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14 Publications

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    07/29/20 | Dense reconstruction of elongated cell lineages: overcoming suboptimum lineage encoding and sparse cell sampling
    Sugino K, Miyares RL, Espinosa-Medina I, Chen H, Potter CJ, Lee T
    bioRxiv. 07/2020:. doi: 10.1101/2020.07.27.223321

    Acquiring both lineage and cell-type information during brain development could elucidate transcriptional programs underling neuronal diversification. This is now feasible with single-cell RNA-seq combined with CRISPR-based lineage tracing, which generates genetic barcodes with cumulative CRISPR edits. This technique has not yet been optimized to deliver high-resolution lineage reconstruction of protracted lineages. Drosophila neuronal lineages are an ideal model to consider, as multiple lineages have been morphologically mapped at single-cell resolution. Here we find the parameter ranges required to encode a representative neuronal lineage emanating from 100 stem cell divisions. We derive the optimum editing rate to be inversely proportional to lineage depth, enabling encoding to persist across lineage progression. Further, we experimentally determine the editing rates of a Cas9-deaminase in cycling neural stem cells, finding near ideal rates to map elongated Drosophila neuronal lineages. Moreover, we propose and evaluate strategies to separate recurring cell-types for lineage reconstruction. Finally, we present a simple method to combine multiple experiments, which permits dense reconstruction of protracted cell lineages despite suboptimum lineage encoding and sparse cell sampling.Competing Interest StatementThe authors have declared no competing interest.

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    10/24/17 | Dual origin of enteric neurons in vagal Schwann cell precursors and the sympathetic neural crest
    Isabel Espinosa-Medina , Ben Jevans , Franck Boismoreau , Zoubida Chettouh , Hideki Enomoto , Thomas Müller , Carmen Birchmeier , Alan J. Burns , Jean-François Brunet
    Proceedings of the National Academy of Sciences. 10/2017;114:11980-11985. doi: 10.1073/pnas.1710308114

    Most of the enteric nervous system derives from the “vagal” neural crest, lying at the level of somites 1–7, which invades the digestive tract rostro-caudally from the foregut to the hindgut. Little is known about the initial phase of this colonization, which brings enteric precursors into the foregut. Here we show that the “vagal crest” subsumes two populations of enteric precursors with contrasted origins, initial modes of migration, and destinations. Crest cells adjacent to somites 1 and 2 produce Schwann cell precursors that colonize the vagus nerve, which in turn guides them into the esophagus and stomach. Crest cells adjacent to somites 3–7 belong to the crest streams contributing to sympathetic chains: they migrate ventrally, seed the sympathetic chains, and colonize the entire digestive tract thence. Accordingly, enteric ganglia, like sympathetic ones, are atrophic when deprived of signaling through the tyrosine kinase receptor ErbB3, while half of the esophageal ganglia require, like parasympathetic ones, the nerve-associated form of the ErbB3 ligand, Neuregulin-1. These dependencies might bear relevance to Hirschsprung disease, with which alleles of Neuregulin-1 are associated.

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    06/12/14 | Parasympathetic ganglia derive from Schwann cell precursors
    I. Espinosa-Medina , E. Outin , C. A. Picard , Z. Chettouh , S. Dymecki , G. G. Consalez , E. Coppola , J.-F. Brunet
    Science. 06/2014;345:87-90. doi: 10.1126/science.1253286

    The parasympathetic nervous system helps regulate the functions of many tissues and organs, including the salivary glands and the esophagus. To do so, it needs to reach throughout the body, connecting central systems to peripheral ones. Dyachuk et al. and Espinosa-Medina et al. explored how these connections are established in mice (see the Perspective by Kalcheim and Rohrer). Progenitor cells that travel along with the developing nerves can give rise to both myelinforming Schwann cells and to parasympathetic neurons. That means the interacting nerves do not have to find each other. Instead, the beginnings of the connections are laid down as the nervous system develops. Science, this issue p. 82, p. 87; see also p. 32 Parasympathetic neurons are born from Schwann cell precursors located in the nerves that carry preganglionic fibers. [Also see Perspective by Kalcheim and Rohrer] Neural crest cells migrate extensively and give rise to most of the peripheral nervous system, including sympathetic, parasympathetic, enteric, and dorsal root ganglia. We studied how parasympathetic ganglia form close to visceral organs and what their precursors are. We find that many cranial nerve-associated crest cells coexpress the pan-autonomic determinant Paired-like homeodomain 2b (Phox2b) together with markers of Schwann cell precursors. Some give rise to Schwann cells after down-regulation of PHOX2b. Others form parasympathetic ganglia after being guided to the site of ganglion formation by the nerves that carry preganglionic fibers, a parsimonious way of wiring the pathway. Thus, cranial Schwann cell precursors are the source of parasympathetic neurons during normal development.

