FOXO1-mediated lipid metabolism maintains mammalian embryos in dormancy

Mammalian developmental timing is adjustable in vivo by preserving pre-implantation embryos in a dormant state called diapause. Inhibition of the growth regulator mTOR (mTORi) pauses mouse development in vitro, yet how embryonic dormancy is maintained is not known. Here we show that mouse embryos in diapause are sustained by using lipids as primary energy source. In vitro, supplementation of embryos with the metabolite l-carnitine balances lipid consumption, puts the embryos in deeper dormancy and boosts embryo longevity. We identify FOXO1 as an essential regulator of the energy balance in dormant embryos and propose, through meta-analyses of dormant cell signatures, that it may be a common regulator of dormancy across adult tissues. Our results lift a constraint on in vitro embryo survival and suggest that lipid metabolism may be a critical metabolic transition relevant for longevity and stem cell function across tissues.

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The number of replicates for each analysis is indicated in the text or figure legends.
For the ESC and TSC proteomics and ESC metabolomics experiment, three biological replicates were used.
For the embryo proteomics experiment, four to five biological replicates were used.At least 5 embryos per condition were used for the MALDI-imaging experiments.Four embryos per condition were used for electron microscopy.For all confocal fluorescence imaging, at least 3 embryos were analyzed per condition.The number of analyzed embryos or cells is indicated in the figures.
No data were excluded from the analysis.

Replication Randomization
The ESC and TSC proteomics and TSC metabolomics were performed with three biological replicates.The embryo proteomics experiment was performed with four to five biological replicates.The embryo supplementation experiments were performed once for supplements that did not improve developmental pausing length, and five times for carnitine supplementation experiments.The embryo staining experiments were performed with at least 3 biological replicates depending on the staining conditions.All attempts at replication were successful.
For cell and embryo culture experiments, treatment groups were randomly attributed.

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No blinding was done because either the phenotype was obvious or treatment material needed to be refreshed often.

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