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1. A dual involvement of Protocadherin 18a in stromal cell development guides the formation of a functional hematopoietic niche

Author: Catherine Vivier, Philippe Herbomel, Anne-Lou Touret, Emi Murayama, Anne Schmidt, Cellules Souches et Développement / Stem Cells and Development, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Ecole Doctorale Complexité du Vivant (ED515), Sorbonne Université (SU), Institut National de la Santé et de la Recherche Médicale (INSERM), This work was funded by Institut Pasteur, CNRS, and by grants from the Fondation pour la Recherche Médicale (Equipe FRM DEQ20160334881, to P.H). and the Fondation ARC pour la Recherche sur le Cancer (to P.H.). A.T.‘s fourth year of PhD fellowship was funded by the Agence Nationale de la Recherche Laboratoire d’Excellence Revive (Investissement d’Avenir, ANR-10-LABX-73)., We thank Stephane Rigaud, Dmitry Ershov and Jean-Yves Tinevez (Image Analysis Hub of Institut Pasteur) and Hannah Julienne (Bioinformatics & Biostatistics Hub of Institut Pasteur) for implementation of Python scripts and a R-script, respectively, that helped us quantify filopodia dynamics and statistics., ANR-10-LABX-0073,REVIVE,Stem Cells in Regenerative Biology and Medicine(2010), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), Département de Biologie du Développement et Cellules souches - Department of Developmental and Stem Cell Biology, Institut Pasteur [Paris] (IP), Biologie du Développement et Cellules souches (CNRS UMR3738), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), and This work was funded by Institut Pasteur and the Centre National de la Recherche Scientifique, and by grants from the Fondation pour la Recherche Médicale (Equipe FRM DEQ20160334881 to P.H.). and the Fondation ARC pour la Recherche sur le Cancer (to P.H.). A.-L.T.’s fourth year of PhD fellowship was funded by the Agence Nationale de la Recherche Laboratoire d'Excellence Revive (Investissement d'Avenir
Subjects: Stromal cell, cell migration, [SDV]Life Sciences [q-bio], Morphogenesis, Protocadherin, Hematopoietic niche, Biology, somite, 03 medical and health sciences, 0302 clinical medicine, Extracellular, Animals, protocadherin, Progenitor cell, Stem Cell Niche, [SDV.BDD]Life Sciences [q-bio]/Development Biology, Molecular Biology, Zebrafish, 030304 developmental biology, Mammals, 0303 health sciences, Hematopoietic Tissue, Endothelial Cells, Venous plexus, Hematopoietic Stem Cells, Protocadherins, Cell biology, Haematopoiesis, Stromal Cells, 030217 neurology & neurosurgery, Developmental Biology
Abstract: Hematopoietic stem and progenitor cells (HSPCs) emerge from the aorta and migrate to the caudal hematopoietic tissue (CHT) of zebrafish larvae, the hematopoietic equivalent of the mammalian fetal liver, for their proliferation and differentiation. We previously reported that somite-derived stromal cells were a key component of the CHT niche. Here we found that the cell adhesion protein protocadherin-18a (Pcdh18a) is expressed in the stromal cell progenitors (SCPs) emigrating from somites toward the future CHT. Deletion of most of the intracellular domain of Pcdh18a caused a decrease in the number of SCPs, the directionality of their migration, and the cell-contact mediated repulsion that normally occurs between migrating SCPs. These defects were followed by abnormal morphogenesis of the venous plexus that forms the CHT framework, and the inability of the resulting CHT to function as a niche for HSPCs. Finally, we found that the extracellular domain of Pcdh18a mediates trans heterophilic adhesion of stromal cells to endothelial cells in vivo and thereby the reticular vs. perivascular fate of SCPs. Our study demonstrates that Pcdh18a expression in SCPs is essential for the proper development of the hematopoietic niche.
Published: 2021
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2. A wave of bipotent T/ILC-restricted progenitors shapes the embryonic thymus microenvironment in a time-dependent manner

Author: Ana Cumano, Elisa Gomez Perdiguero, Pablo Pereira, Rachel Golub, Paulo Vieira, Laina Freyer, Ramy Elsaid, Odile Burlen-Defranoux, Francisca Soares-da-Silva, Thibaut Perchet, Antonio Bandeira, Sylvain Meunier, Lorea Iturri, Lymphocytes et Immunité - Lymphocytes and Immunity, Institut Pasteur [Paris] (IP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris Cité (UPCité), Instituto de Investigação e Inovação em Saúde (I3S), Universidade do Porto = University of Porto, Macrophages et Cellules endothéliales / Macrophages and Endothelial Cells, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Cellule Pasteur UPMC, Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut Pasteur [Paris] (IP), This work was financed by the Institut Pasteur, INSERM, Pasteur-Weizmann Foundation and ANR (grant Twothyme and Epi-Dev) through grants to A.C., by REVIVE (Investissement d'Avenir, ANR-10-LABX-73)to A.C. and R.E., by recurrent funding from the Institut Pasteur, the CNRS, Revive (Investissement d'Avenir, ANR-10-LABX-73) and by an ERC investigator award (2016-StG-715320) from the European Research Council to E.G.P., ANR-14-CE11-0022,Twothyme,Deux progéniteurs hématopoïétiques différents établissent le compartiment de lymphocytes T: tester un nouveau paradigme du développement T.(2014), ANR-10-LABX-0073,REVIVE,Stem Cells in Regenerative Biology and Medicine(2010), Institut Pasteur [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Paris (UP), Universidade do Porto, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut Pasteur [Paris], Vougny, Marie-Christine, Appel à projets générique - Deux progéniteurs hématopoïétiques différents établissent le compartiment de lymphocytes T: tester un nouveau paradigme du développement T. - - Twothyme2014 - ANR-14-CE11-0022 - Appel à projets générique - VALID, and Laboratoires d'excellence - Stem Cells in Regenerative Biology and Medicine - - REVIVE2010 - ANR-10-LABX-0073 - LABX - VALID
Subjects: 0303 health sciences, education.field_of_study, [SDV.IMM] Life Sciences [q-bio]/Immunology, Immunology, Population, Hematopoietic stem cell, Cell Biology, Hematology, Biology, Biochemistry, Embryonic stem cell, Cell biology, 03 medical and health sciences, Haematopoiesis, 0302 clinical medicine, Lymphatic system, Immune system, medicine.anatomical_structure, medicine, [SDV.IMM]Life Sciences [q-bio]/Immunology, Lymphopoiesis, Progenitor cell, education, 030304 developmental biology, 030215 immunology
Abstract: During embryonic development, multiple waves of hematopoietic progenitors with distinct lineage potential are differentially regulated in time and space. Two different waves of thymic progenitors colonize the fetal thymus where they contribute to thymic organogenesis and homeostasis. The origin, the lineage differentiation potential of the first wave, and their relative contribution in shaping the thymus architecture, remained, however, unclear. Here, we show that the first wave of thymic progenitors comprises a unique population of bipotent T and innatel lymphoid cells (T/ILC), generating a lymphoid tissue inducer cells (LTi's), in addition to invariant Vγ5+ T cells. Transcriptional analysis revealed that innate lymphoid gene signatures and, more precisely, the LTi-associated transcripts were expressed in the first, but not in the second, wave of thymic progenitors. Depletion of early thymic progenitors in a temporally controlled manner showed that the progeny of the first wave is indispensable for the differentiation of autoimmune regulator–expressing medullary thymic epithelial cells (mTECs). We further show that these progenitors are of strict hematopoietic stem cell origin, despite the overlap between lymphopoiesis initiation and the transient expression of lymphoid-associated transcripts in yolk sac (YS) erythromyeloid-restricted precursors. Our work highlights the relevance of the developmental timing on the emergence of different lymphoid subsets, required for the establishment of a functionally diverse immune system.
Published: 2021
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3. Mutations involving the SRY-related gene SOX8 are associated with a spectrum of human reproductive anomalies

Author: Aleksandar Rajkovic, S. Christin-Maitre, Anu Bashamboo, Liliana Dain, Caroline Schluth-Bolard, McLean Whi, ossetti R, Caroline Eozenou, Svetlana A. Yatsenko, Inas Mazen, Etienne Patin, Selma F. Witchel, Ralf Jauch, Rajpert-De Meyts E, Joelle Bignon-Topalovic, Kristian Almstrup, Ken McElreavey, Marie-Charlotte Dumargne, Louis-Sylvestre C, Sandra Chantot-Bastaraud, de Malleray Pichard C, Jean-Pierre Siffroi, Célia Ravel, Violeta A. Chiauzzi, Capucine Hyon, John C. Achermann, Hassan Rouba, Stuart A. MacGowan, Reyes-ugica M, Andrew J. Duncan, Eduardo H. Charreau, Leila Fusee, Marie-France Portnoi, Sandra Rojo, Luca Persani, Raja Brauner, Validire P, Yogesh Srivastava, CHU Trousseau [APHP], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU), Maladies génétiques d'expression pédiatrique (U933), Institut National de la Santé et de la Recherche Médicale (INSERM)-CHU Trousseau [APHP], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU), Sorbonne Université - Faculté de Médecine (SU FM), Sorbonne Université (SU), Génétique Evolutive Humaine - Human Evolutionary Genetics, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Children's Hospital of Pittsburgh of UPMC [Etats-Unis], Great Ormond Street Hospital for Children [London] (GOSH), Rigshospitalet [Copenhagen], Copenhagen University Hospital, South China Institute for Stem Cell Biology and Regenerative Medicine [Guangzhou, China], Institut Mutualiste de Montsouris (IMM), Service de gynécologie et d'endocrinologie [CHU Cochin], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Hôpital Cochin [AP-HP], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP), CHU Pontchaillou [Rennes], CHU Saint-Antoine [AP-HP], Fondation Ophtalmologique Adolphe de Rothschild [Paris], Université Paris Descartes - Paris 5 (UPD5), Università degli Studi di Milano = University of Milan (UNIMI), Istituti di Ricovero e Cura a Carattere Scientifico (IRCCS), Consejo Nacional de Investigaciones Científicas y Técnicas [Buenos Aires] (CONICET), National Research Centre [Cairo, Egypt], Institut Pasteur du Maroc, Réseau International des Instituts Pasteur (RIIP), University of Dundee, Actions Concertees Interpasteuriennes (ACIP) and a research grant from the European Society of Pediatric Endocrinology to A.B. A research grant from the EuroDSD in the European Community’s Seventh Framework Programme FP7/2007–2013 under grant agreement No. 201444 as well as grant No. 295097 as part of the EU call FP7-INCO-2011–6 to A.B. and K.McE. A Franco-Egyptian AIRD-STDF grant to A.B., K.M. and I.M. Chinese Government Scholarship and University of the Chinese Academy of Science (UCAS) for financial and infrastructure support to Y.S. 2013 MOST China-EU Science and Technology Cooperation Program, Grant No. 2013DFE33080, by the National Natural Science Foundation of China (Grant No. 31471238) and a 100 talent award of the Chinese Academy of Sciences to R.J. Innovation Fund Denmark (grant # 14–2013-4) to K.A. The human embryonic and fetal material was provided by the Joint MRC/Wellcome Trust (grant # 099175/Z/12/Z) Human Developmental Biology Resource (www.hdbr.org). J.C.A. is a Wellcome Trust Senior Research Fellow in Clinical Science (098513/Z/12/Z) and received support from the National Institute for Health Research Biomedical Research Centre at Great Ormond Street Hospital for Children NHS Foundation Trust and University College London. R.R. is a fellow supported by the Italian Ministry of Health, Rome, Italy (grant # GR-2011–02351636). This work is supported by the COST Action DSDnet BM1303. This work was funded by the Agence Nationale de la Recherche (Laboratoire d’Excellence Revive, Investissement d’Avenir, ANR-10-LABX-73). Funding to pay the Open Access publication charges for this article was provided by Laboratoire d'Excellence Revive, Investissement d'Avenir, ANR-10-LABX-73., ANR-10-LABX-0073,REVIVE,Stem Cells in Regenerative Biology and Medicine(2010), European Project: 201444,EC:FP7:HEALTH,FP7-HEALTH-2007-A,EURODSD(2008), European Project: 295097,EC:FP7:INCO,FP7-INCO-2011-6,GM_NCD_IN_CO(2011), Service de génétique et embryologie médicales [CHU Trousseau], Physiopathologie des maladies génétiques d'expression pédiatrique (UMRS_933), Sorbonne Université (SU)-Institut National de la Santé et de la Recherche Médicale (INSERM), Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Service d'Endocrinologie, diabétologie et endocrinologie de la reproduction [CHU Saint-Antoine], University of Milan, Service de Génétique et d'Embryologie Médicales [CHU Trousseau], Assistance publique - Hôpitaux de Paris (AP-HP) (APHP)-CHU Trousseau [APHP], Physiopathologie des maladies génétiques d'expression pédiatrique, Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU), Assistance publique - Hôpitaux de Paris (AP-HP) (APHP)-CHU Cochin [AP-HP], Service d'Endocrinologie, Diabétologie et d'Endocrinologie de la Reproduction [CHU Saint-Antoine], Assistance publique - Hôpitaux de Paris (AP-HP) (APHP)-CHU Saint-Antoine [APHP], and ANR: 10-LABX-0073,REVIVE,Stem Cells in Regenerative Biology and Medicine(2010)
Subjects: 0301 basic medicine, Male, 46, XX Disorders of Sex Development, Primary Ovarian Insufficiency, Medical and Health Sciences, Male infertility, XY, purl.org/becyt/ford/1 [https], 0302 clinical medicine, Missense mutation, Related gene, Child, MUTATION, Genetics (clinical), Genetics, Genetics & Heredity, SOXE Transcription Factors, Sexual differentiation in humans, General Medicine, Articles, purl.org/becyt/ford/3.1 [https], Bioquímica y Biología Molecular, Biological Sciences, Medicina Básica, medicine.anatomical_structure, Testis determining factor, 030220 oncology & carcinogenesis, Female, purl.org/becyt/ford/3 [https], DISORDER OF SEX DEVELOPMENT, Biología Reproductiva, SOX8, CIENCIAS NATURALES Y EXACTAS, OLIGOSPERMIA, Infertility, Gonad, CIENCIAS MÉDICAS Y DE LA SALUD, Adolescent, Mutation, Missense, Locus (genetics), Biology, INFERTILITY, Ciencias Biológicas, 03 medical and health sciences, XX Disorders of Sex Development, SRY, medicine, Humans, purl.org/becyt/ford/1.6 [https], Molecular Biology, Disorder of Sex Development, 46,XY, Oligospermia, medicine.disease, 030104 developmental biology, [SDV.GEN.GH]Life Sciences [q-bio]/Genetics/Human genetics, Missense
Abstract: SOX8 is an HMG-box transcription factor closely related to SRY and SOX9. Deletion of the gene encoding Sox8 in mice causes reproductive dysfunction but the role of SOX8 in humans is unknown. Here, we show that SOX8 is expressed in the somatic cells of the early developing gonad in the human and influences human sex determination. We identified two individuals with 46, XY disorders/differences in sex development (DSD) and chromosomal rearrangements encompassing the SOX8 locus and a third individual with 46, XY DSD and a missense mutation in the HMG-box of SOX8. In vitro functional assays indicate that this mutation alters the biological activity of the protein. As an emerging body of evidence suggests that DSDs and infertility can have common etiologies, we also analysed SOX8 in a cohort of infertile men (n=274) and two independent cohorts of women with primary ovarian insufficiency (POI; n=153 and n=104). SOX8 mutations were found at increased frequency in oligozoospermic men (3.5%; P < 0.05) and POI (5.06%; P=4.5×10-5) as compared with fertile/normospermic control populations (0.74%). The mutant proteins identified altered SOX8 biological activity as compared with the wild-type protein. These data demonstrate that SOX8 plays an important role in human reproduction and SOX8 mutations contribute to a spectrum of phenotypes including 46, XY DSD, male infertility and 46, XX POI. Fil: Portnoi, Marie France. Inserm; Francia. Sorbonne Université. Faculté de Medecine; Francia. Hôpital Armand Trousseau; Francia Fil: Dumargne, Marie Charlotte. Instituto Pasteur; Francia Fil: Rojo, Sandra. Instituto Pasteur; Francia Fil: Witchel, Selma F.. University of Pittsburgh; Estados Unidos Fil: Duncan, Andrew J.. Great Ormond Street Hospital for Children; Reino Unido Fil: Eozenou, Caroline. Instituto Pasteur; Francia Fil: Bignon Topalovic, Joelle. Instituto Pasteur; Francia Fil: Yatsenko, Svetlana A.. University of Pittsburgh; Estados Unidos Fil: Rajkovic, Aleksandar. University of Pittsburgh; Estados Unidos Fil: Reyes Mugica, Miguel. University of Pittsburgh; Estados Unidos Fil: Almstrup, Kristian. Rigshospitalet; Dinamarca Fil: Fusee, Leila. Instituto Pasteur; Francia Fil: Srivastava, Yogesh. Chinese Academy of Sciences; República de China Fil: Chantot Bastaraud, Sandra. Hôpital Armand Trousseau; Francia Fil: Hyon, Capucine. Hôpital Armand Trousseau; Francia. Inserm; Francia Fil: Louis Sylvestre, Christine. Institut Mutualiste Montsouris; Francia Fil: Validire, Pierre. Institut Mutualiste Montsouris; Francia Fil: de Malleray Pichard, Caroline. Hôpital Cochin; Francia Fil: Ravel, Celia. Centre Hospitalier Universitaire de Rennes; Francia Fil: Christin Maitre, Sophie. Inserm; Francia. Hôpital Saint-Antoine; Francia Fil: Brauner, Raja. Universite de Paris; Francia Fil: Rossetti, Raffaella. Università degli Studi di Milano; Italia. Istituto Auxologico Italiano; Italia Fil: Persani, Luca. Istituto Auxologico Italiano; Italia. Università degli Studi di Milano; Italia Fil: Charreau, Eduardo Hernan. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina. Administracion Nacional de Laboratorios E Institutos de Salud "dr. Carlos G. Malbran". Instituto Nacional de Epidemiologia. Departamento de Investigacion.; Argentina Fil: Dain, Liliana Beatriz. Administracion Nacional de Laboratorios E Institutos de Salud "dr. Carlos G. Malbran". Instituto Nacional de Epidemiologia. Departamento de Investigacion.; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina Fil: Chiauzzi, Violeta Alicia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina. Administracion Nacional de Laboratorios E Institutos de Salud "dr. Carlos G. Malbran". Instituto Nacional de Epidemiologia. Departamento de Investigacion.; Argentina Fil: Mazen, Inas. National Research Centre ; Egipto Fil: Rouba, Hassan. Institut Pasteur du Maroc; Marruecos Fil: Schluth Bolard, Caroline. Hôpital Femme Mère Enfant; Francia Fil: Mac Gowan, Stuart. University of Dundee; Reino Unido Fil: Mc Lean, W. H. Irwin. University of Dundee; Reino Unido Fil: Patin, Etienne. Instituto Pasteur; Francia Fil: Rajpert De Meyts, Ewa. Rigshospitalet; Dinamarca Fil: Jauch, Ralf. Chinese Academy of Sciences; República de China Fil: Achermann, John C.. Great Ormond Street Hospital for Children; Reino Unido Fil: Siffroi, Jean Pierre. Hôpital Armand Trousseau; Francia Fil: Mc Elreavey, Ken. Instituto Pasteur; Francia Fil: Bashamboo, Anu. Inserm; Francia. Instituto Pasteur; Francia
Published: 2018
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4. Early epigenetic reprogramming in fertilized, cloned, and parthenogenetic embryos

Author: Edgar del Llano Solanas, Walid E. Maalouf, Elisa Serrano-Revuelta, Lessly P. Sepulveda-Rincon, Lydia Ruddick, Nathalie Beaujean, School of Medicine, Child Health, Obstetrics and Gynaecology, University of Nottingham, Biologie du développement et reproduction (BDR), École nationale vétérinaire d'Alfort (ENVA)-Institut National de la Recherche Agronomique (INRA)-Centre National de la Recherche Scientifique (CNRS), Laboratoire d'Excellence Revive (Investissement d'Avenir) [ANR-10-LABX-73], CONACyT Mexico, Biologie du Développement et Reproduction (BDR), Institut National de la Recherche Agronomique (INRA), Institut cellule souche et cerveau / Stem Cell and Brain Research Institute (SBRI), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National de la Santé et de la Recherche Médicale (INSERM), École nationale vétérinaire d'Alfort (ENVA)-Institut National de la Recherche Agronomique (INRA), Institut cellule souche et cerveau (U846 Inserm - UCBL1), Institut National de la Recherche Agronomique (INRA)-Université Claude Bernard Lyon 1 (UCBL), Laboratoire d'Excellence Revive (Investissement d'Avenir, ANR-10-LABX-73), and 18th International Congress on Animal Reproduction (Tours, France, 26-30 juin 2016)
Subjects: MESH: Parthenogenesis, 0301 basic medicine, Oocyte, [SDV]Life Sciences [q-bio], Cloning, Organism, Parthenogenesis, Development, Biology, MESH: Cellular Reprogramming, Genome, Epigenesis, Genetic, Histones posttranslational modifications, 03 medical and health sciences, Histones post- translational modifications, Human fertilization, Food Animals, MESH: Cloning, Organism, medicine, Animals, MESH: Animals, mammals, MESH: Epigenesis, Genetic, Small Animals, [SDV.BDD]Life Sciences [q-bio]/Development Biology, ComputingMilieux_MISCELLANEOUS, Genetics, Embryonic genome activation, DNA methylation, Equine, [SDV.BA]Life Sciences [q-bio]/Animal biology, Embryogenesis, MESH: Embryo, Mammalian, Epigenetic, [SDV.BDLR]Life Sciences [q-bio]/Reproductive Biology, Embryo, Cellular Reprogramming, Embryo, Mammalian, Sperm, DNA methylation 2, 030104 developmental biology, medicine.anatomical_structure, Animal Science and Zoology, Nuclear transfer, Reprogramming
Abstract: International audience; Despite ongoing research in a number of species, the efficiency of embryo production by nuclear transfer remains low. Incomplete epigenetic reprogramming of the nucleus introduced in the recipient oocyte is one factor proposed to limit the success of this technique. Nonetheless, knowledge of reprogramming factors has increased-thanks to comparative studies on reprogramming of the paternal genome brought by sperm on fertilization-and will be reviewed here. Another valuable model of reprogramming is the one obtained in the absence of sperm fertilization through artificial activation-the parthenote-and will also be introduced. Altogether the objective of this review is to have a better understanding on the mechanisms responsible for the resistance to reprogramming, not only because it could improve embryonic development but also as it could benefit therapeutic reprogramming research.
Published: 2015
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5. Extraocular muscle stem cells exhibit distinct cellular properties associated with non-muscle molecular signatures

