<i>kin-29</i> acts upstream of ALA and RIS neurons to regulate sleep.

<p>(A) Model by which <i>kin-29</i>/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 <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.3000220#pbio.3000220.s011" target="_blank">S11C–S11F Fig</a> and [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.3000220#pbio.3000220.ref043" target="_blank">43</a>]). (B and C) <i>kin-29</i> null mutation does not affect the body movement quiescence (B) and reduced feeding rate (C) observed in response to EGF OE (<i>n</i> > 10 animals). To induce EGF OE, adult animals expressing a <i>Phsp-16</i>.<i>2</i>::<i>LIN-3C</i> transgene were heat-shocked for 30 min, 2 hr prior to analysis of behavior (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.3000220#sec010" target="_blank">Material and methods</a>). Data are represented as mean ± SEM. *** and ** indicate corrected <i>p</i>-values that are different from WT or <i>kin-29</i> mutants at <i>p</i> < 0.001 and <i>p</i> < 0.01, respectively, by a Kruskal-Wallis with Dunn multiple-comparisons test (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.3000220#pbio.3000220.s018" target="_blank">S1 Data</a>, Sheet 6B and 6C). (D) Optogenetic stimulation of the RIS neuron causes a reduction in feeding rate, which is not dependent on <i>kin-29</i>. WT and <i>kin-29</i> null mutants expressing <i>Paptf-1</i>::<i>ChR2</i> grown either in the presence or absence of ATR were exposed to blue light (ON) (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.3000220#sec010" target="_blank">Material and methods</a>). Pumps were counted during a 10-s window, before, during, and after exposure of transgenic animals (<i>n</i> = 9–13) to blue light. Data are represented as the mean ± SEM for each condition. ns indicates values that are not different between WT <i>Paptf-1</i>::<i>ChR2</i> (+ATR) and <i>kin-29</i>; <i>Paptf-1</i>::<i>ChR2</i> (+ATR) by an ANOVA with Tukey multiple-comparisons test <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.3000220#pbio.3000220.s018" target="_blank">S1 Data</a>, Sheet 6D). ATR, all-<i>trans</i> 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.</p>