Fig. 4. Wg domain regions that give rise to glia and the neurons that guide their migration. (A) The area surrounding the Wg domains was divided into several subdomains on the basis of gene expression patterns. All of the genes categorized (omb, ds, dpp) are regulated by Wg activity. The schematic diagram shows that the Wg domain can be divided into two subdomains (I and III) on the basis of ds expression. wg-expressing cells all express omb, and in one area also express ds. The four subdomains shown are: I, wg/omb/ds; II, wg/omb; III, dpp/omb/ds; and IV, omb. The overlapping expression patterns of these genes are shown in color for the top domain, and in outline form in the bottom domain. The location of the lamina (lam) and lobula (lob) are indicated. An arrow indicates the position of the optic fissure, where the two Wg domains separate during pupal metamorphosis. (B) A horizontal perspective of one Wg domain region. Distal is towards the top. Subdomain I is shown with wg-lacZ positive fascicles extending toward glial migratory destinations. A map of glial subtype origin is overlaid onto subdomain 1, showing the origin of lamina Ma and Ep glia (#1, yellow), medulla neuropile glia (#2, purple), inner chiasm glia (#3, red) and lobula neuropile glia (#4, light blue). The position of these progenitor sites forms a stack on the proximal (#4) to distal (#1) axis. (C) Animal harboring wg-lacZ (anti-ß-gal staining; blue) and a transgenic marker for dpp expression (dpp-GAL4, UAS-CD8::GFP; GFP expression is green). In this lateral view of a late third instar stage optic lobe, the non-overlapping expression of wg and dpp is evident. dpp expression is also found in the inner proliferation center of the lobula (lob). (D) A late third instar optic lobe, like that shown in C, but harboring a ds-lacZ reporter (anti-ß-gal staining in grayscale) and a transgenic marker for omb expression, omb-GAL4, UAS-CD8::GFP (GFP expression in red). Ds expression intersects Omb expression, distinguishing subdomains II and IV from I and III. (E) A late third instar stage optic lobe from an animal harboring the ds-lacZ reporter (anti-ß-gal staining; grayscale), as shown in D, and stained with anti-Dpp antibody (green). The Dpp-positive cells form subdomain III. (F-K) Clonal analysis was performed in order to determine the origins of particular glial subtypes, and the neurons that form their migratory pathways. Random clones were generated using the FLP/FRT system (see Materials and methods) and labeled by membrane-bound GFP (UAS-CD8::GFP, green in F-K). (F) A clone originating within a distal layer of subdomain I (wg/omb/ds) labels lamina glia (Ep, epithelial glia). (G) A clone originating in subdomain I in a slightly more proximal focal plane than in F labels medulla neuropile glia (MNG). The medulla neuropile glia are also labeled by anti-Repo antibody (red). This clone is in the dorsal Wg domain (dorsal towards the top). (H) Clones originating in a more proximal layer of subdomain I than is shown in F or G label inner chiasm glia (Xi). The particular clone shown also labels the scaffold neurons and axons that project along the glial migratory tract (between arrowheads). In this specimen, expression of wg-lacZ is shown in blue. Inset: Xi glia shown double labeled by anti-Repo (red) and repo-GAL4, UAS-CD8::GFP (green) in order to visualize their characteristic size and morphology, which permit these glia to be easily distinguished from many other glial cell types. (I,J) Clones often labeled both scaffold neurons and glia (see Table 1). In these two specimens, single clones labeled both lamina Ma glia (arrows) and a few neurons whose axons project along their migratory pathway (arrowheads). (K) A small (three-or four-cell) clone (green) within the Wg domain (subdomain I, blue) labels neurons (arrowhead) that extend scaffold axons. Scale bars: in C, 20 µm for C-I; in J, 20 µm for J,K. A white or yellow bar indicates the boundary between dorsal and ventral Wg domains in all panels.