Understanding circadian rhythmicity in Neurospora crassa: from behavior to genes and back again

Circadian clocks have been described in organisms ranging in complexity from unicells to mammals, in which they function to control daily rhythms in cellular activities and behavior. The significance of a detailed understanding of the clock can be appreciated by its ubiquity and its established involvement in human physiology, including endocrine function, sleep/wake cycles, psychiatric illness, and drug tolerances and effectiveness. Because the clock in all organisms is assembled within the cell and clock mechanisms are evolutionarily conserved, simple eukaryotes provide appropriate experimental systems for dissecting the clock. Significant progress has been made in deciphering the circadian system in Neurospora crassa using both genetic and molecular approaches, and Neurospora has contributed greatly to our understanding of (1) the feedback cycle that comprises a circadian oscillator, (2) the mechanisms by which the clock is kept in synchrony with the environment, and (3) the genes that reside in rhythmic output pathways. Importantly, the lessons learned in Neurospora are relevant to our understanding of clocks in higher eukaryotes.     

Growth phase-dependent changes in the expression of global regulatory genes and associated metabolic pathways in Escherichia coli

Metabolic regulation in Escherichia coli was studied in terms of the changes in the expression of the global regulatory genes rpoD, rpoS, soxRS, cra, fadR, iclR and arcA at three different growth phases, in batch culture. The expression of rpoS and several rpoS-dependent metabolic pathway genes, such as tktB, talA, fumC, acnA, sucA, acs and sodC, were increased (∼1.5 to 2-fold) as the cells entered the late phase of growth. The changes in the expression of other global regulators and their effects on different metabolic pathway genes were less significant, as compared to rpoS, during the later phases of growth.     

Kinetics of circadian band development in Neurospora crassa

The circadian rhythm of Neurospora crassa can be seen as a conidiation rhythm that produces concentric rings of bands (conidiating regions) alternating with interbands (non-conidiating regions) on the surface of an agar medium. To follow quantitatively this rhythm, densitometric analysis, gravimetric procedures, and video microscopy were employed. The circadian behavior of N. crassa is commonly monitored by cultivation in race tubes; in this work we report different growth kinetics during cultivation in conventional Petri dish cultures. Two different growth parameters were measured: total colony mass (true growth rate) and distance (colony radial expansion or hyphal elongation). Determinations of cellular mass revealed a dramatic circadian oscillation with a marked drop in growth rate during new interband formation followed by a sharp increase during the development of a new conidiation band. On the other hand, we found that the radial expansion of the colony previously reported to decrease periodically seemed unaffected by the circadian clock. Densitometric analysis showed no initial difference in the expanding margin of the colony, independent of whether that area was destined to be a band or an interband. The band areas increased rapidly in density for about 15 h whereas the interband areas maintained an equally rapid rate of increase for only 6h. The density of band areas kept increasing slowly for almost 40 h, along with an increase in the amount of conidia. Video microscopy showed the importance of cytoplasmic flow in colony development with continuous forward flow to support hyphal morphogenesis and reverse flow to support an extended period of conidiogenesis. Our results indicate that the circadian system of Neurospora can be expressed at the level of cellular mass formation, not just as the developmental conidiation rhythm.     

A genetic selection for circadian output pathway mutations in Neurospora crassa

In most organisms, circadian oscillators regulate the daily rhythmic expression of clock-controlled genes (ccgs). However, little is known about the pathways between the circadian oscillator(s) and the ccgs. In Neurospora crassa, the frq, wc-1, and wc-2 genes encode components of the frq-oscillator. A functional frq-oscillator is required for rhythmic expression of the morning-specific ccg-1 and ccg-2 genes. In frq-null or wc-1 mutant strains, ccg-1 mRNA levels fluctuate near peak levels over the course of the day, whereas ccg-2 mRNA remains at trough levels. The simplest model that fits the above observations is that the frq-oscillator regulates a repressor of ccg-1 and an activator of ccg-2. We utilized a genetic selection for mutations that affect the regulation of ccg-1 and ccg-2 by the frq-oscillator. We find that there is at least one mutant strain, COP1-1 (circadian output pathway derived from ccg-1), that has altered expression of ccg-1 mRNA, but normal ccg-2 expression levels. However, the clock does not appear to simply regulate a repressor of ccg-1 and an activator of ccg-2 in two independent pathways, since in our selection we identified three mutant strains, COP1-2, COP1-3, and COP1-4, in which a single mutation in each strain affects the expression levels and rhythmicity of both ccg-1 and ccg-2.