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Snail and the microRNA-200 Family Act in Opposition to Regulate Epithelial-to-Mesenchymal Transition and Germ Layer Fate Restriction in Differentiating ESCs

From the May 2011 Issue of Stem Cells
Paper Commentary by Stuart P. Atkinson

Recent high impact studies have shown that the reprogramming of somatic cells towards pluripotency requires a mesenchymal-to-epithelial transition (MET) (Li et al and Samavarchi-Tehrani et al) which is in part mediated by the function of microRNA (miRNA) species (Liao et al andSubramanyam et al). However, the role of the epithelial-to-mesenchymal transition (EMT) during the differentiation of embryonic stem cells (ESCs) and its potential role in fate commitment have not yet been studied in great detail. Now, in a study published in the May edition ofStem Cells, researchers from the lab of Kenneth M. Murphy at the Washington University School of Medicine, St. Louis, Missouri, USA have begun to unravel the important pathways required for the early differentiation and fate commitment in ESC, and indicate an important role for the tight regulation of EMT controlled by miRNA (Gill et al).

Previous studies by the group (Lindsley et al) had indicated that Mesp1 (mesoderm posterior 1 homolog, expressed in the mesodermal cell lineage during early gastrulation) induced an EMT in differentiating ESC through the induction of Snail (Snai1), which was associated with the downregulation of E-Cadherin (Cdh1) expression. This system was used to examine EMT during ESC differentiation in more detail and showed that E-Cadherin was increasingly lost after 3 days of differentiation, corresponding to the induction of Snail. Inhibition of Snail led to the maintenance of E-Cadherin and the failure of differentiating cells to undergo an EMT, while the over-expression of Snail at day 2 of differentiation led to dramatic changes in morphology, with the cells acquiring spindle-like morphologies and adhered as single cells. Interestingly, this was not observed in day 0 ESC cultures, indicating that other factors may be required for Snails effect. Snail also leads to the upregulation of Fibronectin (Fn1) and induces N-Cadherin (Cdh2) expression at the expense of E-Cadherin. Further, Snail was shown to promote the loss of pluripotency markers (SSEA1, Oct4 and Sox2, but not Nanog) and enhance mesodermal differentiation, revealed by the monitoring of Pdgfra and Flk1 (Kdr) levels. These data combined suggest that Snail promotes features associated with an EMT.

Further more detailed microarray analysis showed that Snail expression led to the global repression of epithelial and ectodermal markers (such as Otx2, Zic5 and Cldn6) and the induction of mesenchymal and mesodermal markers (such as Mmp2, Ncam1, Isl1 and Gata2), indicative of an EMT, as compared to ESC under WNT signalling inhibition, which inhibits endogenous-promoted EMT and mesoderm differentiation. However, these findings were contradictory to the known roles of Snail as a transcriptional repressor, as a strong induction of target genes is observed upon its expression and so miRNAs repressed upon Snail over-expression were sought for as an answer to this contradiction. This led to the discovery of a significant reduction in the expression of members of the miR-200 family, which are known to block EMT, alongside a reduction of many miRNAs associated with the maintenance of pluripotency, but also a significant increase in miR-302 which is associated with the mesodermal lineage. When miR-200 family members were overexpressed in ESC, E-Cadherin expression remained high upon differentiation, whilst mesodermal markers failed to be induced and SSEA1 expression was maintained in around two thirds of differentiating ESCs. Further analysis showed that ESCs overexpressing these miRNAs were similar in their gene expression profiles and morphology to EpiSCs (Epiblast stem cells) and ESD-EpiSCs (ESC-derived epiblast stem cells), which are known to maintain primitive ectoderm markers, and suggests that miR-200 family member expression causes a failure of these cells to progress past the EpiSC stage of differentiation.

EpiSCs and ESD-EpiSCs are maintained through the actions of Activin, which also promotes neuroectodermal differentiation in ESCs, and so it was hypothesised that Activin inhibition in ESC could lead to a downregulation of miR-200 family members and an EMT. Inhibition of Activin did indeed lead to a decrease in E-cadherin and SSEA1 levels indicative of EMT, a reduction in miR-200 family members, but led to the decrease of mesodermal markers. This difference in germ layer differentiation suggests that perhaps Snail expression and Activin signalling act in cooperation to regulate epiblast differentiation and EMT progression and indeed it was found that miR-200 expression in ESC allowed the maintenance of E-Cadherin/SSEA1 cells, but upon Activin inhibition these ESCs underwent differentiation and EMT.

Taken together, this data suggests that Snail, Activin and miR-200 act together to regulate epiblast differentiation, EMT progression and cell fate commitment. This is an important integrative study further highlighting the important role for miRNAs in the differentiation process and in the delineation of EMT in differentiation.



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Mesp1 coordinately regulates cardiovascular fate restriction and epithelial-mesenchymal transition in differentiating ESCs.
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Snail and the microRNA-200 Family Act in Opposition to Regulate Epithelial-to-Mesenchymal Transition and Germ Layer Fate Restriction in Differentiating ESCs.
Gill JG, Langer EM, Lindsley RC, Cai M, Murphy TL, Kyba M, Murphy KM.
Stem Cells. 2011 May;29(5):764-76