Author: Daniela Di Girolamo, Maria Benavente-Diaz, Alexandre Grimaldi, Priscilla Thomas Lopes, Melania Murolo, Brendan Evano, Stamatia Gioftsidi, Vincent Laville, Sebastian Mella, Shahragim Tajbakhsh, Glenda Comai, Cellules Souches et Développement / Stem Cells and Development, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), Ecole Doctorale Complexité du Vivant (ED515), Sorbonne Université (SU), Université Paris-Est (UPE), Freie Universität Berlin, Institut Mondor de Recherche Biomédicale (IMRB), Institut National de la Santé et de la Recherche Médicale (INSERM)-IFR10-Université Paris-Est Créteil Val-de-Marne - Paris 12 (UPEC UP12), Cytometrie et Biomarqueurs – Cytometry and Biomarkers (UTechS CB), Institut Pasteur [Paris] (IP)-Université Paris Cité (UPCité), We acknowledge funding support from the Institut Pasteur, Agence Nationale de la Recerche (Laboratoire d’Exellence Revive, Investissement d’Avenir, ANR-10-LABX-73 to ST and ANR-21-CE13-0005 MUSE to GC), Association Française contre les Myopathies (Grant #20510 to ST and #23201 to GC), and the Centre National de la Recherche Scientifique. M.B.D. was supported by a grant from Laboratoire d’Excellence Revive and La Ligue Contre le Cancer. We gratefully acknowledge the Genomic Patform (Inserm U1016-CNRS UMRS 8104 - Université de Paris Cité) at Institut Cochin, the UtechS Photonic BioImaging (Imagopole), C2RT, Institut Pasteur, supported by the French National Research Agency (France BioImaging, ANR-10–INSB–04, Investments for the Future) and the Center for Translational Science (CRT)-Cytometry and Biomarkers Unit of Technology and Service (CB UTechS) at Institut Pasteur for support in conducting this study., ANR-10-LABX-0073,REVIVE,Stem Cells in Regenerative Biology and Medicine(2010), and ANR-21-CE13-0005,MUSE,Mécanismes définissant l'hétérogénéité fonctionnelle de populations myogéniques anatomiquement distinctes(2021)
Subjects: muscle stem cell diversity, muscle stem, matrisome, extraocular muscles, MuSC sc-RNAseq, TF network, [SDV.BC.BC]Life Sciences [q-bio]/Cellular Biology/Subcellular Processes [q-bio.SC], mesenchymal stromal cells, MuSC expansion, [SDV.BDD]Life Sciences [q-bio]/Development Biology
Abstract: The muscle stem cell (MuSC) population is recognized as functionally heterogeneous. Cranial muscle stem cells, which originate from head mesoderm, can have greater proliferative capacity in culture and higher regenerative potential in transplantation assays when compared to those in the limb. The existence of such functional differences in phenotypic outputs remain unresolved as a comprehensive understanding of the underlying mechanisms is lacking. We addressed this issue using a combination of clonal analysis, live imaging, and scRNA-seq, identifying critical biological features that distinguish extraocular (EOM) and limb (Tibialis anterior, TA) MuSC populations. Time-lapse studies using aMyogenintdTomatoreporter showed that the increased proliferation capacity of EOM MuSCs is accompanied by a differentiation delayin vitro. Unexpectedly, in vitro activated EOM MuSCs expressed a large array of distinct extracellular matrix (ECM) components, growth factors, and signaling molecules that are typically associated with mesenchymal non-muscle cells. These unique features are regulated by a specific set of transcription factors that constitute a coregulating module. This transcription factor network, which includes Foxc1 as one of the major players, appears to be hardwired to EOM identity as it is present in quiescent adult MuSCs, in the activated counterparts during growth and retained upon passages in vitro. These findings provide insights into how high-performing MuSCs regulate myogenic commitment by active remodeling of their local environment.
Published: 2023

6. A gene subset requires CTCF bookmarking during the fast post‐mitotic reactivation of mouse ES cells

Author: Almira Chervova, Nicola Festuccia, Luis Altamirano‐Pacheco, Agnès Dubois, Pablo Navarro, Epigénomique, Prolifération et Identité Cellulaire - Epigenomics, Proliferation and the Identity of Cells (EPIC), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), Collège Doctoral, Sorbonne Université (SU), PN acknowledges the Institut Pasteur, the CNRS and Revive (Investissement d'Avenir, ANR-10-LABX-73) for recurrent funding and the Ligue Nationale Contre le Cancer (LNCC EL2018 NAVARRO) and the European Research Council (ERC-CoG-2017 BIND) for financial support., The authors also acknowledge the Biomics and Flow Cytometry platforms of Institut Pasteur., and ANR-10-LABX-0073,REVIVE,Stem Cells in Regenerative Biology and Medicine(2010)
Subjects: Genetics, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, Molecular Biology, Biochemistry
Abstract: International audience; Mitosis leads to global downregulation of transcription that then needs to be efficiently resumed. In somatic cells, this is mediated by a transient hyper-active state that first reactivates housekeeping and then cell identity genes. Here, we show that mouse embryonic stem cells, which display rapid cell cycles and spend little time in G1, also display accelerated reactivation dynamics. This uniquely fast global reactivation lacks specificity towards functional gene families, enabling the restoration of all regulatory functions before DNA replication. Genes displaying the fastest reactivation are bound by CTCF, a mitotic bookmarking transcription factor. In spite of this, the post-mitotic global burst is robust and largely insensitive to CTCF depletion. There are, however, around 350 genes that respond to CTCF depletion rapidly after mitotic exit. Remarkably, these are characterised by promoter-proximal mitotic bookmarking by CTCF. We propose that the structure of the cell cycle imposes distinct constrains to post-mitotic gene reactivation dynamics in different cell types, via mechanisms that are yet to be identified but that can be modulated by mitotic bookmarking factors.
Published: 2022
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7. Trim33 conditions the lifespan of primitive macrophages and onset of definitive macrophage production

Author: Doris Lou Demy, Anne-Lou Touret, Mylène Lancino, Muriel Tauzin, Lavinia Capuana, Constance Pierre, Philippe Herbomel, Macrophages et Développement de l’Immunité, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), This work was supported by grants to P.H. from the Fondation pour la Recherche Médicale (#DEQ20120323714,#DEQ20160334881) and the Laboratoire d'Excellence Revive (Investissement d'Avenir, ANR-10-LABX-73), and ANR-10-LABX-0073,REVIVE,Stem Cells in Regenerative Biology and Medicine(2010)
Subjects: Longevity, Zebrafish Proteins, Hematopoietic Stem Cells, zebrafish, Trim33, Hematopoiesis, macrophages, Mice, Animals, development, [SDV.BDD]Life Sciences [q-bio]/Development Biology, Molecular Biology, Transcription Factors, Developmental Biology
Abstract: Trim33 (Tif1γ) is a transcriptional regulator that is notably involved in several aspects of hematopoiesis. It is essential for the production of erythrocytes in zebrafish, and for the proper functioning and aging of hematopoietic stem and progenitor cells (HSPCs) in mice. Here, we have found that, in zebrafish development, Trim33 is essential cell-autonomously for the lifespan of the yolk sac-derived primitive macrophages, as well as for the initial production of definitive (HSPC-derived) macrophages in the first niche of definitive hematopoiesis, the caudal hematopoietic tissue. Moreover, Trim33 deficiency leads to an excess production of definitive neutrophils and thrombocytes. Our data indicate that Trim33 radically conditions the differentiation output of aorta-derived HSPCs in all four erythro-myeloid cell types, in a niche-specific manner.
Published: 2022
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8. Alcam-a and Pdgfr-α are essential for the development of sclerotome derived stromal cells that support hematopoiesis in vivo

Author: Emi Murayama, Catherine Vivier, Anne Schmidt, Anne-Lou Touret, Philippe Herbomel, Institut Pasteur [Paris] (IP), This work was supported by Institut Pasteur, CNRS, and by grants to P.H. from the Fondation pour la Recherche Médicale (#DEQ20160334881), the Fondation ARC pour la Recherche sur le Cancer, and the Agence Nationale de la Recherche Laboratoire d’Excellence Revive (Investissement d’Avenir, ANR-10-LABX-73)., ANR-10-LABX-0073,REVIVE,Stem Cells in Regenerative Biology and Medicine(2010), SCHMIDT, Anne, and Laboratoires d'excellence - Stem Cells in Regenerative Biology and Medicine - - REVIVE2010 - ANR-10-LABX-0073 - LABX - VALID
Subjects: [SDV] Life Sciences [q-bio], [SDV]Life Sciences [q-bio], [SDV.BDD] Life Sciences [q-bio]/Development Biology, [SDV.BDD]Life Sciences [q-bio]/Development Biology
Abstract: Mesenchymal stromal cells are essential components of hematopoietic stem and progenitor cell (HSPC) niches, regulating HSPC proliferation and fate decisions. Their developmental origins are largely unknown. In zebrafish, we previously found that the stromal cells of the caudal hematopoietic tissue (CHT), a niche functionally homologous to the fetal liver in mammals, arise from the ventral part of caudal somites. We have now discovered that this ventral domain is actually the sclerotome, and that two typical markers of mammalian mesenchymal stem/stromal cells, Alcam and Pdgfr-α, are distinctively expressed there and instrumental for the emergence and migration of stromal cell progenitors, which in turn conditions the proper assembly of the vascular component of the CHT niche. Furthermore, we find that the trunk somites are similarly dependent on Alcam and Pdgfr-α to produce mesenchymal stromal cells that foster the initial emergence of HSPCs from the dorsal aorta. Thus the sclerotome contributes essential stromal cells for each of the key steps of developmental hematopoiesis, and likely is the embryological origin of most if not all mesenchymal stem/stromal cells found in non-cephalic tissues.
Published: 2022

9. OCT4 activates a Suv39h1-repressive antisense lncRNA to couple histone H3 Lysine 9 methylation to pluripotency

Author: Laure D Bernard, Agnès Dubois, Victor Heurtier, Véronique Fischer, Inma Gonzalez, Almira Chervova, Alexandra Tachtsidi, Noa Gil, Nick Owens, Lawrence E Bates, Sandrine Vandormael-Pournin, José C R Silva, Igor Ulitsky, Michel Cohen-Tannoudji, Pablo Navarro, Epigénomique, Prolifération et Identité Cellulaire - Epigenomics, Proliferation and the Identity of Cells (EPIC), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), Collège Doctoral, Sorbonne Université (SU), Weizmann Institute of Science [Rehovot, Israël], University of Edinburgh, Guangzhou International Bio Island [Guangdong, China] (GIBI), L.B. acknowledges the Ecole Normale Supérieure and Sorbonne Université for funding, P.N. and M.C.-T. acknowledge the Labex Revive [Investissement d’Avenir, ANR-10-LABX-73], Institut Pasteur, CNRS. Funding for open access charge: Pasteur core funding., and ANR-10-LABX-0073,REVIVE,Stem Cells in Regenerative Biology and Medicine(2010)
Subjects: Histones/genetics, Repressor Proteins/genetics, Methyltransferases, Methylation, Chromatin, Histones, Repressor Proteins, Histone Code, Methyltransferases/genetics, Mice, Genetics, Animals, RNA, Long Noncoding, RNA, Long Noncoding/genetics, Octamer Transcription Factor-3, [SDV.BDD]Life Sciences [q-bio]/Development Biology, Octamer Transcription Factor-3/metabolism
Abstract: Histone H3 Lysine 9 (H3K9) methylation, a characteristic mark of heterochromatin, is progressively implemented during development to contribute to cell fate restriction as differentiation proceeds. Accordingly, in undifferentiated and pluripotent mouse Embryonic Stem (ES) cells the global levels of H3K9 methylation are rather low and increase only upon differentiation. How global H3K9 methylation levels are coupled with the loss of pluripotency remains largely unknown. Here, we identify SUV39H1, a major H3K9 di- and tri-methylase, as an indirect target of the pluripotency network of Transcription Factors (TFs). We find that pluripotency TFs, principally OCT4, activate the expression of Suv39h1as, an antisense long non-coding RNA to Suv39h1. In turn, Suv39h1as downregulates Suv39h1 transcription in cis via a mechanism involving the modulation of the chromatin status of the locus. The targeted deletion of the Suv39h1as promoter region triggers increased SUV39H1 expression and H3K9me2 and H3K9me3 levels, affecting all heterochromatic regions, particularly peri-centromeric major satellites and retrotransposons. This increase in heterochromatinization efficiency leads to accelerated and more efficient commitment into differentiation. We report, therefore, a simple genetic circuitry coupling the genetic control of pluripotency with the global efficiency of H3K9 methylation associated with a major cell fate restriction, the irreversible loss of pluripotency.
Published: 2022
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10. H3K9 tri-methylation at Nanog times differentiation commitment and enables the acquisition of primitive endoderm fate

Author: A. Chervova, Michel Cohen-Tannoudji, S. Vandormael-Pournin, L. Vincenti, Agnès Dubois, Pablo Navarro, Institut Pasteur [Paris], Epigénomique, Prolifération et Identité Cellulaire - Epigenomics, Proliferation and the Identity of Cells (EPIC), Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), P.N. and M.C-T. acknowledge the Labex Revive (Investissement d’Avenir, ANR-10-LABX-73), the Institut Pasteur and the CNRS for funding., The authors acknowledge the cytometry platform of Institut Pasteur for technical assistance and L. Bally-Cuif and S. Tajbakhsh for critical reading of the manuscript., ANR-10-LABX-0073,REVIVE,Stem Cells in Regenerative Biology and Medicine(2010), and Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité)
Subjects: Homeobox protein NANOG, 0303 health sciences, biology, Somatic cell, 030302 biochemistry & molecular biology, Embryonic stem cell, Cell biology, 03 medical and health sciences, Histone H3, Histone, embryonic structures, biology.protein, Gene silencing, Enhancer, Transcription factor, [SDV.BDD]Life Sciences [q-bio]/Development Biology, 030304 developmental biology
Abstract: Mouse Embryonic Stem (ES) cells have an inherent propensity to explore distinct gene-regulatory states associated with either self-renewal or differentiation. This property is largely dependent on ERK activity, which promotes silencing of pluripotency genes, most notably of the transcription factor Nanog. Here, we aimed at identifying repressive histone modifications that would mark the Nanog locus for inactivation in response to ERK activity. We found histone H3 lysine 9 tri-methylation (H3K9me3) focally enriched between the Nanog promoter and its −5kb enhancer. While in undifferentiated ES cells H3K9me3 at Nanog depends on ERK activity, in somatic cells it becomes ERK-independent. Moreover, upon deletion of the region harbouring H3K9me3, ES cells display reduced heterogeneity of NANOG expression, delayed commitment into differentiation and impaired ability to acquire a primitive endoderm fate. We suggest that establishment of irreversible H3K9me3 at specific master regulators allows the acquisition of particular cell fates during differentiation.
Published: 2021

11. The activation of a Suv39h1 -repressive antisense lncRNA by OCT4 couples the control of H3K9 methylation to pluripotency

Author: Agnès Dubois, Sandrine Vandormael-Pournin, Pablo Navarro, Michel Cohen-Tannoudji, Victor Heurtier, Laure Bernard, Nick D.L. Owens, Igor Ulitsky, Noa Gil, Almira Chervova, Alexandra Tachtsidi, Institut Pasteur [Paris] (IP), Epigénomique, Prolifération et Identité Cellulaire - Epigenomics, Proliferation and the Identity of Cells (EPIC), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), Collège Doctoral, Sorbonne Université (SU), Weizmann Institute of Science [Rehovot, Israël], Friedrich Miescher Institute for Biomedical Research (FMI), Novartis Research Foundation, University of Exeter, L.B. acknowledges the Ecole Normale Supérieure and Sorbonne Université for funding. P.N. and M.C-T. acknowledge the Labex Revive (Investissement d’Avenir, ANR-10-LABX-73), the Institut Pasteur and the CNRS for funding., ANR-10-LABX-0073,REVIVE,Stem Cells in Regenerative Biology and Medicine(2010), Institut Pasteur [Paris], Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), and Collège doctoral [Sorbonne universités]
Subjects: 0303 health sciences, Methylation, Biology, Embryonic stem cell, Chromatin, Cell biology, 03 medical and health sciences, Histone H3, 0302 clinical medicine, Transcription (biology), Epigenetics, Transcription factor, Reprogramming, [SDV.BDD]Life Sciences [q-bio]/Development Biology, 030217 neurology & neurosurgery, 030304 developmental biology
Abstract: Histone H3 Lysine 9 (H3K9) methylation, a characteristic mark of heterochromatin, is progressively implemented during development to contribute to cell fate restriction as differentiation proceeds. For instance, in pluripotent mouse Embryonic Stem (ES) cells the global levels of H3K9 methylation are rather low and increase only upon differentiation. Conversely, H3K9 methylation represents an epigenetic barrier for reprogramming somatic cells back to pluripotency. How global H3K9 methylation levels are coupled with the acquisition and loss of pluripotency remains largely unknown. Here, we identify SUV39H1, a major H3K9 di- and tri-methylase, as an indirect target of the pluripotency network of Transcription Factors (TFs). We find that pluripotency TFs, principally OCT4, activate the expression of an uncharacterized antisense long non-coding RNA to Suv39h1, which we name Suv39h1as. In turn, Suv39h1as downregulates Suv39h1 transcription in cis via a mechanism involving the modulation of the chromatin status of the locus. The targeted deletion of the Suv39h1as promoter region triggers increased SUV39H1 expression and H3K9me2 and H3K9me3 levels, leading to accelerated and more efficient commitment into differentiation. We report, therefore, a simple genetic circuitry coupling the global levels of H3K9 methylation to pluripotency in mouse ES cells.
Published: 2021

12. Interleukin-10 induces interferon-γ-dependent emergency myelopoiesis

Author: Ana, Cardoso, Ana Catarina, Martins, Ana Raquel, Maceiras, Wei, Liu, Isabel, Castro, António G, Castro, António, Bandeira, James P, Di Santo, Ana, Cumano, Yan, Li, Paulo, Vieira, Margarida, Saraiva, Instituto de Investigação e Inovação em Saúde (I3S), Universidade do Porto = University of Porto, Instituto de Biologia Molecular e Celular - institute for molecular and cell biology [Porto, Portugal] (IBMC), Lymphocytes et Immunité - Lymphocytes and Immunity, Institut Pasteur [Paris] (IP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris Cité (UPCité), Instituto de Ciências Biomédicas de Abel Salazar (ICBAS), Medical School (University of Nanjing), Nanjing University (NJU), University of Minho [Braga], Cellule Pasteur, Université Paris Diderot - Paris 7 (UPD7)-PRES Sorbonne Paris Cité, Immunité Innée - Innate Immunity, A. Cardoso (SFRH/BD/84704/2012) and A.C.M. (SFRH/BD/136800/2018) were supported by the Portuguese Foundation for Science and Technology (FCT) through PhD grants.This study was partially supported by grants from the National Key Research and Development Program of China (2019YFA0802900), National Natural Science Foundation of China (32070942), and Fundamental Research Funds for the Central Universities.The M.S. lab was financed by a FCT-ANR grant (MyeloTEN-FCTANR/ BIM-MEC/0007/2013). We acknowledge the GenomePT project (POCI-01-0145-FEDER-022184), supported by COMPETE 2020 - Operational Programme for Competitiveness and Internationalisation (POCI), Lisboa Portugal Regional Operational Programme (Lisboa2020), Algarve Portugal Regional Operational Programme (CRESC Algarve2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF), and by FCT.This work was funded by National Funds through FCT, I.P., under the project UIDB/04293/2020, and backed by the COST Action BM1404 European Network of Investigators Triggering Exploratory Research on Myeloid Regulatory Cells (http://www.mye-euniter.eu), which is supported by the Horizon 2020—EU Framework Program Research and Innovation Programme. M.S. is funded by FCT through Estímulo Individual ao Emprego Científico.A. Cumano and P.V. were financed by ANR Twothyme and by REVIVE (Investissement d’Avenir, ANR-10-LABX-73). A.B., A. Cumano, and P.V. were also financed by the Institut Pasteur, INSERM, and ANR (project MYELOTEN, ANR-13-ISV1-0003-01)., We thank Vincent Rouilly for statistical advice and Werner Müller (University of Manchester), Rui Appelberg (i3S), and Bruno Silva-Santos (iMM) for providing the IL-10Rα-, IFN-γ-, and TCRγδ-deficient animals, respectively. We thank Anne O’Garra for helpful discussions and for critical reading of the manuscript. We also thank Delfim Duarte and Maria José Teles for helpful discussions and for helping with the hemogram quantification. We thank Caetano Reis e Sousa for help with the preparation of the graphical abstract. We thank the support of the personnel in the animal facilities, the i3S scientific platforms Translational Cytometry (TraCy), and Histology and Electron Microscopy (HEMS). HEMS is a member of the national infrastructure PPBI (Portuguese Platform of Bioimaging, PPBI-POCI-01-0145-FEDER-022122)., ANR-10-LABX-0073,REVIVE,Stem Cells in Regenerative Biology and Medicine(2010), ANR-13-ISV1-0003,MYELOTEN,DEREGULATION DE L'HEMATOPOIESE PAR LA SUREXPRESSION DE L'INTERLEUKINE-10 : IMPLICATIONS DANS LE DEVELOPPEMENT DE PATHOLOGIES HEMATOLOGIQUES(2013), European Project, Vougny, Marie-Christine, Laboratoires d'excellence - Stem Cells in Regenerative Biology and Medicine - - REVIVE2010 - ANR-10-LABX-0073 - LABX - VALID, Blanc – Accords bilatéraux 2013 - DEREGULATION DE L'HEMATOPOIESE PAR LA SUREXPRESSION DE L'INTERLEUKINE-10 : IMPLICATIONS DANS LE DEVELOPPEMENT DE PATHOLOGIES HEMATOLOGIQUES - - MYELOTEN2013 - ANR-13-ISV1-0003 - Blanc – Accords bilatéraux 2013 - VALID, COST Action BM1404 MyeEUNITER - INCOMING, Universidade do Porto, Institut Pasteur [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Paris (UP), and Institut Pasteur [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM)
Subjects: CD4-Positive T-Lymphocytes, Mice, Knockout, Myelopoiesis, [SDV.IMM] Life Sciences [q-bio]/Immunology, T cells, emergency myelopoiesis, CD8-Positive T-Lymphocytes, Interleukin-10, Interferon-gamma, Mice, IL-10, Animals, [SDV.IMM]Life Sciences [q-bio]/Immunology, IFN-γ, Myeloid Progenitor Cells
Abstract: International audience; In emergency myelopoiesis (EM), expansion of the myeloid progenitor compartment and increased myeloid cell production are observed and often mediated by the pro-inflammatory cytokine interferon gamma (IFN-γ). Interleukin-10 (IL-10) inhibits IFN-γ secretion, but paradoxically, its therapeutic administration to humans causes hematologic changes similar to those observed in EM. In this work, we use different in vivo systems, including a humanized immune system mouse model, to show that IL-10 triggers EM, with a significant expansion of the myeloid progenitor compartment and production of myeloid cells. Hematopoietic progenitors display a prominent IFN-γ transcriptional signature, and we show that IFN-γ mediates IL-10-driven EM. We also find that IL-10, unexpectedly, reprograms CD4 and CD8 T cells toward an activation state that includes IFN-γ production by these T cell subsets in vivo. Therefore, in addition to its established anti-inflammatory properties, IL-10 can induce IFN-γ production and EM, opening additional perspectives for the design of IL-10-based immunotherapies.
Published: 2021
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13. Mechanical feedback defines organizing centers to drive digit emergence

Author: Parada, Carolina, Banavar, Samhita, Khalilian, Parisa, Rigaud, Stephane, Michaut, Arthur, Liu, Yucen, Joshy, Dennis Manjaly, Campàs, Otger, Gros, Jerome, Biologie du Développement et Cellules souches (CNRS UMR3738), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), University of California [Santa Barbara] (UC Santa Barbara), University of California (UC), Hub d'analyse d'images - Image Analysis Hub (Platform) (IAH), Institut Pasteur [Paris] (IP), Technische Universität Dresden = Dresden University of Technology (TU Dresden), This work was supported by a Research Grant from Human Frontier Science Program (HFSP, reference no. RGY0067/2015 to O.C. and J.G.) and by Laboratoire d’Excellence Revive (Investissement d’Avenir, ANR-10-LABX-73 to J.G.). O.C. was also partially supported by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) under Germany’s Excellence Strategy – EXC-2068 – 390729961., We thank Francis Corson for critical feedback and for sharing PIV analysis tools, Sangwoo Kim (UCSB) for sharing image analysis codes, Ellen Sletten (UCLA) for sharing custom-made fluorinated dyes, and Yang Chai (USC) for providing us with the plasmids for Sox9 and Activin AB probes., and ANR-10-LABX-0073,REVIVE,Stem Cells in Regenerative Biology and Medicine(2010)
Subjects: digit, Gene Expression Regulation, Developmental, Extremities, Cell Biology, mechanobiology, self-organization, General Biochemistry, Genetics and Molecular Biology, Activins, Feedback, tissue mechanics, limb development, Morphogenesis, mechanical feedback, phalanx-forming region/digit crescent, [SDV.BDD]Life Sciences [q-bio]/Development Biology, Molecular Biology, Chondrogenesis, organizing centers, Developmental Biology
Abstract: International audience; During embryonic development, digits gradually emerge in a periodic pattern. Although genetic evidence indicates that digit formation results from a self-organizing process, the underlying mechanisms are still unclear. Here, we find that convergent-extension tissue flows driven by active stresses underlie digit formation. These active stresses simultaneously shape cartilage condensations and lead to the emergence of a compressive stress region that promotes high activin/p-SMAD/SOX9 expression, thereby defining digit-organizing centers via a mechanical feedback. In Wnt5a mutants, such mechanical feedback is disrupted due to the loss of active stresses, organizing centers do not emerge, and digit formation is precluded. Thus, digit emergence does not result solely from molecular interactions, as was previously thought, but requires a mechanical feedback that ensures continuous coupling between phalanx specification and elongation. Our work, which links mechanical and molecular signals, provides a mechanistic context for the emergence of organizing centers that may underlie various developmental processes.
Published: 2021
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14. The combined action of Esrrb and Nr5a2 is essential for murine naïve pluripotency

