Recently we described the reversible formation of highly phosphorylated tau protein in hibernating European ground squirrels. Hence, mammalian hibernation represents a model system very well suited to study molecular mechanisms of both tau phosphorylation and dephosphorylation under in vivo physiological conditions. Here, we analysed the extent and kinetics of hibernation-state dependent tau phosphorylation in various brain regions of three species of hibernating mammals: arctic ground squirrels, Syrian hamsters and black bears. Overall, tau protein was highly phosphorylated in torpor states and phosphorylation levels decreased after arousal in all species. Differences between brain regions, hibernation-states and phosphosites were observed with respect to degree and kinetics of tau phosphorylation. Furthermore, we tested the phosphate net turnover of tau protein to analyse potential alterations in kinase and/or phosphatase activities during hibernation. Our results demonstrate that the hibernation-state dependent phosphorylation of tau protein is specifically regulated but involves, in addition, passive, temperature driven regulatory mechanisms. By determining the activity-state profile for key enzymes of tau phosphorylation we could identify kinases potentially involved in the differentially regulated, reversible tau phosphorylation that occurs during hibernation. We show that in black bears hibernation is associated with conformational changes of highly phosphorylated tau protein that are typically related to neuropathological alterations. The particular hibernation characteristics of black bears with a continuous torpor period and an only slightly decreased body temperature, therefore, potentially reflects the limitations of this adaptive reaction pattern and, thus, might indicate a transitional state of a physiological process.Abnormal phosphorylation and aggregation of tau protein are hallmarks of a variety of neurological disorders, including Alzheimer's disease (AD). Increased tau phosphorylation is assumed to represent an early event in pathogenesis and a pivotal aspect for aggregation and formation of neurofibrillary tangles. However, the regulation of tau phosphorylation  Tau is an axonally located, microtubule-associated protein that is encoded by a single gene and predominantly expressed in neurons in vitro conditions in vivo models of PHF-like tau phosphorylation so far impedes a proof of this concept.A variety of neurodegenerative disorders is characterised by the formation of intracellular deposits of phosphorylated tau protein aggregated into paired helical filaments (PHF) We have demonstrated a PHF-like phosphorylation of tau in hibernating mammals Therefore, mammalian hibernation represents a model system very well suited to analyse conditions and mechanisms physiologically associated with increased tau phosphorylation and altered synaptic connectivity. In addition, the hibernation model helps to identify potential differences between tau hyperphosphorylation in torpor and AD, thereby contributing to our understanding of the significance of tau phosphorylation for neurodegeneration.Spermophilus parryii), Syrian hamsters (Mesocricetus auratus) and black bears (Ursus americanus). These species were selected since they differ with respect to their hibernation characteristics as well as their degree of body temperature change during torpor. With a body temperature of about 0°C arctic ground squirrels show the most extreme reduction followed by the Syrian hamsters where body temperature is lowered to about 5°C during hibernation. Black bears, however, show only a slight temperature change during hibernation with a body temperature of no lower than 30°C. In arctic ground squirrels and Syrian hamsters hibernation is characterised by regular torpor intervals interrupted by brief arousal episodes, where metabolic rate and body temperature of animals returns to normal levels. On the contrary, black bears do not show such regular arousal episodes.In the present study we analysed the reversible tau phosphorylation in arctic ground squirrels , cyclin dependent kinase 5 (cdk5), stress-activated protein kinase/Jun-amino-terminal kinase (SAPK/JNK) and mitogen activated protein kinase/extracellular regulated protein kinase (MAPK/ERK) during hibernation.The three species were compared using a panel of phosphorylation-dependent tau antibodies that was applied to different brain regions. To analyse whether hibernation-state related tau phosphorylation is a specifically regulated process or passively driven by a brain temperature-dependent shift of kinase- and phosphatase activities, we assessed the phosphate net turnover of tau protein in different hibernation-states. Moreover, to elucidate the molecular mechanisms regulating tau phosphorylation The hibernation-related tau phosphorylation was analysed immunohistochemically and by Western blot using different phosphosite-specific tau antibodies. A summary of the investigated tau phosphorylation sites and the applied antibodies is shown in Generally, tau protein is highly phosphorylated during torpor states and phosphorylation levels decrease after arousal as demonstrated in Antibodies that detect specific phosphorylation sites on tau protein were used to characterise the regional tau phosphorylation pattern during the hibernation cycle by Western blot. In total, seven phosphosites see  were anaThe analysis of results from arctic ground squirrels  and SyriThe tau phosphosite T231/S235 was the only site showing reversible phosphorylation in all brain regions analysed. Interestingly, for this site we also observed aberrant phosphorylation kinetics. In general, tau phosphorylation did not further increase in late torpor. However, phospho levels at T231/S235 were significantly elevated in late torpor compared to early torpor. This increase was detected in both arctic ground squirrels and Syrian hamsters indicating a conserved characteristic of this particular site.In black bears sampled during hibernation all seven of the investigated phosphosites showed an increased phosphorylation level compared to summer active animals, both in frontal cortex and hippocampus , Table 2We applied the Alz-50 antibody that specifically detects a particular tau conformation, typically for PHF-tau Tau protein expression was analysed by Western blot using a phospho-independent tau antibody. The results are listed in Regarding tau mRNA isoform expression we focused on the analysis of a potentially altered splicing of exon 10 that encodes for an additional microtubule binding repeat and thereby alters the binding capacity of tau. The analysis was performed in arctic ground squirrels  whereas prior to the isoform expression analysis Tau gene transcripts of were cloned and sequenced (GenBank accession number: FJ609677). Based on the determined sequence, species-specific primers were designed binding in exon 7 and exon 11 respectively. The PCR revealed two distinct PCR products with a size of about 390 bp and 290 bp corresponding to the predicted PCR product size depending on the presence or absence of exon 10 thereby demonstrating the expression of both 3-repeat and 4-repeat tau isoforms in adult arctic ground squirrels . To deteThe formation of phospho-proteins was determined in neocortical brain homogenates of arctic ground squirrels . There were no differences regarding the levels of phospho-proteins during hibernation .To determine whether tau phosphorylation during hibernation is specifically regulated or a passive process driven by temperature-dependent alterations of kinase- and phosphatase activities, we analysed the net turnover of phosphate in tau protein at different incubation temperatures. This assay is designed to determine both phosphorylation and dephosphorylation of tau by assessing the incorporation of radioactively labelled phosphate residues into the tau protein. Therefore, the assay reflects the result of kinase and phosphatase activities in the analysed brain extracts. The result of enzyme activities was assessed in cortical brain samples of arctic ground squirrels .First, we analysed the phosphate net turnover at various temperatures . ReactioTau protein phosphate net turnover was significantly altered  in both torpid and aroused animals as compared to euthermic animals. Most interestingly, there was no general shift towards increased phosphorylation in torpor and arousal. We rather determined a temperature dependent variation in tau phosphorylation in these hibernation states. The most intense phosphate intake was observed at 30°C in torpid animals resulting in an almost doubled phospho-tau degree.in vitro. Brain samples were taken from euthermic, torpid and aroused animals and subjected to a decreasing and an increasing temperature gradient. As shown in To further analyse this temperature dependency, we measured rate of tau phosphate net turnover at a series of temperatures that simulated the entry into torpor and arousing We determined brain tissue activity of the major tau kinases glycogen synthase kinase 3 beta (GSK3-beta) cyclin dependent kinase 5 (cdk5), stress-activated protein kinase/Jun-amino-terminal kinase (SAPK/JNK), and mitogen activated protein kinases/extracellular regulated protein kinase (MAPK/ERK). Activity of these kinases is regulated by phosphorylation, which increases enzymatic rates, except for GSK3-beta, where phosphorylation of residue S9 is inhibitory Phosphorylation of GSK3-beta and cdk5 was significantly increased in torpid animals, indicating inhibition of GSK3-beta and activation of cdk5 activity during torpor . Phosphoin vivo.In ground squirrels, the microtubule-associated brain protein tau enters a hyperphosphorylated state during hibernation that is fully reversed when animals periodically arouse to normal levels of metabolism and brain temperature Hibernation is a behavioural and physiological adaptation in mammals in which the need for energy is profoundly decreased during conditions of current or anticipated famine. Hibernating animals enter a state of reduced metabolism and decreased body temperature called torpor that can continue for several days to several weeks in small mammals and several months in large mammals. Hibernation is found in a variety of species in several different orders Mammalian hibernation is thought to be based on similar physiological mechanisms Spermophilus parryii), Syrian hamsters (Mesocricetus auratus) and black bears (Ursus americanus). These species were selected to determine whether tau phosphorylation is a general, hibernation-related phenomenon and whether species that hibernate with different body temperatures and patterns show the same patterns of phosphorylation change.Here we analysed the hibernation-state dependent tau phosphorylation in arctic ground squirrels  cyclin dependent kinase 5 (cdk5), stress-activated protein kinase/Jun-amino-terminal kinase (SAPK/JNK) and mitogen activated protein kinases/extracellular regulated protein kinase (MAPK/ERK) in arctic ground squirrels and Syrian hamsters. Interestingly, the results revealed a differentially regulated enzyme activity pattern. Generally, GSK3-beta is supposed to be the primary candidate kinase responsible for tau hyperphosphorylation whereas the other kinases are assumed to modulate tau phosphorylation The phosphorylation of cdk5 at S159, in contrast, indicates an increased activity in the state of torpor Spermophilus richardsonii) no hibernation-related change in JNK activity was found The decreased phosphorylation of SAPK/JNK indicates an inhibited activity during torpor. This finding is consistent with results showing decreased SAPK/JNK activity in torpid arctic ground squirrels The MAP-kinases ERK1 (p44) and ERK2 (p42) showed a differentially regulated activity pattern. ERK1 phosphorylation was increased while ERK2 was less phosphorylated in torpid animals. These results are contrary to findings reporting on a decrease of both ERK1 and ERK2 activity during torpor in arctic ground squirrels To summarise, we found cdk5 and ERK1 positively yet GSK3-beta, SAPK/JNK and ERK2 negatively regulated in torpid animals. The determined kinase activity-state pattern was analogous in both analysed species indicating equivalent regulatory mechanisms. Based on our findings cdk5 and ERK1 may act as kinases that actively phosphorylate tau under physiological conditions. Consequently, since hibernation is not associated with pathological consequences, activation of GSK3-beta and SAPK/JNK as demonstrated in Alzheimer's disease may indicate pathophysiological mechanisms.Unravelling the regulation of hypometabolic states such as hibernation are of great significance for the understanding of cellular and molecular