Pluripotency is established through genome-wide reprogramming during mammalian pre-implantation development, resulting in the formation of the naive epiblast. Reprogramming involves both the resetting of epigenetic marks and the activation of pluripotent-cell-specific genes such as Nanog and Oct4 (also known as Pou5f1)1,2,3,4. The tight regulation of these genes is crucial for reprogramming, but the mechanisms that regulate their expression in vivo have not been uncovered. Here we show that Nanog—but not Oct4—is monoallelically expressed in early pre-implantation embryos. Nanog then undergoes a progressive switch to biallelic expression during the transition towards ground-state pluripotency in the naive epiblast of the late blastocyst. Embryonic stem (ES) cells grown in leukaemia inhibitory factor (LIF) and serum express Nanog mainly monoallelically and show asynchronous replication of the Nanog locus, a feature of monoallelically expressed genes5, but ES cells activate both alleles when cultured under 2i conditions, which mimic the pluripotent ground state in vitro. Live-cell imaging with reporter ES cells confirmed the allelic expression of Nanog and revealed allelic switching. The allelic expression of Nanog is regulated through the fibroblast growth factor–extracellular signal-regulated kinase signalling pathway, and it is accompanied by chromatin changes at the proximal promoter but occurs independently of DNA methylation. Nanog-heterozygous blastocysts have fewer inner-cell-mass derivatives and delayed primitive endoderm formation, indicating a role for the biallelic expression of Nanog in the timely maturation of the inner cell mass into a fully reprogrammed pluripotent epiblast. We suggest that the tight regulation of Nanog dose at the chromosome level is necessary for the acquisition of ground-state pluripotency during development. Our data highlight an unexpected role for allelic expression in controlling the dose of pluripotency factors in vivo, adding an extra level to the regulation of reprogramming.
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We thank M. Okano for providing the triple DNA methyltransferase knockout cells, P. Avner and P. Clerc for hybrid ES cells, H. Schöler for the Oct4 probe (GOF6.1), J. Jaubert for M. musculus castaneus mice, R. Kemler and S. Rudloff for the NanogGt1 mice, G. Charvin for advice on time-lapse analysis. We also thank M. Koch, A. Dierich, M.-C. Birling, E. Heard and I. Okamoto for advice, and A. Burton, E. Heard and O. Pourquie for critical reading of the manuscript. M.-E.T.-P. acknowledges funding from AVENIR/INSERM, ANR-09-Blanc-0114, Epigenesys NoE and FRM Alsace. Y.M. is a recipient of an EMBO long-term fellowship (ALTF864-2008, 2009) and a JSPS postdoctoral fellowship (2010–2011).
This movie shows serial confocal sections (0.3 μm step) of 4-cell stage embryos. Nascent transcripts of Nanog (yellow) and Oct4 (red) were detected by RNA-FISH. DNA was stained with DAPI in blue.
This movie shows serial confocal sections (0.3 μm step) of ICM cells of late blastocyst. Nascent transcript of Nanog (yellow) was visualized by RNA-FISH. DNA was stained with DAPI in blue.
This movie shows serial confocal sections (0.3 μm step) of ES cells cultured with serum and LIF. Nascent transcripts of Nanog (yellow) and Oct4 (red) were visualized by RNA-FISH. DNA was stained with DAPI in blue.
This movie shows serial confocal sections (0.3 μm step) of ES cells cultured with 2i. Nascent transcripts of Nanog (yellow) and Oct4 (red) were visualized by RNA-FISH. DNA was stained with DAPI in blue.
This movie shows time-lapse imaging of NGR ES cells cultured in medium containing LIF. Images are acquired along 7 Z-planes spanning 17.5 μm every 20 min for 38 hrs under a spinning disk confocal. Series of maximum intensity projection at each time point is converted to the movie.