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1.  The Circadian Clock Modulates Enamel Development 
Journal of biological rhythms  2012;27(3):237-245.
Fully mature enamel is about 98% mineral by weight. While mineral crystals appear very early during its formative phase, the newly secreted enamel is a soft gel-like matrix containing several enamel matrix proteins of which the most abundant is amelogenin (Amelx). Histological analysis of mineralized dental enamel reveals markings called cross-striations associated with daily increments of enamel formation, as evidenced by injections of labeling dyes at known time intervals. The daily incremental growth of enamel has led to the hypothesis that the circadian clock might be involved in the regulation of enamel development. To identify daily rhythms of clock genes and Amelx, we subjected murine ameloblast cells to serum synchronization to analyze the expression of the circadian transcription factors Per2 and Bmal1 by real-time PCR. Results indicate that these key genetic regulators of the circadian clock are expressed in synchronized murine ameloblast cell cultures and that their expression profile follows a circadian pattern with acrophase and bathyphase for both gene transcripts in antiphase. Immunohistological analysis confirms the protein expression of Bmal and Cry in enamel cells. Amelx expression in 2-day postnatal mouse molars dissected every 4 hours for a duration of 48 hours oscillated with an approximately 24-hour period, with a significant approximately 2-fold decrease in expression during the dark period compared to the light period. The expression of genes involved in bicarbonate production (Car2) and transport (Slc4a4), as well as in enamel matrix endocytosis (Lamp1), was greater during the dark period, indicating that ameloblasts express these proteins when Amelx expression is at the nadir. The human and mouse Amelx genes each contain a single nonconserved E-box element within 10 kb upstream of their respective transcription start sites. We also found that within 2 kb of the transcription start site of the human NFYA gene, which encodes a positive regulator of amelogenin, there is an E-box element that is conserved in rodents and other mammals. Moreover, we found that Nfya expression in serum-synchronized murine ameloblasts oscillated with a strong 24-hour rhythm. Taken together, our data support the hypothesis that the circadian clock temporally regulates enamel development.
doi:10.1177/0748730412442830
PMCID: PMC3511783  PMID: 22653892
circadian rhythms; enamel development; ameloblast cells; amelogenin
2.  Circadian Rhythms Regulate Amelogenesis 
Bone  2013;55(1):158-165.
Ameloblasts, the cells responsible for making enamel, modify their morphological features in response to specialized functions necessary for synchronized ameloblast differentiation and enamel formation. Secretory and maturation ameloblasts are characterized by the expression of stage-specific genes which follows strictly controlled repetitive patterns. Circadian rhythms are recognized as key regulators of development and diseases of many tissues including bone. Our aim was to gain novel insights on the role of clock genes in enamel formation and to explore the potential links between circadian rhythms and amelogenesis. Our data shows definitive evidence that the main clock genes (Bmal1, Clock, Per1 and Per2) oscillate in ameloblasts at regular circadian (24h) intervals both at RNA and protein levels. This study also reveals that two markers of ameloblast differentiation i.e. amelogenin (Amelx; a marker of secretory ameloblasts) and kallikrein-related peptidase 4 (Klk4, a marker of maturation ameloblasts) are downstream targets of clock genes. Both, Amelx and Klk4 show 24h oscillatory expression patterns and their expression levels are up-regulated after Bmal1 over-expression in HAT-7 ameloblast cells. Taken together, these data suggest that both the secretory and the maturation stage of amelogenesis might be under circadian control. Changes in clock genes expression patterns might result in significant alterations of enamel apposition and mineralization.
doi:10.1016/j.bone.2013.02.011
PMCID: PMC3650122  PMID: 23486183
Clock genes; enamel; amelogenin; kallikrein-related peptidase 4; odontoblasts
3.  Ameloblast Differentiation in the Human Developing Tooth: Effects of Extracellular Matrices 
Matrix Biology  2010;29(5):411-419.
Tooth enamel is formed by epithelially-derived cells called ameloblasts, while the pulp dentin complex is formed by the dental mesenchyme. These tissues differentiate with reciprocal signaling interactions to form a mature tooth. In this study we have characterized ameloblast differentiation in human developing incisors, and have further investigated the role of extracellular matrix proteins on ameloblast differentiation. Histological and immunohistochemical analyses showed that in the human tooth, the basement membrane separating the early developing dental epithelium and mesenchyme was lost shortly before dentin deposition was initiated, prior to enamel matrix secretion. Presecretary ameloblasts elongated as they came into contact with the dentin matrix, and then shortened to become secretory ameloblasts. In situ hybridization showed that at the presecretory stage of odontoblasts expressed type I collagen mRNA, and also briefly expressed amelogenin mRNA. This was followed by upregulation of amelogenin mRNA expression in secretory ameloblasts. In vitro, amelogenin expression was up-regulated in ameloblast lineage cells cultured in Matrigel, and was further up-regulated when these cells/Matrigel were co-cultured with dental pulp cells. Co-culture also up-regulated type I collagen expression by the dental pulp cells. Type I collagen coated culture dishes promoted a more elongated ameloblast lineage cell morphology and enhanced cell adhesion via integrin α2β1. Taken together, these results suggest that the basement membrane proteins and signals from underlying mesenchymal cells coordinate to initiate differentiation of preameloblasts and regulate type I collagen expression by odontoblasts. Type I collagen in the dentin matrix then anchors the presecretary ameloblasts as they further differentiate to secretory cells. These studies show the critical roles of the extracellular matrix proteins in ameloblast differentiation.
doi:10.1016/j.matbio.2010.03.001
PMCID: PMC3296366  PMID: 20211728
basement membrane proteins; type I collagen; integrin; dental epithelial cells; dental pulp cells; ameloblast lineage cells
4.  Genome-Wide and Phase-Specific DNA-Binding Rhythms of BMAL1 Control Circadian Output Functions in Mouse Liver 
PLoS Biology  2011;9(2):e1000595.
Temporal mapping during a circadian day of binding sites for the BMAL1 transcription factor in mouse liver reveals genome-wide daily rhythms in DNA binding and uncovers output functions that are controlled by the circadian oscillator.
The mammalian circadian clock uses interlocked negative feedback loops in which the heterodimeric basic helix-loop-helix transcription factor BMAL1/CLOCK is a master regulator. While there is prominent control of liver functions by the circadian clock, the detailed links between circadian regulators and downstream targets are poorly known. Using chromatin immunoprecipitation combined with deep sequencing we obtained a time-resolved and genome-wide map of BMAL1 binding in mouse liver, which allowed us to identify over 2,000 binding sites, with peak binding narrowly centered around Zeitgeber time 6. Annotation of BMAL1 targets confirms carbohydrate and lipid metabolism as the major output of the circadian clock in mouse liver. Moreover, transcription regulators are largely overrepresented, several of which also exhibit circadian activity. Genes of the core circadian oscillator stand out as strongly bound, often at promoter and distal sites. Genomic sequence analysis of the sites identified E-boxes and tandem E1-E2 consensus elements. Electromobility shift assays showed that E1-E2 sites are bound by a dimer of BMAL1/CLOCK heterodimers with a spacing-dependent cooperative interaction, a finding that was further validated in transactivation assays. BMAL1 target genes showed cyclic mRNA expression profiles with a phase distribution centered at Zeitgeber time 10. Importantly, sites with E1-E2 elements showed tighter phases both in binding and mRNA accumulation. Finally, analyzing the temporal profiles of BMAL1 binding, precursor mRNA and mature mRNA levels showed how transcriptional and post-transcriptional regulation contribute differentially to circadian expression phase. Together, our analysis of a dynamic protein-DNA interactome uncovered how genes of the core circadian oscillator crosstalk and drive phase-specific circadian output programs in a complex tissue.