aspects of neurodegenerative disorders where hypometabolic states of a “vita minima” precede cell death. As shown by functional brain imaging, these hypometabolic states occur very early during the course of AD, even in presymptomatic stages For that reason, physiological adaptations that are observed at the hypometabolic state in hibernation are potentially analogous to neuronal reactions to a hypometabolism in very early stages of AD.Both, the increased tau phosphorylation and the reduced expression of the four-repeat isoforms result in a decreased microtubule binding capacity of tau protein. This coincidence strongly suggests that this particular condition is one prerequisite for neurons to endure the state of torpor. In this regard the biological relevance of an increased phosphorylation of tau protein in preclinical stages of AD might be reconsidered. It is very likely that particular types of neurons respond to a hypometabolic state with an elevated phosphorylation of tau protein. Hence, this reaction represents a physiological mechanism of the cell and not a primary pathological event In vitro studies could demonstrate that the aggregation of tau is inhibited at low temperatures In the neocortex of hibernating black bears we found a conformational change of tau protein. This particular interspecies difference might be the result of the considerably elevated body temperature in torpor of bears. A PHF-like phosphorylation and altered isoform expression of tau protein occurs during torpor in three species of hibernators that differ greatly in body size and in the minimum brain temperatures and metabolic rates they achieve. This phosphorylation is fully reversed when animals return to normal levels of temperature and metabolism whether it is regularly during arousal intervals in small hibernators or seasonally in large hibernators. These findings indicate that a reduced binding capacity of tau may be a precondition to endure the hypometabolic state and reduced tissue temperatures during hibernation. Moreover, the interspecies homogeneity of this reaction pattern suggests that this regulation is subject to a basal physiological mechanism of mammals. Increased neuronal tau phosphorylation in early stages of AD, therefore, may be potentially considered as physiological reaction to a reduced brain metabolic rate. However, as the result of the slow and progressive pathological process in AD, hyperphosphorylated tau protein aggregates to neurofibrillary tangles most likely leading to the degeneration of affected neurons.PHF-like tau phosphorylation in hibernation is paralleled by an aberrant synaptic plasticity and a loss of memory. Due to the co-occurrence of these elementary attributes of AD hibernating mammals represent a unique and useful model organism to study the relevance and coherencies of particular characteristics of AD pathology. This is of utmost importance for the development of potential strategies for a medical treatment and therapy of this disease.Only animals in good health were used.Spermophilus parryii): Animal protocols were approved by the University of Alaska Fairbanks Institutional Animal Care and Use Committee .Arctic ground squirrels (Mesocricetus auratus): All experimental procedures on animals were carried out in accordance with the European Council Directive of November 24th 1986 (86/609/EEC) and had been approved by the local authorities .Syrian hamsters (Ursus americanus): Animal protocols were approved by the University of Alaska Fairbanks Institutional Animal Care and Use Committee  and USAMRMC Animal Care and Use Review Office .American Black bears  were implanted into the peritoneal cavity of each animal (body mass range 650–950 g). Before implant, transmitters were sealed in heat shrink tubing and triple coated in Elvax  creating a package weight of 17–20 g, calibrated to the nearest 0.1°C at 0 and 20°C against a precision mercury glass thermometer, and gas sterilized Arctic ground squirrels (n = 31) were trapped during August in the Alaska Range or on the North Slope of Alaska near Toolik Lake and transferred to University of Alaska Fairbanks. The animals were housed individually and provided with Mazuri Rodent Chow sunflower seeds, carrots, and apple slices and provided with water In this study, animals were compared at five stages of their annual cycle. Euthermic animals  that had ended hibernation and been continuously at high body temperatures for 1–2 months were sampled in May and June after having gone through their reproductive phase and entered spring molt. Animals in hibernation were sampled in mid winter when 3–5 days within a torpor bout  and after 10–12 days in a torpor bout . Animals that naturally rewarmed from prolonged torpor during an interbout arousal were sampled 2–3 h  and 10–12 h  after core body temperature increased above 30°C as indicated by the radiotransmitter. For sampling, summer active and naturally aroused animals were anesthetized with isoflurane, euthanized with sodium pentobarbital and decapitated. Torpid animals were euthanized by decapitation after verification of core body temperatures <7°C. Brains were removed from the skull, rapidly dissected, frozen in liquid nitrogen, and stored at −80°C.2 ventilation and decapitated. Subsequently, the brain was removed, dissected, immediately frozen in liquid nitrogen and stored at −80°C.Syrian hamsters purchased from Harlan Winkelmann GmbH  were bred and housed at the Medizinisch-Experimentelles Zentrum of the Medical Faculty of the University of Leipzig. In total, 41 animals were included in the study and subjected to hibernation conditions st-27th (n = 5). These animals were without food since October 27th and had been offered a diminishing food ration for two weeks prior. For monitoring of physiological conditions, bears used in hibernation experiments were surgically implanted in late November with telemetry transmitters for core body temperature  and transmitters for ECG and EMG monitoring  which transmitted both body core temperature and biopotentials. All transmitters were implanted intra-peritoneally. Beginning in late November, bears were housed in individual outdoor enclosures on the University of Alaska Fairbanks campus that had dens and straw material for nests. Dens were double walled boxes made from 2.5 cm thick plywood or high-density polyethylene with inside dimensions of approximately 0.8×1.0×1.0 m with 5 cm of Styrofoam between walls. Each nest box had a 46×46 cm door opening that was fitted with a double-layered breakaway door sealed by weather stripping and kept in place by metal clamps. The dens were instrumented with antennas and a thermocouple to monitor temperature. Air was continuously drawn from an exit hole at back of the den and oxygen consumption monitored as described previously 2 powered pistol to administer Telazol via a standard Palmer dart. Immobilized bears were transported to a necropsy suite in a nearby building. After blood sampling, bears were euthanised by an intravenous injection of pentobarbital and death by cardiac arrest assessed with a stethoscope. Tissue collection followed immediately with samples frozen in liquid nitrogen and was complete within 15 min.American black bears (31–118 kg) were captured May-July from the field either near McGrath or Anchorage, Alaska. Bears were transferred to Fairbanks, Alaska where they were held individually in a shaded outdoor holding facility. In summer and fall, bears were fed dog chow and had continuous access to water. Bears representing the non-hibernating condition were still feeding and active when they were euthanized and sampled for tissues between late May and late July (n = 6). Food was withdrawn 24 hours before these animals were sacrificed. Bears in the hibernating condition were euthanised for tissue sampling between March 1Animals were anesthetized with carbon dioxide and perfused transcardialy with physiological saline supplemented Heparin and afterwards with 4% paraformaldehyde and 0.1% glutaraldehyde in phosphate buffered saline (PBS). Subsequently the brain was removed, postfixed for 24 h in 4% paraformaldehyde in PBS, and cryoprotected by immersion in 30% sucrose in PBS for 48 h. The brains where cut into 30 µm thick sagittal sections and stored at 4°C in PBS with 0.01% sodium azide added as a preservative. Briefly, free-floating sections were processed for immunoperoxidase labelling: sections were rinsed in PBS, and incubated 30 min in 0.3% hydrogen peroxide in PBS. Sections were rinsed in TBS-T  and incubated for 60 min in 5% normal donkey serum . The primary antibody  was applied overnight. Subsequently, sections were rinsed, incubated 2 h with biotinylated donkey-anti-mouse , rinsed, incubated for 45 min with ExtrAvidin , rinsed, and developed with nickel-enhanced diaminobenzidine. Afterwards the sections were mounted, dehydrated, cleared, and coverslipped.2; 2 mM EDTA; 2 mM EGTA; 1% NP40; 5 mM NaF; 1 mM Na3VO4; 5% glycerol; 1 mM PMSF; 1 µg/ml leupeptin; Complete protease inhibitor cocktail ) using Ultra Turrax. Homogenates were centrifuged , and supernatants were transferred into new tubes.Brain tissue samples were ground in liquid nitrogen, aliquoted and stored at −80°C. Tissue samples for Western blot analysis were homogenised in 9 volumes (w/v) protein extraction buffer B . The immunoreactivity of phosphosite-specific tau antibodies was related to the expression of tau determined using an antibody directed against the non-phosphorylated protein. The uniformity of sample loading was confirmed by immunodetection of beta-actin and Coomassie-stain of the corresponding membrane after stripping.A potential, hibernation-state related alternative splicing of the tau exon 10 was determined in arctic ground squirrels . For this purpose neocortical brain tissue samples (70–100 mg) were homogenised in 1 ml Trizol reagent  and mRNA was isolated according to the instructions of the product supplier. Quality and integrity of isolated mRNA was assessed by electrophoresis. Prior to the expression analysis a RT-PCR using degenerated primer  were separated by SDS-PAGE and transferred to a PVDF membrane (see 2.2.1). The proteins were stained using the Pro-Q Diamond phosphoprotein blot stain kit  according to the instructions of the product supplier.2; 10 mM MnCl2; 80 ng/µl recombinant tau protein; 100 µm ATP [cold]; 50 nCi/µl 32P ATP) and incubated at various temperatures and times. Reactions for the analysis of tau phosphate net turnover were started at 37°C and aliquots were taken successively after incubation at temperatures that were lowered stepwise. To determine the tau phosphate net turnover in an increasing temperature gradient the pre-phosphorylation of tau was performed by 15 min incubation at 30°C and samples were immediately chilled on ice. The temperature was increased stepwise and aliquots were taken successively from the samples after incubation. The record of reaction was started after incubation at 5°C. Reactions were terminated by adding SDS-sample buffer followed by heat denaturation at 95°C for 10 min. Samples were separated by SDS-PAGE; gels were dried and exposed to X-ray film. Optical density was analysed using the 1D module of TotalLab software (Nonlinear Dynamics) based on 16-bit grey-level film scans.To determine the temperature sensitivity of enzyme activity we used cortical brain samples from arctic ground squirrels  that were homogenized with an Ultra Turrax in five volumes of buffer K . Protein concentrations were determined according to Bradford The activity state of the tau kinases glycogen synthase kinase 3 beta (GSK3-beta), cyclin dependent kinase 5 (cdk5), stress-activated protein kinase/Jun-amino-terminal kinase (SAPK/JNK) and mitogen activated protein kinase/extracellular regulated protein kinase (MAPK/ERK), was determined by Western blot as described previously (2.2.1). Immunoreactivities of the phosphosite-specific antibodies were related to the expression of the according proteins determined applying antibodies directed against the non-modified molecule . The conTable S1Spermophilus parryii) and Syrian hamsters (Mesocricetus auratus). The table lists the increase of tau phosphorylation at specific sites in different brain regions during the hibernation cycle . Data are based on Western blot experiments. Increase factors are related to the level of phosphorylation in euthermic animals. One way ANOVA (unblocked) was performed and italic p-values indicate significant alterations (p≤0.05). Newman-Keuls contrasts are listed to indicate the difference in tau phosphorylation level between all analysed groups.Summary of hibernation-dependent tau phosphorylation in arctic ground squirrels ((0.04 MB DOC)Click here for additional data file.


