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The differentiation of embryonic stem cells is associated with extensive changes in gene expression. It is not yet clear whether these changes are the result of binary switch‐like mechanisms or that of continuous and progressive variation. Here, I have used immunostaining and single molecule RNA fluorescence in situ hybridization (FISH) to assess changes in the expression of the well‐known pluripotency‐associated gene  Pou5f1  (also known as  Oct4 ) and early differentiation markers  Sox1  and  T‐brachyury  in single cells during the early steps of differentiation of mouse embryonic stem cells. I found extensive overlap between the expression of  Pou5f1 / Sox1  or  Pou5f1 / T‐brachyury  shortly after the initiation of differentiation towards either the neuronal or the mesendodermal lineage, but no evidence of correlation between their respective expression levels. Quantitative analysis of transcriptional output at the sites of nascent transcription revealed that  Pou5f1  and  Sox1  were transcribed in pulses and that embryonic stem cell differentiation was accompanied by changes in pulsing frequencies. The progressive induction of  Sox1  was further associated with an increase in the average size of individual transcriptional bursts. Surprisingly, single cells that actively and simultaneously transcribe both the pluripotency‐ and the lineage‐associated genes could easily be found in the differentiating population. The results presented here show for the first time that lineage priming can occur in cells that are actively transcribing a pluripotent marker. Furthermore, they suggest that this process is associated with changes in transcriptional dynamics. S tem  C ells   2015;33:2949–2960

stem cells

Single Cell Analysis Reveals Concomitant Transcription of Pluripotent and Lineage Markers During the Early Steps of Differentiation of Embryonic Stem Cells