The chromatin regulator MLL2 (KMT2B) is the primary histone 3 lysine 4 (H3K4) trimethyltransferase acting at bivalent promoters in embryonic stem cells (ESCs) and is required for differentiation toward neuroectoderm. Here, we demonstrate that this requirement occurs during exit from naive pluripotency, days before neuroectoderm differentiation is impaired. During exit, the effect of MLL2 on transcription is subtle, increasing the expression of a few important neuroectodermal transcription factors. In contrast, MLL2's effect on chromatin architecture is substantial, stabilising loops associated with bivalent promoters in primed ESCs. MLL2 H3K4 catalytic activity is dispensable for stabilising these loops during ESC exit and for neuroectoderm differentiation. We therefore identify a non-catalytic function for MLL2 in stabilising 3D chromatin architecture, which has implications for lineage specification. Because MLL2 shares features with all four MLLs, we propose that chromatin tethering, rather than H3K4 methylation, represents a primary function for MLLs during lineage commitment decisions. ### Competing Interest Statement The authors have declared no competing interest.

During mouse embryo compaction, outer cells become trophectoderm, while inner cells form the inner cell mass (ICM), later differentiating into primitive endoderm and epiblast - source of embryonic stem cells (ESCs) - during blastocyst formation. Trophectoderm specification is driven by position-governed polarisation, while primitive endoderm specification is positively regulated by FGF4 signalling from the ICM and epiblast. When injected into an 8-cell stage morula, ESCs can exclude host cells from the epiblast, leading to mice derived entirely from these cells. While evidence suggests roles for ESC-produced FGF4 and physical crowding in host cell displacement from the ICM, the interplay between these possible mechanisms has yet to be dissected, in part due to the lack of studies using Fgf4-/- ESCs. Here, we combine chimaera titration assays with mathematical modelling to study these mechanisms of host cell displacement. Both Fgf4+/+ and Fgf4-/- ESCs displaced host cells from the epiblast, while only Fgf4-/- injected embryos reduced primitive endoderm and increased trophectoderm, indicating sequential exclusion by displacement crowding followed by FGF4 signalling. ### Competing Interest Statement The authors have declared no competing interest.

A crucial aspect of embryology is relating the position of individual cells to the broader geometry of the embryo. A classic example of this is the first cell-fate decision of the mouse embryo, where interior cells become inner cell mass and exterior cells become trophectoderm. Fluorescent labelling, imaging, and quantification of tissue-specific proteins have advanced our understanding of this dynamic process. However instances arise where these markers are either not available, or not reliable, and we are left only with the cells’ spatial locations. Therefore, a simple, robust method for classifying interior and exterior cells of an embryo using spatial information is required. Here, we describe a simple mathematical framework and an unsupervised machine learning approach, termed insideOutside , for classifying interior and exterior points of a three-dimensional point-cloud, a common output from imaged cells within the early mouse embryo. We benchmark our method against other published methods to demonstrate that it yields greater accuracy in classification of nuclei from the pre-implantation mouse embryos and greater accuracy when challenged with local surface concavities. We have made MATLAB and Python implementations of the method freely available. This method should prove useful for embryology, with broader applications to similar data arising in the life sciences.

Single-molecule localization microscopy (SMLM) allows the super-resolved imaging of proteins within mammalian nuclei at spatial resolutions comparable to that of a nucleosome itself (~20 nm). The technique is therefore well suited to the study of chromatin structure. Fixed-cell SMLM has already allowed temporal "snapshots" of how proteins are arranged on chromatin within mammalian nuclei. In this chapter, we focus on how recent developments, for example in selective plane illumination, 3D SMLM, and protein labeling, have led to a range of live-cell SMLM studies. We describe how to carry out single-particle tracking (SPT) of single proteins and, by analyzing their diffusion parameters, how to determine whether proteins interact with chromatin, diffuse freely, or do both. We can study the numbers of proteins that interact with chromatin and also determine their residence time on chromatin. We can determine whether these proteins form functional clusters within the nucleus as well as whether they form specific nuclear structures.

Gastrulation controls the emergence of cellular diversity and axis patterning in the early embryo. In mammals, this transformation is orchestrated by dynamic signalling centres at the interface of embryonic and extraembryonic tissues1–3. Elucidating the molecular framework of axis formation in vivo is fundamental for our understanding of human development4–6 and to advance stem-cell-based regenerative approaches7. Here we illuminate early gastrulation of marmoset embryos in utero using spatial transcriptomics and stem-cell-based embryo models. Gaussian process regression-based 3D transcriptomes delineate the emergence of the anterior visceral endoderm, which is hallmarked by conserved (HHEX, LEFTY2, LHX1) and primate-specific (POSTN, SDC4, FZD5) factors. WNT signalling spatially coordinates the formation of the primitive streak in the embryonic disc and is counteracted by SFRP1 and SFRP2 to sustain pluripotency in the anterior domain. Amnion specification occurs at the boundaries of the embryonic disc through ID1, ID2 and ID3 in response to BMP signalling, providing a developmental rationale for amnion differentiation of primate pluripotent stem cells (PSCs). Spatial identity mapping demonstrates that primed marmoset PSCs exhibit the highest similarity to the anterior embryonic disc, whereas naive PSCs resemble the preimplantation epiblast. Our 3D transcriptome models reveal the molecular code of lineage specification in the primate embryo and provide an in vivo reference to decipher human development. 3D transcriptomes reveal the molecular code of lineage specification in the primate embryo and provide an in vivo reference to decipher human development.

