Robo-3--mediated repulsive interactions guide R8 axons during Drosophila visual system development

Abstract

The formation of neuronal connections requires the precise guidance of developing axons toward their targets. In the Drosophila visual system, photoreceptor neurons (R cells) project from the eye into the brain. These cells are grouped into some 750 clusters comprised of eight photoreceptors or R cells each. R cells fall into three classes: R1 to R6, R7, and R8. Posterior R8 cells are the first to project axons into the brain. How these axons select a specific pathway is not known. Here, we used a microarray-based approach to identify genes expressed in R8 neurons as they extend into the brain. We found that Roundabout-3 (Robo3), an axon-guidance receptor, is expressed specifically and transiently in R8 growth cones. In wild-type animals, posterior-most R8 axons extend along a border of glial cells demarcated by the expression of Slit, the secreted ligand of Robo3. In contrast, robo3 mutant R8 axons extend across this border and fasciculate inappropriately with other axon tracts. We demonstrate that either Robo1 or Robo2 rescues the robo3 mutant phenotype when each is knocked into the endogenous robo3 locus separately, indicating that R8 does not require a function unique to the Robo3 paralog. However, persistent expression of Robo3 in R8 disrupts the layer-specific targeting of R8 growth cones. Thus, the transient cell-specific expression of Robo3 plays a crucial role in establishing neural circuits in the Drosophila visual system by selectively regulating pathway choice for posterior-most R8 growth cones.

Fig. 1.

Robo3 is expressed specifically in the R8 growth cones. (A and B) Schematic representations of the developing 3rd instar optic lobe (A) and the adult optic lobe (B), showing the relative orientation of the lamina and medulla neuropils and projections of the posterior R8 axons. R, R cells; La, lamina; Gl, glia; Lo, lobula; Lp, lobula plate; Med, medial; Lat, lateral; Mn, medulla; A, anterior; P, posterior; C+T, C2, C3, T2, and T3 neurons. (C) A third instar eye disc costained with antibodies against Robo3 protein (green) and Sens (magenta). An apico-basal section through the eye disc reveals punctate Robo3 staining around the Sens positive nucleus. As shown in Fig. 2B, staining was not observed using the antibody to Robo3 in robo3³ mutant tissue. (D) A third instar, robo3-GAL4/UAS-CD8-GFP, eye disc costained with antibodies against GFP protein (green) and Run (magenta) reveal Run positive basal nuclei surrounded by membrane localized GFP (white arrowhead). (E and E') Robo3 is expressed in third instar R8 growth cones as they enter the optic lobe, but down-regulated shortly after the R8 axons enter the medulla neuropil. Arrowhead and arrow indicate the youngest and oldest part of the medulla neuropil, respectively. (F and F') Robo3 expression is not detectable in the R8 axons by 30% APF at which time all the R8 axons have reached the R8 temporary layer. Residual Robo3 staining is observed in the growth cones of a group of optic lobe neurons that project into more proximal regions of the developing medulla neuropil (white arrow in F and F'). (All scale bars: 10 μm.) See SI Materials and Methods for a list of genotypes.

Fig. 2.

Defects in axon pathways in robo3 mutant optic lobes. (A) Schematic of the robo3 locus and the position of the NP4968 P-element insertion (robo3-GAL4). The robo3³ allele was generated by imprecise excision of the NP4968 P-element and deletes exon1, which contains both the transcriptional and translational start sites for robo3. (B and B“) robo3³ mutant clones in a 3rd instar eye disc. The mutant patches lack GFP expression and are demarcated by the dotted white lines. GFP negative patches also lack Robo3 protein staining (B‘ and B“). (C and D) Adult optic lobes stained with the mAb-24B10 antibody. (C) A robo3³ heterozygous optic lobe shows the wild-type organization of visual neuropils. la, lamina; oc, optic chiasm; dme, distal medulla; pme, proximal medulla. The R8 axons terminate in the M3 layer and the R7 axons terminate in the M6 layer (dashed lines). (D) Posterior photoreceptor axons take an aberrant path to the proximal medulla in robo3³ homozygous optic lobes (white arrowhead). (E and F) Adult optic lobes from robo3 heterozygous (E) or robo3 homozygous animals (F) in the background of the Rhodopsin-5-LacZ (Rh5-LacZ) transgene costained with mAb-24B10 (magenta) and -galactosidase (green) antibodies. Misprojecting robo3³ mutant fascicles contain R8 axons (white arrowhead in F). As previously reported, Rh5-LacZ (like the endogenous gene) is only expressed in some 30% of wild-type R8 neurons. -Galactosidase staining is not continuous and thus projections appear punctate. (G and H) Schematic representations of wild-type (G) and robo3³ mutant (H) optic lobes, highlighting the normal and aberrant paths taken by the posterior R8 axons. R, R cells; La, lamina; Gl, glia; A, anterior; P, posterior; Mn, medulla; C+T, C2, C3, T2, and T3 neurons. (I and J) Adult optic lobes from animals with the whole eye rendered mutant by mitotic recombination of a control, wild-type, chromosome (I) or a robo3³ mutant chromosome (J). robo3³ mutant photoreceptor axons take an aberrant path to the proximal medulla (white arrowhead in J). (K and L) Thirty percent APF optic lobes containing control or MARCM mutant R8 clones with persistent Robo3 expression, colabeled with anti-GFP (green) and mAb-24B10 (magenta) antibodies. sens-GAL4 was used in conjunction with MARCM to promote persistent expression of UAS-robo3 in R8 (green). Persistent expression of Robo3 beyond early pupal stages causes R8 axons to extend through the R8 temporary layer and, instead, terminate in deeper layers close to the R7 temporary layer (arrowheads in L). Control clones show wild-type R8 targeting (K). (Scale bars: 10μm.) See SI Materials and Methods for a list of genotypes.