Author Summary
The circadian clock is a timing system that allows organisms to keep behavioral, physiological, and cellular rhythms in resonance with daily environmental cycles. In mammals, such clocks use transcriptional regulatory loops in which the heterodimeric transcription factor BMAL1/CLOCK plays a central role. While defects in circadian clock function have been associated with diabetes, obesity, and cancer, the molecular links between the circadian clock and such output pathways are poorly characterized. Here, we mapped DNA-binding sites of BMAL1 in mouse liver during one circadian cycle. Our temporal analysis revealed widespread daily rhythms in DNA binding, with maximum levels peaking at midday. In the list of candidates, core circadian genes stood out as the most strongly bound, often showing multiple binding sites. Interestingly, BMAL1 targets were highly enriched in genes involved in carbohydrate and lipid metabolism, and also in transcription factors, in particular nuclear receptors. Our results suggest that the mammalian clock uses BMAL1 to control transcriptional output programs both directly and indirectly. Additionally, the DNA specificity of BMAL1 binding revealed the importance of tandem E-box elements, which may favor strong binding and precise timing of daily gene expression. Taken together, our work confirms BMAL1's primary function as a master regulator of the core circadian oscillator, while demonstrating that it also contributes in a more distributed fashion to a variety of output programs.
doi:10.1371/journal.pbio.1000595
PMCID: PMC3043000  PMID: 21364973
5.  Targeted Expression of csCSF-1 in op/op Mice Ameliorates Tooth Defects 
Archives of oral biology  2006;52(5):432-443.
Objective
The aim of this study was to characterize the tooth phenotype of CSF-1-deficient op/op mice and determine whether expression of csCSF-1 in these mice has a role in primary tooth matrix formation.
Design
Ameloblasts and odontoblasts, isolated from wt/wt frozen sections using laser capture microdissection, were analyzed for csCSF-1, sCSF-1 and CSF-1R mRNA by RT-PCR. Mandibles, excised from 8 day op/op and wt/wt littermates, were examined for tooth morphology as well as amelogenin and DMP1 expression using in situ hybridization. Op/opCS transgenic mice, expressing csCSF-1 in teeth and bone using the osteocalcin promoter, were generated. Skeletal x-rays and histomorphometry were performed; teeth were analyzed for morphology and matrix proteins.
Results
Normal dental cells in vivo express both CSF-1 isoforms and CSF-1R. Compared to wt/wt, op/op teeth prior to eruption showed altered dental cell morphology and dramatic reduction in DMP1 transcripts. Op/opCS mice showed marked resolution of osteopetrosis, tooth eruption and teeth that resembled amelogenesis imperfecta-like phenotype. At 3 weeks, op/op teeth showed severe enamel and dentin defects and barely detectable amelogenin and DMP1. In op/opCS mice, DMP1 in odontoblasts increased to near normal and dentin morphology was restored; amelogenin also increased. Enamel integrity improved in op/opCS, although it was thinner than wt enamel.
Conclusions
Results demonstrate that ameloblasts and odontoblasts are a source and potential target of CSF-1 isoforms in vivo. Expression of csCSF-1 within the tooth microenvironment is essential for normal tooth morphogenesis and may provide a mechanism for coordinating the process of tooth eruption with endogenous matrix formation.
doi:10.1016/j.archoralbio.2006.10.018
PMCID: PMC1890041  PMID: 17126805
Macrophage colony-stimulating factor (CSF-1); osteopetrosis; transgenic mice; osteocalcin promoter; teeth
6.  TGF-β1 sensitizes TRPV1 through Cdk5 signaling in odontoblast-like cells 
Molecular Pain  2013;9:24.
Background
Odontoblasts are specialized cells that form dentin and they are believed to be sensors for tooth pain. Transforming growth factor-β1 (TGF-β1), a pro-inflammatory cytokine expressed early in odontoblasts, plays an important role in the immune response during tooth inflammation and infection. TGF-β1 is also known to participate in pain signaling by regulating cyclin-dependent kinase 5 (Cdk5) in nociceptive neurons of the trigeminal and dorsal root ganglia. However, the precise role of TGF-β1 in tooth pain signaling is not well characterized. The aim of our present study was to determine whether or not in odontoblasts Cdk5 is functionally active, if it is regulated by TGF-β1, and if it affects the downstream pain receptor, transient receptor potential vanilloid-1 (TRPV1).
Results
We first determined that Cdk5 and p35 are indeed expressed in an odontoblast-enriched primary preparation from murine teeth. For the subsequent analysis, we used an odontoblast-like cell line (MDPC-23) and found that Cdk5 is functionally active in these cells and its kinase activity is upregulated during cell differentiation. We found that TGF-β1 treatment potentiated Cdk5 kinase activity in undifferentiated MDPC-23 cells. SB431542, a specific inhibitor of TGF-β1 receptor 1 (Tgfbr1), when co-administered with TGF-β1, blocked the induction of Cdk5 activity. TGF-β1 treatment also activated the ERK1/2 signaling pathway, causing an increase in early growth response-1 (Egr-1), a transcription factor that induces p35 expression. In MDPC-23 cells transfected with TRPV1, Cdk5-mediated phosphorylation of TRPV1 at threonine-407 was significantly increased after TGF-β1 treatment. In contrast, SB431542 co-treatment blocked TRPV1 phosphorylation. Moreover, TGF-β1 treatment enhanced both proton- and capsaicin-induced Ca2+ influx in TRPV1-expressing MDPC-23 cells, while co-treatment with either SB431542 or roscovitine blocked this effect.
Conclusions
Cdk5 and p35 are expressed in a murine odontoblast-enriched primary preparation of cells from teeth. Cdk5 is also functionally active in odontoblast-like MDPC-23 cells. TGF-β1 sensitizes TRPV1 through Cdk5 signaling in MDPC-23 cells, suggesting the direct involvement of odontoblasts and Cdk5 in dental nociceptive pain transduction.
doi:10.1186/1744-8069-9-24
PMCID: PMC3680294  PMID: 23668392
TGF-β1; Cdk5; p35; TRPV1; MDPC-23 cells
7.  New Roles and Mechanism of Action of BMP4 in Postnatal Tooth Cytodifferentiation 
Bone  2010;46(6):1533-1545.
During the phase of overt tooth cytodifferentiation that occurs after birth in the mouse and using the 3.6Collagen1a-Cre, the BMP4 floxed and BMP4 knock-out mice, the BMP4 gene was deleted in early collagen producing odontoblasts around postnatal day 1. BMP4 expression was reduced over 90% in alveolar osteoblasts and odontoblasts. There was decreased rate of predentin to dentin formation and decreased mature odontoblast differentiation reflected in reduced DMP1 expression and proper dentinal tubule formation, as well as reduced Collagen type I and Osteocalcin expression. We observed mutant dysmorphogenic odontoblasts that failed to properly elongate and differentiate. The consequence of this failed differentiation process lead to permanent loss of dentin thickness, apparent enlarged pulp chambers in the molars and reduced bone supporting the tooth structures in mice as old as 10–12 months. Deletion of the BMP4 gene in odontoblasts also indirectly disrupted the process of enamel formation that persisted throughout life. The mechanism for this altered differentiation program in the absence of the BMP4 gene in odontoblasts is from decreased BMP signaling, and decreased expression of three key transcription factors, Dlx3, Dlx5, and Osterix. BMP signaling, as well as Dlx3 and Amelogenin expression, are also indirectly reduced in the ameloblasts of the odontoblast BMP4 cKO mice. This supports a key paracrine or endocrine role of odontoblasts derived BMP4 postnatally on the proper amelogenesis and formation of the enamel.
doi:10.1016/j.bone.2010.02.024
PMCID: PMC2875306  PMID: 20206312
8.  Molecular and circadian controls of ameloblasts 
European journal of oral sciences  2011;119(Suppl 1):35-40.
Stage-specific expression of ameloblast-specific genes is controlled by differential expression of transcription factors. In addition, ameloblasts follow daily rhythms in their main activities i.e. enamel protein secretion and enamel mineralization. This time related control is orchestrated by oscillations of clock proteins involved in circadian rhythms regulation. Our aim was to identify the potential links between daily rhythms and developmental controls of ameloblast differentiation. The effects of selected transcriptional factors Distal-less homeobox 3 (Dlx3) and Runt related transcription factor 2 (Runx2) and clock gene Nuclear receptor subfamily 1, group D, member 1 (Nr1d1) on secretory and maturation ameloblasts [using stage-specific markers amelogenin (Amel), enamelin (Enam) and kallikrein related-peptidase 4 (Klk4)] were evaluated in HAT-7 ameloblast cell line. Amel and Enam steady-state RNA expression levels were down-regulated in Runx2 over-expressing cells and up-regulated in Dlx3 over-expressing cells. In contrast, Klk4 was up-regulated by both Dlx3 and Runx2. Furthermore, a temporal and spatial relationship between clock genes and ameloblast differentiation markers was detected. Of interest, clock genes not only affected rhythmic expression of ameloblast specific genes but also influenced the expression of Runx2. Multi-scale mathematical modeling is being explored to further understand the temporal and developmental controls of ameloblast differentiation. Our study provides novel insights into the regulatory mechanisms sustaining ameloblast differentiation.
doi:10.1111/j.1600-0722.2011.00918.x
PMCID: PMC3516856  PMID: 22243224
Ameloblast gene regulation; circadian rhythms; clock genes; multi-scale modeling; enamel
9.  THE CYSTIC FIBROSIS TRANSMEMBRANE CONDUCTANCE REGULATOR (CFTR) IS EXPRESSED IN MATURATION STAGE AMELOBLASTS, ODONTOBLASTS AND BONE CELLS 
Bone  2009;46(4):1188-1196.
