Koh-hei Masumoto

Melanopsin-dependent photo-perturbation reveals desynchronization underlying the singularity of mammalian circadian clocks.

Singularity behaviour in circadian clocks — the loss of robust circadian rhythms following exposure to a stimulus such as a pulse of bright light — is one of the fundamental but mysterious properties of clocks. To quantitatively perturb and accurately measure the dynamics of cellular clocks, we synthetically produced photo-responsiveness within mammalian cells by exogenously introducing the photoreceptor melanopsin and continuously monitoring the effect of photo-perturbation on the state of cellular clocks. Here we report that a critical light pulse drives cellular clocks into singularity behaviour. Our theoretical analysis consistently predicts and subsequent single-cell level observation directly proves that desynchronization of individual cellular clocks underlies singularity behaviour. Our theoretical framework also explains why singularity behaviours have been experimentally observed in various organisms, and it suggests that desynchronization is a plausible mechanism for the observable singularity of circadian clocks. Importantly, these in vitro and in silico findings are further supported by in vivo observations that desynchronization underlies the multicell-level amplitude decrease in the rat suprachiasmatic nucleus induced by critical light pulses.

Analysis and synthesis of high-amplitude Cis-elements in the mammalian circadian clock

Mammalian circadian clocks consist of regulatory loops mediated by Clock/Bmal1-binding elements, DBP/E4BP4 binding elements, and RevErbA/ROR binding elements. As a step toward system-level understanding of the dynamic transcriptional regulation of the oscillator, we constructed and used a mammalian promoter/enhancer database (http://promoter.cdb.riken.jp/) with computational models of the Clock/Bmal1-binding elements, DBP/E4BP4 binding elements, and RevErbA/ROR binding elements to predict new targets of the clock and subsequently validated these targets at the level of the cell and organism. We further demonstrated the predictive nature of these models by generating and testing synthetic regulatory elements that do not occur in nature and showed that these elements produced high-amplitude circadian gene regulation. Biochemical experiments to characterize these synthetic elements revealed the importance of the affinity balance between transactivators and transrepressors in generating high-amplitude circadian transcriptional output. These results highlight the power of comparative genomics approaches for system-level identification and knowledge-based design of dynamic regulatory circuits.

Variations in PROKR2, But Not PROK2, Are Associated With Hypopituitarism and Septo-optic Dysplasia

Context: Loss-of-function mutations in PROK2 and PROKR2 have been implicated in Kallmann syndrome (KS), characterized by hypogonadotropic hypogonadism and anosmia. Recent data suggest overlapping phenotypes/genotypes between KS and congenital hypopituitarism (CH), including septo-optic dysplasia (SOD). Objective: We screened a cohort of patients with complex forms of CH (n = 422) for mutations in PROK2 and PROKR2. Results: We detected 5 PROKR2 variants in 11 patients with SOD/CH: novel p.G371R and previously reported p.A51T, p.R85L, p.L173R, and p.R268C—the latter 3 being known functionally deleterious variants. Surprisingly, 1 patient with SOD was heterozygous for the p.L173R variant, whereas his phenotypically unaffected mother was homozygous for the variant. We sought to clarify the role of PROKR2 in hypothalamopituitary development through analysis of Prokr2−/− mice. Interestingly, these revealed predominantly normal hypothalamopituitary development and terminal cell differentiation, with the exception of reduced LH; this was inconsistent with patient phenotypes and more analogous to the healthy mother, although she did not have KS, unlike the Prokr2−/− mice. Conclusions: The role of PROKR2 in the etiology of CH, SOD, and KS is uncertain, as demonstrated by no clear phenotype-genotype correlation; loss-of-function variants in heterozygosity or homozygosity can be associated with these disorders. However, we report a phenotypically normal parent, homozygous for p.L173R. Our data suggest that the variants identified herein are unlikely to be implicated in isolation in these disorders; other genetic or environmental modifiers may also impact on the etiology. Given the phenotypic variability, genetic counseling may presently be inappropriate.

