Liprin-alpha is required for photoreceptor target selection in Drosophila

Abstract

Classical cadherin-mediated interactions between axons and dendrites are critical to target selection and synapse assembly. However, the molecular mechanisms by which these interactions are controlled are incompletely understood. In the Drosophila visual system, N-cadherin is required in both photoreceptor (R cell) axons and their targets to mediate stabilizing interactions required for R cell target selection. Here we identify the scaffolding protein Liprin-alpha as a critical component in this process. We isolated mutations in Liprin-alpha in a genetic screen for mutations affecting the pattern of synaptic connections made by R1-R6 photoreceptors. Using eye-specific mosaics, we demonstrate a previously undescribed, axonal function for Liprin-alpha in target selection: Liprin-alpha is required to be cell-autonomous in all subtypes of R1-R6 cells for their axons to reach their targets. Because Liprin-alpha, the receptor tyrosine phosphatase LAR, and N-cadherin share qualitatively similar mutant phenotypes in R1-R6 cells and are coexpressed in R cells and their synaptic targets, we infer that these three genes act at the same step in the targeting process. However, unlike N-cadherin, neither Liprin-alpha nor LAR is required postsynaptically for R cells to project to their correct targets. Thus, these two proteins, unlike N-cadherin, are functionally asymmetric between axons and dendrites. We propose that the adhesive mechanisms that link pre- and postsynaptic cells before synapse formation may be differentially regulated in these two compartments.

Fig. 1.

Liprin-α is required for both cartridge assembly and layer-specific targeting in the visual system and displays phenotypes indistinguishable from those associated with mutations in LAR and N-cadherin. (A–F) Cross-sectional views of the lamina. R1–R6 axons express LacZ under the control of the Rh1 promoter (green); all R cells are counterstained with the R cell-specific antibody mAb24B10 (red). (A) WT R1–R6 axons assemble into fascicles, denoted cartridges (circled), containing 6 R cell axons. R7 and R8 sit outside of each cartridge. (B–D) Liprin-α somatic mosaic animals in which photoreceptor axons are homozygous mutant. Individual cartridges are of unequal size and contain variable numbers of R1–R6 termini. (E) Lar²¹²⁷. (F) N-cadherin^Δ14. The phenotypes observed in E and F are indistinguishable from those seen in B–D. (G–N) Horizontal section of the medulla in eye-specific mosaic adult flies. (G–K) R7 axons express lacZ under the control of the Rh3 promoter (green); all R cell axons are counterstained with mAb24B10 (red). Note that not all R7 cells express Rh3lacZ; a subset express the R4 opsin and, thus, are labeled only with mAb24B10 (red). (G and H) WT. R7 axons invariably stop in a layer more proximal than R8; hash marks denote each layer. (I) Liprin-α^E. (J) Lar²¹²⁷. (K) N-cadherin^Δ14. In these three mutant backgrounds, R7 axons sometimes stop in the R8 recipient layer instead of the R7 recipient layer (arrowheads), leaving gaps in the array of otherwise regular R7 termini. (L–N) R8 axons are labeled with lacZ expressed under the control of the Rh5 promoter (green); all R cell axons are stained with mAb24B10 (red). Note that not all R8 axons express Rh5lacZ; some normally express Rh6 and, thus, are labeled only with mAB24B10 (red). (L and M) WT. (N) Liprin-α^E. Layer-specific targeting of R8 is unaffected by the loss of Liprin-α. (O–Q) R1–R6 axons are labeled with lacZ under the control of the Rh1 promoter; all R cell axons are stained with mAb24B10 (red). (O and P) WT. (Q) Liprin-α^E. The ganglion-specific targeting of R1–R6 axons to the lamina occurs normally in Liprin-α mutants. (Scale bars: A–F, 5 μm; G–N, 30 μm.)

Fig. 2.

R cell fate determination, axon guidance into the brain, and brain development are normal in Liprin-α mutants. (A and B) Cross-sections of the retina visualized during mid-pupal development, labeling R cells with the R cell-specific antibody mAb24B10 (green). (A) WT. (B) Liprin-α¹. R cell shape and rhabdomere morphology are unaffected by the loss of Liprin-α. (C and D) Horizontal views of the brain during the third instar larval stage. R cell axons are visualized with mAb24B10 (red); neuronal nuclei are marked with antibodies directed against Elav (green); L5 lamina neurons and some medullar neurons are labeled with antibodies directed against the Brain-Specific Homeobox (blue). Lp, lamina plexus; Md, medulla. (C) WT. (D) Liprin-α¹. (E and F) En face views of R cell axons entering the lamina; R cell axons are labeled with mAb24B10 (red); R2-R5 axons express tau-lacZ under the control of the rough promoter (green). (G and H) Horizontal views of the brain during the third instar larval stage. Neuronal processes are labeled with anti-horseradish peroxidase-FITC (green); glial nuclei are stained with an antibody directed against Repo (red). StG, satellite glia; MgG, marginal glia; EpG, epithelial glia; MdG, medulla glia. (G) WT. (H) Liprin-α¹. (Scale bar: A and B, 10 μm; C–F, 20 μm; G and H, 30 μm.)

