記述言語：英語   掲載種別：研究論文（学術雑誌）  

Despite recent improvements in microscope technologies, segmenting and tracking cells in three-dimensional time-lapse images (3D + T images) to extract their dynamic positions and activities remains a considerable bottleneck in the field. We developed a deep learning-based software pipeline, 3DeeCellTracker, by integrating multiple existing and new techniques including deep learning for tracking. With only one volume of training data, one initial correction, and a few parameter changes, 3DeeCellTracker successfully segmented and tracked ~100 cells in both semi-immobilized and 'straightened' freely moving worm's brain, in a naturally beating zebrafish heart, and ~1000 cells in a 3D cultured tumor spheroid. While these datasets were imaged with highly divergent optical systems, our method tracked 90-100% of the cells in most cases, which is comparable or superior to previous results. These results suggest that 3DeeCellTracker could pave the way for revealing dynamic cell activities in image datasets that have been difficult to analyze.

DOI： 10.7554/eLife.59187

記述言語：英語  

BACKGROUND: Annotation of cell identity is an essential process in neuroscience that allows comparison of cells, including that of neural activities across different animals. In Caenorhabditis elegans, although unique identities have been assigned to all neurons, the number of annotatable neurons in an intact animal has been limited due to the lack of quantitative information on the location and identity of neurons. RESULTS: Here, we present a dataset that facilitates the annotation of neuronal identities, and demonstrate its application in a comprehensive analysis of whole-brain imaging. We systematically identified neurons in the head region of 311 adult worms using 35 cell-specific promoters and created a dataset of the expression patterns and the positions of the neurons. We found large positional variations that illustrated the difficulty of the annotation task. We investigated multiple combinations of cell-specific promoters driving distinct fluorescence and generated optimal strains for the annotation of most head neurons in an animal. We also developed an automatic annotation method with human interaction functionality that facilitates annotations needed for whole-brain imaging. CONCLUSION: Our neuron ID dataset and optimal fluorescent strains enable the annotation of most neurons in the head region of adult C. elegans, both in full-automated fashion and a semi-automated version that includes human interaction functionalities. Our method can potentially be applied to model species used in research other than C. elegans, where the number of available cell-type-specific promoters and their variety will be an important consideration.

DOI： 10.1186/s12915-020-0745-2

記述言語：その他  

<title>Abstract</title>Annotation of cell identity is an essential process in neuroscience that allows for comparing neural activities across different animals. In <italic>C. elegans</italic>, although unique identities have been assigned to all neurons, the number of annotatable neurons in an intact animal is limited in practice and comprehensive methods for cell annotation are required. Here we propose an efficient annotation method that can be integrated with the whole-brain imaging technique. We systematically identified neurons in the head region of 311 adult worms using 35 cell-specific promoters and created a dataset of the expression patterns and the positions of the neurons. The large positional variations illustrated the difficulty of the annotation task. We investigated multiple combinations of cell-specific promoters to tackle this problem. We also developed an automatic annotation method with human interaction functionality that facilitates annotation for whole-brain imaging.

DOI： 10.1101/698241

記述言語：その他  

Deep-learning-based flexible pipeline for segmenting and tracking cells in 3D image time series for whole brain imaging

DOI： 10.1101/385567

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Environmental responsiveness of tubulin glutamylation in sensory cilia is regulated by the p38 MAPK pathway.

