Role of FGF10/FGFR2b Signaling in Homeostasis and Regeneration of Adult Lacrimal Gland and Corneal Epithelium Proliferation.

Purpose: Fibroblast growth factor 10 (FGF10) is involved in eye, meibomian, and lacrimal gland (LG) development, but its function in adult eye structures remains unknown. This study aimed to characterize the role of FGF10 in homeostasis and regeneration of adult LG and corneal epithelium proliferation. Methods: Quantitative reverse transcription PCR was used for analysis of FGF10 expression in both early postnatal and adult mouse LG, and RNA sequencing was used to analyze gene expression during LG inflammation. FGF10 was injected into the LG of two mouse models of Sjögren's syndrome and healthy controls. Flow cytometry, BrdU cell proliferation assay, immunostaining, and hematoxylin and eosin staining were used to evaluate the effects of FGF10 injection on inflammation and cell proliferation in vivo. Mouse and human epithelial cell cultures were treated with FGF10 in vitro, and cell viability was assessed using WST-8 and adenosine triphosphate (ATP) quantification assays. Results: The level of Fgf10 mRNA expression was lower in adult LG compared to early postnatal LG and was downregulated in chronic inflammation. FGF10 injection into diseased LGs significantly increased cell proliferation and decreased the number of B cells. Mouse and human corneal epithelial cell cultures treated with FGF10 showed significantly higher cell viability and greater cell proliferation. Conclusions: FGF10 appears to promote regeneration in damaged adult LGs. These findings have therapeutic potential for developing new treatments for dry eye disease targeting the ability of the cornea and LG to regenerate.

The Effect of Anti-Inflammatory Topical Ophthalmic Treatments on In Vitro Corneal Epithelial Cells.

Purpose: To compare the effect of three commonly prescribed anti-inflammatory eye drops on corneal epithelial cells in vitro. Methods: Three different lines of human corneal epithelial cells were tested: primary cells cultured from donor tissue, commercially available primary cells, and immortalized cells. Cells were seeded on 96-well plates and treated with the following eye drops: cyclosporine 0.05%, lifitegrast 5%, and tacrolimus 0.03% or 0.1%. Exposure times tested were 30 seconds, 1 minute, 2 minutes, 1 hour, 2 hours, 4 hours, and 24 hours. Brightfield images and viability assays were analyzed 48 to 72 hours after the initiation of treatments. At least five replicates were tested per drug and time exposure. Results: Commercially obtained primary cells showed reduced viability following 1 hour with tacrolimus 0.1% (8%; P = 0.043%) and 4 hours with tacrolimus 0.03% (17%; P = 0.042%). Lifitegrast exposure reduced primary cell viability after 4 hours (10%; P = 0.042). Cell viability in primary cells was not deleteriously affected following exposure to cyclosporine for up to 4 hours. A similar trend was observed in both primary cells cultured from donor tissue and immortalized human corneal epithelial cells, demonstrating greater decreases in cell viability in tacrolimus compared to lifitegrast and cyclosporine. Light microscopy imaging for analysis of cell morphology and confluence supported the results. Conclusions: Tacrolimus showed the highest impact on corneal epithelium survival in vitro, and cyclosporine proved the most protective. Translational Relevance: Comparing anti-inflammatory eye drops on corneal epithelial cells in vitro may inform eye drop selection and development for clinical purposes.

Production of chitin nanoparticles by bottom-up approach from alkaline chitin solution.

Most of the series of nanochitins have been produced by the break-down process. In this study, chitin nanoparticles were prepared by a bottom-up process. Chitin was treated with sodium hydroxide to obtain an alkaline chitin aqueous solution. The alkaline chitin was regenerated by neutralization and then vigorously stirred to obtain chitin nanoparticles. The average particle size of the chitin nanoparticles was 7 nm. The individual particles were stably dispersed in water. Chitin nanoparticles had lower crystallinity than the raw material chitin and the surface of the chitin nanoparticles regenerated in water were presumed to be hydrophilic. The low crystallinity and the high hydrophilicity of the surface contributed to the high dispersibility of the chitin nanoparticles in water. Chitin nanoparticles had higher heat resistance than the raw material chitin, suggesting a large change in the higher-order structure associated with dissolution and subsequent regeneration of chitin. Since chitin nanoparticles interact with each other less than chitin nanofibers produced by mechanical treatment, the viscosity of nanoparticles was smaller than that of nanofibers. Therefore, it can be prepared at a high concentration. In addition, the chitin nanoparticles can be easily redispersed in water after being concentrated by centrifugation.

