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75 Publications

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    01/11/24 | Epigenetic priming of embryonic lineages in the mammalian epiblast
    Miquel Sendra , Katie McDole , Daniel Jimenez-Carretero , Juan de Dios Hourcade , Susana Temiño , Léo Guignard , Philipp J Keller , Fátima Sánchez-Cabo , Jorge N. Domínguez , Miguel Torres
    bioRxiv. 2024 Jan 11:. doi: 10.1101/2024.01.11.575188

    Understanding the diversification of mammalian cell lineages is an essential to embryonic development, organ regeneration and tissue engineering. Shortly after implantation in the uterus, the pluripotent cells of the mammalian epiblast generate the three germ layers: ectoderm, mesoderm and endoderm1. Although clonal analyses suggest early specification of epiblast cells towards particular cell lineages24, single-cell transcriptomes do not identify lineage-specific markers in the epiblast511 and thus, the molecular regulation of such specification remains unknow. Here, we studied the epigenetic landscape of single epiblast cells, which revealed lineage priming towards endoderm, ectoderm or mesoderm. Unexpectedly, epiblast cells with mesodermal priming show a strong signature for the endothelial/endocardial fate, suggesting early specification of this lineage aside from other mesoderm. Through clonal analysis and live imaging, we show that endothelial precursors show early lineage divergence from the rest of mesodermal derivatives. In particular, cardiomyocytes and endocardial cells show limited lineage relationship, despite being temporally and spatially co-recruited during gastrulation. Furthermore, analysing the live tracks of single cells through unsupervised classification of cell migratory activity, we found early behavioral divergence of endothelial precursors shortly after the onset of mesoderm migration towards the cardiogenic area. These results provide a new model for the phenotypically silent specification of mammalian cell lineages in pluripotent cells of the epiblast and modify current knowledge on the sequence and timing of cardiovascular lineages diversification.

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    01/01/23 | Automated reconstruction of whole-embryo cell lineages by learning from sparse annotations.
    Malin-Mayor C, Hirsch P, Guignard L, McDole K, Wan Y, Lemon WC, Kainmueller D, Keller PJ, Preibisch S, Funke J
    Nature Biotechnology. 2023 Jan 01;41(1):44-49. doi: 10.1038/s41587-022-01427-7

    We present a method to automatically identify and track nuclei in time-lapse microscopy recordings of entire developing embryos. The method combines deep learning and global optimization. On a mouse dataset, it reconstructs 75.8% of cell lineages spanning 1 h, as compared to 31.8% for the competing method. Our approach improves understanding of where and when cell fate decisions are made in developing embryos, tissues, and organs.

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    09/05/22 | Automated reconstruction of whole-embryo cell lineages by learning from sparse annotations.
    Malin-Mayor C, Hirsch P, Guignard L, McDole K, Wan Y, Lemon WC, Kainmueller D, Keller PJ, Preibisch S, Funke J
    Nat Biotechnology. 2022 Sep 05:. doi: 10.1038/s41587-022-01427-7

    We present a method to automatically identify and track nuclei in time-lapse microscopy recordings of entire developing embryos. The method combines deep learning and global optimization. On a mouse dataset, it reconstructs 75.8% of cell lineages spanning 1 h, as compared to 31.8% for the competing method. Our approach improves understanding of where and when cell fate decisions are made in developing embryos, tissues, and organs.

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    Keller LabLooger Lab
    06/22/21 | In vivo glucose imaging in multiple model organisms with an engineered single-wavelength sensor.
    Keller JP, Marvin JS, Lacin H, Lemon WC, Shea J, Kim S, Lee RT, Koyama M, Keller PJ, Looger LL
    Cell Reports. 2021 Jun 22;35(12):109284. doi: 10.1016/j.celrep.2021.109284

    Glucose is arguably the most important molecule in metabolism, and its dysregulation underlies diabetes. We describe a family of single-wavelength genetically encoded glucose sensors with a high signal-to-noise ratio, fast kinetics, and affinities varying over four orders of magnitude (1 μM to 10 mM). The sensors allow mechanistic characterization of glucose transporters expressed in cultured cells with high spatial and temporal resolution. Imaging of neuron/glia co-cultures revealed ∼3-fold faster glucose changes in astrocytes. In larval Drosophila central nervous system explants, intracellular neuronal glucose fluxes suggested a rostro-caudal transport pathway in the ventral nerve cord neuropil. In zebrafish, expected glucose-related physiological sequelae of insulin and epinephrine treatments were directly visualized. Additionally, spontaneous muscle twitches induced glucose uptake in muscle, and sensory and pharmacological perturbations produced large changes in the brain. These sensors will enable rapid, high-resolution imaging of glucose influx, efflux, and metabolism in behaving animals.

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    01/07/21 | Characterization of a common progenitor pool of the epicardium and myocardium.
    Tyser RC, Ibarra-Soria X, McDole K, A Jayaram S, Godwin J, van den Brand TA, Miranda AM, Scialdone A, Keller PJ, Marioni JC, Srinivas S
    Science. 2021 Jan 07:. doi: 10.1126/science.abb2986

    The mammalian heart is derived from multiple cell lineages; however, our understanding of when and how the diverse cardiac cell types arise is limited. We mapped the origin of the embryonic mouse heart at single-cell resolution using a combination of transcriptomic, imaging, and genetic lineage labeling approaches. This provided a transcriptional and anatomic definition of cardiac progenitor types. Furthermore, it revealed a cardiac progenitor pool that is anatomically and transcriptionally distinct from currently known cardiac progenitors. Besides contributing to cardiomyocytes, these cells also represent the earliest progenitor of the epicardium, a source of trophic factors and cells during cardiac development and injury. This study provides detailed insights into the formation of early cardiac cell types, with particular relevance to the development of cell-based cardiac regenerative therapies.