Angiopoietin-Like 2, a Circadian Gene, Improves Type 2 Diabetes Through Potentiation of Insulin Sensitivity in Mice Adipocytes

Angiopoietin-like (Angptl)2, a member of the Angptl protein family, is predominantly secreted from adipose tissue and the heart. Here, we demonstrate that the expression of Angptl2 in epididymal adipose tissue of C57BL/6J mice shows pulsatility and circadian rhythmicity and that the rhythmicity was disrupted in high-fat-fed and leptin receptor-deficient diabetic db/db mice with insulin resistance. To investigate whether the reduction in Angptl2 expression was related to the progression of diabetes, we treated db/db mice with recombinant Angptl2 for 4 wk during the peak period of Angptl2 expression in C57BL/6J mice. Angptl2-treated mice showed decreases in plasma glucose, insulin, triglyceride, and fatty acid levels and an increase in plasma adiponectin, a therapeutic regulator of insulin resistance, leading to improvements in glucose tolerance. In cultured adipocytes, recombinant Angptl2 increased adiponectin expression and stimulated insulin sensitivity partially by reducing the levels of tribbles homolog 3, a specific Akt kinase inhibitory protein. Conversely, Angptl2 small interfering RNA reduced adiponectin expression, resulting in insulin resistance. In preadipocytes, treatment with Angptl2 small interfering RNA inhibited differentiation to adipocytes and reduced adiponectin expression. Taken together, our results suggest that replenishment of Angptl2 stimulates insulin sensitivity and improves the type 2 diabetic state.

Regional circadian period difference in the suprachiasmatic nucleus of the mammalian circadian center

The suprachiasmatic nucleus (SCN) is the mammalian circadian rhythm center. Individual oscillating neurons have different endogenous circadian periods, but they are usually synchronized by an intercellular coupling mechanism. The differences in the period of each oscillating neuron have been extensively studied; however, the clustering of oscillators with similar periods has not been reported. In the present study, we artificially disrupted the intercellular coupling among oscillating neurons in the SCN and observed regional differences in the periods of the oscillating small‐latticed regions of the SCN using a transgenic rat carrying a luciferase reporter gene driven by regulatory elements from a per2 clock gene (Per2::dluc rat). The analysis divided the SCN into two regions – a region with periods shorter than 24 h (short‐period region, SPR) and another with periods longer than 24 h (long‐period region, LPR). The SPR was located in the smaller medial region of the dorsal SCN, whereas the LPR occupied the remaining larger region. We also found that slices containing the medial region of the SCN generated shorter circadian periods than slices that contained the lateral region of the SCN. Interestingly, the SPR corresponded well with the region where the SCN phase wave is generated. We numerically simulated the relationship between the SPR and a large LPR. A mathematical model of the SCN based on our findings faithfully reproduced the kinetics of the oscillators in the SCN in synchronized conditions, assuming the existence of clustered short‐period oscillators.

FRNK, the Autonomously Expressed C-Terminal Region of Focal Adhesion Kinase, Is Uniquely Regulated in Vascular Smooth Muscle: Analysis of Expression in Transgenic Mice

FRNK, the autonomously expressed carboxyl‐terminal region of focal adhesion kinase (FAK), is expressed in tissues that are rich in vascular smooth muscle cells (VSMCs). Here we report the generation of transgenic mice harboring the putative FRNK promoter fused to LacZ and examine the promoter activity in situ via expression of β‐galactosidase. The transgenic mice exhibited expression of β‐galactosidase predominantly in arterial VSMCs in large and small blood vessels of major organs. Upregulation of β‐galactosidase activity was observed in tunica media following carotid injury, indicating that the FRNK promoter is activated in VSMCs in response to injury. Robust expression of β‐galactosidase in blood vessels was also detected in the developing embryo. However, expression was also observed in the midline, the nose and skin epidermis, indicating distinct transcriptional regulation of the FRNK promoter in embryogenesis. To analyze FRNK expression in vitro, we identified a 116 bp sequence in the FRNK promoter that was sufficient to function as an enhancer when fused to the minimal actin promoter and expressed in cultured smooth muscle cells. Mutation of AP‐1 and NF‐E2 binding consensus sequences within this element abrogated enhancer activity, supporting the involvement of this promoter element in VSMC expression of FRNK.