Patients with cystic fibrosis (CF) have mild defects in dental enamel. The gene mutated in these patients is CFTR, a Cl− channel involved in transepithelial salt- and water transport and bicarbonate secretion. We tested the hypothesis that Cftr channels are present and operating in the plasma membranes of mouse ameloblasts.
Tissue sections of young mouse jaws and fetal human jaws were immunostained with various anti-Cftr antibodies. Specificity of the antibodies was validated in Cftr-deficient murine and human tissues. Immunostaining for Cftr was obtained in the apical plasma membranes of mouse maturation ameloblasts of both incisor and molar tooth germs. A granular intracellular immunostaining of variable intensity was also noted in bone cells and odontoblasts. In Cftr-deficient mice the incisors were chalky white and eroded much faster than in wild type mice. Histologically, only maturation ameloblasts of incisors were structurally affected in Cftr-deficient mice. Some antibody species gave also a positive cytosolic staining in Cftr-deficient cells. Transcripts of Cftr were found in maturation ameloblasts, odontoblasts and bone cells. Similar data were obtained in forming human dentin and bone.
We conclude that Cftr protein locates in the apical plasma membranes of mouse maturation ameloblasts. In mouse incisors Cftr is critical for completion of enamel mineralization and conceivably functions as a regulator of pH during rapid crystal growth. Osteopenia found in CF patients as well as in Cftr-deficient mice is likely associated with defective Cftr operating in bone cells.
doi:10.1016/j.bone.2009.12.002
PMCID: PMC2842452  PMID: 20004757
CFTR; immunostaining; ameloblasts; osteoblasts; osteoclasts; Cftr-knockout
10.  Transcriptional Repression of the Dspp Gene Leads to Dentinogenesis Imperfecta Phenotype in Col1a1-Trps1 Transgenic Mice 
Journal of Bone and Mineral Research  2012;27(8):1735-1745.
Dentinogenesis imperfecta (DGI) is a hereditary defect of dentin, a calcified tissue that is the most abundant component of teeth. Most commonly, DGI is manifested as a part of osteogenesis imperfecta (OI) or the phenotype is restricted to dental findings only. In the latter case, DGI is caused by mutations in the DSPP gene, which codes for dentin sialoprotein (DSP) and dentin phosphoprotein (DPP). Although these two proteins together constitute the majority of noncollagenous proteins of the dentin, little is known about their transcriptional regulation. Here we demonstrate that mice overexpressing the Trps1 transcription factor (Col1a1-Trps1 mice) in dentin-producing cells, odontoblasts, present with severe defects of dentin formation that resemble DGI. Combined micro–computed tomography (µCT) and histological analyses revealed tooth fragility due to severe hypomineralization of dentin and a diminished dentin layer with irregular mineralization in Col1a1-Trps1 mice. Biochemical analyses of noncollagenous dentin matrix proteins demonstrated decreased levels of both DSP and DPP proteins in Col1a1-Trps1 mice. On the molecular level, we demonstrated that sustained high levels of Trps1 in odontoblasts lead to dramatic decrease of Dspp expression as a result of direct inhibition of the Dspp promoter by Trps1. During tooth development Trps1 is highly expressed in preodontoblasts, but in mature odontoblasts secreting matrix its expression significantly decreases, which suggests a Trps1 role in odontoblast development. In these studies we identified Trps1 as a potent inhibitor of Dspp expression and the subsequent mineralization of dentin. Thus, we provide novel insights into mechanisms of transcriptional dysregulation that leads to DGI. © 2012 American Society for Bone and Mineral Research.
doi:10.1002/jbmr.1636
PMCID: PMC3399940  PMID: 22508542
MINERALIZATION; DENTINOGENESIS; TRPS1; DSPP; TRANSCRIPTION
11.  Transcriptional Repression of the Dspp Gene Leads to Dentinogenesis Imperfecta Phenotype in Col1a1-Trps1 Transgenic Mice 
Dentinogenesis imperfecta (DGI) is a hereditary defect of dentin, a calcified tissue that is the most abundant component of teeth. Most commonly, DGI is manifested as a part of the osteogenesis imperfecta (OI) or the phenotype is restricted to dental findings only. In the latter case, DGI is caused by mutations in the DSPP gene, which codes for dentin sialoprotein (DSP) and dentin phosphoprotein (DPP). Although these two proteins together constitute the majority of non-collagenous proteins of the dentin, little is known about their transcriptional regulation. Here we demonstrate that mice overexpressing the Trps1 transcription factor (Col1a1-Trps1 mice) in dentin-producing cells, odontoblasts, present with severe defects of dentin formation that resemble DGI. Combined μCT and histological analyses revealed tooth fragility due to severe hypomineralization of dentin and a diminished dentin layer with irregular mineralization in Col1a1-Trps1 mice. Biochemical analyses of non-collagenous dentin matrix proteins demonstrated decreased levels of both DSP and DPP proteins in Col1a1-Trps1 mice. On the molecular level, we demonstrated that sustained high levels of Trps1 in odontoblasts lead to dramatic decrease of Dspp expression as a result of direct inhibition of the Dspp promoter by Trps1. During tooth development Trps1 is highly expressed in preodontoblasts, but in mature odontoblasts secreting matrix its expression significantly decreases, which suggests a Trps1 role in odontoblast development. In these studies we identified Trps1 as a potent inhibitor of Dspp expression and the subsequent mineralization of dentin. Thus we provide novel insights into mechanisms of transcriptional dysregulation that leads to dentinogenesis imperfecta.
doi:10.1002/jbmr.1636
PMCID: PMC3399940  PMID: 22508542
mineralization; dentinogenesis; Trps1; Dspp; transcription
12.  Accurate timekeeping is controlled by a cycling activator in Arabidopsis 
eLife  2013;2:e00473.
Transcriptional feedback loops are key to circadian clock function in many organisms. Current models of the Arabidopsis circadian network consist of several coupled feedback loops composed almost exclusively of transcriptional repressors. Indeed, a central regulatory mechanism is the repression of evening-phased clock genes via the binding of morning-phased Myb-like repressors to evening element (EE) promoter motifs. We now demonstrate that a related Myb-like protein, REVEILLE8 (RVE8), is a direct transcriptional activator of EE-containing clock and output genes. Loss of RVE8 and its close homologs causes a delay and reduction in levels of evening-phased clock gene transcripts and significant lengthening of clock pace. Our data suggest a substantially revised model of the circadian oscillator, with a clock-regulated activator essential both for clock progression and control of clock outputs. Further, our work suggests that the plant clock consists of a highly interconnected, complex regulatory network rather than of coupled morning and evening feedback loops.
DOI: http://dx.doi.org/10.7554/eLife.00473.001
eLife digest
We live in a world with a 24-hr cycle in which day follows night follows day with complete predictability. Life on earth has evolved to take advantage of this predictability by using circadian clocks to prepare for the coming of night (or day), and plants are no exception. Even in constant darkness, characteristics such as leaf movements show a constant cycle of around 24 hr.
Most circadian clocks rely on negative feedback loops involving various genes and proteins to keep track of time. In one of these feedback loops, certain genes—called morning-phased genes—are expressed as proteins during the day, and these proteins prevent other genes—called evening-phased genes—from producing proteins. As night approaches, however, a second feedback loop acts to stop the morning-phased genes being expressed, thus allowing the evening-phased genes to produce proteins. And as day approaches, expression of these genes is stopped and the whole cycle starts again.
Many of the genes and proteins involved in the circadian system of Arabidopsis thaliana, a small flowering plant that is widely used as a model organism, have been identified, and its circadian clock was thought to rely almost entirely on proteins called repressors that block the transcription of genes. Now, Hsu et al. have shown that the Arabidopsis clock also involves proteins that increase the expression of certain genes at specific times of the day.
