SPECIFIC AIMS A primary goal of this proposal is to understand the molecular genetic mechanisms regulating glia differentiation. Traditionally, glia were thought of mainly as support cells for neuronal function. In recent years, however, it has become increasingly clear that glia are pivotal for proper neuronal development and function. Glia mediate a remarkable array of cellular functions including axon ensheathment, establishment of blood brain barrier, trophic response, ionic equilibrium, synaptogenesis, axon pruning, engulfment and neuronal plasticity. To carry out these important functions, glia must themselves differentiate and function properly. Indeed, glia malfunction often precedes neuronal/axonal degeneration in many human neurodegenerative diseases (BAUMANN and PHAM-DINH 2001; BENARROCH 2005; FREEMAN 2005b; FREEMAN 2005c; KIM and DE VELLIS 2005; SCHWABE et a/. 2005; SHAHAM 2005; WYSS-CORAY and MUCKE 2002). Despite their immense importance in neurobiology, glia are remarkably understudied and the molecular genetic mechanisms that direct the differentiation of glia are poorly understood. The developing nervous system of Drosophila offers a superb experimental system in which to understand these mechanisms. Drosophila glia are remarkably similar to the mammalian glia in their development, structure and function (FREEMAN and DOHERTY 2005). In Drosophila, molecular and genetic manipulations are readily feasible and the cellular signaling pathways that regulate nervous system development are highly conserved between Drosophila and mammalian systems. Thus an understanding of the regulatory mechanisms that direct glia differentiation in Drosophila will provide important insights into mammalian glia differentiation and will be invaluable in our understanding of human neurological disorders. The focus of our proposal is on the role of ubiquitination as a regulatory mechanism during glia differentiation. Tagging of specific proteins for degradation by ubiquitination has emerged as an important regulatory mechanism in nervous system development and disease. We previously identified and molecularly characterized Rap/Fzr, an activator of the multi-subunit ubiquitin ligase, APC (Anaphase promoting complex). Our work has shown that Rap/Fzr regulates cell cycle progression and is required for proper neuronal patterning in the developing eye (JACOBS etal. 2002; KARPILOW et a/. 1989; KARPILOW etal. 1996; PIMENTEL and VENKATESH 2005b). To identify novel cellular functions of Rap/Fzr we carried out genetic studies which showed that Rap/Fzr interacts with several genes required for glia differentiation (Kaplow et al. 2006 submitted). We have recently made the novel observation that Rap/Fzr regulates glia differentiation by interacting with an Ets domain transcription factor, Pointed, already known to be required for glia differentiation and with Apc2. a catalytic subunit of the APC ubiquitin ligase. Our working hypothesis is that glia differentiation is regulated by novel interactions involving Rap/Fzr, Pointed and Apc2. Rap/Fzr binds Pointed and targets it to the ubiquitin ligase complex (APC), where it is ubiquitinated. Pointed is eventually degraded by the 26S proteosome. In our model, the level of Pointed is regulated by Rap/Fzr, and is a key determinant in the regulation of glia differentiation (Figure 1). The specific aims of this proposal are: Specific Aim I. To test whether glia differentiation is negatively regulated by Rap/Fzr. Our preliminary results suggest that Rap/Fzr is a negative regulator of glia differentiation, a) We will test at the cellular level the effects of loss-of-function Rap/Fzr mutations on glia differentiation in the developing larval brain and the eye. We will generate homozygous rap/fzr- mutant clones in a wild type tissue background using the FLP-FRT technique, b) To directly assess the effects of Rap/Fzr loss-of-function on glia differentiation in a spatially and temporally restricted manner, we will use RNAi and UAS-GAL4 techniques to knockdown Rap/Fzr function in specific tissues at specific times, c) To test the effects of gain-of-function of Rap/Fzr on glia differentiation, we will generate random clones of cells expressing Rap/Fzr, using UAS-GAL4 as well as FLP-FRT systems. We will determine the role of Rap/Fzr in regulating the number and position of glia in the developing larval nervous system. These experiments will address whether the Rap/Fzr function is necessary for glia differentiation in a cell autonomous manner. Specific aim II. To test whether Rap/Fzr regulates glia differentiation by direct interaction with Pointed: Our working hypothesis is that Rap/Fzr targets Pointed for ubiquitination by the ubiquitin ligase APC. Pointed is an ETS domain transcription factor which is required for glia differentiation, a) We will test whether Rap/Fzr binds Pointed using in vitro and in vivo biochemical assays. We will use tissue extracts from larval central nervous system (CMS) and tissue culture S2 cell extracts and assay for binding interactions between Rap/Fzr and Pointed by co-immunoprecipitation assays, b) We will test whether Rap/Fzr and Pointed co-localize in glia in vivo using confocal microscopy in combination with immunohistochemistry. c) We will test whether Rap/Fzr and Pointed interact physically using yeast two-hybrid assays, d) We will perform in vitro ubiquitination assays and test whether Pointed can serve as a substrate for ubiquitination by Rap/Fzr and APC. Specific Aim III. To test whether Apc2/Morula regulates glia differentiation: Apc2 is the catalytic subunit of the ubiquitin ligase complex, APC, encoded by the morula gene. Our preliminary studies suggest that Apc2 is a negative regulator of glia differentiation. To directly assess the role of Apc2 in glia differentiation at the cellular level: a) We will test the effects of loss-of-function of APC on glia differentiation. We will induce clones of Apc2-/Apc2- tissue in the developing larval brain and the eye and determine whether Apc2 is required cell autonomously for glia differentiation, b) We will test the effect of loss-of-function of Apc2 on glia differentiation using the RNAi technique, c) We will test the effects of the gain-of-function of Apc2 on glia differentiation by generating random clones of cells expressing Apc2 using the UAS-GAL4 and the FLP-FRT techniques.