Dscam diversity is essential for neuronal wiring and self-recognition

Abstract

Neurons are thought to use diverse families of cell-surface molecules for cell recognition during circuit assembly. In Drosophila, alternative splicing of the Down syndrome cell adhesion molecule (Dscam) gene potentially generates 38,016 closely related transmembrane proteins of the immunoglobulin superfamily, each comprising one of 19,008 alternative ectodomains linked to one of two alternative transmembrane segments. These ectodomains show isoform-specific homophilic binding, leading to speculation that Dscam proteins mediate cell recognition. Genetic studies have established that Dscam is required for neural circuit assembly, but the extent to which isoform diversity contributes to this process is not known. Here we provide conclusive evidence that Dscam diversity is essential for circuit assembly. Using homologous recombination, we reduced the entire repertoire of Dscam ectodomains to just a single isoform. Neural circuits in these mutants are severely disorganized. Furthermore, we show that it is crucial for neighbouring neurons to express distinct isoforms, but that the specific identity of the isoforms expressed in an individual neuron is unimportant. We conclude that Dscam diversity provides each neuron with a unique identity by which it can distinguish its own processes from those of other neurons, and that this self-recognition is essential for wiring the Drosophila brain.

Figure 1. Generation and molecular characterization of Dscam^single alleles

a, Schematic representation of the genomic organization and proteins encoded by Dscam alleles used in this paper. Numbers above bars in the genomic structure indicate exons. Alternatively spliced exons are shown in colour. cDNA encoding a single isoform in the Dscam^single includes exons 3–11 (asterisk). Pink triangles indicate the FRT site between exons 16 and 17. b, Using a bead-aggregation assay, Dscam^10.27.25, Dscam^3.31.8, and Dscam^6.5.9 ectodomains show homophilic binding similar to the Dscam^7.27.25 control. Aggregation of fluorescent beads decorated with ectodomain–Fc fusion proteins was measured as an increase in the mean fluorescence intensity (MFI) of each particle. Binding experiments were performed twice. Error bars represent±1 s.d. c, Dscam^single alleles express Dscam protein at wild-type levels. The level of Dscam protein in extracts of the larval central nervous system was assessed by immunoblotting. Actin was used as a loading control. d, Dscam protein expression pattern (green) is normal in Dscam^single embryonic ventral nerve cord (stage 16). Anti-HRP (horseradish peroxidase) staining (purple) was used to visualize the neuropil. Scale bar, 10 μm. e, Expression of two alternative transmembrane (TM) domains in Dscam^single animals. RT-PCR across exons encoding each of the alternative TM domains was performed using RNA extracted from the third instar larval brains. Gel electrophoresis separates products encoding TM1 and TM2 as indicated.

Figure 2. Viability and neuronal wiring defects in Dscam^single mutants

a, Survival of different genotypes to late pupal stages. Dscam, Dscam and Dscam^Df6055 are protein-null alleles. b, Dscam^single embryos show defects in embryonic central nervous system organization. Stage-16 embryos were examined for neuropil structure (anti-HRP, purple) and for three distinct longitudinal axon tracts (monoclonal antibody 1D4, anti FasII, green). Dscam^single/Dscam^single embryos (>95%; n=41) show severely disrupted longitudinal tracts (arrow) and aberrant midline crossing (arrowhead). Scale bar, 10 μm. c–e, Dscam diversity is required in the olfactory system. Antennal lobes (AL) were visualized with the presynaptic marker nc82 (purple) and synaptotagmin-GFP (green fluorescent protein) expression (green) in three classes of olfactory receptor neurons (see schematic). c, Dscam^FRT/Dscam^null controls show normal organization (n=88 antennal lobes). Scale bar, 10 μm. d, Loss of Dscam (Dscam^null/Dscam^null) results in mistargeting of ORNs to multiple glomeruli. The position and morphology of glomeruli are also abnormal (n=28). e, Loss of diversity (Dscam^single/Dscam^null) leads to loss of glomerular boundaries, as well as formation of ectopic ORN termini throughout the antennal lobe. Severe phenotypes were observed for all three Dscam^single alleles (100% penetrance: n=28, 128 and 16 for Dscam^3.31.8, Dscam^10.27.25 and Dscam^6.5.9, respectively).

Figure 3. Dscam diversity is required for mushroom body development

a, Schematic of mushroom body development (P, peduncle; D, dorsal lobe; M, medial lobe). Two representative mushroom body neurons are shown (yellow and pink). b–d, Left panels show mushroom body lobes from late pupae visualized by staining with monoclonal antibody 1D4 (anti-FasII). Only the lobe region is shown. Arrowhead indicates the location of the branch point. Dashed line, midline. Scale bar, 10 μm. Dscam isoforms expressed on sister branches (pink) and a non-self branch (yellow) are represented as coloured bars in panels on the right. Arrows indicate repulsive signals resulting from Dscam isoform-specific homophilic binding. b, Control animals (Dscam^FRT/Dscam^null). c, Dscam^single/Dscam⁺ heterozygous animals. d, Dscam^single/Dscam^null mutant animals. e, Quantification of mushroom body (MB) phenotypes.

Figure 4. Dscam^single is sufficient to promote branch segregation with high fidelity at the single-cell level

a, Schematic of intragenic MARCM strategy to generate and label single Dscam^single cells in an otherwise wild-type background (that is, transheterozygous with the Dscam^FRT and Dscam loss-of-function (Dscam^LOF) alleles). FLP recombinase induces mitotic recombination between FRT sites within the Dscam locus. Chromosomes can segregate in two ways. In one way (pattern 2), GFP-labelled (green) Dscam^single/Dscam^5′HR mutant cells are generated. The Dscam^5′HR allele (Supplementary Fig. 1) is a loss-of-function allele. If chromosomes segregate in the alternative fashion (pattern 1), no labelled cells will be produced. One of these cells will carry an intact Dscam^single allele. b, Branch segregation phenotypes. Labelled cells for Dscam^{wild type} and Dscam^single were generated using intragenic MARCM, and Dscam^null labelled cells were produced using conventional MARCM. The mushroom body was visualized by staining with monoclonal antibody 1D4, anti-FasII (purple), and the clones were labelled with membrane-targeted chimeric GFP (mCD8GFP, green). The genotype of each clone is indicated above the panel. Arrows indicate the branch point. Quantification is shown as a bar graph (using a two-tailed Fisher's exact test; n.s., not significant).