Central Circadian Oscillator Research Articles

Adrenergic Agonists Activate Transcriptional Activity in Immortalized Neuronal Cells From the Mouse Suprachiasmatic Nucleus.

The suprachiasmatic nucleus of the hypothalamus (SCN) houses the central circadian oscillator of mammals. The main neurotransmitters produced in the SCN are γ-amino-butyric acid, arginine-vasopressin (AVP), vasoactive intestinal peptide (VIP), pituitary-derived adenylate cyclase-activating peptide (PACAP), prokineticin 2, neuromedin S, and gastrin-releasing peptide (GRP). Apart from these, catecholamines and their receptors were detected in the SCN as well. In this study, we confirmed the presence of β-adrenergic receptors in SCN and a mouse SCN-derived immortalized cell line by immunohistochemical, immuno-cytochemical, and pharmacological techniques. We then characterized the effects of β-adrenergic agonists and antagonists on cAMP-regulated element (CRE) signaling. Moreover, we investigated the interaction of β-adrenergic signaling with substances influencing parallel signaling pathways. Our findings have potential implications on the role of stress (elevated adrenaline) on the biological clock and may explain some of the side effects of β-blockers applied as anti-hypertensive drugs.

Artificial light at night suppresses the day-night cardiovascular variability: evidence from humans and rats.

Artificial light at night (ALAN) affects most of the population. Through the retinohypothalamic tract, ALAN modulates the activity of the central circadian oscillator and, consequently, various physiological systems, including the cardiovascular one. We summarised the current knowledge about the effects of ALAN on the cardiovascular system in diurnal and nocturnal animals. Based on published data, ALAN reduces the day-night variability of the blood pressure and heart rate in diurnal and nocturnal animals by increasing the nocturnal values of cardiovascular variables in diurnal animals and decreasing them in nocturnal animals. The effects of ALAN on the cardiovascular system are mainly transmitted through the autonomic nervous system. ALAN is also considered a stress-inducing factor, as glucocorticoid and glucose level changes indicate. Moreover, in nocturnal rats, ALAN increases the pressure response to load. In addition, ALAN induces molecular changes in the heart and blood vessels. Changes in the cardiovascular system significantly depend on the duration of ALAN exposure. To some extent, alterations in physical activity can explain the changes observed in the cardiovascular system after ALAN exposure. Although ALAN acts differently on nocturnal and diurnal animals, we can conclude that both exhibit a weakened circadian coordination among physiological systems, which increases the risk of future cardiovascular complications and reduces the ability to anticipate stress.

Two central circadian oscillators OsPRR59 and OsPRR95 modulate magnesium homeostasis and carbon fixation in rice

Photosynthesis, which provides oxygen and energy for all living organisms, is circadian regulated. Photosynthesis-associated metabolism must tightly coordinate with the circadian clock to maximize the efficiency of the light-energy capture and carbon fixation. However, the molecular basis for the interplay of photosynthesis and the circadian clock is not fully understood, particularly in crop plants. Here, we report two central oscillator genes of circadian clock, OsPRR95 and OsPRR59 in rice, which function as transcriptional repressors to negatively regulate the rhythmic expression of OsMGT3 encoding a chloroplast-localized Mg2+ transporter. OsMGT3-dependent rhythmic Mg fluctuations modulate carbon fixation and consequent sugar output in rice chloroplasts. Furthermore, sugar triggers the increase of superoxide, which may act as a feedback signal to positively regulate the expression of OsPRR95 and OsPRR59. Taken together, our results reveal a negative-feedback loop that strengthens the crosstalk between photosynthetic carbon fixation and the circadian clock, which may improve plan adaptation and performance in fluctuating environments.

The central circadian regulator CCA1 functions in Glycine max during defense to a root pathogen, regulating the expression of genes acting in effector triggered immunity (ETI) and cell wall metabolism

