-EGFRAP (red) and Hoechst (DNA, blue). (G’-G”) Confocal yz sections, along the white dotted lines on the anterior (A) and posterior regions (P) of the wing disc shown in G. Apical side of the wing disc is to the left. The statistical significance of differences was assessed with a t-test, **** and ** P values <0.0001 and <0.01, respectively. Scale bars, 50 μm (A-C’, G).
Fig 8.
Model of EGFRAP function as a modulator of EGFR/Ras-dependent tissue hyperplasia.
Schematic drawing depicting the mechanisms by which EGFRAP could limit EGFR/Ras activity in normal (A) and RasV12-dependent oncogenic cells (B). (A) In normal cells EGFRAP and PVRAP act as negative regulator of the EGFR. Expression of EGRAP is confined to cells with high levels of EGFR activity via the Notch pathway. EGFRAP elimination results in a slight increase in EGFR activity, which does not seem to affect normal morphogenesis. However, the simultaneous elimination of EGFRAP and PVRAP leads to a further enhancement of EGFR signaling with consequences in normal morphogenesis. (B) Oncogenic EGFR/Ras activity promotes, via activation of the Notch pathway, an increase in EGFRAP expression that, in turn, restrains both EGFR activity and its capacity to induce hyperplasia. Downregulation of EGFRAP releases this restraint leading to a further increase in EGFR/Ras pathway activity and tumor growth, which is enhanced by elimination of PVRAP.