Tissue regeneration and recovery in the adult body depends on self-renewal and differentiation of stem and progenitor cells. Mesenchymal stem cells (MSCs) that have the ability to differentiate into various cell types, have been isolated from the stromal fraction of virtually all tissues. However, little is known about the true identity of MSCs. MSC populations exhibit great tissue-, location- and patient-specific variation in gene expression and are heterogeneous in cell composition.Our aim was to analyze the dynamics of differentiation of two closely related stromal cell types, adipose tissue-derived MSCs (AdMSCs) and dermal fibroblasts (FBs) along adipogenic, osteogenic and chondrogenic lineages using multiplex RNA-seq technology. We found that undifferentiated donor-matched AdMSCs and FBs are distinct populations that stay different upon differentiation into adipocytes, osteoblasts and chondrocytes. The changes in lineage-specific gene expression occur early in differentiation and persist over time in both AdMSCs and FBs. Further, AdMSCs and FBs exhibit similar dynamics of adipogenic and osteogenic differentiation but different dynamics of chondrogenic differentiation.Our findings suggest that stromal stem cells including AdMSCs and dermal FBs exploit different molecular mechanisms of differentiation to reach a common cell fate. The early mechanisms of differentiation are lineage-specific and are similar for adipogenic and osteogenic differentiation but are distinct for chondrogenic differentiation between AdMSCs and FBs. In vitro studies have demonstrated that MSCs can be expanded in culture and differentiated into several cell types under appropriate conditions. In addition to fat, bone and cartilage cells, MSCs have been demonstrated to give rise to muscle and nerve cells in vitroTissue regeneration is dependent on progenitor cells that self-renew and differentiate into different cell types with specialized functions. Mesenchymal stem cells (MSCs) have been isolated from many different adult organs and tissues including skin, lung, liver and fat In contrast, differentiation of dermal fibroblasts (FBs) into various mesodermal cell types under similar conditions has produced contradictory results. In some experimental settings FBs were shown to lack multilineage differentiation potential The molecular characterization of MSCs is hampered by the lack of biomarkers that would allow their selective isolation from different tissue sources with heterogeneity of cell populations. MSCs are currently isolated as plastic-adherent cells with fibroblast-like morphology that can be differentiated into several mesodermal cell types Here we aimed to analyze the transcriptome profiles of several differentiated cells starting from AdMSCs and dermal FBs obtained from two matching donors and differentiated under similar experimental conditions towards adipocytes, osteoblasts and chondrocytes . RNA-seqIn vitro differentiation of AdMSCs and FBs was confirmed by detection of formation of lipid droplets with Oil Red O staining , matrix mineralization with Alizarin Red S staining  or formation of proteoglycan-rich matrix with Alcian Blue staining . Induced AdMSCs and FBs (from both donors) differentiated into cells with positive staining for ORO, ARS and AB confirming the similar developmental capacity of these cell types  on complete gene expression data without prior statistical filtering . The cirThe analysis above was performed with unfiltered data. However, PCA can be used to visualize filtered data. We used multi-group ANOVA to compare gene expression between defined groups and then used the ANOVA-filtered data in subsequent PCAs to visualize differences between other groups (not between those used in the ANOVA). The genes were selected for ANOVA based on false discovery rate (FDR) to control the effects for multiple testing. The step-wise filtering and vizualisation of the data was performed with Qlucore Omics Explorer.in vitro development of mature cell types usually takes 2–4 weeks. It is thus possible that the differences between AdMSCs and FBs that are evident after one week of differentiation may disappear after longer differentiation.Next, we analyzed how different cell types  are related to each other based on filtered differences in gene expression. A multi-group ANOVA with a FDR of 0.1% recovered 792 differentially expressed genes between different cell types. PCA was then used to visualize the relationship of the individual samples . The edgThe observation that undifferentiated AdMSCs and FBs clustered separately based on the expression of 792 lineage-specific genes raised the question how different are AdMSCs and FBs before differentiation. Heat map-view of differentially expressed genes (including 9000 genes) was generated using all replicate samples (5 of AdMSCs and 6 of FBs). The scale in S100A4, vesicular trafficking - CAV1, DNM1) had higher expression in FBs compared to AdMSCs. However, expression of genes associated with either BMP (GREM1), VEGF (MYOF) or Wnt signaling (ZRANB1) was significantly higher in AdMSCs compared to FBs. Most of the genes that participate in the biosynthetic processes or in the regulation of extracellular matrix organization and adhesion had higher expression in AdMSCs than FBs. Interestingly, we identified high expression of developmentally important gene chromobox homolog 8 (CBX8) in AdMSCs but not in FBs  and PDLIM7  involved in developmental processes were common to AdMSCs and chondrocytes. Both of these pathways play important role in cartilage development The observation that AdMSCs are closely connected to chondrocytes . The genGene expression patterns of AdMSCs and FBs become more similar upon differentiation, but they still remain distinguishable within differentiated cell clusters indicating that cells ‘remember’ their origin . We askeTo answer the question whether genes that are initially distinctly expressed in AdMSCs and FBs also remain differentially expressed in differentiated cells, the comparison of genes differentially expressed in undifferentiated and differentiated AdMSCs and FBs was done and the extent of overlap was determined for each AdMSC- and FB-derived differentiated cell type. Results of the analysis were visualized using Venn diagram, where the size of a circle is proportional to the number of genes it represents . A fractCOL1A1, COL1A2, EFEMP1 (fibulin 3), FN1 (fibronectin 1), GGT5 (gamma-glutamyltransferase 5) and TPM2 (tropomyosin 2) is characteristic for AdMSCs and AdMSC-derived cells. On the other hand, expression of S100A4 (fibroblast-specific protein 1) and TK1 (thymidine kinase 1) is characteristic for FBs and FB-derived cell types. It would be of interest to learn whether after longer period of differentiation the differential expression of those ’memory’ genes in AdMSC- and FB-derived mature adipocytes, osteoblasts and chondrocytes will remain present or disappear.The fact that several genes that are differently expressed in AdMSCs and FBs remain differently expressed in AdMSC- and FB-derived differentiated cells raises the possibility that the cells express so called ’source’-specific cell ’memory’ genes that are not regulated during the differentiation. Our data show that high expression of It is well known that cell differentiation is a process of sequential induction of regulatory genes that in turn initiate the expression of a pile of tissue-specific target genes. Still, each developmental process requires the activation of a specific transcriptional program. Our data show that global changes in cell type-specific gene expression take place quickly upon differentiation of AdMSCs and FBs . Next weTo visualize transcriptome profiles of differentiating AdMSCs and FBs along adipogenic, osteogenic and chondrogenic lineages over time, the daily time points except day 0 as ‘undif.’ and ‘day 7’, were assembled into the following groups: ‘early’ including days 1–3 and ‘late’ including days 4–6. Gene expression at different time points was compared using ANOVA (FDR of 1%) and significantly differentially expressed genes were used to visualize the linkage of different samples in the PCA plot based on time group, and AdMSC or FB annotations . In totaThe analysis of above described gene expression data shows that approximately 70% of adipogenesis-related and 43% of osteogenesis-related genes are down regulated in the process of differentiation of both AdMSCs and FBs . These rStem cells are promising tools to study mechanisms of development and regeneration. Molecular characterization of MSCs is held back by the lack of marker genes that would distinguish them from other cell types in different tissues. MSCs are similar to FBs in growth properties, morphology, surface marker expression and developmental potential as well as origin. The global gene expression analysis of AdMSCs and FBs, both in the undifferentiated state and in the process of differentiation along adipogenic, osteogenic and chondrogenic lineages using cells from the same donors, allowed the identification of cell type-specific gene expression dynamics of two closely related stromal stem cells.First, our study reveals that the transcriptome profiles of undifferentiated AdMSCs and FBs are distinct and stay distinct upon differentiation despite the similar general characteristics of the cells. In previous studies the comparison of gene expression profiles between AdMSCs and FBs has been carried out using cells from different donors, body locations and developmental stage , leading to possible variation in gene expression that is not directly related to the differences between these cell types Secondly, the analysis of gene expression profiles over time shows that the differences in lineage-specific gene expression occur early in differentiation of both AdMSCs and FBs. Interestingly, changes in the gene expression of AdMSCs and FBs upon induction are related initially to rapid down-regulation of gene expression, whereas up regulation occurs slowly over the week. It has been suggested that gene repression is a predominant early mechanism before final cell commitment and that lineage-specific molecular processes are transcriptionally up regulated only after commitment vice versa, many of those genes that become down regulated over the week, are initially up regulated upon differentiation. The distinct pattern of gene regulation upon chondrogenesis in AdMSCs could be related to the observation that AdMSCs are more similar to chondrocytes in the undifferentiated state than FBs. It is intruiging to speculate that AdMSCs are pre-committed to chondrocyte development and initiation of differentiation does not involve global transcriptional reprogramming. Such pre-commitment of AdMSCs seems not to affect their ability to differentiate into other cell types similarly with FBs. It has been shown that lineage-committed MSCs can transdifferentiate into other cell types in response to inducive extracellular cues Thirdly, dynamics of chondrogenic differentiation is different in AdMSCs and FBs. Unlike in FBs, in AdMSCs several genes that become up regulated along chondrogenesis are initially down-regulated and Together, our study shows that stromal stem cells including adipose-derived AdMSCs and dermal FBs exhibit distinct dynamics of differentiation into mesodermal cell types under similar experimental conditions. AdMSCs and FBs exploit similar early mechanisms for differentiation into adipocytes and osteoblasts but show different molecular mechanisms for chondrogenic differentiation. Further finding suggests that the switch from stromal regulation to adipocyte regulation is faster than the switch to osteoblast and chondrocyte regulation. The results of the global study provide relevant insight to the molecular mechanisms of differentiation of stromal stem cells that can be used in further studies.Experiments with human tissues were approved by National Institute for Health Development and Ethics Committee in Estonia .12, biotin, and ascorbic acid, Gibco 32571) at 37°C for 1.5 h, followed by neutralization of enzyme activity with 20% fetal bovine serum (FBS) and 1% penicillin-streptomycin alphaMEM growth medium. Following centrifugation, stromal cell pellet was passed through a 100 µm nylon mesh (BD Biosciences) and resuspended in 10% FBS growth medium, plated at a density of 10 000 cells/cm2 and incubated at 37°C with 5% CO2. After 48 h medium was replaced to remove non-adherent cells. Further cultivation was performed under standard cell culture conditions. Fibroblasts were isolated from dermal skin of the same donors as AdMSCs, using a method described before AdMSCs were isolated from human subcutaneous adipose tissue according to Lin et al. and Yamamoto et al. 2 72 hours prior to induction of differentiation. 