The Notch signalling pathway plays fundamental roles in diverse developmental processes in metazoans, where it is important in driving cell fate and directing differentiation of various cell types. However, we still have limited knowledge about the role of Notch in early preimplantation stages of mammalian development, or how it interacts with other signalling pathways active at these stages such as Hippo. By using genetic and pharmacological tools in vivo, together with image analysis of single embryos and pluripotent cell culture, we have found that Notch is active from the 4-cell stage. Transcriptomic analysis in single morula identified novel Notch targets, such as early naïve pluripotency markers or transcriptional repressors such as TLE4. Our results reveal a previously undescribed role for Notch in driving transitions during the gradual loss of potency that takes place in the early mouse embryo prior to the first lineage decisions.

The Notch signalling pathway plays fundamental roles in diverse developmental processes in metazoans, where it is important in driving cell fate and directing differentiation of various cell types. However, we still have limited knowledge about the role of Notch in early preimplantation stages of mammalian development, or how it interacts with other signalling pathways active at these stages such as Hippo. By using genetic and pharmacological tools in vivo, together with image analysis of single embryos and pluripotent cell culture, we have found that Notch is active from the 4-cell stage. Transcriptomic analysis in single morula identified novel Notch targets, such as early naïve pluripotency markers or transcriptional repressors such as TLE4. Our results reveal a previously undescribed role for Notch in driving transitions during the gradual loss of potency that takes place in the early mouse embryo prior to the first lineage decisions.Menchero et al. 3 IntroductionThe totipotent mammalian zygote has the self-organising capacity of generating embryonic and extraembryonic structures to build a complete organism [1]. This undifferentiated cell will proliferate and its descendants will take lineage decisions that entail a progressive loss of potency. The first differentiation event that leads to distinct lineages takes place during preimplantation development at the morula to blastocyst transition, resulting in the formation of the trophectoderm (TE, extraembryonic population) and the inner cell mass (ICM, embryonic population). How the establishment of these early lineages is achieved has been widely studied and we now know that a combination of morphogenetic cues breaks the symmetry in the embryo [2][3][4]. The first morphological sign of differentiation is evident in the compacting morula, 2.5 days after fertilization (embryonic day E2.5), when blastomeres increase their intercellular interactions and outer cells acquire an apical-basal polarity. These polarized cells on the surface enclose an inner group of apolar cells [5,6]. The outer versus inner position of the blastomeres correlates with their fate, becoming TE or ICM respectively, although cells can change their position within the embryo [7-9]. Prior to compaction, blastomeres appear morphologically equivalent. However, transcriptional differences among blastomeres have been described as early as in the 4-cell embryo [10][11][12]. Although cells at this stage are not committed to a specific fate, these early heterogeneities correlate with specific fate biases before lineage commitment.However, how these early heterogeneities arise and their implications in cell plasticity are still unclear [13].Once the embryo compacts, differences in contractility and the activity of signalling pathways orchestrate the lineage-commitment of cell populations [14][15][16][17][18][19][20]. The initial stochastic expression of the main lineage-specific transcription factors (such as CDX2 or GATA3 for the TE, and OCT4 or NANOG for the ICM) is gradually restricted to their ...

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