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    10/28/21 | TEMPO: A system to sequentially label and genetically manipulate vertebrate cell lineages
    Espinosa-Medina I, Feliciano D, Belmonte-Mateos C, Garcia-Marques J, Foster B, Miyares RL, Pujades C, Koyama M, Lee T
    bioRxiv. 10/2021:. doi: 10.1101/2021.10.27.466134

    During development, regulatory factors appear in a precise order to determine cell fates over time. To investigate complex tissue development, one should not just label cell lineages but further visualize and manipulate cells with temporal control. Current strategies for tracing vertebrate cell lineages lack genetic access to sequentially produced cells. Here we present TEMPO (Temporal Encoding and Manipulation in a Predefined Order), an imaging-readable genetic tool allowing differential labelling and manipulation of consecutive cell generations in vertebrates. TEMPO is based on CRISPR and powered by a cascade of gRNAs that drive orderly activation/inactivation of reporters/effectors. Using TEMPO to visualize zebrafish and mouse neurogenesis, we recapitulated birth-order-dependent neuronal fates. Temporally manipulating cell-cycle regulators in mouse cortex progenitors altered the proportion and distribution of neurons and glia, revealing the effects of temporal gene perturbation on serial cell fates. Thus, TEMPO enables sequential manipulation of molecular factors, crucial to study cell-type specification.One-Sentence Summary Gaining sequential genetic access to vertebrate cell lineages.Competing Interest StatementThe authors have declared no competing interest.

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    02/28/18 | The "sacral parasympathetic": ontogeny and anatomy of a myth.
    Espinosa-Medina I, Saha O, Boismoreau F, Brunet J
    Clin Auton Res. 2018 02;28(1):13-21. doi: 10.1007/s10286-017-0478-7

    We recently defined genetic traits that distinguish sympathetic from parasympathetic neurons, both preganglionic and ganglionic (Espinosa-Medina et al., Science 354:893-897, 2016). By this set of criteria, we found that the sacral autonomic outflow is sympathetic, not parasympathetic as has been thought for more than a century. Proposing such a belated shift in perspective begs the question why the new criterion (cell types defined by their genetic make-up and dependencies) should be favored over the anatomical, physiological and pharmacological considerations of long ago that inspired the "parasympathetic" classification. After a brief reminder of the former, we expound the weaknesses of the latter and argue that the novel genetic definition helps integrating neglected anatomical and physiological observations and clearing the path for future research.

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    11/18/16 | The sacral autonomic outflow is sympathetic
    I. Espinosa-Medina , O. Saha , F. Boismoreau , Z. Chettouh , F. Rossi , W. D. Richardson , J.-F. Brunet
    Science. 11/2016;354:893-897. doi: 10.1126/science.aah5454

    The autonomic nervous system regulates the function of internal organs such as the gut. The parasympathetic and sympathetic arms of this system tend to operate antagonistically. Espinosa-Medina et al. used anatomical and molecular analyses to reevaluate the assignment of neurons in the sacral autonomic nervous system (see the Perspective by Adameyko). Previously categorized as parasympathetic, these neurons are now identified as sympathetic. The results resolve a persistent confusion about how the two systems developed and open the avenue to more predictable outcomes in developing treatments targeted to the pelvic autonomic nervous system. Science, this issue p. 893; see also p. 833 Contrary to a century-old dogma, the pelvic nerves and ganglia do not belong to the parasympathetic nervous system but to the sympathetic one. A kinship between cranial and pelvic visceral nerves of vertebrates has been accepted for a century. Accordingly, sacral preganglionic neurons are considered parasympathetic, as are their targets in the pelvic ganglia that prominently control rectal, bladder, and genital functions. Here, we uncover 15 phenotypic and ontogenetic features that distinguish pre- and postganglionic neurons of the cranial parasympathetic outflow from those of the thoracolumbar sympathetic outflow in mice. By every single one, the sacral outflow is indistinguishable from the thoracolumbar outflow. Thus, the parasympathetic nervous system receives input from cranial nerves exclusively and the sympathetic nervous system from spinal nerves, thoracic to sacral inclusively. This simplified, bipartite architecture offers a new framework to understand pelvic neurophysiology as well as development and evolution of the autonomic nervous system.