Circadian rhythm bifurcation induces flexible phase resetting by reducing circadian amplitude.

Shift-work and jet-lag-related disorders are caused by the limited flexibility of the suprachiasmatic nucleus (SCN), a master circadian clock in the hypothalamus, to adjust to new light-dark (LD) cycles. Recent findings confirmed here establish that behavioral jet lag after simulated time-zone travel is virtually eliminated following bifurcated circadian entrainment under a novel and atypical 24-h light:dark:light:dark (LDLD) cycle. To investigate the mechanisms of this fast resetting, we examined the oscillatory stability of the SCN and peripheral tissues in LDLD-bifurcated mice employing the dissection procedure as a perturbing resetting stimulus. SCN, lung, liver, and adrenal tissue were extracted at times throughout the day from female and male PER2::Luciferase knock-in mice entrained to either LDLD or a normal LD cycle. Except for adrenals, the phase of the cultured explants was more strongly set by dissection under LDLD than under normal LD. Acute bioluminescence levels of SCN explants indicate that the rhythm amplitude of PER2 is reduced and phase is altered in LDLD. Real-time quantitative PCR suggests that amplitude and rhythmicity of canonical clock genes in the lung, liver, and kidney are also significantly reduced in LDLD in vivo. Furthermore, spatiotemporal patterns of PER2 peak time in cultured SCN were altered in LDLD. These results suggest that altered gene expression patterns in the SCN caused by bifurcation likely result in fast resetting of behavior and cultured explants, consistent with previously reported mathematical models. Thus, non-invasive, simple light manipulations can make circadian rhythms more adaptable to abrupt shifts in the environmental LD cycle.

A novel hydrochloride-free chitosan oligosaccharide production method to improve taste.

Chitosan oligosaccharide hydrochloride (COS-HCl) has an unpleasant taste. To improve this taste, we used an enzymatic hydrolysis with chitosan to manufacture hydrochloride-free chitosan oligosaccharide (HFCOS). We found that HFCOS powder with weight-average molecular weight (Mw) of approximately 1000 and a high degree of deacetylation can be obtained from amorphous chitosan in carbonated water by enzymatic hydrolysis because amorphous chitosan slightly dissolves in carbonated water. HFCOS was stable enough to be atomized by spray-drying. However, HFCOS yield was quite low (15.7%). There were few chloride ions in HFCOS (0.1% ±â€¯0.1%), in contrast to the number of chloride ions in COS-HCl (16.6% ±â€¯0.6%) with statistical significance (p < 0.01). The molar ratio of anion/amino group of HFCOS was only 0.05. Therefore, it appeared that the amino groups of HFCOS were mostly free bases. In sensory evaluations, 5% w/w HFCOS was significantly sweeter than 5% w/w COS-HCl (p < 0.01); saltiness, acidity, bitterness were significantly weak (p < 0.01); and aftertaste and overall taste were significantly improved (p < 0.01). These results suggest that HFCOS can be obtained by a novel method of enzymatically hydrolyzing amorphous chitosan dissolved in carbonated water and this is one way to improve its taste.

Calcium Circadian Rhythmicity in the Suprachiasmatic Nucleus: Cell Autonomy and Network Modulation.

