Staining with an anti-PDM3 antibody confirmed a drastic reduction in brain protein levels with knockdown ( Figure 1-figure supplement 2 Chen et al., 2012). In Drosophila, PDM3 is a broadly expressed neuronal protein that coordinates axon targeting in several areas of the brain ( Chen et al., 2012 Tichy et al., 2008). Pdm3 is part of the POU domain transcription factor family, a gene group with essential roles in nervous system patterning across species ( Badea et al., 2012 Dominguez et al., 2013 Olsson-Carter and Slack, 2011). This phenotype was driven by attenuation of daytime sleep ontogeny ( Figure 1F) and less night sleep in young compared to mature flies with pdm3 knockdown ( Figure 1G). Following outcrossing to a uniform genetic background, we found that while genetic controls had robust ontogenetic change in total sleep time, pan-neuronal pdm3 knockdown abolished this transition ( Figure 1D,E). Knockdown of the gene pdm3 with either one of two RNAi lines, each targeting a different part of the gene, resulted in a consistent loss of sleep ontogenetic change ( Figure 1B,C). Graphs in this figure and all others unless otherwise specified are presented as means ± SEM. ( H) Total sleep time with re-expression of PDM3 (right) versus a control UAS-GFP construct (middle) (n = 24, 24, 19, 16, 30, 31 left to right). Comparison of ( E) total sleep, ( F) day sleep and ( G) night sleep in pdm3 RNAi and controls at day 1 versus day 4–5 (n = 97, 106, 119, 95, 140, 145 left to right in E-G). Young flies are shown in blue and mature flies are shown in orange. ( D) Representative sleep traces of genetic controls (top) and pdm3 knockdown (bottom). ( C) Secondary screen of primary hits (n ≥ 8 flies per genotype/age in B and C). Ontogeny ratio = (min daytime sleep, young) / (min daytime sleep, mature). ( A) Ontogeny screen design ( B) Primary sleep ontogeny screen. Thus, miswiring during early development leads to a brain structural abnormality that disrupts the normal ontogeny of sleep behavior. Transcriptional profiling of mid-pupal brains and a subsequent genetic modifier screen reveal that pdm3 regulates expression of the synapse assembly gene, Msp300, to control sleep ontogeny. Blocking DA signaling to the dFSB rescues sleep ontogeny in flies lacking PDM3, demonstrating that greater inhibitory DA signaling to the sleep center prevents young flies from achieving high sleep amounts. Pdm3 knockdown specifically during pupal development prematurely increases wake-promoting DA input to the dFSB. In contrast to all other characterized sleep mutants, flies lacking PDM3 do not attain appropriately high juvenile sleep amounts. Thus, genes regulating sleep ontogenetic change are likely distinct from those that control sleep duration.įrom an RNAi-based screen, we identified the transcription factor pdm3 as a genetic regulator of sleep ontogeny. We previously found that all studied short and long-sleeping Drosophila mutants sleep more when young ( Dilley et al., 2018). More broadly, there are no known genes that regulate sleep ontogenetic change. The mechanistic underpinnings of how this central sleep circuit develops are not defined. At the circuit level, activity of wake-promoting dopaminergic (DA) neurons increases as flies mature, exerting greater inhibitory influence on the sleep-promoting dorsal fan shaped body (dFSB) ( Donlea et al., 2014 Kayser et al., 2014 Liu et al., 2012 Ueno et al., 2012). Like other animals, the fruit fly, Drosophila melanogaster, exhibits increased sleep duration in young adulthood that tapers with maturity ( Dilley et al., 2018 Kayser et al., 2014 Shaw et al., 2000). Although mechanisms controlling mature adult sleep have been uncovered ( Allada et al., 2017), the regulation of early life sleep remains poorly understood. In humans, childhood sleep disturbances portend later neurocognitive deficits, possibly because sleep loss impinges on neural circuit formation ( Kotagal, 2015 O'Brien, 2009). Increasing evidence suggests early life sleep may represent a distinct behavioral state, uniquely evolved for the needs of a developing nervous system ( Blumberg, 2015 Clawson et al., 2016 Dilley et al., 2018 Frank, 2011). Across species, sleep amounts are highest in early life and decrease as animals mature ( Kayser and Biron, 2016 Roffwarg et al., 1966).
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