Generation of BAC Transgenic Tadpoles Enabling Live Imaging of Motoneurons by Using the Urotensin II-Related Peptide (ust2b) Gene as a Driver

Autor: Frédéric Relaix, Didier Le Ray, François M. Lambert, Hervé Tostivint, Frédéric Auradé, Marion Bougerol, Nicolas Pollet, Muriel Thoby-Brisson, Denis Combes
Přispěvatelé: Evolution des régulations endocriniennes (ERE), Muséum national d'Histoire naturelle (MNHN)-Centre National de la Recherche Scientifique (CNRS), Centre de recherche en myologie, Université Pierre et Marie Curie - Paris 6 (UPMC)-Association française contre les myopathies (AFM-Téléthon)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), CHU Pitié-Salpêtrière [AP-HP], Sorbonne Université (SU)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP), Institut de Neurosciences cognitives et intégratives d'Aquitaine (INCIA), Université Bordeaux Segalen - Bordeaux 2-Université Sciences et Technologies - Bordeaux 1-SFR Bordeaux Neurosciences-Centre National de la Recherche Scientifique (CNRS), Institute of Systems and Synthetic Biology, Genopole, Universite d’Evry Val d’Essonne, Genavenir 3, Centre National de la Recherche Scientifique (CNRS), Université Bordeaux Segalen - Bordeaux 2-Université Sciences et Technologies - Bordeaux 1 (UB)-SFR Bordeaux Neurosciences-Centre National de la Recherche Scientifique (CNRS), Institut de biologie systémique et synthétique (ISSB), Université d'Évry-Val-d'Essonne (UEVE)-Centre National de la Recherche Scientifique (CNRS), Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU), HAL UPMC, Gestionnaire, Thérapie des maladies du muscle strié, Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Pierre et Marie Curie - Paris 6 (UPMC), Etablissement Français du Sang [Créteil], Epigenomics Project, Genopole, Université d'Évry-Val-d'Essonne (UEVE), Centre de recherche en Myologie – U974 SU-INSERM, Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU), Développement et évolution (DE), Université Paris-Sud - Paris 11 (UP11)-Centre National de la Recherche Scientifique (CNRS), Centre de Recherche en Myologie, LE RAY, Didier
Jazyk: angličtina
Rok vydání: 2015
Předmět:
Chromosomes
Artificial
Bacterial
Embryo
Nonmammalian
[SDV.NEU.NB]Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC]/Neurobiology
[SDV]Life Sciences [q-bio]
Xenopus
lcsh:Medicine
MESH: Microscopy
Fluorescence
[SDV.GEN] Life Sciences [q-bio]/Genetics
Green fluorescent protein
MESH: Spinal Cord
Animals
Genetically Modified
Genes
Reporter
Gene expression
MESH: Animals
MESH: Xenopus
MESH: Electrophysiological Phenomena
lcsh:Science
ComputingMilieux_MISCELLANEOUS
In Situ Hybridization
Motor Neurons
Multidisciplinary
MESH: Peptides
medicine.anatomical_structure
Spinal Cord
[SDV.NEU]Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC]
Brainstem
MESH: Motor Neurons
Research Article
MESH: Chromosomes
Artificial
Bacterial
Urotensins
Central nervous system
Hindbrain
Biology
MESH: Animals
Genetically Modified
MESH: In Situ Hybridization
medicine
Animals
[SDV.NEU] Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC]
Reporter gene
[SDV.GEN]Life Sciences [q-bio]/Genetics
MESH: Urotensins
lcsh:R
MESH: Genes
Reporter
fungi
[SDV.NEU.NB] Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC]/Neurobiology
MESH: Embryo
Nonmammalian
biology.organism_classification
Spinal cord
Molecular biology
Electrophysiological Phenomena
Microscopy
Fluorescence
lcsh:Q
Peptides
Zdroj: PLoS ONE
PLoS ONE, Public Library of Science, 2015, 10 (2), pp.e0117370. ⟨10.1371/journal.pone.0117370⟩
PLoS ONE, 2015, 10 (2), pp.e0117370. ⟨10.1371/journal.pone.0117370⟩
PLoS ONE, Public Library of Science, 2015, ⟨10.1371/journal.pone.0117370⟩
PLoS ONE, Vol 10, Iss 2, p e0117370 (2015)
PLoS ONE, 2015, ⟨10.1371/journal.pone.0117370⟩
ISSN: 1932-6203
DOI: 10.1371/journal.pone.0117370⟩
Popis: International audience; Xenopus is an excellent tetrapod model for studying normal and pathological motoneuron ontogeny due to its developmental morpho-physiological advantages. In mammals, the uro-tensin II-related peptide (UTS2B) gene is primarily expressed in motoneurons of the brain-stem and the spinal cord. Here, we show that this expression pattern was conserved in Xenopus and established during the early embryonic development, starting at the early tail-bud stage. In late tadpole stage, uts2b mRNA was detected both in the hindbrain and in the spinal cord. Spinal uts2b + cells were identified as axial motoneurons. In adult, however, the uts2b expression was only detected in the hindbrain. We assessed the ability of the uts2b promoter to drive the expression of a fluorescent reporter in motoneurons by recombineer-ing a green fluorescent protein (GFP) into a bacterial artificial chromosome (BAC) clone containing the entire X. tropicalis uts2b locus. After injection of this construction in one-cell stage embryos, a transient GFP expression was observed in the spinal cord of about a quarter of the resulting animals from the early tailbud stage and up to juveniles. The GFP expression pattern was globally consistent with that of the endogenous uts2b in the spinal cord but no fluorescence was observed in the brainstem. A combination of histological and electrophysiological approaches was employed to further characterize the GFP + cells in the larvae. More than 98% of the GFP + cells expressed choline acetyltransferase, while their projections were co-localized with α-bungarotoxin labeling. When tail myotomes were injected with rhodamine dextran amine crystals, numerous double-stained GFP + cells were observed. In addition, intracellular electrophysiological recordings of GFP + neurons revealed locomotion-related rhythmic discharge patterns during fictive swimming. Taken together our results provide evidence that uts2b is an appropriate driver to express reporter genes in larval motoneurons of the Xenopus spinal cord.
Databáze: OpenAIRE
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