10% FBS and 1% penicillin-streptomycin containing growth medium was supplemented with:Passage three or four cells were plated at density of 15 000 cells/cm1 µM dexamethasone, 500 µM IBMX (3-isobutyl-1methylxanthine), 100 µM indomethacin and 10 µg/ml insulin for adipogenic induction,100 nM dexamethasone, 50 µM L-ascorbic acid 2-phosphate and 10 mM glycerol 2-phosphate for osteogenic induction,50 µM L-ascorbic acid 2-phosphate, 6,25 µg/ml insulin and 10 ng/ml TGFbeta-1 (Peprotech) for chondrogenic induction.Treatment media was changed once (on day 3) during the 7-day differentiation assay or twice a week during a long-term differentiation assay. All chemicals, if not specified differently, were purchased from Sigma. Accumulation of lipid droplets in adipocytes was determined by Oil Red O (ORO) staining as previously described Cells were lyzed at day 0, 1, 2, 3, 4, 5, 6 and 7 of adipogenic, osteogenic and chondrogenic differentiation for total RNA extraction using Trizol reagent (Invitrogen). Following a phenol/chloroform extraction and isopropanol precipitation, RNA samples were treated with DNase I using DNA-free ™ kit (Ambion). The resulting 96 RNA samples were applied to sample preparation for deep sequencing.Gene expression analysis was performed as previously described for single-cells Figure S1Differences in gene expression of AdMSCs and FBs. ANOVA (with FDR of 5%) between undifferentiated AdMSCs and FBs resulted in 178 differentially expressed genes, 59 with higher and 119 with lower expression in AdMSCs than in FBs. The scale shows the up (light red) or down regulation (light blue) in standard deviations from the mean expression for each gene.(TIF)Click here for additional data file.Table S1The list of samples used in the study.(DOCX)Click here for additional data file.Table S2The list of lineage-specific genes.(XLSX)Click here for additional data file.Table S3Up and down regulation of genes during differentiation.(DOCX)Click here for additional data file.


The pan-protein kinase C (PKC) inhibitor sotrastaurin (AEB071) is a novel immunosuppressant currently in phase II trials for immunosuppression after solid organ transplantation. Besides T-cell activation, PKC affects numerous cellular processes that are potentially important for the replication of hepatitis B virus (HBV) and hepatitis C virus (HCV), major blood-borne pathogens prevalent in solid organ transplant recipients. This study uses state of the art virological assays to assess the direct, non-immune mediated effects of sotrastaurin on HBV and HCV. Most importantly, sotrastaurin had no pro-viral effect on either HBV or HCV. In the presence of high concentrations of sotrastaurin, well above those used clinically and close to levels where cytotoxic effects become detectable, there was a reduction of HCV and HBV replication. This reduction is very likely due to cytotoxic and/or anti-proliferative effects rather than direct anti-viral activity of the drug. Replication cycle stages other than genome replication such as viral cell entry and spread of HCV infection directly between adjacent cells was clearly unaffected by sotrastaurin. These data support the evaluation of sotrastaurin in HBV and/or HCV infected transplant recipients. Worldwide about half a billion people are chronically infected with the hepatitis B virus (HBV), hepatitis C virus (HCV) or both and thus at risk for cirrhosis and hepatocellular carcinoma. HBV and HCV pose a particularly vexing clinical problem in organ transplant recipients Immunosuppression itself results in enhanced viral replication, but as shown by us and others some immunosuppressive agents have additional direct non-immune mediated effects on viral replication: while cyclosporine A (CsA) and its derivatives are potent inhibitors of HCV-replication and are now being developed as anti-viral drugs Hepadnaviridae family, and HCV, a plus-strand RNA-virus of the Flaviviridae family, is intricately linked to PKC-dependent cellular processes Sotrastaurin (AEB071) is a novel immunosuppressive drug currently in phase I and II clinical trials for post transplant immunosuppression in vitro infection systems capable of recapitulating the entire replication cycle of these viruses we observed no stimulatory effect of sotrastaurin on HBV or HCV at any drug concentration tested. These data indicate that sotrastaurin may be a viable option to achieve immunosuppression in HBV and/or HCV infected solid organ transplant recipients.Using cell based Sotrastaurin (AEB071) and cyclosporin A were provided by Novartis, Basel, Switzerland. Tacrolimus and Bisindolylmaleimide I (BIM-I) were purchased from Sigma-Aldrich . All were dissolved in dimethyl sulfoxide (DMSO).The subgenomic JFH1 NS3-5B HCV reporter genome (SG-JFH1-Luc) that encodes firefly luciferase as a sensitive marker of viral genome replication and the full length HCV reporter genome Jc1-Luc also encoding firefly luciferase and a chimeric JFH1  and J6/CF  HCV genome have been described recently 5 cells per well. Two weeks prior to infection differentiation was induced by adding 0.5% DMSO Huh-7.5 4 cells per well into 12-well plates. The co-culture was overlaid with 1% Seaplaque low melting temperature agarose to prevent virus spread through the cell-free route. After 96 hours agarose was removed, the cells were co-stained with DAPI and the percentage of infected target cells was determined.Assays for HCV genome replication, production of newly formed HCVcc and infection of naive cells were performed using HCV strains Jc1-Luc (containing a luciferase reporter) or chimeric H77/JFH1 genomes and highly HCV-susceptible Huh-7.5 human hepatoma cells as previously described Pseudoparticles were generated as previously described in 293T cells by co-transfection of (1) a lentigenome with a GFP reporter; (2) HIV gag-pol and (3) the HCV glycoproteins E1 and E2 of strain H77 or JFH-1 with Fugene  To probe for cytotoxic effects, Huh-7.5 cells were stained with a LIVE/DEAD fixable far red dead cell stain kit from Invitrogen  according to the manufactures protocol. The cells were washed twice and immediately analysed on a fluorescence-activated cell sorter . Data were analysed using FloJo software . Alternatively, the Cytotoxicity Detection Kit PLUS that measures lactate dehydrogenase (LDH) release from damaged cells was used . To measure the percentage of proliferating cells we used the BrdU Labeling kit I  according to the manufacturer'’s instructions. Cell viability and proliferation assay were done at the same time as the corresponding replication or infectivity measurements unless stated otherwise.g, 60 min, 4°C), resuspended in 1/100 of the initial volume of PBS with 12.5% FCS, and frozen in aliquots at −80°C. For infection, differentiated HepaRG cells (see cell culture section above) were inoculated with 500 µl of medium containing 4% PEG 8000, 2% DMSO and 25 µl of the concentrated virus stock. After overnight incubation the cells were washed three times with PBS and further cultivated for at least nine days. HBsAg and HBeAg in the culture supernatant were determined at day 9 by using Abbott AxSYM assays for HBeAg and HBsAg .HBV virions were generated in HepAD38 cells that contain a Tet-regulated HBV genome in vitro transcribed HCV Jc1-Luc genome and 4 hours later increasing amounts of sotrastaurin were added to the medium. Replication efficiency was assessed 4, 24, 48 and 72 hours after transfection  and observed an equal percentage of infected target cells in the presence or absence of 10 µg/ml sotrastaurin  indicatiTransplant recipients are usually treated with a combination of two or more immunosuppressive drugs. Among potential combination partners of sotrastaurin, CsA is known to have a strong inhibitory effect on HCV RNA replication. To rule out that the combination of CsA with sotrastaurin might influence the antiviral effect of CsA and in order to mimic real-life conditions we tested combinations of sotrastaurin with CsA or tacrolimus, a calcineurin inhibitor with no direct effect on HCV replication. As shown in myr, a peptide that efficiently blocks HBV entry To address the question if the novel PKC inhibitor sotrastaurin has an influence on HBV we infected differentiated HepaRGs in the presence or absence of sotrastaurin. HBeAg and HBsAg in the supernatant were measured by ELISA 9 days after inoculation (The pan-PKC inhibitor sotrastaurin (AEB071) is a novel immunosuppressive drug. Currently phase II trials evaluate the combination of sotrastaurin and other immunosuppressive drugs in the post solid organ transplant setting (liver and kidney). Patients with viral hepatitis as yet are excluded from these trials.To our knowledge this is the first experimental study testing for non-immune mediated effects of sotrastaurin on HBV or HCV replication. The most important finding of this study is that sotrastaurin does not upregulate HBV or HCV replication in any of our assay. These results support that sotrastaurin may be a viable option for immunosuppression in HBV and/or HCV infected organ transplant recipients.We found that sotrastaurin reduces HCV replication at levels of 10 µg/ml and above which is equivalent to the concentrations where anti-proliferative effects become detectable (10 µg/ml). Overt cytotoxicity was seen at only slightly higher concentration (50-100 µg/ml). Hence we believe that sotrastaurin has no specific activity against HCV, but rather affects the intracellular milieu in a way that indirectly reduces viral replication. It is even conceivable that the presence of the replicating viral genome makes cells more vulnerable to pharmacologically induced apoptosis as has been shown for HCV and actinomycin D and staurosporine In the case of HBV 5 or 10 µg/ml sotrastaurin reduced intracellular genome replication while cytotoxic effects became apparent at 25 µg/ml. Presence of the drug only during cell entry had no effect and its administration only after cell entry has been completed (16 hours) had a very small effect. Given this as well as the negligible effect on HBV in AD38 cells where