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First published online 21 April 2004
doi: 10.1242/dev.01111

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An axon scaffold induced by retinal axons directs glia to destinations in the Drosophila optic lobe

Department of Molecular and Cellular Biology, Harvard University, Cambridge MA 02138, USA

View larger version (70K): [in a new window]   Fig. 2. Developmental profile of scaffold axon outgrowth, glial migration and retinal innervation. Wg expression commences in the mid-1st instar larval stage in a pair of domains in the outer anlagen, preceding retinal innervation by several days. Scaffold axon fascicle elaboration was visualized by wg-lacZ expression (blue in A-E or white in A'-E'). Glia were visualized by anti-Repo staining (red in all panels). Neuronal cell bodies and neuropiles were visualized by anti-HRP staining (green in A-E). Above each optic lobe image, which shows a different time point (youngest at left in A), is a schematic diagram indicating the state of ommatidial development in terms of the number of ommatidial columns. The developing optic lobe is outlined in white in A-E and yellow in A'-E'. (A,A') An early 2nd instar stage specimen, lacking any retinal innervation (0 columns), in which the two Wg domains contain a small number of cells. A small island of Repo-positive glia are observed in the prospective photoreceptor axon target region (arrowhead). Scaffold axon fascicles are not detected. (B,B') A mid-2nd instar specimen, a time point just prior to ommatidial development. The anlage have grown in size, but scaffold axon tracts are not yet detectable. (C,C') A late 2nd instar specimen in which one ommatidial column is detected in the retina, and a small amount of retinal innervation has occurred. The medulla neuropile has just begun formation. Scaffold axon fascicles are clearly visible emanating from the Wg domains and extending towards the neuropile (green arrowheads in C'). There is an increase in glia number within the neuropile region at this time point (yellow arrowhead). (D,D') An early 3rd instar stage animal in which three columns of developing ommatidia were present in the retina. Scaffold axon fascicles begin to elaborate an architecture reminiscent of the late third instar (compare with Fig. 1C), in which tracts for medulla neuropile glia and lobula neuropile glia are distinguishable (green arrowheads in D'). Considerably more glia are visible in the neuropile region (yellow arrowhead in D') in the focal plane shown. (E,E') A mid-3rd instar stage specimen in which seven columns of ommatidia were detected in the retina. Scaffold axon fascicles for MNG glial migration are indicated (green arrowheads in E').

View larger version (73K): [in a new window]   Fig. 1. Axons from Wg-expressing neurons form pathways for glial migration to different optic lobe target layers. (A) Schematic diagram of developing optic lobe architecture and glial migration viewed from a lateral perspective in a late third instar larval stage brain. Dorsal is upwards and posterior rightwards. Inner chiasm glia (Xi, red), medulla neuropile glia (MNG, purple), marginal and epithelial lamina glia (Ma/Ep, yellow), and lobula neuropile glia (LoG, light blue) exit the Wg domains (Wg, dark blue) via distinct scaffold axon fascicles. The eye disc (ed), photoreceptor axons (R-cell) and optic stalk (os) are indicated. The medulla (med, dark green), lamina (lam, light green) and lobula (lob, brown) neuropiles are indicated. (A') An enlarged schematic view of the ventral Wg domain, showing the distinct scaffold axon pathways for different glial cell types. (B) Optic lobe architecture and glial migration viewed from the horizontal perspective. Distal is upwards. The scaffold axon fascicles are distributed on the proximal distal axis. The pathway for lamina glia is the most distally positioned, while the tract for lobula neuropile glia is most proximal. Labeling as in A. (B') An enlarged view of the ventral Wg domain in horizontal view, illustrating the lamination of scaffold axon fascicles and glial cell types. (C) Two scaffold axon fascicles (orange arrowheads) are observed extending from each of the dorsal and ventral Wg domains (Wg; dorsoventral boundary indicated by the orange line in C-F), which are visualized by expression of wg-lacZ (anti-ß-gal; blue) in this late third instar stage brain. The two pairs of scaffold axon fascicles visible in this plane extend to the medulla neuropile (med n'pil, tract for MNG glia) and lobula neuropile (lob n'pil, tract for lobula neuropile glia). The neuropiles are visualized by anti-HRP antibody staining (green). The location of medulla cortex (med cortex) is indicated. (C') Image in C showing only wg-lacZ expression, to permit visualization of the scaffold axon fascicle pairs (orange arrows). (D) Glia (anti-Repo staining; red) seen during migration (white arrowheads) from the Wg domains (wg-lacZ visualized by anti-ß-gal staining; blue). The layer of lamina marginal glia (Ma) is visible in this plane, as well as some inner chiasm glia (Xi) headed towards a more proximal destination. (D') Image shown in D with only wg-lacZ expression shown in order to detail the scaffold axon fascicles (orange arrowheads). (E) On their pathway towards the lamina (lam), glial differentiation is marked by the onset of Gcm expression (anti-Gcm, white, cells between arrowheads). The glia emerge from a region in which Optomotor Blind (Omb; anti-Omb, purple) is expressed under Wg control, and continue to express Omb as they migrate toward neuropile destinations. The area demarcated by white arrowheads, in which most cells express Gcm, can be compared with the onset of Repo expression in the same region, indicated by white arrowheads in D. (F) Scaffold axon fascicles (orange arrowheads) originate from a subset of Wg expressing neurons (wg-lacZ; anti-ß-gal staining; white), which co-express the neuronal marker Elav (anti-Elav; red). Neuronal populations and neuropiles are labeled as in C. Scale bar: 20 µm.