Circadian rhythms of mammalian physiology and behavior are coordinated by the suprachiasmatic nucleus (SCN) in the hypothalamus. Within SCN neurons, various aspects of cell physiology exhibit circadian oscillations, including circadian clock gene expression, levels of intracellular Ca2+ ([Ca2+]i), and neuronal firing rate. [Ca2+]i oscillates in SCN neurons even in the absence of neuronal firing. To determine the causal relationship between circadian clock gene expression and [Ca2+]i rhythms in the SCN, as well as the SCN neuronal network dependence of [Ca2+]i rhythms, we introduced GCaMP3, a genetically encoded fluorescent Ca2+ indicator, into SCN neurons from PER2::LUC knock-in reporter mice. Then, PER2 and [Ca2+]i were imaged in SCN dispersed and organotypic slice cultures. In dispersed cells, PER2 and [Ca2+]i both exhibited cell autonomous circadian rhythms, but [Ca2+]i rhythms were typically weaker than PER2 rhythms. This result matches the predictions of a detailed mathematical model in which clock gene rhythms drive [Ca2+]i rhythms. As predicted by the model, PER2 and [Ca2+]i rhythms were both stronger in SCN slices than in dispersed cells and were weakened by blocking neuronal firing in slices but not in dispersed cells. The phase relationship between [Ca2+]i and PER2 rhythms was more variable in cells within slices than in dispersed cells. Both PER2 and [Ca2+]i rhythms were abolished in SCN cells deficient in the essential clock gene Bmal1. These results suggest that the circadian rhythm of [Ca2+]i in SCN neurons is cell autonomous and dependent on clock gene rhythms, but reinforced and modulated by a synchronized SCN neuronal network.

Cellular circadian oscillators in the suprachiasmatic nucleus remain coupled in the absence of connexin-36.

In mammals, the master circadian clock resides in the suprachiasmatic nucleus (SCN). The SCN is characterized by robust circadian oscillations of clock gene expression and neuronal firing. The synchronization of circadian oscillations among individual cells in the SCN is attributed to intercellular coupling. Previous studies have shown that gap junctions, specifically those composed of connexin-36 (Cx36) subunits, are required for coupling of electrical firing among SCN neurons at a time scale of milliseconds. However, it remains unknown whether Cx36 gap junctions also contribute to coupling of circadian (∼24h) rhythms of clock gene expression. Here, we investigated circadian expression patterns of the clock gene Period 2 (Per2) in the SCN of Cx36-deficient mice using luminometry and single-cell bioluminescence imaging. Surprisingly, we found that synchronization of circadian PER2 expression rhythms is maintained in SCN explants from Cx36-deficient mice. Since Cx36 expression levels change with age, we also tested circadian running-wheel behavior of juvenile (3-4weeks old) and adult (9-30weeks old) Cx36-deficient mice. We found that impact of connexin-36 expression on circadian behavior changes greatly during postnatal development. However, consistent with the intact synchrony among SCN cells in cultured explants, Cx36-deficient mice had intact locomotor circadian rhythms, although adults displayed a lengthened period in constant darkness. Our data indicate that even though Cx36 may be required for electrical coupling of SCN cells, it does not affect coupling of molecular clock gene rhythms. Thus, electrical coupling of neurons and coupling of circadian clock gene oscillations can be regulated independently in the SCN.

Cardiomyocyte Circadian Oscillations Are Cell-Autonomous, Amplified by ß-Adrenergic Signaling, and Synchronized in Cardiac Ventricle Tissue.

Circadian clocks impact vital cardiac parameters such as blood pressure and heart rate, and adverse cardiac events such as myocardial infarction and sudden cardiac death. In mammals, the central circadian pacemaker, located in the suprachiasmatic nucleus of the hypothalamus, synchronizes cellular circadian clocks in the heart and many other tissues throughout the body. Cardiac ventricle explants maintain autonomous contractions and robust circadian oscillations of clock gene expression in culture. In the present study, we examined the relationship between intrinsic myocardial function and circadian rhythms in cultures from mouse heart. We cultured ventricular explants or dispersed cardiomyocytes from neonatal mice expressing a PER2::LUC bioluminescent reporter of circadian clock gene expression. We found that isoproterenol, a ß-adrenoceptor agonist known to increase heart rate and contractility, also amplifies PER2 circadian rhythms in ventricular explants. We found robust, cell-autonomous PER2 circadian rhythms in dispersed cardiomyocytes. Single-cell rhythms were initially synchronized in ventricular explants but desynchronized in dispersed cells. In addition, we developed a method for long-term, simultaneous monitoring of clock gene expression, contraction rate, and basal intracellular Ca2+ level in cardiomyocytes using PER2::LUC in combination with GCaMP3, a genetically encoded fluorescent Ca2+ reporter. In contrast to robust PER2 circadian rhythms in cardiomyocytes, we detected no rhythms in contraction rate and only weak rhythms in basal Ca2+ level. In summary, we found that PER2 circadian rhythms of cardiomyocytes are cell-autonomous, amplified by adrenergic signaling, and synchronized by intercellular communication in ventricle explants, but we detected no robust circadian rhythms in contraction rate or basal Ca2+.