replication is already well established before the drug is administered, one could speculate that it is the initiation of viral replication that is most sensitive to sotrastaurin. The HBV assays employed do not permit sensitive detection of effects on new particle production since new particle assembly in differentiated HepaRG cells is thought to be very low. The effect of sotrastaurin on HBV appears small and its mechanism is unclear. Although there was no cytotoxicity detectable at the concentrations where the reduction in HBV replication was seen cytotoxicity became apparent at only slightly higher concentrations and it is certainly possible that non-specific perturbations of the intracellular milieu at slightly sub-toxic concentrations adversely affect viral replication. However, the viral transcriptional activators HBx and PreS2-activator large surface protein have both been reported to trigger signaling processes involving PKC so that an anti-viral effect of PKC-inhibition by sotrastaurin would also be plausible Based on our data we can rule out a direct pro-viral effect of sotrastaurin on either HBV or HCV at any concentration tested. This is important information when considering the use of this immunosuppressant in HBV- and/or HCV-positive recipients of solid organ transplants. Differently from what has been reported for the PKC inhibitor BIM-I we have no evidence for any anti-HCV effect of sotrastaurin even at drug concentrations well above what is clinically achieved in transplant recipients taking sotrastaurin for immunosuppression. Based on our data a small inhibitory effect of sotrastaurin on HBV replication is possible albeit only at concentrations well above what is clinically achieved. Thus based on our findings we do not expect any clinically significant non-immune mediated pro- or anti-viral effect of sotrastaurin on either HBV or HCV.We are grateful to Takaji Wakita for the JFH1 isolate, to Jens Bukh for the J6CF and the H77/JFH1 genomes, and to Charles Rice for Huh-7.5 cells. We thank Heiner Wedemeyer for critical reading of the manuscript and Novartis  for providing cyclosporin A and sotrastaurin (AEB071).


Shiftwork is associated with adverse metabolic pathophysiology, and the rising incidence of shiftwork in modern societies is thought to contribute to the worldwide increase in obesity and metabolic syndrome. The underlying mechanisms are largely unknown, but may involve direct physiological effects of nocturnal light exposure, or indirect consequences of perturbed endogenous circadian clocks. This study employs a two-week paradigm in mice to model the early molecular and physiological effects of shiftwork. Two weeks of timed sleep restriction has moderate effects on diurnal activity patterns, feeding behavior, and clock gene regulation in the circadian pacemaker of the suprachiasmatic nucleus. In contrast, microarray analyses reveal global disruption of diurnal liver transcriptome rhythms, enriched for pathways involved in glucose and lipid metabolism and correlating with first indications of altered metabolism. Although altered food timing itself is not sufficient to provoke these effects, stabilizing peripheral clocks by timed food access can restore molecular rhythms and metabolic function under sleep restriction conditions. This study suggests that peripheral circadian desynchrony marks an early event in the metabolic disruption associated with chronic shiftwork. Thus, strengthening the peripheral circadian system by minimizing food intake during night shifts may counteract the adverse physiological consequences frequently observed in human shift workers. Shiftwork refers to a job schedule in which employees work hours other than the standard hours, e.g. in the early morning or during the night. Chronic shiftwork is correlated to increased body mass index (BMI) and risk of developing metabolic syndrome Circadian clocks provide a mechanism by which external time is interpreted and translated into time-of-day appropriate physiology, with the overall aim of promoting fitness and energy efficiency of an organism cis-elements on target gene promoters. Transcription of three period (Per1-3) and two cryptochrome genes (Cry1 and 2) is initiated by the CLOCK:BMAL1 complex. The resulting CRY and PER proteins inhibit CLOCK:BMAL1 activity, thus forming a negative feedback loop impinging on their own transcription. The clock machinery translates time information into physiologically meaningful signals by the regulation of hundreds of clock-controlled output genes (CCGs). While the nature of core clock genes is preserved among different tissues, CCGs are highly tissue-specific. It is estimated that up to 10% of the transcriptome of each cell is regulated in a circadian fashion At the cellular level circadian clocks are organized into a system of interlocking transcriptional translational feedback loops (TTLs) Bmal1−/− mice show impaired glucose tolerance, gluconeogenesis, altered triglyceride rhythms and increased body fat ClockΔ19 mice display impaired gluconeogenesis in addition to hyperphagy and obesity Per2 mutant mice are also obese The rhythmic mammalian transcriptome of various tissues is enriched for genes involved in metabolic processes, indicating a major role for the clock in metabolic regulation Given that sleep and clock dysfunction share the same metabolic endpoint, it was hypothesized that shiftwork-induced metabolic deregulation may be a direct result of circadian clock disturbance. This study demonstrates that two weeks of timed sleep restriction (TSR) leads to clock disruption and global diurnal desynchrony at the transcriptional level in the liver, while metabolic functions are still only moderately affected. Molecular and metabolic effects of TSR can be alleviated by timed food access, a strong synchronizer of peripheral clocks, emphasizing the therapeutic potential of behavioral chronotherapy to combat the metabolic consequences of shiftwork.ad libitum at all times. During TSR animals were kept awake during the first 6 hours of the light phase (Zeitgeber time (ZT) 0–6) on Days 1 through 5 and Days 8 through 12 using a ‘gentle handling’ approach designed to minimize stress effects and intervention by the experimenter 35S-UTP labeled ISH on sections. An upregulation of mRNA levels was observed in TSR animals in the early light phase (ZT1) for Bmal1, Per1 and Dbp, and mid light phase (ZT7) for Rev-erbα  conditions with food and water given Rev-erbα . However into DD .Bmal1, Per1 and Npas2 with changes in expression peak times for Bmal1 and Per1, while little effect was seen for Rev-erbα . Under control conditions, and in accordance with previous reports, we found a large number of genes with diurnal changes in expression levels . After TPrevious studies in humans and rodents show that chronic shiftwork has profound effects on metabolism. Supporting this, gene ontology (GO) analysis of our array data revealed that the genes that lost rhythmicity following TSR were enriched for transcripts involved in metabolic processes . To furt(Slc2a2), glucose-responsive forkhead box O1 (Foxo1) and insulin receptor substrate 2 (Irs2). There appeared to be a particular emphasis on regulators of the glycolysis/gluconeogenesis pathway, such as liver pyruvate kinase (Pklr), pyruvate dehydrogenase kinase 4 (Pdk4), the pyruvate transporter Slc16a7, the glycerol transporter aquaporin 9 (Aqp9), fructose-2,6-biphosphatase 1 (Pfkfb1) and pyruvate carboxylase (Pcx). Glycerol phosphate dehydrogenase 2 (Gpd2) and glycerol kinase (Gyk), regulators of glycerol biosynthesis, and glutamic-pyruvate transaminase (Gpt), a regulator of pyruvate production from alanine, were also affected. Together, these findings suggested a marked effect of TSR on glucose metabolism, particularly gluconeogenesis. Supporting this, TSR animals showed profoundly impaired gluconeogenic capacity, determined by a pyruvate tolerance test  marked effects on both the molecular and metabolic level were observed. In NF-TSR animal diurnal expression peak times for reserved . Moreovereserved  togetherreserved . Finallyreserved . In summreserved , followereserved , indicatThe current study employs a 2 week schedule of TSR in mice to examine the early molecular and physiological responses to altered activity timing preceding the metabolic consequences of chronic shiftwork. Profound disruption in the diurnal regulation of core clock genes and CCGs was observed in liver, with a strong emphasis on metabolic transcripts, while only moderate effects were seen at the level of the circadian pacemaker of the SCN. The physiological significance of these changes was demonstrated by early alterations in lipid and glucose handling, while bodyweight regulation was not yet affected. Both peripheral clock gene rhythmicity and metabolic physiology were rescued by concurrent dark phase restricted food access, suggesting meal timing as a possible therapeutic intervention for the treatment of shiftwork-associated metabolic disorders.Bmal1, Per1, Rev-erbα and Dbp were observed, while overall rhythmicity was preserved. At the behavioral level, however, decreased activity was observed in the dark phase, accompanied by a shift of food intake to the light phase. These activity phase shifts appear to be acute effects of the TSR procedure, perhaps in response to increased light input, and alterations in activity rhythms ceased when animals were released into DD conditions after the end of TSR , high Pcx , high Gpd2 and Gyk (glycerol de novo biosynthesis), and low PDK4 mRNA levels (inhibition of glycolysis). TSR resulted in most of these genes being reduced, in some cases remaining at basal expression levels throughout the day, indicating profound changes to diurnal carbohydrate utilization  ClockΔ19 and Bmal1−/− mice, suggesting a direct effect of the circadian clock on this process L-Foxo1−/− mice similarly display reduced hepatic glucose production Genes involved in carbohydrate metabolism were deregulated in the liver. Under normal conditions hepatic glucose uptake and glycolysis are enhanced during the active phase, corresponding with high lization . DownregCirculating glycerol, a product of lipolysis and a substrate for gluconeogenesis, was high directly after the sleep restriction period (ZT7) following TSR , suggestob/ob mice, showing that circadian disruption precedes the development of obesity in these animals Collectively, the observed molecular disruption seen in the liver can be directly correlated to changes in physiology. However, given the short duration of the model used, the full-blown metabolic phenotype characterized by obesity, insulin and leptin resistance seen in more chronic models of sleep restriction and in human shiftwork were not observed. Similarly for rodents, in which 4 weeks of night work results in hyperphagy and obesity, after only 2 weeks the effects on food intake are still relatively mild In order to strengthen the link between the circadian clock and shiftwork-induced metabolic effects, a regime of normocaloric dark phase restricted feeding was employed concurrent to TSR to stabilize the peripheral clock machinery. Dark phase restricted feeding has been shown previously to reset clock gene rhythms in subsidiary oscillators, but not the SCN master clock This study examines the early effects of extended timed sleep restriction as an experimental model of shiftwork on behavior, physiology and transcriptional