Abstract
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.
Highlights
Cellular plasticity is an essential requirement for multicellular organisms
Totipotent-like cells resembling 2-cell-stage mouse embryos arise spontaneously in embryonic stem cell (ESC) cultures, but only in very low proportions of around 0.5%6. 2CLCs recapitulate several molecular features of the totipotent cells in mouse embryos and display expanded potency, including higher ability to be reprogrammed upon nuclear transfer[6–8]
Analysis of replication fork speed in 2CLCs revealed a significantly slower fork speed compared with ESCs (Fig. 1b)
Summary
Cells in the early mammalian embryo are most plastic because they can generate every cell type in the body. The mouse zygote and each of the blastomeres in 2-cell-stage embryos are totipotent[1,2], because they can generate a new organism on their own without the need for carrier cells. This contrasts with pluripotent cells, which can generate all the cells in the body, but not extraembryonic tissues[3,4]. How the early mammalian embryo replicates its DNA and whether the acquisition of totipotency is regulated through DNA-replication-dependent mechanisms is unknown. As the molecular properties of the replication fork are central to the regulation of replication[5], we set out to investigate replication fork dynamics in totipotent cells in vivo and totipotent-like cells in culture
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