Lithium effects on circadian rhythms in fibroblasts and suprachiasmatic nucleus slices from Cry knockout mice.

Lithium is widely used as a treatment of bipolar disorder, a neuropsychiatric disorder associated with disrupted circadian rhythms. Lithium is known to lengthen period and increase amplitude of circadian rhythms. One possible pathway for these effects involves inhibition of glycogen synthase kinase-3ß (GSK-3ß), which regulates degradation of CRY2, a canonical clock protein determining circadian period. CRY1 is also known to play important roles in regulating circadian period and phase, although there is no evidence that it is similarly phosphorylated by GSK-3ß. In this paper, we tested the hypothesis that lithium affects circadian rhythms through CRYs. We cultured fibroblasts and slices of the suprachiasmatic nucleus (SCN), the master circadian pacemaker of the brain, from Cry1-/-, Cry2-/-, or wild-type (WT) mice bearing the PER2:LUC circadian reporter. Lithium was applied in the culture medium, and circadian rhythms of PER2 expression were measured. In WT and Cry2-/- fibroblasts, 10mM lithium increased PER2 expression and rhythm amplitude but not period, and 1mM lithium did not affect either period or amplitude. In non-rhythmic Cry1-/- fibroblasts, 10mM lithium increased PER2 expression. In SCN slices, 1mM lithium lengthened period ∼1h in all genotypes, but did not affect amplitude except in Cry2-/- SCN. Thus, the amplitude-enhancing effect of lithium in WT fibroblasts was unaffected by Cry2 knockout and occurred in the absence of period-lengthening, whereas the period-lengthening effect of lithium in WT SCN was unaffected by Cry1 or Cry2 knockout and occurred in the absence of rhythm amplification, suggesting that these two effects of lithium on circadian rhythms are independent of CRYs and of each other.

Light evokes rapid circadian network oscillator desynchrony followed by gradual phase retuning of synchrony.

Circadian neural circuits generate near 24-hr physiological rhythms that can be entrained by light to coordinate animal physiology with daily solar cycles. To examine how a circadian circuit reorganizes its activity in response to light, we imaged period (per) clock gene cycling for up to 6 days at single-neuron resolution in whole-brain explant cultures prepared from per-luciferase transgenic flies. We compared cultures subjected to a phase-advancing light pulse (LP) to cultures maintained in darkness (DD). In DD, individual neuronal oscillators in all circadian subgroups are initially well synchronized but then show monotonic decrease in oscillator rhythm amplitude and synchrony with time. The small ventral lateral neurons (s-LNvs) and dorsal lateral neurons (LNds) exhibit this decrease at a slower relative rate. In contrast, the LP evokes a rapid loss of oscillator synchrony between and within most circadian neuronal subgroups, followed by gradual phase retuning of whole-circuit oscillator synchrony. The LNds maintain high rhythmic amplitude and synchrony following the LP along with the most rapid coherent phase advance. Immunocytochemical analysis of PER shows that these dynamics in DD and LP are recapitulated in vivo. Anatomically distinct circadian neuronal subgroups vary in their response to the LP, showing differences in the degree and kinetics of their loss, recovery and/or strengthening of synchrony, and rhythmicity. Transient desynchrony appears to be an integral feature of light response of the Drosophila multicellular circadian clock. Individual oscillators in different neuronal subgroups of the circadian circuit show distinct kinetic signatures of light response and phase retuning.