regulation, with an emphasis on the circadian aspects of metabolism. While it is clear that causal interpretations from studies on complex interacting processes such as diurnal activities, feeding, sleep, metabolism, and circadian mechanisms are always limited, our liver transcriptome analyses indicate a direct effect of sleep timing on the molecular circadian machinery and on clock controlled genes involved in carbohydrate and lipid utilization. Importantly, disruption to circadian gene rhythmicity appears early compared to the adverse metabolic disturbances seen with chronic shiftwork in both rodents and humans, suggesting a causal role of clock disruption in the progression of shiftwork-induced metabolic syndrome. Resetting peripheral circadian clocks by nighttime feeding rescues physiological parameters altered during timed sleep restriction, implying that peripheral clocks may be attractive therapeutic targets for combating the metabolic effects of shiftwork.ad libitum access to standard chow  and water. All animal experiments were approved by the Office for Consumer Protection and Food Safety (LAVES) of the State of Lower Saxony and executed in accordance with the German Law on Animal Welfare.Male C67Bl6/J mice, 10–14 weeks old, were entrained to a 12 h (50 lux) light: 12 h dark (LD) schedule with lights on (ZT0) at 7:00am. Activity measurements were recorded using custom-made infra-red detectors fitted to the roof of the cages and analyzed using ClockLab software . Mice were housed in groups of 3–6 animals per cage, and kept under constant temperature (20.0+/−0.5°C) and humidity (50–60%) conditions with ad libitum. Control mice were kept under the same conditions in separate compartments, without intervention. Starting in the evening of Day 11, control and TSR mice were sacrificed by cervical dislocation at 6 hour intervals. In the dark phase, a 5 W safety red light was used to avoid light effects. Plasma, liver, epididymal adipose (WAT) and brains were collected and frozen. In the case of TSR with high light intensity, 500 lux light was used in the same LD schedule. In the case of TSR with dark phase restricted feeding (DF-TSR), food was removed from the cages at ZT0 (7:00am) on each day and replaced at ZT12 (7:00pm) on each day. In the case of altered food rhythmicity in control mice, mice were provided with 1.6 g of food at the start of the light/inactive phase and 2.2 g of food at the start of the dark/active phase. Each time food was given, any remaining uneaten food was removed to prevent hoarding.During TSR mice were kept awake using a gentle handling method between ZT0 and ZT6 from Days 1 to 5, and Days 8 to 12 Pyruvate tolerance tests were performed in the week prior to TSR, and on Day 12 of TSR. Mice were starved for 13 h overnight, and injected with 2 g/kg pyruvate i.p. at ZT1. Glucose measurements were taken from tail vein blood at indicated time points.Leptin was determined using the Mouse Leptin ELISA Kit , and insulin was determined using the Ultrasensitive Mouse Insulin ELISA . Circulating glycerol and triglycerides were determined using the Serum Triglyceride Determination Kit , and glycogen was determined using anthrone reagent as described previously In situ hybridization using digoxigenin or 35S-labeled probes on frozen sections was performed as described Eef1a was used as a reference gene, and data was expressed as relative quantitation (ΔΔ-CT method).Relative quantification of mRNA levels by qPCR was done as previously described PER2::LUC (n = 6) Control and TSR heterozygous Microarray experiments using liver and adipose tissue total RNA preparations were performed at the Transcriptome Analysis Lab (TAL) of the University of Göttingen, Germany. cRNA samples were hybridized to MoGene ST1.0 gene expression arrays . Three chips per time point, condition and tissue were used. Gene expression was normalized using d-Chip Software Unless otherwise noted in the figure legend, all data are shown as mean +/− SEM. Statistical comparisons were performed using GraphPad Prism software  for single gene expression profiles and physiological parameters or using the Bioconductor/R software package Figure S1Body weight of mice during and following TSR (n  = 10).(TIF)Click here for additional data file.Figure S2Comparison of qPCR (data replotted from ) and array fluorescence measures  during control and TSR conditions in the liver. Arrays are represented by the solid lines, qPCR data by the broken lines. * indicates p<0.05 determined by 2-way ANOVA (n  = 3 per time point and condition).(TIF)Click here for additional data file.Figure S3Peak time phase relationships of metabolic genes that were rhythmic under both control and TSR conditions (Classes I and II) in the liver. Peak times were assessed by sine wave fitting. Circles indicate number of genes per 20° wedge. The black arrow represents the normalization vector for all shifts/genes.(TIF)Click here for additional data file.Table S1Full gene lists from , including Probe ID, Gene ID, and peak time in both control and TSR mice.(PDF)Click here for additional data file.Table S2Category 1 enriched GO processes (DAVID) of Class III TSR-regulated genes  in the liver.(PDF)Click here for additional data file.


Genome-wide association mapping is highly sensitive to environmental changes, but network analysis allows rapid causal gene identification. Arabidopsis spp. can serve as a model system to understand the genomic architecture of adaptive quantitative traits. GSL are key anti-herbivory defenses that impart adaptive advantages within field trials. While little is known about how variation in the external or internal environment of an organism may influence the efficiency of GWA, GSL variation is known to be highly dependent upon the external stresses and developmental processes of the plant lending it to be an excellent model for studying conditional GWA.Genome-wide association (GWA) is gaining popularity as a means to study the architecture of complex quantitative traits, partially due to the improvement of high-throughput low-cost genotyping and phenotyping technologies. Glucosinolate (GSL) secondary metabolites within Arabidopsis thaliana accessions, >40 GSL phenotypes across three conditions  and ∼230,000 SNPs. Developmental stage had dramatic effects on the outcome of GWA, with each stage identifying different loci associated with GSL traits. Further, while the molecular bases of numerous quantitative trait loci (QTL) controlling GSL traits have been identified, there is currently no estimate of how many additional genes may control natural variation in these traits. We developed a novel co-expression network approach to prioritize the thousands of GWA candidates and successfully validated a large number of these genes as influencing GSL accumulation within A. thaliana using single gene isogenic lines.To understand how development and environment can influence GWA, we conducted a study using 96 Together, these results suggest that complex traits imparting environmentally contingent adaptive advantages are likely influenced by up to thousands of loci that are sensitive to fluctuations in the environment or developmental state of the organism. Additionally, while GWA is highly conditional upon genetics, the use of additional genomic information can rapidly identify causal loci en masse. Arabidopsis thaliana to begin exploring these questions. We show that both external and internal environments significantly alter the identified genes, such that using different tissues can lead to the identification of nearly completely different gene sets. Given the large number of potential false-positives, we developed an orthogonal approach to filtering the possible genes, by identifying co-functioning networks using the nominal candidate gene list derived from genome-wide association studies. This allowed us to rapidly identify and validate a large number of novel and unexpected genes that affect Arabidopsis thaliana defense metabolism within phenotypic ranges that have been shown to be selectable within the field. These genes and the associated networks suggest that Arabidopsis thaliana defense metabolism is more readily similar to the infinite gene hypothesis, according to which there is a vast number of causative genes controlling natural variation in this phenotype. It remains to be seen how frequently this is true for other organisms and other phenotypes.Understanding how genetic variation can control phenotypic variation is a fundamental goal of modern biology. A major push has been made using genome-wide association mapping in all organisms to attempt and rapidly identify the genes contributing to phenotypes such as disease and nutritional disorders. But a number of fundamental questions have not been answered about the use of genome-wide association: for example, how does the internal or external environment influence the genes found? Furthermore, the simple question of how many genes may influence a trait is unknown. Finally, a number of studies have identified significant false-positive and -negative issues within genome-wide association studies that are not solvable by direct statistical approaches. We have used genome-wide association mapping in the plant  Biologists across fields possess a common need to identify the genetic variation causing natural phenotypic variation. Genome-wide association (GWA) studies are a promising route to associate phenotypes with genotypes, at a genome-wide level, using “unrelated” individuals Repeated detection of a genotype-phenotype association across populations or experiments has been proposed to increase support for the biological reality of that association, and has even been proposed as a requirement for validation of trait-phenotype associations Integrating GWA mapping results with additional forms of genome-scale data, such as transcript profiling or proteomics datasets, has also been proposed to strengthen support for detected gene-trait associations and reduce the incidence of false-positive associations Arabidopsis thaliana. A. thaliana has become a key model for advancing genetic technologies and analytical approaches for studying complex quantitative genetics in wild species A. thaliana has long provided a model system for applying concepts surrounding GWA mapping To evaluate the influence of environmental or developmentally conditional genetics on GWA mapping and the utility of network filtering in identifying candidate causal genes, we focused on defense metabolism within the plant A. thaliana secondary metabolite pathways, responsible for aliphatic and indolic glucosinolate (GSL) biosynthesis. These pathways have become useful models for quantitative genetics and ecology  to mimic pathogen attack. In addition, we measured seedling glucosinolates from the same accessions to provide a tissue comparison as well as a treatment comparison. Seedlings were measured at 2 d post-germination at a stage where the GSL are largely representative of the GSL present within the mature seed 3-treated samples, we detected only 16 aliphatic GSL and four indolic GSL, but also were able to measure camalexin, which is related to indolic GSL  accounted for 3 times more of the variation in GSL than environmental effects (average 5%). In particular, only three GSL  were affected more strongly by AgNOccession , whereasccession . This agUsing 229,940 SNP available for this collection of 96 