A light-entrainable circadian clock controls development and physiology in Neurospora crassa. Existing simple models for resetting based on light pulses (so-called nonparametric entrainment) predict that constant light should quickly send the clock to an arrhythmic state; however, such a clock would be of little use to an organism in changing photoperiods in the wild, and we confirm that true, albeit dampened, rhythmicity can be observed in extended light. This rhythmicity requires the PAS/LOV protein VIVID (VVD) that acts, in the light, to facilitate expression of an oscillator that is related to, but distinguishable from, the classic FREQUENCY/WHITE-COLLAR complex (FRQ/WCC)-based oscillator that runs in darkness. VVD prevents light resetting of the clock at dawn but, by influencing frq RNA turnover, promotes resetting at dusk, thereby allowing the clock to run through the dawn transition and take its phase cues from dusk. Consistent with this, loss of VVD yields a clock whose performance follows the simple predictions of earlier models, and overexpression of VVD restores rhythmicity in the light and sensitivity of phase to the duration of the photoperiod.[Keywords: Circadian clock; vivid; white-collar; frequency; limit cycle; Neurospora] Supplemental material is available at http://www.genesedev.org.

The photoreceptor and PAS/LOV protein VIVID (VVD) modulates blue-light signaling and influences light and temperature responses of the circadian clock in Neurospora crassa. One of the main actions of VVD on the circadian clock is to influence circadian clock phase by regulating levels of the transcripts encoded by the central clock gene frequency (frq). How this regulation is achieved is unknown. Here we show that VVD interacts with complexes central for circadian clock and blue-light signaling, namely the WHITE-COLLAR complex (WCC) and FREQUENCY-interacting RNA helicase (FRH), a component that complexes with FRQ to mediate negative feedback control in Neurospora. VVD interacts with FRH in the absence of WCC and FRQ but does not seem to control the exosome-mediated negative feedback loop. Instead, VVD acts to modulate the transcriptional activity of the WCC.ight, in addition to providing an energy source for many life forms on Earth, acts as a signal that may trigger development or serve as a repetitive cue that marks the passing of external time. External time cues are used by cellular timers such as circadian clocks to lock their periods to that of the external day. The process of period locking is called "entrainment" and ensures that cellular and behavioral activities happen at times of day when their adaptive value is highest (1-3). Blue light plays a central role in the entrainment of circadian clocks. Indeed blue-light photoreceptors and circadian clocks may have coevolved from a mechanism that originally served to detect (photoreceptor) and avoid (timer) harmful radiation (4-6). Our understanding of the molecular bases of circadian clocks and their responses to light has improved dramatically during the last decade or so, and the eukaryotic model organism Neurospora crassa has become one of the best-studied systems for understanding both processes (7-9).The key components of the Neurospora circadian clock are the products of the white collar (wc-1 and wc-2), frequency (frq), and frq-interacting helicase (frh) genes (4, 10, 11). The blue-light photoreceptor WC-1, and its interaction partner WC-2, form the transcriptionally and photoactive WHITE COLLAR complex (WCC) that activates frq expression (4, 12). FRQ protein, in turn, complexes with FRH to form an FRQ-FRH complex (FFC) that represses WCC activity (9, 11). Thus, photoreception and temporal organization of gene expression are linked via the WCC (4,(12)(13)(14). Hyperphosphorylated WCC is transcriptionally less active, and repression of WCC by FRQ occurs via FRQ-mediated phosphorylation of WCC by Casein kinase 1 and 2 (CK1 and 2) (14, 15).A second feedback loop that acts to repress WCC activity involves the product of the vivid (vvd) gene (16). Like WC-1, VVD is a PAS/LOV protein and blue-light photoreceptor; however, unlike WC-1, its presence is not essential for circadian rhythmicity in constant darkness (DD) (16)(17)(18)(19). Nevertheless, VVD has important roles within the Neurospora circadian system. Without VVD the organism is more sensitive to ...

BackgroundRNAi technology by feeding of E. coli containing dsRNA in C. elegans has significantly contributed to further our understanding of many different fields, including genetics, molecular biology, developmental biology and functional genomics. Most of this research has been carried out in a single genotype or genetic background. However, RNAi effects in one genotype do not reveal the allelic effects that segregate in natural populations and contribute to phenotypic variation.ResultsHere we present a method that allows for rapidly comparing RNAi effects among diverse genotypes at an improved high throughput rate. It is based on assessing the fitness of a population of worms by measuring the rate at which E. coli is consumed. Critically, we demonstrate the analytical power of this method by QTL mapping the loss of RNAi sensitivity (in the germline) in a recombinant inbred population derived from a cross between Bristol and a natural isolate from Hawaii. Hawaii has lost RNAi sensitivity in the germline. We found that polymorphisms in ppw-1 contribute to this loss of RNAi sensitivity, but that other loci are also likely to be important.ConclusionsIn summary, we have established a fast method that improves the throughput of RNAi in liquid, that generates quantitative data, that is easy to implement in most laboratories, and importantly that enables QTL mapping using RNAi.

Circadian clocks are cellular timekeepers that regulate aspects of temporal organization on daily and seasonal time scales. To allow accurate time measurement, the period lengths of clocks are conserved in a range of temperatures-a phenomenon known as temperature compensation. Temperature compensation of circadian clock period aids in maintaining a stable "target time" or phase of clock-controlled events. Here we show that the Neurospora protein VIVID (VVD) buffers the circadian system against temperature fluctuations. In vvd-null mutants, the circadian period of clock-controlled events such as asexual sporulation (conidiation) is temperature compensated, but the phase of this clock time marker is not. Consistent with delayed conidiation at lower temperatures in vvd KO strains, the levels of vvd gene products in the wild type increase with decreasing temperatures. Moreover, vvd C108A mutants that lack the light function of VVD maintain a dark activity that transiently influences the phase of conidiation, indicating that VVD influences the time of conidiation downstream from the clock. FREQUENCY (FRQ) phosphorylation is altered in a vvdKO strain, suggesting a mechanism by which VVD can influence the timing of clock-controlled processes in the dark. Thus, temperature compensation of clock-controlled output is a key factor in maintaining temperature compensation of the entire circadian system.[Keywords: Neurospora; circadian clock; temperature compensation; period; phase; VVD] Supplemental material is available at http://www.genesdev.org.

Complex traits, including common disease-related traits, are affected by many different genes that function in multiple pathways and networks. The apoptosis, MAPK, Notch, and Wnt signalling pathways play important roles in development and disease progression. At the moment we have a poor understanding of how allelic variation affects gene expression in these pathways at the level of translation. Here we report the effect of natural genetic variation on transcript and protein abundance involved in developmental signalling pathways in Caenorhabditis elegans. We used selected reaction monitoring to analyse proteins from the abovementioned four pathways in a set of recombinant inbred lines (RILs) generated from the wild-type strains N2 (Bristol) and CB4856 (Hawaii) to enable quantitative trait locus (QTL) mapping. About half of the cases from the 44 genes tested showed a statistically significant change in protein abundance between various strains, most of these were however very weak (below 1.3-fold change). We detected a distant QTL on the left arm of chromosome II that affected protein abundance of the phosphatidylserine receptor protein PSR-1, and two separate QTLs that influenced embryonic and ionizing radiation-induced apoptosis on chromosome IV. Our results demonstrate that natural variation in C. elegans is sufficient to cause significant changes in signalling pathways both at the gene expression (transcript and protein abundance) and phenotypic levels.