Highly sensitive luciferase reporter assay using a potent destabilization sequence of calpain 3.

Reporter assays that use luciferases are widely employed for monitoring cellular events associated with gene expression in vitro and in vivo. To improve the response of the luciferase reporter to acute changes of gene expression, a destabilization sequence is frequently used to reduce the stability of luciferase protein in the cells, which results in an increase of sensitivity of the luciferase reporter assay. In this study, we identified a potent destabilization sequence (referred to as the C9 fragment) consisting of 42 amino acid residues from human calpain 3 (CAPN3). Whereas the half-life of Emerald Luc (ELuc) from the Brazilian click beetle Pyrearinus termitilluminans was reduced by fusing PEST (t1/2=9.8 to 2.8h), the half-life of C9-fused ELuc was significantly shorter (t1/2=1.0h) than that of PEST-fused ELuc when measurements were conducted at 37°C. In addition, firefly luciferase (luc2) was also markedly destabilized by the C9 fragment compared with the humanized PEST sequence. These results indicate that the C9 fragment from CAPN3 is a much more potent destabilization sequence than the PEST sequence. Furthermore, real-time bioluminescence recording of the activation kinetics of nuclear factor-κB after transient treatment with tumor necrosis factor α revealed that the response of C9-fused ELuc is significantly greater than that of PEST-fused ELuc, demonstrating that the use of the C9 fragment realizes a luciferase reporter assay that has faster response speed compared with that provided by the PEST sequence.

Concentrations of trace elements in American alligators (Alligator mississippiensis) from Florida, USA.

Concentrations of 28 trace elements (Li, Mg, Al, V, Cr, Mn, Fe, Co, Ni, Cu, Zn, Ga, As, Se, Rb, Sr, Mo, Ag, Cd, In, Sn, Sb, Cs, Tl, Hg, Pb, and Bi) in the livers of juvenile and adult American alligators inhabiting two central Florida lakes, Lake Apopka (LA), and Lake Woodruff National Wildlife Refuge (LW) and one lagoon population located in Merritt Island National Wildlife Refuge (MINWR; NASA), were determined. In juveniles from MINWR, concentrations of nine elements (Li, Fe, Ni, Sr, In, Sb, Hg, Pb and Bi) were significantly higher, whereas six elements (V, Fe, As, Sr, Hg and Bi) were elevated in adults (p<0.05) obtained from MINWR. Significant enrichment of some trace elements in adults, relative to juveniles, was observed at all three sampling areas. Specifically, Fe, Pb and Hg were significantly elevated in adults when compared to juveniles, suggesting age-dependent accumulation of these elements. Further, As, Se and Sn showed the same trend but only in animals collected from MINWR. Mean Fe concentrations in the livers of adults from LA, LW and MINWR were 1770 µg g(-1) DW, 3690 µg g(-1) DW and 5250 µg g(-1) DW, respectively. More than half of the adult specimens from LW and MINWR exhibited elevated hepatic Fe concentrations that exceed the threshold value for toxic effects in donkey, red deer and human. These results prompted us to express our concern on possible exposure and health effects in American alligators by some trace elements derived from NASA activities.

Fibroblast PER2 circadian rhythmicity depends on cell density.