accessions, we conducted GWA-mapping for GLS traits in both the Seedling and Silver datasets using a maximum likelihood approach that accounts for genetic similarity (EMMA) p = 0.001). This is not explainable by differential heritability as the short-chain aliphatic GSLs have the highest heritability in numerous studies including this one  wherein a large set of non-causal genes are physically linked with the causal AOP2/3 and MAM1/3 genes AOP and MAM loci, the hotspot at the MAM locus was much more pronounced than the AOP locus in the seedling GWA dataset   . This suAOP2/3 and MAM1/2/3 genes GWA studies generally produce large lists of candidate genes, presumed to contain a significant fraction of false-positive associations. One proposed strategy refines these results by searching for enrichment of candidate genes within pre-defined proteomic or transcriptomic networks A. thaliana secondary metabolism  and novel candidate  genes that were associated with the core-biosynthetic part of the network. RML1 , a biosynthetic enzyme for glutathione, has previously been shown to control GSL accumulation either via a signaling role or actual biosynthesis of glutathione ATSFGH , At1g06640 (unknown 2-oxoacid dependent dioxygenase – 2-ODD), or At1g04770 (tetratricopeptide containing protein) may play a role in GSL accumulation, we obtained insertional mutants. This showed that the disruption of At1g06640 led to significantly increased accumulation of the short-chain methylsulfinyl GSL but not the corresponding methylthio or long-chain GSL , a gene co-expressed with the GSL core but not a candidate from the GWA analysis, had no detectable impact upon GSL accumulation , Erecta (ER) ELF3/GI  network and specifically the ER locus had previously been queried for the ability to alter GSL accumulation using two Arabidopsis RIL populations (Ler×Col-0 and Ler×Cvi) that segregate for a loss-of-function allele at the ER locus ER locus was linked to seed/seedling GSL accumulation in only one of the two populations and not linked to mature leaf GSL accumulation ER mutant within the Col-0 genotype showed that the Erecta gene does influence GSL content within leaves as suggested by the GWA results  was associated with increased levels of short-chain aliphatic GSL and a corresponding decrease in long-chain aliphatic GSL , is likely involved in chlorophyll catabolism and possibly also chloroplast senescence CLPX with several catabolism genes. Analysis of mutants deficient in function for two of these genes showed that they all possessed increased aliphatic GSL in comparison to wild-type controls. These results suggest that natural variation in this putative network could influence GSL content in A. thaliana. The majority (12 of 13) of genes in this network show significant variation in transcript abundance across A. thaliana accessions, a significantly greater proportion than expected by chance (X2p<0.001) The final network examined here, represented by AtPTR3  and DPL1 . T-DNA mutants in both genes appeared to be lethal as we could not identify homozygous progeny. However, comparison of the heterozygous progeny to wildtype homozygotes showed that mutants in both genes led to elevated levels of aliphatic GSL , suggesting that combining GWA-identified candidate genes with co-expression networks successfully identifies genes with the capacity to cause natural variation in GSL content. Identifying the specific mechanisms involved will require significant future research.While GSL are considered “secondary” metabolites, these compounds are affected by many aspects of plant metabolism, thus GSL phenotyping is sensitive to any genetic perturbation that affects plant physiology. As such, we identified six genes that were expressed in mature leaves but did not show any significant association of DNA sequence polymorphism with GSL phenotypes and were additionally not identified within any of the above co-expression networks. Insertional mutants disrupted at these loci were designated as random mutant controls . AnalyziThe influence of conditional genetics, i.e. interaction of genotypes with environment or development, has been intensively studied within structured mapping populations and shown to exert considerable influence on the accumulation of small metabolites 3 treatment in influencing genetic variation across this collection of accessions, as displayed by the distribution of phenotypes and their variance across the datasets  across a wide range of developmental and environmental states A. thaliana genes based upon non-genetic variation. This provides an orthogonal grouping to that provided by the GWA mapping which associates genes to phenotypes via natural genetic variation. This approach is similar to other filtering approaches that utilize complementary datasets to rank candidate genes A limiting factor for the utility of GWA studies has been the preponderance of false-positive and false-negative associations which makes the accurate prediction of biologically valid genotype-phenotype associations very difficult. In this report, we describe the implementation and validation of a candidate gene co-expression filter that has given us a high success rate in candidate gene validation (>75%). The co-expression dataset is derived from transcript accumulation within a single gi mutants in Ler v Col-0, A. thaliana, echoing recent findings regarding the genetics of human height, and maize flowering time The use of multiple tissues and treatment conditions, as well as a large set of different but related GSL phenotypes, led to the identification of several thousand candidate genes. Even after decreasing this number by using the network expression filter approach, several hundred candidate genes of interest remained. Analysis of a set of these genes via plants bearing single gene mutations showed that disruption of many of these genes can alter the amount or pattern of GSL accumulation  and 7. GA. thaliana in natural settings. Thus, it may be more useful to consider, instead of indirect versus direct effects of a locus, a continuous distribution that describes the number of molecular steps required to link a particular gene to the most proximal controller of the phenotype—in this case, an enzyme in the biosynthetic pathway. This raises the distinct problem of adaptive constraint wherein natural variation at a locus is limited by its indirect consequences upon other phenotypes. For instance, a phototropin allele with a beneficial effect on seedling phototropic behavior may be limited in its selective advantage due to a deleterious effect on GSL accumulation The potential existence of thousands of polymorphic genes influencing a phenotype raises a common concern that these effects actually represent indirect pleiotropy, where moderate to small effects of a locus upon a phenotype are not biologically significant and do not reflect direct molecular control of the trait. However, numerous studies on GSL variation within wild populations have shown that changes in GSL accumulation similar to those identified here have selective consequences in field studies A more mundane but significant experimental challenge of generating a list of thousands of candidate genes potentially causing natural variation in a phenotype is validation. Even after our expression network filtering, we were left with hundreds of likely candidates that would take decades to rigorously validate. Given that it is likely that at least several hundred genes lead to natural variation in GSL accumulation We identified significant differences in GSL accumulation between two different developmental stages and this led to the identification of GWA candidate genes. While previous work on structured mapping populations, such as RILs, has shown that each tissue may be viewed as a distinct genetic module for both development and biochemistry In this report, we show that GWA-mapping, like QTL-mapping using structured populations, is sensitive to interaction of genetic variation with the environment and the developmental stage of phenotype measurement. This has not often been considered as a critical factor influencing GWA studies, given the difficulty of obtaining replicated analyses within organisms such as humans. Future work incorporating systematic analysis of how GWA studies are influenced by developmental or environmental gradients will be critical to understanding how the genomic architecture of a species controls its phenotypes. We have developed and validated a new approach to identifying GWA candidate genes and shown that the use of orthogonal genomic network datasets can lead to a very high success rate in the biological validation of candidate genes. This new approach, in combination with the observation of conditional GWA results, suggests that large numbers of genes can have a causal connection to variation within GSL and other phenotypes.A. thaliana accessions was used to measure GSL accumulation for GWA mapping with existing SNP data from these same lines 3 soil volume per cell). Four plantings of the 96 accessions provided four independent replicates for each accession. At 1 wk of age, seedlings were thinned to leave one plant per cell and glucosinolates were extracted from 10 of the removed seedlings. For all experiments, plants were maintained under short day conditions in controlled environment growth chambers. At 35 d post-germination, two fully expanded mature leaves were harvested, digitally photographed, and one was directly analyzed for GSL content as described below 3 for 48 h prior to harvest for GSL analysis. AgNO3 induces plant responses to pathogens by interfering with ethylene hormone-signaling and inducing reactive oxygen species. We utilized AgNO3 as a treatment to estimate the effect of variation in plant defense response upon GWA mapping 3 treatment of leaves (Silver Dataset). The data for the control dataset is reported elsewhere as the “2008 dataset” A previously described collection of 96 natural 2 of leaf area for the mature leave data or per seedling for the seedling data. There was no significant variation detected for leaf density within these accessions (unpublished data). In addition to the content of individual GSL, we developed a series of summation and ratio traits based on prior knowledge of the GSL pathways GSL content of excised leaves and seedlings was measured using a previously described high-throughput analytical system sar = μ+Ss+A(S)sa+Tt+R(T)tr+Tt:Ss+Tt:A(S)sa+εsart where s = 1,…,8; r = 1,…4; t = 1,2; and a = 1,…,95. The main effects are denoted as S, A, T, and R and represent structure, accession, treatment (or tissue), and replicate block, respectively. Here, the variable T may refer to (1) treatment corresponding to the two factors with or without AgNO3 treatment or (2) tissue corresponding to the two factors' mature leaves or seedlings. Population structure is represented as s = 1,…,8, corresponding to eight distinct groups into which these 96 accessions have previously been assigned sartε, is assumed to be normally distributed with mean 0 and variance σε2. Broad-sense heritability was estimated as the percent of total variance attributable to accession nested within structure and that for structure was estimated as the percent of total variance attributable to structure. The data were analyzed independently for the two treatments or conditions: control versus AgNO3 and control versus seedling  algorithm