DOI： 10.1038/s41598-018-26694-w

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Sensory processing is regulated by the coordinated excitation and inhibition of neurons in neuronal circuits. The analysis of neuronal activities has greatly benefited from the recent development of genetically encoded Ca²⁺ indicators (GECIs). These molecules change their fluorescence intensities or colours in response to changing levels of Ca²⁺ and can, therefore, be used to sensitively monitor intracellular Ca²⁺ concentration, which enables the detection of neuronal excitation, including action potentials. These GECIs were developed to monitor increases in Ca²⁺ concentration; therefore, neuronal inhibition cannot be sensitively detected by these GECIs. To overcome this difficulty, we hypothesised that an inverse-type of GECI, whose fluorescence intensity increases as Ca²⁺ levels decrease, could sensitively monitor reducing intracellular Ca²⁺ concentrations. We, therefore, developed a Ca²⁺ indicator named inverse-pericam 2.0 (IP2.0) whose fluorescent intensity decreases 25-fold upon Ca²⁺ binding in vitro. Using IP2.0, we successfully detected putative neuronal inhibition by monitoring the decrease in intracellular Ca²⁺ concentration in AWC^ON and ASEL neurons in Caenorhabditis elegans. Therefore, IP2.0 is a useful tool for studying neuronal inhibition and for the detailed analysis of neuronal activities in vivo.

DOI： 10.1371/journal.pone.0194707

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Tracking many cells in time-lapse 3D image sequences is an important challenging task of bioimage informatics. Motivated by a study of brain-wide 4D imaging of neural activity in C. elegans, we present a new method of multi-cell tracking. Data types to which the method is applicable are characterized as follows: (i) cells are imaged as globular-like objects, (ii) it is difficult to distinguish cells on the basis of shape and size only, (iii) the number of imaged cells in the several-hundred range, (iv) movements of nearly-located cells are strongly correlated, and (v) cells do not divide. We developed a tracking software suite that we call SPF-CellTracker. Incorporating dependency on the cells&#039; movements into the prediction model is the key for reducing the tracking errors: the cell switching and the coalescence of the tracked positions. We model the target cells&#039; correlated movements as a Markov random field and we also derive a fast computation algorithm, which we call spatial particle filter. With the live-imaging data of the nuclei of C. elegans neurons in which approximately 120 nuclei of neurons were imaged, the proposed method demonstrated improved accuracy compared to the standard particle filter and the method developed by Tokunaga et al. (2014).

DOI： 10.1109/TCBB.2017.2782255

記述言語：英語   掲載種別：研究論文（その他学術会議資料等）  

Large Graph Laplacian Matrix and Functional Map of Whole Brain of C. elegans

記述言語：その他  

Gonadal Maturation Changes Chemotaxis Behavior and Neural Processing in the Olfactory Circuit of Caenorhabditis elegans.

DOI： 10.1016/j.cub.2016.04.058

記述言語：英語   掲載種別：研究論文（学術雑誌）  

To measure the activity of neurons using whole-brain activity imaging, precise detection of each neuron or its nucleus is required. In the head region of the nematode C. elegans, the neuronal cell bodies are distributed densely in three-dimensional (3D) space. However, no existing computational methods of image analysis can separate them with sufficient accuracy. Here we propose a highly accurate segmentation method based on the curvatures of the iso-intensity surfaces. To obtain accurate positions of nuclei, we also developed a new procedure for least squares fitting with a Gaussian mixture model. Combining these methods enables accurate detection of densely distributed cell nuclei in a 3D space. The proposed method was implemented as a graphical user interface program that allows visualization and correction of the results of automatic detection. Additionally, the proposed method was applied to time-lapse 3D calcium imaging data, and most of the nuclei in the images were successfully tracked and measured.

DOI： 10.1371/journal.pcbi.1004970

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Motivation: Automated fluorescence microscopes produce massive amounts of images observing cells, often in four dimensions of space and time. This study addresses two tasks of time-lapse imaging analyses; detection and tracking of the many imaged cells, and it is especially intended for 4D live-cell imaging of neuronal nuclei of Caenorhabditis elegans. The cells of interest appear as slightly deformed ellipsoidal forms. They are densely distributed, and move rapidly in a series of 3D images. Thus, existing tracking methods often fail because more than one tracker will follow the same target or a tracker transits from one to other of different targets during rapid moves.
Results: The present method begins by performing the kernel density estimation in order to convert each 3D image into a smooth, continuous function. The cell bodies in the image are assumed to lie in the regions near the multiple local maxima of the density function. The tasks of detecting and tracking the cells are then addressed with two hill-climbing algorithms. The positions of the trackers are initialized by applying the cell-detection method to an image in the first frame. The tracking method keeps attacking them to near the local maxima in each subsequent image. To prevent the tracker from following multiple cells, we use a Markov random field (MRF) to model the spatial and temporal covariation of the cells and to maximize the image forces and the MRF-induced constraint on the trackers. The tracking procedure is demonstrated with dynamic 3D images that each contain 4100 neurons of C. elegans.

