Glia are the most abundant cells in the human brain and they play key roles in CNS function and health. Glial cells regulate synaptic signaling, ensheath axonal projections and, importantly, protect the brain by serving as the first line of defense against neuronal damage. The adult brain contains a striking array of diverse glial subtypes, but little is known about the unique genetic profiles of distinct classes of glia that alow them to carry out their important and varied functions. Moreover, determining how the transcriptional profile of glial cells are altered in response to neural injury has presented a unique set of challenges, since the process of isolating glia from the brain for transcriptional analysis is, in and of itself, highly stressful to the cells. Recent work has shown that the adult Drosophila melanogaster brain contains a variety of glial subtypes that are strikingly similar to those described in vertebrates. In addition, acute neural injury induces glial immune responses in flies that are highly reminiscent of those triggered in mammalian glia, including upregulation of essential glial immune genes. This project will take advantage of these evolutionarily conserved features of glia and integrate cutting-edge advances in the fields of in vivo RNA labeling and high throughput deep sequencing to generate a comprehensive transcriptome of Drosophila glial cells in the intact adult brain before and after injury. We will use novel genetic drivers that are expressed in discrete glial subtypes in the adult fly brain to genetically label RNA in each class of glia in vivo and then biochemically isolate the labeled RNA to sequence glial subtype transcriptomes by RNA-seq. Using a well-established axotomy assay, we will perform these experiments in uninjured and injured flies to compare the transcriptional profiles of glia before and after acute axon injury. Finally, we will validate the expression of glial genes identified by RNA-seq and begin to characterize the functional role of the newly discovered immune genes that are acutely regulated in glia responding to axotomy. This work (a) will provide critical mechanistic insight into the function of diverse glial subtypes in the adult brain (b) offers a unique opportunity to investigate how gene expression is altered in glia responding to neurodegeneration in the intact CNS and (c) will generate a valuable genetic toolkit for the scientific community to investigate many unexplored aspects of glial cell biology.