Our previous studies led us to propose that a sex determination regulatory gene, fruitless (fru), is responsible for building the potential for male sexual behavior into the CNS during Drosophila development. The transcripts of the distal (P1) fru promoter are sex-specifically spliced to produce female- and male-specific mRNAs. It is the P1 derived male-specific FRU proteins (FRU/M) that are needed for male sexual behavior. FRU/M proteins are required for all, or nearly all, aspects of male sexual behavior, from the initial recognition of a potential mate, through to the transfer of bodily fluids and the duration of copulation. That fru functions during developmental to establish the potential for male sexual behavior is suggested by the temporal and spatial patterns of expression of the FRU/M proteins. These proteins are expressed almost exclusively in the CNS. Expression is maximal in the mid-pupal period when they are expressed in ca. 1700 cells (2% of the CNS); this time coincides with the period of major morphogenetic events that shape the adult CNS. It is on the developmental origins of these neurons, the characteristics that distinguish them, and the roles they play in male sexual behavior that this grant is focused. We address the following topics. (1) The behavioral roles of individual groups of FRU/M-expressing neurons. (2) To being to understand these neurons functions we are (A) identifying the types of neurons they are, (B) addressing whether homologously positioned groups of fru P1-expressing neurons in males and females differ in their projection patterns, branching patterns, or synapses, and (C) for selected groups of neurons, determine which cells they are connected to so as to begin to elucidate the neuronal circuitry underlying male courtship behavior. (3) We will determine when the FRUM cells are born during development, whether FRUM cells found in clusters are clonally related to one another, and the reasons why some groups of cells expressing the P1 promoter are found in only one sex. (4) The FRU proteins are putative BTB Zn-finger transcription factors, and thus likely regulate the expression of other genes; therefore we will (A) determine whether FRU proteins function as homo- or heterodimers with other BTB domain proteins, (B) investigate the role of the unique 101 amino acid N-terminus of the male-specific FRU proteins, (C) determine the consensus DNA binding sites of the different FRU Zn-finger pairs, and (D) identify genes that are regulated by FRU proteins. (5) We will determine whether fru is the "master" gene for male sexual behavior: is FRUM both necessary and sufficient for male courtship behavior.