Hsu et al. focused on the promoter regions of evening-phased genes: these regions are stretches of DNA that proteins called transcription factors bind to and either encourage the expression of a gene (if the protein is a transcriptional activator) or block its expression (as a transcriptional repressor). In particular, they focused on a protein called RVE8 that is most strongly expressed in the afternoon and, based on previous research, is thought to activate the transcription of genes. Using genetically modified plants in which the gene for RVE8 can be turned on and off, they found that this protein led to increases in the expression of some genes, and reductions in the expression of others.
Further analysis showed that RVE8 was able to activate the expression of evening-phased genes directly, without requiring that new proteins be made first. By contrast, morning-expressed genes were likely to be suppressed by RVE8 via an indirect mechanism that involved other proteins that had previously been activated by RVE8. The expression of RVE8 itself is regulated by other clock genes and also by an undefined post-transcriptional process. Therefore rather than consisting of a morning feedback loop coupled to an evening feedback loop, with both loops being based on repressors, the plant clock is instead better viewed as a highly connected network of activators and repressors. Further research is clearly necessary to understand this unexpected complexity in the circadian clock of Arabidopsis.
DOI: http://dx.doi.org/10.7554/eLife.00473.002
doi:10.7554/eLife.00473
PMCID: PMC3639509  PMID: 23638299
circadian rhythm; transcription factors; evening element; phase; Arabidopsis
13.  CLOCK and NPAS2 have overlapping roles in the circadian oscillation of arylalkylamine N-acetyltransferase (Aanat) mRNA in chicken cone photoreceptors 
Journal of neurochemistry  2010;113(5):1296-1306.
Circadian clocks in vertebrates are thought to be composed of transcriptional-translational feedback loops involving a highly conversed set of “clock genes”: namely, period (Per1–3) and cryptochrome (Cry1–2), which encode negative transcriptional regulators; and Bmal1, Clock, and Npas2, which encode positive regulators. Aanat, which encodes arylalkylamine N-acetyltransferase (AANAT), the key regulatory enzyme that drives the circadian rhythm of melatonin synthesis, contains a circadian E-box element (CACGTG) in its proximal promoter that is potentially capable of binding CLOCK: BMAL1 and NPAS2: BMAL1 heterodimers. The present study was conducted to investigate whether CLOCK and/or NPAS2 regulates Aanat expression in photoreceptor cells. Npas2 and Clock are both expressed in photoreceptor cells in vivo and in vitro. To assess the roles of CLOCK and NPAS2 in Aanat expression, gene specific microRNA (miR) vectors were used to knock down expression of these clock genes in photoreceptor-enriched cell cultures. The knockdown of CLOCK protein significantly reduced the circadian expression of Npas2, Per2, and Aanat transcripts but had no effect on the circadian rhythm of Bmal1 transcript level. The knockdown of NPAS2 significantly damped the circadian rhythm of Aanat mRNAs but had no effect on circadian expression of any of clock genes examined, except Npas2 itself. Chromatin immunoprecipitation studies indicated that both CLOCK and NPAS2 bound to the Aanat promoter in situ. Thus, CLOCK and NPAS2 have overlapping roles in the clock output pathway that regulates the rhythmic expression of Aanat in photoreceptors. However, CLOCK plays the predominant role in the chicken photoreceptor circadian clockwork mechanism, including the regulation of NPAS2 expression.
doi:10.1111/j.1471-4159.2010.06698.x
PMCID: PMC2950611  PMID: 20345751
circadian clock genes; circadian rhythms; retina; melatonin; transcription factors; RNA interference
14.  Light Directs Zebrafish period2 Expression via Conserved D and E Boxes 
PLoS Biology  2009;7(10):e1000223.
A highly conserved promoter module in a vertebrate clock gene confers light-regulated gene expression.
For most species, light represents the principal environmental signal for entraining the endogenous circadian clock. The zebrafish is a fascinating vertebrate model for studying this process since unlike mammals, direct exposure of most of its tissues to light leads to local clock entrainment. Importantly, light induces the expression of a set of genes including certain clock genes in most zebrafish cell types in vivo and in vitro. However, the mechanism linking light to gene expression remains poorly understood. To elucidate this key mechanism, here we focus on how light regulates transcription of the zebrafish period2 (per2) gene. Using transgenic fish and stably transfected cell line–based assays, we define a Light Responsive Module (LRM) within the per2 promoter. The LRM lies proximal to the transcription start site and is both necessary and sufficient for light-driven gene expression and also for a light-dependent circadian clock regulation. Curiously, the LRM sequence is strongly conserved in other vertebrate per2 genes, even in species lacking directly light-sensitive peripheral clocks. Furthermore, we reveal that the human LRM can substitute for the zebrafish LRM to confer light-regulated transcription in zebrafish cells. The LRM contains E- and D-box elements that are critical for its function. While the E-box directs circadian clock regulation by mediating BMAL/CLOCK activity, the D-box confers light-driven expression. The zebrafish homolog of the thyrotroph embryonic factor binds efficiently to the LRM D-box and transactivates expression. We demonstrate that tef mRNA levels are light inducible and that knock-down of tef expression attenuates light-driven transcription from the per2 promoter in vivo. Together, our results support a model where a light-dependent crosstalk between E- and D-box binding factors is a central determinant of per2 expression. These findings extend the general understanding of the mechanism whereby the clock is entrained by light and how the regulation of clock gene expression by light has evolved in vertebrates.
Author Summary
Light is the principal signal used by animals to synchronize their circadian clocks with the day/night environment. Central to this vital property is the ability of light to trigger changes in gene expression. However, we still lack a complete understanding of how this occurs. The zebrafish is particularly interesting in this regard since direct light exposure induces the expression of clock genes in most of its tissues and in turn adjusts the phase of the intrinsic clocks. Here, by studying the promoter of one key light-regulated zebrafish clock gene, per2, we have identified a Light Responsive Module (LRM) that is necessary and sufficient for light controlled expression. Interestingly, the LRM is also highly conserved in the per2 genes of other vertebrates that lack widespread light-sensing tissues. In addition, the human LRM can substitute for its zebrafish counterpart to confer direct light regulation of gene expression in zebrafish cells. The LRM contains E- and D-box enhancers critical for its function. While the E-box is a target of clock regulation, the D-box directs light driven expression. We show that the expression of the D-box binding transcription factor, tef, is itself induced by light and is essential for normal light-induced per2 expression. These results advance our understanding of the mechanisms underlying entrainment by light and how light-regulated clock gene expression has evolved in vertebrates.
doi:10.1371/journal.pbio.1000223
PMCID: PMC2759001  PMID: 19859524
15.  Expression Patterns and Subcellular Localization of Carbonic Anhydrases Are Developmentally Regulated during Tooth Formation 
PLoS ONE  2014;9(5):e96007.
Carbonic anhydrases (CAs) play fundamental roles in several physiological events, and emerging evidence points at their involvement in an array of disorders, including cancer. The expression of CAs in the different cells of teeth is unknown, let alone their expression patterns during odontogenesis. As a first step towards understanding the role of CAs during odontogenesis, we used immunohistochemistry, histochemistry and in situ hybridization to reveal hitherto unknown dynamic distribution patterns of eight CAs in mice. The most salient findings include expression of CAII/Car2 not only in maturation-stage ameloblasts (MA) but also in the papillary layer, dental papilla mesenchyme, odontoblasts and the epithelial rests of Malassez. We uncovered that the latter form lace-like networks around incisors; hitherto these have been known to occur only in molars. All CAs studied were produced by MA, however CAIV, CAIX and CARPXI proteins were distinctly enriched in the ruffled membrane of the ruffled MA but exhibited a homogeneous distribution in smooth-ended MA. While CAIV, CAVI/Car6, CAIX, CARPXI and CAXIV were produced by all odontoblasts, CAIII distribution displayed a striking asymmetry, in that it was virtually confined to odontoblasts in the root of molars and root analog of incisors. Remarkably, from initiation until near completion of odontogenesis and in several other tissues, CAXIII localized mainly in intracellular punctae/vesicles that we show to overlap with LAMP-1- and LAMP-2-positive vesicles, suggesting that CAXIII localizes within lysosomes. We showed that expression of CAs in developing teeth is not confined to cells involved in biomineralization, pointing at their participation in other biological events. Finally, we uncovered novel sites of CA expression, including the developing brain and eye, the olfactory epithelium, melanoblasts, tongue, notochord, nucleus pulposus and sebaceous glands. Our study provides important information for future single or multiple gene targeting strategies aiming at deciphering the function of CAs during odontogenesis.
doi:10.1371/journal.pone.0096007
PMCID: PMC4006843  PMID: 24789143
16.  Data assimilation constrains new connections and components in a complex, eukaryotic circadian clock model 
Integrating molecular time-series data resulted in a more robust model of the plant clock, which predicts that a wave of inhibitory PRR proteins controls the morning genes LHY and CCA1.PRR5 is experimentally validated as a late-acting component of this wave.The family of sequentially expressed PRR proteins allows flexible entrainment of the clock, whereas a single protein could not, suggesting that the duplication of clock genes might confer this generic, functional advantage.The observed post-translational regulation of the evening protein TOC1 by interaction with ZTL and GI remains consistent with an indirect activation of TOC1 mRNA expression by GI, which was previously postulated from modelling.
