Endogenous clocks (circadian pacemakers) that regulate the temporal expression patterns of physiological, endocrine, and behavioral processes exist within the central nervous systems of all animals. Such pacemakers have been the subject of intensive molecular genetic investigations in a number of organisms including Drosophila. In Drosophila, at least two different processes are modulated by a circadian pacemaker and are therefore expressed as daily rhythms: (1) locomotor activity and (2) eclosion, which is defined as the emergence of the adult after pupal metamorphosis. This latter rhythm, which is a consequence of the precise regulation of a developmental event (eclosion) by an endogenous pacemaker, has been extremely well characterized from decades of studies. The present proposal is directed towards elucidating the physiological mechanisms governing the """"""""temporal gating"""""""" of eclosion. More precisely, we are proposing molecular genetic studies of Drosophila eclosion-rhythm mutants to define the neuroendocrine signals that comprise the output pathway leading from the circadian pacemaker (in the brain) to the neural targets responsible for the initiation of eclosion. To distinguish specific lesions of this output pathway from more general effects on the circadian system, we are selecting mutants that have abnormal patterns of eclosion, but normal locomotor activity rhythms. The new mutants are being generated by transposon insertional mutagenesis in order to 'tag' the identified genes for molecular analysis. We hypothesize that such mutants will identify components of the pacemaker output pathway regulating eclosion.
The specific aims of this application emphasize studies of a gene known as lark. This gene was identified by a transposon-induced allele that alters the timing of eclosion peaks (they occur abnormally early in the day), but has no effect on activity rhythms. The lark mutation, unlike any other existing rhythm variant, identifies a vital function; it has semidominant effects on the timing of eclosion, but is a recessive embryonic lethal. The lark gene has been cloned by virtue of the transposon tag, and is being subjected to molecular analysis. Preliminary spatial expression studies suggest that the gene product is required for an essential neural function. This application proposes genetic, molecular, and cellular localization studies that will emphasize the cellular function(s) of the lark product within the context of the temporal regulation of the eclosion event. An understanding of the cellular and molecular bases of circadian regulation in any organism has important implications for the etiology and treatment of certain human psychiatric and sleep disorders.
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