We propose that a rhythmic molecular clock in the DN1s of per01; DN1 > per larvae drives rhythmic signals from DN1s that regulate LNv neuronal activity. Because DN1s seem to be most active at dusk, this would allow LNvs to promote light avoidance at dawn even in the absence of their own functional clock. This result directly

parallels observations from adult flies, in which restoring per to only non-LNv clock neurons in per01 mutant flies restored the morning peak of locomotor activity ( Stoleru et al., 2004). Conversely, we propose that larvae lacking per expression in DN1s (per01; Pdf > per; Figure 4B) remain rhythmic because high CLK/CYC activity in per01 DN1s ( Figure 3C) renders them excitable and able to release their essential signal, while the functional LNv SCH727965 clock controls the timing of behavior. This contrasts with DN1 ablation, which prevents rhythms ( Figure 4A). Therefore, the DN1 signal is both necessary (ablated DN1s; Figure 4A) and sufficient (per+ DN1s with per mutant LNvs; Figure 4B) for light avoidance rhythms. If CLK/CYC activity regulates DN1 excitability (Figure 3), low CLK/CYC activity should block release of the essential DN1 signal and be phenotypically similar to ablating DN1s. To test this,

we assayed the effect of stopping the DN1 molecular clock with low CLK/CYC activity on light avoidance rhythms at 150 lux (Figure 4C). We found that DN1 > ClkDN larvae lost light avoidance rhythms, with larvae constitutively sensitive to light at both 150 lux and 50 lux, similar to DN1 ablation. It should be noted that the experiments in Galunisertib clinical trial Figures 4B and 4C are complementary rather than identical because expression of ClkDN or cycDN in a single neuronal group blocks the clock in those cells but leaves the other clock neurons wild-type, whereas restoration of per Carnitine dehydrogenase to a single neuronal group leaves the rest of the larva in a mutant

per01 state. Overall, our LD and DD data suggest that the DN1 molecular clock regulates DN1 neuronal activity, with DN1s least active when CLK/CYC activity is lowest at dawn. Next, we sought to directly test when DN1s normally signal by using a transgene that expresses the heat-activated cation channel, TrpA1 (Hamada et al., 2008). Because TrpA1 is activated at temperatures >25°C, it can be used to transiently activate neurons in which it is expressed (Pulver et al., 2009). We used TrpA1 to transiently stimulate DN1s at CT12 and CT24 and measure the effect on light avoidance (Figure 4D). At 20°C, DN1 > TrpA1 larvae displayed normal light avoidance rhythms. However, activating DN1s via TrpA1 at 26°C blocked the rhythm, with levels of light avoidance constitutively low at both CT12 and CT24. No reduction in light avoidance at CT24 was observed between 20°C and 26°C for either UAS-TrpA1 / + or DN1 / + control larvae ( Figure S3).