Expression of the central circadian oscillator components CIRCADIAN CLOCK ASSOCIATED 1 (CCA1), TIMING OF CAB1 (TOC1), GIGANTEA (GI), and CONSTANS (CO) occurs in Glycine max root cells (syncytia) parasitized by the nematode Heterodera glycines while undergoing resistance, indicating a defense role. GmCCA1-1 relative transcript abundance (RTA) in roots experiencing overexpression (OE) or RNA interference (RNAi) is characterized by rhythmic oscillations, compared to a ribosomal protein gene (GmRPS21) control. A GmCCA1-1 RTA change, advancing by 12 h, exists in H. glycines-infected as compared to uninfected controls in wild-type, H. glycines-resistant, G. max[Peking/PI 548402]. The G. max[Peking/PI 548402] transgenic controls exhibit the RTA change by 4 h post infection (hpi), not consistently occurring in the H. glycines-susceptible G. max[Williams 82/PI 518671] until 56 hpi. GmCCA1-1 expression is observed to be reduced in H. glycines-infected GmCCA1-1-OE roots as compared to non-infected transgenic roots with no significant change observed among RNAi roots. The GmCCA1-1 expression in transgenic GmCCA1-1-OE roots remains higher than control and RNAi roots. Decreased GmCCA1-1 mRNA among infected roots shows the altered expression is targeted by H. glycines. Gene expression of proven defense genes including 9 different mitogen activated protein kinases (GmMAPKs), NON-RACE SPECIFIC DISEASE RESISTANCE 1 (GmNDR1-1), RPM1-INTERACTING PROTEIN 4 (GmRIN4-4), and the secreted xyloglucan endotransglycosylase/hydrolase 43 (GmXTH43) in GmCCA1-1-OE and GmCCA1-1-RNAi roots, compared to controls, reveal a significant role of GmCCA1-1 expression in roots undergoing defense to H. glycines parasitism. The observation that GmCCA1-1 regulates GmXTH43 expression links the central circadian oscillator to the functionality of the secretion system.

A central circadian oscillator confers defense heterosis in hybrids without growth vigor costs

Plant immunity frequently incurs growth penalties, which known as the trade-off between immunity and growth. Heterosis, the phenotypic superiority of a hybrid over its parents, has been demonstrated for many traits but rarely for disease resistance. Here, we report that the central circadian oscillator, CCA1, confers heterosis for bacterial defense in hybrids without growth vigor costs, and it even significantly enhances the growth heterosis of hybrids under pathogen infection. The genetic perturbation of CCA1 abrogated heterosis for both defense and growth in hybrids. Upon pathogen attack, the expression of CCA1 in F1 hybrids is precisely modulated at different time points during the day by its rhythmic histone modifications. Before dawn of the first infection day, epigenetic activation of CCA1 promotes an elevation of salicylic acid accumulation in hybrids, enabling heterosis for defense. During the middle of every infection day, diurnal epigenetic repression of CCA1 leads to rhythmically increased chlorophyll synthesis and starch metabolism in hybrids, effectively eliminating the immunity-growth heterosis trade-offs in hybrids.

Novel Approaches for Assessing Circadian Rhythmicity in Humans: A Review.

The temporal organization of molecular and physiological processes is driven by environmental and behavioral cycles as well as by self-sustained molecular circadian oscillators. Quantification of phase, amplitude, period, and disruption of circadian oscillators is essential for understanding their contribution to sleep-wake disorders, social jet lag, interindividual differences in entrainment, and the development of chrono-therapeutics. Traditionally, assessment of the human circadian system, and the output of the SCN in particular, has required collection of long time series of univariate markers such as melatonin or core body temperature. Data were collected in specialized laboratory protocols designed to control for environmental and behavioral influences on rhythmicity. These protocols are time-consuming, expensive, and not practical for assessing circadian status in patients or in participants in epidemiologic studies. Novel approaches for assessment of circadian parameters of the SCN or peripheral oscillators have been developed. They are based on machine learning or mathematical model-informed analyses of features extracted from 1 or a few samples of high-dimensional data, such as transcriptomes, metabolomes, long-term simultaneous recording of activity, light exposure, skin temperature, and heart rate or in vitro approaches. Here, we review whether these approaches successfully quantify parameters of central and peripheral circadian oscillators as indexed by gold standard markers. Although several approaches perform well under entrained conditions when sleep occurs at night, the methods either perform worse in other conditions such as shift work or they have not been assessed under any conditions other than entrainment and thus we do not yet know how robust they are. Novel approaches for the assessment of circadian parameters hold promise for circadian medicine, chrono-therapeutics, and chrono-epidemiology. There remains a need to validate these approaches against gold standard markers, in individuals of all sexes and ages, in patient populations, and, in particular, under conditions in which behavioral cycles are displaced.

