![]() ( B) Diagram of CRISPR-Cas9 mediated DNA damage and repair pathways. ( A) Schematic of the transcriptional/translational negative feedback loop that drives rhythmic expression and activity of the four core circadian regulators: Period (Per), Timeless (Tim), Clock (Clk), and Cycle (Cyc). Importantly, the core components of the molecular clock in Drosophila are conserved in humans ( Ch and Takahashi, 2006). In brief, Clock and Cycle activate transcription of period and timeless which, once translated, dimerize and translocate into the nucleus where they bind to Clock and Cycle, thereby inhibiting their own transcription this molecular feedback loop repeats with a 24-hour periodicity ( Figure 1A). ![]() Work in Drosophila has been crucial for understanding the molecular clock, a transcriptional negative feedback loop with four core proteins: Clock, Cycle, Period, and Timeless ( Figure 1A) ( Allada et al., 1998 Hunter-Ensor et al., 1996 Rutila et al., 1998 Sehgal et al., 1994 Sehgal et al., 1995 Vosshall et al., 1994). Circadian rhythms are driven by ‘molecular clocks,’ or proteins that regulate rhythmic gene expression. Disruption in circadian regulation is a common feature of aging and is associated with a variety of adverse health outcomes such as diabetes and cancer ( Rosbash and Takahashi, 2002 Maury et al., 2010 Turek et al., 1995 Wulff et al., 2010). This suggests that clock neurons do not act in a hierarchy but as a distributed network to regulate circadian activity.Ĭircadian rhythms are 24-hour oscillations in physiological functions and behaviors, including locomotor activity, immune system function, metabolism, and sleep ( Allen et al., 2016 Ulgherait et al., 2016 Stone et al., 2012 Hill et al., 2018 Shirasu-Hiza et al., 2007 Panda, 2016 Vaccaro et al., 2017). ![]() While loss of the molecular clock in both the morning and evening oscillators eliminates circadian locomotor activity, the molecular clock in either oscillator alone is sufficient to rescue circadian locomotor activity in the absence of the other. To test this, we developed and validated Gal4-UAS based CRISPR tools for cell-specific disruption of key molecular clock components, period and timeless. It has been assumed that the molecular clock in Pdf + neurons is required for these functions. ![]() A subset of Pdf + neurons (the morning oscillator) regulates morning activity and communicates with other non-Pdf + neurons, including a subset called the evening oscillator. Sixteen of these neurons secrete the neuropeptide Pdf and have been called ‘master pacemakers’ because they are essential for circadian rhythms. In Drosophila, ~150 neurons expressing molecular clock proteins regulate circadian behavior. ![]()
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