Two distinct mechanisms segregate Prospero in the longitudinal glia underlying the timing of interactions with axons.

Prospero is required in dividing longitudinal glia (LG) during axon guidance; initially to enable glial division in response to neuronal contact, and subsequently to maintain glial precursors in a quiescent state with mitotic potential. Only Prospero-positive LG respond to neuronal ablation by over-proliferating, mimicking a glial-repair response. Prospero is distributed unequally through the progeny cells of the longitudinal glioblast lineage. Just before axon contact the concentration of Prospero is higher in two of the four progeny cells, and after axon guidance Prospero is present only in six out of ten progeny LG. Here we ask how Prospero is distributed unequally in these two distinct phases. We show that before neuronal contact, longitudinal glioblasts undergo invaginating divisions, perpendicular to the ectodermal layer. Miranda is required to segregate Prospero asymmetrically up to the four glial-progeny stage. After neuronal contact, Prospero is present in only the LG that activate Notch signalling in response to Serrate provided by commissural axons, and Numb is restricted to the glia that do not contain Prospero. As a result of this dual regulation of Prospero deployment, glia are coupled to the formation and maintenance of axonal trajectories.