TThe Drosophila circadian clock is comprised of transcriptional feedback loops, which control rhythmic gene expression and are also responsible for daily rhythms in physiology, metabolism and behavior. The core feedback loop, which employs CLOCK-CYCLE (CLK-CYC) activators and PERIOD-TIMELESS (PER-TIM) repressors to drive rhythmic transcription peaking at dusk, is required for circadian timekeeping and overt behavioral rhythms. CLK-CYC also activates an interlocked feedback loop, which uses PAR DOMAIN PROTEIN 1? (PDP1?) activator and VRILLE (VRI) repressor to drive Clk and other rhythmic transcription peaking at dawn. In addition to its role in the clock, vri controls many developmental processes which are essential for early embryonic development, making vri null mutants embryonic lethal. This has hindered the ability to determine the role that vri plays within the circadian clock. The goal of my study is to determine if vri is necessary for circadian time keeping and/or output. To achieve this goal, I first determined which transcripts were important for clock function. I generated an isoform specific vri mutant, which eliminated the expression of mRNAs that were thought to be essential for clock function. It turned out that these mutants are hypomorphic, as residual VRI expression was still observed in the clock cells of these mutants. Further analysis demonstrated that another vri mRNA isoform is expressed rhythmically under the control of the clock. Interestingly, the mutation impacted the abundance of short and long proteins, since the mutated mRNA can produce both VRI protein isoforms using an alternative translation initiation site.
Furthermore, the E-boxes that are used by CLK-CYC are important for development, since deletion of these E-boxes leads to embryonic lethality.
Then using a conditionally inactivatable transgene to rescue vri developmental lethality, I showed that clock function persists after vri inactivation, but activity rhythms were abolished. The inactivation of vri disrupts multiple output pathways thought to be important for activity rhythms, including Pigment-Dispersing Factor (PDF) accumulation and arborization rhythms in the small ventrolateral neuron (sLNvv) dorsal projection. These results demonstrate that vri acts as a key regulator of clock output, and suggests that the primary function of vri in Drosophila is to drive rhythmic transcription, which is required for overt rhythms.
- Hardin, Paul Distinguished Professor