View larger version (76K): [in a new window]   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.

View larger version (135K): [in a new window]   Fig. 3. Fate of Wg-expressing neurons in the mature optic lobe. Specimens harboring the transgene wg-lacZ were examined during the late pupal stage [about 200 hours AEL (after egg laying)] for the location and axonal projection pattern of the Wg-positive neurons. (A) Most Wg-positive neurons are located at dorsal and ventral positions in the medulla and lobula cortices. In this image, a dorsal cluster of neurons is visible in the upper right corner of the yellow box (anti-ß-gal staining; blue). MNG and Xi glia (anti-Repo staining; red) are indicated in their distinct positions relative to the medulla and lobula neuropiles (med n'pil, lob n'pil; anti-HRP staining; green). The medulla cortex (med ctx) is also indicated. (A') A higher-magnification view of the yellow-boxed region in A. Clusters of wg-lacZ-positive neurons (anti-ß-gal staining; grayscale) extend axons (yellow arrowheads) towards the edge of the medulla neuropile and into the proximal medulla layer (green arrowhead). (B) A late pupal stage specimen with wg-lacZ-positive neurons visible (blue) along the dorsal edge of the cortices. In this image, glial layers of the lamina (lam) are visible. (B') The image in B with anti-HRP staining omitted and wg-lacZ expression shown in grayscale. wg-lacZ-positive neurons elaborate long axons that project tangentially into the medulla and lobula neuropiles (yellow and green arrowheads). Glia (red; yellow arrows) are associated with the wg-lacZ-positive axons In this specimen, the lamina marginal (Ma) and epithelial (Ep) glial layers are indicated, but wg-lacZ-positive axon tracts are not found extending toward these layers at this stage. Scale bars: in A, 15 µm for A,B,B'; in A', 10 µm.

View larger version (105K): [in a new window]   Fig. 5. Retinal innervation is locally required for scaffold axon outgrowth. (A-A'') A wild-type late third instar stage optic lobe (lateral view) in which the Wg domains (indicated by a pair of arrows) are marked by wg-lacZ (anti-ß-gal staining; blue). Glia are marked by anti-Repo staining (red). MNG and Xi glia are visible in this focal plane. These two glial types are more easily discerned when anti-HRP staining (green, A) is omitted in A' and A''. (A'') An enlarged view of the area boxed in A', where the alignment of MNG glia (yellow arrowheads) with a scaffold axon fascicle is evident. (A'') wg-lacZ staining is shown in grayscale. (B-B'') An so1, wg-lacZ late third instar stage optic lobe in which all photoreceptor axon innervation is absent. The medulla neuropile (arrow in B) is somewhat disorganized (med n'pil; anti-HRP, green). Glia (red) are mostly absent from the neuropile region, and appear accumulated at the edge of the Wg domains (yellow arrowheads in B',B''). Scaffold axons are completely absent, as is clear in the higher magnification view (B'') of the boxed area in B'. (C-C'') An so1, wg-lacZ late third instar specimen in which photoreceptor axons have innervated only the dorsal half of the optic lobe. The dorsal scaffold axon fascicles (anti-ß-gal staining, blue) have extended (yellow arrow in C', C'') and glia (red) migrate into the neuropile. By contrast, ventral scaffold axons are missing and the glia stall (yellow arrowhead) as in B. Medulla neuropile organization is disrupted in the ventral half of the optic lobe. White or yellow lines indicate the boundary between dorsal and ventral Wg domains. Scale bars: 20 µm in all panels.