Author: Festuccia, Nicola, Owens, Nick, Chervova, Almira, Dubois, Agnès, Navarro, Pablo, Epigénomique, Prolifération et Identité Cellulaire - Epigenomics, Proliferation and the Identity of Cells (EPIC), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), Imperial College London, University of Exeter Medical School, University of Exeter, N.F., during his stay at the LMS, was funded by an Imperial College London Research Fellowship and a Medical Research Council Career Development Award. P.N. acknowledges the European Research Council (Erc-cog-2017, BIND), the Labex Revive (Investissement d'Avenir, ANR-10-LABX-73), the Institut Pasteur and the Centre National de la Recherche Scientifique for funding. Deposited in PMC for immediate release., We thank the MRC London Institute of Medical Sciences flow cytometry and microscopy facilities for providing technical assistance., and ANR-10-LABX-0073,REVIVE,Stem Cells in Regenerative Biology and Medicine(2010)
Subjects: Pluripotency, Embryonic stem cells, Orphan nuclear receptors, [SDV]Life Sciences [q-bio], SOXB1 Transcription Factors, Stem Cells & Regeneration, Receptors, Cytoplasmic and Nuclear, Cell Differentiation, Mouse Embryonic Stem Cells, Nanog Homeobox Protein, Stem Cells and Regeneration, Mice, Receptors, Estrogen, Nr5a2, embryonic structures, Animals, Gene Regulatory Networks, biological phenomena, cell phenomena, and immunity, Esrrb, Cell Self Renewal, Octamer Transcription Factor-3, reproductive and urinary physiology, Protein Binding
Abstract: The maintenance of pluripotency in mouse embryonic stem cells (ESCs) is governed by the action of an interconnected network of transcription factors. Among them, only Oct4 and Sox2 have been shown to be strictly required for the self-renewal of ESCs and pluripotency, particularly in culture conditions in which differentiation cues are chemically inhibited. Here, we report that the conjunct activity of two orphan nuclear receptors, Esrrb and Nr5a2, parallels the importance of that of Oct4 and Sox2 in naïve mouse ESCs. By occupying a large common set of regulatory elements, these two factors control the binding of Oct4, Sox2 and Nanog to DNA. Consequently, in their absence the pluripotency network collapses and the transcriptome is substantially deregulated, leading to the differentiation of ESCs. Altogether, this work identifies orphan nuclear receptors, previously thought to be performing supportive functions, as a set of core regulators of naïve pluripotency., Summary: Esrrb and Nr5a2, two orphan nuclear receptors, are identified as essential regulators of pluripotency in mouse embryonic stem cells.
Published: 2021
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15. Erythro-myeloid progenitor origin of Hofbauer cells in the early mouse placenta

Author: Laina Freyer, Yvan Lallemand, Pascal Dardenne, Alina Sommer, Anne Biton, Elisa Gomez Perdiguero, Macrophages et Cellules endothéliales / Macrophages and Endothelial Cells, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), Sorbonne Université (SU), Hub Bioinformatique et Biostatistique - Bioinformatics and Biostatistics HUB, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), This work was funded by the Institut Pasteur, by the Centre national de la recherche scientifique, by Revive (Investissement d'Avenir, ANR-10-LABX-73), by a European Research Council investigator award (2016-StG-715230 to E.G.P.), by Fondation Schlumberger (FRM FSER 2017), by the Emergence(s) program from Ville de Paris (2016 DAE 190), and by the Florence Gould Foundation and the Pasteur Foundation (fellowship to L.F.). Open access funding provided by European Research Council., and ANR-10-LABX-0073,REVIVE,Stem Cells in Regenerative Biology and Medicine(2010)
Subjects: [SDV]Life Sciences [q-bio], Macrophages, Placenta, Hematopoietic Stem Cells, Mice, Pregnancy, embryonic structures, [SDV.IMM]Life Sciences [q-bio]/Immunology, Animals, Female, Molecular Biology, [SDV.BDD]Life Sciences [q-bio]/Development Biology, Myeloid Progenitor Cells, Developmental Biology, Yolk Sac
Abstract: Hofbauer cells (HBCs) are tissue macrophages of the placenta thought to be important for fetoplacental vascular development and innate immune protection. The developmental origins of HBCs remain unresolved and could implicate functional diversity of HBCs in placenta development and disease. In this study, we used flow cytometry and paternally inherited reporters to phenotype placenta macrophages and to identify fetal-derived HBCs and placenta-associated maternal macrophages in the mouse. In vivo pulse-labeling traced the ontogeny of HBCs from yolk sac-derived erythro-myeloid progenitors, with a minor contribution from fetal hematopoietic stem cells later on. Single-cell RNA-sequencing revealed transcriptional similarities between placenta macrophages and erythro-myeloid progenitor-derived fetal liver macrophages and microglia. As with other fetal tissue macrophages, HBCs were dependent on the transcription factor Pu.1, the loss-of-function of which in embryos disrupted fetoplacental labyrinth morphology, supporting a role for HBC in labyrinth angiogenesis and/or remodeling. HBC were also sensitive to Pu.1 (Spi1) haploinsufficiency, which caused an initial deficiency in the numbers of macrophages in the early mouse placenta. These results provide groundwork for future investigation into the relationship between HBC ontogeny and function in placenta pathophysiology.
Published: 2021
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16. LocalZProjector and DeProj: a toolbox for local 2D projection and accurate morphometrics of large 3D microscopy images

Author: Nathalie Aulner, Spencer L. Shorte, Sébastien Herbert, Elric Esposito, Romain Levayer, Laure Bally-Cuif, Jean-Yves Tinevez, Jerome Gros, Paolo Caldarelli, Laure Mancini, Léo Valon, Nicolas Dray, Hub d'analyse d'images - Image Analysis Hub (Platform) (IAH), Institut Pasteur [Paris] (IP), Mort cellulaire et homéostasie des épithéliums / Cell death and epithelial homeostasis, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Neurogénétique du Poisson zébré / Zebrafish Neurogenetics, Collège Doctoral, Sorbonne Université (SU), Régulation Dynamique de la Morphogénèse - Dynamic Regulation of Morphogenesis, BioImagerie Photonique – Photonic BioImaging (UTechS PBI), This work was supported by the Labex REVIVE (Investissement d’Avenir, ANR-10-LABX-73), France BioImaging (Investissement d’Avenir, ANR-10–INSB–04) and the Région Île-de-France in the framework of DIM ELICIT. Work in the R. L. laboratory is supported by the Institut Pasteur (G5 starting package), a ERC starting grant (CoSpaDD, #758457), the 'Cercle FSER' and the CNRS (UMR 3738). L. V. is supported by a Post-doctoral grant 'Aide au 224 Retour en France' from the Fondation pour la Recherche Médicale (ARF20170938651) and a Marie Sklodowska-Curie postdoctoral fellowship (MechDeath, 789573). Work in the J. G. laboratory was supported by the European Research Council under the European Union’s Seventh Framework Program (FP7/2007-2013) / ERC Grant Agreement no. 337635. P. C. is sponsored by the Institut Pasteur and the European Union’s Horizon 2020 research and innovation program under the Marie Sklodowska Curie (665807). Work in the L. B-C. laboratory was supported by the Ligue Nationale Contre le Cancer and the European Research Council (AdG 322936). L. M. was recipient of a PhD student fellowship from the Fondation pour la Recherche Médicale., ANR-10-LABX-0073,REVIVE,Stem Cells in Regenerative Biology and Medicine(2010), European Project: 758457,H2020-EU.1.1. - EXCELLENT SCIENCE - European Research Council (ERC),ERC-2017-STG,CoSpaDD(2018), European Project: 789573,FP7-IDEAS-ERC - Specific programme: 'Ideas' implementing the Seventh Framework Programme of the European Community for research, technological development and demonstration activities (2007 to 2013),H2020-MSCA-IF-2017,MechDeath(2019), European Project: 337635,EC:FP7:ERC,ERC-2013-StG,LIMBCELLDYNAMICS(2014), European Project: 665807,H2020,H2020-MSCA-COFUND-2014,PASTEURDOC(2015), European Project: 322936,EC:FP7:ERC,ERC-2012-ADG_20120314,SYSTEMATICS(2013), ANR-10-LABX-73), France BioImaging (Investissement d’ Avenir, ANR-10–INSB–04) and the Région Île-de-France in the framework of DIM ELICIT. Work in the R. L. laboratory is supported by the Institut Pasteur (G5 starting package), a ERC starting grant (CoSpaDD, #758457), the 'Cercle FSER' and the CNRS (UMR 3738). L. V. is supported by a Postdoctoral grant 'Aide au 224 Retour en France' from the Fondation pour la Recherche Médicale (ARF20170938651) and a Marie Sklodowska-Curie postdoctoral fellowship (MechDeath, 789573). Work in the J. G. laboratory was supported by the European Research Council under the European Union’s Seventh Framework Program (FP7/2007-2013) / ERC Grant Agreement no. 337635. P. C. is sponsored by the Institut Pasteur and the European Union’s Horizon 2020 research and innovation program under the Marie Sklodowska Curie (665807). Work in the L. B-C. laboratory was supported by the Ligue Nationale Contre le Cancer and the European Research Council (AdG 322936). L. M. was recipient of a PhD student fellowship from the Fondation pour la Recherche Médicale., ANR-10-INBS-0004,France-BioImaging,Développment d'une infrastructure française distribuée coordonnée(2010), Centre de Ressources et de Recherche Technologique - Center for Technological Resources and Research (C2RT), Institut Pasteur [Paris]-Institut Pasteur [Paris], Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Neurogénétique du Poisson zébré - Zebrafish Neurogenetics, Collège doctoral [Sorbonne universités], Sorbonne Université, Institut Pasteur [Paris], and Département de Biologie du Développement et Cellules souches - Department of Developmental and Stem Cell Biology
Subjects: Morphology, [SDV.BIO]Life Sciences [q-bio]/Biotechnology, QH301-705.5, Computer science, [SDV]Life Sciences [q-bio], [SDV.BC]Life Sciences [q-bio]/Cellular Biology, [SDV.BC.BC]Life Sciences [q-bio]/Cellular Biology/Subcellular Processes [q-bio.SC], Signal, Tissue imaging, Image analysis, 03 medical and health sciences, Imaging, Three-Dimensional, 0302 clinical medicine, 3D imaging, Segmentation, Computer vision, Biology (General), Projection (set theory), Process (anatomy), 030304 developmental biology, Microscopy, 0303 health sciences, Plane (geometry), business.industry, Distortion (optics), Visibility (geometry), Range (mathematics), [SDV.MP]Life Sciences [q-bio]/Microbiology and Parasitology, Artificial intelligence, business, Software, 030217 neurology & neurosurgery
Abstract: BackgroundQuantitative imaging of epithelial tissues prompts for bioimage analysis tools that are widely applicable and accurate. In the case of imaging 3D tissues, a common pre-processing step consists in projecting the acquired 3D volume on a 2D plane mapping the tissue surface. Indeed, while segmenting the tissue cells is amenable on 2D projections, it is still very difficult and cumbersome in 3D. However, for many specimen and models used in Developmental and Cell Biology, the complex content of the image volume surrounding the epithelium in a tissue often reduces the visibility of the biological object in the projection, compromising its subsequent analysis. In addition, the projection may distort the geometry of the tissue and can lead to strong artifacts in the morphology measurement.ResultsHere we introduce a user-friendly toolbox built to robustly project epithelia on their 2D surface from 3D volumes, and to produce accurate morphology measurement corrected for the projection distortion, even for very curved tissues. Our toolbox is built upon two components. LocalZProjector is a user-friendly and configurable Fiji plugin that generates 2D projections and height-maps from potentially large 3D stacks (larger than 40 GB per time-point) by only incorporating signal of the planes with local highest variance/mean intensity, despite a possibly complex image content. DeProj is a MATLAB tool that generates correct morphology measurements by combining the height-map output (such as the one offered by LocalZProjector) and the results of a cell segmentation on the 2D projection, hence effectively deprojecting the 2D segmentation in 3D. In this paper we demonstrate their effectiveness over a wide range of different biological samples. We then compare its performance and accuracy against similar existing tools.ConclusionsWe find that LocalZProjector performs well even in situations where the volume to project also contains un-wanted signal in other layers. We show that it can process large images without a pre-processing step. We study the impact of geometrical distortions on morphological measurements induced by the projection. We measured very large distortions which are then corrected by DeProj, providing accurate outputs.
Published: 2021
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17. The molecular logic of Nanog-induced self-renewal in mouse embryonic stem cells

Author: Philippe Clerc, Agnès Dubois, Victor Heurtier, Nick D.L. Owens, Florian Mueller, Damien Mornico, Pablo Navarro, Caroline Proux, Inma Gonzalez, Epigénétique des Cellules Souches - Epigenetics of Stem Cells, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Imagerie et Modélisation - Imaging and Modeling, Transcriptome et Epigénome (PF2), Institut Pasteur [Paris], Hub Bioinformatique et Biostatistique - Bioinformatics and Biostatistics HUB, This work was supported by recurrent funding from the Institut Pasteur, the CNRS, and Revive (Investissement d’Avenir, ANR-10-LABX-73). P.N. acknowledges financial support from the Fondation Schlumberger (FRM FSER 2017), the Fondation ARC pour la recherche sur le cancer (ARC NAVARRO CA 14/12/2016), the Agence Nationale de la Recherche (ANR 16 CE12 0004 01 MITMAT), and the Ligue contre le Cancer (LNCC EL2018 NAVARRO). V.H. is supported by the Fondation ARC and Revive. N.O. is supported by Revive., We thank Dr. Pentao Liu and Dr. Di Croce for kindly providing PiggyBack plasmids and Phf19 knock-down vectors, respectively, Dr. Domingos Henrique for fruitful discussions regarding the involvement of Polycomb activity in Nanog function, Carla Mulas for critical reading of the manuscript., ANR-10-LABX-0073,REVIVE,Stem Cells in Regenerative Biology and Medicine(2010), ANR-16-CE12-0004,MitMAT,Mémoire mitotique de l'activité transcriptionnelle dans les cellules ES(2016), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), and Institut Pasteur [Paris] (IP)
Subjects: 0301 basic medicine, Homeobox protein NANOG, Science, General Physics and Astronomy, 02 engineering and technology, [SDV.BC.BC]Life Sciences [q-bio]/Cellular Biology/Subcellular Processes [q-bio.SC], Leukemia Inhibitory Factor, Article, General Biochemistry, Genetics and Molecular Biology, Cell Line, Mice, 03 medical and health sciences, [SDV.BBM.GTP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Genomics [q-bio.GN], Animals, Gene Regulatory Networks, Cell Self Renewal, Enhancer, lcsh:Science, Transcription factor, reproductive and urinary physiology, Epigenomics, Regulation of gene expression, Otx Transcription Factors, Multidisciplinary, biology, Mouse Embryonic Stem Cells, Nanog Homeobox Protein, General Chemistry, 021001 nanoscience & nanotechnology, Embryonic stem cell, Chromatin, Cell biology, Histone Code, Enhancer Elements, Genetic, 030104 developmental biology, Histone, [SDV.BDD.EO]Life Sciences [q-bio]/Development Biology/Embryology and Organogenesis, Gene Expression Regulation, embryonic structures, biology.protein, lcsh:Q, 0210 nano-technology
Abstract: Transcription factor networks, together with histone modifications and signalling pathways, underlie the establishment and maintenance of gene regulatory architectures associated with the molecular identity of each cell type. However, how master transcription factors individually impact the epigenomic landscape and orchestrate the behaviour of regulatory networks under different environmental constraints is only partially understood. Here, we show that the transcription factor Nanog deploys multiple distinct mechanisms to enhance embryonic stem cell self-renewal. In the presence of LIF, which fosters self-renewal, Nanog rewires the pluripotency network by promoting chromatin accessibility and binding of other pluripotency factors to thousands of enhancers. In the absence of LIF, Nanog blocks differentiation by sustaining H3K27me3, a repressive histone mark, at developmental regulators. Among those, we show that the repression of Otx2 plays a preponderant role. Our results underscore the versatility of master transcription factors, such as Nanog, to globally influence gene regulation during developmental processes., Transcription factor (TF) networks are essential for the molecular identity of each cell type. Here, the authors show that TF Nanog utilises multiple molecular strategies to enhance embryonic stem cell self-renewal, which include regulation of chromatin accessibility in the presence of LIF or maintenance of H3K27me3 at developmental regulators in its absence.
Published: 2019
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18. Amphiregulin mediates non-cell-autonomous effect of senescence on reprogramming

Author: Mathieu von Joest, Cheng Chen, Thibaut Douché, Jeremy Chantrel, Aurélie Chiche, Quentin Giai Gianetto, Mariette Matondo, Han Li, Plasticité cellulaire et Modélisation des Maladies / Cellular Plasticity and Disease Modelling, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), Plateforme de Protéomique / Proteomics platform, Université Paris Cité (UPCité)-Spectrométrie de Masse pour la Biologie – Mass Spectrometry for Biology (UTechS MSBio), Institut Pasteur [Paris] (IP)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité)-Institut Pasteur [Paris] (IP)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Hub Bioinformatique et Biostatistique - Bioinformatics and Biostatistics HUB, Institut Pasteur [Paris] (IP)-Université Paris Cité (UPCité), Work in the laboratory of H.L. is funded by Institut Pasteur, Centre National pour la Recherche Scientific, and the Agence Nationale de la Recherche, France (Laboratoire d’Excellence Revive, Investissement d’Avenir, ANR-10-LABX-73, ANR-16-CE13-0017, ANR-21-CE13-0006-01). A.C. was funded by the postdoctoral fellowships from the Revive Consortium., We are indebted to Jun Zhang for his excellent technical support in image analysis. We are grateful to the Central Animal Facility, the Proteomic Platform, the Cytometry Platform, and the Bioinformatics and Biostatistics Hub of the Institut Pasteur., ANR-10-LABX-0073,REVIVE,Stem Cells in Regenerative Biology and Medicine(2010), ANR-16-CE13-0017,iCELLPLASTICITY,Plasticité cellulaire dans le vieillissement(2016), and ANR-21-CE13-0006,SenoMuscle,Conséquence de la sénescence dans la régénération musculaire et les myopathies dégénératives(2021)
Subjects: muscle regeneration, quantitative proteomics, EGFR, [SDV]Life Sciences [q-bio], aging, Cell Plasticity, cellular plasticity, SASP, Amphiregulin, General Biochemistry, Genetics and Molecular Biology, in vitro reprogramming, Phenotype, in vivo reprogramming, CP: Cell biology, Cellular Senescence, Signal Transduction
Abstract: International audience; Cellular senescence is an irreversible growth arrest with a dynamic secretome, termed the senescence-associatedsecretory phenotype (SASP). Senescence is a cell-intrinsic barrier for reprogramming, whereas theSASP facilitates cell fate conversion in non-senescent cells. However, the mechanisms by which reprogramming-induced senescence regulates cell plasticity are not well understood. Here, we investigate how the heterogeneityof paracrine senescence impacts reprogramming. We show that senescence promotes in vitro reprogrammingin a stress-dependent manner. Unbiased proteomics identifies a catalog of SASP factorsinvolved in the cell fate conversion. Amphiregulin (AREG), frequently secreted by senescent cells, promotesin vitro reprogramming by accelerating proliferation and the mesenchymal-epithelial transition via EGFRsignaling. AREG treatment diminishes the negative effect of donor age on reprogramming. Finally, AREG enhances in vivo reprogramming in skeletal muscle. Hence, various SASP factors can facilitate cellular plasticityto promote reprogramming and tissue repair.
Published: 2022
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19. Overlapping Definitive Progenitor Waves Divide and Conquer to Build a Layered Hematopoietic System

Author: Lorea Iturri, Elisa Gomez Perdiguero, Anne Biton, Laina Freyer, Macrophages et Cellules endothéliales / Macrophages and Endothelial Cells, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Hub Bioinformatique et Biostatistique - Bioinformatics and Biostatistics HUB, This work was funded by the Institut Pasteur, the CNRS, Revive (Investissement d'Avenir, ANR-10-LABX-73) and the European Research Council ERC investigator award (2016-StG-715320 to E.G.P.) E.G.P. also acknowledges financial support from the Fondation Schlumberger (FRM FSER 2017) and the Emergence(s) program from Ville de Paris (2016 DAE 190). L.I. was supported by a PhD fellowship from the Revive Labex and L.F. by a Florence Gould-Pasteur Foundation fellowship., ANR-10-LABX-0073,REVIVE,Stem Cells in Regenerative Biology and Medicine(2010), European Project: 715320,ERC-2016-STG,ResidentMacroPhage(2016), and Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)
Subjects: 0303 health sciences, Cell type, Myeloid, [SDV]Life Sciences [q-bio], Biology, Granulopoiesis, Cell biology, 03 medical and health sciences, Haematopoiesis, 0302 clinical medicine, medicine.anatomical_structure, medicine, [SDV.IMM]Life Sciences [q-bio]/Immunology, Myelopoiesis, Stem cell, Progenitor cell, [SDV.BDD]Life Sciences [q-bio]/Development Biology, 030217 neurology & neurosurgery, 030304 developmental biology, Progenitor
Abstract: We are indebted to Baptiste Saudemont and Yann Loe-Mie for critical advice, training, reagents and quality control for the MARS-Seq pipeline.We would like tothankRebecca Gentek, Marc Bajénoff (CIML in Marseille) and Kémy Adefor providing and acquiring exploratory data for Cdh5CreERT2experiments, Pascal Dardenne and Yvan Lallemand for handling and injections of mice, Sébastien Bastide and Caroline Kaiser for advice on scRNA-Seq analysis with Seurat, Tobias Weinberger and Rebeca Ponce Landete for isolation of adult tissues, Anne Dejean for transfer of the Cdh5CreERT2strainand other members of the Gomez Perdiguero and Cumano groups for critical discussions and feedback. We appreciate the support and advice of the Cytometry and Biomarkers (UTECHS CB) platform (Sophie Novault and Sandrine Schmutz), the Center of Bioinformatics, Biostatistics and Integrative Biology (C3BI), the Institute PasteurSingle Cell Initiative (Heather Marlow) and the Animalerie Centrale.; ABSTRACT Adult innate immune cells are part of a layered hematopoietic system constructed from definitive hematopoietic stem and progenitor cells (HSPC) with diverse origins during development. One source of HSPC are fetal hematopoietic stem cells (HSC) that provide long-term reconstitution throughout life. However, the extent to which HSC produce mature cells in utero is only recently being uncovered. This is in part due to the added complexity of an overlapping wave of definitive progenitors that derive from yolk sac erythro-myeloid progenitors (EMP). HSC and EMP are generated from spatiotemporally distinct hemogenic endothelia, yet they both migrate to the fetal liver niche where they co-habitate and are presumed to reach their full potential. Delineation of the respective HSC and EMP pathways towards developmental immune cell differentiation has been confounded by challenges in ontogeny-specific cell labeling. In this study, in vivo inducible pulse chase labeling revealed that HSC contribute little to fetal myelopoiesis and that EMP are the predominant source of mature myeloid cells until birth. This is similar to what has been reported for the erythroid branch of hematopoiesis thereby establishing a developmentally-restricted privilege for erythro-myeloid differentiation from EMP compared to HSC. Tracing the origins of mature cells to the progenitor level by immunophenotyping and single cell RNA sequencing uncovered a dichotomy in the allocation of fetal liver EMP and HSC to myeloid progenitor subsets, both in timing and lineage bias. This has exposed an uncoupling between developmental granulopoiesis and monopoiesis from EMP and HSC pathways, and provides a framework for future studies of HSC-dependent and -independent hematopoiesis. HIGHLIGHTS EMP-to-HSC switch in fetal liver myelopoiesis occurs late in gestation EMP are efficient at producing early transit amplifying erythroid and myeloid intermediates scRNA-seq reveals three trajectories of EMP myelopoiesis Myeloid lineage commitment during development is cell type and ontogeny specific
Published: 2021

20. Megakaryocyte production is sustained by direct differentiation from erythromyeloid progenitors in the yolk sac until midgestation