Establishment of TSH β real-time monitoring system in mammalian photoperiodism

Organisms have seasonal physiological changes in response to day length. Long‐day stimulation induces thyroid‐stimulating hormone beta subunit (TSHβ) in the pars tuberalis (PT), which mediates photoperiodic reactions like day‐length measurement and physiological adaptation. However, the mechanism of TSHβ induction for day‐length measurement is largely unknown. To screen candidate upstream molecules of TSHβ, which convey light information to the PT, we generated Luciferase knock‐in mice, which quantitatively report the dynamics of TSHβ expression. We cultured brain slices containing the PT region from adult and neonatal mice and measured the bioluminescence activities from each slice over several days. A decrease in the bioluminescence activities was observed after melatonin treatment in adult and neonatal slices. These observations indicate that the experimental system possesses responsiveness of the TSHβ expression to melatonin. Thus, we concluded that our experimental system monitors TSHβ expression dynamics in response to external stimuli.

Transcriptome Tomography for Brain Analysis in the Web-Accessible Anatomical Space

Increased information on the encoded mammalian genome is expected to facilitate an integrated understanding of complex anatomical structure and function based on the knowledge of gene products. Determination of gene expression-anatomy associations is crucial for this understanding. To elicit the association in the three-dimensional (3D) space, we introduce a novel technique for comprehensive mapping of endogenous gene expression into a web-accessible standard space: Transcriptome Tomography. The technique is based on conjugation of sequential tissue-block sectioning, all fractions of which are used for molecular measurements of gene expression densities, and the block- face imaging, which are used for 3D reconstruction of the fractions. To generate a 3D map, tissues are serially sectioned in each of three orthogonal planes and the expression density data are mapped using a tomographic technique. This rapid and unbiased mapping technique using a relatively small number of original data points allows researchers to create their own expression maps in the broad anatomical context of the space. In the first instance we generated a dataset of 36,000 maps, reconstructed from data of 61 fractions measured with microarray, covering the whole mouse brain (ViBrism: http://vibrism.riken.jp/3dviewer/ex/index.html) in one month. After computational estimation of the mapping accuracy we validated the dataset against existing data with respect to the expression location and density. To demonstrate the relevance of the framework, we showed disease related expression of Huntington’s disease gene and Bdnf. Our tomographic approach is applicable to analysis of any biological molecules derived from frozen tissues, organs and whole embryos, and the maps are spatially isotropic and well suited to the analysis in the standard space (e.g. Waxholm Space for brain-atlas databases). This will facilitate research creating and using open-standards for a molecular-based understanding of complex structures; and will contribute to new insights into a broad range of biological and medical questions.

Prokr2-deficient mice display vascular dysmorphology of the fetal testes: potential implications for Kallmann syndrome aetiology.

Kallmann syndrome is a form of hypogonadotropic hypogonadism also associated with the loss of smell. It is a phenotypically and genetically heterogeneous disorder, with mutations in several known causative genes now accounting for approximately 30% of cases. The prevalence for the disease is also much higher in males than in females, a phenomenon that remains to be fully explained. Here, we show that loss of Prokr2, which is linked to autosomal recessive Kallmann syndrome type 3 (KAL3; OMIM 244200), affects fetal testis differentiation in mice. We find that Prokr2 is specifically expressed in the XY gonads during sex determination and fetal sexual differentiation, and knockout mice display a variable degree of compromised vasculature in the fetal testes. This phenotype offers potential insight into the clinical heterogeneity observed within familial cases, and may contribute to the gender bias in Kallmann syndrome patients.

Circadian expression and specific localization of a sialyltransferase gene in the suprachiasmatic nucleus.

Polysialic acids are implicated in various biological processes such as neural cell migration, axonal growth, synaptogenesis and resetting of the circadian rhythm. Recently, polysialation has been reported to be involved in the formation and resetting of the circadian clock. However, the genes that control the circadian rhythm of polysialation have not been elucidated. In the present study, we investigated the expression profile of ST8 alpha-N-acetyl-neuraminide alpha-2,8-sialyltransferase 6 (ST8Sia VI) in the suprachiasmatic nucleus (SCN), which is one of the modification transferases that add sialic acids to type O carbohydrate chains. ST8Sia VI mRNA showed strong expression in the SCN with dynamic circadian rhythm. Further, the amount of ST8Sia VI mRNA in the SCN was increased by brief light exposure. Interestingly, the localization of ST8Sia VI mRNA in the SCN differs from those of arginine vasopressin and vasoactive intestinal peptide mRNAs, which are typical SCN subregion markers showing shell and core, dorsomedial and ventrolateral, or light-responsive and unresponsive regions, respectively. The present findings suggest that ST8siVI is involved in rhythmic polysialation in the SCN and that ST8siVI expression provides a novel compartmentation of the mammalian circadian center.