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First published online 3 August 2006
doi: 10.1242/dev.02511

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Actin capping protein maintains vestigial-expressing cells within the Drosophila wing disc epithelium

Florence Janody^1,*, and Jessica E. Treisman²

¹ Developmental Biology Institute of Marseille Luminy, UMR6216 - Case 907, Parc Scientific de Luminy, 13288 Marseille Cedex 09, France.
² Skirball Institute of Biomolecular Medicine and Department of Cell Biology, NYU School of Medicine, New York, NY 10016, USA.

View larger version (126K): [in a new window]   Fig. 1. cpa or cpb mutant clones are extruded from the wing blade epithelium. All panels show third instar wing discs. (A-C) Standard confocal sections. (A) cpa107E, (B) tsr110M and (C) captE636 mutant clones are marked by the absence of GFP (green). (D-H) Optical cross-sections through the wing disc epithelium. Mutant clones are positively labeled with GFP (green) and sections are stained with anti-Dlg (red) and anti-Arm (blue) to outline apical cell membranes. (D) cpa107E; (E) cpbM143; (F) tsr110M; (G) captE593; (H) cpa107E mutant clones overexpressing full-length cpa. cpa or cpb mutant clones are extruded basally in the wing blade primordium (red arrows), but not the notum; this extrusion is rescued by full-length cpa. tsr and capt mutant clones are not extruded. The white arrows define the wing blade region. Dorsal is towards the left on optical cross-sections in this and subsequent figures.

View larger version (110K): [in a new window]   Fig. 2. Extrusion of cpa mutant cells is independent of programmed cell death. All panels show third instar wing imaginal discs. (A-C) Optical cross-sections of discs stained with anti-Dlg (blue in A,B'',C) to outline apical cell membranes and anti-Caspase 3 (red in A,B',B'') or anti-ß-Galactosidase to reveal puc-lacZ expression (red in C). Anti-Caspase 3 antibody gives a non-specific background staining seen at the apical surface of the discs. (A) T155-Gal4; UAS-flp induced cpa69E mutant clones marked by the absence of GFP (green). (B-C) hs>flp induced cpa69E (B) or cpa107E (C) mutant clones, positively labeled with GFP (green). (A,B'',C) The overlay of all three channels. cpa mutant clones express Caspase 3 and puc-lacZ cell autonomously. Cell death is seen when FLP is induced either by heat shock or by the epithelial driver T155-GAL4, and is therefore not due to stressed conditions induced by heat shock, as described for clones mutant for the Dpp receptor thickveins (tkv) (Gibson and Perrimon, 2005). (D-I) Standard confocal sections (D-F) or optical cross sections (G-I) of clones positively labelled with GFP (green) and stained with anti-Dlg (red) and anti-Arm (blue). (D,G) cpa107E mutant clones; (E,H) clones overexpressing th; (F,I) cpa107E mutant clones overexpressing th. th overexpression promotes survival of cpa mutant cells, but fails to prevent their extrusion. The white arrows define the wing blade region.

View larger version (113K): [in a new window]   Fig. 3. cpa maintains the localization of adherens junction components. (A-F) Optical cross-sections through third instar wing imaginal discs, stained with anti-Arm (red in A,B'',B''', green in C,D,E,F or white in B',C',D',E',F') and anti-HA, reflecting UAS-HA-cpa expression (green in A,B'',B''' or white in B) or anti-Dlg (red in C,D,E,F or white in C'',D'',E'',F''). (A,B) cpa69E mutant clones overexpressing HA-tagged full-length cpa, positively labeled with GFP (blue in A,B'''). The white arrow in A defines the wing blade region. (B-B''') Magnification of the blade primordium. HA-cpa accumulates at the apical membrane, partly co-localizes with Arm in all regions of the wing disc and rescues extrusion of cpa mutant clones in the wing blade primordium. (C,D,F) cpa69E mutant clones positively labeled with GFP (blue in C,D,F) in the wing blade (C,D) or notum (F) primordium, induced both at second or early third instar and dissected at the late third instar stage either 60 hours (C) or 36 hours (D,F) after clone induction. Although we could recover mutant clones within the disc epithelium 36 hours after clone induction, all mutant cells were extruded by 60 hours. (E) tsr99E mutant clones in the blade primordium, dissected 36 hours after clone induction and positively labeled with GFP (blue in E). Arm is mislocalized to basolateral regions in extruding cpa mutant cells and in tsr mutant cells that are maintained within the epithelium (red arrows in D' and E'). Following extrusion of cpa mutant cells, expression of both Arm and Dlg are lost (C). The white arrows in A define the wing blade region.

