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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.

Author information

1
Institute of Anatomy and Cell Biology, University of Würzburg, Würzburg, Germany.
2
Institute of Reconstructive Neurobiology, University of Bonn Medical School, Bonn, Germany.
3
LIMES Institute, University of Bonn, Bonn, Germany.
4
Institute of Pharmacology and Toxicology, Bonn, Germany.
5
Department of Developmental Pathology, University of Bonn Medical School, Bonn, Germany.
6
Department of Genomics, Stem Cell Biology & Regenerative Medicine, Institute of Molecular Biology, Leopold-Franzens-University Innsbruck (LFUI), Innsbruck, Austria.

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.

KEYWORDS:

Connexin; Embryoid body; Embryonic stem cells; Gap junction; NFAT; Primitive endoderm

PMID:
27870307
DOI:
10.1002/stem.2545
[Indexed for MEDLINE]
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