Author: Iturri, Lorea, Freyer, Laina, Biton, Anne, Dardenne, Pascal, Lallemand, Yvan, Gomez Perdiguero, Elisa, Macrophages et Cellules endothéliales / Macrophages and Endothelial Cells, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Collège doctoral [Sorbonne universités], Sorbonne Université (SU), Hub Bioinformatique et Biostatistique - Bioinformatics and Biostatistics HUB, This work was supported by recurrent funding from the Institut Pasteur , the CNRS , and Revive (Investissement d’Avenir, ANR-10-LABX-73 ), and by an ERC investigator award ( 2016-StG-715320 ) from the European Research Council to E.G.P. E.G.P. also acknowledges financial support from the Fondation Schlumberger (FRM FSER 2017) and the Emergence(s) program from Ville de Paris (2016 DAE 190). L.I. was supported by a fellowship from the Revive Labex and L.F. by a Florence Gould-Pasteur Foundation fellowship., We gratefully acknowledge the Plateforme de Cytometrie (Sandrine Schmutz and Sophie Novault) at the Center for Innovation and Technological Research of the Institut Pasteur for support in conducting this study. We thank Yann Loe Mie and Baptiste Saudemont for their help with the MARS-Seq pipeline., ANR-10-LABX-0073,REVIVE,Stem Cells in Regenerative Biology and Medicine(2010), European Project: 715320,ERC-2016-STG,ResidentMacroPhage(2016), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), and Collège Doctoral
Subjects: Male, resident macrophages, Erythrocytes, [SDV]Life Sciences [q-bio], embryo, mast cells, Article, Monocytes, Mice, Pregnancy, Animals, Cell Lineage, Myeloid Cells, [SDV.BDD]Life Sciences [q-bio]/Development Biology, Cells, Cultured, yolk sac, Macrophages, Multipotent Stem Cells, Stem Cells, Cell Differentiation, scRNAseq, Embryo, Mammalian, Hematopoietic Stem Cells, hematopoiesis, Mice, Inbred C57BL, embryonic structures, Female, fate mapping, Megakaryocytes, megakaryopoiesis, erythropoiesis, Granulocytes, fetal liver
Abstract: Summary The extra-embryonic yolk sac contains the first definitive multipotent hematopoietic cells, denominated erythromyeloid progenitors. They originate in situ prior to the emergence of hematopoietic stem cells and give rise to erythroid, monocytes, granulocytes, mast cells and macrophages, the latter in a Myb transcription factor-independent manner. We uncovered here the heterogeneity of yolk sac erythromyeloid progenitors, at the single cell level, and discriminated multipotent from committed progenitors, prior to fetal liver colonization. We identified two temporally distinct megakaryocyte differentiation pathways. The first occurs in the yolk sac, bypasses intermediate bipotent megakaryocyte-erythroid progenitors and, similar to the differentiation of macrophages, is Myb-independent. By contrast, the second originates later, from Myb-dependent bipotent progenitors expressing Csf2rb and colonize the fetal liver, where they give rise to megakaryocytes and to large numbers of erythrocytes. Understanding megakaryocyte development is crucial as they play key functions during vascular development, in particular in separating blood and lymphatic networks., Graphical abstract, Highlights • EMP produce locally committed progenitors prior to fetal liver colonization • Two sequential waves of EMP differentiation revealed at the single cell level • Yolk sac Mk develop mainly from EMPs in a Myb-independent manner • CD131 identifies Myb-dependent EMP-derived MEP progenitors in the yolk sac, The yolk sac produces the first blood cells during embryonic development. Iturri et al. now show the extent of local definitive hematopoietic differentiation occurring in this organ before colonization of the fetal liver and demonstrate the existence of a direct pathway of megakaryopoiesis produced by EMPs, independently of hematopoietic stem cells.
Published: 2021
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21. Identification des progéniteurs bipotents qui donnent naissance aux tissus myogéniques et conjonctifs chez la souris

Author: Alexandre Grimaldi, Shahragim Tajbakhsh, Glenda Comai, Sébastien Mella, Cellules Souches et Développement / Stem Cells and Development, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), We acknowledge funding support from the Institut Pasteur, Association Française contre le Myopathies, Agence Nationale de la Recherche (Laboratoire d’Excellence Revive, Investissement d’Avenir, ANR-10-LABX-73) and MyoHead, Association Française contre les Myopathies (Grant #20510), Fondation pour la Recherche Médicale (Grant # FDT201904008277), and the Centre National de la Recherche Scientifique. We gratefully acknowledge the UtechS Photonic BioImaging, C2RT, Institut Pasteur, supported by the French National Research Agency (France BioImaging, ANR-10–INSB–04, Investments for the Future)., ANR-10-LABX-0073,REVIVE,Stem Cells in Regenerative Biology and Medicine(2010), ANR-10-INBS-0004,France-BioImaging,Développment d'une infrastructure française distribuée coordonnée(2010), and Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS)
Subjects: Cell type, Mesoderm, [SDV]Life Sciences [q-bio], Cell, Connective tissue, regenerative medicine, Biology, Muscle Development, General Biochemistry, Genetics and Molecular Biology, cranial muscles, 03 medical and health sciences, developmental biology, Mice, 0302 clinical medicine, stem cells, medicine, Animals, Progenitor cell, Muscle, Skeletal, mouse, 030304 developmental biology, 0303 health sciences, General Immunology and Microbiology, General Neuroscience, Neural crest, Skeletal muscle, Gene Expression Regulation, Developmental, [SDV.BDD.MOR]Life Sciences [q-bio]/Development Biology/Morphogenesis, Cell Differentiation, General Medicine, scRNAseq, Cell biology, medicine.anatomical_structure, [SDV.BDD.EO]Life Sciences [q-bio]/Development Biology/Embryology and Organogenesis, Connective Tissue, Neural Crest, cranial mesoderm, MYF5, myogenesis, 030217 neurology & neurosurgery
Abstract: How distinct cell fates are manifested by direct lineage ancestry from bipotent progenitors, or by specification of individual cell types is a key question for understanding the emergence of tissues. The interplay between skeletal muscle progenitors and associated connective tissue cells provides a model for examining how muscle functional units are established. Most craniofacial structures originate from the vertebrate-specific neural crest cells except in the dorsal portion of the head, where they arise from cranial mesoderm. Here, using multiple lineage-tracing strategies combined with single cell RNAseq and in situ analyses, we identify bipotent progenitors expressing Myf5 (an upstream regulator of myogenic fate) that give rise to both muscle and juxtaposed connective tissue. Following this bifurcation, muscle and connective tissue cells retain complementary signalling features and maintain spatial proximity. Disrupting myogenic identity shifts muscle progenitors to a connective tissue fate. The emergence of Myf5-derived connective tissue is associated with the activity of several transcription factors, including Foxp2. Interestingly, this unexpected bifurcation in cell fate was not observed in craniofacial regions that are colonised by neural crest cells. Therefore, we propose that an ancestral bi-fated program gives rise to muscle and connective tissue cells in skeletal muscles that are deprived of neural crest cells.
Published: 2021
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22. Dynamic extrinsic pacing of the HOX clock in human axial progenitors controls motor neuron subtype specification

Author: Hynek Wichterle, Virginie Rouiller-Fabre, Nour Nicolas, Gist F. Croft, Vincent Mouilleau, Vanessa Ribes, Léa Lesueur, Célia Vaslin, Mathieu Daynac, Stéphane Nedelec, Angélique Terray, Margot Jarrige, Simona Gribaudo, Mackenzie W. Mathis, Cécile Martinat, Rémi Robert, Institut du Fer à Moulin (IFM - Inserm U1270 - SU), Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU), Stem cells in neurodevelopment (SCN), Institut du Fer à Moulin (UMR-S 1270), Institut des cellules souches pour le traitement et l'étude des maladies monogéniques (I-STEM), Université d'Évry-Val-d'Essonne (UEVE)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris-Saclay-Généthon, Institut de Biologie François JACOB (JACOB), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Columbia University [New York], Institut Jacques Monod (IJM (UMR_7592)), Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), This project was supported by grants from AVENIR/ATIP, Association Française contre les Myopathies (AFM) and Laboratoire d’Excellence (Labex) Biopsy and Revive (Investissement d’Avenir (ANR-10-LABX-73 and 11-LABX-0035, respectively). V.M has received fellowships from Domaine d’Investissement Majeur (DIM) 'Biothérapies' of the region Ile-de-France and from the Fondation pour la Recherche Médicale (FRM). C.V. is supported by a PhD fellowship of the French Ministry of Research. V.R. and S.N salaries are funded by the INSERM, ANR-10-LABX-0073,REVIVE,Stem Cells in Regenerative Biology and Medicine(2010), ANR-11-LABX-0035,BIOPSY,Laboratoire de Psychiatrie Biologique(2011), Université Paris Cité, Equipe HAL, Laboratoires d'excellence - Stem Cells in Regenerative Biology and Medicine - - REVIVE2010 - ANR-10-LABX-0073 - LABX - VALID, Laboratoire de Psychiatrie Biologique - - BIOPSY2011 - ANR-11-LABX-0035 - LABX - VALID, and Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)
Subjects: MESH: Signal Transduction, MESH: Mammalian, MESH: Bone Morphogenetic Proteins, [SDV]Life Sciences [q-bio], Fibroblast growth factor, MESH: Spinal Cord, MESH: Benzamides, 0302 clinical medicine, MESH: Gene Expression Regulation, Developmental, MESH: Embryonic Development, Induced pluripotent stem cell, Hox gene, Motor Neurons, 0303 health sciences, patterning, Gene Expression Regulation, Developmental, Cell Differentiation, axial, HOX genes, ES cells, Chromatin, Growth Differentiation Factors, [SDV] Life Sciences [q-bio], medicine.anatomical_structure, Benzamides, Bone Morphogenetic Proteins, embryonic structures, MESH: Pluripotent Stem Cells, Stem cell, pluripotent stem cells, MESH: Fibroblast Growth Factors, MESH: Motor Neurons, Signal Transduction, MESH: Cell Differentiation, animal structures, MESH: Circadian Clocks, Human Development, Embryonic Development, Biology, 03 medical and health sciences, Circadian Clocks, MESH: Homeodomain Proteins, medicine, Humans, human, Progenitor cell, motor neuron, Molecular Biology, 030304 developmental biology, Homeodomain Proteins, MESH: Humans, MESH: Embryo, Diphenylamine, spinal cord, Motor neuron, Embryo, Mammalian, NMP, MESH: Growth Differentiation Factors, MESH: Diphenylamine, Fibroblast Growth Factors, Pyrimidines, MESH: Pyrimidines, GDF11, Neuroscience, 030217 neurology & neurosurgery, Developmental Biology
Abstract: Rostro-caudal patterning of vertebrates depends on the temporally progressive activation of HOX genes within axial stem cells that fuel axial embryo elongation. Whether the pace of sequential activation of HOX genes, the 'HOX clock', is controlled by intrinsic chromatin-based timing mechanisms or by temporal changes in extrinsic cues remains unclear. Here, we studied HOX clock pacing in human pluripotent stem cell-derived axial progenitors differentiating into diverse spinal cord motor neuron subtypes. We show that the progressive activation of caudal HOX genes is controlled by a dynamic increase in FGF signaling. Blocking the FGF pathway stalled induction of HOX genes, while a precocious increase of FGF, alone or with GDF11 ligand, accelerated the HOX clock. Cells differentiated under accelerated HOX induction generated appropriate posterior motor neuron subtypes found along the human embryonic spinal cord. The pacing of the HOX clock is thus dynamically regulated by exposure to secreted cues. Its manipulation by extrinsic factors provides synchronized access to multiple human neuronal subtypes of distinct rostro-caudal identities for basic and translational applications. This article has an associated ‘The people behind the papers’ interview.
Published: 2021
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23. Longitudinal high-resolution imaging through a flexible intravital imaging window

Author: Bethan Lloyd-Lewis, Didier Surdez, Silvia Fre, Aurélien Bore, Maria Benavente-Diaz, Virginie Dangles-Marie, Shahragim Tajbakhsh, Guillaume Jacquemin, Samir Djaber, Génétique et Biologie du Développement, Institut Curie [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS), Cellules Souches et Développement / Stem Cells and Development, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Ecole Doctorale Complexité du Vivant (ED515), Sorbonne Université (SU), Plates-formes mutualisées du centre de recherche pharmaceutique de Paris (P-MIM - UMS 3612), Institut de Recherche pour le Développement (IRD)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), Unité de génétique et biologie des cancers (U830), Institut Curie [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM), University of Bristol [Bristol], This work was supported by grants to S.F., B.L.-L., and G.J. from Paris Sciences et Lettres University (PSL Research University), the Institut Carnot, the French National Research Agency (ANR, grant no. ANR-15-CE13-0013-01), the Cancéropôle Île-de-France (grant no. 2015-2-APD-01-ICR-1), the Ligue contre le cancer (grant no. RS19/75-101), the LABEX DEEP ANR, number 11-LBX-0044, and by the 'FRM Equipes' EQU201903007821 and the FSER (La Fondation Schlumberger pour l’Education et la Recherche) FSER20200211117. The PICT-IBiSA facility is supported by the Fondation pour la Recherche Médicale (FRM no. DGE20111123020), the Cancéropôle Île-de-France (no. 2012-2-EML-04-IC-1), and the INCA (Cancer National Institute, no. 2011-1-LABEL-IC-4). D.S. and A.B. are supported by the Institut Curie SIRIC (Site de Recherche Intégrée en Cancérologie, INCa-DGOS-Inserm_12554, ITMO Cancer AVIESAN). B.L.-L. acknowledges support from the Academy of Medical Sciences, Wellcome Trust, and the University of Bristol. S.T. acknowledges funding support from the Institut Pasteur, Agence Nationale de la Recherche (Laboratoire d’Excellence Revive, Investissement d’Avenir, ANR-10-LABX-73), M.B.-D. was supported by the Laboratoire d’Excellence Revive and La Ligue Contre le cancer., ANR-15-CE13-0013,LumStemCell,Etude de la plasticité et du potentiel de différentiation des cellules souches luminales dans la glande mammaire pendant le développement et l'homéostasie adulte(2015), ANR-11-LABX-0044,DEEP,Développement, Epigénèse, Epigénétique et potentiel de vie(2011), ANR-10-LABX-0073,REVIVE,Stem Cells in Regenerative Biology and Medicine(2010), Gestionnaire, HAL Sorbonne Université 5, Etude de la plasticité et du potentiel de différentiation des cellules souches luminales dans la glande mammaire pendant le développement et l'homéostasie adulte - - LumStemCell2015 - ANR-15-CE13-0013 - AAPG2015 - VALID, Développement, Epigénèse, Epigénétique et potentiel de vie - - DEEP2011 - ANR-11-LABX-0044 - LABX - VALID, Laboratoires d'excellence - Stem Cells in Regenerative Biology and Medicine - - REVIVE2010 - ANR-10-LABX-0073 - LABX - VALID, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), and Institut de Recherche pour le Développement (IRD)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université de Paris (UP)
Subjects: [SDV.GEN]Life Sciences [q-bio]/Genetics, 0303 health sciences, Multidisciplinary, Materials science, integumentary system, ComputingMethodologies_IMAGEPROCESSINGANDCOMPUTERVISION, SciAdv r-articles, Window (computing), [SDV.GEN] Life Sciences [q-bio]/Genetics, Cell Biology, Intravital Imaging, 03 medical and health sciences, 0302 clinical medicine, Research Methods, Implantation procedure, High resolution imaging, Research Articles, 030217 neurology & neurosurgery, Intravital microscopy, Preclinical imaging, Research Article, 030304 developmental biology, Biomedical engineering
Abstract: A fully flexible imaging window acting as an implantable transparent “second skin” upgrades in vivo imaging., Intravital microscopy (IVM) is a powerful technique that enables imaging of internal tissues at (sub)cellular resolutions in living animals. Here, we present a silicone-based imaging window consisting of a fully flexible, sutureless design that is ideally suited for long-term, longitudinal IVM of growing tissues and tumors. Crucially, we show that this window, without any customization, is suitable for numerous anatomical locations in mice using a rapid and standardized implantation procedure. This low-cost device represents a substantial technological and performance advance that facilitates intravital imaging in diverse contexts in higher organisms, opening previously unattainable avenues for in vivo imaging of soft and fragile tissues.
Published: 2021
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24. Dynamics of myogenic differentiation using a novel Myogenin knock-in reporter mouse

Author: Benavente-Diaz, Maria, Comai, Glenda, Di Girolamo, Daniela, Langa, Francina, Tajbakhsh, Shahragim, Cellules Souches et Développement / Stem Cells and Development, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), ED 515 - Complexité du vivant, Sorbonne Université (SU), Centre d'Ingénierie génétique murine - Mouse Genetics Engineering Center (CIGM), Institut Pasteur [Paris] (IP), We acknowledge funding support from the Institut Pasteur, Agence Nationale de la Recherche (Laboratoire d’Excellence Revive, Investissement d’Avenir, ANR-10-LABX-73), Association Française contre les Myopathies (Grant #20510), and the Centre National de la Recherche Scientifique. M.B.D. was supported by a grant from Laboratoire d’Excellence Revive and La Ligue Contre le Cancer., ANR-10-LABX-0073,REVIVE,Stem Cells in Regenerative Biology and Medicine(2010), Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Institut Pasteur [Paris], Benavente-Diaz, Maria, Comai, Glenda, Di Girolamo, Daniela, Langa, Francina, and Tajbakhsh, Shahragim
Subjects: lcsh:Diseases of the musculoskeletal system, [SDV]Life Sciences [q-bio], Methodology, Skeletal muscle, Cell Differentiation, Muscle Development, musculoskeletal system, Knock-in mouse, Myoblasts, Mice, Intravital imaging, Animals, Myogenin, lcsh:RC925-935, Muscle, Skeletal, tissues, [SDV.BDD]Life Sciences [q-bio]/Development Biology, tdTOMATO
Abstract: Background Myogenin is a transcription factor that is expressed during terminal myoblast differentiation in embryonic development and adult muscle regeneration. Investigation of this cell state transition has been hampered by the lack of a sensitive reporter to dynamically track cells during differentiation. Results Here, we report a knock-in mouse line expressing the tdTOMATO fluorescent protein from the endogenous Myogenin locus. Expression of tdTOMATO in MyogntdTom mice recapitulated endogenous Myogenin expression during embryonic muscle formation and adult regeneration and enabled the isolation of the MYOGENIN+ cell population. We also show that tdTOMATO fluorescence allows tracking of differentiating myoblasts in vitro and by intravital imaging in vivo. Lastly, we monitored by live imaging the cell division dynamics of differentiating myoblasts in vitro and showed that a fraction of the MYOGENIN+ population can undergo one round of cell division, albeit at a much lower frequency than MYOGENIN− myoblasts. Conclusions We expect that this reporter mouse will be a valuable resource for researchers investigating skeletal muscle biology in developmental and adult contexts. Supplementary Information The online version contains supplementary material available at 10.1186/s13395-021-00260-x.
Published: 2021
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25. Myogenesis modelled by human pluripotent stem cells: a multi‐omic study of Duchenne myopathy early onset

Author: Maxime R. F. Gosselin, Etienne Kornobis, Jean-Baptiste Dupont, Spiros D. Garbis, Christian Pinset, Alan R L Dorval, Antigoni Manousopoulou, Virginie Mournetas, Margot Jarrige, Dariusz C. Górecki, Hélène Polvèche, Emmanuelle Massouridès, Institut des cellules souches pour le traitement et l'étude des maladies monogéniques (I-STEM), Université d'Évry-Val-d'Essonne (UEVE)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris-Saclay-Généthon, Hub Bioinformatique et Biostatistique - Bioinformatics and Biostatistics, Institut Pasteur [Paris] (IP), Centre de Ressources et de Recherche Technologique - Center for Technological Resources and Research (C2RT), University of Portsmouth, Beckman Research Institute [Duarte, CA], University of Southampton, Military Institute of Hygiene and Epidemiology (WIHE), We thank the Fondation Maladies Rares (GenOmics grant), Labex Revive (Investissement d’Avenir, ANR-10-LABX-73),and the AFM Téléthon for funding this project., ANR-10-LABX-0073,REVIVE,Stem Cells in Regenerative Biology and Medicine(2010), Kornobis, Etienne, and Laboratoires d'excellence - Stem Cells in Regenerative Biology and Medicine - - REVIVE2010 - ANR-10-LABX-0073 - LABX - VALID
Subjects: 0301 basic medicine, Male, Proteomics, Duchenne muscular dystrophy, lcsh:Diseases of the musculoskeletal system, Induced Pluripotent Stem Cells, Omics, [SDV.GEN.GH] Life Sciences [q-bio]/Genetics/Human genetics, Bioinformatics, Muscle Development, lcsh:QM1-695, Dystrophin, 03 medical and health sciences, 0302 clinical medicine, Physiology (medical), medicine, Myocyte, Humans, Orthopedics and Sports Medicine, Human pluripotent stem cells, human pluripotent stem cells, Myopathy, Induced pluripotent stem cell, biology, business.industry, Myogenesis, Skeletal muscle, lcsh:Human anatomy, Original Articles, medicine.disease, Embryonic stem cell, omics, Muscular Dystrophy, Duchenne, 030104 developmental biology, medicine.anatomical_structure, [SDV.GEN.GH]Life Sciences [q-bio]/Genetics/Human genetics, 030220 oncology & carcinogenesis, biology.protein, Original Article, myogenesis, lcsh:RC925-935, medicine.symptom, business
Abstract: Background: Duchenne muscular dystrophy (DMD) causes severe disability of children and death of young men, with an incidence of approximately 1/5000 male births. Symptoms appear in early childhood, with a diagnosis made mostly around 4 years old, a time where the amount of muscle damage is already significant, preventing early therapeutic interventions that could be more efficient at halting disease progression. In the meantime, the precise moment at which disease phenotypes arise—even asymptomatically—is still unknown. Thus, there is a critical need to better define DMD onset as well as its first manifestations, which could help identify early disease biomarkers and novel therapeutic targets.Methods: We have used both human tissue‐derived myoblasts and human induced pluripotent stem cells (hiPSCs) from DMD patients to model skeletal myogenesis and compared their differentiation dynamics with that of healthy control cells by a comprehensive multi‐omic analysis at seven time points. Results were strengthened with the analysis of isogenic CRISPR‐edited human embryonic stem cells and through comparisons against published transcriptomic and proteomic datasets from human DMD muscles. The study was completed with DMD knockdown/rescue experiments in hiPSC‐derived skeletal muscle progenitor cells and adenosine triphosphate measurement in hiPSC‐derived myotubes.Results: Transcriptome and miRnome comparisons combined with protein analyses demonstrated that hiPSC differentiation (i) leads to embryonic/foetal myotubes that mimic described DMD phenotypes at the differentiation endpoint and (ii) homogeneously and robustly recapitulates key developmental steps—mesoderm, somite, and skeletal muscle. Starting at the somite stage, DMD dysregulations concerned almost 10% of the transcriptome. These include mitochondrial genes whose dysregulations escalate during differentiation. We also describe fibrosis as an intrinsic feature of DMD skeletal muscle cells that begins early during myogenesis. All the omics data are available online for exploration through a graphical interface at https://muscle‐dmd.omics.ovh/.Conclusions: Our data argue for an early developmental manifestation of DMD whose onset is triggered before the entry into the skeletal muscle compartment, data leading to a necessary reconsideration of dystrophin roles during muscle development. This hiPSC model of skeletal muscle differentiation offers the possibility to explore these functions as well as find earlier DMD biomarkers and therapeutic targets.
Published: 2021
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26. Long-range GABAergic projections contribute to cortical feedback control of sensory processing

