Transcriptome analysis of unfulfilled-dependent gene expression in the mushroom body neurons. Janos Molnar, Karen Bates, Steven Robinow. Biology, University of Hawaii, Honolulu, HI.

   The mushroom body (MB) of Drosophila is required for memory, learning, and other complex behaviors. The adult MB consists of 5 lobes that are the result of the sequential development and differentiation of three subtypes of neurons, the neurons, the / neurons, and the / neurons. The transcription factor UNFULFILLED, a nuclear receptor, is required for the development of all three neuronal subtypes although the role in each subtype is likely to be unique. While hundreds of genes are known to be expressed in the MB and many have been shown to be involved in various aspects of development and differentiation, the genetic hierarchies that regulate MB development and differentiation are only partially understood. To better define the genetic hierarchies that regulate MB development and differentiation, we are identifying genes that act in the MB downstream of unfulfilled. We are taking both classical genetic (see abstract by Bates et al., this volume) and comparative transcriptomic approaches to this problem. The RNA of hand-dissected mutant and wild type MBs from second instar larvae was purified and sequenced. Sixteen million paired end reads were mapped and processed with Tophat and Cufflinks. We have detected the expression of 66% of the transcriptome with read counts >1 read per kilobase per million reads (RPKM), with an additional 11% of the genes showing marginal expression below 1 RPKM indicating good coverage of the transcriptome. 837 genes showed significant differential expression (p0.05). Included in this list are genes of the ecdysone biosynthetic pathway, ubiquitination-related genes, axon-guidance genes, and neuronal fate determination genes. These findings are being validated by in situ hybridization and genetic screening. We are now analyzing data of the transcriptomes from the developmental stage when the wild type and unfulfilled mutant and / neurons are first morphologically distinguishable.