Orthodenticle Is Required for the Expression of Principal Recognition Molecules That Control Axon Targeting in the Drosophila Retina

Abstract

Parallel processing of neuronal inputs relies on assembling neural circuits into distinct synaptic-columns and layers. This is orchestrated by matching recognition molecules between afferent growth cones and target areas. Controlling the expression of these molecules during development is crucial but not well understood. The developing Drosophila visual system is a powerful genetic model for addressing this question. In this model system, the achromatic R1-6 photoreceptors project their axons in the lamina while the R7 and R8 photoreceptors, which are involved in colour detection, project their axons to two distinct synaptic-layers in the medulla. Here we show that the conserved homeodomain transcription factor Orthodenticle (Otd), which in the eye is a main regulator of rhodopsin expression, is also required for R1-6 photoreceptor synaptic-column specific innervation of the lamina. Our data indicate that otd function in these photoreceptors is largely mediated by the recognition molecules flamingo (fmi) and golden goal (gogo). In addition, we find that otd regulates synaptic-layer targeting of R8. We demonstrate that during this process, otd and the R8-specific transcription factor senseless/Gfi1 (sens) function as independent transcriptional inputs that are required for the expression of fmi, gogo and the adhesion molecule capricious (caps), which govern R8 synaptic-layer targeting. Our work therefore demonstrates that otd is a main component of the gene regulatory network that regulates synaptic-column and layer targeting in the fly visual system.

Fig 1. Schematic representation of the Drosophila visual system.

(A) Schematic representation of Drosophila axonal photoreceptor projections in the third instar larva optic lobe. The outer R1-R6 (indicated in light blue) from each ommatidium in the eye disc project their axons into the lamina part of the brain. At this early developmental stage, the inner photoreceptor R8s (yellow) project through the lamina and establish a regular retinotopic array of terminals in the medulla. (B) Schematic representation of the adult Drosophila visual system. R-cell axons are organized into synaptic-columns and layers. Six different photoreceptors (indicated in light blue) from six neighbouring ommatidia share the same optical axis and pool their axons in the same synaptic-column in the lamina [2]. R8 (orange) and R7 (green) photoreceptor axons pass through the lamina and terminate in distinct synaptic-layers M3 (R8) and M6 (R7).

Fig 2. The early pattern of photoreceptor axon projections is affected in otd mutants.

Wild-type (A) and otd ^uvi mutant (B) eye imaginal discs from third instar larvae stained for the neural marker Elav (blue) and for the R7- and R8-specific markers Pros (red) and Sens (green) respectively. Asterisks indicate the position of the bristle cell. Wild-type (C) and otd ^uvi (D) photoreceptor axon projections in third-instar larva were visualized by expressing UAS-mCD8::GFP under the control of the GMR-Gal4 driver [43]. The growth cones of R1-R6 form a neural plexus (chevrons) at the lamina (L). R8 axons project to the medulla neuropile (M). bn stands for the Bolwig’s nerve.

Fig 3. otd is required for synaptic-column targeting of R1-6 in the lamina.

Electron micrographs of lamina cross-sections showing the normal organization of synaptic-columns in a wild-type lamina (A) and the defects in lamina column innervation observed in the case of otd mutant retina (B). Photoreceptor terminals are colored in pink (R), central lamina neurons are labeled ‘L’. Confocal sections of wild-type (C-C’,E,E’) and otd mutant (D,D’,F,F’) lamina at 40% after puparium formation. R1-6 axons are labeled by mCD8::GFP (green in C,D and E,F) and stained for Fmi (C-D’) (red) or NCad (E-F’) (red). (G) Frequency distribution polygon showing the numbers of R1-6 axons terminals innervating each synaptic column in otd ^uvi mutant lamina (black line) and in otd ^uvi mutants where either fmi (red line) or gogo (green line) are expressed using the GMR-Gal4 driver line. Data were gathered from EM micrographs. A Levene's test for the equality of variances, was applied. The variance in the number of axon terminals per cartridge is significantly lower in the otd ^uvi; GMR-Gal4; UAS-fmi and otd ^uvi ; GMR-Gal4; UAS-gogo lamina (1.66 and 1.70 respectively) compared to otd ^uv mutant flies (2.83, p<0.05 in both cases). (H) Real-time PCR quantification of caps, gogo, fmi, lar, NCad and InR mRNA normalized to GAPDH mRNA levels comparing wild-type and otd mutant retina at 40% after puparium formation. n = at least three independent mRNA extracts from wild-type and otd-mutant retinas. Error bars represent SEM.

Fig 4. Caps expression is downregulated in otd ^uvi mutant R8 photoreceptors.

Side view of wild-type (A) and otd ^uvi mutant (B) third instar larva eye disc revealing that caps expression is strongly reduced in otd mutant R8. The expression of mCD8-GFP is under the control of caps-Gal4 [8]. Elav (blue) and 24B10 (red) stain the full set of photoreceptors. (C) Representation of an ommatidium showing the basal localization of the R8 photoreceptor in green. Expression of Caps (green) in wild-type (D, D’) and otd ^uvi mutant (E, E’) optic lobes (60% after puparium formation). Photoreceptor-cell-axons are stained with the 24B10 antibody. The R8 (M3) and R7 (M6) recipient layers are indicated by dashed lines in this and the following figures. When compared to wild-type, otd ^uvi mutant retina show a reproducible gap pattern detected in the R8 layer (M3) (D’, E’). These gaps correspond to a loss of Caps expression in the afferent R8 axons (boxed in E and magnified in E’).