DOI： 10.1093/bioinformatics/btu271

記述言語：英語  

DOI： 10.1093/bioinformatics/btu271

記述言語：英語  

DOI： 10.1016/j.celrep.2013.02.019

記述言語：英語   掲載種別：研究論文（学術雑誌）  

An Expanded Palette of Genetically Encoded Ca2+ Indicators
Engineered fluorescent protein (FP) chimeras that modulate their fluorescence in response to changes in calcium ion (Ca2+) concentration are powerful tools for visualizing intracellular signaling activity. However, despite a decade of availability, the palette of single FP-based Ca2+ indicators has remained limited to a single green hue. We have expanded this palette by developing blue, improved green, and red intensiometric indicators, as well as an emission ratiometric indicator with an 11,000&#37; ratio change. This series enables improved single-color Ca2+ imaging in neurons and transgenic Caenorhabditis elegans. In HeLa cells, Ca2+ was imaged in three subcellular compartments, and, in conjunction with a cyan FP-yellow FP-based indicator, Ca2+ and adenosine 5'-triphosphate were simultaneously imaged. This palette of indicators paints the way to a colorful new era of Ca2+ imaging.

DOI： 10.1126/science.1208592

記述言語：英語  

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Cilia are important sensory organelles, which are thought to be essential regulators of numerous signaling pathways. In Caenorhabditis elegans, defects in sensory cilium formation result in a small-body phenotype, suggesting the role of sensory cilia in body size determination. Previous analyses suggest that lack of normal cilia causes the small-body phenotype through the activation of a signaling pathway which consists of the EGL-4 cGMP-dependent protein kinase and the GCY-12 receptor-type guanylyl cyclase. By genetic suppressor screening of the small-body phenotype of a cilium defective mutant, we identified a chb-3 gene. Genetic analyses placed chb-3 in the same pathway as egl-4 and gcy-12 and upstream of egl-4. chb-3 encodes a novel protein, with a zf-MYND motif and ankyrin repeats, that is highly conserved from worm to human. In chb-3 mutants, GCY-12 guanylyl cyclase visualized by tagged GFP (GCY-12::GFP) fails to localize to sensory cilia properly and accumulates in cell bodies. Our analyses suggest that decreased GCY-12 levels in the cilia of chb-3 mutants may cause the suppression of the small-body phenotype of a cilium defective mutant. By observing the transport of GCY-12::GFP particles along the dendrites to the cilia in sensory neurons, we found that the velocities and the frequencies of the particle movement are decreased in chb-3 mutant animals. How membrane proteins are trafficked to cilia has been the focus of extensive studies in vertebrates and invertebrates, although only a few of the relevant proteins have been identified. Our study defines a new regulator, CHB-3, in the trafficking process and also shows the importance of ciliary targeting of the signaling molecule, GCY-12, in sensory-dependent body size regulation in C. elegans. Given that CHB-3 is highly conserved in mammal, a similar system may be used in the trafficking of signaling proteins to the cilia of other species.