Circadian rhythms are present in most eukaryotic organisms including plants. The core genes of the circadian clock are very important for plant physiology as they drive the rhythmic expression of around 30% of Arabidopsis genes (Edwards et al, 2006; Michael et al, 2008). The clock is normally entrained by daily environmental changes in light and temperature. Oscillations also persist under constant environmental conditions in a laboratory. The clock gene circuit in Arabidopsis is based on multiple interlocked feedback loops, which are typical of circadian genetic networks in other organisms (Dunlap and Loros, 2004; Bell-Pedersen et al, 2005). Mechanistic, mathematical models are increasingly useful in analysing and understanding how the observed molecular components give rise to the rhythmic behaviour of this dynamic, non-linear system.
Our previous model of Arabidopsis circadian clock (Locke et al, 2006) presented the core, three-loop structure of the clock, which comprised morning and evening oscillators and coupling between them (Figure 1). The morning loop included the dawn-expressed LATE ELONGATED HYPOCOTYL (LHY) and CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) genes, which negatively regulate their expression through activation of the inhibitor proteins, PSEUDO-RESPONSE REGULATOR 9 (PRR9) and PRR7. These were described by a single, combined model component, PRR9/7. The evening loop included the dusk-expressed gene TIMING OF CAB EXPRESSION 1 (TOC1), which negatively regulates itself through inhibition of a hypothetical activator, gene Y. The evening-expressed gene GIGANTEA (GI) contributes to Y function. The morning and evening loops were connected through inhibition of the evening genes by LHY/CCA1 and activation of LHY/CCA1 expression by a hypothetical evening gene X. Here, we extend the previous model of circadian gene expression (Locke et al, 2006) based on recently published data (Figure 1). The new model retains the good match of our previous model to the large volume of molecular time-series data, and improves the behaviour of the model clock system under a range of light conditions and in a wider range of mutants.
The morning loop was extended by adding a hypothetical clock component, the night inhibitor (NI), which acts together with PRR9 and PRR7 to keep the expression of LHY and CCA1 at low levels over a broad interval spanning dusk. This regulation is important to set the phase of LHY/CCA1 expression at dawn. Data from the literature suggested that the PRR5 gene was a candidate for NI, leading us to predict that the sequentially expressed PRR9, PRR7 and PRR5 proteins together formed a wave of inhibitors of LHY and CCA1. This hypothesis was tested under discriminating light conditions, in which the light interval is replaced with the dawn and dusk pulses of light to form a ‘skeleton photoperiod'. Combining this protocol with mutation of the PRR7 and/or PRR5 genes, our new experimental results validated the model predictions and confirmed that PRR5 contributes to the function that we modelled as NI. During revision of this paper, that result received further experimental support (Nakamichi et al, 2010).
Model simulations revealed the functional importance of the inhibitor wave in entraining the clock to the light/dark cycle. Separating PRR9 from the other inhibitors in the model showed how the strong light activation observed for this gene contributes to more rapid entrainment. The observed, post-translation regulation of all three inhibitor proteins by light (Farre and Kay, 2007; Ito et al, 2007; Kiba et al, 2007) was also included in the model. Light-regulated degradation provides a molecular mechanism to explain the later phase of LHY and CCA1 expression under long photoperiods compared with short photoperiods, in line with experimental observations.
The connection between evening and morning loops was revised by including the inhibition of the morning gene PRR9 by the evening component TOC1, based on the data on TOC1-overexpressing plants (Makino et al, 2002; Ito et al, 2005). This inhibition causes a delay of PRR9 expression relative to LHY/CCA1, which allows LHY/CCA1 to reach a higher expression level at dawn. Our simulations showed that a partial mutant that lacks this inhibition of PRR9 by TOC1 is sufficient to cause the higher level of PRR9 and the short circadian period observed in toc1 mutant plants.
The evening loop was extended by introducing the observed, post-translational regulation of the TOC1 protein by the F-box protein ZEITLUPE (ZTL) and stabilization of ZTL by its interaction with GI in the presence of light (Kim et al, 2007). GI's function in the clock model has thus been revised according to the data: GI promotes an inhibition of TOC1 protein function through positive regulation of ZTL. This results, together with negative regulation of Y by TOC1, in indirect activation of TOC1 mRNA expression by GI, which agrees with our earlier experimental data (Locke et al, 2006). Simulations showed that the post-translational regulation of TOC1 by ZTL and GI results in the observed long period of the ztl mutant and fast dampening of rhythms in the lhy/cca1/gi triple mutant.
This is the first mathematical model that incorporates the observed post-translational regulation into the genetic network of the Arabidopsis clock. In addition to specific, mechanistic insights, the model shows a generic advantage from the duplication of clock genes and their expression at different phases. Such clock gene duplications are observed in eukaryotes with larger genomes, such as the mouse. Analogous, functional duplication can be achieved by differential regulation of a single clock gene in distinct cells, as in Drosophila.
Circadian clocks generate 24-h rhythms that are entrained by the day/night cycle. Clock circuits include several light inputs and interlocked feedback loops, with complex dynamics. Multiple biological components can contribute to each part of the circuit in higher organisms. Mechanistic models with morning, evening and central feedback loops have provided a heuristic framework for the clock in plants, but were based on transcriptional control. Here, we model observed, post-transcriptional and post-translational regulation and constrain many parameter values based on experimental data. The model's feedback circuit is revised and now includes PSEUDO-RESPONSE REGULATOR 7 (PRR7) and ZEITLUPE. The revised model matches data in varying environments and mutants, and gains robustness to parameter variation. Our results suggest that the activation of important morning-expressed genes follows their release from a night inhibitor (NI). Experiments inspired by the new model support the predicted NI function and show that the PRR5 gene contributes to the NI. The multiple PRR genes of Arabidopsis uncouple events in the late night from light-driven responses in the day, increasing the flexibility of rhythmic regulation.
doi:10.1038/msb.2010.69
PMCID: PMC2964123  PMID: 20865009
Arabidopsis thaliana; biological clocks; circadian rhythms; mathematical model; systems biology
17.  Profiling of Circadian Genes Expressed in the Uterus Endometrial Stromal Cells of Pregnant Rats as Revealed by DNA Microarray Coupled with RNA Interference 
The peripheral circadian oscillator plays an essential role in synchronizing local physiology to operate in a circadian manner via regulation of the expression of clock-controlled genes. The present study aimed to evaluate the circadian rhythms of clock genes and clock-controlled genes expressed in the rat uterus endometrial stromal cells (UESCs) during the stage of implantation by a DNA microarray. Of 12,252 genes showing significantly expression, 7,235 genes displayed significant alterations. As revealed by the biological pathway analysis using the database for annotation, visualization, and integrated discovery online annotation software, genes were involved in cell cycle, glutathione metabolism, MAPK signaling pathway, fatty acid metabolism, ubiquitin mediated proteolysis, focal adhesion, and PPAR signaling pathway. The clustering of clock genes were mainly divided into four groups: the first group was Rorα, Timeless, Npas2, Bmal1, Id2, and Cry2; the second group Per1, Per2, Per3, Dec1, Tef, and Dbp; the third group Bmal2, Cry1, E4bp4, Rorβ, and Clock; the fourth group Rev-erbα. Eleven implantation-related genes and 24 placenta formation-related genes displayed significant alterations, suggesting that these genes involved in implantation and placenta formation are controlled under circadian clock. Some candidates as clock-controlled genes were evaluated by using RNA interference to Bmal1 mRNA. Down-regulation of Igf1 gene expression was observed by Bmal1 silencing, whereas the expression of Inhβa was significantly increased. During active oscillation of circadian clock, the apoptosis-related genes Fas and Caspase3 remained no significant changes, but they were significantly increased by knockdown of Bmal1 mRNA. These results indicate that clock-controlled genes are up- or down-regulated in rat UESCs during the stage of decidualization. DNA microarray analysis coupled with RNA interference will be helpful to understand the physiological roles of some oscillating genes in blastocyst implantation and placenta formation.
doi:10.3389/fendo.2013.00082
PMCID: PMC3703733  PMID: 23847593
uterus endometrial stromal cells; clock genes; clock-controlled genes; DNA microarray; implantation; decidualization; Bmal1 siRNA; Per2-dLuc reporter gene
18.  Adult Circadian Behavior in Drosophila Requires Developmental Expression of cycle, But Not period 
PLoS Genetics  2011;7(7):e1002167.
