DAF-16/FOXO regulates homeostasis of essential sleep-like behavior during larval transitions in C. elegans

Abstract

Sleep homeostasis, which refers to the maintenance of sleep amount or depth following sleep deprivation, indicates that sleep and sleep-like states serve fundamental functions that cannot be bypassed [1]. Homeostasis of sleep-like behavior is observed during C. elegans lethargus, a 2-3 hr behavioral quiescent period that occurs during larval state transitions [2]. Here, we report a role for DAF-16/FOXO, a transcription factor that is active under conditions of stress [3], in the response to deprivation of lethargus quiescence. Forced locomotion during lethargus results in nuclear translocation of DAF-16. The formation of dauer larvae, a developmental state promoted by daf-16, is increased in response to quiescence deprivation. daf-16 mutants show an impaired homeostatic response to deprivation of lethargus quiescence and are hypersensitive to the lethal effects of forced locomotion during lethargus. DAF-16 expression in muscle cells, but not in neurons, is sufficient to restore a homeostatic response to deprivation of quiescence, pointing to a role for muscle in sleep homeostasis. These findings are relevant to clinical observations of altered metabolic signaling in response to sleep deprivation and suggest that these signaling pathways may act in nonneuronal tissue to regulate sleep behaviors.

Figure 1

Nuclear translocation of DAF-16 as a function of developmental time (C) and in response to deprivation of lethargus quiescence in intestine (A), and body muscle (B). DEP denotes worms that had been stimulated during lethargus (Protocol 2) for 30 minutes. White arrows in the fluorescent images point to nuclei showing DAF-16∷GFP localization. The average pixel fluorescent intensity ratio between the nucleus and the cytoplasm is shown. The p value (2-tailed t test) is shown between conditions compared. N=15 for each condition. (D) The nuclear/cytoplasmic ratio decreases with time after completion of deprivation. Shown is the average of 3 worms, each mounted for imaging within 5 minutes of being deprived of L4 lethargus quiescence for 30 minutes, and then imaged for 20 minutes. Error bars denote standard deviation.

Figure 2

Dauer formation is increased following deprivation of lethargus quiescence. Forced swimming of daf-8(e1393) and daf-7(e1372) mutants during L1 lethargus leads to a greater percentage of dauers. Introducing the daf-16(mu86) into the daf-7 mutant attenuates the dauer-inducing effects of lethargus quiescence deprivation. N was 86-181 in each condition. P values were calculated with a twotailed Fisher’s Exact Test.

Figure 3

Deprivation of early L4 lethargus quiescence does not delay the timing of lethargus end points but does affect subsequent sleep quality. (A) Duration from onset of pumping cessation to ecdysis of individual worms. Blue vertical lines denote resumption of pharyngeal pumping and red vertical lines denote resumption of defecation movements. (B) Mean durations of the data presented in A. Filled bars denote deprived animals. NS denotes not significant p>0.1 (two-tailed Student’s T test). Two worms, which did not recover from the deprivation and were therefore censored (Table S2), were observed to resume body movements but not pharyngeal pumping or defecation movements and remained trapped in their prior stage cuticle. (C) Wild-type worms have shortened 1-octanol response latencies following strong stimulation during lethargus but then return to baseline elevated response latencies over 10 minutes. In contrast, worms that had been stimulated for 30 minutes during lethargus (dotted line, Protocol 2) show an accelerated return to baseline response latencies after a strong stimulus. P values shown at each time point were calculated with a two-tailed Student’s t test. N=10 worms. Error bars denote SEM. NS denotes p>0.05.

Figure 4

daf-16 mutants are defective in the homeostatic behavioral response to deprivation of lethargus quiescence. A. daf-16(mgDf50) mutants have 1-octanol response latencies following 30-minutes of stimulation (Protocol 2) during lethargus (dotted line) similar to those of non-deprived animals (solid line). At all times points, p>0.2, Student’s t test. N=10 worms. Error bars denote SEM. B. 1-octanol response latencies at baseline and at 4 minutes after strong stimulation without (CON) and with (DEP) a 30-minute deprivation of lethargus quiescence (Protocol 2). ˆAverage of 3-5 trials, with 10 worms per trial. *Significant after Bonferroni correction for multiple testing.