Publications by authors named "Etsuo A Susaki"

23 Publications

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Activation of Sympathetic Signaling in Macrophages Blocks Systemic Inflammation and Protects against Renal Ischemia-Reperfusion Injury.

J Am Soc Nephrol 2021 Apr 19. Epub 2021 Apr 19.

Division of CKD Pathophysiology, University of Tokyo Graduate School of Medicine, Tokyo, Japan.

Background: The sympathetic nervous system regulates immune cell dynamics. However, the detailed role of sympathetic signaling in inflammatory diseases is still unclear because it varies according to the disease situation and responsible cell types. This study focused on identifying the functions of sympathetic signaling in macrophages in LPS-induced sepsis and renal ischemia-reperfusion injury (IRI).

Methods: We performed RNA sequencing of mouse macrophage cell lines to identify the critical gene that mediates the anti-inflammatory effect of 2-adrenergic receptor (Adrb2) signaling. We also examined the effects of salbutamol (a selective Adrb2 agonist) in LPS-induced systemic inflammation and renal IRI. Macrophage-specific conditional knockout (cKO) mice and the adoptive transfer of salbutamol-treated macrophages were used to assess the involvement of macrophage Adrb2 signaling.

Results: , activation of Adrb2 signaling in macrophages induced the expression of T cell Ig and mucin domain 3 (), which contributes to anti-inflammatory phenotypic alterations. , salbutamol administration blocked LPS-induced systemic inflammation and protected against renal IRI; this protection was mitigated in macrophage-specific cKO mice. The adoptive transfer of salbutamol-treated macrophages also protected against renal IRI. Single-cell RNA sequencing revealed that this protection was associated with the accumulation of -expressing macrophages in the renal tissue.

Conclusions: The activation of Adrb2 signaling in macrophages induces anti-inflammatory phenotypic alterations partially via the induction of expression, which blocks LPS-induced systemic inflammation and protects against renal IRI.
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http://dx.doi.org/10.1681/ASN.2020121723DOI Listing
April 2021

Generation of a p16 Reporter Mouse and Its Use to Characterize and Target p16 Cells In Vivo.

Cell Metab 2020 11 18;32(5):814-828.e6. Epub 2020 Sep 18.

Division of Molecular Regulation of Inflammatory and Immune Diseases, Research Institute of Biomedical Sciences, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba 278-0022, Japan.

Cell senescence plays a key role in age-associated organ dysfunction, but the in vivo pathogenesis is largely unclear. Here, we generated a p16-Cre-tdTomato mouse model to analyze the in vivo characteristics of p16 cells at a single-cell level. We found tdTomato-positive p16 cells detectable in all organs, which were enriched with age. We also found that these cells failed to proliferate and had half-lives ranging from 2.6 to 4.2 months, depending on the tissue examined. Single-cell transcriptomics in the liver and kidneys revealed that p16 cells were present in various cell types, though most dominant in hepatic endothelium and in renal proximal and distal tubule epithelia, and that these cells exhibited heterogeneous senescence-associated phenotypes. Further, elimination of p16 cells ameliorated nonalcoholic steatohepatitis-related hepatic lipidosis and immune cell infiltration. Our new mouse model and single-cell analysis provide a powerful resource to enable the discovery of previously unidentified senescence functions in vivo.
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http://dx.doi.org/10.1016/j.cmet.2020.09.006DOI Listing
November 2020

Versatile whole-organ/body staining and imaging based on electrolyte-gel properties of biological tissues.

Nat Commun 2020 04 27;11(1):1982. Epub 2020 Apr 27.

Department of Systems Pharmacology, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-0033, Japan.