Author: Pierre-Marie Lledo, Soham Saha, Antoine Nissant, Enzo Peroni, Camille Mazo, Gabriel Lepousez, Perception et Mémoire / Perception and Memory, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), This work was supported by the life insurance company 'AG2R-La-Mondiale', the Agence Nationale de la Recherche (ANR-15-CE37-0004 'SmellBrain' and ANR-16-CE37-0010 'ORUPS') and the Laboratoire d’Excellence Revive (Investissement d’Avenir, ANR-10-LABX-73). Our laboratory is part of the Ecole des Neurosciences de Paris (ENP) Ile-de-France network and is affiliated with the Bio-Psy Laboratory of Excellence. C.M. is a recipient of a fellowship from the French Ministère de l’Education Supérieure et de la Recherche and was also supported by the Fondation de la Recherche Médicale (FDT20160435483). S.S. acknowledges funding from the ENP Graduate program and the Labex Revive consortium for his doctoral program. C.M. and S.S acknowledge the ED3C doctoral school of the Sorbonne Université., We thank Sara Moberg for comments on the manuscript. We also thank Carine Moigneu for viral injections, Julien Grimaud and Lucie Dixsaut for early works. We also wish to thank Uwe Maskos and David DiGregorio from the Institut Pasteur for the gift of SST-Cre, PV-Cre and VIP-Cre mice. We also thank the Genetically-Encoded Neuronal Indicator and Effector (GENIE) Project and the Janelia Farm Research Campus of the Howard Hughes Medical Institute for sharing GCaMP6f constructs, ANR-15-CE37-0004,SmellBrain,Dissection fonctionnelle des circuits codant pour la récompense dans le système olfactif(2015), ANR-16-CE37-0010,ORUPS,Représentation sensorielle lors d'états psychotiques(2016), ANR-10-LABX-0073,REVIVE,Stem Cells in Regenerative Biology and Medicine(2010), and Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité)
Subjects: Olfactory system, [SDV]Life Sciences [q-bio], Population, General Physics and Astronomy, Sensory system, Optogenetics, Biology, Inhibitory postsynaptic potential, Somatosensory system, General Biochemistry, Genetics and Molecular Biology, Feedback, Centrifugal, GABAergic Neurons, education, education.field_of_study, Multidisciplinary, [SCCO.NEUR]Cognitive science/Neuroscience, Sensory circuits, Olfactory Pathways, General Chemistry, Top-down, Olfactory Bulb, Inhibitory, Olfactory bulb, Smell, Odorants, GABAergic, Perception, [SDV.NEU]Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC], Barrel cortex, Neuroscience
Abstract: In sensory systems, cortical areas send excitatory projections back to subcortical areas to dynamically adjust sensory processing. Here, we uncover for the first time the existence of a cortical inhibitory feedback to subcortical sensory areas. Investigating the olfactory system, we reveal that a subpopulation of GABAergic neurons in the anterior olfactory cortex target the olfactory bulb. Analogous inhibitory cortico-thalamic projections were also present in the somatosensory system. Long-range inhibitory inputs synapsed with both local and output neurons of the olfactory bulb. At the functional level, optogenetic activation of cortical GABAergic projections caused a net subtractive inhibition of both spontaneous and odor-evoked activity in local as well as output projection neurons, mitral and tufted cells. In tufted cells, but not mitral cells, this resulted in an enhanced separation of population odor responses. Furthermore, GABAergic corticofugal projections entrained network oscillations in the communication band between the cortex and the olfactory bulb. Targeted pharmacogenetic silencing of the cortical GABAergic outputs in the olfactory bulb impaired discrimination of similar odor mixtures. Thus, cortical GABAergic feedback represents a new circuit motif in sensory systems involved in refining sensory processing and perception.
Published: 2020
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27. Local retinoic acid signaling directs emergence of the extraocular muscle functional unit

Author: Comai, G., Tesařová, Markéta, Dupé, Valerie, Rhinn, Muriel, Vallecillo-García, Pedro, Da Silva, Fabio, Feret, Betty, Exelby, Katherine, Dollé, Pascal, Carlsson, Leif, Pryce, Brian, Spitz, François, Stricker, Sigmar, Zikmund, Tomáš, Kaiser, Jozef, Briscoe, James, Schedl, Andreas, Ghyselinck, Norbert, Schweitzer, Ronen, Tajbakhsh, Shahragim, Cellules Souches et Développement / Stem Cells and Development, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Brno University of Technology [Brno] (BUT), Institut de Génétique et Développement de Rennes (IGDR), Université de Rennes 1 (UR1), Université de Rennes (UNIV-RENNES)-Université de Rennes (UNIV-RENNES)-Centre National de la Recherche Scientifique (CNRS)-Structure Fédérative de Recherche en Biologie et Santé de Rennes ( Biosit : Biologie - Santé - Innovation Technologique ), Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA), Freie Universität Berlin, Institut de Biologie Valrose (IBV), Université Nice Sophia Antipolis (... - 2019) (UNS), COMUE Université Côte d'Azur (2015 - 2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015 - 2019) (COMUE UCA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Côte d'Azur (UCA)-Centre National de la Recherche Scientifique (CNRS), The Francis Crick Institute, Umeå University, Shriners Hospital for Children [Portland], Génomique et Epigénomique du Développement des Animaux - Genomics and Epigenomics of Animal Development, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Université de Rennes (UR)-Centre National de la Recherche Scientifique (CNRS)-Structure Fédérative de Recherche en Biologie et Santé de Rennes ( Biosit : Biologie - Santé - Innovation Technologique ), Université de Strasbourg (UNISTRA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Université Nice Sophia Antipolis (1965 - 2019) (UNS), COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Côte d'Azur (UCA), The Francis Crick Institute [London], Génomique et Epigénomique du Développement des Vertébrés - Genomics and Epigenomics of Vertebrates Development, Institut Pasteur, French Muscular Dystrophy Association, ANR-10-LABX-73, Laboratoire d’Excellence Revive, Investissement d’Avenir, CNRS, ANR-10-LABX-0073,REVIVE,Stem Cells in Regenerative Biology and Medicine(2010), Comai, Glenda, Laboratoires d'excellence - Stem Cells in Regenerative Biology and Medicine - - REVIVE2010 - ANR-10-LABX-0073 - LABX - VALID, Structure Fédérative de Recherche en Biologie et Santé de Rennes ( Biosit : Biologie - Santé - Innovation Technologique )-Centre National de la Recherche Scientifique (CNRS)-Université de Rennes 1 (UR1), Université de Rennes (UNIV-RENNES)-Université de Rennes (UNIV-RENNES), COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Côte d'Azur (UCA)-Centre National de la Recherche Scientifique (CNRS), and Génomique et Epigénomique du Développement des Vertébrés - Genomics and Epigenomics of Vertebrate Development
Subjects: Cell- och molekylärbiologi, [SDV]Life Sciences [q-bio], Retinoic Acid, Immunostaining, Eye, Biochemistry, Tendons, Mice, Cell Signaling, Retinoic Acid Signaling Cascade, Medicine and Health Sciences, Morphogenesis, Metabolites, Biology (General), [SDV.BDD]Life Sciences [q-bio]/Development Biology, Staining, [SDV.BDD.EO] Life Sciences [q-bio]/Development Biology/Embryology and Organogenesis, Eye Muscles, Muscle Differentiation, eye, Signaling Cascades, [SDV.BDD.MOR] Life Sciences [q-bio]/Development Biology/Morphogenesis, [SDV] Life Sciences [q-bio], Chemistry, Connective Tissue, Mice, Inbred DBA, extraocular muscles, Physical Sciences, embryogenesis, Anatomy, Research Article, Signal Transduction, QH301-705.5, Ocular Anatomy, Muscle Tissue, Embryonic Development, Tretinoin, Research and Analysis Methods, Imaging, Three-Dimensional, Ocular System, [SDV.BDD] Life Sciences [q-bio]/Development Biology, Animals, Retinoid Signaling, development, EOM, Chemical Compounds, Biology and Life Sciences, [SDV.BDD.MOR]Life Sciences [q-bio]/Development Biology/Morphogenesis, Cell Biology, eye diseases, Mice, Inbred C57BL, Biological Tissue, Metabolism, [SDV.BDD.EO]Life Sciences [q-bio]/Development Biology/Embryology and Organogenesis, Specimen Preparation and Treatment, Oculomotor Muscles, sense organs, Acids, Cell and Molecular Biology, Developmental Biology
Abstract: Coordinated development of muscles, tendons, and their attachment sites ensures emergence of functional musculoskeletal units that are adapted to diverse anatomical demands among different species. How these different tissues are patterned and functionally assembled during embryogenesis is poorly understood. Here, we investigated the morphogenesis of extraocular muscles (EOMs), an evolutionary conserved cranial muscle group that is crucial for the coordinated movement of the eyeballs and for visual acuity. By means of lineage analysis, we redefined the cellular origins of periocular connective tissues interacting with the EOMs, which do not arise exclusively from neural crest mesenchyme as previously thought. Using 3D imaging approaches, we established an integrative blueprint for the EOM functional unit. By doing so, we identified a developmental time window in which individual EOMs emerge from a unique muscle anlage and establish insertions in the sclera, which sets these muscles apart from classical muscle-to-bone type of insertions. Further, we demonstrate that the eyeballs are a source of diffusible all-trans retinoic acid (ATRA) that allow their targeting by the EOMs in a temporal and dose-dependent manner. Using genetically modified mice and inhibitor treatments, we find that endogenous local variations in the concentration of retinoids contribute to the establishment of tendon condensations and attachment sites that precede the initiation of muscle patterning. Collectively, our results highlight how global and site-specific programs are deployed for the assembly of muscle functional units with precise definition of muscle shapes and topographical wiring of their tendon attachments., The extraocular muscles (EOMs) are an evolutionarily conserved group of muscles that are precisely engineered for fine displacement of the eyeball, and thus crucial for visual acuity. This study presents the first integrative blueprint for morphogenesis of the EOM functional unit, providing genetic evidence for the existence of a retinoic acid signaling module that coordinates the emergence of individual EOMs, their tendons and insertion sites.
Published: 2020
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28. The cationic amino acid exporter Slc7a7 is induced and vital in zebrafish tissue macrophages with sustained efferocytic activity

Author: Mireille Carrère, Marion Le Bris, Philippe Herbomel, Ignaty Leshchiner, Chooyoung Baek, Doris Lou Demy, Ramil Noche, Wolfram Goessling, Muriel Tauzin, Cellules Souches et Développement / Stem Cells and Development, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Broad Institute of MIT and Harvard (BROAD INSTITUTE), Harvard Medical School [Boston] (HMS)-Massachusetts Institute of Technology (MIT)-Massachusetts General Hospital [Boston], Harvard Medical School [Boston] (HMS), Macrophages et Développement de l’Immunité, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), This work was supported by grants to P.H. from the European Commission through the FP6 ‘ZF-Models’ Integrated Project, from the Fondation pour la Recherche Médicale (‘FRM 2012 team’ #DEQ20120323714, ‘FRM 2016 team’ #DEQ20160334881) and from the Agence Nationale de la Recherche Laboratoire d'Excellence Revive (Investissement d'Avenir, ANR-10-LABX-73), ANR-10-LABX-0073,REVIVE,Stem Cells in Regenerative Biology and Medicine(2010), and European Project: 26837,ZF-MODELS
Subjects: Programmed cell death, [SDV]Life Sciences [q-bio], Population, microglia, Biology, 03 medical and health sciences, 0302 clinical medicine, medicine, Macrophage, Animals, Amino acid transporter, Amino Acids, education, Efferocytosis, Zebrafish, 030304 developmental biology, efferocytosis, 0303 health sciences, education.field_of_study, Microglia, Cell Biology, Zebrafish Proteins, biology.organism_classification, zebrafish, Cell biology, SLC7A7 Gene, macrophages, medicine.anatomical_structure, 030217 neurology & neurosurgery
Abstract: International audience; Most tissues harbor a substantial population of resident macrophages. Here, we elucidate a functional link between the Slc7a7 cationic amino acid transporter and tissue macrophages. We identified a mutant zebrafish devoid of microglia due to a mutation in the slc7a7 gene. We found that in Slc7a7-deficient larvae, macrophages do enter the retina and brain to become microglia, but then die during the developmental wave of neuronal apoptosis, which triggers intense efferocytic work from them. A similar macrophage demise occurs in other tissues, at stages where macrophages have to engulf many cell corpses, whether due to developmental or experimentally triggered cell death. We found that Slc7a7 is the main cationic amino acid transporter expressed in macrophages of zebrafish larvae, and that its expression is induced in tissue macrophages within 1-2 h upon efferocytosis. Our data indicate that Slc7a7 is vital not only for microglia but also for any steadily efferocytic tissue macrophages, and that slc7a7 gene induction is one of the adaptive responses that allow them to cope with the catabolism of numerous dead cells without compromising their own viability.
Published: 2020
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29. SIX1 and SIX4 homeoproteins regulate PAX7+ progenitor cell properties during fetal epaxial myogenesis

Author: Fabien Le Grand, Josiane Demignon, Stephen J. Tapscott, Athanassia Sotiropoulos, Pascal Maire, Elisa Negroni, Rouba Madani, Benjamin Saintpierre, Vincent Mouly, Carmen Birchmeier, Helge Amthor, Nathalie Chaverot, Maud Wurmser, Hiroshi Sakai, Shahragim Tajbakhsh, Institut Cochin (IC UM3 (UMR 8104 / U1016)), Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université de Paris (UP), Cellules Souches et Développement / Stem Cells and Development, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Ehime University [Matsuyama], Institut de Myologie, Centre National de la Recherche Scientifique (CNRS)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Association française contre les myopathies (AFM-Téléthon)-Sorbonne Université (SU), Centre de Recherche en Myologie, Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU), Handicap neuromusculaire : Physiopathologie, Biothérapie et Pharmacologies appliquées (END-ICAP), Université de Versailles Saint-Quentin-en-Yvelines (UVSQ)-Institut National de la Santé et de la Recherche Médicale (INSERM), Fred Hutchinson Cancer Research Center [Seattle] (FHCRC), Max Delbrück Center for Molecular Medicine [Berlin] (MDC), Helmholtz-Gemeinschaft = Helmholtz Association, Institut NeuroMyoGène (INMG), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM), M.W was supported by successive fellowships from the University de Paris, the Association Française contre les Myopathies (AFM), and the Agence Nationale pour la Recherche (ANR-16-CE14-0032-01). Financial support was provided by the AFM (n°16427 and 17406), the ANR-16-CE14-0002-01, the Institut National de la Santé et de la Recherche Médicale (INSERM) and the Centre National de la Recherche Scientifique (CNRS). S.T. acknowledges funding support from Institut Pasteur, Agence Nationale de la Recherche (Laboratoire d’Excellence Revive, Investissement d’Avenir, ANR-10-LABX-73)., ANR-16-CE14-0032,MYOLINC,Contrôles génétique et épigénétique des types de fibres musculaires squelettiques(2016), ANR-16-CE14-0002,BMP-MYOSTEM,Régulation des cellules souches du muscle squelettique adulte par la signalisation des ' Bone morphogenetic proteins '(2016), ANR-10-LABX-0073,REVIVE,Stem Cells in Regenerative Biology and Medicine(2010), Centre de recherche en Myologie – U974 SU-INSERM, Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Ehime University [Matsuyama, Japon], and Université de Lyon-Université de Lyon-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)
Subjects: Six1, Six4, Homing, Stem cells, Biology, Muscle Development, Transcriptome, 03 medical and health sciences, Mice, 0302 clinical medicine, Myocyte, Animals, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Progenitor cell, Muscle, Skeletal, Molecular Biology, 030304 developmental biology, Homeodomain Proteins, Mice, Knockout, 0303 health sciences, Myogenesis, PAX7 Transcription Factor, Pax7, Cell biology, Transplantation, Trans-Activators, Homeobox, PAX7, Stem cell, Function and Dysfunction of the Nervous System, 030217 neurology & neurosurgery, Developmental Biology
Abstract: International audience; Pax7 expression marks stem cells in developing skeletal muscles and adult satellite cells during homeostasis and muscle regeneration. The genetic determinants that control the entrance into the myogenic program and the appearance of PAX7+ cells during embryogenesis are poorly understood. SIX homeoproteins are encoded by the Sine oculis homeobox related Six1-Six6 genes in vertebrates. Six1, Six2, Six4 and Six5 are expressed in the muscle lineage. Here we tested the hypothesis that Six1 and Six4 could participate in the genesis of myogenic stem cells. We show that fewer PAX7+ cells occupy a satellite cell position between the myofiber and its associated basal lamina in Six1 and Six4 (s1s4KO) at E18. However, PAX7+ cells are detected in remaining muscle masses present in the epaxial region of the double mutant embryos and are able to divide and contribute to muscle growth. To further characterize the properties of s1s4KO PAX7+ cells, we analyzed their transcriptome and tested their properties after transplantation in adult regenerating tibialis anterior (TA) muscle. Mutant stem cells form hypotrophic myofibers that are not innervated but retain the ability to self-renew.
Published: 2020
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30. Dynamics of Asymmetric and Symmetric Divisions of Muscle Stem Cells In Vivo and on Artificial Niches

Author: Evano, Brendan, Khalilian, Sara, Le Carrou, Gilles, Almouzni, Geneviève, Tajbakhsh, Shahragim, Cellules Souches et Développement / Stem Cells and Development, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Dynamique du noyau [Institut Curie], Institut Curie [Paris]-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS), This work was supported by Institut Pasteur and grants from Agence Nationale de la Recherche ANR, (Laboratoire d’Excellence Revive, Investissement d’Avenir, ANR-10-LABX-73, ANR-16-CE12-0024) and Association Française contre les Myopathies, AFM, le Centre national de la recherche scientifique Centre national de la recherche scientifique, (CNRS), and the European Research Council (Advanced Research Grant 332893).G.A. received support from the French National Research Agency (ANR), Investissement D’avenir Labex développement, épigenèse, épigénétique et potentiel (DEEP), and the European Research Council Advanced (grant ChromAdict)., ANR-10-LABX-0073,REVIVE,Stem Cells in Regenerative Biology and Medicine(2010), ANR-16-CE12-0024,CHIFT,Chaperons et Histones Déterminants pour l'Identité Cellulaire, le Destin de Lignage et les Transitions(2016), ANR-10-IDEX-0001,PSL,Paris Sciences et Lettres(2010), European Project: 332893,332893, European Project: 694694,Chromatin Adaptations through Interactions of Chaperones in Time (ChromADICT), and Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS)
Subjects: SNAP-tag, muscle regeneration, Histone 3, [SDV]Life Sciences [q-bio], Muscle stem cells, symmetric cell division, Myogenin, non-random DNA segregation, asymmetric cell division, Pax7
Abstract: International audience; Stem cells can be maintained through symmetric cell divisions (SCDs) and asymmetric cell divisions (ACDs). How and when these divisions occur in vivo in vertebrates is poorly understood. Here, we developed a clonogenic cell tracing method that demonstrates the asymmetric distribution of transcription factors along with old and new DNA in mouse muscle stem cells during skeletal muscle regeneration. Combining single-cell tracking and artificial niches ex vivo, we show how cells switch from ACDs to SCDs, suggesting that they are not engaged in an obligate mode of cell division. Further, we generated SNAP-tagged histone H3-reporter mice and find that, unlike fly germline stem cells, differential fate outcomes are associated with a symmetric distribution of the H3.1 and H3.3 histone variants in mouse muscle stem cells. This versatile and efficient H3-SNAP labeling system will allow an investigation of mechanisms underlying the maintenance of epigenomic identity and plasticity in a variety of tissues.; Les cellules souches peuvent être maintenues par des divisions cellulaires symétriques (DCS) et des divisions cellulaires asymétriques (DCA). On comprend mal comment et quand ces divisions se produisent in vivo chez les vertébrés. Ici, nous avons développé une méthode de traçage cellulaire clonogène qui démontre la distribution asymétrique des facteurs de transcription ainsi que de l'ADN ancien et nouveau dans les cellules souches de muscles de souris pendant la régénération des muscles squelettiques. En combinant le traçage d'une seule cellule et des niches artificielles ex vivo, nous montrons comment les cellules passent des ACD aux SCD, ce qui suggère qu'elles ne sont pas engagées dans un mode de division cellulaire obligatoire. En outre, nous avons généré des souris indicatrices d'histone H3 marquées par SNAP et nous avons découvert que, contrairement aux cellules souches germinales de mouches, les résultats différentiels du destin sont associés à une distribution symétrique des variantes d'histone H3.1 et H3.3 dans les cellules souches musculaires de souris. Ce système de marquage H3-SNAP polyvalent et efficace permettra d'étudier les mécanismes sous-jacents au maintien de l'identité et de la plasticité épigénomiques dans une variété de tissus.
Published: 2020
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31. Hematopoiesis: A Layered Organization Across Chordate Species

Author: Ramy Elsaid, Francisca Soares-da-Silva, Marcia Peixoto, Dali Amiri, Nathan Mackowski, Pablo Pereira, Antonio Bandeira, Ana Cumano, Instituto de Investigação e Inovação em Saúde, Lymphocytes et Immunité - Lymphocytes and Immunity, Institut Pasteur [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Paris (UP), Instituto de Investigação e Inovação em Saúde (I3S), Universidade do Porto, Instituto de Ciências Biomédicas de Abel Salazar (ICBAS), This work was financed by the PPU Pasteur Ph.D. program. This work was financed by the Institut Pasteur, INSERM, Pasteur-Weizmann Foundation and ANR (grant Twothyme and Epi-Dev) through grants to AC, by REVIVE (Investissement d’Avenir, ANR-10-LABX-73) through grants to AC and RE, by FCT through the grants PD/BD/114128/2015 and POCI-0100145-FEDER-01638 to FS, and the grant SFRH/BD/143605/2019 to MP, ANR-10-LABX-0073,REVIVE,Stem Cells in Regenerative Biology and Medicine(2010), Institut Pasteur [Paris] (IP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris Cité (UPCité), Universidade do Porto = University of Porto, Vougny, Marie-Christine, and Laboratoires d'excellence - Stem Cells in Regenerative Biology and Medicine - - REVIVE2010 - ANR-10-LABX-0073 - LABX - VALID
Subjects: Cell type, [SDV.IMM] Life Sciences [q-bio]/Immunology, Hematopoieisis, embryo, lymphopoieis, Review, Biology, 03 medical and health sciences, Cell and Developmental Biology, 0302 clinical medicine, Immune system, layered, evo devo biology, Progenitor cell, lcsh:QH301-705.5, Tissue homeostasis, 030304 developmental biology, 0303 health sciences, hematopoieisis, Innate lymphoid cell, Cell Biology, Layered, Cell biology, Haematopoiesis, Lymphopoieis, lcsh:Biology (General), Embryo, 030220 oncology & carcinogenesis, [SDV.IMM]Life Sciences [q-bio]/Immunology, Stem cell, Evo devo biology, Developmental Biology, Homing (hematopoietic)
Abstract: The identification of distinct waves of progenitors during development, each corresponding to a specific time, space, and function, provided the basis for the concept of a “layered” organization in development. The concept of a layered hematopoiesis was established by classical embryology studies in birds and amphibians. Recent progress in generating reliable lineage tracing models together with transcriptional and proteomic analyses in single cells revealed that, also in mammals, the hematopoietic system evolves in successive waves of progenitors with distinct properties and fate. During embryogenesis, sequential waves of hematopoietic progenitors emerge at different anatomic sites, generating specific cell types with distinct functions and tissue homing capacities. The first progenitors originate in the yolk sac before the emergence of hematopoietic stem cells, some giving rise to progenies that persist throughout life. Hematopoietic stem cell-derived cells that protect organisms against environmental pathogens follow the same sequential strategy, with subsets of lymphoid cells being only produced during embryonic development. Growing evidence indicates that fetal immune cells contribute to the proper development of the organs they seed and later ensure life-long tissue homeostasis and immune protection. They include macrophages, mast cells, some ¿d T cells, B-1 B cells, and innate lymphoid cells, which have “non-redundant” functions, and early perturbations in their development or function affect immunity in the adult. These observations challenged the view that all hematopoietic cells found in the adult result from constant and monotonous production from bone marrow-resident hematopoietic stem cells. In this review, we evaluate evidence for a layered hematopoietic system across species. We discuss mechanisms and selective pressures leading to the temporal generation of different cell types. We elaborate on the consequences of disturbing fetal immune cells on tissue homeostasis and immune development later in life. This work was financed by the PPU Pasteur Ph.D. program. This work was financed by the Institut Pasteur, INSERM, Pasteur-Weizmann Foundation and ANR (grant Twothyme and Epi-Dev) through grants to AC; by REVIVE (Investissement d’Avenir; ANR-10-LABX-73) through grants to AC and RE; by FCT through the grants PD/BD/114128/2015 and POCI-0100145-FEDER-01638 to FS; and the grant SFRH/BD/143605/2019 to MP.
Published: 2020
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32. Systemic GDF11 stimulates the secretion of adiponectin and induces a calorie restriction‐like phenotype in aged mice

