Robust discrimination between self and non-self neurites requires thousands of Dscam1 isoforms

In fruitfly nerve cells, alternative splicing of the Dscam gene's messenger RNA produces a remarkable variety of cell-surface proteins — presenting some 19,000 different extracellular domains. Each isoform is capable of binding exclusively to itself, thus promoting repulsion among neuronal processes extended by the same neuron. This process of neurite self-avoidance is important in helping to direct dendritic and axonal branching appropriately. By generating mutant flies with graded reductions in the number of Dscam isoforms, Larry Zipursky and his team have now determined that at least 1,000 but no more than 5,000 isoforms are essential to provide a robust mechanism of neuronal self-avoidance during brain wiring. How this translates into vertebrate brain development remains unclear, as vertebrate Dscam genes produce just a handful of distinct isoforms. The tendency of branches from the same neuron (self-branches) to selectively avoid one another is known as self-avoidance. The expression of different isoforms of the Down Syndrome cell adhesion molecule (Dscam) gene, which expresses a neuronal cell recognition protein of the immunoglobulin superfamily, ensures that repulsion is restricted to self-branches. Here, in Drosophila, the number of isoforms of Dscam1 required to prevent non-self branches from recognizing each other is determined. Down Syndrome cell adhesion molecule (Dscam) genes encode neuronal cell recognition proteins of the immunoglobulin superfamily1,2. In Drosophila, Dscam1 generates 19,008 different ectodomains by alternative splicing of three exon clusters, each encoding half or a complete variable immunoglobulin domain3. Identical isoforms bind to each other, but rarely to isoforms differing at any one of the variable immunoglobulin domains4,5. Binding between isoforms on opposing membranes promotes repulsion6. Isoform diversity provides the molecular basis for neurite self-avoidance6,7,8,9,10,11. Self-avoidance refers to the tendency of branches from the same neuron (self-branches) to selectively avoid one another12. To ensure that repulsion is restricted to self-branches, different neurons express different sets of isoforms in a biased stochastic fashion7,13. Genetic studies demonstrated that Dscam1 diversity has a profound role in wiring the fly brain11. Here we show how many isoforms are required to provide an identification system that prevents non-self branches from inappropriately recognizing each other. Using homologous recombination, we generated mutant animals encoding 12, 24, 576 and 1,152 potential isoforms. Mutant animals with deletions encoding 4,752 and 14,256 isoforms14 were also analysed. Branching phenotypes were assessed in three classes of neurons. Branching patterns improved as the potential number of isoforms increased, and this was independent of the identity of the isoforms. Although branching defects in animals with 1,152 potential isoforms remained substantial, animals with 4,752 isoforms were indistinguishable from wild-type controls. Mathematical modelling studies were consistent with the experimental results that thousands of isoforms are necessary to ensure acquisition of unique Dscam1 identities in many neurons. We conclude that thousands of isoforms are essential to provide neurons with a robust discrimination mechanism to distinguish between self and non-self during self-avoidance.