Whole-organ/body three-dimensional (3D) staining and imaging have been enduring challenges in histology. By dissecting the complex physicochemical environment of the staining system, we developed a highly optimized 3D staining imaging pipeline based on CUBIC. Based on our precise characterization of biological tissues as an electrolyte gel, we experimentally evaluated broad 3D staining conditions by using an artificial tissue-mimicking material. The combination of optimized conditions allows a bottom-up design of a superior 3D staining protocol that can uniformly label whole adult mouse brains, an adult marmoset brain hemisphere, an ~1 cm tissue block of a postmortem adult human cerebellum, and an entire infant marmoset body with dozens of antibodies and cell-impermeant nuclear stains. The whole-organ 3D images collected by light-sheet microscopy are used for computational analyses and whole-organ comparison analysis between species. This pipeline, named CUBIC-HistoVIsion, thus offers advanced opportunities for organ- and organism-scale histological analysis of multicellular systems.
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http://dx.doi.org/10.1038/s41467-020-15906-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7184626PMC
April 2020

The oral hypoxia-inducible factor prolyl hydroxylase inhibitor enarodustat counteracts alterations in renal energy metabolism in the early stages of diabetic kidney disease.

Kidney Int 2020 05 25;97(5):934-950. Epub 2019 Dec 25.

Division of Nephrology and Endocrinology, The University of Tokyo Graduate School of Medicine, Tokyo, Japan. Electronic address:

Hypoxia-inducible factor (HIF) prolyl hydroxylase inhibitors, also known as HIF stabilizers, increase endogenous erythropoietin production and serve as novel therapeutic agents against anemia in chronic kidney disease. HIF induces the expression of various genes related to energy metabolism as an adaptive response to hypoxia. However, it remains obscure how the metabolic reprogramming in renal tissue by HIF stabilization affects the pathophysiology of kidney diseases. Previous studies suggest that systemic metabolic disorders such as hyperglycemia and dyslipidemia cause alterations of renal metabolism, leading to renal dysfunction including diabetic kidney disease. Here, we analyze the effects of enarodustat (JTZ-951), an oral HIF stabilizer, on renal energy metabolism in the early stages of diabetic kidney disease, using streptozotocin-induced diabetic rats and alloxan-induced diabetic mice. Transcriptome analysis revealed that enarodustat counteracts the alterations in diabetic renal metabolism. Transcriptome analysis showed that fatty acid and amino acid metabolisms were upregulated in diabetic renal tissue and downregulated by enarodustat, whereas glucose metabolism was upregulated. These symmetric changes were confirmed by metabolome analysis. Whereas glycolysis and tricarboxylic acid cycle metabolites were accumulated and amino acids reduced in renal tissue of diabetic animals, these metabolic disturbances were mitigated by enarodustat. Furthermore, enarodustat increased the glutathione to glutathione disulfide ratio and relieved oxidative stress in renal tissue of diabetic animals. Thus, HIF stabilization counteracts alterations in renal energy metabolism occurring in incipient diabetic kidney disease.
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http://dx.doi.org/10.1016/j.kint.2019.12.007DOI Listing
May 2020

Visualization and molecular characterization of whole-brain vascular networks with capillary resolution.

Nat Commun 2020 02 27;11(1):1104. Epub 2020 Feb 27.

Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan.

Structural elucidation and molecular scrutiny of cerebral vasculature is crucial for understanding the functions and diseases of the brain. Here, we introduce SeeNet, a method for near-complete three-dimensional visualization of cerebral vascular networks with high signal-to-noise ratios compatible with molecular phenotyping. SeeNet employs perfusion of a multifunctional crosslinker, vascular casting by temperature-controlled polymerization of hybrid hydrogels, and a bile salt-based tissue-clearing technique optimized for observation of vascular connectivity. SeeNet is capable of whole-brain visualization of molecularly characterized cerebral vasculatures at the single-microvessel level. Moreover, SeeNet reveals a hitherto unidentified vascular pathway bridging cerebral and hippocampal vessels, thus serving as a potential tool to evaluate the connectivity of cerebral vasculature.
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http://dx.doi.org/10.1038/s41467-020-14786-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7046771PMC
February 2020

[Recent Developments in a Automated Diagnosis of Pathological Images and Three-dimensional Histopathology].

Authors:
Etsuo A Susaki

Brain Nerve 2019 Jul;71(7):723-732

Department of Systems Pharmacology, The University of Tokyo Graduate School of Medicine.

