Project Summary Astrocytes are crucial regulators of brain development and function. Astrocytes acquire a remarkably complex morphology that allows them to associate with each other, other cell types (e.g. the vasculature) and synapses where they regulate synaptogenesis, neurotransmitter reuptake, metabolic support, ion balance, and ultimately animal behavior. While it is believed that the elaborate morphology of astrocytes is absolutely essential for efficient astrocyte function, how astrocytes acquire this unusually complex architecture remains poorly defined. This is surprising in light of their crucial roles in neural circuit formation and function, and the fact that disruption of astrocyte growth control results in the most intractable and deadly human brain tumor, glioblastoma. How do astrocytes acquire their remarkably morphology, and how do they organize their subcellular architecture to enable their diverse functions? We will attempt to answer these central questions using Drosophila astrocytes as a model. Fly astrocytes are remarkably similar to their mammalian counterparts by morphological, developmental, molecular, and functional criteria, and Drosophila offers a battery of powerful molecular-genetic tools with which to explore fundamental questions in astrocyte biology that are not available in other organisms. We will begin by comprehensively characterizing the cell-wide organellar landscape of astrocytes by examining the distribution of ~30 genetically encodable markers that label cellular organelles (Aim 1). This will allow us to define, with single-cell precision, the basic organellar architecture of astrocytes. This will be an essential first step toward understanding how their intricate morphology is arranged ultrastructurally and how it may dictate, or be regulate by, their functions. These cellular landmarks will also enable a rigorous analysis of mutants that affect astrocyte morphology. In Aim 2, we will perform the first unbiased forward genetic screen for astrocyte growth control pathways. We have established a unique genetic screening platform for this purpose in Drosophila based on MARCM technology, which allows for rapid screening with single-cell resolution for mutants that alter a variety of phenotypes including cell morphology (growth, tiling, association with synapses), changes in proliferation, or other changes in astrocyte properties. In preliminary work we have optimized our screening system, along with imaging methods to maximally facilitate our work. This is part of a long term effort to understand how astrocytes are built in vivo. Defining how astrocytes control their cell growth, infiltration, and tiling will be critical for us to gain a better understanding how astrocytes affect brain health and disease. Since this will be the first forward genetic screen for astrocyte growth control pathways, a wealth of exciting mutants await discovery. We will focus our subsequent efforts on pathways conserved in mammal...