Circadian clocks have evolved as internal time keeping mechanisms that allow anticipation of daily environmental changes and organization of a daily program of physiological and behavioral rhythms. To better examine the mechanisms underlying circadian clocks in animals and to ask whether clock gene expression and function during development affected subsequent daily time keeping in the adult, we used the genetic tools available in Drosophila to conditionally manipulate the function of the CYCLE component of the positive regulator CLOCK/CYCLE (CLK/CYC) or its negative feedback inhibitor PERIOD (PER). Differential manipulation of clock function during development and in adulthood indicated that there is no developmental requirement for either a running clock mechanism or expression of per. However, conditional suppression of CLK/CYC activity either via per over-expression or cyc depletion during metamorphosis resulted in persistent arrhythmic behavior in the adult. Two distinct mechanisms were identified that may contribute to this developmental function of CLK/CYC and both involve the ventral lateral clock neurons (LNvs) that are crucial to circadian control of locomotor behavior: (1) selective depletion of cyc expression in the LNvs resulted in abnormal peptidergic small-LNv dorsal projections, and (2) PER expression rhythms in the adult LNvs appeared to be affected by developmental inhibition of CLK/CYC activity. Given the conservation of clock genes and circuits among animals, this study provides a rationale for investigating a possible similar developmental role of the homologous mammalian CLOCK/BMAL1 complex.
Author Summary
The fruit fly Drosophila melanogaster is an excellent model system for studying the internal circadian clocks that animals use for daily time keeping. Since clocks exist and function in animals not only in adults, but also during prior development, the question arises if and how adult circadian rhythms depend on developmental clock circuits and components. To address this question we created transgenic flies in which the essential clock components CLOCK/CYCLE (CLK/CYC) and PERIOD (PER) can be manipulated via environmental temperature. Stopping the clock during development by depleting the negative regulator PER did not prevent restoration of circadian time keeping in the adult. However, a developmental arrest of the clock due to either depletion of the positive regulator CYC or overproduction of PER resulted in a persistent loss of clock-controlled behavior function in adults. Taken together, these observations indicate that adult clock function developmentally requires activity of the CLK/CYC transcription complex rather than a ticking clock. Based on the behavioral, molecular, and anatomical consequences of inhibiting CLK/CYC in circadian pacemaker neurons, we propose that the developmental requirement maps to these cells. It will be interesting to determine whether there is a comparable developmental requirement for the equivalent clock genes in humans.
doi:10.1371/journal.pgen.1002167
PMCID: PMC3131292  PMID: 21750685
19.  Quantitative analysis of regulatory flexibility under changing environmental conditions 
Day length changes with the seasons in temperate latitudes, affecting the many biological rhythms that entrain to the day/night cycle: we measure these effects on the expression of Arabidopsis clock genes, using RNA and reporter gene readouts, with a new method of phase analysis.Dusk sensitivity is proposed as a simple, natural and general mathematical measure to analyse and manipulate the changing phase of a clock output relative to the change in the day/night cycle.Dusk sensitivity shows how increasing the numbers of feedback loops in the Arabidopsis clock models allows more flexible regulation, consistent with a previously-proposed, general operating principle of biological networks.The Arabidopsis clock genes show flexibility of regulation that is characteristic of a three-loop clock model, validating aspects of the model and the operating principle, but some clock output genes show greater flexibility arising from direct light regulation.
The analysis of dynamic, non-linear regulation with the aid of mechanistic models is central to Systems Biology. This study compares the predictions of mechanistic, mathematical models of the circadian clock with molecular time-series data on rhythmic gene expression in the higher plant Arabidopsis thaliana. Analysis of the models helps us to understand (explain and predict) how the clock gene circuit balances regulation by external and endogenous factors to achieve particular behaviours. Such multi-factorial regulation is ubiquitous in, and characteristic of, living systems.
The Earth's rotation causes predictable changes in the environment, notably in the availability of sunlight for photosynthesis. Many biological processes are driven by the environmental input via sensory pathways, for example, from photoreceptors. Circadian clocks provide an alternative strategy. These endogenous, 24-h rhythms can drive biological processes that anticipate the regular environmental changes, rather than merely responding. Many rhythmic processes have both light and clock control. Indeed, the clock components themselves must balance internal timing with external inputs, because circadian clocks are reset daily through light regulation of one or more clock components. This process of entrainment is complicated by the change in day length. When the times of dawn and dusk move apart in summer, and closer together in winter, does the clock track dawn, track dusk or interpolate between them?
In plants, the clock controls leaf and petal movements, the opening and closing of stomatal pores, the discharge of floral fragrances, and many metabolic activities, especially those associated with photosynthesis. Centuries of physiological studies have shown that these rhythms can behave differently. Flowering in Ipomoea nil (Pharbitis nil, Japanese morning glory) is controlled by a rhythm that tracks the time of dusk, to give a classic example. We showed that two other rhythms associated with vegetative growth track dawn in this species (Figure 5A), so the clock system allows flexible regulation.
The relatively small number of components involved in the circadian clockwork makes it an ideal candidate for mathematical modelling. Molecular genetic studies in a variety of model eukaryotes have shown that the circadian rhythm is generated by a network of 6–20 genes. These genes form feedback loops generating a rhythm in mRNA production. A single negative feedback loop in which a gene encodes a protein that, after several hours, turns off transcription is capable of generating a circadian rhythm, in principle. A single light input can entrain the clock to ‘local time', synchronised with a light–dark cycle. However, real circadian clocks have proven to be more complicated than this, with multiple light inputs and interlocked feedback loops.
We have previously argued from mathematical analysis that multi-loop networks increase the flexibility of regulation (Rand et al, 2004) and have shown that appropriately deployed flexibility can confer functional robustness (Akman et al, 2010). Here we test whether that flexibility can be demonstrated in vivo, in the model plant, A. thaliana. The Arabidopsis clock mechanism comprises a feedback loop in which two partially redundant, myb transcription factors, LATE ELONGATED HYPOCOTYL (LHY) and CIRCADIAN CLOCK ASSOCIATED 1 (CCA1), repress the expression of their activator, TIMING OF CAB EXPRESSION 1 (TOC1). We previously modelled this single-loop circuit and showed that it was not capable of recreating important data (Locke et al, 2005a). An extended, two-loop model was developed to match observed behaviours, incorporating a hypothetical gene Y, for which the best identified candidate was the GIGANTEA gene (GI) (Locke et al, 2005b). Two further models incorporated the TOC1 homologues PSEUDO-RESPONSE REGULATOR (PRR) 9 and PRR7 (Locke et al, 2006; Zeilinger et al, 2006). In these circuits, a morning oscillator (LHY/CCA1–PRR9/7) is coupled to an evening oscillator (Y/GI–TOC1) via the original LHY/CCA1–TOC1 loop.
These clock models, like those for all other organisms, were developed using data from simple conditions of constant light, darkness or 12-h light–12-h dark cycles. We therefore tested how the clock genes in Arabidopsis responded to light–dark cycles with different photoperiods, from 3 h light to 18 h light per 24-h cycle (Edinburgh, 56° North latitude, has 17.5 h light in midsummer). The time-series assays of mRNA and in vivo reporter gene images showed a range of peak times for different genes, depending on the photoperiod (Figure 5C). A new data analysis method, mFourfit, was introduced to measure the peak times, in the Biological Rhythms Analysis Software Suite (BRASS v3.0). None of the genes showed the dusk-tracking behaviour characteristic of the Ipomoea flowering rhythm. The one-, two- and three-loop models were analysed to understand the observed patterns. A new mathematical measure, dusk sensitivity, was introduced to measure the change in timing of a model component versus a change in the time of dusk. The one- and two-loop models tracked dawn and dusk, respectively, under all conditions. Only the three-loop model (Figure 5B) had the flexibility required to match the photoperiod-dependent changes that we found in vivo, and in particular the unexpected, V-shaped pattern in the peak time of TOC1 expression. This pattern of regulation depends on the structure and light inputs to the model's evening oscillator, so the in vivo data supported this aspect of the model. LHY and CCA1 gene expression under short photoperiods showed greater dusk sensitivity, in the interval 2–6 h before dawn, than the three-loop model predicted, so these data will help to constrain future models.