CIRCADIAN CLOCK ASSOCIATED 1 gates morning phased auxin response in Arabidopsis thaliana

Circadian clock controls plant behaviors to anticipate day-night switch and keeps plant fitness. Here, we reported that plant response to auxin is also strictly governed by clock. The amplitude of auxin-responsive gene expressions gradually declined from morning to the dusk, and then enhanced from dusk to dawn. Plants with defects in both CIRCADIAN CLOCK ASSOCIATED1 (CCA1) and its closest homologue LATE ELONGATED HYPOCOTYL (LHY) (cca1 lhy) showed comparable responses to auxin at different time points in consecutive days, suggesting that CCA1 and LHY were required for gating auxin responses. Moreover, CCA1/LHY physically interacted with the core transcriptional repressors (Aux/IAA proteins), which might further modulate plant sensitivity to auxin. Taken together, we demonstrate that the central morning phased circadian oscillator CCA1 plays a pivotal role in gating auxin response.

Kalanchoë PPC1 Is Essential for Crassulacean Acid Metabolism and the Regulation of Core Circadian Clock and Guard Cell Signaling Genes.

Unlike C3 plants, Crassulacean acid metabolism (CAM) plants fix CO2 in the dark using phosphoenolpyruvate carboxylase (PPC; EC 4.1.1.31). PPC combines phosphoenolpyruvate with CO2 (as HCO3 -), forming oxaloacetate. The oxaloacetate is converted to malate, leading to malic acid accumulation in the vacuole, which peaks at dawn. During the light period, malate decarboxylation concentrates CO2 around Rubisco for secondary fixation. CAM mutants lacking PPC have not been described. Here, we employed RNA interference to silence the CAM isogene PPC1 in Kalanchoë laxiflora Line rPPC1-B lacked PPC1 transcripts, PPC activity, dark period CO2 fixation, and nocturnal malate accumulation. Light period stomatal closure was also perturbed, and the plants displayed reduced but detectable dark period stomatal conductance and arrhythmia of the CAM CO2 fixation circadian rhythm under constant light and temperature free-running conditions. By contrast, the rhythm of delayed fluorescence was enhanced in plants lacking PPC1 Furthermore, a subset of gene transcripts within the central circadian oscillator was upregulated and oscillated robustly in this line. The regulation of guard cell genes involved in controlling stomatal movements was also perturbed in rPPC1-B These findings provide direct evidence that the regulatory patterns of key guard cell signaling genes are linked with the characteristic inverse pattern of stomatal opening and closing during CAM.

Age-Induced Differential Changes in the Central and Colonic Human Circadian Oscillators.

Aging modifies not only multiple cellular and homeostatic systems, but also biological rhythms. The circadian system is driven by a central hypothalamic oscillator which entrains peripheral oscillators, in both cases underlain by circadian genes. Our aim was to characterize the effect of aging in the circadian expression of clock genes in the human colon. Ambulatory recordings of the circadian rhythms of skin wrist temperature, motor activity and the integrated variable TAP (temperature, activity and position) were dampened by aging, especially beyond 74 years of age. On the contrary, quantitative analysis of genes expression in the muscle layer of colonic explants during 24 h revealed that the circadian expression of Bmal1, Per1 and Clock genes, was larger beyond that age. In vitro experiments showed that aging induced a parallel increase in the myogenic contractility of the circular colonic muscle. This effect was not accompanied by enhancement of Ca2+ signals. In conclusion, we describe here for the first time the presence of a molecular oscillator in the human colon. Aging has a differential effect on the systemic circadian rhythms, that are impaired by aging, and the colonic oscillator, that is strengthened in parallel with the myogenic contractility.

The endogenous redox rhythm is controlled by a central circadian oscillator in cyanobacterium Synechococcus elongatus PCC7942.

The intracellular redox and the circadian clock in photosynthetic organisms are two major regulators globally affecting various biological functions. Both of the global control systems have evolved as systems to adapt to regularly or irregularly changing light environments. Here, we report that the two global regulators mutually interact in cyanobacterium Synechococcus elongatus PCC7942, a model photosynthetic organism whose clock molecular mechanism is well known. Electrochemical assay using a transmembrane electron mediator revealed that intracellular redox of S. elongatus PCC7942 cell exhibited circadian rhythms under constant light conditions. The redox rhythm disappeared when transcription/translation of clock genes is defunctionalized, indicating that the transcription/translation controlled by a core KaiABC oscillator generates the circadian redox rhythm. Importantly, the amplitude of the redox rhythm at a constant light condition was large enough to affect the KaiABC oscillator. The findings indicated that the intracellular redox state is actively controlled to change in a 24-h cycle under constant light conditions by the circadian clock system.