Rapid improvements in computing power are advancing machine learning technology using neural networks, revolutionizing the field of image analysis and allowing for automated diagnosis of pathological images. In addition, the recent development of tissue clearing technology introduces the possibility of three-dimensional (3D) pathology. The scheme of 3D pathology allows for the collection of the 3D context of tissue and would contribute to improved accuracy of automatic pathological diagnosis by machine learning.
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http://dx.doi.org/10.11477/mf.1416201344DOI Listing
July 2019

Novel 3D analysis using optical tissue clearing documents the evolution of murine rapidly progressive glomerulonephritis.

Kidney Int 2019 08 15;96(2):505-516. Epub 2019 Mar 15.

Department of Nephrology and Clinical Immunology, RWTH Aachen University Clinic, Aachen, Germany; Interdisciplinary Centre for Clinical Research (IZKF Aachen), RWTH Aachen University Hospital, Aachen, Germany; Heisenberg Chair for Preventive and Translational Nephrology, Division of Nephrology, RWTH Aachen University, Aachen, Germany. Electronic address:

Recent developments in optical tissue clearing have been difficult to apply for the morphometric analysis of organs with high cellular content and small functional structures, such as the kidney. Here, we establish combinations of genetic and immuno-labelling for single cell identification, tissue clearing and subsequent de-clarification for histoimmunopathology and transmission electron microscopy. Using advanced light microscopy and computational analyses, we investigated a murine model of crescentic nephritis, an inflammatory kidney disease typified by immune-mediated damage to glomeruli leading to the formation of hypercellular lesions and the rapid loss of kidney function induced by nephrotoxic serum. Results show a graded susceptibility of the glomeruli, significant podocyte loss and capillary injury. These effects are associated with activation of parietal epithelial cells and formation of glomerular lesions that may evolve and obstruct the kidney tubule, thereby explaining the loss of kidney function. Thus, our work provides new high-throughput endpoints for the analysis of complex tissues with single-cell resolution.
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http://dx.doi.org/10.1016/j.kint.2019.02.034DOI Listing
August 2019

Comprehensive three-dimensional analysis (CUBIC-kidney) visualizes abnormal renal sympathetic nerves after ischemia/reperfusion injury.

Kidney Int 2019 07 9;96(1):129-138. Epub 2019 Apr 9.

Division of Nephrology and Endocrinology, The University of Tokyo Graduate School of Medicine, Tokyo, Japan.

The sympathetic nervous system is critical in maintaining the homeostasis of renal functions. However, its three-dimensional (3D) structures in the kidney have not been elucidated due to limitation of conventional imaging methods. CUBIC (Clear, Unobstructed Brain/Body Imaging Cocktails and Computational analysis) is a newly developed tissue-clearing technique, which enables whole-organ 3D imaging without thin-sectioning. Comprehensive 3D imaging by CUBIC found that sympathetic nerves are primarily distributed around arteries in the mouse kidney. Notably, the sympathetic innervation density was significantly decreased 10 days after ischemia-reperfusion injury (voluminal ratio of innervation area to kidney) by about 70%. Moreover, norepinephrine levels in kidney tissue (output of sympathetic nerves) were significantly reduced in injured kidneys by 77%, confirming sympathetic denervation after ischemia-reperfusion injury. Time-course imaging indicated that innervation partially recovered although overall denervation persisted 28 days after injury, indicating a continuous sympathetic nervous abnormality during the progression of chronic kidney disease. Thus, CUBIC-kidney, the 3D imaging analysis, can be a strong imaging tool, providing comprehensive, macroscopic perspectives for kidney research.
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http://dx.doi.org/10.1016/j.kint.2019.02.011DOI Listing
July 2019

Chemical Landscape for Tissue Clearing Based on Hydrophilic Reagents.