Until recently, naïve pluripotent stem cell lines were not captured from human embryos because protocols were based upon those devised for murine embryonic stem cells. In contrast with early lineage segregation in mouse embryos, human hypoblast specification is not solely dependent upon FGF signaling; consequently, its maturation during embryo explant culture may provide inductive signals to drive differentiation of the epiblast. To overcome this potential risk, here we describe how cells of the immature inner cell mass of human embryos can be physically separated during derivation, achieved via "immunosurgery", to eliminate the trophectoderm, followed by disaggregation of the remaining inner cell mass cells. A modification of a culture regime developed for propagation of human pluripotent stem cells reset to the naïve state is used, which comprises serum-free medium supplemented with various inhibitors of signaling pathways, polarization, and differentiation. Colonies arising from the first plating of an inner cell mass may be pooled for ease of handling, or propagated separately to allow establishment of clonal human naïve embryonic stem cell lines.

OCT4 is a fundamental component of the molecular circuitry governing pluripotency in vivo and in vitro. To determine how OCT4 establishes and protects the pluripotent lineage in the embryo, we used comparative single-cell transcriptomics and quantitative immunofluorescence on control and OCT4 null blastocyst inner cell masses at two developmental stages. Surprisingly, activation of most pluripotency-associated transcription factors in the early mouse embryo occurs independently of OCT4, with the exception of the JAK/STAT signaling machinery. Concurrently, OCT4 null inner cell masses ectopically activate a subset of trophectoderm-associated genes. Inspection of metabolic pathways implicates the regulation of rate-limiting glycolytic enzymes by OCT4, consistent with a role in sustaining glycolysis. Furthermore, up-regulation of the lysosomal pathway was specifically detected in OCT4 null embryos. This finding implicates a requirement for OCT4 in the production of normal trophectoderm. Collectively, our findings uncover regulation of cellular metabolism and biophysical properties as mechanisms by which OCT4 instructs pluripotency.

Classic embryological experiments have established that the early mouse embryo develops via sequential lineage bifurcations. The first segregated lineage is the trophectoderm, essential for blastocyst formation. Mouse naive epiblast and derivative embryonic stem cells are restricted accordingly from producing trophectoderm. Here we show, in contrast, that human naive embryonic stem cells readily make blastocyst trophectoderm and descendant trophoblast cell types. Trophectoderm was induced rapidly and efficiently by inhibition of ERK/mitogen-activated protein kinase (MAPK) and Nodal signaling. Transcriptome comparison with the human embryo substantiated direct formation of trophectoderm with subsequent differentiation into syncytiotrophoblast, cytotrophoblast, and downstream trophoblast stem cells. During pluripotency progression lineage potential switches from trophectoderm to amnion. Live-cell tracking revealed that epiblast cells in the human blastocyst are also able to produce trophectoderm. Thus, the paradigm of developmental specification coupled to lineage restriction does not apply to humans. Instead, epiblast plasticity and the potential for blastocyst regeneration are retained until implantation.

Mouse embryonic stem (ES) cells are derived from the epiblast of the preimplantation embryo and retain the capacity to give rise to all embryo lineages. ES cells can be released into differentiation from a near-homogeneous maintenance condition. Exit from the ES cell state can be accurately monitored using the Rex1-GFPd2 transgenic reporter, providing a powerful system for examining a mammalian cell fate transition. Here, we performed live-cell imaging and tracking of ES cells during entry into differentiation for 48 hours in defined conditions. We observed a greater cell surface area and a modest shortening of the cell cycle prior to exit and subsequently a reduction in cell size and increase in motility. We did not see any instance of cells regaining ES cell identity, consistent with unidirectional developmental progression. Transition occurred asynchronously across the population but genealogical tracking revealed a high correlation in cell-cycle length and Rex1-GFPd2 expression between daughter cells. A population dynamics model was consistent with symmetric divisions during exit from naive pluripotency. Collapse of ES cell identity occurred acutely in individual cells but after a variable delay. The variation in lag period can extend up to three generations, creating marked population asynchrony.

Classical mouse embryology has established a sequence of lineage bifurcations underpinning early mammalian development. Consistent with this paradigm, mouse embryonic stem cells have lost the capacity to generate extraembryonic trophectoderm. We show here, however, that human naive epiblast stem cells readily produce this lineage. Inhibition of ERK signalling, fundamental to naive cell propagation, is unexpectedly instrumental in trophectoderm induction. Transcriptome analyses authenticate trophoblast fate and expose a trajectory via reversion to inner cell mass (ICM). Nodal inhibition enhances differentiation and BMP signalling is not engaged. Strikingly, after formative transition primed stem cells cannot make trophectoderm but respond to BMP and form amnion. Gene perturbations in naive cells reveal that YAP and TFAP2C promote trophectoderm as in mouse, while NANOG suppresses distinctively in human. Finally, ICMs from expanded human blastocysts efficiently regenerate trophectoderm. Thus in human, retained trophectoderm potential is an integral feature of emergent pluripotency that confers higher regulative plasticity.