Author: Erzsebet Kokovay, Nicolas Kuperwasser, Virginie Tolle, Pierre-Marie Lledo, Han Li, Carine Moigneu, Lida Katsimpardi, Aurélie Chiche, Claire Camus, Perception et Mémoire / Perception and Memory, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Institut Necker Enfants-Malades (INEM - UM 111 (UMR 8253 / U1151)), Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université de Paris (UP), Plasticité cellulaire et Modélisation des Maladies / Cellular Plasticity and Disease Modelling, INSERM UMR894-Centre de Psychiatrie et Neurosciences (CPN), Universite´ Paris Descartes, Sorbonne Paris Cite, The University of Texas at San Antonio (UTSA), L. K. received a fellowship from LabEx/Revive and a research grant from Inserm 'Transversal program on Aging' (AGEMED). A. C. was funded by a postdoctoral fellowship from the Revive Consortium. The Lledo laboratory was supported by national funds (ANR-10-LABX-73, ANR-15-CE37-0004-01, ANR-11-IDEX-0004-02), the life insurance company 'AG2R-La-Mondiale' and Laboratoire d'Excellence Revive (Investissement d'Avenir). The Kokovay laboratory acknowledges the Morrison Trust Foundation and the Barshop Institute Nathan Shock Center. The Li laboratory was funded by IP, CNRS and the Agence Nationale de la Recherche (ANR-10-LABX-73, ANR-16-CE13-0017-01), Fondation ARC (PJA 20161205028, 20181208231) and AFM-Telethon Foundation., ANR-10-LABX-0073,REVIVE,Stem Cells in Regenerative Biology and Medicine(2010), ANR-15-CE37-0004,SmellBrain,Dissection fonctionnelle des circuits codant pour la récompense dans le système olfactif(2015), ANR-11-IDEX-0004,SUPER,Sorbonne Universités à Paris pour l'Enseignement et la Recherche(2011), ANR-16-CE13-0017,iCELLPLASTICITY,Plasticité cellulaire dans le vieillissement(2016), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), Chiche, Aurélie, Laboratoires d'excellence - Stem Cells in Regenerative Biology and Medicine - - REVIVE2010 - ANR-10-LABX-0073 - LABX - VALID, Dissection fonctionnelle des circuits codant pour la récompense dans le système olfactif - Dissection fonctionnelle des circuits codant pour la récompense dans le système olfactif - - SmellBrain2015 - ANR-15-CE37-0004 - AAPG2015 - VALID, Sorbonne Universités à Paris pour l'Enseignement et la Recherche - - SUPER2011 - ANR-11-IDEX-0004 - IDEX - VALID, and Plasticité cellulaire dans le vieillissement - - iCELLPLASTICITY2016 - ANR-16-CE13-0017 - AAPG2016 - VALID
Subjects: 0301 basic medicine, medicine.medical_specialty, Parabiosis, media_common.quotation_subject, medicine.medical_treatment, MESH: Bone Morphogenetic Proteins, Calorie restriction, rejuvenation, White adipose tissue, [SDV.BC.BC]Life Sciences [q-bio]/Cellular Biology/Subcellular Processes [q-bio.SC], Biology, MESH: Phenotype, 03 medical and health sciences, Mice, 0302 clinical medicine, Internal medicine, medicine, [SDV.BC.BC] Life Sciences [q-bio]/Cellular Biology/Subcellular Processes [q-bio.SC], Animals, heterochronic parabiosis, Adiponectin secretion, MESH: Aging, MESH: Animals, MESH: Mice, media_common, Caloric Restriction, MESH: Caloric Restriction, Adiponectin, adiponectin, Insulin, [SCCO.NEUR]Cognitive science/Neuroscience, [SCCO.NEUR] Cognitive science/Neuroscience, aging, Appetite, Cell Biology, Original Articles, calorie restriction, MESH: Adiponectin, MESH: Growth Differentiation Factors, Growth Differentiation Factors, 030104 developmental biology, Endocrinology, Phenotype, Bone Morphogenetic Proteins, GDF11, Original Article, 030217 neurology & neurosurgery, Homeostasis
Abstract: Aging is a negative regulator of general homeostasis, tissue function, and regeneration. Changes in organismal energy levels and physiology, through systemic manipulations such as calorie restriction and young blood infusion, can regenerate tissue activity and increase lifespan in aged mice. However, whether these two systemic manipulations could be linked has never been investigated. Here, we report that systemic GDF11 triggers a calorie restriction‐like phenotype without affecting appetite or GDF15 levels in the blood, restores the insulin/IGF‐1 signaling pathway, and stimulates adiponectin secretion from white adipose tissue by direct action on adipocytes, while repairing neurogenesis in the aged brain. These findings suggest that GDF11 has a pleiotropic effect on an organismal level and that it could be a linking mechanism of rejuvenation between heterochronic parabiosis and calorie restriction. As such, GDF11 could be considered as an important therapeutic candidate for age‐related neurodegenerative and metabolic disorders., GDF11 stimulates adipocytes to release adiponectin in the bloodstream of aged mice and induces a calorie restriction‐like phenotype without affecting appetite. At the same time, these same GDF11‐treated aged mice exhibit enhanced neurogenesis. This shows that GDF11 has pleiotropic effects in both the brain and the periphery of aged mice, linking the beneficial effects of both heterochronic parabiosis and calorie restriction.
Published: 2019
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33. A distinct cardiopharyngeal mesoderm genetic hierarchy establishes antero-posterior patterning of esophagus striated muscle

Author: Francesca Pala, Shahragim Tajbakhsh, Francina Langa, Glenda Comai, Gabrielle Kardon, Sebastian Mella, Sylvain Paisant, Eglantine Heude, Swetha Gopalakrishnan, Mirialys Gallardo, Cellules Souches et Développement / Stem Cells and Development, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Département Adaptations du vivant (AVIV), Muséum national d'Histoire naturelle (MNHN), Laboratory of Clinical Immunology and Microbiology [Bethesda, MA, USA] (LCIM), National Institute of Allergy and Infectious Diseases [Bethesda] (NIAID-NIH), National Institutes of Health [Bethesda] (NIH)-National Institutes of Health [Bethesda] (NIH), Department of Human Genetics [Salt Lake City, UT, USA], University of Utah, Centre d'Ingénierie génétique murine - Mouse Genetics Engineering Center (CIGM), Institut Pasteur [Paris], HiLIFE - Neuroscience Center (NC), Helsinki Institute of Life Science (HiLIFE), University of Helsinki-University of Helsinki, We acknowledge funding support from the Institut Pasteur, Association Franc¸ aise contre le Myopathies, Agence Nationale de la Recherche (Laboratoire d’Excellence Revive, Investissement d’Avenir, ANR-10-LABX-73)., ANR-10-LABX-0073,REVIVE,Stem Cells in Regenerative Biology and Medicine(2010), Heude, Eglantine, Laboratoires d'excellence - Stem Cells in Regenerative Biology and Medicine - - REVIVE2010 - ANR-10-LABX-0073 - LABX - VALID, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Physiologie moléculaire et adaptation (PhyMA), Muséum national d'Histoire naturelle (MNHN)-Centre National de la Recherche Scientifique (CNRS), Institut Pasteur [Paris] (IP), Helsingin yliopisto = Helsingfors universitet = University of Helsinki-Helsingin yliopisto = Helsingfors universitet = University of Helsinki, Centre of Excellence in Stem Cell Metabolism, and Institute of Biotechnology
Subjects: Mouse, Tbx1-Isl1-Met/HGF, [SDV.BBM.BM] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, Mesoderm, Mice, 0302 clinical medicine, SKELETAL MYOGENESIS, Biology (General), [SDV.BDD]Life Sciences [q-bio]/Development Biology, 0303 health sciences, Hepatocyte Growth Factor, Myogenesis, General Neuroscience, C-MET RECEPTOR, 1184 Genetics, developmental biology, physiology, Gene Expression Regulation, Developmental, General Medicine, Proto-Oncogene Proteins c-met, Cell biology, DIFFERENTIATION, medicine.anatomical_structure, Medicine, MOUSE ESOPHAGUS, Signal Transduction, Research Article, EXPRESSION, TBX1, GROWTH-FACTOR, QH301-705.5, PHARYNGEAL ARCH, Science, LIM-Homeodomain Proteins, Biology, General Biochemistry, Genetics and Molecular Biology, 03 medical and health sciences, Esophagus, REVEALS, [SDV.BDD] Life Sciences [q-bio]/Development Biology, myogenic migration, medicine, Animals, HEAD, Body Patterning, 030304 developmental biology, Progenitor, Evolutionary Biology, General Immunology and Microbiology, antero-posterior patterning, cardiopharyngeal mesoderm, Skeletal muscle, [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, Muscle, Striated, PROGENITOR CELLS, esophagus striated muscles, ISL1, T-Box Domain Proteins, 030217 neurology & neurosurgery, Pharyngeal arch, Transcription Factors, Developmental Biology
Abstract: International audience; In most vertebrates, the upper digestive tract is composed of muscularized jaws linked to the esophagus that permits food ingestion and swallowing. Masticatory and esophagus striated muscles (ESM) share a common cardiopharyngeal mesoderm (CPM) origin, however ESM are unusual among striated muscles as they are established in the absence of a primary skeletal muscle scaffold. Using mouse chimeras, we show that the transcription factors Tbx1 and Isl1 are required cell-autonomously for myogenic specification of ESM progenitors. Further, genetic loss-of-function and pharmacological studies point to MET/HGF signaling for antero-posterior migration of esophagus muscle progenitors, where Hgf ligand is expressed in adjacent smooth muscle cells. These observations highlight the functional relevance of a smooth and striated muscle progenitor dialogue for ESM patterning. Our findings establish a Tbx1-Isl1-Met genetic hierarchy that uniquely regulates esophagus myogenesis and identify distinct genetic signatures that can be used as framework to interpret pathologies arising within CPM derivatives.
Published: 2019
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34. Ageing affects DNA methylation drift and transcriptional cell-to-cell variability in mouse muscle stem cells

Author: Shahragim Tajbakhsh, Wolf Reik, Brendan Evano, Thomas M. Stubbs, Pierre-Henri Commere, Marc Jan Bonder, Stephen J. Clark, Simon Andrews, Irene Hernando-Herraez, The Babraham Institute [Cambridge, UK], Cellules Souches et Développement / Stem Cells and Development, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Cytometrie et Biomarqueurs – Cytometry and Biomarkers (UTechS CB), Institut Pasteur [Paris] (IP), European Bioinformatics Institute [Hinxton] (EMBL-EBI), EMBL Heidelberg, This project was supported by grants from Institut Pasteur, Agence Nationale de la Recherche (Laboratoire d’Excellence Revive, Investissement d’Avenir, ANR-10-LABX-73), Association Française contre les Myopathies (21857), CNRS, the European Research Council (Advanced Research Grant 332893), and the Biotechnology and Biological Sciences Research Council (BBSRC, CBBS/E/B/000C0425). I.H.-H. was supported by a Marie Sklodowska-Curie Individual Fellowship (751439). M.J.B. was supported by a fellowship from the EMBL Interdisciplinary Postdoc (EI3POD) program under Marie Skłodowska-Curie Actions COFUND (grant number 664726)., ANR-10-LABX-0073,REVIVE,Stem Cells in Regenerative Biology and Medicine(2010), European Project: 332893,332893, European Project: 664726,H2020,H2020-MSCA-COFUND-2014,EI3POD(2015), European Project: 751439,H2020-MSCA-IF-2016,MeGa(2018), Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Institut Pasteur [Paris], Clark, Stephen James [0000-0002-6183-491X], Andrews, Simon Richard [0000-0002-5006-3507], Reik, Wolf [0000-0003-0216-9881], and Apollo - University of Cambridge Repository
Subjects: 0301 basic medicine, Epigenomics, MESH: Gene Ontology, Aging, MESH: Muscles, [SDV]Life Sciences [q-bio], Cell, MESH: Epigenomics, General Physics and Astronomy, Epigenesis, Genetic, Transcriptome, 0302 clinical medicine, MESH: DNA Methylation, MESH: Aging, MESH: Animals, MESH: Epigenesis, Genetic, Promoter Regions, Genetic, lcsh:Science, Cells, Cultured, Cellular Senescence, Multidisciplinary, Muscles, Stem Cells, Methylation, Cell biology, medicine.anatomical_structure, Mice, Inbred DBA, DNA methylation, Stem cell, Single-Cell Analysis, Transcription, MESH: Cells, Cultured, MESH: Mice, Transgenic, Science, Mice, Transgenic, MESH: Stem Cells, Biology, Article, General Biochemistry, Genetics and Molecular Biology, 03 medical and health sciences, MESH: Gene Expression Profiling, MESH: Mice, Inbred C57BL, MESH: Promoter Regions, Genetic, medicine, Animals, Humans, Epigenetics, Gene, MESH: Humans, MESH: Mice, Inbred DBA, Gene Expression Profiling, MESH: Transcriptome, Promoter, MESH: Cellular Senescence, General Chemistry, DNA Methylation, Computational biology and bioinformatics, Mice, Inbred C57BL, Ageing, 030104 developmental biology, Gene Ontology, lcsh:Q, Gene expression, 030217 neurology & neurosurgery, MESH: Single-Cell Analysis
Abstract: Age-related tissue alterations have been associated with a decline in stem cell number and function. Although increased cell-to-cell variability in transcription or epigenetic marks has been proposed to be a major hallmark of ageing, little is known about the molecular diversity of stem cells during ageing. Here we present a single cell multi-omics study of mouse muscle stem cells, combining single-cell transcriptome and DNA methylome profiling. Aged cells show a global increase of uncoordinated transcriptional heterogeneity biased towards genes regulating cell-niche interactions. We find context-dependent alterations of DNA methylation in aged stem cells. Importantly, promoters with increased methylation heterogeneity are associated with increased transcriptional heterogeneity of the genes they drive. These results indicate that epigenetic drift, by accumulation of stochastic DNA methylation changes in promoters, is associated with the degradation of coherent transcriptional networks during stem cell ageing. Furthermore, our observations also shed light on the mechanisms underlying the DNA methylation clock., Age-related tissue alterations have been associated with a decline in stem cell number and function. Here the authors report a single cell multi-omics study of mouse muscle stem cells, combining single cell transcriptome and DNA methylome profiling and find that aged cells have a global increase of uncoordinated transcriptional heterogeneity biased towards genes regulating cell-niche interactions.
Published: 2019
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35. CTCF confers local nucleosome resiliency after DNA replication and during mitosis

Author: Nicola Festuccia, Alexandra Tachtsidi, Inma Gonzalez, Thaleia Papadopoulou, Agnès Dubois, Pablo Navarro, Elphège P. Nora, Michel Cohen-Tannoudji, Sandrine Vandormael-Pournin, Benoit G. Bruneau, Nick D.L. Owens, Epigénomique, Prolifération et Identité Cellulaire - Epigenomics, Proliferation and the Identity of Cells (EPIC), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Collège Doctoral, Sorbonne Université (SU), Embryon précoce de mammifères et cellules souches, Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), University of California [San Francisco] (UC San Francisco), University of California (UC), This work was supported by recurrent funding from the Institut Pasteur, the CNRS, and Revive (Investissement d’Avenir, ANR-10-LABX-73). E.P.N. was supported by EMBO (ALTF523-2013), HSFP, and the Roddenberry Stem Cell Center at Gladstone. N.O. is supported by Revive. P.N. acknowledges financial support from the Fondation Schlumberger (FRM FSER 2017), the Agence Nationale de la Recherche (ANR 16 CE120004 01 MITMAT), the Ligue contre le Cancer (LNCC EL2018 NAVARRO) and the European Research Council (ERC-CoG-2017 BIND)., ANR-10-LABX-0073,REVIVE,Stem Cells in Regenerative Biology and Medicine(2010), ANR-16-CE12-0004,MitMAT,Mémoire mitotique de l'activité transcriptionnelle dans les cellules ES(2016), European Project: 773083,ERC-CoG-2017,BIND(2018), Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Collège doctoral [Sorbonne universités], Embryon précoce de mammifères et cellules souches - Early Mammalian Development & Stem Cell Biology, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS)-Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), University of California [San Francisco] (UCSF), University of California, Epigénétique des Cellules Souches - Epigenetics of Stem Cells, Génétique fonctionnelle de la Souris, Génétique Fonctionnelle de la Souris, ANR-10-LABX-0073/10-LABX-0073,REVIVE,Stem Cells in Regenerative Biology and Medicine(2010), and European Project
Subjects: 0301 basic medicine, Nucleosome organization, CCCTC-Binding Factor, Cell division, Mouse, MESH: DNA Replication, [SDV.BC.BC]Life Sciences [q-bio]/Cellular Biology/Subcellular Processes [q-bio.SC], chemistry.chemical_compound, Mice, 0302 clinical medicine, MESH: Animals, MESH: CCCTC-Binding Factor, Biology (General), MESH: Embryonic Stem Cells, Cells, Cultured, 0303 health sciences, Cultured, General Neuroscience, General Medicine, Chromosomes and Gene Expression, MESH: Gene Expression Regulation, 3. Good health, Cell biology, Nucleosomes, Medicine, nucleosome positioning, MESH: Cells, Cultured, Research Article, DNA Replication, Transcriptional Activation, chromosomes, replication, QH301-705.5, Science, Cells, Biology, General Biochemistry, Genetics and Molecular Biology, 03 medical and health sciences, MESH: Nucleosomes, [SDV.BBM.GTP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Genomics [q-bio.GN], transcription factors, Genetics, Nucleosome, Animals, Mitosis, Transcription factor, Gene, MESH: Mice, mouse, Embryonic Stem Cells, 030304 developmental biology, mitosis, General Immunology and Microbiology, fungi, DNA replication, MESH: Mitosis, Stem Cell Research, CTCF, pluripotency, 030104 developmental biology, [SDV.BDD.EO]Life Sciences [q-bio]/Development Biology/Embryology and Organogenesis, chemistry, Gene Expression Regulation, gene expression, MESH: Transcriptional Activation, Generic health relevance, Biochemistry and Cell Biology, 030217 neurology & neurosurgery, DNA
Abstract: The access of Transcription Factors (TFs) to their cognate DNA binding motifs requires a precise control over nucleosome positioning. This is especially important following DNA replication and during mitosis, both resulting in profound changes in nucleosome organization over TF binding regions. Using mouse Embryonic Stem (ES) cells, we show that the TF CTCF displaces nucleosomes from its binding site and locally organizes large and phased nucleosomal arrays, not only in interphase steady-state but also immediately after replication and during mitosis. Correlative analyses suggest this is associated with fast gene reactivation following replication and mitosis. While regions bound by other TFs (Oct4/Sox2), display major rearrangement, the post-replication and mitotic nucleosome positioning activity of CTCF is not unique: Esrrb binding regions are also characterized by persistent nucleosome positioning. Therefore, selected TFs such as CTCF and Esrrb act as resilient TFs governing the inheritance of nucleosome positioning at regulatory regions throughout the cell-cycle., eLife digest A single cell contains several meters of DNA which must be tightly packaged to fit inside. Typically, the DNA is wound around proteins, like a thread around many spools, to form more compact structures called nucleosomes. Before a cell divides in two, however, it needs first to access and replicate its DNA so that each new cell can get a copy of the genetic material. The cell then needs to condense the DNA again so that the two copies can be easily separated via a process called mitosis. These two processes – DNA replication and mitosis – entail major rearrangements of the nucleosomes, which then need to be returned to their original positions. Nucleosomes are also repositioned when cells need to access the coded instructions written in genes. Molecules called transcription factors bind to targets within the DNA to make sure genes are active or inactive at the right times of a cell’s life, but many are evicted from the DNA during its replication and during cell division. Most transcription factors also require nucleosomes to be specifically organized to bind to the DNA, and it remains unclear how the factors re-engage with the DNA and how nucleosomes are managed during and after DNA replication and mitosis. Owens, Papadopoulou et al. set out to understand how nucleosomes are organized immediately after DNA is replicated and while cells divide. Experiments with mouse cells grown in the laboratory showed that certain transcription factors can rebind to their targets within minutes of replication finishing, remain bound to the DNA during cell division, and displace nucleosomes from their binding sites. Owens, Papadopoulou et al. refer to these factors as “resilient transcription factors” and identified two examples, named CTCF and Esrrb. Further experiments showed that, by maintaining the structure of nearby nucleosomes while a cell divides, these resilient transcription factors could quickly reactivate genes immediately after DNA replication and mitosis are complete. These findings show that transcription factors play a fundamental role in maintaining gene regulation from one generation of cells to the next. Further studies on this topic may eventually foster progress in research areas where cell division is paramount, such as regenerative medicine and cancer biology.
Published: 2019
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36. Dynamics of Asymmetric and Symmetric Divisions of Muscle Stem Cells In Vivo and on Artificial Niches

Author: Shahragim Tajbakhsh, Brendan Evano, Gilles Le Carrou, Sara Khalilian, Geneviève Almouzni, Cellules Souches et Développement / Stem Cells and Development, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Dynamique du noyau [Institut Curie], Institut Curie [Paris]-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS), This work was supported by Institut Pasteur and grants from Agence Nationale de la Recherche ANR, (Laboratoire d’Excellence Revive, Investissement d’Avenir, ANR-10-LABX-73, ANR-16-CE12-0024) and Association Française contre les Myopathies, AFM, le Centre national de la recherche scientifique Centre national de la recherche scientifique, (CNRS), and the European Research Council (Advanced Research Grant 332893).G.A. received support from the French National Research Agency (ANR), Investissement D’avenir Labex développement, épigenèse, épigénétique et potentiel (DEEP), and the European Research Council Advanced (grant ChromAdict)., ANR-10-LABX-0073,REVIVE,Stem Cells in Regenerative Biology and Medicine(2010), ANR-16-CE12-0024,CHIFT,Chaperons et Histones Déterminants pour l'Identité Cellulaire, le Destin de Lignage et les Transitions(2016), ANR-10-IDEX-0001,PSL,Paris Sciences et Lettres(2010), European Project: 332893,332893, European Project: 694694,Chromatin Adaptations through Interactions of Chaperones in Time (ChromADICT), and Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)
Subjects: 0301 basic medicine, Cell division, [SDV]Life Sciences [q-bio], Symmetric cell division, Biology, General Biochemistry, Genetics and Molecular Biology, Germline, Histones, Mice, 03 medical and health sciences, 0302 clinical medicine, Genes, Reporter, Muscle stem cells, symmetric cell division, Asymmetric cell division, medicine, Animals, Cell Lineage, non-random DNA segregation, Transgenes, Stem Cell Niche, lcsh:QH301-705.5, Epigenomics, SNAP-tag, muscle regeneration, Muscles, Stem Cells, Regeneration (biology), Asymmetric Cell Division, Skeletal muscle, DNA, Pax7, Cell biology, Histone 3, 030104 developmental biology, medicine.anatomical_structure, lcsh:Biology (General), Myogenin, Stem cell, 030217 neurology & neurosurgery
Abstract: International audience; Stem cells can be maintained through symmetric cell divisions (SCDs) and asymmetric cell divisions (ACDs). How and when these divisions occur in vivo in vertebrates is poorly understood. Here, we developed a clonogenic cell tracing method that demonstrates the asymmetric distribution of transcription factors along with old and new DNA in mouse muscle stem cells during skeletal muscle regeneration. Combining single-cell tracking and artificial niches ex vivo, we show how cells switch from ACDs to SCDs, suggesting that they are not engaged in an obligate mode of cell division. Further, we generated SNAP-tagged histone H3-reporter mice and find that, unlike fly germline stem cells, differential fate outcomes are associated with a symmetric distribution of the H3.1 and H3.3 histone variants in mouse muscle stem cells. This versatile and efficient H3-SNAP labeling system will allow an investigation of mechanisms underlying the maintenance of epigenomic identity and plasticity in a variety of tissues.; Les cellules souches peuvent être maintenues par des divisions cellulaires symétriques (DCS) et des divisions cellulaires asymétriques (DCA). On comprend mal comment et quand ces divisions se produisent in vivo chez les vertébrés. Ici, nous avons développé une méthode de traçage cellulaire clonogène qui démontre la distribution asymétrique des facteurs de transcription ainsi que de l'ADN ancien et nouveau dans les cellules souches de muscles de souris pendant la régénération des muscles squelettiques. En combinant le traçage d'une seule cellule et des niches artificielles ex vivo, nous montrons comment les cellules passent des ACD aux SCD, ce qui suggère qu'elles ne sont pas engagées dans un mode de division cellulaire obligatoire. En outre, nous avons généré des souris indicatrices d'histone H3 marquées par SNAP et nous avons découvert que, contrairement aux cellules souches germinales de mouches, les résultats différentiels du destin sont associés à une distribution symétrique des variantes d'histone H3.1 et H3.3 dans les cellules souches musculaires de souris. Ce système de marquage H3-SNAP polyvalent et efficace permettra d'étudier les mécanismes sous-jacents au maintien de l'identité et de la plasticité épigénomiques dans une variété de tissus.
Published: 2020
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37. Differential cell fates of muscle stem cells are accompanied by symmetric segregation of canonical H3 histones in vivo