View larger version (84K): [in a new window]   Fig. 4. Loss of cpa causes excessive actin polymerization. (A-E) Third instar wing discs in which clones are marked by the absence of GFP (green in A',D,E or blue in B,C) and stained with TRITC-phalloidin to reveal F-actin (red in A,A',B,B',C,C',D,D',E,E') and anti-Arm (green in B,C or white in B'',C''). (A) Standard confocal sections; (B-E) optical cross-sections. (A-C) cpa69E mutant clones in the notum (C) or the blade (B) primordium. cpa mutant clones accumulate actin filaments near the apical cell membrane in the notum but throughout the cell in the blade primordium. (D) tsr110M or (E) captE636 mutant clones.

View larger version (129K): [in a new window]   Fig. 5. cpa is required in vestigial expressing cells. (A-G) Third instar wing imaginal discs. (A-A'') Standard confocal sections of wing discs with cpa mutant clones marked by the absence of GFP (green in A,A'') and stained with anti-Vg (magenta in A',A''). Vg is expressed in cpa mutant clones. (B-G) Discs in which clones are positively labeled with GFP (green) and stained with anti-Dlg (blue) and anti-Caspase 3 (red). (B-D) Standard confocal sections; (E-G) optical cross sections. (B,E) cpa43D mutant clones are extruded and die in the wing blade region; (C,F) clones overexpressing vg are not extruded and survive; (D,G) cpa43D mutant clones overexpressing vg are extruded and die in all regions of the disc. Red arrows in E,G indicate clones within the notum primordium. The white arrows define the wing blade region.

View larger version (68K): [in a new window]   Fig. 6. Vg and Notch upregulate cpa transcription in the wing blade primordium. (A-F') Third instar wing imaginal discs. (A,B,D-F) Standard confocal sections. (C) Optical cross section, stained with anti-Arm to outline the apical membrane. Clones are positively labeled with GFP (green in D',E',F'). (A,C,D,D';E,E') cpa antisense probe to reveal cpa mRNA expression (white in A,C,D,E and magenta in D',E') in wild type (A,C) or in clones overexpressing vg (white arrow in D,D') or in clones overexpressing Nintra (white arrow in E,E'). As cpa mRNA accumulates on the basal surface of the wing disc epithelium, standard confocal sections fail to reveal cpa accumulation in all vg and N- overexpressing clones. (B) cpa sense probe: the background level is very low. (F) Clones overexpressing Nintra, stained with anti-Vg (white in F or magenta in F'). Vg is misexpressed.

View larger version (32K): [in a new window]   Fig. 7. Models for the effect of CPs and Vg on wing morphogenesis. (A) The phenotypic differences between cpa, tsr and capt could be due to different structures of the actin network in mutant cells. CPs are represented by green bars, Capt by blue crescents binding to monomeric actin (black circles) and Cofilin activity by pink arrows. Loss of cpa would result in extension of each branch of the network. Loss of tsr would make the core filament longer, while the length of branches would be unchanged as they have no free pointed ends. Loss of capt would free more actin monomers for incorporation into networks with the wild-type structure. (B) Cpa (green) is expressed at the apical membrane and co-localized with junctional complexes (orange squares) that link the actin cytoskeleton (in red) of neighboring cells. Vg expression differentiates the wing blade from the notum, enhances cpa expression and also alters the cytoskeleton in such a way as to make the cells dependent on cpa. These changes may contribute to morphogenesis of the adult wing blade. In the notum, Cpa contributes to bristle development. (C) cpa mutant cells (green) accumulate actin filaments near the apical membrane and are maintained in the epithelium in the notum, causing defects in bristle development. However, in the wing primordium, owing to other cytoskeletal properties induced by Vg, cpa mutant cells mislocalize junctional components, accumulate actin filaments throughout the cell, are extruded and die.