Diurnal regulation of SDG2 and JMJ14 by circadian clock oscillators orchestrates histone modification rhythms in Arabidopsis

BackgroundCircadian rhythms modulate growth and development in all organisms through interlocking transcriptional-translational feedback loops. The transcriptional loop involves chromatin modifications of central circadian oscillators in mammals and plants. However, the molecular basis for rhythmic epigenetic modifications and circadian regulation is poorly understood.ResultsHere we report a feedback relationship between diurnal regulation of circadian clock genes and histone modifications in Arabidopsis. On one hand, the circadian oscillators CCA1 and LHY regulate diurnal expression of genes coding for the eraser (JMJ14) directly and writer (SDG2) indirectly for H3K4me3 modification, leading to rhythmic H3K4me3 changes in target genes. On the other hand, expression of circadian oscillator genes including CCA1 and LHY is associated with H3K4me3 levels and decreased in the sdg2 mutant but increased in the jmj14 mutant. At the genome-wide level, diurnal rhythms of H3K4me3 and another histone mark H3K9ac are associated with diurnal regulation of 20–30% of the expressed genes. While the majority (86%) of H3K4me3 and H3K9ac target genes overlap, only 13% of morning-phased and 22% of evening-phased genes had both H3K4me3 and H3K9ac peaks, suggesting specific roles of different histone modifications in diurnal gene expression.ConclusionsCircadian clock genes promote diurnal regulation of SDG2 and JMJ14 expression, which in turn regulate rhythmic histone modification dynamics for the clock and its output genes. This reciprocal regulatory module between chromatin modifiers and circadian clock oscillators orchestrates diurnal gene expression that governs plant growth and development.

Measuring Phytochrome-Dependent Light Input to thePlant Circadian Clock.

The circadian clock allows plants to synchronize their internal processes with the external environment. This synchronization occurs through daily cues, one of which is light. Phytochromes are well established as light-sensing proteins and have been identified in forming multiple signaling networks with the central circadian oscillator. However, the precise details of how these networks are formed are yet to be established. Using established promoter-luciferase lines for clock genes crossed into mutant lines, it is possible to use luciferase-based imaging technologies to determine whether specific proteins are involved in phytochrome signaling to the circadian oscillator. The methods presented here use two automated methods of luciferase imaging in Arabidopsis to allow for high-throughput measurement of circadian clock components under a range of different light conditions.

MTOR signaling regulates central and peripheral circadian clock function.

The circadian clock coordinates physiology and metabolism. mTOR (mammalian/mechanistic target of rapamycin) is a major intracellular sensor that integrates nutrient and energy status to regulate protein synthesis, metabolism, and cell growth. Previous studies have identified a key role for mTOR in regulating photic entrainment and synchrony of the central circadian clock in the suprachiasmatic nucleus (SCN). Given that mTOR activities exhibit robust circadian oscillations in a variety of tissues and cells including the SCN, here we continued to investigate the role of mTOR in orchestrating autonomous clock functions in central and peripheral circadian oscillators. Using a combination of genetic and pharmacological approaches we show that mTOR regulates intrinsic clock properties including period and amplitude. In peripheral clock models of hepatocytes and adipocytes, mTOR inhibition lengthens period and dampens amplitude, whereas mTOR activation shortens period and augments amplitude. Constitutive activation of mTOR in Tsc2–/–fibroblasts elevates levels of core clock proteins, including CRY1, BMAL1 and CLOCK. Serum stimulation induces CRY1 upregulation in fibroblasts in an mTOR-dependent but Bmal1- and Period-independent manner. Consistent with results from cellular clock models, mTOR perturbation also regulates period and amplitude in the ex vivo SCN and liver clocks. Further, mTOR heterozygous mice show lengthened circadian period of locomotor activity in both constant darkness and constant light. Together, these results support a significant role for mTOR in circadian timekeeping and in linking metabolic states to circadian clock functions.

Shining a light on the Arabidopsis circadian clock.