DOI： 10.1371/journal.pgen.1001211

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Cilia are important sensory organelles, which are thought to be essential regulators of numerous signaling pathways. In Caenorhabditis elegans, defects in sensory cilium formation result in a small-body phenotype, suggesting the role of sensory cilia in body size determination. Previous analyses suggest that lack of normal cilia causes the small-body phenotype through the activation of a signaling pathway which consists of the EGL-4 cGMP-dependent protein kinase and the GCY-12 receptor-type guanylyl cyclase. By genetic suppressor screening of the small-body phenotype of a cilium defective mutant, we identified a chb-3 gene. Genetic analyses placed chb-3 in the same pathway as egl-4 and gcy-12 and upstream of egl-4. chb-3 encodes a novel protein, with a zf-MYND motif and ankyrin repeats, that is highly conserved from worm to human. In chb-3 mutants, GCY-12 guanylyl cyclase visualized by tagged GFP (GCY-12::GFP) fails to localize to sensory cilia properly and accumulates in cell bodies. Our analyses suggest that decreased GCY-12 levels in the cilia of chb-3 mutants may cause the suppression of the small-body phenotype of a cilium defective mutant. By observing the transport of GCY-12::GFP particles along the dendrites to the cilia in sensory neurons, we found that the velocities and the frequencies of the particle movement are decreased in chb-3 mutant animals. How membrane proteins are trafficked to cilia has been the focus of extensive studies in vertebrates and invertebrates, although only a few of the relevant proteins have been identified. Our study defines a new regulator, CHB-3, in the trafficking process and also shows the importance of ciliary targeting of the signaling molecule, GCY-12, in sensory-dependent body size regulation in C. elegans. Given that CHB-3 is highly conserved in mammal, a similar system may be used in the trafficking of signaling proteins to the cilia of other species. © 2010 Fujiwara et al.

DOI： 10.1371/journal.pgen.1001211

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Systemic magnesium homeostasis in mammals is primarily governed by the activities of the TRPM6 and TRPM7 cation channels, which mediate both uptake by the intestinal epithelial cells and reabsorption by the distal convoluted tubule cells in the kidney. In the nematode, C. elegans, intestinal magnesium uptake is dependent on the activities of the TRPM channel proteins, GON-2 and GTL-1. In this paper we provide evidence that another member of the TRPM protein family, GTL-2, acts within the C. elegans excretory cell to mediate the excretion of excess magnesium. Thus, the activity of GTL-2 balances the activities of the paralogous TRPM channel proteins, GON-2 and GTL-1.

DOI： 10.1371/journal.pone.0009589

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Intercellular calcium waves can be observed in adult tissues, but whether they are instructive, permissive, or even required for behavior is predominantly unknown. In the nematode Caenorhabditis elegans, a periodic calcium spike in a pacemaker cell initiates a calcium wave in the intestine [1, 2]. The calcium wave is followed by three muscle contractions that comprise the defecation motor program [1]. Normal wave propagation requires the pannexin gap-junction subunit INX-16 at the interfaces of the intestinal cells. In the absence of this gap-junction subunit, calcium waves are frequently absent. The remaining waves are slow, initiate at abnormal locations, or travel in the opposite direction. Abnormal waves are associated with parallel effects in the first step of the motor program: The contractions of the overlying muscles fail to propagate beyond the pacemaker cell, are slow, initiate in abnormal locations, or are reversed. Moreover, the last two motor steps are predominantly absent. Finally, the absence of this gap-junction subunit also affects the reliability of the pacemaker cell; cycle timing is often irregular. These data demonstrate that pannexin gap junctions propagate calcium waves in the C. elegans intestine. The calcium waves instruct the motor steps and regulate the pacemaker cell's authority and reliability.