Cell Rep 2018 08;24(8):2196-2210.e9

Department of Systems Pharmacology, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan; Laboratory for Synthetic Biology, RIKEN Center for Biosystems Dynamics Research, 1-3 Yamadaoka, Suita, Osaka 565-5241, Japan; International Research Center for Neurointelligence (WPI-IRCN), UTIAS, The University of Tokyo, Tokyo, Japan. Electronic address:

We describe a strategy for developing hydrophilic chemical cocktails for tissue delipidation, decoloring, refractive index (RI) matching, and decalcification, based on comprehensive chemical profiling. More than 1,600 chemicals were screened by a high-throughput evaluation system for each chemical process. The chemical profiling revealed important chemical factors: salt-free amine with high octanol/water partition-coefficient (logP) for delipidation, N-alkylimidazole for decoloring, aromatic amide for RI matching, and protonation of phosphate ion for decalcification. The strategic integration of optimal chemical cocktails provided a series of CUBIC (clear, unobstructed brain/body imaging cocktails and computational analysis) protocols, which efficiently clear mouse organs, mouse body including bone, and even large primate and human tissues. The updated CUBIC protocols are scalable and reproducible, and they enable three-dimensional imaging of the mammalian body and large primate and human tissues. This strategy represents a future paradigm for the rational design of hydrophilic clearing cocktails that can be used for large tissues.
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http://dx.doi.org/10.1016/j.celrep.2018.07.056DOI Listing
August 2018

Comparison of the 3-D patterns of the parasympathetic nervous system in the lung at late developmental stages between mouse and chicken.

Dev Biol 2018 12 21;444 Suppl 1:S325-S336. Epub 2018 May 21.

Department of Zoology, Graduate School of Science, Kyoto University, Kitashirakawa, Sakyo-ku, Kyoto 606-8502, Japan; AMED Core Research for Evolutional Science and Technology (AMED-CREST), Japan Agency for Medical Research and Development (AMED), Chiyoda-ku, Tokyo 100-0004, Japan. Electronic address:

Although the basic schema of the body plan is similar among different species of amniotes (mammals, birds, and reptiles), the lung is an exception. Here, anatomy and physiology are considerably different, particularly between mammals and birds. In mammals, inhaled and exhaled airs mix in the airways, whereas in birds the inspired air flows unidirectionally without mixing with the expired air. This bird-specific respiration system is enabled by the complex tubular structures called parabronchi where gas exchange takes place, and also by the bellow-like air sacs appended to the main part of the lung. That the lung is predominantly governed by the parasympathetic nervous system has been shown mostly by physiological studies in mammals. However, how the parasympathetic nervous system in the lung is established during late development has largely been unexplored both in mammals and birds. In this study, by combining immunocytochemistry, the tissue-clearing CUBIC method, and ink-injection to airways, we have visualized the 3-D distribution patterns of parasympathetic nerves and ganglia in the lung at late developmental stages of mice and chickens. These patterns were further compared between these species, and three prominent similarities emerged: (1) parasympathetic postganglionic fibers and ganglia are widely distributed in the lung covering the proximal and distal portions, (2) the gas exchange units, alveoli in mice and parabronchi in chickens, are devoid of parasympathetic nerves, (3) parasympathetic nerves are in close association with smooth muscle cells, particularly at the base of the gas exchange units. These observations suggest that despite gross differences in anatomy, the basic mechanisms underlying parasympathetic control of smooth muscles and gas exchange might be conserved between mammals and birds.
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http://dx.doi.org/10.1016/j.ydbio.2018.05.014DOI Listing
December 2018

Neuronal signals regulate obesity induced β-cell proliferation by FoxM1 dependent mechanism.

Nat Commun 2017 12 5;8(1):1930. Epub 2017 Dec 5.

Department of Metabolism and Diabetes, Tohoku University Graduate School of Medicine, Sendai, 980-8575, Japan.

Under insulin-resistant conditions such as obesity, pancreatic β-cells proliferate to prevent blood glucose elevations. A liver-brain-pancreas neuronal relay plays an important role in this process. Here, we show the molecular mechanism underlying this compensatory β-cell proliferation. We identify FoxM1 activation in islets from neuronal relay-stimulated mice. Blockade of this relay, including vagotomy, inhibits obesity-induced activation of the β-cell FoxM1 pathway and suppresses β-cell expansion. Inducible β-cell-specific FoxM1 deficiency also blocks compensatory β-cell proliferation. In isolated islets, carbachol and PACAP/VIP synergistically promote β-cell proliferation through a FoxM1-dependent mechanism. These findings indicate that vagal nerves that release several neurotransmitters may allow simultaneous activation of multiple pathways in β-cells selectively, thereby efficiently promoting β-cell proliferation and maintaining glucose homeostasis during obesity development. This neuronal signal-mediated mechanism holds potential for developing novel approaches to regenerating pancreatic β-cells.
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http://dx.doi.org/10.1038/s41467-017-01869-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5717276PMC
December 2017

CUBIC pathology: three-dimensional imaging for pathological diagnosis.