The circadian clock provides essential timing information to ensure optimal growth to prevailing external environmental conditions. A major time-setting mechanism (zeitgeber) in clock synchronization is light. Differing light wavelengths, intensities, and photoperiodic duration are processed for the clock-setting mechanism. Many studies on light-input pathways to the clock have focused on Arabidopsis thaliana. Photoreceptors are specific chromic proteins that detect light signals and transmit this information to the central circadian oscillator through a number of different signalling mechanisms. The most well-characterized clock-mediating photoreceptors are cryptochromes and phytochromes, detecting blue, red, and far-red wavelengths of light. Ultraviolet and shaded light are also processed signals to the oscillator. Notably, the clock reciprocally generates rhythms of photoreceptor action leading to so-called gating of light responses. Intermediate proteins, such as Phytochrome interacting factors (PIFs), constitutive photomorphogenic 1 (COP1) and EARLY FLOWERING 3 (ELF3), have been established in signalling pathways downstream of photoreceptor activation. However, the precise details for these signalling mechanisms are not fully established. This review highlights both historical and recent efforts made to understand overall light input to the oscillator, first looking at how each wavelength of light is detected, this is then related to known input mechanisms and their interactions.

Lithium lengthens circadian period of cultured brain slices in area specific manner

Lithium has been used for the treatment of bipolar disorder (BD). However, the mechanisms how lithium exerts its mood stabilizing effects remain to be studied. The disorder in circadian pacemaking has been suggested as an underlying mechanism of the characteristic mood instability of the BD. Lithium is also known to lengthen the circadian periods. We recently proposed that chronic methamphetamine treatment induced circadian oscillation as a complex oscillator including multiple dopaminergic brain areas, and the complex oscillator regulates behavior rhythm independent from the central circadian oscillator in the suprachiasmatic nucleus (SCN). Sleep-wake pattern of rapid cycling BD exhibits similar rhythm disorganization to methamphetamine treated animals. Therefore, we hypothesized that the dysregulated circadian rhythm in BD patients is caused by desynchronization of sleep-wake rhythms from the central clock in the SCN, and that mood stabilizing effect of lithium is achieved through their resynchronization. In the present experiment, we examined how lithium affects the circadian rhythms of brain areas involved in the complex oscillator as well as the SCN. Here we report that lithium lengthens the circadian periods in the SCN, olfactory bulb, median eminence and substantia nigra with dose and area specific manner. The effective lithium dose was much higher than the plasma levels that are required for lengthening the circadian behavior rhythms as well for therapeutic use. Low dose of lithium did not lengthen the period but enhanced the amplitude of circadian rhythms, which may exert therapeutic effects on BD.

Monitoring light/dark association dynamics of multi-protein complexes in cyanobacteria using size exclusion chromatography-based proteomics

Diurnal rhythms are recurring 24h patterns such as light/dark cycles that affect many natural environmental and biological processes. The cyanobacterium Synechococcus elongatus PCC 7942 (S. elongatus) produces its energy through photosynthesis and therefore its internal molecular machinery is strongly influenced by these diurnal rhythms. Moreover, it has one of the simplest, self-sustained, circadian rhythms, extensively studied functionally and structurally. These characteristics together with the relatively small genome of S. elongatus, make it an ideal model system for the study of diurnal and circadian rhythms. Although expression of many gene transcripts has been shown to fluctuate in phase with the circadian rhythm, fluctuations at the protein level were less pronounced. This led us to hypothesize that the diurnal adaptation occurs at the level of higher organization of protein complexes. Therefore, we probed the abundance and constituency of S. elongatus protein complexes during the day and night. Following several well-known complexes such as the RNA polymerase, the ribosome and photosynthetic protein complexes, we observe for the first time that these complexes change not only in abundance but also in constituency. Therefore, we conclude that the dynamic assembly of protein complexes is indeed also a key-player in the processes governing the diurnal rhythm. SignificanceThe succession of day and night periods imposes drastic changes in all living organisms. Cyanobacteria produce their energy through photosynthesis and are therefore strongly influenced by diurnal rhythms. The cyanobacteria, Synechococcus elongatus PCC 7942 (S. elongatus), also exhibit a self-sustained biological clock. The connection between the central circadian oscillator and its output to the rest of the cell is not completely known. It has been shown that the expression of many gene transcripts heavily fluctuates in phase with the circadian rhythm; however, our recent global proteomics investigation revealed that the diurnal fluctuations seemed to be less pronounced at the protein level. As many known regulatory functions depend on protein-protein interactions (PPIs) and/or protein assemblies and the fact that so few fluctuations in protein abundances were observed earlier, here we investigated the diurnal adaptation at the level of dynamic changes in protein assembly.The paper demonstrates that the combination of native protein complex fractionation and high-resolution proteomics provides insight in the regulation of megadalton protein assemblies in cyanobacteria, including the ribosomal and photosynthetic complexes. The differences observed between the light and dark conditions in these complexes indicate a cyclic regulation of essential cellular processes.