cessions  and S4. p value distributions of the GWA analysis were not uniform. Accepting an inherently elevated false-positive rate, we identified SNP within the bottom 0.1 percentile of each p value distribution, corresponding to each trait, as significant for EMMA. Given previous observations that multiple SNPs per gene are typically associated with a trait for true-positives We utilized a previously reported criterion for calling significant gene-trait associations in these three datasets We estimated the variance explained by the candidate GWA mapping genes identified in this study using the GenABEL package in R AOP and MAM loci were then excluded to reduce detection of false associations due to linkage with the causal AOP2/3 and MAM1/2/3 genes Co-expression data were obtained from ATTED II To test if GWA-identified candidate genes showed tighter linkage to known GSL networks than expected by chance, the shortest paths between each candidate or randomly selected control gene and all verified GSL genes within the full co-expression network were compared using the R/igraph package We conducted two statistical tests to compare the null distribution (distances from non-significant genes to known GSL genes) with the GWA mapping candidate distribution (distances from GWA candidate genes to known GSL genes). The Wilcoxon Rank Sum Test tests the probability of a location shift between the distribution of the shortest paths of all GWA mapping candidate genes (from one of the three datasets) to all known GSL genes and the distribution of the shortest paths of all non-significantly associated genes to the all known GSL genes. The Ansari-Bradley Test examines the probability that the two aforementioned distributions are differently dispersed. Both statistic tests were conducted using the full GSL network list as well as each individual biosynthetic pathway  and S8.http://signal.salk.edu/tdnaprimers.2.html). Of the 18 T-DNA mutants surveyed, homozygous mutants could not be obtained for 11 mutants, likely from lethality. In these cases, heterozygote lines were allowed to self-pollinate, and homozygous seed stocks were obtained by single seed decent following PCR-based genotyping of the progeny. In the absence of a homozygous line, we tested GSL content within the adult rosette leaves within PCR-confirmed heterozygous individuals. We also obtained mutants deficient in function at the following loci: phototropin1/phototropin2 (phot1/phot2) (4 lines), Gigantea (gi) , Erecta (er) in Col-0, and early flowering 3-1 (elf3-1) t-test was conducted to test the statistical significance of differences in GSL content between the mutant and wild-type while correcting for multiple comparisons using the R/multcomp package . Criteria for selection of candidate genes from these networks for testing were connectedness (the gene had to show correlated expression levels (MR rank of <16) with multiple candidate genes within the network) and availability of viable mutants. These mutants were either a pre-existing characterized mutant line or a homozygous T-DNA mutation within an early exon of the candidate gene available from the Arabidopsis Biological Resource Center (ABRC)  package  [141]. G package . Only wiELF3 locus did alter GSL accumulation elf3:Bay-0 and elf3:Sha transgenic T1 seeds were planted on soil including elf3.1 mutants and wild-type Col-0 as a control elf3.1 mutants were assessed to distinguish transformed from untransformed plants elf3:Bay-0 and 44 elf3:Sha independent T1 plants. GSL differences between the two ELF3 alleles were tested as described above.We utilized previously generated quantitative complementation lines to validate that natural variation in the Dataset S1GWA network candidate results. This dataset contains the GWA network candidate output results in a .net file ready for import into Pajek.(TXT)Click here for additional data file.Figure S13, and seedling datasets. Distributions of total aliphatic (left) and total indolic (right) glucosinolates are shown as examples to illustrate the differences between the three datasets. Seedling glucosinolates are presented in amount per seedling to control for differences in cellular expansion.Trait distributions from leaf-control, leaf-AgNO(TIF)Click here for additional data file.Figure S2VENN Diagram of positive calls and trait groups. VENN diagrams showing the numbers of GWAS significantly associated genes for each dataset, Silver, Control, and Seedling, are shown. The GSL were separated into four trait groups based on previous biochemical analysis; INDOLE, indolic GSL; OHBUT, 2-hydroxy-but-3-enyl GSL traits; LC, 7 and 8 C long methionine derived GSL; SC, 3 and 4 C long methionine derived GSL. Two Seedling specific trait groups were also included for seedling specific GSL; BZO, benzoyloxy GSL and Benzyl are phenylalanine derived GSL. The bottom right VENN diagram displays overlap between the four common trait groups and the seedling specific groups.(TIF)Click here for additional data file.Figure S3Core GSL co-expression network. The known or predicted GSL genes generate a core GSL co-expression network that is expanded in this presentation for legibility. The general biochemical functions of the four major clusters within this super network are labeled. Three of the major clusters are further magnified to provide gene identification.(PDF)Click here for additional data file.Figure S4p value comparing the probability of a location shift between the distribution of the shortest paths of all GWA candidate genes (from one of the three datasets) to the corresponding glucosinolate gene and the distribution of the shortest paths of all non-significantly associated genes to the corresponding glucosinolate gene. Pa is the Ansari-Bradley Test probability assessing the difference in dispersion between the two aforementioned distributions. This was done for all GSL genes as well as for each of the specific biosynthetic networks as defined.Distributions of shortest distances between known GSL genes and GWA candidates. Shown are plots comparing the distributions of the shortest distances between known GSL genes and GWA candidates for the control (red), silver (green), and seedling (blue) datasets. For comparison similar distributions derived from non-GWA-candidates  are also shown (black lines). Pw is the Wilcoxon Rank Sum test (PDF)Click here for additional data file.Table S13 study. For each glucosinolate trait the following model was examined: ysar∼μ+Ss+A(S)sa+Tt+R(T)tr+Tt:Ss+Tt:A(S)sa, where μ is the intercept, s is the K ∈ {1,…,8) value for the corresponding accession sa is the effect of accession nested in structure, Tt is the effect of AgNO3-treatment, and R(T)tr is the biological/technical replicate of the measure. The model was evaluated by combining the control (untreated mature leaves) and AgNO3-treated datasets. F, F-statistic of the model; P(F), nominal p value of the F-statistic; DF(num), numerator degrees of freedom; DF(denom), denominator d.f.; R2, fraction of total variance explained by the model; η2(x), partial R2 of the corresponding predictor variable; and P(x), p value of the corresponding predictor variable.Estimates of variance components for GSL in AgNO(XLS)Click here for additional data file.Table S2sar∼μ+Ss+A(S)sa+Tt+R(T)tr+Tt:Ss+Tt:A(S)sa, where μ is the intercept, s is the K ∈ {1,…,8) value for the corresponding accession sa is the effect of accession nested in structure, Tt is the effect of tissue type (mature leaves versus seedlings), and R(T)tr is the biological/technical replicate of the measure. The model was evaluated by combining the control (mature leaves) and seedling datasets. F, F-statistic of the model; P(F), nominal p value of the F-statistic; DF(num), numerator degrees of freedom; DF(denom), denominator d.f.; R2, fraction of total variance explained by the model; η2(x), partial R2 of the corresponding predictor variable; and P(x), p value of the corresponding predictor variable.Estimates of variance components for GSL in seedling study. For each glucosinolate trait the following model was examined: y(XLS)Click here for additional data file.Table S3sar = μ+Aa+rR+sarε. Treated and untreated camalexin values are presented and are considered related to the indole GSL metabolites.Genetic means of glucosinolate abundance per accession for silver treated accessions. All metabolite values are in nmol per mg fresh weight tissue. Shown are the predicted means from four independent plants treated with silver nitrate per accession as per the statistical model: y(XLS)Click here for additional data file.Table S4sar = μ+Aa+rR+sarε.Genetic means of glucosinolate abundance per accession for seedlings. All metabolite values are in nmol per seedling. Shown are the predicted means from four independent samples per accession as per the statistical model: y(XLS)Click here for additional data file.Table S5p value in the bottom 0.1 percentile of the p value distribution. T, is significant; F, not significant and genes with no significances are not listed.Gene-to-trait associations as identified using silver treated samples. Logical table indicating whether each of 31,505 genes is significantly associated to each of the 46 traits within the seedling samples. AGI is the gene code, Chr is the chromosome, and Start and End are the position of the gene in basepairs. For each trait, a gene is significantly associated if at least two SNP within ±1 kb flanking the coding region has a (XLS)Click here for additional data file.Table S6p value in the bottom 0.1 percentile of the p value distribution. T, is significant; F, not significant and genes with no significances are not listed.Gene-to-trait associations as identified using seedling material. Logical table indicating whether each of 31,505 genes is significantly associated to each of the 46 traits within the seedling samples. AGI is the gene code, Chr is the chromosome, and Start and End are the position of the gene in basepairs. For each trait, a gene is significantly associated if at least two SNP within ±1 kb flanking the coding region has a (XLS)Click here for additional data file.Table S7Known and putative genes involved in the GSL pathway. List of genes either known or predicted to play a role in GSL metabolism and regulation. AGI, the AGI (Arabidopsis Genome Initiative) code for each gene; Pathway, specific part of the GSL metabolic system the gene is thought function; Pseudogene, whether or not the gene is predicted to be a pseudogene; Evidence, experimental evidence  or sequence evidence base on homology to validated GSL gene (Homology).(XLS)Click here for additional data file.Table S8GSL abbreviations.(XLS)Click here for additional data file.Table S9p value comparing the two genotypes is on the line labeled p value and n shows the number of independent plants measured per line.Mutant analysis for altered GSL accumulation. Chemical and statistical analysis for the various single gene mutants and genotypes queried within the manuscript. The wildtype to mutant comparison being conducted is shown in bold at the start of each subtable. The average value for mutant and control are shown in the top table for each mutant while the standard error is shown in the second table. The (XLS)Click here for additional data file.Table S10Estimated phenotypic variance determined by significant GWAS candidates. Abbreviations per glucosinolate are as described in (XLS)Click here for additional data file.