Jeffrey C. Hall - US grants

Reproductive neurogenetics in Drosophila involves a widening array of behavioral and neural mutants as well as molecular manipulations involving factors that influence sex-specific features of the fly's nervous system. To extend the analysis of semi-classical courtship genes, the roles played by calcium-channel polypeptides and nucleic-acid binding proteins will be dissected. For the former of these two genes, cacophony, the experiments include molecular- genetic analysis of courtship-song defects and visual-system ones as well (the latter being caused by certain of the cac-locus mutations). The significance of posttranscriptional variation in channel quality will be assessed, including molecular manipulations and behavioral bioassays of putative RNA editing events involving cac s primary product. The no-on-transient-A gene also can be mutated to cause singing and visual abnormalities. The significance of nonA's spatial expression will be dissected, principally in terms of a novel abnormality of courtship-humming sounds associated with transgene manipulations of the normal allele. How nonA and the putative RNA-binding protein it encodes may interact with cac will be determined, in part by determining whether in vivo an and engineered nonA mutations affect post-transcriptional processing of the Ca2+-channel RNA cac's behavioral and molecular-genetics suggests one way that courtship songs diverge evolutionarily: by modulations of patterned neuronal outputs caused by calcium- channel variations; this will be investigated by analyzing inter- specific relatives of cac and bioassays of the molecular variants in transgenic males. The genetics of sex determination will be further merged with courtship studies by behavioral and pheromonal analysis of fruitless and doublesex mutants; the experiments include molecular neurogenetic dissection of courtship-hum abnormalities and analysis of sex-specific locomotor behavior. To move in certain new directions, the genetics of female receptivity will be analyzed by neurobiological and molecular studies of a new mutant; also, possible resets of a biological clock, induced by conditioned courtship stimuli, will be investigated to see if parallels can be drawn to a novel finding in this area from mammals. -- Reproduction seems on the face of it to have broad significance. Variations of these phenomena, caused genetically in organisms ranging up to humans, are being increasingly appreciated, analyzed, and manufactured experimentally. It is suggested that neurogenetic and molecular neurobiological findings and principles, stemming in part from studies of Drosophila courtship and mating, may contribute to the design and interpretation of investigations in this area, which would involve a variety of different organisms.

Circadian rhythms are organismically ubiquitous biological cycles whose propr daily regulation is strongly connected to the well being of species ranging from microbes to mammals. The 25- year-old genetic approach toward understanding these rhythms has recently begun to reveal elements of the clock mechanisms that underlie daily rhythmicities. This sub-proposal in a Program Project application revolves around the behavioral genetic, neuro- genetics, and molecular neurobiology of circadian rhythms in Drosophila. The experiments proposed stress more of a ~systems~ approach than one that would concentrate upon the hard core of molecular pacemaking. Thus, the neural substrates of behavioral rhythms, and a periodic feature of late development, will be delved into by application of rhythm mutants and transgenic strains carrying manipulated forms of clock-genes; these studies include descriptions of anatomic output pathways from neurons that are candidate for CNS-pacemaker cells, and selectively effected perturbations of the structure and function of such cells in conjunction with bioassaying the effects of the molecularly mediated neuronal damage. Input paths to the central pacemakers, which bring in environmental cues to effect crucial daily re-sets of the clock, will be dissected both in terms of anatomy and elements of signal-transduction pathways that putatively participate in processing the resetting stimuli. Output pathways will be investigated, with respect to varying physiological parameters that are hypothesize to be the first- or second-stage targets of clock- gene functions. The latter includes cyclically varying levels of the encoded mRNAs and proteins; these molecular cyclings will be tracked in conjunction with physiological recordings and perturbations, by applying a transgene in which portions of a clock gene (called period) have been fused to DNA sequences encoding a real-time reporter; this is luciferase activity, which as recently been shown to permit non-invasive monitoring of molecular rhythms in live adult flies and in per-expressing tissues explanted from animals late in development. Genetic and molecular-genetic studies are proposed in two areas: (1) analysis of the behavioral and neurobiological consequences of manipulating a clock-controlled gene~s expression, and of effecting the same kinds of perturbations of a neuropeptide-encoding gene whose product is co-expressed with clock genes in a subset of the CNA pacemaking neurons; (2) genetic and biological studies of rhythm mutants, recently isolated on the basis of defects in circadian behavioral rhythms: these phenogenetic studies will proceed into cloning of the genes defined by mutations that appear to be the most promising candidates for disrupting important elements of Drosophila~s circadian system.

[unreadable] DESCRIPTION (provided by applicant): Experiments are proposed to delve into the cellular neurobiology of Drosophila's rhythm system--the cells, molecules, and systems that underlie the animal's rest-activity cycles. One focus will involve certain adult-brain neurons, within which actions of and interactions among clock-gene products are hypothesized to form the molecular and cellular substrates of circadian behavioral rhythms: How do these proteins physically interact in vivo? To this end, FRET-based analyses will be performed to assess such biologically meaningful interactions. This will complement and may even modify the conventional view of clockwork protein-protein interactions, because the latter stems largely from characterizations of the protein interactions in tissue extracts or cultured non-neuronal cells. In experiments to be carried out at more of a systems level, THE FUNCTIONS [sic] of the brain neurons in question will be dissected by manipulations of transgenes derived from in-hand clock genes, and from the identification of others by virtue of their presumed specification of pacemaker-output factors. Which subsets of these neurons carry out functions that are output into rhythmic behavior per se, and which are involved in communication among cells within the pacemaking system? Do the presumed neurochemical outputs (alluded to above) function in that manner only, or do they also mediate feedback functions back into the pacemakers (considered both cellularly and molecularly)? With regard to the known rhythmic oscillations of certain clock-gene products and putative clock-output molecules--which of these systematic fluctuations are important for the behavioral rhythmicity that can be sustained in constant environmental conditions, and is such molecular maintenance in part a systems property (involving interactions among certain neurons, cellular feedback phenomena, or both)? Daily oscillations of behavior in Drosophila represent a feature of circadian rhythmicity that is among the most prominent and widespread among metazoan organisms. Moreover, the kinds of gene products and the ways they form the clockworks are analogous across a broad array of species. Therefore, brain-behavior studies of chronobiology in this model system may provide insights into the nature of certain rhythm-related disorders of humans, some of which are associated with genetic variations of Drosophila clock gene orthologs [unreadable] [unreadable]