Stem Cells. 2017 Apr;35(4):859-871. doi: 10.1002/stem.2545. Epub 2016 Dec 20.
Abrogation of Gap Junctional Communication in ES Cells Results in a Disruption of Primitive Endoderm Formation in Embryoid Bodies.
Wörsdörfer P1,2, Bosen F3, Gebhardt M1,2, Russ N2, Zimmermann K4, Komla Kessie D1, Sekaran T2, Egert A5, Ergün S1, Schorle H5, Pfeifer A4, Edenhofer F1,2,6, Willecke K3.
Institute of Anatomy and Cell Biology, University of Würzburg, Würzburg, Germany.
Institute of Reconstructive Neurobiology, University of Bonn Medical School, Bonn, Germany.
Institute of Pharmacology and Toxicology, Bonn, Germany.
Department of Developmental Pathology, 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.
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.
© 2016 The Authors STEM CELLS published by Wiley Periodicals, Inc. on behalf of AlphaMed Press.