Author: Brendan Evano, Geneviève Almouzni, Gilles Le Carrou, Shahragim Tajbakhsh, Cellules Souches et Développement / Stem Cells and Development, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Dynamique du noyau [Institut Curie], Institut Curie [Paris]-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS), This work was supported by InstitutPasteur and grants from Agence Nationale de la Recherche (Laboratoire d'Excellence Revive, Investissement d'Avenir, ANR-10-LABX-73, ANR-16-CE12-0024) and Association Française contre les Myopathies, CNRS, and the European Research Council (Advanced Research Grant 332893)., ANR-10-LABX-0073,REVIVE,Stem Cells in Regenerative Biology and Medicine(2010), and ANR-16-CE12-0024,CHIFT,Chaperons et Histones Déterminants pour l'Identité Cellulaire, le Destin de Lignage et les Transitions(2016)
Subjects: 0303 health sciences, biology, [SDV]Life Sciences [q-bio], Cell, [SDV.BC.BC]Life Sciences [q-bio]/Cellular Biology/Subcellular Processes [q-bio.SC], Germline, Cell biology, 03 medical and health sciences, Histone H3, 0302 clinical medicine, Histone, medicine.anatomical_structure, biology.protein, medicine, Epigenetics, Stem cell, Mitosis, 030217 neurology & neurosurgery, 030304 developmental biology, Adult stem cell
Abstract: Posté le 18 décembre 2018 sur BioRxiv; Stem cells are maintained through symmetric or asymmetric cell divisions. While various mechanisms initiate asymmetric cell fates during mitosis, possible epigenetic control of this process has emerged recently. The asymmetrical distribution of a canonical histone H3 variant during mitosis in fly germline has suggested a role for partitioning old and new nucleosomes in asymmetric cell fates. Here, we provide resources for single cell assays and show the asymmetric segregation of transcription factors along with old and new DNA in mouse muscle stem cells ex vivo and in vivo . However, these differential fate outcomes contrast with a symmetric distribution of the canonical H3.1 vertebrate variant. These findings point to different evolutionary mechanisms operating in fly germline stem cells and vertebrate somatic stem cells to mitigate epigenetic regulation of asymmetric cell fates.; Les cellules souches sont maintenues par des divisions cellulaires symétriques ou asymétriques. Alors que divers mécanismes déclenchent des destins cellulaires asymétriques pendant la mitose, un éventuel contrôle épigénétique de ce processus est apparu récemment. La distribution asymétrique d'une variante canonique de l'histone H3 pendant la mitose dans la lignée germinale de la mouche a suggéré un rôle pour la partition des anciens et des nouveaux nucléosomes dans les destins cellulaires asymétriques. Ici, nous fournissons des ressources pour les essais sur cellules individuelles et montrons la ségrégation asymétrique des facteurs de transcription avec l'ancien et le nouvel ADN dans les cellules souches musculaires de souris ex vivo et in vivo . Cependant, ces résultats de destins différentiels contrastent avec une distribution symétrique de la variante canonique des vertébrés H3.1. Ces résultats indiquent que différents mécanismes d'évolution opèrent dans les cellules souches germinales de mouches et les cellules souches somatiques de vertébrés pour atténuer la régulation épigénétique des destins cellulaires asymétriques.
Published: 2018
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38. Skeletal muscle stem cells in comfort and stress

Author: Brendan Evano, Shahragim Tajbakhsh, Cellules Souches et Développement / Stem Cells and Development, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), We acknowledge support from the Institut Pasteur, Association Française contre les Myopathies, Agence Nationale de la Recherche (Laboratoire d’Excellence Revive,Investissement d’Avenir, ANR-10-LABX-73), and the European Research Council(Advanced Research Grant 332893), ANR-10-LABX-0073,REVIVE,Stem Cells in Regenerative Biology and Medicine(2010), European Project: 332893,332893, and Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS)
Subjects: 0301 basic medicine, Senescence, Stromal cell, Myogenesis, Regeneration (biology), [SDV]Life Sciences [q-bio], lcsh:R, Cell, Biomedical Engineering, lcsh:Medicine, Medicine (miscellaneous), Skeletal muscle, [SDV.BDD.MOR]Life Sciences [q-bio]/Development Biology/Morphogenesis, Cell Biology, Review Article, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, Biology, Cell biology, 03 medical and health sciences, 030104 developmental biology, medicine.anatomical_structure, Ageing, medicine, Stem cell, Developmental Biology
Abstract: Investigations on developmental and regenerative myogenesis have led to major advances in decrypting stem cell properties and potential, as well as their interactions within the evolving niche. As a consequence, regenerative myogenesis has provided a forum to investigate intrinsic regulators of stem cell properties as well as extrinsic factors, including stromal cells, during normal growth and following injury and disease. Here we review some of the latest advances in the field that have exposed fundamental processes including regulation of stress following trauma and ageing, senescence, DNA damage control and modes of symmetric and asymmetric cell divisions. Recent studies have begun to explore the nature of the niche that is distinct in different muscle groups, and that is altered from prenatal to postnatal stages, and during ageing. We also discuss heterogeneities among muscle stem cells and how distinct properties within the quiescent and proliferating cell states might impact on homoeostasis and regeneration. Interestingly, cellular quiescence, which was thought to be a passive cell state, is regulated by multiple mechanisms, many of which are deregulated in various contexts including ageing. These and other factors including metabolic activity and genetic background can impact on the efficiency of muscle regeneration.
Published: 2018
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39. SUMO Safeguards Somatic and Pluripotent Cell Identities by Enforcing Distinct Chromatin States

Author: Han Li, Ilan Theurillat, Jack-Christophe Cossec, Claudia Chica, Guillaume Bossis, Ane Iturbide, Jacob-Sebastian Seeler, Anne Dejean, Sabela Búa Aguín, Alexandra Andrieux, Grégory Jouvion, Xavier Gaume, Maria-Elena Torres-Padilla, Organisation Nucléaire et Oncogenèse / Nuclear Organization and Oncogenesis, Institut Pasteur [Paris] (IP)-Institut National de la Santé et de la Recherche Médicale (INSERM), Sorbonne Université (SU), Hub Bioinformatique et Biostatistique - Bioinformatics and Biostatistics HUB, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Plasticité cellulaire et Modélisation des Maladies / Cellular Plasticity and Disease Modelling, Helmholtz Zentrum München = German Research Center for Environmental Health, Neuropathologie expérimentale / Experimental neuropathology, Institut Pasteur [Paris] (IP)-Université Paris Cité (UPCité), Institut de Génétique Moléculaire de Montpellier (IGMM), Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS), This work was supported by grants from European Research Council (Advanced Research Grant 294341), LNCC (Equipe labellisée), INCa (n°2017-1 PL BIO-05-IP-1), and Odyssey to A.D. and from Association pour la Recherche sur le Cancer (ARC) (PJA 20161205028) and Revive (Laboratoire d’Excellence Revive, Investissement d’Avenir, ANR-10-LABX- 73) to H.L. J-C.C. was supported by INCa, I.T. by MESR and FRM, and X.G. by the Helmholtz Association., ANR-10-LABX-0073,REVIVE,Stem Cells in Regenerative Biology and Medicine(2010), European Project: 294341,EC:FP7:ERC,ERC-2011-ADG_20110310,SUMOSTRESS(2013), Institut Pasteur [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM), Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Helmholtz-Zentrum München (HZM), Institut Pasteur [Paris]-Université de Paris (UP), and Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM)
Subjects: 0301 basic medicine, MESH: Mouse Embryonic Stem Cells / metabolism, transdifferentiation, Cellular differentiation, SUMO protein, MESH: Small Ubiquitin-Related Modifier Proteins / metabolism, Biology, MESH: Cellular Reprogramming, MESH: Chromatin / metabolism, 03 medical and health sciences, 0302 clinical medicine, 2c-like Cells, Dux, Sumo, Cell Fate Change, Chromatin, Embryonic Stem Cells, Pluripotency, Reprogramming, Totipotency, Transdifferentiation, SOX2, MESH: Mice, Inbred C57BL, Genetics, totipotency, MESH: Animals, Induced pluripotent stem cell, MESH: Mice, 2C-like cells, MESH: Transcription Factors / metabolism, [SDV.BA]Life Sciences [q-bio]/Animal biology, reprogramming, cell fate change, Cell Biology, embryonic stem cells, pluripotency, MESH: Mouse Embryonic Stem Cells / cytology, Embryonic stem cell, Cell biology, 030104 developmental biology, KLF4, SUMO, embryonic structures, Molecular Medicine, chromatin, 030217 neurology & neurosurgery, MESH: Cells, Cultured
Abstract: International audience; Understanding general principles that safeguard cellular identity should reveal critical insights into common mechanisms underlying specification of varied cell types. Here, we show that SUMO modification acts to stabilize cell fate in a variety of contexts. Hyposumoylation enhances pluripotency reprogramming in vitro and in vivo, increases lineage transdifferentiation, and facilitates leukemic cell differentiation. Suppressing sumoylation in embryonic stem cells (ESCs) promotes their conversion into 2-cell-embryo-like (2C-like) cells. During reprogramming to pluripotency, SUMO functions on fibroblastic enhancers to retain somatic transcription factors together with Oct4, Sox2, and Klf4, thus impeding somatic enhancer inactivation. In contrast, in ESCs, SUMO functions on heterochromatin to silence the 2C program, maintaining both proper H3K9me3 levels genome-wide and repression of the Dux locus by triggering recruitment of the sumoylated PRC1.6 and Kap/Setdb1 repressive complexes. Together, these studies show that SUMO acts on chromatin as a glue to stabilize key determinants of somatic and pluripotent states.
Published: 2018
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40. The role of Pitx2 and Pitx3 in muscle stem cells gives new insights into P38α MAP kinase and redox regulation of muscle regeneration

Author: Aurore L'honoré, Pierre-Henri Commère, Elisa Negroni, Giorgia Pallafacchina, Bertrand Friguet, Jacques Drouin, Margaret Buckingham, Didier Montarras, Adaptation Biologique et Vieillissement = Biological Adaptation and Ageing (B2A), Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)-Institut de Biologie Paris Seine (IBPS), Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Cellules Souches et Développement / Stem Cells and Development, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Cytométrie (Plate-forme), Institut Pasteur [Paris], Centre de recherche en Myologie – U974 SU-INSERM, Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU), Department of Biomedical Sciences [Padova, Italy] (Neuroscience Institute), Italian National Research Council [Padova, Italy]-University of Padova [Padova, Italy], Laboratory of Molecular Genetics [Montréal, Canada], Institut De Recherches Cliniques De Montreal - IRCM [Canada], This work was supported by the Institut Pasteur, the CNRS (UMR 3738 and UMR 8256) and Sorbonne Universités, with grants to MB, DM and BF from the AFM, grants to MB and DM from the ANR (REGSAT), the Laboratoire d’Excellence REVIVE (Investissement d’Avenir ANR-10-LABX-73) and the EU Optistem project (Health, FP7-2007, 223098) and grant to AL from the DIM Stem Pôle. A L’honoré was supported by a postdoctoral fellowship from the Fondation pour la Recherche Médicale (FRM) and a Marie Curie IRG grant (MC-IRG248496/SATELLITE CELL). G Pallafacchina was supported by a postdoctoral fellowship from the Fondation pour la Recherche Médicale., ANR-10-LABX-0073,REVIVE,Stem Cells in Regenerative Biology and Medicine(2010), European Project: 223098,EC:FP7:HEALTH,FP7-HEALTH-2007-B,OPTISTEM(2009), Cellules Souches et Développement, Centre de recherche en myologie, Association française contre les myopathies (AFM-Téléthon)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS), ANR-10-LABX-0073/10-LABX-0073,REVIVE,Stem Cells in Regenerative Biology and Medicine(2010), Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Institut Pasteur [Paris] (IP), Università degli Studi di Padova = University of Padua (Unipd)-Italian National Research Council [Padova, Italy], HAL UPMC, Gestionnaire, Laboratoires d'excellence - Stem Cells in Regenerative Biology and Medicine - - REVIVE2010 - ANR-10-LABX-0073 - LABX - VALID, Optimization of stem cell therapy for clinical trials of degenerative skin and muscle diseases - OPTISTEM - - EC:FP7:HEALTH2009-01-01 - 2013-12-31 - 223098 - VALID, and Centre de Recherche en Myologie
Subjects: Satellite Cells, Skeletal Muscle, Mouse, QH301-705.5, Science, [SDV]Life Sciences [q-bio], Mitogen-Activated Protein Kinase 14, Mice, Animals, Biology (General), Muscle, Skeletal, muscle stem cell, p38α MAPK, Cellular Senescence, Homeodomain Proteins, Stem Cells, Cell Differentiation, Cell Biology, Stem Cells and Regenerative Medicine, Acetylcysteine, [SDV] Life Sciences [q-bio], redox state, regeneration, Mutation, Medicine, Pitx2/3, cell therapy, Reactive Oxygen Species, Oxidation-Reduction, Transcription Factors, Research Article
Abstract: International audience; Skeletal muscle regeneration depends on satellite cells. After injury these muscle stem cells exit quiescence, proliferate and differentiate to regenerate damaged fibres. We show that this progression is accompanied by metabolic changes leading to increased production of reactive oxygen species (ROS). Using Pitx2/3 single and double mutant mice that provide genetic models of deregulated redox states, we demonstrate that moderate overproduction of ROS results in premature differentiation of satellite cells while high levels lead to their senescence and regenerative failure. Using the ROS scavenger, N-Acetyl-Cysteine (NAC), in primary cultures we show that a physiological increase in ROS is required for satellite cells to exit the cell cycle and initiate differentiation through the redox activation of p38a MAP kinase. Subjecting cultured satellite cells to transient inhibition of P38a MAP kinase in conjunction with NAC treatment leads to their rapid expansion, with striking improvement of their regenerative potential in grafting experiments.
Published: 2018
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41. Distinct metabolic states govern skeletal muscle stem cell fates during prenatal and postnatal myogenesis

Author: Shahragim Tajbakhsh, Siham Yennek, Francesca Pala, Daniela Di Girolamo, Miria Ricchetti, Sébastien Mella, Laurent Chatre, Cellules Souches et Développement / Stem Cells and Development, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Università degli studi di Napoli Federico II, Institut Pasteur, Centre National pour la RechercheScientific and the Agence Nationale de la Recherche (Laboratoire d’Excellence Revive, Investissement d’Avenir, ANR-10-LABX- 73) and the EuropeanResearch Council (Advanced Research Grant 332893)., ANR-10-LABX-0073,REVIVE,Stem Cells in Regenerative Biology and Medicine(2010), European Project: 332893,332893, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), University of Naples Federico II = Università degli studi di Napoli Federico II, Pala, Francesca, Di Girolamo, Daniela, Mella, Sébastien, Yennek, Siham, Chatre, Laurent, Ricchetti, Miria, and Tajbakhsh, Shahragim
Subjects: 0301 basic medicine, Satellite Cells, Skeletal Muscle, [SDV]Life Sciences [q-bio], Oxidative phosphorylation, Mitochondrion, Biology, Peroxisome, Muscle Development, Mice, 03 medical and health sciences, Peroxisomes, medicine, Animals, Regeneration, Muscle, Skeletal, 10. No inequality, [SDV.BDD]Life Sciences [q-bio]/Development Biology, Cell Proliferation, Myogenesis, Stem Cells, Regeneration (biology), Fatty Acids, Skeletal muscle, Cell Biology, Cell biology, Mitochondria, Metabolic pathway, Ageing, 030104 developmental biology, medicine.anatomical_structure, Metabolic state, Stem cell, Skeletal muscle stem cells, Oxidation-Reduction, Research Article
Abstract: During growth, homeostasis and regeneration, stem cells are exposed to different energy demands. Here, we characterise the metabolic pathways that mediate the commitment and differentiation of mouse skeletal muscle stem cells, and how their modulation can influence the cell state. We show that quiescent satellite stem cells have low energetic demands and perturbed oxidative phosphorylation during ageing, which is also the case for cells from post-mortem tissues. We show also that myogenic fetal cells have distinct metabolic requirements compared to those proliferating during regeneration, with the former displaying a low respiration demand relying mostly on glycolysis. Furthermore, we show distinct requirements for peroxisomal and mitochondrial fatty acid oxidation (FAO) in myogenic cells. Compromising peroxisomal but not mitochondrial FAO promotes early differentiation of myogenic cells. Acute muscle injury and pharmacological block of peroxisomal and mitochondrial FAO expose differential requirements for these organelles during muscle regeneration. Taken together, these observations indicate that changes in myogenic cell state lead to significant alterations in metabolic requirements. In addition, perturbing specific metabolic pathways impacts on myogenic cell fates and the regeneration process., Summary: Distinct energy metabolism pathways act during mouse skeletal muscle stem cell commitment and differentiation in different physiological states.
Published: 2018
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42. 2i, or Not 2i: The Soliloquy of Nanog-Negative Mouse Embryonic Stem Cells

Author: Pablo Navarro, Epigénétique des Cellules Souches - Epigenetics of Stem Cells, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Research in P.N.’s laboratory is supported by recurrent funding from the Institut Pasteur, the CNRS, and Revive (Investissement d’Avenir, ANR-10-LABX-73). P.N. acknowledges financial support from the Fondation Schlumberger (FRM FSER 2017), the Fondation ARC pour la recherche sur le cancer (ARC NAVARRO CA 14/12/2016), the Agence Nationale de la Recherche (ANR 16 CE12 0004 01 MITMAT), and the Ligue contre le Cancer (LNCC EL2018 NAVARRO)., The author is grateful to Agnès Dubois and Alexandra Tachtsidi for providing the images in Figure 1 and to Nick Owens for comments., ANR-10-LABX-0073,REVIVE,Stem Cells in Regenerative Biology and Medicine(2010), ANR-16-CE12-0004,MitMAT,Mémoire mitotique de l'activité transcriptionnelle dans les cellules ES(2016), and Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)
Subjects: 0301 basic medicine, Homeobox protein NANOG, MAPK/ERK pathway, [SDV]Life Sciences [q-bio], Cell Culture Techniques, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, [SDV.BC.BC]Life Sciences [q-bio]/Cellular Biology/Subcellular Processes [q-bio.SC], Biology, Biochemistry, Gene Expression Regulation, Enzymologic, Mice, 03 medical and health sciences, [SDV.BBM.GTP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Genomics [q-bio.GN], Genetics, Animals, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Extracellular Signal-Regulated MAP Kinases, lcsh:QH301-705.5, Transcription factor, GSK3B, [SDV.BDD]Life Sciences [q-bio]/Development Biology, Cells, Cultured, reproductive and urinary physiology, Regulation of gene expression, lcsh:R5-920, Glycogen Synthase Kinase 3 beta, Mouse Embryonic Stem Cells, Nanog Homeobox Protein, Cell Biology, Preview, Embryonic stem cell, Cell biology, 030104 developmental biology, [SDV.BDD.EO]Life Sciences [q-bio]/Development Biology/Embryology and Organogenesis, lcsh:Biology (General), Cell culture, embryonic structures, Stem cell, biological phenomena, cell phenomena, and immunity, lcsh:Medicine (General), Developmental Biology
Abstract: In this issue of Stem Cell Reports, Hastreiter et al. (2018) use continuous time-lapse imaging of mouse embryonic stem cells to investigate how the inhibition of GSK3b and MEK/ERK (2i) leads to homogeneous expression of the transcription factor Nanog. They show that both induction of Nanog expression and selection against cells expressing low levels of Nanog contribute to the homogeneous appearance of 2i cultures. : In this issue of Stem Cell Reports, Hastreiter et al. (2018) use continuous time-lapse imaging of mouse embryonic stem cells to investigate how the inhibition of GSK3b and MEK/ERK (2i) leads to homogeneous expression of the transcription factor Nanog. They show that both induction of Nanog expression and selection against cells expressing low levels of Nanog contribute to the homogeneous appearance of 2i cultures.
Published: 2018
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43. Regulation of neurogenesis in the adult and aging brain

Author: Lida Katsimpardi, Pierre-Marie Lledo, Perception et Mémoire / Perception and Memory, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), This work was supported by Institut Pasteur, Centre National pour la Recherche Scientifique, the life insurance company 'AG2R-La-Mondiale', Agence Nationale de la Recherche (ANR-15-CE37-0004-01), NIH US-French Research Proposal Grants #1R01DC015137-01 and ANR-15-NEUC-0004 (Circuit-OPL), the Laboratory for Excellence Programme 'Revive' (Investissement d'Avenir, ANR-10-LABX-73) and 'Biopsy' (Investissement d'Avenir, ANR-11-IDEX-0004-02)., We apologize to colleagues whose work could not be cited due to space limitations. We thank Nicolas Kuperwasser for proofreading the manuscript. L.K. received a fellowship from the Laboratory for Excellence Programme 'Revive'., ANR-15-CE37-0004,SmellBrain,Dissection fonctionnelle des circuits codant pour la récompense dans le système olfactif(2015), ANR-15-NEUC-0004,CIRCUIT-OPL,US-French Research Proposal- Neural Circuits and Plasticity- Olfactory Perception and Learning(2015), and ANR-10-LABX-0073,REVIVE,Stem Cells in Regenerative Biology and Medicine(2010)
Subjects: 0301 basic medicine, Aging, [SDV]Life Sciences [q-bio], [SDV.NEU.NB]Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC]/Neurobiology, General Neuroscience, Neurogenesis, Biology, Neural stem cell, 03 medical and health sciences, 030104 developmental biology, Neural Stem Cells, Aging brain, Animals, Humans, [SDV.NEU]Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC], Stem cell, Neuroscience
Abstract: International audience; Neural stem cells (NSCs) represent a remarkable developmental unit, necessary for the proper functioning of neurogenesis, by retaining their plasticity to self-renew and give rise to progeny throughout life in specific regions of the adult brain. Although NSCs were thought to merely represent a stem cell type in the brain, recent advances have demonstrated the incredible complexity of NSC identity and functions. Ranging between quiescence, activation and intermediary subtypes, NSCs choose their fate through their developmental inheritance, regional positioning within the niche, as well as dynamic transcriptional and metabolic states. The plasticity of their developmental program is reflected in the tremendous changes they undergo upon external environmental cues and extrinsic manipulations, and harnessing these potentials can open new avenues to fight against brain injury, neurodegenerative and age-related diseases.
Published: 2018
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44. Small-RNA sequencing identifies dynamic microRNA deregulation during skeletal muscle lineage progression