Fig. 3.

Posterior R8 axons take an inappropriate pathway into the medulla. (A and B) Schematic of the relationship between the posterior R8 axons (red), glial cells (dark blue), and C+T axons (light blue) in early third-instar optic lobes of wild-type (A) or robo3³ mutant (B) animals. C+T, C2, C3, T2, and T3 neurons; Gl, Glia; La, Lamina; Lat, Lateral; Med, Medial; Mn, Medulla; R- R Cells. (C and D) Third-instar larval optic lobes from GMR-GFP flies in a wild-type (C), or slit^dui homozygous mutant background (D) (anti-Slit, magenta; anti-GFP, green). Slit expression is detected at the posterior margin of the lamina in wild-type optic lobes (arrowhead in C). Slit expression is lost in slit^dui homozygous mutant optic lobes (arrowhead in D). (E and F) Third-instar larval optic lobes from sens-GAL4, UAS-CD8-GFP flies colabeled with anti-FasII and GFP antibodies showing the relationship of the earliest arriving R cell and the C+T axons. FasII labels photoreceptors, lamina neurons, and C+T axons. (E and E′) Wild-type R8 axons project along glial-cell boundaries but never cross them but robo3³ mutant posterior R8 (F and F′) axons cross glial-cell boundaries and project along the paths taken by the C+T axons (arrowhead in F′). (G, G′, and H) Glial cells mark the posterior boundary of the lamina and separate posterior R8 axons from C+T axons (arrowhead in H). Third-instar optic lobes from C3-GAL4, UAS-CD8-GFP larvae (G and G′) and repo-GAL4, UAS-CD8-GFP larvae (H) showing the relationship of the earliest arriving R cell axons (FasII, magenta), glia (anti-GFP, green in H), and the C3 axons (anti-GFP, green in G). (I and J) Inappropriately projecting posterior R8 axons can be visualized in robo3³ mutant pupal optic lobes (arrowheads in J and J′). Twenty-percent APF optic lobes with sens-GAL4/ UAS-CD8-GFP in WT (I and I′), or robo3³ mutant backgrounds (J and J′); R-cell axons, anti-24B10 (magenta); R8 axons, anti-GFP (green). (K and L) Inappropriately projecting posterior R8 axons grow along the C3 axons tracts before entering the distal medulla (arrowhead in L). robo3³ heterozygous (K) or homozygous (L) adult optic lobes that also contain the C3-Gal4/ UAS-CD8GFP transgenes. R-cell axons, mAB-24B10 (magenta); C3 cell bodies and axons, anti-GFP (green). (Scale bars, 10 μm.)

Fig. 4.

Both robo1 and robo2 rescue the robo3 R8 axon guidance defects. (A–C) Representative eye discs from robo3^robo1 (A), robo3^robo2 (B), and robo3^robo3(C) third-instar larvae, costained with antibodies against HA and Sens. In each case HA staining colocalizes around the R8 nucleus labeled by Sens. The superscript indicates the coding sequence knocked into the endogenous robo3 locus. (D–F) Robo1 and Robo2 receptors can substitute for Robo3 to prevent posterior R8 axons from joining C+T axon tracts. Adult optic lobes from robo3^robo1/robo3³ (D), robo3^robo2/robo3³ (E), and robo3^robo3/robo3³ (F) animals stained with the mAb-24B10 antibody. (Scale bars, 10 μm.)