(A–B′) Optical section of late second instar wing imaginal disc (∼72 hr AEL, schematic in A with AMPs in green), showing monostratified (single layer) arrangement of AMPs (marked in green and denoted as M for mesoderm, B) stained for Twi (anti-Twist, green), Dcad2 (anti-Dcad2, red) (Disc-epithelium [E]) and TO-PRO3 (a nuclear stain, blue). n = 10, Schematic in B′. (C–C′) Late third instar wing disc showing multistratified (3–4 layers) of AMPs in presumptive notum region. n = 20 Scale bar 50 μm. AMPs are stained for Twi (anti-Twi, green), epithelium (anti-Dcad2, red) and TOPRO (Nuclear stain, blue) (D–D″) A late third instar wing imaginal disc showing multistratified arrangement of AMP using Dmef2-Gal4 > UASmCD8GFP (anti-GFP, green) and co-stained for mitotic marker PH-3 (anti-phosphohistone, red) and TO-PRO3 (Nuclear stain, blue). C′ shows a schematic representation. D′ shows an optical section of image D showing active mitotic divisions (red dotted circles) exclusively in the layer most-proximal to the disc epithelium (marked as E). n = 20. D″ represents this schematically. (E, F and G) Late second instar wing discs (∼70 hr AEL) marked for centrosomin-GFP (CNN-GFP, a pericentrosomal marker, green) using Dmef2-Gal4, PH3 (anti-phosphohistone, red) and TO-PRO3 (Nuclear stain, blue). In each panel, E is the Epidermis and M the AMPs. (H–H′) Schematic (H) of a late second instar disc (as shown in F) showing active mitotic division (Black arrow) parallel to the disc epithelium. Pie-chart representation (H′) (Blue: 10°–20° with 0° being parallel to the epidermis. n = 6 preps. (I, J and K) Third instar wing imaginal disc (∼140 hr AEL) showing a more orthogonal orientation in AMP marked using centrosomin-GFP (CNN-GFP) (a centrosome marker, green) using Dmef2-Gal4, PH3 (anti-phosphohistone, red) and TO-PRO3 (Nuclear stain, blue). n = 10. Scale bar, 10 μm. (L–L′) A schematic representation (L) and pie-chart representation (L′) of late third instar disc showing mitotic division axis (black arrow, in L) of AMP (green) oblique to disc surface (blue). The angle of division at this stage is in the range of 50–80° as represented in L′.