Self-renewal and pluripotency are hallmarks of embryonic stem cells (ESCs). However, the signaling pathways that trigger their transition from selfrenewal to differentiation remain elusive. Here, we report that calcineurin-NFAT signaling is both necessary and sufficient to switch ESCs from an undifferentiated state to lineage-specific cells and that the inhibition of this pathway can maintain long-term ESC self-renewal independent of leukemia inhibitory factor. Mechanistically, this pathway converges with the Erk1/2 pathway to regulate Src expression and promote the epithelial-mesenchymal transition (EMT), a process required for lineage specification in response to differentiation stimuli. Furthermore, calcineurin-NFAT signaling is activated when the earliest differentiation event occurs in mouse embryos, and its inhibition disrupts extraembryonic lineage development. Collectively, our results demonstrate that the NFAT and Erk1/2 cascades form a signaling switch for early lineage segregation in mouse ESCs and provide significant insights into the regulation of the balance between ESC selfrenewal and early lineage specification.

Totipotency emerges in early embryogenesis, but its molecular underpinnings remain poorly characterized. In the present study, we employed DNA fiber analysis to investigate how pluripotent stem cells are reprogrammed into totipotent-like 2-cell-like cells (2CLCs). We show that totipotent cells of the early mouse embryo have slow DNA replication fork speed and that 2CLCs recapitulate this feature, suggesting that fork speed underlies the transition to a totipotent-like state. 2CLCs emerge concomitant with DNA replication and display changes in replication timing (RT), particularly during the early S-phase. RT changes occur prior to 2CLC emergence, suggesting that RT may predispose to gene expression changes and consequent reprogramming of cell fate. Slowing down replication fork speed experimentally induces 2CLCs. In vivo, slowing fork speed improves the reprogramming efficiency of somatic cell nuclear transfer. Our data suggest that fork speed regulates cellular plasticity and that remodeling of replication features leads to changes in cell fate and reprogramming.

Various types of stem cell lines have been derived from preimplantation or postimplantation mouse embryos: embryonic stem cell lines, epiblast stem cell lines, and trophoblast stem cell lines. It is not known if extraembryonic endoderm stem (XEN) cell lines can be derived from postimplantation mouse embryos. Here, we report the derivation of 77 XEN cell lines from 85 postimplantation embryos at embryonic day E5.5 or E6.5, in parallel to the derivation of 41 XEN lines from 69 preimplantation embryos at the blastocyst stage. We attain a success rate of 100% of XEN cell line derivation with our E5.5 whole-embryo and E6.5 disaggregated-embryo methods. Immunofluorescence and NanoString gene expression analyses indicate that the XEN cell lines that we derived from postimplantation embryos (post-XEN) are very similar to the XEN cell lines that we derived from preimplantation embryos (pre-XEN) using a conventional method. After injection into blastocysts, post-XEN cells contribute to extraembryonic endoderm in chimeras at E6.5 and E7.5.

SummaryGene targeting in embryonic stem (ES) cells remains best practice for introducing complex mutations into the mouse germline. One aspect in this multistep process that has not been streamlined with regard to the logistics and ethics of mouse breeding is the efficiency of germline transmission: the transmission of the ES cell‐derived genome through the germline of chimeras to their offspring. A method whereby male chimeras transmit exclusively the genome of the injected ES cells to their offspring has been developed. The new technology, referred to as goGermline, entails injecting ES cells into blastocysts produced by superovulated homozygous Tsc22d3 floxed females mated with homozygous ROSA26‐Cre males. This cross produces males that are sterile due to a complete cell‐autonomous defect in spermatogenesis. The resulting male chimeras can be sterile but when fertile, they transmit the ES cell‐derived genome to 100% of their offspring. The method was validated extensively and in two laboratories for gene‐targeted ES clones that were derived from the commonly used parental ES cell lines Bruce4, E14, and JM8A3. The complete elimination of the collateral birth of undesired, non‐ES cell‐derived offspring in goGermline technology fulfills the reduction imperative of the 3R principle of humane experimental technique with animals. genesis 54:326–333, 2016. © 2016 The Authors. Genesis Published by Wiley Periodicals, Inc.

The low success rate of somatic nuclear transfer (NT) is hypothesized to be mainly due to functional defects in the trophoblast cell lineage rather than the inner cell mass (ICM); this hypothesis, however, remains to be tested directly. Here we separated the ICMs from cloned blastocysts and aggregated the cloned ICM with two fertilization-derived (FD) tetraploid (4N) embryos. We found that the full-term development of cloned ICMs was dramatically improved after the trophoblast cells in the cloned blastocysts were replaced by cells from tetraploid embryos, thus providing direct evidence that defects in trophoblast cell lineage underlie the low success rate of somatic NT.

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SummaryExtraembryonic endoderm stem (XEN) cell lines can be derived and maintained in vitro and reflect the primitive endoderm lineage. Platelet-derived growth factor receptor alpha (PDGFRA) is thought to be essential for the derivation and maintenance of mouse XEN cell lines. Here, we have re-evaluated this requirement for PDGFRA. We derived multiple PDGFRA-deficient XEN cell lines from postimplantation and preimplantation embryos of a PDGFRA-GFP knockout strain. We also converted PDGFRA-deficient embryonic stem cell lines into XEN cell lines chemically by transient culturing with retinoic acid and Activin A. We confirmed the XEN profile of our 12 PDGFRA-deficient cell lines by immunofluorescence with various markers, by NanoString gene expression analyses, and by their contribution to the extraembryonic endoderm of chimeric embryos produced by injecting these cells into blastocysts. Thus, PDGFRA is not essential for the derivation and maintenance of XEN cell lines.

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