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First published online 3 November 2004
doi: 10.1242/dev.01465

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Compartmentalization of visual centers in the Drosophila brain requires Slit and Robo proteins

Timothy D. Tayler, Myles B. Robichaux^* and Paul A. Garrity

Department of Biology, Massachusetts Institute of Technology, 77 Massachusetts Avenue 68-230B, Cambridge, MA 02139, USA

View larger version (93K): [in a new window]   Fig. 1. Developing Drosophila visual system. (A) Horizontal view of wild-type third instar visual system (anterior to left) of animal expressing CD8:GFP under the control of Sca-Gal4 (Sca:GFP). GFP is expressed in the OPC, IPC, medulla cortex and portions of the lobula cortex. Sca:GFP (green). Neuronal nuclei are visualized using anti-Elav (blue); photoreceptor axons, lamina monopolar axons and axons from neurons of the lobula cortex (a subset of which contact the medulla neuropil) are visualized using anti-Fasciclin 2 (Fas2) (red). Dotted line indicates anterior edge of lobula cortex. (B) Lateral view (anterior at bottom) of Sca:GFP animal in which neuronal nuclei have been visualized using anti-Elav (blue) and glial nuclei using anti-Repo (red). Schematics of (C) horizontal view and (D) lateral view, indicating cell populations and axons described in the text. ep, epithelial glia; IPC, inner proliferation center; lamc, lamina cortex; lamn, lamina neuropil; lobc, lobula cortex; ma, marginal glia; medc, medulla cortex; mg, medulla glia; medn, medulla neuropil; OPC, outer proliferation center; os, optic stalk.

View larger version (92K): [in a new window]   Fig. 2. Slit is required for optic lobe development. (A,B,E-H) Third instar visual systems, photoreceptor axons visualized with anti-Chaoptin. (A) In wild type, photoreceptor axons grow into the brain through the optic stalk. The R1-R6 subset of photoreceptor axons stop in the lamina neuropil while R7 and R8 continue into the medulla neuropil. (B) In slitdui mutants, there are gaps in the lamina neuropil (arrow) and increased numbers of axons enter the medulla (arrowhead). (C) Wild type and (D) slitdui visual systems in which R2-R5 photoreceptor axons are visualized using Ro--lacZ (Garrity et al., 1999). (C) In wild type, all R2-R5 axons stop in the lamina neuropil. (D) In slitdui mutants, many R2-R5 axons pass through the lamina and enter the medulla (arrowheads). (E) slitdui/slitl(2)k05248, (F) slitdui/slitE158 and (G) slitdui/slit2 animals show photoreceptor axon targeting defects indistinguishable from slitdui homozygotes, with gaps in the lamina (arrow) and increased numbers of axons entering the medulla (arrowhead). (H) Omb-Gal4;UAS-Slit; slitdui/slit2 visual system. Slit cDNA expression controlled by Omb-Gal4 largely rescues slit targeting defects, restoring even layer of photoreceptor growth cones in the lamina (arrow). (I,J) Animals carrying loco:lacZ enhancer trap (which is strongly expressed in epithelial and marginal glia) stained with anti-lacZ. (I) In wild type, continuous layers of epithelial and marginal glia are observed in the lamina. (J) In slit mutants, there are clumps of glia (arrowhead) and gaps (arrow) in the glial layers. (K,L) Photoreceptors axons are visualized with GMR:GFP (green) and glial nuclei with Repo:lacZ (magenta). (K) In wild type, R1-R6 axons stop in the lamina between layers of glia (open arrowheads). (L) In slit mutants, there are gaps in the photoreceptor innervation of the lamina, correlated with regions of the lamina devoid of glia (asterisk) and uneven innervation in regions containing clumps of glia (arrow). The clear separation between glia at the base of the lamina and glia surrounding the medulla observed in wild type is missing in slit mutants (arrowhead). ep, epithelial glia; lamn, lamina neuropil; ma, marginal glia; medn, medulla neuropil.

View larger version (140K): [in a new window]   Fig. 3. Distal cell neurons invade the lamina in slit mutants. (A-D) Third instar visual systems in which IPC neuroblasts and distal cell neurons are visualized using anti-Fasciclin 3 (Fas3, red), photoreceptor axons using GMR:GFP (green), and neuronal nuclei using anti-Elav (blue). (A,B) Horizontal view (anterior to left). (A) In wild type, IPC neuroblasts (which express Fas3) and their distal cell neuron progeny (which express Fas3 and Elav) are adjacent to the posterior edge of the lamina (arrowhead). (B) In slitdui/slit2 mutants, distal cell neurons enter the base of the lamina (arrow) and reach the lamina's anterior edge (arrowhead). Distal cell neurons also enter the neuropil of the lamina (asterisk) and photoreceptor innervation is disrupted. (C,D) Lateral view (anterior at bottom). (C) In wild type, distal cell neurons are immediately adjacent to the posterior face of the lamina. (D) In slitdui/slit2 mutants, distal cell neurons enter the posterior face of the lamina (arrow) and reach its anterior edge (arrowhead). dcn, distal cell neuron progeny; IPC, inner proliferation center; lamc, lamina cortex; lamn, lamina neuropil; lobc, lobula cortex; medc, medulla cortex; medn, medulla neuropil.