Fig 5. otd is required for R8 synaptic-layer targeting.

Wild-type (A,A’) and otd ^uvi mutant (B,B’) adult optic lobes expressing the R8 specific marker Rh6-lacZ stained with anti-β-galactosidase (green). In wild-type (A,A’) Rh6-lacZ-positive R8 axons terminate in the M3 layer. otd ^uvi mutants (B-B”) display a strong R8 axon misprojection phenotype. R8 axons specifically overshoot to the M6 layer (boxed in B’ and quantified in Fig 5H) while some R8 axons invade neighbouring columns cross-laterally and terminate in abnormal positions (boxed in B”). Several R8 axons terminals can also be seen to stall at more superficial layers and fail to innervate the medulla (double arrows in b”‘). All panels (C-F) show photoreceptor axon projections stained with the 24B10 antibody (red). R8 axons in wild-type (c,c’) and otd ^uvi mutant (D,D’) optic lobes (40% after puparium formation) are visualized using the ato-τ-myc transgene stained with anti-Myc antibody (green). At this early stage of development, in otd ^uvi mutant retina, R8 growth cones fail to stop in the M1 layer and extend specifically to the R7 temporary layer (n = 438 misprojecting axons of 798 R8 axons quantified in the two temporary layers). The staining for ato-τ-Myc is magnified in (C’,D’) and arrows in (D’) indicate misprojecting R8 axons. boss ¹ mutant (E) and otd ^uvi /boss ¹ double-mutant (F) retina. Adult optic lobes stained with 24B10 antibody. In (E), residual 24B10 staining in the R7 M6 layer is derived from medulla neurons [44]. (E) boss ¹ mutant R8 terminate in the R8 recipient layer M3, with only a few R8 targeting to the M6 layer. (F) Most of the otd ^uvi /boss ¹ double-mutants display a strong R8 mis-projection phenotype in the R7 layer, M6. (G) otd ^uvi mutant flies where Otd expression has been restored specifically in the photoreceptors using the GMRGal4 driver. In this context, staining of the R8 specific marker Rh6-lacZ (green) demonstrates that normal R8 photoreceptor targeting is almost completely restored. (H) Quantification of the misprojections of Rh6-lacZ-positive R8 axons in wild-type, otd ^uvi mutant and otd ^uvi mutant flies where Otd expression has been restored using the GMR-Gal4 driver.

Fig 6. otd and sens are required for R8 synaptic layer targeting.

All panels (A-F) show photoreceptor cell projections stained with 24B10 (red). (A) Expression of UAS-sens ^RNAi (GFP-negative ommatidia, encircled by a dotted line) in wild-type tissue (GFP positive) 48 h after clone induction using the tub>GFP>Gal4 system. UAS-sens ^RNAi expressing cells show a clear reduction in Sens protein levels (blue). (B) Expression of UAS-sens ^RNAi transgene (GFP-negative ommatidia, encircled by a dotted line) in wild-type tissue (GFP positive) 48 h after clone induction using the tub>GFP>Gal4 system. Otd expression (red) is unaffected in UAS-sens ^RNAi expressing R8 cells (indicated by asterisks). (C) Rh6-lacZ-positive R8 axons in sens ^RNAi (R8-specific driver-Gal4 ^109–68;UAS-sens ^RNAi) and in otd ^uvi mutant combined with sens ^RNAi retina (D), stained with anti-β-galactosidase (green). sens knockdown in the retina leads to a few defects in R8 axon projection, however the combination of the sens knockdown and otd ^uvi mutant leads to a complete failure of R8 layer-specific targeting, indicating that sens and otd act in parallel. (E,F) Layer-specific targeting of the R7 photoreceptors is assessed using the R7 specific transgene Rh4-lacZ. As in wild-type (E), Rh4-lacZ positive R7 terminals (green) correctly target to the M6 layer in otd ^uvi mutants flies (F). (G) Quantification of the misprojections of Rh6-lacZ-positive R8 axons in wild-type, otd ^uvi mutant and otd ^uvi mutant flies where either caps, fmi or gogo expression has been restored. Since Caps is present only in R8, UAS-caps is expressed under the control of an R8-specific Gal4 driver, while UAS-gogo and UAS-fmi are expressed using the pan-photoreceptor GMR-Gal4 driver. Restoring caps expression in otd-mutant R8 photoreceptors partially rescues the otd ^uvi mutant R8 mis-targeting phenotype (from 59% R8 misprojection in otd ^uvi to 25% in otd ^uvi + UAS-caps). Similarly, expression of fmi and gogo in otd ^uvi mutant photoreceptors partially suppress the R8 misprojection phenotype (36% R8 axons misprojecting in otd ^uvi + UAS-fmi, n = 374 of 1039; 40% R8 axons misprojecting in otd ^uvi + UAS-gogo, n = 436 of 1090). Percentages indicate quantitative assessment of R8 axons as detected in M3 and M6 layers only. In wild-type, all R8 neurons target to the M3 layer, thus the mistargeting percentage is zero.

Fig 7. Transcriptional inputs required for proper photoreceptor targeting in the Drosophila visual system.

Summary of the transcription factors that regulate column- and layer-specific targeting of the R1-6 and R7 and R8 photoreceptors. Predicted indirect regulation is shown with a double arrowhead (Sens binds directly on the caps promoter). Although all of the currently known transcriptional regulators (i.e., seq, sens and otd) are presented, for clarity, only those CAMs and adhesion molecules most relevant to our findings are shown here.