The approach described here could act as a template for experimental biologists seeking to understand biological regulation using dynamic, experimental perturbations and time-series data. Simulation of mathematical models (despite known imperfections) can provide contrasting hypotheses that guide understanding. The system's detailed behaviour is complex, so a natural and general measure such as dusk sensitivity is helpful to focus on one property of the system. We used the measure to compare models, and to predict how this property could be manipulated. To enable additional analysis of this system, we provide the time-series data and experimental metadata online.
The circadian clock controls 24-h rhythms in many biological processes, allowing appropriate timing of biological rhythms relative to dawn and dusk. Known clock circuits include multiple, interlocked feedback loops. Theory suggested that multiple loops contribute the flexibility for molecular rhythms to track multiple phases of the external cycle. Clear dawn- and dusk-tracking rhythms illustrate the flexibility of timing in Ipomoea nil. Molecular clock components in Arabidopsis thaliana showed complex, photoperiod-dependent regulation, which was analysed by comparison with three contrasting models. A simple, quantitative measure, Dusk Sensitivity, was introduced to compare the behaviour of clock models with varying loop complexity. Evening-expressed clock genes showed photoperiod-dependent dusk sensitivity, as predicted by the three-loop model, whereas the one- and two-loop models tracked dawn and dusk, respectively. Output genes for starch degradation achieved dusk-tracking expression through light regulation, rather than a dusk-tracking rhythm. Model analysis predicted which biochemical processes could be manipulated to extend dusk tracking. Our results reveal how an operating principle of biological regulators applies specifically to the plant circadian clock.
doi:10.1038/msb.2010.81
PMCID: PMC3010117  PMID: 21045818
Arabidopsis thaliana; biological clocks; dynamical systems; gene regulatory networks; mathematical models; photoperiodism
20.  System-Driven and Oscillator-Dependent Circadian Transcription in Mice with a Conditionally Active Liver Clock  
PLoS Biology  2007;5(2):e34.
The mammalian circadian timing system consists of a master pacemaker in neurons of the suprachiasmatic nucleus (SCN) and clocks of a similar molecular makeup in most peripheral body cells. Peripheral oscillators are self-sustained and cell autonomous, but they have to be synchronized by the SCN to ensure phase coherence within the organism. In principle, the rhythmic expression of genes in peripheral organs could thus be driven not only by local oscillators, but also by circadian systemic signals. To discriminate between these mechanisms, we engineered a mouse strain with a conditionally active liver clock, in which REV-ERBα represses the transcription of the essential core clock gene Bmal1 in a doxycycline-dependent manner. We examined circadian liver gene expression genome-wide in mice in which hepatocyte oscillators were either running or arrested, and found that the rhythmic transcription of most genes depended on functional hepatocyte clocks. However, we discovered 31 genes, including the core clock gene mPer2, whose expression oscillated robustly irrespective of whether the liver clock was running or not. By contrast, in liver explants cultured in vitro, circadian cycles of mPer2::luciferase bioluminescence could only be observed when hepatocyte oscillators were operational. Hence, the circadian cycles observed in the liver of intact animals without functional hepatocyte oscillators were likely generated by systemic signals. The finding that rhythmic mPer2 expression can be driven by both systemic cues and local oscillators suggests a plausible mechanism for the phase entrainment of subsidiary clocks in peripheral organs.
Author Summary
In contrast to previously held belief, molecular circadian oscillators are not restricted to specialized pacemaker tissues, such as the brain's suprachiasmatic nucleus (SCN), but exist in virtually all body cells. Although the circadian clocks operative in peripheral cell types are as robust as those residing in SCN neurons, they quickly become desynchronized in vitro due to variations in period length. Hence, in intact animals, the phase coherence between peripheral oscillators must be established by daily signals generated by the SCN master clock. Although the hierarchy between master and slave oscillators is now well established, the respective roles of these clocks in governing the circadian transcription program in a given organ have never been examined. In principle, the circadian expression of genes in a peripheral tissue could be driven either by cyclic systemic cues, by peripheral oscillators, or by both. In order to discriminate between genes regulated by local oscillators and systemic cues in liver, we generated mice in which hepatocyte clocks can be turned on and off at will. These studies suggest that 90% of the circadian transcription program in the liver is abolished or strongly attenuated when hepatocyte clocks are turned off, indicating that the expression of most circadian liver genes is orchestrated by local cellular clocks. The remaining 10% of cyclically expressed liver genes continue to be transcribed in a robustly circadian fashion in the absence of functional hepatocyte oscillators. These genes, which unexpectedly include the bona fide clock gene mPer2, must therefore be regulated by oscillating systemic signals, such as hormones, metabolites, or body temperature. Although temperature rhythms display only modest amplitudes, they appear to play a significant role in the phase entrainment of mPer2 transcription.
Research on mice engineered with an inducible liver clock enabled identification of some genes with expression controlled by the local clock, and other genes (includingmPer2) that maintained circadian oscillations thanks to cues from the SCN.
doi:10.1371/journal.pbio.0050034
PMCID: PMC1783671  PMID: 17298173
21.  Heterogeneous Expression of the Core Circadian Clock Proteins among Neuronal Cell Types in Mouse Retina 
PLoS ONE  2012;7(11):e50602.
Circadian rhythms in metabolism, physiology, and behavior originate from cell-autonomous circadian clocks located in many organs and structures throughout the body and that share a common molecular mechanism based on the clock genes and their protein products. In the mammalian neural retina, despite evidence supporting the presence of several circadian clocks regulating many facets of retinal physiology and function, the exact cellular location and genetic signature of the retinal clock cells remain largely unknown. Here we examined the expression of the core circadian clock proteins CLOCK, BMAL1, NPAS2, PERIOD 1(PER1), PERIOD 2 (PER2), and CRYPTOCHROME2 (CRY2) in identified neurons of the mouse retina during daily and circadian cycles. We found concurrent clock protein expression in most retinal neurons, including cone photoreceptors, dopaminergic amacrine cells, and melanopsin-expressing intrinsically photosensitive ganglion cells. Remarkably, diurnal and circadian rhythms of expression of all clock proteins were observed in the cones whereas only CRY2 expression was found to be rhythmic in the dopaminergic amacrine cells. Only a low level of expression of the clock proteins was detected in the rods at any time of the daily or circadian cycle. Our observations provide evidence that cones and not rods are cell-autonomous circadian clocks and reveal an important disparity in the expression of the core clock components among neuronal cell types. We propose that the overall temporal architecture of the mammalian retina does not result from the synchronous activity of pervasive identical clocks but rather reflects the cellular and regional heterogeneity in clock function within retinal tissue.
doi:10.1371/journal.pone.0050602
PMCID: PMC3506613  PMID: 23189207
22.  Combined effects of simvastatin and enamel matrix derivative on odontoblastic differentiation of human dental pulp cells 
Journal of endodontics  2012;39(1):10.1016/j.joen.2012.10.013.
Introduction
We previously reported that simvastatin and enamel matrix derivative (EMD) have a dentinogenic effect. However, there is little information about the combined effects of these 2 agents on odontoblastic differentiation. The aim of this study was to investigate the effects of combined treatment with simvastatin and EMD on odontoblastic differentiation of human dental pulp cells (hDPCs). This study further explored the role of extracellular signal-regulated kinase (ERK) as a target and mediator of the differentiation induced by simvastatin in hDPCs.
Methods
The odontoblastic differentiation was analyzed by alkaline phosphatase activity, real-time polymerase chain reaction (PCR) for odontoblastic/osteoblastic markers (ie, dentin sialophosphoprotein, dentin matrix protein 1, and osteonectin), and alizarin red S staining. We also explored the role of ERK signaling as a mediator of simvastatin by Western blotting and real-time PCR. The expression of osteoblast-specific transcription factors was detected by reverse-transcription PCR.