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    10/06/20 | Nuclear crowding and nonlinear diffusion during interkinetic nuclear migration in the zebrafish retina.
    Azizi A, Herrmann A, Wan Y, Buse SJ, Keller PJ, Goldstein RE, Harris WA
    eLife. 2020 Oct 06;9:. doi: 10.7554/eLife.58635

    An important question in early neural development is the origin of stochastic nuclear movement between apical and basal surfaces of neuroepithelia during interkinetic nuclear migration. Tracking of nuclear subpopulations has shown evidence of diffusion - mean squared displacements growing linearly in time - and suggested crowding from cell division at the apical surface drives basalward motion. Yet, this hypothesis has not yet been tested, and the forces involved not quantified. We employ long-term, rapid light-sheet and two-photon imaging of early zebrafish retinogenesis to track entire populations of nuclei within the tissue. The time-varying concentration profiles show clear evidence of crowding as nuclei reach close-packing and are quantitatively described by a nonlinear diffusion model. Considerations of nuclear motion constrained inside the enveloping cell membrane show that concentration-dependent stochastic forces inside cells, compatible in magnitude to those found in cytoskeletal transport, can explain the observed magnitude of the diffusion constant.

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    05/06/20 | Whole-brain profiling of cells and circuits in mammals by tissue clearing and light-sheet microscopy.
    Ueda HR, Dodt H, Osten P, Economo MN, Chandrashekar J, Keller PJ
    Neuron. 2020 May 06;106(3):369-387. doi: 10.1016/j.neuron.2020.03.004

    Tissue clearing and light-sheet microscopy have a 100-year-plus history, yet these fields have been combined only recently to facilitate novel experiments and measurements in neuroscience. Since tissue-clearing methods were first combined with modernized light-sheet microscopy a decade ago, the performance of both technologies has rapidly improved, broadening their applications. Here, we review the state of the art of tissue-clearing methods and light-sheet microscopy and discuss applications of these techniques in profiling cells and circuits in mice. We examine outstanding challenges and future opportunities for expanding these techniques to achieve brain-wide profiling of cells and circuits in primates and humans. Such integration will help provide a systems-level understanding of the physiology and pathology of our central nervous system.

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    05/06/20 | Whole-brain profiling of cells and circuits in mammals by tissue clearing and light-sheet microscopy.
    Ueda HR, Dodt H, Osten P, Economo MN, Chandrashekar J, Keller PJ
    Neuron. 2020 May 06;106(3):369-387. doi: 10.1016/j.neuron.2020.03.004

    Tissue clearing and light-sheet microscopy have a 100-year-plus history, yet these fields have been combined only recently to facilitate novel experiments and measurements in neuroscience. Since tissue-clearing methods were first combined with modernized light-sheet microscopy a decade ago, the performance of both technologies has rapidly improved, broadening their applications. Here, we review the state of the art of tissue-clearing methods and light-sheet microscopy and discuss applications of these techniques in profiling cells and circuits in mice. We examine outstanding challenges and future opportunities for expanding these techniques to achieve brain-wide profiling of cells and circuits in primates and humans. Such integration will help provide a systems-level understanding of the physiology and pathology of our central nervous system.

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    02/01/20 | Tissue clearing and its applications in neuroscience
    Ueda HR, Ertürk A, Chung K, Gradinaru V, Chédotal A, Tomancak P, Keller PJ
    Nature Reviews Neuroscience. 2020 Feb 1:. doi: 10.1038/s41583-019-0250-1

    State-of-the-art tissue-clearing methods provide subcellular-level optical access to intact tissues from individual organs and even to some entire mammals. When combined with light-sheet microscopy and automated approaches to image analysis, existing tissue-clearing methods can speed up and may reduce the cost of conventional histology by several orders of magnitude. In addition, tissue-clearing chemistry allows whole-organ antibody labelling, which can be applied even to thick human tissues. By combining the most powerful labelling, clearing, imaging and data-analysis tools, scientists are extracting structural and functional cellular and subcellular information on complex mammalian bodies and large human specimens at an accelerated pace. The rapid generation of terabyte-scale imaging data furthermore creates a high demand for efficient computational approaches that tackle challenges in large-scale data analysis and management. In this Review, we discuss how tissue-clearing methods could provide an unbiased, system-level view of mammalian bodies and human specimens and discuss future opportunities for the use of these methods in human neuroscience.

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    01/02/20 | Tissue clearing and its applications in neuroscience.
    Ueda HR, Ertürk A, Chung K, Gradinaru V, Chédotal A, Tomancak P, Keller PJ
    Nature Reviews Neuroscience. 2020 Jan 02;21(2):61-79. doi: 10.1038/s41583-019-0250-1

    State-of-the-art tissue-clearing methods provide subcellular-level optical access to intact tissues from individual organs and even to some entire mammals. When combined with light-sheet microscopy and automated approaches to image analysis, existing tissue-clearing methods can speed up and may reduce the cost of conventional histology by several orders of magnitude. In addition, tissue-clearing chemistry allows whole-organ antibody labelling, which can be applied even to thick human tissues. By combining the most powerful labelling, clearing, imaging and data-analysis tools, scientists are extracting structural and functional cellular and subcellular information on complex mammalian bodies and large human specimens at an accelerated pace. The rapid generation of terabyte-scale imaging data furthermore creates a high demand for efficient computational approaches that tackle challenges in large-scale data analysis and management. In this Review, we discuss how tissue-clearing methods could provide an unbiased, system-level view of mammalian bodies and human specimens and discuss future opportunities for the use of these methods in human neuroscience.

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