title: Abrogation of Gap Junctional Communication in ES Cells Results in a Disruption of Primitive Endoderm Formation in Embryoid Bodies.
abstract: Gap junctional intercellular communication (GJIC) has been suggested to be involved in early embryonic development but the actual functional role remained elusive. Connexin (Cx) 43 and Cx45 are co-expressed in embryonic stem (ES) cells, form gap junctions and are considered to exhibit adhesive function and/or to contribute to the establishment of defined communication compartments. Here, we describe the generation of Cx43/Cx45-double deficient mouse ES cells to achieve almost complete breakdown of GJIC. Cre-loxP induced deletion of both, Cx43 and Cx45, results in a block of differentiation in embryoid bodies (EBs) without affecting pluripotency marker expression and proliferation in ES cells. We demonstrate that GJIC-incompetent ES cells fail to form primitive endoderm in EB cultures, representing the inductive key step of further differentiation events. Lentiviral overexpression of either Cx43 or Cx45 in Cx43/45 mutants rescued the observed phenotype, confirming the specificity and indicating a partially redundant function of both connexins. Upon differentiation GJIC-incompetent ES cells exhibit a strikingly altered subcellular localization pattern of the transcription factor NFATc3. Control EBs exhibit significantly more activated NFATc3 in cellular nuclei than mutant EBs suggesting that Cx-mediated communication is needed for synchronized NFAT activation to induce orchestrated primitive endoderm formation. Moreover, pharmacological inhibition of NFATc3 activation by Cyclosporin A, a well-described inhibitor of calcineurin, phenocopies the loss of GJIC in control cells. Stem Cells 2017;35:859-871.
authors.affiliation: Institute of Anatomy and Cell Biology, University of Würzburg, Würzburg, Germany.|Institute of Reconstructive Neurobiology, University of Bonn Medical School, Bonn, Germany.
authors.forename: Philipp
authors.initials: P
authors.lastname: Wörsdörfer
authors.affiliation: LIMES Institute, University of Bonn, Bonn, Germany.
authors.forename: Felicitas
authors.initials: F
authors.lastname: Bosen
authors.affiliation: Institute of Anatomy and Cell Biology, University of Würzburg, Würzburg, Germany.|Institute of Reconstructive Neurobiology, University of Bonn Medical School, Bonn, Germany.
authors.forename: Martina
authors.initials: M
authors.lastname: Gebhardt
authors.affiliation: Institute of Reconstructive Neurobiology, University of Bonn Medical School, Bonn, Germany.
authors.forename: Nicole
authors.initials: N
authors.lastname: Russ
authors.affiliation: Institute of Pharmacology and Toxicology, Bonn, Germany.
authors.forename: Katrin
authors.initials: K
authors.lastname: Zimmermann
authors.affiliation: Institute of Anatomy and Cell Biology, University of Würzburg, Würzburg, Germany.
authors.forename: David
authors.initials: D
authors.lastname: Komla Kessie
authors.affiliation: Institute of Reconstructive Neurobiology, University of Bonn Medical School, Bonn, Germany.
authors.forename: Thileepan
authors.initials: T
authors.lastname: Sekaran
authors.affiliation: Department of Developmental Pathology, University of Bonn Medical School, Bonn, Germany.
authors.forename: Angela
authors.initials: A
authors.lastname: Egert
authors.affiliation: Institute of Anatomy and Cell Biology, University of Würzburg, Würzburg, Germany.
authors.forename: Süleyman
authors.initials: S
authors.lastname: Ergün
authors.affiliation: Department of Developmental Pathology, University of Bonn Medical School, Bonn, Germany.
authors.forename: Hubert
authors.identifier: 0000-0001-8272-0076
authors.initials: H
authors.lastname: Schorle
authors.affiliation: Institute of Pharmacology and Toxicology, Bonn, Germany.
authors.forename: Alexander
authors.initials: A
authors.lastname: Pfeifer
authors.affiliation: Institute of Anatomy and Cell Biology, University of Würzburg, Würzburg, Germany.|Institute of Reconstructive Neurobiology, University of Bonn Medical School, Bonn, Germany.|Department of Genomics, Stem Cell Biology & Regenerative Medicine, Institute of Molecular Biology, Leopold-Franzens-University Innsbruck (LFUI), Innsbruck, Austria.
authors.forename: Frank
authors.initials: F
authors.lastname: Edenhofer
authors.affiliation: LIMES Institute, University of Bonn, Bonn, Germany.
authors.forename: Klaus
authors.initials: K
authors.lastname: Willecke
mesh_terms: D000818:Animals; D017209:Apoptosis; D015415:Biomarkers / Q000378:metabolism; D019703:Calcineurin / Q000378:metabolism; D002450:Cell Communication; D002454:Cell Differentiation; D049109:Cell Proliferation; D018031:Connexin 43 / Q000378:metabolism; D017630:Connexins / Q000378:metabolism; D058732:Embryoid Bodies / Q000166:cytology* / Q000378:metabolism*; D004707:Endoderm / Q000166:cytology / Q000196:embryology* / Q000378:metabolism*; D017629:Gap Junctions / Q000378:metabolism*; D054262:Gastrulation; D016086:Lentivirus / Q000378:metabolism; D051379:Mice; D016296:Mutagenesis / Q000235:genetics; D050778:NFATC Transcription Factors / Q000378:metabolism; D015398:Signal Transduction
chemical_list: D015415:Biomarkers; D018031:Connexin 43; D017630:Connexins; D050778:NFATC Transcription Factors; C082920:connexin 45; D019703:Calcineurin
keywords: Connexin; Embryoid body; Embryonic stem cells; Gap junction; NFAT; Primitive endoderm