Results
The alkaline phosphatase activity and the expression of odontoblastic markers (ie, dentin sialophosphoprotein and dentin matrix protein 1) increased in simvastatin/EMD-treated cells. Mineralized nodule formation increased in EMD- and simvastatin/EMD-treated cells. Notably, the combined use of both simvastatin and EMD resulted in more potent differentiation than that observed after a single therapy. Simvastatin activated ERK phosphorylation and treatment with ERK inhibitor blocked the messenger RNA expression of odontoblastic markers. However, in simvastatin/EMD-treated cells, the expression of these genes did not decrease significantly. Compared with other groups, the EMD- and simvastatin/EMD-treated group showed a greater expression of osterix.
Conclusions
Simvastatin promotes odontoblastic differentiation of hDPCs via the ERK signaling pathway. In addition, simvastatin-induced differentiation is facilitated by co-treatment with EMD. Collectively, these results suggest a new strategy to induce odontoblastic differentiation of hDPCs.
doi:10.1016/j.joen.2012.10.013
PMCID: PMC3812675  PMID: 23228261
Combination; enamel matrix derivative; extracellular signal–regulated kinase; simvastatin; odontoblastic
23.  Regulation of reactionary dentin formation by odontoblasts in response to polymicrobial invasion of dentin matrix 
Bone  2011;50(1):265-275.
Odontoblast synthesis of dentin proceeds through discrete but overlapping phases characterized by formation of a patterned organic matrix followed by remodelling and active mineralization. Microbial invasion of dentin in caries triggers an adaptive response by odontoblasts, culminating in formation of a structurally altered reactionary dentin, marked by biochemical and architectonic modifications including diminished tubularity. Scanning electron microscopy of the collagen framework in reactionary dentin revealed a radically modified yet highly organized meshwork as indicated by fractal and lacunarity analyses. Immuno-gold labelling demonstrated increased density and regular spatial distribution of dentin sialoprotein (DSP) in reactionary dentin. DSP contributes putative hydroxyapatite nucleation sites on the collagen scaffold. To further dissect the formation of this altered dentin matrix, the associated enzymatic machinery was investigated. Analysis of extracted dentin matrix indicated increased activity of matrix metalloproteinase-2 (MMP-2) in the reactionary zone referenced to physiologic dentin. Likewise, gene expression analysis of micro-dissected odontoblast layer revealed up-regulation of MMP-2. Parallel up-regulation of tissue inhibitor of metalloproteinase-2 (TIMP-2) and membrane type 1- matrix metalloproteinase (MT1-MMP) was observed in response to caries. Next, modulation of odontoblastic dentinogenic enzyme repertoire was addressed. In the odontoblast layer expression of Toll-like receptors was markedly altered in response to bacterial invasion. In carious teeth TLR-2 and the gene encoding the corresponding adaptor protein MyD88 were down-regulated whereas genes encoding TLR-4 and adaptor proteins TRAM and Mal/TIRAP were up-regulated. TLR-4 signalling mediated by binding of bacterial products has been linked to up-regulation of MMP-2. Further, increased expression of genes encoding components of the TGF-β signalling pathway, namely SMAD-2 and SMAD-4, may explain the increased synthesis of collagen by odontoblasts in caries. These findings indicate a radical adaptive response of odontoblasts to microbial invasion of dentin with resultant synthesis of modified mineralized matrix.
doi:10.1016/j.bone.2011.10.031
PMCID: PMC3246533  PMID: 22079283
Reactionary dentin; collagen network; DSP; matrix metalloproteinases; Toll-like receptors
24.  Disruption of Wnt/β-catenin Signaling in Odontoblasts and Cementoblasts Arrests Tooth Root Development in Postnatal Mouse Teeth 
Tooth development undergoes a series of complex reciprocal interactions between dental epithelium and the underlying mesenchymal cells. Compared with the study in tooth crown formation, little is known about the molecular mechanism underlying the development of tooth roots. In the present study, we conditionally knock out β-catenin gene (Ctnnb1) within developing odontoblasts and cementoblasts during the development of tooth roots, and observed rootless molars as well as incomplete incisors. Histological analyses revealed intact structure of molar crown and labial side of incisor, however, as for the molar roots and the lingual portion of incisor, the formation of dentin and periodontal tissues were greatly hampered. In situ hybridization experiments using probes of odontoblastic marker genes collagen type I, alpha 1 (Col1a1), osteocalcin (OC) and dentin sialophosphoprotein (Dspp) manifested striking undifferentiation of root odontoblasts in which Ctnnb1 was eliminated. Bromodeoxyuridine (BrdU) labeling and proliferating cell nuclear antigen (PCNA) immunohistochemical experiments also showed retarded proliferation of pre-odontoblasts in mutant mice. However, cell apoptosis was not affected. Additionally, a disrupted formation of cementoblasts, suggested by the absence of transcripts of bone sialoprotein (Bsp) in follicle mesenchyme, was also evident in mutant mice. Our study provides strong in vivo evidence to confirm that Wnt/β-catenin signaling is functionally significant to root odontogenesis and cementogenesis during the tooth root development.
doi:10.7150/ijbs.5476
PMCID: PMC3596708  PMID: 23494738
Tooth root development; Odontoblast; Wnt/β-catenin signaling; mouse model.
25.  Usf1, a suppressor of the circadian Clock mutant, reveals the nature of the DNA-binding of the CLOCK:BMAL1 complex in mice 
eLife  2013;2:e00426.
Genetic and molecular approaches have been critical for elucidating the mechanism of the mammalian circadian clock. Here, we demonstrate that the ClockΔ19 mutant behavioral phenotype is significantly modified by mouse strain genetic background. We map a suppressor of the ClockΔ19 mutation to a ∼900 kb interval on mouse chromosome 1 and identify the transcription factor, Usf1, as the responsible gene. A SNP in the promoter of Usf1 causes elevation of its transcript and protein in strains that suppress the Clock mutant phenotype. USF1 competes with the CLOCK:BMAL1 complex for binding to E-box sites in target genes. Saturation binding experiments demonstrate reduced affinity of the CLOCKΔ19:BMAL1 complex for E-box sites, thereby permitting increased USF1 occupancy on a genome-wide basis. We propose that USF1 is an important modulator of molecular and behavioral circadian rhythms in mammals.
DOI: http://dx.doi.org/10.7554/eLife.00426.001
eLife digest
Circadian rhythms are biochemical, physiological and behavioral processes that follow a 24-hr cycle, responding primarily to the periods of light and dark, and they have been observed in bacteria, fungi, plants and animals. The circadian clock that drives these rhythms—which dictate our sleep patterns and other processes—involves a set of genes and proteins that participate in a collection of positive and negative feedback loops.
Previous research has mainly focused on identifying core clock genes—that is, genes that make up the molecular clock—and studying the functions of these genes and the proteins they code for. However, it has become clear that other clock genes are also involved in circadian behavior, and it has been proposed that polymorphisms in these non-core clock genes could contribute to the variations in circadian behavior displayed by different mammals.
One important feedback loop in mammals involves two key transcription factors, CLOCK and BMAL1, that combine to form a complex that initiates the transcription of the negative feedback genes, Period and Cryptochrome. Shimomura et al. discovered that Usf1, a gene that codes for a transcription factor that is typically involved in lipid and carbohydrate metabolism, as well as other cellular processes, is also important. In particular, this transcription factor is capable of partially rescuing an abnormal circadian rhythm caused by a mutation in the Clock gene in mice.
Shimomura et al. showed that the proteins expressed by the mutant Clock gene can bind to the same regulatory sites in the genome as the normal CLOCK:BMAL1 complex, but that gene expression of these targets is reduced because transcriptional activation is lower and binding of the complex is not as strong. However, proteins expressed by the Usf1 gene are able to counter this by binding to the same sites in the genome and compensating for the mutant CLOCK protein. Further experiments are needed to explore how the interactions between the USF1 and CLOCK:BMAL1 transcriptional networks regulate circadian rhythms and, possibly, carbohydrate and lipid metabolism as well.
DOI: http://dx.doi.org/10.7554/eLife.00426.002
doi:10.7554/eLife.00426
PMCID: PMC3622178  PMID: 23580255
Clock gene; USF1; genetic modifier; circadian rhythms; mouse genetics; ChIP-Seq; Mouse

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