View larger version (109K): [in a new window]   Fig. 4. Slit is expressed in the developing optic lobe. (A,B) Third instar nervous system stained with anti-Slit. (A) Slit is expressed in the medulla neuropil and the base of the lamina (arrow), as well as the ventral ganglion midline and mushroom bodies. (B) In slitdui mutants, Slit expression is greatly reduced in the optic lobe and ventral ganglion, although robust mushroom body staining is still observed. (C) Slit mRNA is expressed by cells surrounding the medulla. (D,E) Third instar visual systems stained with anti-Slit (magenta) and anti-Repo (green). (D) Slit protein is found throughout the medulla neuropil (arrow), which is surrounded by medulla neuropil glia. (E) Slit is present in the lamina neuropil and surrounds the epithelial, marginal and medulla glia. (F) Similar view as in E, stained with anti-Slit (magenta) and Sca:GFP (green). (G) Horizontal view stained with anti-Slit (red), anti-Elav (blue) and Sca:GFP (green). Slit protein localizes immediately adjacent to distal cell neurons at the base of the lamina and the optic chiasm. (H) Summary of Slit expression. (I) Third instar eya2 mutant visual system stained with anti-Slit. Slit protein is expressed in the medulla neuropil (arrow) in the absence of photoreceptor innervation. (I,J) Expression of the slitl(2)k05248 (Slit:lacZ) enhancer trap. (J,K) Optic lobes stained with anti-lacZ (red) and anti-Repo (blue). (J) Slit:lacZ is expressed in medulla glia (arrowhead) and cells in the medulla cortex (arrow). (K) Slit:lacZ cells in medulla cortex (arrow) lie adjacent to medulla neuropil glia. (L) Optic lobe stained with anti-lacZ (upper panel), anti-Elav (middle panel) and a merged image (lower panel) with anti-lacZ in magenta and anti-Elav in green. Slit:lacZ cells in medulla cortex (arrows) coexpress varying levels of neuronal marker Elav. ep, epithelial glia; lamc, lamina cortex; lamn, lamina neuropil; lobc, lobula cortex; ma, marginal glia; mb, mushroom bodies; medc, medulla cortex; medn, medulla neuropil; mg, medulla glia; mng, medulla neuropil glia; vg, ventral ganglion midline.

View larger version (125K): [in a new window]   Fig. 5. Robo, Robo2 and Robo3 are expressed in overlapping patterns in the visual system. (A,D,G) Third instar nervous systems stained with antisera against indicated Robo family member. (B,E,H) Lateral view of optic lobe stained with antisera against indicated Robo family member (magenta), neuronal nuclei stained with anti-Elav (blue), and photoreceptor axons visualized with GMR:GFP (green). (C,F,I) Robo family staining alone. (A) Robo is expressed in the developing optic lobes (arrowheads). (B,C) Robo is expressed by IPC neuroblasts, by distal cell neurons, and in the medulla cortex. (D) Robo2 is expressed in the developing optic lobes (arrowheads). (E,F) Robo2 is expressed by IPC neuroblasts and distal cell neurons. (G) Robo3 is expressed in the developing optic lobes (arrowheads). (H,I) Robo3 expression is not detected in IPC neuroblasts, but is detected in distal cell neurons and in the medulla cortex as well as in photoreceptor axons. IPC, inner proliferation center; lamn, lamina neuropil; medc, medulla cortex.

