Abrogation Of Gap Junctional Communication In ES Cells Results In A Disruption Of Primitive Endoderm Formation In Embryoid Bodies

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 without affecting pluripotency marker expression and proliferation in ES cells. We demonstrate that GJIC-incompetent ES cells fail to form primitive endoderm in embryoid body cultures, representing the inductive key step of further differentiation events in this system. 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.

间隙连接细胞间通讯（Gap junctional intercellular communication, GJIC）被认为参与早期胚胎发育，但其实际功能角色仍未明确。连接蛋白（Connexin, Cx）43与Cx45在胚胎干细胞（embryonic stem, ES）中共表达，可形成缝隙连接，被认为具有黏附功能，或有助于构建特定的通讯区室。本研究构建了Cx43/Cx45双缺陷型小鼠胚胎干细胞，实现了GJIC的近乎完全阻断。通过Cre-loxP重组系统诱导同时缺失Cx43与Cx45，会导致拟胚体（embryoid bodies）的分化受阻，但不影响ES细胞的多能性标志物表达与增殖能力。研究证实，缺乏GJIC的ES细胞无法在拟胚体培养中形成原始内胚层，而这正是该系统中后续分化事件的关键诱导步骤。在Cx43/Cx45缺陷细胞中，通过慢病毒过表达Cx43或Cx45均可挽救该观测到的表型，证实了该效应的特异性，并提示两种连接蛋白存在部分冗余的功能。在分化过程中，缺乏GJIC的ES细胞的转录因子NFATc3的亚细胞定位模式发生显著改变。对照拟胚体的细胞核内活化型NFATc3水平显著高于缺陷型拟胚体，提示Cx介导的细胞间通讯对于同步化NFAT激活以协同诱导原始内胚层形成是必需的。此外，通过钙调磷酸酶的经典抑制剂环孢菌素A（Cyclosporin A）药理学抑制NFATc3激活，可在对照细胞中模拟GJIC缺失的表型。