Differential adhesion determines the organization of synaptic fascicles in the Drosophila visual system

Abstract

Background: Neuronal circuits in worms, flies, and mammals are organized so as to minimize wiring length for a functional number of synaptic connections, a phenomenon called wiring optimization. However, the molecular mechanisms that establish optimal wiring during development are unknown. We addressed this question by studying the role of N-cadherin in the development of optimally wired neurite fascicles in the peripheral visual system of Drosophila.

Results: Photoreceptor axons surround the dendrites of their postsynaptic targets, called lamina cells, within a concentric fascicle called a cartridge. N-cadherin is expressed at higher levels in lamina cells than in photoreceptors, and all genetic manipulations that invert these relative differences displace lamina cells to the periphery and relocate photoreceptor axon terminals into the center.

Conclusions: Differential expression of a single cadherin is both necessary and sufficient to determine cartridge structure because it positions the most-adhesive elements that make the most synapses at the core and the less-adhesive elements that make fewer synapses at the periphery. These results suggest a general model by which differential adhesion can be utilized to determine the relative positions of axons and dendrites to establish optimal wiring.

Figure 1. Development of the lamina cartridge and Ncad expression in the lamina

(A) Single cartridge cross-sections at 28, 38 and 48% apf, R cells labeled by mAb24B10 (magenta) and single L cells with mCD8GFP (green). All cartridges imaged are from the left dorsal hemisphere, except for L1–5, L1/2 and L5 at 28% apf. L4 cells also form lateral dendrite branches deeper in the lamina (see Figure S1). (B) Schematic organization of the R cell axons and L cell neurites in the lamina cartridge (R cells: magenta, L cells: green). (C, E) Plan view of the lamina at 28% apf (C) and 48% apf (E); R cells are labeled with mAb24B10 (red in merge) and Ncad (blue in merge). L cells are labeled by myr-EGFP (L cells, green in merge) driven by gcm-Gal4 (C) or GH146-Gal4 (E). (D) Intensity profile plot along the white scan line in (C) shows that Ncad (blue) is expressed at higher levels in L cells (green) than in R cells (magenta). Green and magenta bars denote L cell and R cell territory, respectively.

Figure 2. Changes in Ncad expression levels affects cartridge organization

(A–H) Adult laminas were labeled with the R cell marker Csp2a. Insets show magnified views of a single, representative cartridge. (A) Control: mCD8GFP. (B) L cell- specific Ncad RNAi. (C) R cell- specific Ncad RNAi. (D) Ncad RNAi in both cell types. (E) L cell - specific Ncad loss of function. (F) R cell- specific Ncad loss of function. (G) R cell- specific overexpression (OE) of Ncad-13b. (H) R cell- specific OE of Ncad-13a. (I) Schemata of observed phenotypes. (J) Quantification of Ncad expression in R cells vs. L cells (see Supplemental Experimental Procedures). If Ncad levels were relatively higher in R cells than L cells (dark gray), values were made negative. Light gray denotes genotypes where Ncad is relatively enriched in L cells. All groups are significantly different from control with at least p<0.05 (ANOVA, n=6 brains per group). (K) Quantification of cartridge defects, n= 151–288 cartridges per genotype. All groups, except Ncad overexpression (OE) in L cells are significantly different from control with p<0.001 (Fisher’s exact test).

Figure 3. Ncad loss in L cells, but not in R cells separates R cell and L cell fascicles

Large Ncad mutant MARCM clones either in R cells (A) or L cells (B), positively labeled with mCD8GFP at 48% apf (green). All R cells are labeled with mAb24B10 (magenta). Insets show single, representative cartridges.

Figure 4. EM analysis of Ncad mutant cartridges

(A–F) Individual cartridge cross sections, (A, C, E) or their corresponding traced and color-coded profiles (B, D, F). Primary neurites of L1 and L2 are indicated by asterisks. (A, B) Cartridges with Ncad FLICK mutant L cells, including supernumerary L2 cells. (Bi) Same reconstruction as in B, but only showing R cell terminals and the primary neurites of L1 and L2. (C, D) Cartridges innervated by Ncad⁹³⁶ mutant eye containing one of each identified L-cell type (L1–L4). (E, F) Ncad FLICK control cartridges. (G–P) 3D reconstructions of R cell axon terminals and L cell dendrites in the Ncad L cell FLICK mutant (G–K), or the R cell mutant Ncad⁹³⁶ (L–P). (Q, R) Synapses in cartridges with Ncad FLICK mutant L-cells, L-cell elements color-coded as in A–F. (Q) Normal tetrad synapse with T-bar ribbon (arrow). (R) Abnormal synapse formed between two Ncad FLICK mutant L2 cells with T-bar ribbon. Scale bars: (C) 2 μm; (H) 200 nm.

Figure 5. Ncad cell autonomously regulates L cell neurite position

(A) Schemata showing L1–L5 neurites in wild-type cartridges. (B–D) Single mCD8GFP- labeled (green) L cells either wild-type (B), Ncad mutant (C) or overexpressing Ncad using elav^C155-Gal4 (D) using MARCM in an Ncad heterozygous background, counterstained with mAb24B10 (all R cells, magenta). Single cartridge profiles are outlined by dashed white line. All cartridges, except L4 in control and L5 in Ncad OE are from the dorsal hemisphere, the equator is to the right in all panels. (E) Percentage of L cells localized at the core (inside) or periphery (outside) of the cartridge, control n=19–44, Ncad loss n=28–38, Ncad overexpression n=18–87. Ncad mutant L1 and L2, as well as Ncad overexpressing L3 differ significantly different from control with p<0.05 (Fisher’s exact test).

Figure 6. Differential adhesion between R cells and L cells guides cartridge assembly

The experimentally observed neurite configurations of the lamina cartridge can be predicted by differences in adhesive work (W) [6].