Temperature and light entrainment of the Drosophila circadian clock

Drosophila melanogaster locomotor activity responds to seasonal conditions by modulating the “evening” activity component. During simulated winters of cold temperature and short days an advanced evening locomotor peak occurs with more daytime locomotor activity; on the other hand long photoperiods and warm temperatures give a delay in the evening peak, thereby avoiding a possible desiccation during the hottest times of the day. This pattern of activity is related to a thermosensitive splicing event that occurs in a 3’ intron in the period gene, with a higher level of splicing and earlier accumulation of PERIOD in short days and low temperatures. A mutation in norpA which encodes a phospholipase C, generates a high level of spliced per at warmer temperature, so mutants behave as if it is colder than it actually is. The relation between norpA, per splicing and the circadian neurons has been analysed. Initially, norpA expression has been investigated via in situ hybridisation and immunocytochemistry. norpA transcript has been localised among the clock pacemakers but not NORPA. Subsequently, norpA expression has been knocked-down by RNAi in specific subset of neurons. The resulting locomotor behaviour shows seasonally related effects implicating the photoreceptors, lateral and dorsal clock neurons as structures involved in timing the locomotor behaviour. In parallel, the thermal role of a second PLCβ, plc21C, has been investigated via RNAi among circadian pacemakers. It has been possible to show that plc21C expression in the photoreceptors, lateral and dorsal neurons is required to set different locomotor behaviours at different temperatures, but not via per and tim splicing. Finally, in contrast to reports that the double photoreceptor mutants involving glass and cryptochrome are “circadian blind”, these flies have been observed to entrain to light-dark cycles at moderate temperatures. Candidate orphan G protein coupled receptors have been screened in order to identify a further set of putative circadian-relevant photoreceptors contributing to this residual entrainment in glass60jcryb mutants. In constant light conditions, the RNAi of CG7497 and CG16958 generates rhythmic or arrhythmic flies depending on the genetic background tested.