Fig. 1. R8, R7, and L1-L5 axons sequentially project into distinct layers in the medulla during the first layer-selection stage. (A,A',B,B',B'') 17% APF; (C,C',D,D') 35% APF. (A,A') The initial projections of R7 and R8 axons were assessed at 17% APF, using PM181-Gal4, UAS-mCD8-GFP (green) and Mab24B10 (red), respectively. (A') A high magnification view of medulla of A. The developing medulla is viewed in cross section to reveal the developmental sequence of R7 and R8 innervation at 17% APF. In this view, the younger R7 and R8 growth cones are to the left and the older ones to the right. The newly arriving R7 growth cones (arrow) project past the R8 growth cones and then expand (arrowhead) to terminate at the layer below the R8 growth cones. (B,B',B'') The L1-L5 projections viewed in the same direction and at the same stage as in A,A'. The L1-L5 axons expressed Gcm-Gal4, UAS-lacZ, and were visualized with anti-lacZ antibody (blue). At this stage, a subset of young medulla neurons (MN) also expresses Gcm although their axons have yet to enter the medulla. Both R7 and R8 axons were labeled using a pan-R-cell marker, GMR-GFP (green). R8 axons and older R7 axons are visualized with Mab24B10 (red). (B') A high magnification view of the medulla of B. The newly arriving R7 growth cones (arrow) express GFP but not Mab24B10. (B'') The green channel (GFP) is removed from B' to facilitate the visualization of the L1-L5 projections (blue). Based on the R7 and L1-L5 markers, we estimated that the newly differentiating L1-L5 growth cone (arrow) reaches the medulla 1-2 rows behind R7s. The older L1-L5 growth cones (arrowhead) terminate between R7 and R8 growth cones. (C,C') R7 and R8 growth cones (red) form two separate medulla layers at 35% APF. R7 axons were labeled using PM181-Gal4, UAS-mCD8-GFP marker, which was prolonged with the UAS-Gal4 method. This method also labels subsets of medulla (MN) and lobular (lo) neurons, which normally express PM181 at a very low level. (D,D') L1-L5 growth cones form two separate layers between R7 and R8 growth cones at 35% APF. The L1-L5 neurons were labeled as in B. At this stage, medulla glia (MG), which wrap around external medulla, express Gcm. The presumptive R7-temporary layers are indicated by dotted lines in B',B''. (A'-D') High magnification views of A-D, respectively. ed, eye disc; la, lamina; me, medulla: lo, lobula; LN, L1-L5 neurons; MN, medulla neurons; MG, medulla glia. Scale bars: in A, 30 µm for A-D; in A' 10 µm for A'-D'. (E) A schematic diagram illustrating the order of R-cell and L1-L5 afferents innervating the medulla. The developmental sequences in the eye disc, lamina, and medulla are indicated by arrows. R8 (red), R7 (green), and L1-L5 (blue) axons sequentially innervate the medulla (gray shaded box) to reach their temporary layers (as indicated).