DOI： 10.1016/j.cub.2007.08.031

記述言語：英語  

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Periodic behavioral motor patterns are normally controlled by neural circuits, such as central pattern generators. We here report a novel mechanism of motor pattern generation by non-neural cells. The defecation motor program in Caenorhabditis elegans consists of three stereotyped motor steps with precise timing and this behavior has been studied as a model system of a ultradian biological clock [J.H. Thomas, Genetic analysis of defecation in C. elegans, Genetics 124 (1990) 855-872; D.W. Liu, J.H. Thomas, Regulation of a periodic motor program in C elegans, J. Neurosci. 14 (1994) 1953-1962; K. Iwasaki, D.W. Liu, J.H. Thomas, Genes that control a temperature-compensated ultradian clock in Caenorhabditis elegans, Proc. Nad. Acad. Sci. USA 92 (1995), 10317-10321]. It was previously implied that the inositol-1,4,5-trisphosphate (IP3) receptor in the intestine was necessary for this periodic behavior [P. Dal Santo, M.A. Logan, A.D. Chisholm, E.M. Jorgensen, The inositol trisphosphate receptor regulates a 50 s behavioral rhythm in C. elegans, Cell 98 (1999) 757-767]. Therefore, we developed a new assay system to study a relationship between this behavioral timing and intestinal Ca2+ dynamics. Using this assay system, we found that the timing between the first and second motor steps is coordinated by intercellular Ca2+-wave propagation in the intestine. Lack of the Ca2+-wave propagation correlated with no coordination of the motor steps in the CaMKII mutant. Also, when the Ca2+-wave propagation was blocked by the IP3 receptor inhibitor heparin at the mid-intestine in wild type, the second/third motor steps were eliminated, which phenocopied ablation of the motor neurons AVL and DVB. These observations suggest that an intestinal Ca2+-wave propagation governs the timing of neural activities that controls specific behavioral patterns in C. elegans. (c) 2006 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.ceca.2006.04.009

記述言語：英語   掲載種別：研究論文（学術雑誌）  

The transient receptor potential (TRP) channels are implicated in various cellular processes, including sensory signal transduction and electrolyte homeostasis. We show here that the GTL-1 and GON-2 TRPM channels regulate electrolyte homeostasis in the C. elegans intestine. GON-2 is responsible for a large outwardly rectifying current of intestinal cells, and its activity is tightly regulated by intracellular Mg2+ levels, while GTL-1 mainly contributes to appropriate Mg2+ responsiveness of the outwardly rectifying current. We also used nickel cytotoxicity to study the function of these channels. Both GON-2 and GTL-1 are necessary for intestinal uptake of nickel, but GTL-1 is continuously active while GON-2 is inactivated at higher Mg2+ levels. This type of differential regulation of intestinal electrolyte absorption ensures a constant supply of electrolytes through GTL-1, while occasional bursts of GON-2 activity allow rapid return to normal electrolyte concentrations following physiological perturbations.

DOI： 10.1016/j.cmet.2005.04.007

記述言語：その他  

記述言語：英語  

記述言語：英語  

記述言語：英語  

開催年月日： 2014年7月

記述言語：英語   会議種別：口頭発表（一般）  

開催地：University of Wisconsin - Madison, USA   国名：日本国  

開催年月日： 2014年7月

記述言語：英語   会議種別：口頭発表（一般）  

開催地：University of Wisconsin - Madison, USA   国名：日本国  

開催年月日： 2014年6月

記述言語：英語   会議種別：口頭発表（一般）  

開催地：九州工業大学   国名：日本国  

記述言語：日本語  

国名：日本国  

Tracking multiple cells with a spatial particle filter

記述言語：日本語  

国名：日本国  

4Dライブセルイメージングデータ内の非常に多数の細胞を自動追跡するための空間粒子フィルタ手法の開発

記述言語：日本語  

国名：日本国  

生命動態システム科学 I.定量生物学 2.組織・個体の計測 1)神経(1)神経系の丸ごと観測による神経回路の解析

記述言語：日本語  

国名：日本国  

逐次的カーネル密度推定法を用いた神経細胞カルシウムイオン分布の定量化

記述言語：日本語  

国名：日本国  

<p>細胞動画像の典型的な特徴として多数の追跡対象物が密に存在し視覚的に類似していることが挙げられる．このような動画像に対し標準的な粒子フィルタを利用した場合，本来の追跡対象を別の対象物と誤認し追跡に容易に失敗する．この問題に対し多数の細胞の共変動性に着目し追跡のための補助情報としてこれを利用することで追跡精度の向上を目指す．</p>

記述言語：英語