Like neurons in the suprachiasmatic nucleus (SCN), the master circadian pacemaker in the brain, single fibroblasts can function as independent oscillators. In the SCN, synaptic and paracrine signaling among cells creates a robust, synchronized circadian oscillation, whereas there is no evidence for such integration in fibroblast cultures. However, interactions among single-cell fibroblast oscillators cannot be completely excluded, because fibroblasts were not isolated in previous work. In this study, we tested the autonomy of fibroblasts as single-cell circadian oscillators in high- and low-density culture, by single-cell imaging of cells from PER2::LUC circadian reporter mice. We found greatly reduced PER2::LUC rhythmicity in low-density cultures, which could result from lack of either constitutive or rhythmic paracrine signals from neighboring fibroblasts. To discriminate between these 2 possibilities, we mixed PER2::LUC wild-type (WT) cells with nonluminescent, nonrhythmic Bmal1-/- cells, so that density of rhythmic cells was low but overall cell density remained high. In this condition, WT cells showed clear rhythmicity similar to high-density cultures. We also mixed PER2::LUC WT cells with nonluminescent, long period Cry2-/- cells. In this condition, WT cells showed a period no different from cells cultured with rhythmic WT cells or nonrhythmic Bmal1-/- cells. In previous work, we found that low K⁺ suppresses fibroblast rhythmicity, and we and others have found that either low K⁺ or low Ca²âº suppresses SCN rhythmicity. Therefore, we attempted to rescue rhythmicity of low-density fibroblasts with high K⁺ (21 mM), high Ca²âº (3.6 mM), or conditioned medium. Conditioned medium from high-density fibroblast cultures rescued rhythmicity of low-density cultures, whereas high K⁺ or Ca²âº medium did not consistently rescue rhythmicity. These data suggest that fibroblasts require paracrine signals from adjacent cells for normal expression of rhythmicity, but that these signals do not have to be rhythmic, and that rhythmic signals from other cells do not affect the intrinsic periods of fibroblasts.

Cellular bioluminescence imaging.

Bioluminescence imaging of live cells has recently been recognized as an important alternative to fluorescence imaging. Fluorescent probes are much brighter than bioluminescent probes (luciferase enzymes) and, therefore, provide much better spatial and temporal resolution and much better contrast for delineating cell structure. However, with bioluminescence imaging there is virtually no background or toxicity. As a result, bioluminescence can be superior to fluorescence for detecting and quantifying molecules and their interactions in living cells, particularly in long-term studies. Structurally diverse luciferases from beetle and marine species have been used for a wide variety of applications, including tracking cells in vivo, detecting protein-protein interactions, measuring levels of calcium and other signaling molecules, detecting protease activity, and reporting circadian clock gene expression. Such applications can be optimized by the use of brighter and variously colored luciferases, brighter microscope optics, and ultrasensitive, low-noise cameras. This article presents a review of how bioluminescence differs from fluorescence, its applications to cellular imaging, and available probes, optics, and detectors. It also gives practical suggestions for optimal bioluminescence imaging of single cells.

Identification of small molecule activators of cryptochrome.

Impairment of the circadian clock has been associated with numerous disorders, including metabolic disease. Although small molecules that modulate clock function might offer therapeutic approaches to such diseases, only a few compounds have been identified that selectively target core clock proteins. From an unbiased cell-based circadian phenotypic screen, we identified KL001, a small molecule that specifically interacts with cryptochrome (CRY). KL001 prevented ubiquitin-dependent degradation of CRY, resulting in lengthening of the circadian period. In combination with mathematical modeling, our studies using KL001 revealed that CRY1 and CRY2 share a similar functional role in the period regulation. Furthermore, KL001-mediated CRY stabilization inhibited glucagon-induced gluconeogenesis in primary hepatocytes. KL001 thus provides a tool to study the regulation of CRY-dependent physiology and aid development of clock-based therapeutics of diabetes.

Fibroblast circadian rhythms of PER2 expression depend on membrane potential and intracellular calcium.

The suprachiasmatic nucleus (SCN) of the hypothalamus synchronizes circadian rhythms of cells and tissues throughout the body. In SCN neurons, rhythms of clock gene expression are suppressed by manipulations that hyperpolarize the plasma membrane or lower intracellular Ca(2+). However, whether clocks in other cells also depend on membrane potential and calcium is unknown. In this study, the authors investigate the effects of membrane potential and intracellular calcium on circadian rhythms in mouse primary fibroblasts. Rhythms of clock gene expression were monitored using a PER2::LUC knockin reporter. Rhythms were lost or delayed at lower (hyperpolarizing) K(+) concentrations. Bioluminescence imaging revealed that this loss of rhythmicity in cultures was due to loss of rhythmicity of single cells rather than loss of synchrony among cells. In lower Ca(2+) concentrations, rhythms were advanced or had shorter periods. Buffering intracellular Ca(2+) by the calcium chelator 1,2-Bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis acetoxymethyl ester (BAPTA-AM) or manipulation of inositol triphosphate (IP(3))-sensitive intracellular calcium stores by thapsigargin delayed rhythms. These results suggest that the circadian clock in fibroblasts, as in SCN neurons, is regulated by membrane potential and Ca(2+). Changes in intracellular Ca(2+) may mediate the effects of membrane potential observed in this study.