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    04/14/19 | Theoretical modeling on CRISPR-coded cell lineages: efficient encoding and optimal reconstruction
    Sugino K, Garcia-Marques J, Espinosa-Medina I, Lee T
    bioRxiv. 04/2019:. doi: 10.1101/538488

    Delineating cell lineages is a prerequisite for interrogating the genesis of cell types. CRISPR/Cas9 can edit genomic sequence during development which enables to trace cell lineages. Recent studies have demonstrated the feasibility of this idea. However, the optimality of the encoding or reconstruction processes has not been adequately addressed. Here, we surveyed a multitude of reconstruction algorithms and found hierarchical clustering, with a metric based on the number of shared Cas9 edits, delivers the best reconstruction. However, the trackable depth is ultimately limited by the number of available coding units that typically decrease exponentially across cell generations. To overcome this limit, we established two strategies that better sustain the coding capacity. One involves controlling target availability via use of parallel gRNA cascades, whereas the other strategy exploits adjustable Cas9/gRNA editing rates. In summary, we provide a theoretical basis in understanding, designing, and analyzing robust CRISPR barcodes for dense reconstruction of protracted cell lineages.

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    07/23/21 | YAP1 nuclear efflux and transcriptional reprograming follow membrane diminution upon VSV-G-induced cell fusion.
    Feliciano D, Ott CM, Isabel Espinosa Medina , Weigel AV, Benedetti L, Milano KM, Tang Z, Lee T, Kliman HJ, Guller SM, Lippincott-Schwartz J
    Nature Communications. 2021 Jul 23;12(1):4502. doi: 10.1038/s41467-021-24708-2

    Cells in many tissues, such as bone, muscle, and placenta, fuse into syncytia to acquire new functions and transcriptional programs. While it is known that fused cells are specialized, it is unclear whether cell-fusion itself contributes to programmatic-changes that generate the new cellular state. Here, we address this by employing a fusogen-mediated, cell-fusion system to create syncytia from undifferentiated cells. RNA-Seq analysis reveals VSV-G-induced cell fusion precedes transcriptional changes. To gain mechanistic insights, we measure the plasma membrane surface area after cell-fusion and observe it diminishes through increases in endocytosis. Consequently, glucose transporters internalize, and cytoplasmic glucose and ATP transiently decrease. This reduced energetic state activates AMPK, which inhibits YAP1, causing transcriptional-reprogramming and cell-cycle arrest. Impairing either endocytosis or AMPK activity prevents YAP1 inhibition and cell-cycle arrest after fusion. Together, these data demonstrate plasma membrane diminishment upon cell-fusion causes transient nutrient stress that may promote transcriptional-reprogramming independent from extrinsic cues.

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    04/01/21 | The art of lineage tracing: From worm to human.
    Garcia-Marques J, Isabel Espinosa Medina , Lee T
    Progress in Neurobiology. 2021 Apr;199:101966. doi: 10.1016/j.pneurobio.2020.101966

    Reconstructing the genealogy of every cell that makes up an organism remains a long-standing challenge in developmental biology. Besides its relevance for understanding the mechanisms underlying normal and pathological development, resolving the lineage origin of cell types will be crucial to create these types on-demand. Multiple strategies have been deployed towards the problem of lineage tracing, ranging from direct observation to sophisticated genetic approaches. Here we discuss the achievements and limitations of past and current technology. Finally, we speculate about the future of lineage tracing and how to reach the next milestones in the field.

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    12/01/20 | A programmable sequence of reporters for lineage analysis.
    Garcia-Marques J, Isabel Espinosa Medina , Ku K, Yang C, Koyama M, Yu H, Lee T
    Nature Neuroscience. 2020 Dec 01;23(12):1618-28. doi: 10.1038/s41593-020-0676-9

    We present CLADES (cell lineage access driven by an edition sequence), a technology for cell lineage studies based on CRISPR-Cas9 techniques. CLADES relies on a system of genetic switches to activate and inactivate reporter genes in a predetermined order. Targeting CLADES to progenitor cells allows the progeny to inherit a sequential cascade of reporters, thereby coupling birth order to reporter expression. This system, which can also be temporally induced by heat shock, enables the temporal resolution of lineage development and can therefore be used to deconstruct an extended cell lineage by tracking the reporters expressed in the progeny. When targeted to the germ line, the same cascade progresses across animal generations, predominantly marking each generation with the corresponding combination of reporters. CLADES therefore offers an innovative strategy for making programmable cascades of genes that can be used for genetic manipulation or to record serial biological events.

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