Axon bifurcation results in the formation of sister branches, and divergent segregation of the sister branches is essential for efficient innervation of multiple targets. From a genetic mosaic screen, we find that a lethal mutation in the Drosophila Down syndrome cell adhesion molecule (Dscam) specifically perturbs segregation of axonal branches in the mushroom bodies. Single axon analysis further reveals that Dscam mutant axons generate additional branches, which randomly segregate among the available targets. Moreover, when only one target remains, branching is suppressed in wild-type axons while Dscam mutant axons still form multiple branches at the original bifurcation point. Taken together, we conclude that Dscam controls axon branching and guidance such that a neuron can innervate multiple targets with minimal branching.

Besides 19,008 possible ectodomains, Drosophila Dscam contains two alternative transmembrane/juxtamembrane segments, respectively, derived from exon 17.1 and exon 17.2. We wondered whether specific Dscam isoforms mediate formation and segregation of axonal branches in the Drosophila mushroom bodies (MBs). Removal of various subsets of the 12 exon 4s does not affect MB neuronal morphogenesis, while expression of a Dscam transgene only partially rescues Dscam mutant phenotypes. Interestingly, differential rescuing effects are observed between two Dscam transgenes that each possesses one of the two possible exon 17s. Axon bifurcation/segregation abnormalities are better rescued by the exon 17.2-containing transgene, but coexpression of both transgenes is required for rescuing mutant viability. Meanwhile, exon 17.1 targets ectopically expressed Dscam-GFP to dendrites while Dscam[exon 17.2]-GFP is enriched in axons; only Dscam[exon 17.2] affects MB axons. These results suggest that exon 17.1 is minimally involved in axonal morphogenesis and that morphogenesis of MB axons probably involves multiple distinct exon 17.2-containing Dscam isoforms.

Polyhomeotic (Ph), which forms complexes with other Polycomb-group (PcG)proteins, is widely required for maintenance of cell identity by ensuring differential gene expression patterns in distinct types of cells. Genetic mosaic screens in adult fly brains allow for recovery of a mutation that simultaneously disrupts the tandemly duplicated Drosophila phtranscriptional units. Distinct clones of neurons normally acquire different characteristic projection patterns and can be differentially labeled using various subtype-specific drivers in mosaic brains. Such neuronal diversity is lost without Ph. In response to ecdysone, ph mutant neurons are transformed into cells with unidentifiable projection patterns and indistinguishable gene expression profiles during early metamorphosis. Some subtype-specific neuronal drivers become constitutively activated, while others are constantly suppressed. By contrast, loss of other PcG proteins,including Pc and E(z), causes different neuronal developmental defects; and,consistent with these phenomena, distinct Hox genes are differentially misexpressed in different PcG mutant clones. Taken together, Drosophila Ph is essential for governing neuronal diversity,especially during steroid hormone signaling.