View larger version (58K): [in a new window]   Fig. 6. Evidence that scaffold axons are required for glia migration and direct glia along specific pathways. (A,A') Wild-type scaffold axon fascicles (anti-ß-gal; grayscale) in a wg-lacZ late third instar optic lobe project in a stereotyped fashion toward glial destinations. MNG and Xi glia (anti-Repo; red) are seen following along fascicles towards their appropriate destinations. A higher magnification view of the yellow boxed area in A is shown in A'. (B,B') In ds1 homozygous animals, scaffold axon fascicles project aberrantly, and the distribution of glia is correspondingly aberrant. A higher magnification image of the yellow-boxed region of B is shown in B'; glia (yellow arrowheads) on an abnormal trajectory are closely associated with the misprojecting scaffold axon fascicle (anti-ß-gal; grayscale). (C,C') Scaffold axon projections are likewise aberrant in ds33k homozygous animals. In this specimen, dorsal scaffold axon fascicles bifurcate onto aberrant trajectories, and glia are likewise misdirected. The higher magnification image of the boxed region of C shown in C' reveals mispositioned glia (anti-Repo; red) in association with the misrouted scaffold axon fascicles (anti-ß-gal staining in grayscale). (D,D') Random somatic clones were generated expressing both a GFP marker and an activated Ras protein (Ras1N17) that inhibits axon extension. wg-lacZ (anti-ß-gal staining; blue) was used to mark the migratory axon scaffold. In the late third instar optic lobe shown, a clone encompasses part of the dorsal Wg domain (white outline in D and yellow outline in D'). Glia (anti-Repo, red), which do not express the UAS-Ras1N17 transgene, stalled at the edge of this Wg domain (yellow arrowheads in D,D'), much as they do in `eyeless' mutant strains (see Fig. 5). Scaffold axon fascicles cannot be detected emanating from the dorsal Wg domain, but are clearly visible extending from the ventral Wg domain (yellow arrows); ventral glia migrate normally. (E,E') A specimen like that shown in D,D' in which somatic clones (white outline in E, yellow outline in E') express UAS-Ras1N17 (labeled by co-expression of GFP, green in E). Clones of particular interest encompass both the dorsal and ventral Wg domains. Scaffold axons are absent from both and glia stall at both the dorsal and ventral margins of the Wg domains (yellow arrowheads in E'). Scale bars: in A, 20 µm (for A-D,D',E,E'); in A', 8 µm (for A',B',C').

View larger version (122K): [in a new window]   Fig. 7. A normal glial distribution is required for cell survival in the developing medulla. Cell death was compared in wild-type and so1 animals in relation to the distribution of glia. (A-A'') A wild-type late third instar optic lobe is stained to reveal a normal organization of the medulla neuropile (med. n'pil, anti-HRP staining, green) and distribution of MNG and Xi glia (anti-Repo, red). Very few apoptotic cells are evident by staining with anti-activated caspase 3 antibody (blue in A,A', shown in grayscale in A''). (B-B'') In so1 animals, an increased frequency of apoptosis was detected in the late third instar optic lobe (see the text for details). In the specimen shown, retinal axons have innervated the dorsal (dorsal is upwards) optic lobe, resulting in a relatively normally organized medulla neuropile. The ventral optic lobe lacks innervation and glia accumulate in the vicinity of the Wg domain (white arrow in B'). Few glia are found in the ventral neuropile region. An increased frequency of anti-activated caspase 3-positive cells (blue in B and B', grayscale in B'') is seen throughout the medulla cortex. More apoptotic cells are found in the ventral region (regions between white, yellow and blue arrowheads; see text for quantification), where glia are rare. (C-C'') An animal in which glial migration was blocked by expression of UAS-Ras1N17 in somatic clones (marked by co-expression of UAS-CD8::GFP (green) (see the legend to Fig. 6, and Materials and methods for experimental details). A Ras1N17-expressing clone in the dorsal Wg domain region has blocked glial migration into the dorsal optic lobe in this late third instar stage specimen. Glia (red) accumulate at the position of the yellow arrowhead shown in the boxed area in C. A locally increased frequency of activated Caspase 3-positive cells is visible (blue in C; grayscale in C', shown alone in grayscale between arrowheads in C''). Scale bar: 20 µm.

View larger version (12K): [in a new window]   Fig. 8. A cellular model for the control of glial migration by retinal innervation. A small number of glia (red) migrate into the target region for photoreceptor axons prior to ommatidial development, and are ready to provide initial guidance cues to the first photoreceptor axons (green; top two panels). These early photoreceptor axons trigger the elaboration of the axonal scaffold for glial migration (blue; third panel from top), which extends in stereotypical fashion to establish the multiple pathways (not depicted). Glia generated in the Wg subdomain 1 (yellow) can then migrate to target destinations, such as the lamina (shown, bottom panel). Subsequent migration may be independent of continued retinal axon ingrowth (see the text). Through this mechanism, the distribution of glia throughout the optic lobe is coordinated with innervation by the photoreceptor axons.