Sci Rep 2017 08 24;7(1):9269. Epub 2017 Aug 24.

Department of Systems Pharmacology, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-0033, Japan.

The examination of hematoxylin and eosin (H&E)-stained tissues on glass slides by conventional light microscopy is the foundation for histopathological diagnosis. However, this conventional method has some limitations in x-y axes due to its relatively narrow range of observation area and in z-axis due to its two-dimensionality. In this study, we applied a CUBIC pipeline, which is the most powerful tissue-clearing and three-dimensional (3D)-imaging technique, to clinical pathology. CUBIC was applicable to 3D imaging of both normal and abnormal patient-derived, human lung and lymph node tissues. Notably, the combination of deparaffinization and CUBIC enabled 3D imaging of specimens derived from paraffin-embedded tissue blocks, allowing quantitative evaluation of nuclear and structural atypia of an archival malignant lymphoma tissue. Furthermore, to examine whether CUBIC can be applied to practical use in pathological diagnosis, we performed a histopathological screening of a lymph node metastasis based on CUBIC, which successfully improved the sensitivity in detecting minor metastatic carcinoma nodules in lymph nodes. Collectively, our results indicate that CUBIC significantly contributes to retrospective and prospective clinicopathological diagnosis, which might lead to the establishment of a novel field of medical science based on 3D histopathology.
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http://dx.doi.org/10.1038/s41598-017-09117-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5571108PMC
August 2017

Next-generation mammalian genetics toward organism-level systems biology.

NPJ Syst Biol Appl 2017 5;3:15. Epub 2017 Jun 5.

Department of Systems Pharmacology, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, , Bunkyo-ku, Tokyo 113-0033 Japan.

Organism-level systems biology in mammals aims to identify, analyze, control, and design molecular and cellular networks executing various biological functions in mammals. In particular, system-level identification and analysis of molecular and cellular networks can be accelerated by next-generation mammalian genetics. Mammalian genetics without crossing, where all production and phenotyping studies of genome-edited animals are completed within a single generation drastically reduce the time, space, and effort of conducting the systems research. Next-generation mammalian genetics is based on recent technological advancements in genome editing and developmental engineering. The process begins with introduction of double-strand breaks into genomic DNA by using site-specific endonucleases, which results in highly efficient genome editing in mammalian zygotes or embryonic stem cells. By using nuclease-mediated genome editing in zygotes, or ~100% embryonic stem cell-derived mouse technology, whole-body knock-out and knock-in mice can be produced within a single generation. These emerging technologies allow us to produce multiple knock-out or knock-in strains in high-throughput manner. In this review, we discuss the basic concepts and related technologies as well as current challenges and future opportunities for next-generation mammalian genetics in organism-level systems biology.
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http://dx.doi.org/10.1038/s41540-017-0015-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5459797PMC
June 2017

Knockout-Rescue Embryonic Stem Cell-Derived Mouse Reveals Circadian-Period Control by Quality and Quantity of CRY1.

Mol Cell 2017 Jan 22;65(1):176-190. Epub 2016 Dec 22.

Department of Systems Pharmacology, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan; Laboratory for Synthetic Biology, RIKEN Quantitative Biology Center, 1-3 Yamadaoka, Suita, Osaka 565-0871, Japan. Electronic address:

To conduct comprehensive characterization of molecular properties in organisms, we established an efficient method to produce knockout (KO)-rescue mice within a single generation. We applied this method to produce 20 strains of almost completely embryonic stem cell (ESC)-derived mice ("ES mice") rescued with wild-type and mutant Cry1 gene under a Cry1:Cry2 background. A series of both phosphorylation-mimetic and non-phosphorylation-mimetic CRY1 mutants revealed that multisite phosphorylation of CRY1 can serve as a cumulative timer in the mammalian circadian clock. KO-rescue ES mice also revealed that CRY1-PER2 interaction confers a robust circadian rhythmicity in mice. Surprisingly, in contrast to theoretical predictions from canonical transcription/translation feedback loops, the residues surrounding the flexible P loop and C-lid domains of CRY1 determine circadian period without changing the degradation rate of CRY1. These results suggest that CRY1 determines circadian period through both its degradation-dependent and -independent pathways.
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http://dx.doi.org/10.1016/j.molcel.2016.11.022DOI Listing
January 2017

Involvement of Ca(2+)-Dependent Hyperpolarization in Sleep Duration in Mammals.

Neuron 2016 Apr 17;90(1):70-85. Epub 2016 Mar 17.

Department of Systems Pharmacology, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan; Laboratory for Synthetic Biology, RIKEN Quantitative Biology Center, 2-2-3 Minatojima-minamimachi, Chuo-ku, Kobe, Hyogo 650-0047, Japan. Electronic address:

The detailed molecular mechanisms underlying the regulation of sleep duration in mammals are still elusive. To address this challenge, we constructed a simple computational model, which recapitulates the electrophysiological characteristics of the slow-wave sleep and awake states. Comprehensive bifurcation analysis predicted that a Ca(2+)-dependent hyperpolarization pathway may play a role in slow-wave sleep and hence in the regulation of sleep duration. To experimentally validate the prediction, we generate and analyze 21 KO mice. Here we found that impaired Ca(2+)-dependent K(+) channels (Kcnn2 and Kcnn3), voltage-gated Ca(2+) channels (Cacna1g and Cacna1h), or Ca(2+)/calmodulin-dependent kinases (Camk2a and Camk2b) decrease sleep duration, while impaired plasma membrane Ca(2+) ATPase (Atp2b3) increases sleep duration. Pharmacological intervention and whole-brain imaging validated that impaired NMDA receptors reduce sleep duration and directly increase the excitability of cells. Based on these results, we propose a hypothesis that a Ca(2+)-dependent hyperpolarization pathway underlies the regulation of sleep duration in mammals.
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http://dx.doi.org/10.1016/j.neuron.2016.02.032DOI Listing
April 2016

Whole-body and Whole-Organ Clearing and Imaging Techniques with Single-Cell Resolution: Toward Organism-Level Systems Biology in Mammals.

Cell Chem Biol 2016 Jan;23(1):137-157

Department of Systems Pharmacology, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan; Laboratory for Synthetic Biology, RIKEN Quantitative Biology Center (QBiC), 1-3, Yamadaoka, Suita, Osaka 565-0874, Japan; AMED-CREST, Japan Agency for Medical Research and Development (AMED), 1-7-1, Ohte-machi, Chiyoda-ku, Tokyo, 100-0004, Japan. Electronic address:

Organism-level systems biology aims to identify, analyze, control and design cellular circuits in organisms. Many experimental and computational approaches have been developed over the years to allow us to conduct these studies. Some of the most powerful methods are based on using optical imaging in combination with fluorescent labeling, and for those one of the long-standing stumbling blocks has been tissue opacity. Recently, the solutions to this problem have started to emerge based on whole-body and whole-organ clearing techniques that employ innovative tissue-clearing chemistry. Here, we review these advancements and discuss how combining new clearing techniques with high-performing fluorescent proteins or small molecule tags, rapid volume imaging and efficient image informatics is resulting in comprehensive and quantitative organ-wide, single-cell resolution experimental data. These technologies are starting to yield information on connectivity and dynamics in cellular circuits at unprecedented resolution, and bring us closer to system-level understanding of physiology and diseases of complex mammalian systems.
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http://dx.doi.org/10.1016/j.chembiol.2015.11.009DOI Listing
January 2016

Sleep as a biological problem: an overview of frontiers in sleep research.

J Physiol Sci 2016 Jan 5;66(1):1-13. Epub 2015 Nov 5.

International Institute for Integrative Sleep Medicine (WPI-IIIS), University of Tsukuba, Ibaraki, 305-8575, Japan.