Author: Christophe Antoniewski, Shahragim Tajbakhsh, Jérôme Cavaillé, David Castel, Virginie Marty, Meryem B. Baghdadi, Sébastien Mella, Barbara Gayraud-Morel, Cellules Souches et Développement / Stem Cells and Development, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Cellule Pasteur UPMC, Institut Pasteur [Paris]-Université Pierre et Marie Curie - Paris 6 (UPMC), Laboratoire de biologie moléculaire eucaryote (LBME), Centre National de la Recherche Scientifique (CNRS)-Centre de Biologie Intégrative (CBI), Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS), Génétique et épigénétique de la Drosophile [IBPS] = Drosophila genetics and epigenetics [IBPS] (LBD-E16), Laboratoire de Biologie du Développement [Paris] (LBD), Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)-Institut de Biologie Paris Seine (IBPS), Sorbonne Université (SU)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)-Institut de Biologie Paris Seine (IBPS), Sorbonne Université (SU)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Bioinformatique [IBPS] (IBPS-Artbio), Institut de Biologie Paris Seine (IBPS), Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), S.T. was funded by Institut Pasteur, Centre National pour la Recherche Scientifique and the Agence Nationale de la Recherche (Laboratoire d’Excellence Revive, Investissement d’Avenir, ANR-10-LABX- 73), the European Research Council (Advanced Research Grant 332893) and the French Muscular Dystrophy Association (AFM-Téléthon). M.B.B. was funded by the Fondation pour la Recherche Médicale (FRM)., We acknowledge the Flow Cytometry Platform of the Technology Core-Center for Translational Science (CRT) and the Transcriptome and EpiGenome Platform of the Center for Innovation & Technological Research at Institut Pasteur for support in conducting this study, We thank the ABC Facility of ANEXPLO Toulouse for mouse husbandry., ANR-10-LABX-0073,REVIVE,Stem Cells in Regenerative Biology and Medicine(2010), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut Pasteur [Paris] (IP), Université de Toulouse (UT)-Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS)-Centre de Biologie Intégrative (CBI), Université de Toulouse (UT)-Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS), Laboratoire de Biologie du Développement [IBPS] (LBD), Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)-Institut de Biologie Paris Seine (IBPS), Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), HAL UPMC, Gestionnaire, Laboratoires d'excellence - Stem Cells in Regenerative Biology and Medicine - - REVIVE2010 - ANR-10-LABX-0073 - LABX - VALID, Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS)-Centre de Biologie Intégrative (CBI), and Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS)
Subjects: 0301 basic medicine, Small RNA, Satellite Cells, Skeletal Muscle, lcsh:Medicine, Biology, Muscle Development, Article, Transcriptome, 03 medical and health sciences, Mice, microRNA, Muscle stem cells, [SDV.BBM] Life Sciences [q-bio]/Biochemistry, Molecular Biology, medicine, Myocyte, Animals, Homeostasis, Regeneration, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Cell Lineage, Cell Self Renewal, lcsh:Science, Transcriptomics, Tissue homeostasis, Multidisciplinary, Myogenesis, Sequence Analysis, RNA, Gene Expression Profiling, lcsh:R, Skeletal muscle, Chromosomes, Mammalian, Cell biology, MicroRNAs, 030104 developmental biology, medicine.anatomical_structure, miRNAs, Self-renewal, lcsh:Q, Stem cell
Abstract: Skeletal muscle satellite cells are quiescent adult resident stem cells that activate, proliferate and differentiate to generate myofibres following injury. They harbour a robust proliferation potential and self-renewing capacity enabling lifelong muscle regeneration. Although several classes of microRNAs were shown to regulate adult myogenesis, systematic examination of stage-specific microRNAs during lineage progression from the quiescent state is lacking. Here we provide a genome-wide assessment of the expression of small RNAs during the quiescence/activation transition and differentiation by RNA-sequencing. We show that the majority of small RNAs present in quiescent, activated and differentiated muscle cells belong to the microRNA class. Furthermore, by comparing expression in distinct cell states, we report a massive and dynamic regulation of microRNAs, both in numbers and amplitude, highlighting their pivotal role in regulation of quiescence, activation and differentiation. We also identify a number of microRNAs with reliable and specific expression in quiescence including several maternally-expressed miRNAs generated at the imprinted Dlk1-Dio3 locus. Unexpectedly, the majority of class-switching miRNAs are associated with the quiescence/activation transition suggesting a poised program that is actively repressed. These data constitute a key resource for functional analyses of miRNAs in skeletal myogenesis, and more broadly, in the regulation of stem cell self-renewal and tissue homeostasis.
Published: 2018
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45. Loss of Function of the Nuclear Receptor NR2F2, Encoding COUP-TF2, Causes Testis Development and Cardiac Defects in 46,XX Children

Author: Caroline Eozenou, David Rodriguez-Buritica, Zita Halász, Jean-Pierre Siffroi, Joelle Bignon-Topalovic, Anne Jorgensen, Sophie Lambert, Rajpert-De Meyts E, János Sólyom, Anu Bashamboo, Ken McElreavey, Paye-Jaouen A, Rita Bertalan, Attila Tar, Capucine Hyon, John C. Achermann, Peter Nagy, Laetitia Martinerie, Génétique du développement humain, Institut Pasteur [Paris], Department of Growth and Reproduction [Rigshospitalet], Rigshospitalet [Copenhagen], Copenhagen University Hospital-Copenhagen University Hospital, CHU Trousseau [APHP], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU), Pál Heim Children's Hospital = Heim Pál Gyermekkórház [Budapest], First Department of Pathology and Experimental Cancer Research, Semmelweis University [Budapest], Chirurgie viscérale et urologie pédiatriques [AP-HP Robert Debré], AP-HP Hôpital universitaire Robert-Debré [Paris], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP), Service d'Endocrinologie et Diabétologie Pédiatriques, Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Hôpital Robert Debré, McGovern Medical School [Houston, Texas], The University of Texas Health Science Center at Houston (UTHealth), Copenhagen University Hospital, Institute of Child Health [London], University College of London [London] (UCL), A.B. is funded in part by the program Actions Concertees Interpasteuriennes (ACIP) and a research grant from the European Society of Pediatric Endocrinology. A.B. and K.McE. are funded by a research grant from the EuroDSD in the European Community’s Seventh Framework Programme FP7/2007-2013 under grant agreement no. 201444 as well as grant no. 295097 as part of the EU call FP7-INCO-2011-6. J.C.A. is a Wellcome Trust Senior Research Fellow in Clinical Science (grant 098513/Z/12/Z, 209328/Z/17/Z) with research support from Great Ormond Street Hospital Children's Charity (grant V2518) and the NIHR GOSH BRC (IS-BRC-1215-20012)., This work is supported by the COST Action DSDnet BM1303., This work was funded by the Agence Nationale de la Recherche (Laboratoire d’Excellence Revive, Investissement d’Avenir, ANR-10-LABX-73)., The authors wish to thank Dr. Etienne Patin for comments on the manuscript., ANR-10-LABX-0073,REVIVE,Stem Cells in Regenerative Biology and Medicine(2010), European Project: 201444,EC:FP7:HEALTH,FP7-HEALTH-2007-A,EURODSD(2008), European Project: 295097,EC:FP7:INCO,FP7-INCO-2011-6,GM_NCD_IN_CO(2011), Génétique du Développement humain - Human developmental genetics, and Institut Pasteur [Paris] (IP)
Subjects: Forkhead Box Protein L2, Male, 0301 basic medicine, new syndrome, 46, XX Disorders of Sex Development, [SDV]Life Sciences [q-bio], MESH: 46, XX Disorders of Sex Development / genetics, sex determination, MESH: Amino Acid Sequence, MESH: Loss of Function Mutation / genetics, MESH: Base Sequence, Bioinformatics, medicine.disease_cause, COUP Transcription Factor II, 0302 clinical medicine, Loss of Function Mutation, MESH: Ovary / growth & development, MESH: Child, Testis, MESH: Testis / growth & development, nuclear receptor, Child, Frameshift Mutation, Exome, Genetics (clinical), MESH: Heterozygote, 0303 health sciences, education.field_of_study, Mutation, MESH: Testis / abnormalities, MESH: COUP Transcription Factor II / genetics, Cell biology, Phenotype, medicine.anatomical_structure, 030220 oncology & carcinogenesis, Female, medicine.symptom, Heterozygote, medicine.drug_class, Population, Ovary, Biology, MESH: COUP Transcription Factor II / chemistry, MESH: Phenotype, Frameshift mutation, [SDV.BDLR.RS]Life Sciences [q-bio]/Reproductive Biology/Sexual reproduction, 03 medical and health sciences, MESH: Forkhead Box Protein L2 / metabolism, Report, Genetics, medicine, Humans, Amino Acid Sequence, education, testicular DSD, Loss function, 030304 developmental biology, MESH: Humans, Base Sequence, Virilization, MESH: Frameshift Mutation / genetics, Androgen, MESH: Ovary / metabolism, NR2F2, MESH: Male, 030104 developmental biology, Nuclear receptor, [SDV.GEN.GH]Life Sciences [q-bio]/Genetics/Human genetics, COUP-TF2, Cardiac defects, MESH: Female
Abstract: International audience; Emerging evidence from murine studies suggests that mammalian sex determination is the outcome of an imbalance between mutually antagonistic male and female regulatory networks that canalize development down one pathway while actively repressing the other. However, in contrast to testis formation, the gene regulatory pathways governing mammalian ovary development have remained elusive. We performed exome or Sanger sequencing on 79 46,XX SRY-negative individuals with either unexplained virilization or with testicular/ovotesticular disorders/differences of sex development (TDSD/OTDSD). We identified heterozygous frameshift mutations in NR2F2, encoding COUP-TF2, in three children. One carried a c.103_109delGGCGCCC (p.Gly35Argfs∗75) mutation, while two others carried a c.97_103delCCGCCCG (p.Pro33Alafs∗77) mutation. In two of three children the mutation was de novo. All three children presented with congenital heart disease (CHD), one child with congenital diaphragmatic hernia (CDH), and two children with blepharophimosis-ptosis-epicanthus inversus syndrome (BPES). The three children had androgen production, virilization of external genitalia, and biochemical or histological evidence of testicular tissue. We demonstrate a highly significant association between the NR2F2 loss-of-function mutations and this syndromic form of DSD (p = 2.44 × 10-8). We show that COUP-TF2 is highly abundant in a FOXL2-negative stromal cell population of the fetal human ovary. In contrast to the mouse, these data establish COUP-TF2 as a human "pro-ovary" and "anti-testis" sex-determining factor in female gonads. Furthermore, the data presented here provide additional evidence of the emerging importance of nuclear receptors in establishing human ovarian identity and indicate that nuclear receptors may have divergent functions in mouse and human biology.
Published: 2018
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46. Modeling amyotrophic lateral sclerosis in pure human iPSc-derived motor neurons isolated by a novel FACS double selection technique

Author: Dorothée Buttigieg, Stéphane Blanchard, Diana Toli, Delphine Bohl, Georg Haase, Gilbert Baillat, Sarah Bellouze, Thomas Lemonnier, Boris Lamotte d'Incamps, Biothérapies pour les Maladies Neurodégénératives, Institut Pasteur [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM), Institut de Neurosciences de la Timone (INT), Centre National de la Recherche Scientifique (CNRS)-Aix Marseille Université (AMU), Centre de neurophysique, physiologie, pathologie (UMR 8119), Université Paris Descartes - Paris 5 (UPD5)-Centre National de la Recherche Scientifique (CNRS), This work was supported by grants from Agence Nationale pour la Recherche (Modeling ALS with IPS-derived motor neurons, IPSOALS, ANR-08-NEUR-005-02) in the frame of the ERANET-NEURON program. Work in the laboratory of Georg Haase is further financed by CNRS, Aix-Marseille University, Fédération pour la Recherche sur le Cerveau (FRC/Rotary Grant 'Protein aggregates in motoneuron subpopulations: the therapeutic target?'), Association Française contre les Myopathies (AFM grant, 'Motoneuron subpopulations in development and disease'). Work in the laboratory of D. Bohl is supported by INSERM, Institut Pasteur, Agence Nationale de la Recherche (Laboratoire d’Excellence Revive, Investissement d’Avenir, ANR-10-LABX-73), Association Française contre les Myopathies (AFM grant 'Characterisation of pathological defects in motor neurons derived from patients with amyotrophic lateral sclerosis'), Association pour la Recherche sur la Sclerose Laterale Amyotrophique et autres Maladies du Motoneurone (ARS grant 'Human motor neurons genetically reprogrammed from patient fibroblasts: a new approach to analyse ALS physiopathology'), and the Thierry Latran foundation. D. Toli was the recipient of an A.S. Onassis Foundation Scholarship., ANR-09-NEUR-0005,MODDIFSYN(2009), ANR-10-LABX-0073,REVIVE,Stem Cells in Regenerative Biology and Medicine(2010), Institut Pasteur [Paris] (IP)-Institut National de la Santé et de la Recherche Médicale (INSERM), and Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS)
Subjects: Cell, MESH: Flow Cytometry, MESH: Nerve Degeneration, Superoxide Dismutase-1, Genes, Reporter, MESH: Child, MESH: Nerve Tissue Proteins, Amyotrophic lateral sclerosis (ALS), Axon, Amyotrophic lateral sclerosis, Child, Induced pluripotent stem cell, Cells, Cultured, MESH: Amyotrophic Lateral Sclerosis, MESH: Superoxide Dismutase, MESH: Lentivirus, MESH: Superoxide Dismutase-1, Motor Neurons, Myogenesis, Flow Cytometry, medicine.anatomical_structure, MESH: Cell Survival, Neurology, [SDV.NEU]Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC], MESH: Motor Neurons, MESH: Cells, Cultured, Adult, Induced pluripotent stem cells (iPSc), Cell death, MESH: Axons, Cell type, Programmed cell death, MESH: Mutation, Axon degeneration, Cell Survival, Induced Pluripotent Stem Cells, Nerve Tissue Proteins, Receptors, Nerve Growth Factor, MESH: Induced Pluripotent Stem Cells, Biology, lcsh:RC321-571, MESH: Coculture Techniques, p75NTR, MESH: Receptors, Nerve Growth Factor, medicine, Humans, Motor neuron disease, lcsh:Neurosciences. Biological psychiatry. Neuropsychiatry, Fluorescent-activated cell sorting (FACS), MESH: Humans, Superoxide Dismutase, Amyotrophic Lateral Sclerosis, Lentivirus, MESH: Genes, Reporter, MESH: Adult, Motor neuron, medicine.disease, Axons, Coculture Techniques, MESH: Astrocytes, HB9, nervous system, Astrocytes, Mutation, Nerve Degeneration, Superoxide dismutase 1 (SOD1), Neuroscience
Abstract: International audience; Amyotrophic lateral sclerosis (ALS) is a severe and incurable neurodegenerative disease. Human motor neurons generated from induced pluripotent stem cells (iPSc) offer new perspectives for disease modeling and drug testing in ALS. In standard iPSc-derived cultures, however, the two major phenotypic alterations of ALS--degeneration of motor neuron cell bodies and axons--are often obscured by cell body clustering, extensive axon criss-crossing and presence of unwanted cell types. Here, we succeeded in isolating 100% pure and standardized human motor neurons by a novel FACS double selection based on a p75(NTR) surface epitope and an HB9::RFP lentivirus reporter. The p75(NTR)/HB9::RFP motor neurons survive and grow well without forming clusters or entangled axons, are electrically excitable, contain ALS-relevant motor neuron subtypes and form functional connections with co-cultured myotubes. Importantly, they undergo rapid and massive cell death and axon degeneration in response to mutant SOD1 astrocytes. These data demonstrate the potential of FACS-isolated human iPSc-derived motor neurons for improved disease modeling and drug testing in ALS and related motor neuron diseases.
Published: 2015
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47. Alternative splicing regulates the expression of G9A and SUV39H2 methyltransferases, and dramatically changes SUV39H2 functions

Author: Eric Allemand, Roscoe Klinck, Oriane Mauger, Benoit Chabot, Christian Muchardt, Eric Batsché, Régulation Epigénétique, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Université Pierre et Marie Curie - Paris 6 (UPMC), Laboratory of Functional Genomics of the Université de Sherbrooke, Université de Sherbrooke (UdeS), Department of Microbiology and Infectious Diseases [Sherbrooke], Ministry of Research (MENRT, O.M.), Institut National de la Santé et de la Recherche Médicale (INSERM, E.A.), The Centre National de Recherche Scientifique (CNRS, E.B. and C.M.). B.C. is the Canada Research Chair in Functional Genomics. Agence Nationale de la Recherche (ANR-11-BSV8–0013), REVIVE—Investissement d'Avenir (to C.M. and O.M.), Canadian Institute of Health Research (to B.C.). Funding for open access charge: CNRS recurrent funding, and Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS)
Subjects: Genetics, [SDV.GEN]Life Sciences [q-bio]/Genetics, Alternative splicing, Exonic splicing enhancer, Intron, Computational biology, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, Biology, Cell Line, Splicing factor, Exon, Alternative Splicing, Histone methyltransferase, RNA splicing, RNA, Humans, DNA Modification Methylases, Minigene
Abstract: Alternative splicing is the main source of proteome diversity. Here, we have investigated how alternative splicing affects the function of two human histone methyltransferases (HMTase): G9A and SUV39H2. We show that exon 10 in G9A and exon 3 in SUV39H2 are alternatively included in a variety of tissues and cell lines, as well as in a different species. The production of these variants is likely tightly regulated because both constitutive and alternative splicing factors control their splicing profiles. Based on this evidence, we have assessed the link between the inclusion of these exons and the activity of both enzymes. We document that these HMTase genes yield several protein isoforms, which are likely issued from alternative splicing regulation. We demonstrate that inclusion of SUV39H2 exon 3 is a determinant of the stability, the sub-nuclear localization, and the HMTase activity. Genome-wide expression analysis further revealed that alternative inclusion of SUV39H2 exon 3 differentially modulates the expression of target genes. Our data also suggest that a variant of G9A may display a function that is independent of H3K9 methylation. Our work emphasizes that expression and function of genes are not collinear; therefore alternative splicing must be taken into account in any functional study.
Published: 2015
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48. Notchless defines a stage-specific requirement for ribosome biogenesis during lineage progression in adult skeletal myogenesis

Author: Barbara Gayraud-Morel, Marie Le Bouteiller, Michel Cohen-Tannoudji, Shahragim Tajbakhsh, Pierre-Henri Commere, Cellules Souches et Développement / Stem Cells and Development, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Embryon précoce de mammifères et cellules souches, Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Cytométrie (Plate-forme), Institut Pasteur [Paris] (IP), S.T. and M.C.-T. acknowledge support from the Institut Pasteur and the Agence Nationale de la Recherche (Laboratoire d'Excellence Revive, Investissement d'Avenir, ANR-10-LABX-73)., We thank Patricia Flamant and Clémire Cimper for the histology and cryosections and Jean-Yves Tinevez at the Imaging Platform for assistance with cell tracking. We also thank Sophie Novault (Flow Cytometry Platform of the Technology Core at the Center for Translational Science at Institut Pasteur), ANR-10-LABX-0073,REVIVE,Stem Cells in Regenerative Biology and Medicine(2010), Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), and Institut Pasteur [Paris]
Subjects: 0301 basic medicine, Mouse, Satellite Cells, Skeletal Muscle, Ribosome biogenesis, Quiescence, Cell fate determination, Biology, Muscle Development, Myoblasts, 03 medical and health sciences, Notchless, Cyclin E, Animals, Regeneration, Cell Lineage, Progenitor cell, Muscle, Skeletal, [SDV.BDD]Life Sciences [q-bio]/Development Biology, Molecular Biology, Transcription factor, Cells, Cultured, Mice, Knockout, Organelle Biogenesis, Myogenesis, Cell Cycle, Gene Expression Regulation, Developmental, Membrane Proteins, Translation (biology), Cell Differentiation, Cell biology, Haematopoiesis, 030104 developmental biology, Mutation, Stem cell, Skeletal muscle stem cells, Ribosomes, Developmental Biology
Abstract: Cell fate decisions occur through the action of multiple factors, including signalling molecules and transcription factors. Recently, the regulation of translation has emerged as an important step for modulating cellular function and fate, as exemplified by ribosomes that play distinct roles in regulating cell behaviour. Notchless (Nle) is a conserved nuclear protein that is involved in a crucial step in ribosome biogenesis, and is required for the maintenance of adult haematopoietic and intestinal stem/progenitor cells. Here, we show that activated skeletal muscle satellite cells in conditional Nle mutant mice are arrested in proliferation; however, deletion of Nle in myofibres does not impair myogenesis. Furthermore, conditional deletion of Nle in satellite cells during homeostasis did not impact on their fate for up to 3 months. In contrast, loss of Nle function in primary myogenic cells blocked proliferation because of major defects in ribosome formation. Taken together, we show that muscle stem cells undergo a stage-specific regulation of ribosome biogenesis, thereby underscoring the importance of differential modulation of mRNA translation for controlling cell fate decisions.
Published: 2017
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49. Evaluation of Injury-induced Senescence and In Vivo Reprogramming in the Skeletal Muscle

Author: Cazin, Coralie, Chiche, Aurelie, Li, Han, Plasticité cellulaire et Modélisation des Maladies / Cellular Plasticity and Disease Modelling, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Work in the laboratory of H.L. was funded by Institut Pasteur, Centre National pour la Recherche Scientific, and the Agence Nationale de la Recherche (Laboratoire d'Excellence Revive, Investissement d'Avenir, ANR-10-LABX- 73), the Agence Nationale de la Recherche (ANR-16-CE13-0017-01) and Fondation ARC (PJA 20161205028). C.C. and A.C. are funded by the Ph.D. and postdoctoral fellowships from the Revive Consortium., ANR-10-LABX-0073,REVIVE,Stem Cells in Regenerative Biology and Medicine(2010), and ANR-16-CE13-0017,iCELLPLASTICITY,Plasticité cellulaire dans le vieillissement(2016)
Subjects: Pluripotent Stem Cells, Disease Models, Animal, Mice, Cell Plasticity, Animals, Cellular Reprogramming Techniques, Mice, Transgenic, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, Muscle, Skeletal, beta-Galactosidase, Cellular Senescence, Developmental Biology, Cell Proliferation
Abstract: International audience; Cellular senescence is a stress response that is characterized by a stable cellular growth arrest, which is important for many physiological and pathological processes, such as cancer and ageing. Recently, senescence has also been implicated in tissue repair and regeneration. Therefore, it has become increasingly critical to identify senescent cells in vivo. Senescence-associated β-galactosidase (SA-β-Gal) assay is the most widely used assay to detect senescent cells both in culture and in vivo. This assay is based on the increased lysosomal contents in the senescent cells, which allows the histochemical detection of lysosomal β-galactosidase activity at suboptimum pH (6 or 5.5). In comparison with other assays, such as flow cytometry, this allows the identification of senescent cells in their resident environment, which offers valuable information such as the location relating to the tissue architecture, the morphology, and the possibility of coupling with other markers via immunohistochemistry (IHC). The major limitation of the SA-β-Gal assay is the requirement of fresh or frozen samples. Here, we present a detailed protocol to understand how cellular senescence promotes cellular plasticity and tissue regeneration in vivo. We use SA-β-Gal to detect senescent cells in the skeletal muscle upon injury, which is a well-established system to study tissue regeneration. Moreover, we use IHC to detect Nanog, a marker of pluripotent stem cells, in a transgenic mouse model. This protocol enables us to examine and quantify cellular senescence in the context of induced cellular plasticity and in vivo reprogramming.
Published: 2017
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50. Mitotic bookmarking in development and stem cells

Author: Pablo Navarro, Inma Gonzalez, Nicola Festuccia, Nick D.L. Owens, Epigénétique des Cellules Souches - Epigenetics of Stem Cells, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Research in the P.N. laboratory is supported by the Institut Pasteur, the Centre National de la Recherche Scientifique, the Agence Nationale de la Recherche (ANR) Laboratoire d'Excellence Revive (Investissement d'Avenir, ANR-10-LABX-73), the ANR DS0405-2016 program (MitMAT, ANR-16-CE12-0004), the Fondation ARC pour la Recherche sur le Cancer (PJA20161204705), and the Fondation Schlumberger pour l'Education et la Recherche (cercle FSER-2016). N.F. was funded by an European Molecular Biology Organization Long Term Fellowship (ALTF 876-2013) and a Marie-Curie Actions Intra-European Fellowship (EFIMB – 626705). I.G. and N.O. are supported by Revive., We acknowledge Shahragim Tajbakhsh, François Schweisguth and Alfonso Martinez-Arias for stimulating discussions., ANR-16-CE12-0004,MitMAT,Mémoire mitotique de l'activité transcriptionnelle dans les cellules ES(2016), ANR-10-LABX-0073,REVIVE,Stem Cells in Regenerative Biology and Medicine(2010), European Project: 626705,EC:FP7:PEOPLE,FP7-PEOPLE-2013-IEF,EFIMB(2015), and Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS)
Subjects: 0301 basic medicine, Cell division, Mitosis, Stem cells, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, Biology, Cell fate determination, Mitotic inheritance, Epigenesis, Genetic, 03 medical and health sciences, Mice, Transcription factors, Animals, Humans, Cell Lineage, Epigenetics, Molecular Biology, Transcription factor, Cell Nucleus, [SDV.GEN]Life Sciences [q-bio]/Genetics, Bookmarking, Cell Cycle, Gene Expression Regulation, Developmental, Cell Differentiation, Chromatin, Cell biology, 030104 developmental biology, Gene Expression Regulation, Stem cell, Developmental Biology, Mitotic bookmarking
Abstract: International audience; The changes imposed on the nucleus, chromatin and its regulators during mitosis lead to the dismantlement of most gene regulatory processes. However, an increasing number of transcriptional regulators are being identified as capable of binding their genomic targets during mitosis. These so-called 'mitotic bookmarking factors' encompass transcription factors and chromatin modifiers that are believed to convey gene regulatory information from mother to daughter cells. In this Primer, we review mitotic bookmarking processes in development and stem cells and discuss the interest and potential importance of this concept with regard to epigenetic regulation and cell fate transitions involving cellular proliferation.
Published: 2017
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