RBF- and RBF^D253A-induced apoptosis as well as RBF^D253A-induced overgrowth depend on the JNK pathway activity.

<p>(A–C) Anti-◯P-JNK staining in young third instar larvae wing imaginal discs. (A) No staining is observed in <i>ptc-Gal4/+</i> control. (B, C) <i>UAS-RBF/ptc-Gal4</i> and <i>UAS-RBF^D253A/X; ptc-Gal4/+</i> discs present anti-◯P-JNK staining at the dorso-ventral boundary. All discs are shown with posterior to the top. (D) Notch phenotypes in adult wings of <i>vg-Gal4/+</i>; <i>UAS-RBF</i> and <i>vg-Gal4/+</i>; <i>UAS-RBF/UAS-bsk-RNAi</i> flies are grouped into four categories (wild type, weak, intermediate and strong) according to the number and size of notches observed on the wing margin (asterisks). <i>Bsk-RNAi</i> partially suppresses <i>RBF</i>-induced notch phenotypes (Wilcoxon test, α<10⁻¹⁵, n = 540). (E) The frequency of RBF^D253A-induced ectopic tissue growth is strongly decreased in <i>UAS-bsk-RNAi</i> co-expressing flies (Chi² test, α = 8.8 10⁻¹⁵). (F) TUNEL-labeling of apoptotic cells in wing imaginal discs; specific staining of apoptotic cells corresponds to bright patches in wing discs <i>of vg-Gal4/+</i>; <i>UAS-bsk-RNAi/+</i> (left panel), <i>vg-Gal4/+</i>; <i>UAS-RBF^D253A/+</i> (center panel), <i>vg-Gal4/+</i>; <i>UAS-RBF^D253A/UAS-bsk-RNAi</i> (right panel) <i>larvae</i>. RNAi-mediated knockdown of <i>bsk</i> strongly decreased RBF^D253A-induced apoptosis. All discs are shown with posterior to the top.</p

RBF^D253A is pro-apoptotic in the ZNC and induces more apoptosis than RBF in third instar larvae wing imaginal discs.

<p>(A, E) <i>C96-Gal4</i> and <i>vg-Gal4</i> expression patterns are visualized by <i>UAS-mtGFP</i> expression in third instar larvae wing imaginal discs. (B-D, F-H) Apoptotic cells are labeled by TUNEL in wing imaginal discs; specific staining of apoptotic cells corresponds to bright white patches. (B, F) <i>C96-Gal4/+</i> and <i>vg-Gal4/+</i> control discs have few apoptotic cells. (C) <i>C96-Gal4/UAS-RBF</i> wing discs are similar to control. (D) Some apoptotic cells are observed within the <i>C96-Gal4</i> expression domain in <i>UAS-RBF^D253A/X</i>; <i>C96-Gal4/+</i> discs (white arrow). (G, H) Apoptotic cells are observed within the <i>vg-Gal4</i> expression domain in <i>vg-Gal4/+</i>; <i>UAS-RBF/+</i> and <i>UAS-RBF^D253A/X</i>; <i>vg-Gal4/+</i> wing discs (white arrows). (G) The white arrowhead indicates a zone at the center of the pouch where cells are not TUNEL-labeled in <i>vg-Gal4/+</i>; <i>UAS-RBF/+</i> wing discs. All discs are shown with posterior to the top.</p

<i>RBF^D253A</i> expression induces proliferation of neighboring cells.

<p>(A–F) From left to right, phenotypes of the larvae are: <i>en-Gal4/+</i>, <i>en-Gal4/+</i>; <i>UAS-RBF/+</i> and <i>UAS-RBF^D253AX; en-Gal4/+</i>. (A–C) S phase staining by BrdU (green), and RBF immuno-staining (red). (D–F) BrdU staining (white) of the discs shown in (A–C). (B, E) In RBF expressing discs, as in the control disc shown in (A,D), BrdU staining is homogeneous in the whole disc, except in the ZNC (Zone of Non-proliferating Cells) (white arrow). (C–F) In RBF^D253A expressing discs, cells surrounding the strong RBF staining exhibit an enhanced BrdU staining, indicating that these cells have an increased proliferation rate. (G–L) From left to right, genotypes of the larvae are: <i>ptc-Gal4/+</i>, <i>ptc-Gal4/UAS-RBF</i>, and <i>UAS-RBF^D253A</i> X; <i>ptc-Gal4/+</i>. (G–I) S phase staining by BrdU (green), and RBF staining (red). (J–L) BrdU staining (white) of the discs shown in (G–I) with enlarged view of boxed area. (G, J) BrdU staining is homogeneous in the whole disc, except in the ZNC (white arrow). (I–L) In <i>ptc>RBF^D253A</i> discs, cells within the ZNC that are adjacent to RBF^D253A expressing cells are labeled with BrdU, indicating an abnormal proliferation of these cells. All discs are shown with posterior to the top.</p

RBF^D253A-induced overgrowth does not depend on Wg.

<p>(A, B) Anti-Wg staining in wing imaginal discs of control (A) or <i>en-Gal4, UAS-wg-RNAi</i> third instar larvae (B). No staining is observed in the posterior compartment of discs that express <i>wg-RNAi</i>. (C) The frequency of RBF^D253A-induced ectopic tissue growth is not affected in <i>UAS-wg-RNAi</i> co-expressing flies (Chi² test, α = 0.15). All discs are shown with posterior to the top.</p