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      The single cell transcriptional landscape of mammalian organogenesis

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          Abstract

          Mammalian organogenesis is an astonishing process. Within a short window of time, the cells of the three germ layers transform into an embryo that includes most major internal and external organs. Here we set out to investigate the transcriptional dynamics of mouse organogenesis at single cell resolution. With sci-RNA-seq3, we profiled ~2 million cells, derived from 61 embryos staged between 9.5 and 13.5 days of gestation, in a single experiment. The resulting ‘mouse organogenesis cell atlas’ (MOCA) provides a global view of developmental processes during this critical window. We identify hundreds of cell types and 56 trajectories, many of which are detected only because of the depth of cellular coverage, and collectively define thousands of corresponding marker genes. With Monocle 3, we explore the dynamics of gene expression within cell types and trajectories over time, including focused analyses of the apical ectodermal ridge, limb mesenchyme and skeletal muscle.

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          Most cited references38

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          p63 is essential for regenerative proliferation in limb, craniofacial and epithelial development.

          The p63 gene, a homologue of the tumour-suppressor p53, is highly expressed in the basal or progenitor layers of many epithelial tissues. Here we report that mice homozygous for a disrupted p63 gene have major defects in their limb, craniofacial and epithelial development. p63 is expressed in the ectodermal surfaces of the limb buds, branchial arches and epidermal appendages, which are all sites of reciprocal signalling that direct morphogenetic patterning of the underlying mesoderm. The limb truncations are due to a failure to maintain the apical ectodermal ridge, a stratified epithelium, essential for limb development. The embryonic epidermis of p63-/- mice undergoes an unusual process of non-regenerative differentiation, culminating in a striking absence of all squamous epithelia and their derivatives, including mammary, lacrymal and salivary glands. Taken together, our results indicate that p63 is critical for maintaining the progenitor-cell populations that are necessary to sustain epithelial development and morphogenesis.
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            Single-cell mapping of gene expression landscapes and lineage in the zebrafish embryo

            High-throughput mapping of cellular differentiation hierarchies from single-cell data promises to empower systematic interrogations of vertebrate development and disease. Here, we applied single-cell RNA sequencing to >92,000 cells from zebrafish embryos during the first day of development. Using a graph-based approach, we mapped a cell state landscape that describes axis patterning, germ layer formation, and organogenesis. We tested how clonally related cells traverse this landscape by developing a transposon-based barcoding approach ("TracerSeq") for reconstructing single-cell lineage histories. Clonally related cells were often restricted by the state landscape, including a case in which two independent lineages converge on similar fates. Cell fates remained restricted to this landscape in chordin-deficient embryos. We provide web-based resources for further analysis of the single-cell data.
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              Massively multiplex single-cell Hi-C

              We present single-cell combinatorial indexed Hi-C (sciHi-C), which applies the concept of combinatorial cellular indexing to chromosome conformation capture. In this proof-of-concept, we generate and sequence six sciHi-C libraries comprising a total of 10,696 single cells. We use sciHi-C data to separate cells by karytoypic and cell-cycle state differences and identify cell-to-cell heterogeneity in mammalian chromosomal conformation. Our results demonstrate that combinatorial indexing is a generalizable strategy for single-cell genomics.
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                Author and article information

                Journal
                0410462
                6011
                Nature
                Nature
                Nature
                0028-0836
                1476-4687
                10 January 2019
                20 February 2019
                February 2019
                20 August 2019
                : 566
                : 7745
                : 496-502
                Affiliations
                [1. ]Department of Genome Sciences, University of Washington, Seattle, Washington, USA
                [2. ]Molecular and Cellular Biology Program, University of Washington, Seattle, WA, USA.
                [3. ]Department of Computer Science, University of Washington, Seattle, Washington, USA
                [4. ]Max Planck Institute for Molecular Genetics, RG Development & Disease, Berlin, Germany
                [5. ]Institute for Medical and Human Genetics, Charité Universitätsmedizin Berlin, Berlin, Germany
                [6. ]Illumina, San Diego, California, USA
                [7. ]Brotman Baty Institute for Precision Medicine, Seattle, WA 98195
                [8. ]Howard Hughes Medical Institute, Seattle, Washington, USA
                Author notes
                [†]

                Equally contributing

                Author Contributions J.C. developed techniques and performed sci-RNA-seq3 experiments with assistance from M.S., F.Z., L.C., F.S.; M.S. performed embryo collection and in-situ hybridization validations with assistance from D.I. and S.M.; J.C. and C.T. performed computation analysis with assistance from M.S., X.Q. and A.H.; X.Q. and C.T. developed Monocle 3. X.H. developed website with assistance from J.C.; J.S. and C.T. supervised the project; J.S., C.T., J.C. and M.S. conceived the project and wrote the manuscript.

                Author Information Reprints and permissions information is available at www.nature.com/reprints. L.C., F.Z. and F.S. declare competing financial interests in the form of stock ownership and paid employment by Illumina, Inc. One or more embodiments of one or more patents and patent applications filed by Illumina may encompass the methods, reagents, and data disclosed in this manuscript. Some work in this study may be related to technology described in the following exemplary published patent applications: WO2010/0120098 and WO2011/0287435. Readers are welcome to comment on the online version of the paper. Correspondence and requests for materials should be addressed to J.S. ( shendure@ 123456uw.edu ) or C.T. ( coletrap@ 123456uw.edu ).

                Article
                NIHMS1518381
                10.1038/s41586-019-0969-x
                6434952
                30787437
                2e01e2d4-0ae1-4e79-83bf-70dfc03ee87d

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