Sleep is a physiological process not only for the rest of the body but also for several brain functions such as mood, memory, and consciousness. Nevertheless, the nature and functions of sleep remain largely unknown due to its extremely complicated nature and lack of optimized technology for the experiments. Here we review the recent progress in the biology of the mammalian sleep, which covers a wide range of research areas: the basic knowledge about sleep, the physiology of cerebral cortex in sleeping animals, the detailed morphological features of thalamocortical networks, the mechanisms underlying fluctuating activity of autonomic nervous systems during rapid eye movement sleep, the cutting-edge technology of tissue clearing for visualization of the whole brain, the ketogenesis-mediated homeostatic regulation of sleep, and the forward genetic approach for identification of novel genes involved in sleep. We hope this multifaceted review will be helpful for researchers who are interested in the biology of sleep.
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http://dx.doi.org/10.1007/s12576-015-0414-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4742504PMC
January 2016

Advanced CUBIC protocols for whole-brain and whole-body clearing and imaging.

Nat Protoc 2015 Nov 8;10(11):1709-27. Epub 2015 Oct 8.

Department of Systems Pharmacology, The University of Tokyo, Tokyo, Japan.

Here we describe a protocol for advanced CUBIC (Clear, Unobstructed Brain/Body Imaging Cocktails and Computational analysis). The CUBIC protocol enables simple and efficient organ clearing, rapid imaging by light-sheet microscopy and quantitative imaging analysis of multiple samples. The organ or body is cleared by immersion for 1-14 d, with the exact time required dependent on the sample type and the experimental purposes. A single imaging set can be completed in 30-60 min. Image processing and analysis can take <1 d, but it is dependent on the number of samples in the data set. The CUBIC clearing protocol can process multiple samples simultaneously. We previously used CUBIC to image whole-brain neural activities at single-cell resolution using Arc-dVenus transgenic (Tg) mice. CUBIC informatics calculated the Venus signal subtraction, comparing different brains at a whole-organ scale. These protocols provide a platform for organism-level systems biology by comprehensively detecting cells in a whole organ or body.
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http://dx.doi.org/10.1038/nprot.2015.085DOI Listing
November 2015

Whole-body imaging with single-cell resolution by tissue decolorization.

Cell 2014 Nov;159(4):911-24

Department of Systems Pharmacology, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan; Laboratory for Synthetic Biology, RIKEN Quantitative Biology Center, 2-2-3 Minatojima-minamimachi, Chuo-ku, Kobe, Hyogo 650-0047, Japan; CREST, Japan Science and Technology Agency, 4-1-8 Honcho, Kawaguchi, Saitama 332-0012, Japan. Electronic address:

The development of whole-body imaging at single-cell resolution enables system-level approaches to studying cellular circuits in organisms. Previous clearing methods focused on homogenizing mismatched refractive indices of individual tissues, enabling reductions in opacity but falling short of achieving transparency. Here, we show that an aminoalcohol decolorizes blood by efficiently eluting the heme chromophore from hemoglobin. Direct transcardial perfusion of an aminoalcohol-containing cocktail that we previously termed CUBIC coupled with a 10 day to 2 week clearing protocol decolorized and rendered nearly transparent almost all organs of adult mice as well as the entire body of infant and adult mice. This CUBIC-perfusion protocol enables rapid whole-body and whole-organ imaging at single-cell resolution by using light-sheet fluorescent microscopy. The CUBIC protocol is also applicable to 3D pathology, anatomy, and immunohistochemistry of various organs. These results suggest that whole-body imaging of colorless tissues at high resolution will contribute to organism-level systems biology.
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http://dx.doi.org/10.1016/j.cell.2014.10.034DOI Listing
November 2014

Whole-brain imaging with single-cell resolution using chemical cocktails and computational analysis.

Cell 2014 Apr 17;157(3):726-39. Epub 2014 Apr 17.

