Project description:The Eastern North American monarch butterfly, Danaus plexippus, is notorious for its spectacular seasonal long-distance migration. In recent years, it has also emerged as a novel system to study how animal circadian clocks keep track of time and regulate ecologically relevant daily rhythmic activities and seasonal behavioral outputs. However, unlike Drosophila and the mouse, little work has been undertaken in the monarch to identify clock-controlled output genes and elucidate the regulation of their rhythmic expression. Here, we used RNA-sequencing and Assay for Transposase-Accessible Chromatin (ATAC)-sequencing to profile the diurnal transcriptome, open chromatin regions, and transcription factor (TF) footprints in the brain of wild-type monarchs and Cryptochrome 2 (Cry2), Clock (Clk), and Bmal1 (named DCyc-like) butterfly mutants with impaired clock function. We identified 366 rhythmic transcripts under circadian clock control belonging to biological processes key to brain function, such as neurotransmission, neuropeptide signaling, and glucose metabolism. Surprisingly, we found no significant time of day and genotype-dependent changes in chromatin accessibility (i.e., cis-regulatory elements) in the brain. Instead, we found the existence of a temporal regulation of TFs occupancy within open chromatin regions in the vicinity of rhythmic genes in the brains of wild-type monarchs, which is abolished in clock deficient mutants. Our data suggest that TFs binding specifically in the middle of the day display pioneer-like activity by increasing the accessibility of the surrounding chromatin, while TFs binding specifically in the middle of the night would bind DNA with a longer residency time without affecting accessibility of the surrounding chromatin. Together, this work identifies for the first time the clock-controlled genes and modes of regulation by which diurnal transcription rhythms are regulated in the monarch brain. It also illustrates the power of ATAC-seq to profile genome-wide regulatory elements and TF binding in unconventional organisms.

Project description:Seasonal daylength, or circadian photoperiod, is a pervasive environmental signal that profoundly influences physiology and behavior. In mammals, the central circadian clock resides in the suprachiasmatic nuclei (SCN) of the hypothalamus where it receives retinal input and synchronizes, or entrains, organismal physiology and behavior to the prevailing light cycle. The process of entrainment induces sustained plasticity in the SCN, but the molecular mechanisms underlying SCN plasticity are incompletely understood. Entrainment to different photoperiods persistently alters the timing, waveform, period, and light resetting properties of the SCN clock and its driven rhythms. To elucidate novel molecular mechanisms of photoperiod plasticity, we performed RNAseq on whole SCN dissected from mice raised in Long (LD 16:8) and Short (LD 8:16) photoperiods. Fewer rhythmic genes were detected in Long photoperiod and in general the timing of gene expression rhythms was advanced 4-6 hours. However, a few genes showed significant delays, including Gem. There were significant changes in the expression clock-associated gene Timeless and in SCN genes related to light responses, neuropeptides, GABA, ion channels, and serotonin. Particularly striking were differences in the expression of the neuropeptide signaling genes Prokr2 and Cck, as well as convergent regulation of the expression of three SCN light response genes, Dusp4, Rasd1, and Gem. Transcriptional modulation of Dusp4 and Rasd1, and phase regulation of Gem, are compelling candidate molecular mechanisms for plasticity in the SCN light response through their modulation of the critical NMDAR-MAPK/ERK-CREB/CRE light signaling pathway in SCN neurons. Modulation of Prokr2 and Cck may critically support SCN neural network reconfiguration during photoperiodic entrainment. Our findings identify the SCN light response and neuropeptide signaling gene sets as rich substrates for elucidating novel mechanisms of photoperiod plasticity. Data is also available on our corresponding website, where users can search and view the expression and rhythmic properties of genes across these photoperiod conditions.

Project description:Annual cycles in daylength provide an initial predictive environmental cue that plants and animals use to time seasonal biology. Seasonal changes in photoperiodic information acts to entrain endogenous programs in physiology to optimize an animal’s fitness. Attempts to identify the neural and molecular substrates of photoperiodic time measurement in birds have, to date, focussed on blunt changes in light exposure during a restricted period of photoinducibility. The objectives of these studies were first to characterise a molecular seasonal clock in Japanese quail and second, to identify the key transcripts involved in endogenously generated interval timing that underlies photosensitivity in birds. We hypothesized that the mediobasal hypothalamus (MBH) provides the neuroendocrine control of photoperiod-induced changes in reproductive physiology, and that the pars distalis of the pituitary gland contains an endogenous internal timer for the short photoperiod dependent development of reproductive photosensitivity. Here we report distinct seasonal waveforms of transcript expression in the MBH, and pituitary gland and discovered the patterns were not synchronized across tissues. Follicle-stimulating hormone-β (FSHβ) expression increased during the simulated spring equinox, prior to photoinduced increases in prolactin, thyrotropin-stimulating hormone-β and testicular growth. Diurnal analyses of transcript expression showed sustained elevated levels of FSHβ under conditions of the spring equinox, compared to autumnal equinox, short (<12L) and long (>12L) photoperiods. FSHβ expression increased in quail held in non-stimulatory short photoperiod, indicative of the initiation of an endogenously programmed interval timer. These data identify that FSHβ establishes a state of photosensitivity for the external coincidence timing of seasonal physiology. The independent regulation of FSHβ expression provides an alternative pathway through which other supplementary environmental cues, such as temperature, can fine tune seasonal reproductive maturation and involution.

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Circadian clock genes and the vitamin A pathway regulate seasonal photoperiodic responsiveness in an insect

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