Spatial and temporal regulation of biosynthesis of the plant immune signal salicylic acid

The plant hormone salicylic acid (SA) is essential for local defense and systemic acquired resistance (SAR). When plants, such as Arabidopsis, are challenged by different pathogens, an increase in SA biosynthesis generally occurs through transcriptional induction of the key synthetic enzyme isochorismate synthase 1 (ICS1). However, the regulatory mechanism for this induction is poorly understood. Using a yeast one-hybrid screen, we identified two transcription factors (TFs), NTM1-like 9 (NTL9) and CCA1 hiking expedition (CHE), as activators of ICS1 during specific immune responses. NTL9 is essential for inducing ICS1 and two other SA synthesis-related genes, phytoalexin-deficient 4 (PAD4) and enhanced disease susceptibility 1 (EDS1), in guard cells that form stomata. Stomata can quickly close upon challenge to block pathogen entry. This stomatal immunity requires ICS1 and the SA signaling pathway. In the ntl9 mutant, this response is defective and can be rescued by exogenous application of SA, indicating that NTL9-mediated SA synthesis is essential for stomatal immunity. CHE, the second identified TF, is a central circadian clock oscillator and is required not only for the daily oscillation in SA levels but also for the pathogen-induced SA synthesis in systemic tissues during SAR. CHE may also regulate ICS1 through the known transcription activators calmodulin binding protein 60g (CBP60g) and systemic acquired resistance deficient 1 (SARD1) because induction of these TF genes is compromised in the che-2 mutant. Our study shows that SA biosynthesis is regulated by multiple TFs in a spatial and temporal manner and therefore fills a gap in the signal transduction pathway between pathogen recognition and SA production.

Marine systems biology.

EDITORIAL article Front. Genet., 13 May 2015Sec. Systems Biology Archive Volume 6 - 2015 | https://doi.org/10.3389/fgene.2015.00181

Clock-Talk: Interactions between Central and Peripheral Circadian Oscillators in Mammals.

In mammals, including humans, nearly all physiological processes are subject to daily oscillations that are governed by a circadian timing system with a complex hierarchical structure. The central pacemaker, residing in the suprachiasmatic nucleus (SCN) of the ventral hypothalamus, is synchronized daily by photic cues transmitted from the retina to SCN neurons via the retinohypothalamic tract. In turn, the SCN must establish phase coherence between self-sustained and cell-autonomous oscillators present in most peripheral cell types. The synchronization signals (Zeitgebers) can be controlled more or less directly by the SCN. In mice and rats, feeding-fasting rhythms, which are driven by the SCN through rest-activity cycles, are the most potent Zeitgebers for the circadian oscillators of peripheral organs. Signaling through the glucocorticoid receptor and the serum response factor also participate in the phase entrainment of peripheral clocks, and these two pathways are controlled by the SCN independently of feeding-fasting rhythms. Body temperature rhythms, governed by the SCN directly and indirectly through rest-activity cycles, are perhaps the most surprising cues for peripheral oscillators. Although the molecular makeup of circadian oscillators is nearly identical in all cells, these oscillators are used for different purposes in the SCN and in peripheral organs.

Scientists’ use of diagrams in developing mechanistic explanations

We explore the crucial role of diagrams in scientific reasoning, especially reasoning directed at developing mechanistic explanations of biological phenomena. We offer a case study focusing on one research project that resulted in a published paper advancing a new understanding of the mechanism by which the central circadian oscillator in Synechococcus elongatus controls gene expression. By examining how the diagrams prepared for the paper developed over the course of multiple drafts, we show how the process of generating a new explanation vitally involved the development and integration of multiple versions of different types of diagrams, and how reasoning about the mechanism proceeded in tandem with the development of the diagrams used to represent it.