Laboratory for Synthetic Biology, RIKEN Quantitative Biology Center, 2-2-3 Minatojima-minamimachi, Chuo-ku, Kobe, Hyogo 650-0047, Japan; Laboratory for Systems Biology, RIKEN Center for Developmental Biology, 2-2-3 Minatojima-minamimachi, Chuo-ku, Kobe, Hyogo 650-0047, Japan; Department of Systems Pharmacology, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan; CREST, Japan Science and Technology Agency, 4-1-8 Honcho, Kawaguchi, Saitama, 332-0012, Japan. Electronic address:

Systems-level identification and analysis of cellular circuits in the brain will require the development of whole-brain imaging with single-cell resolution. To this end, we performed comprehensive chemical screening to develop a whole-brain clearing and imaging method, termed CUBIC (clear, unobstructed brain imaging cocktails and computational analysis). CUBIC is a simple and efficient method involving the immersion of brain samples in chemical mixtures containing aminoalcohols, which enables rapid whole-brain imaging with single-photon excitation microscopy. CUBIC is applicable to multicolor imaging of fluorescent proteins or immunostained samples in adult brains and is scalable from a primate brain to subcellular structures. We also developed a whole-brain cell-nuclear counterstaining protocol and a computational image analysis pipeline that, together with CUBIC reagents, enable the visualization and quantification of neural activities induced by environmental stimulation. CUBIC enables time-course expression profiling of whole adult brains with single-cell resolution.
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http://dx.doi.org/10.1016/j.cell.2014.03.042DOI Listing
April 2014

Non-enzymatic DNA cleavage reaction induced by 5-ethynyluracil in methylamine aqueous solution and application to DNA concatenation.

PLoS One 2014 19;9(3):e92369. Epub 2014 Mar 19.

Laboratory for Synthetic Biology, Quantitative Biology Center, RIKEN, Chuo-ku, Kobe, Japan; Laboratory for Systems Biology, Center for Developmental Biology, RIKEN, Chuo-ku, Kobe, Japan; Department of Systems Pharmacology, Graduate School of Medicine, The University of Tokyo, Bunkyo-ku, Tokyo, Japan; Department of Biological Sciences, Graduate School of Science, Osaka University, Toyonaka, Osaka, Japan; Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka, Japan.

DNA can be concatenated by hybridization of DNA fragments with protruding single-stranded termini. DNA cleavage occurring at a nucleotide containing a DNA base analogue is a useful method to obtain DNA with designed protruding termini. Here, we report a novel non-enzymatic DNA cleavage reaction for DNA concatenation. We found that DNA is cleaved at a nucleotide containing 5-ethynyluracil in a methylamine aqueous solution to generate 5'-phosphorylated DNA fragment as a cleavage product. We demonstrated that the reaction can be applied to DNA concatenation of PCR-amplified DNA fragments. This novel non-enzymatic DNA cleavage reaction is a simple practical approach for DNA concatenation.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0092369PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3960239PMC
November 2014

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

Genes Cells 2013 Jul 12;18(7):575-88. Epub 2013 Jun 12.

Laboratory for Systems Biology, RIKEN Center for Developmental Biology, Kobe, Hyogo 650-0047, Japan.

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.
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http://dx.doi.org/10.1111/gtc.12063DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3738941PMC
July 2013

Challenges in synthetically designing mammalian circadian clocks.

Curr Opin Biotechnol 2010 Aug 12;21(4):556-65. Epub 2010 Aug 12.

Laboratory for Systems Biology, RIKEN Center for Developmental Biology, 2-2-3 Minatojima-minamimachi, Chuo-ku, Kobe, Hyogo 650-0047, Japan.

Synthetic biology, in which complex, dynamic biological systems are designed or reconstructed from basic biological components, can help elucidate the design principles of such systems. However, this engineering approach has only been applied to a few simple biological systems. The circadian clock is appropriate for this approach, since it is a dynamic system with complex transcriptional and post-transcriptional circuits that have been comprehensively described. Rational synthesis of the properties of the suprachiasmatic nucleus, the central clock tissue of the circadian system that controls many dynamic behaviors, will be important for understanding the neural-circuit systems that control physiological behaviors. These approaches will provide a deeper understanding of the biological clock, and of clinical problems associated with it, such as sleep disorders.
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http://dx.doi.org/10.1016/j.copbio.2010.07.011DOI Listing
August 2010