Fig. 3.

Liprin-α is required cell autonomously in R1–R6 axons for target selection in the lamina. Single-mutant R cells were generated by mitotic recombination and visualized at 42 h after puparium formation to capture axonal extension. R cell clones were labeled with GFP (green), and cartridges were visualized by staining all R cells with mAb24B10 (red). (A–D) Schematic cross-sectional views of the retina. Each R cell type is identifiable by its characteristic morphology and position. (E–H) Cross-sectional images of the retina. (I–L) Schematic representation of the projection of each R cell subtype. Each R cell axon makes a projection that is invariant in morphology, and which projects in an orientation that can be predicted from the position of its cell body within the ommatidium. Each R cell extends from one cartridge (arrowhead) to a neighboring cartridge (arrow). (M–P) WT. (Q–T) Liprin-α^E. Most Liprin-α mutant R cell growth cones fail to extend across the surface of the lamina. (A, E, I, M, and Q) R1. (B, F, J, N, and R) R2. (C, G, K, O, and S) R3. The asterisk in o denotes an R4 axon from a neighboring ommatidium (which extends normally). (D, H, L, P, and T) R4. (Scale bars: 5 μm.)

Fig. 4.

Liprin-α function is required in all R cell subtypes. Each axonal projection was categorized as absent (no extension, black bars), present but abnormal in morphology (aberrant, gray bars), or normal (white bars). n, number of fibers of each type examined; the lower three graphs (Liprin-α^E, Liprin-α¹, and Liprin-α^F) combine data across all R cell subtypes. These data demonstrate that Liprin-α function is critical to R cell axon extension and that this requirement is not restricted to particular R cell subtypes.

Fig. 5.

Liprin-α and LAR, unlike N-cadherin, are not required in lamina neurons for normal R cell target selection. (A) Schematic view of the retina and lamina. R cells (red) extend axons out of the retina in fascicles before choosing targets in the lamina. Each fascicle is associated with a column of five lamina neurons (green) and contains all eight R cells from the same ommatidium. Within the lamina plexus (gray plane), R1–R6 axons extend laterally across the surface, choosing targets arranged in invariant relative positions. The R4 axon is highlighted (white). Upon reaching their target, each R cell axon joins a new fascicle, the cartridge, comprising R cell axon termini surrounding lamina neuron processes. (B) Schematic cross-sectional views of lamina cartridges. R1–R6 (red) surround, and are flanked by, lamina neuron processes (green). (C) Schematic cross-sectional view illustrating R4 axon projections (white) within a group of lamina neuron processes (green). R4 axons extend downward from one column (white dot), terminating in a growth cone adjacent to a second column (green). (D, I, N, and S) Single cross-sections of the adult lamina in which target neurons are homozygous for a control chromosome, and R cell axons termini are labeled red. (E–I) Liprin-α¹. (J–N) LAR^omb451. (O–S) N-cadherin^Δ14. (E, J, and O) Single cross-sections of the lamina plexus. Large clones of mutant lamina neurons labeled with GFP (green) were generated by using the MARCM system; photoreceptor axons were stained by using mAb24B10 (red). In this section plane, lamina neuron processes are visible as small profiles, whereas clusters of R cell axon termini label each cartridge. In Liprin-α and LAR mutant target clones, as in wild type, the overall spacing of cartridges is regular, and each cartridge contains R cell axon termini closely associated with lamina neuron processes. In N-cadherin clones, the spacing of cartridges is disrupted, and many lamina neuron processes lack associated R cell axons (arrowhead). (F–H, K–M, and P–R) Cross-sections of the lamina plexus within large target cell clones in which single R4 axons were labeled with mδ-lacZ. (F, K, and P) Target clone (green). (G, L, and Q) mδ-lacZ (white). (H, M, and R) Merge. In Liprin-α and LAR mutant clones, extended R4 axons form parallel rows of fibers both inside and outside the clone. In N-cadherin mutant clones, R4 axons frequently fail to extend or extend to inappropriate targets. (Scale bars: 10 μm.)