View larger version (86K): [in a new window]   Fig. 6. Knockdown of Robo family proteins using transgenic RNAi. (A) Western blot analysis of adult heads, showing that animals homozygous for robo31 express full-length Robo3 protein (caret) as well as a truncated Robo3 (asterisk). Anti-Elav used as loading control. (B) Western blot analysis of adult heads, showing that expression of UAS:RoboRNAi in the nervous system controlled by c155-Gal4 reduces Robo protein levels. (C) Ubiquitous expression of UAS:Robo2RNAi under the control of tubulin-Gal4 reduces anti-Robo2 staining in the visual system. (D) Western blot analysis of adult heads, showing that expression of UAS-Robo3RNAi controlled by c155-Gal4 reduces Robo3 protein levels. (E-M) RNAi of an individual Robo family member does not detectably reduce expression of other Robo family proteins. (E-G) Anti-Robo staining in magenta. (H-J) Anti-Robo2 expression in magenta. (K-M) Anti-Robo3 expression in magenta. (E,H,K) Robo RNAi detectably reduces Robo expression (open arrowhead), but not Robo2 or Robo3 (closed arrowheads). (F,I,L) Robo2 RNAi detectably reduces Robo2 expression (open arrowhead), but not Robo or Robo3 (closed arrowheads). (G,J,M) Robo3 RNAi detectably reduces Robo3 expression (open arrowhead), but not Robo or Robo2 (closed arrowheads).

View larger version (113K): [in a new window]   Fig. 7. Distal cell neurons intermingle with lamina glia in Robo family knockdowns. (A,B) Lateral view (anterior at bottom). IPC neuroblasts and distal cell neurons are visualized with anti-Fas3 (red), c155:GFP is most strongly observed in IPC neuroblasts and photoreceptors (green), and neuronal nuclei are visualized with anti-Elav (blue). (A) c155-Gal4;UAS-GFP. (B) Distal cell neurons enter the lamina in c155-Gal4;UAS-GFP;UAS-RoboRNAi;UAS-Robo2RNAi;UAS-Robo3RNAi animals (arrow), reaching anterior edge of lamina (arrowhead). (C-F) Animals express GFP (green) under control of Sca-Gal4, labeling the IPC, distal cell neurons and medulla cortex. Neuronal nuclei are visualized with anti-Elav (blue). (C,D) Photoreceptor axons are visualized using anti-Chaoptin (red). (C) Sca-Gal4;UAS-GFP animal. (D) Distal cell neurons enter the lamina in Sca-Gal4;UAS-GFP;UAS-RoboRNAi;UAS-Robo2RNAi;UAS-Robo3RNAi animals (arrow). (E,F) Lamina glia are visualized using anti-Repo (red). Distal cell neurons intermingle (arrows) with lamina glia in Robo family knockdown animals. (E) Sca-Gal4;UAS-GFP;UAS-RoboRNAi,UAS-Robo3RNAi;UAS-RoboRNAi,UAS-Robo3RNAi animal. (While Sca-Gal4;UAS-RoboRNAi;UAS-Robo3RNAi animals had no defects, animals containing two copies of both UAS-RoboRNAi and UAS-Robo3RNAi had modest defects, consistent with overlapping roles of Robo family members.) (F) Sca-Gal4;UAS-GFP;UAS-RoboRNAi;UAS-Robo2RNAi;UAS-Robo3RNAi animal. dcn, distal cell neuron progeny; IPC, inner proliferation center; lam, lamina; medc, medulla cortex.

View larger version (80K): [in a new window]   Fig. 8. Robo family knockdown disrupts photoreceptor axon targeting. (A-C) Photoreceptor axons visualized using anti-Chaoptin. (A) Tubulin-Gal4;UAS-GFP control. (B) Tubulin-Gal4;UAS-GFP;UAS-RoboRNAi;UAS-Robo2RNAi;UAS-Robo3RNAi animal, showing many photoreceptor axons extending through the lamina (arrow) and too many photoreceptor axons entering the medulla (arrowhead). (C) GMR-Gal4;UAS-GFP;UAS-RoboRNAi;UAS-Robo2RNAi;UAS-Robo3RNAi animal. (D-F) Animals express GFP (green) under control of Sca-Gal4, while photoreceptor axons are visualized using anti-Chaoptin (magenta). (D) Sca-Gal4;UAS-GFP animal. (E) Sca-Gal4;UAS-GFP;UAS-RoboRNAi,UAS-Robo3RNAi;UAS-RoboRNAi,UAS-Robo3RNAi animal in which GFP-expressing cells in the lamina correspond to regions of photoreceptor axon mistargeting (arrow). (F) Sca-Gal4;UAS-GFP;UAS-RoboRNAi;UAS-Robo2RNAi;UAS-Robo3RNAi animal. (G) Schematic of observed disruptions in visual system development. In wild type, distal cell neurons (blue) express Robos (orange outline), while Slit protein (red) surrounds glia (yellow with black outline) at the base of the lamina. Lamina glia serve as initial targets of incoming R1-R6 photoreceptor axons (green). When expression of all three Robo family members is inhibited in distal cell neurons (robo, robo2, robo3), distal cell neurons intermingle with the lamina glia and photoreceptor axon targeting is disrupted. Loss of Slit expression (slit) causes an indistinguishable defect.