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

Abstract

Tissue patterning must be translated into morphogenesis through cell shape changes mediated by remodeling of the actin cytoskeleton. We have found that Capping protein alpha (Cpa) and Capping protein beta (Cpb), which prevent extension of the barbed ends of actin filaments, are specifically required in the wing blade primordium of the Drosophila wing disc. cpa or cpb mutant cells in this region, but not in the remainder of the wing disc, are extruded from the epithelium and undergo apoptosis. Excessive actin filament polymerization is not sufficient to explain this phenotype, as loss of Cofilin or Cyclase-associated protein does not cause cell extrusion or death. Misexpression of Vestigial, the transcription factor that specifies the wing blade, both increases cpa transcription and makes cells dependent on cpa for their maintenance in the epithelium. Our results suggest that Vestigial specifies the cytoskeletal changes that lead to morphogenesis of the adult wing.

Figure 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. cpa^107E (A); tsr^110M (B) and capt^E636 (C) 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 and stained with anti-Dlg (red) and anti-Arm (blue) to outline apical cell membranes. (D) cpa^107E; (E) cpb^M143; (F) tsr^110M; (G) capt^E593; (H) cpa^107E 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 to the left on optical cross-sections in this and subsequent figures.

Figure 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-β-Galactosidase to reveal puc-lacZ expression (red in C). Note that anti-Caspase 3 antibody gives a non-specific background staining seen at the apical surface of the discs. (A) T155-Gal4; UAS-flp induced cpa^69E mutant clones marked by the absence of GFP (green). (B–C) hs>flp induced cpa^69E (B) or cpa^107E (C) mutant clones, positively labelled with GFP (green). (A, B”, C) show 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) stained with anti-Dlg (red) and anti-Arm (blue). (D, G) cpa^107E mutant clones; (E, H) clones overexpressing DIAP1; (F, I) cpa^107E mutant clones overexpressing DIAP1. DIAP1 overexpression promotes survival of cpa mutant cells, but fails to prevent their extrusion. The white arrows define the wing blade region.

Figure 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”‘, C,D,E,F or white in B’,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 (green in C,D,E,F or white in C”,D”,E”,F”). (A–B) cpa^69E mutant clones overexpressing HA-tagged full-length cpa, positively labelled 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) cpa^69E 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. While we could recover mutant clones within the disc epithelium 36 hours after clone induction, all mutant cells were extruded by 60 hours. (E) tsr^99E 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).

Figure 4. Loss of cpa causes excessive actin polymerization

All panels show 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) cpa^69E 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) tsr^110M or (E) capt^E636 mutant clones.

Figure 5. cpa is required in vestigial expressing cells

All panels show 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) cpa^43D mutant clones are extruded and die in the wing blade region; (C, F) clones overexpressing vg are not extruded and survive; (D, G) cpa^43D 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.

Figure 6. Vg and Notch upregulate cpa transcription in the wing blade primordium

All panels show 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 anti-sense probe to reveal cpa mRNA expression (white in A,C,D,E and magenta in D’,E’) in wildtype (A, C) or in clones overexpressing vg (white arrow in D, D’) or in clones overexpressing N^intra (white arrow in E, E’). Since 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. Note that the background level is very low. (F) clones overexpressing N^intra, stained with anti-Vg (white in F or magenta in F’). Vg is misexpressed.

Figure 7. Models for the effect of CP 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. CP is represented by green bars, Capt by blue crescents binding to monomeric actin, 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 wildtype 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, due to other cytoskeletal properties induced by Vg, cpa mutant cells mislocalize junctional components, accumulate actin filaments throughout the cell, are extruded and die.

Supplementary Figure S1. Dynein/Dynactin-based transport, JNK and blistered are not essential for extrusion of cpa mutant cells

All panels show optical cross sections through the wing disc epithelium of clones positively labeled with GFP (green) and stained with anti-Dlg (blue in A,B,C,F,G,H or red in D,E)) and anti-Caspase 3 (red in A,B,C,G,H) or anti-Arm (red in D,E). (A) cpa^69E mutant clones. (B) khc^K13314 mutant clones (c) cpa^69E; khc^K13314 double mutant clones. (D) Clones overexpressing bsk^DN. (E) cpa^69E mutant clones overexpressing bsk^DN. Removal of bsk or khc fails to rescue extrusion of cpa mutant clones. (F) Clones expressing Mal-D-ΔN. Removal of the N-terminus of Mal-D renders it nuclear and active (Somogyi and Rorth, 2004). (G) Clones expressing dia^CA. (H) cpa^69E, bs² double mutant clones. Expression of Mal-D or dia^CA causes extrusion and death of epithelial cells, but removing dSRF fails to rescue extrusion of cpa mutant cells. The white arrows define the wing blade region.