A dual-color luciferase assay system reveals circadian resetting of cultured fibroblasts by co-cultured adrenal glands.

In mammals, circadian rhythms of various organs and tissues are synchronized by pacemaker neurons in the suprachiasmatic nucleus (SCN) of the hypothalamus. Glucocorticoids released from the adrenal glands can synchronize circadian rhythms in other tissues. Many hormones show circadian rhythms in their plasma concentrations; however, whether organs outside the SCN can serve as master synchronizers to entrain circadian rhythms in target tissues is not well understood. To further delineate the function of the adrenal glands and the interactions of circadian rhythms in putative master synchronizing organs and their target tissues, here we report a simple co-culture system using a dual-color luciferase assay to monitor circadian rhythms separately in various explanted tissues and fibroblasts. In this system, circadian rhythms of organs and target cells were simultaneously tracked by the green-emitting beetle luciferase from Pyrearinus termitilluminans (ELuc) and the red-emitting beetle luciferase from Phrixothrix hirtus (SLR), respectively. We obtained tissues from the adrenal glands, thyroid glands, and lungs of transgenic mice that expressed ELuc under control of the promoter from a canonical clock gene, mBmal1. The tissues were co-cultured with Rat-1 fibroblasts as representative target cells expressing SLR under control of the mBmal1 promoter. Amplitudes of the circadian rhythms of Rat-1 fibroblasts were potentiated when the fibroblasts were co-cultured with adrenal gland tissue, but not when co-cultured with thyroid gland or lung tissue. The phases of Rat-1 fibroblasts were reset by application of adrenal gland tissue, whereas the phases of adrenal gland tissue were not influenced by Rat-1 fibroblasts. Furthermore, the effect of the adrenal gland tissue on the fibroblasts was blocked by application of a glucocorticoid receptor (GR) antagonist. These results demonstrate that glucocorticoids are strong circadian synchronizers for fibroblasts and that this co-culture system is a useful tool to analyze humoral communication between different tissues or cell populations.

Dual-color luciferase mouse directly demonstrates coupled expression of two clock genes.

We have established a dual-color transgenic mouse that simultaneously reports the expression of two clock genes, Bmal1 and Per2, in a single tissue. The expression of the two genes is monitored with green- and red-emitting beetle luciferases with a single luminescent substrate. Antiphasic oscillations of Bmal1 and Per2, consistent with their endogenous mRNA profiles, were clearly monitored in the suprachiasmatic nucleus (SCN), the master circadian pacemaker, and in the peripheral tissues, demonstrating that the system allows the long-term, quantitative, and simultaneous monitoring of the expression of the two genes. We also showed that although the expression patterns of Bmal1 and Per2 in each organ are strictly antiphasic, the recorded circadian phases and periods of both genes varied between organs. The phase shifts in the expression of both genes in the SCN, induced by a change of medium, also occurred in a similar manner. Therefore, this dual-color luciferase mouse allows noninvasive and continuous monitoring of the coupled expression of two clock genes. This system provides a simple technique with which to unravel the complex interactions of two genes in the body.

Enhanced beetle luciferase for high-resolution bioluminescence imaging.

We developed an enhanced green-emitting luciferase (ELuc) to be used as a bioluminescence imaging (BLI) probe. ELuc exhibits a light signal in mammalian cells that is over 10-fold stronger than that of the firefly luciferase (FLuc), which is the most widely used luciferase reporter gene. We showed that ELuc produces a strong light signal in primary cells and tissues and that it enables the visualization of gene expression with high temporal resolution at the single-cell level. Moreover, we successfully imaged the nucleocytoplasmic shuttling of importin alpha by fusing ELuc at the intracellular level. These results demonstrate that the use of ELuc allows a BLI spatiotemporal resolution far greater than that provided by FLuc.