kin-29 acts upstream of ALA and RIS neurons to regulate sleep.

(A) Model by which kin-29/SIK functions in energy-sensitive sensory neurons upstream of the sleep-promoting RIS and ALA neurons. ALA and RIS activation by LIN-3/EGF or RIS activation by ChR2 bypasses the requirement for KIN-29 function in sleep. RIS is also required for movement quiescence during SIS (see S11C–S11F Fig and [43]). (B and C) kin-29 null mutation does not affect the body movement quiescence (B) and reduced feeding rate (C) observed in response to EGF OE (n > 10 animals). To induce EGF OE, adult animals expressing a Phsp-16.2::LIN-3C transgene were heat-shocked for 30 min, 2 hr prior to analysis of behavior (see Material and methods). Data are represented as mean ± SEM. *** and ** indicate corrected p-values that are different from WT or kin-29 mutants at p < 0.001 and p < 0.01, respectively, by a Kruskal-Wallis with Dunn multiple-comparisons test (S1 Data, Sheet 6B and 6C). (D) Optogenetic stimulation of the RIS neuron causes a reduction in feeding rate, which is not dependent on kin-29. WT and kin-29 null mutants expressing Paptf-1::ChR2 grown either in the presence or absence of ATR were exposed to blue light (ON) (see Material and methods). Pumps were counted during a 10-s window, before, during, and after exposure of transgenic animals (n = 9–13) to blue light. Data are represented as the mean ± SEM for each condition. ns indicates values that are not different between WT Paptf-1::ChR2 (+ATR) and kin-29; Paptf-1::ChR2 (+ATR) by an ANOVA with Tukey multiple-comparisons test S1 Data, Sheet 6D). ATR, all-trans retinal; ChR2, channelrhodopsin2; EGF, epidermal growth factor; EGFR, EGF receptor; ns, not significant; OE, overexpression; SIK, salt-inducible kinase; SIS, sleep-induced stress; WT, wild type.