Axon growth across the Drosophila midline requires Comm to downregulate Robo, the receptor for the midline repellent Slit. We show here that comm is required in neurons, not in midline cells as previously thought, and that it is expressed specifically and transiently in commissural neurons. Comm acts as a sorting receptor for Robo, diverting it from the synthetic to the late endocytic pathway. A conserved cytoplasmic LPSY motif is required for endosomal sorting of Comm in vitro and for Comm to downregulate Robo and promote midline crossing in vivo. Axon traffic at the CNS midline is thus controlled by the intracellular trafficking of the Robo guidance receptor, which in turn depends on the precisely regulated expression of the Comm sorting receptor.

A prominent feature of glial cells is their ability to migrate along axons to finally wrap and insulate them. In the embryonic Drosophila PNS, most glial cells are born in the CNS and have to migrate to reach their final destinations. To understand how migration of the peripheral glia is regulated, we have conducted a genetic screen looking for mutants that disrupt the normal glial pattern. Here we present an analysis of two of these mutants: Notch and numb. Complete loss of Notch function leads to an increase in the number of glial cells. Embryos hemizygous for the weak Notch(B-8X) allele display an irregular migration phenotype and mutant glial cells show an increased formation of filopodia-like structures. A similar phenotype occurs in embryos carrying the Notch(ts1) allele when shifted to the restrictive temperature during the glial cell migration phase, suggesting that Notch must be activated during glial migration. This is corroborated by the fact that cell-specific reduction of Notch activity in glial cells by directed numb expression also results in similar migration phenotypes. Since the glial migration phenotypes of Notch and numb mutants resemble each other, our data support a model where the precise temporal and quantitative regulation of Numb and Notch activity is not only required during fate decisions but also later during glial differentiation and migration.

The analysis of mutants is an indispensable approach towards characterizing gene function. Combining several tools of Drosophila genetics, we designed a new strategy for a mutagenesis screen which is fast, easy-toapply, and cheap. The combination of a cell-specific Gal4 line with an upstream activating sequence-green fluorescent protein (UAS-GFP) allows the in vivo detection of the cells or tissues of interest without the need for fixation and staining. To further simplify and accelerate the screening procedure, we generated recombinant flies that carry the Gal80 transgene in balancer chromosomes. Gal80 inactivates Gal4; and thus prevents GFP-expression during embryonic and postembryonic development in all individuals carrying the balancer chromosomes. This allows for an easy distinction in vivo between heterozygous and homozygous mutants, the latter being the only ones expressing GFP. Since most of the fly strains and balancer chromosomes can be substituted, this method is suitable for nearly any mutagenesis screen that does not have major restrictions.