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Size control is fundamental in tissue development and homeostasis 1 , 2 . Although the role of cell proliferation in these processes has been widely studied, the mechanisms that control embryo size—and how these mechanisms affect cell fate—remain unknown. Here we use the mouse blastocyst as a model to unravel a key role of fluid-filled lumen in the control of embryo size and specification of cell fate. We find that there is a twofold increase in lumenal pressure during blastocyst development, which translates into a concomitant increase in cell cortical tension and tissue stiffness of the trophectoderm that lines the lumen. Increased cortical tension leads to vinculin mechanosensing and maturation of functional tight junctions, which establishes a positive feedback loop to accommodate lumen growth. When the cortical tension reaches a critical threshold, cell–cell adhesion cannot be sustained during mitotic entry, which leads to trophectoderm rupture and blastocyst collapse. A simple theory of hydraulically gated oscillations recapitulates the observed dynamics of size oscillations, and predicts the scaling of embryo size with tissue volume. This theory further predicts that disrupted tight junctions or increased tissue stiffness lead to a smaller embryo size, which we verified by biophysical, embryological, pharmacological and genetic perturbations. Changes in lumenal pressure and size can influence the cell division pattern of the trophectoderm, and thereby affect cell allocation and fate. Our study reveals how lumenal pressure and tissue mechanics control embryo size at the tissue scale, which is coupled to cell position and fate at the cellular scale. A mouse blastocyst model reveals how lumenal pressure, cell cortical tension and tissue stiffness act at the tissue scale to regulate embryo size, which in turn influences the division pattern of trophectoderm cells and their fate specification.

The epiblast (EPI) is the origin of all somatic and germ cells in mammals, and of pluripotent stem cells in vitro . To explore the ontogeny of human and primate pluripotency, here we perform comprehensive single-cell RNA sequencing for pre- and post-implantation EPI development in cynomolgus monkeys ( Macaca fascicularis ). We show that after specification in the blastocysts, EPI from cynomolgus monkeys (cyEPI) undergoes major transcriptome changes on implantation. Thereafter, while generating gastrulating cells, cyEPI stably maintains its transcriptome over a week, retains a unique set of pluripotency genes and acquires properties for ‘neuron differentiation’. Human and monkey pluripotent stem cells show the highest similarity to post-implantation late cyEPI, which, despite co-existing with gastrulating cells, bears characteristics of pre-gastrulating mouse EPI and epiblast-like cells in vitro . These findings not only reveal the divergence and coherence of EPI development, but also identify a developmental coordinate of the spectrum of pluripotency among key species, providing a basis for better regulation of human pluripotency in vitro . Using a single-cell sequencing analysis in monkey embryos, and comparing the genes expressed during early development in this species with those in mice and in human pluripotent stem cells, the authors define characteristics of pluripotency ontogeny across mammalian species. Species differences in developing pluripotent stem cells Using a single-cell-sequencing-based analysis in monkey embryos, and comparing the genes expressed during early development in this species and what is known from mouse and human studies, Mitinori Saitou and colleagues define characteristics of pluripotency ontogeny across mammalian species. They show that, surprisingly, monkey cells undergoing neuronal differentiation continue to express genes associated with pluripotency during gastrulation. The analysis also provides insights into the comparative properties of developmental-stage pluripotent stem cells in key species that will help to establish a basis for better regulation of human pluripotency in vitro .

Current understandings of cell specification in early mammalian pre-implantation development are based mainly on mouse studies. The first lineage differentiation event occurs at the morula stage, with outer cells initiating a trophectoderm (TE) placental progenitor program. The inner cell mass arises from inner cells during subsequent developmental stages and comprises precursor cells of the embryo proper and yolk sac 1 . Recent gene-expression analyses suggest that the mechanisms that regulate early lineage specification in the mouse may differ in other mammals, including human 2 – 5 and cow 6 . Here we show the evolutionary conservation of a molecular cascade that initiates TE segregation in human, cow and mouse embryos. At the morula stage, outer cells acquire an apical–basal cell polarity, with expression of atypical protein kinase C (aPKC) at the contact-free domain, nuclear expression of Hippo signalling pathway effectors and restricted expression of TE-associated factors such as GATA3, which suggests initiation of a TE program. Furthermore, we demonstrate that inhibition of aPKC by small-molecule pharmacological modulation or Trim-Away protein depletion impairs TE initiation at the morula stage. Our comparative embryology analysis provides insights into early lineage specification and suggests that a similar mechanism initiates a TE program in human, cow and mouse embryos. Comparative analysis of human, cow and mouse embryos shows that a mechanism involving atypical protein kinase C initiates the trophectoderm program during the morula stage in these three species.

In mammalian development, epigenetic modifications, including DNA methylation patterns, play a crucial role in defining cell fate but also represent epigenetic barriers that restrict developmental potential. At two points in the life cycle, DNA methylation marks are reprogrammed on a global scale, concomitant with restoration of developmental potency. DNA methylation patterns are subsequently re-established with the commitment towards a distinct cell fate. This reprogramming of DNA methylation takes place firstly on fertilization in the zygote, and secondly in primordial germ cells (PGCs), which are the direct progenitors of sperm or oocyte. In each reprogramming window, a unique set of mechanisms regulates DNA methylation erasure and re-establishment. Recent advances have uncovered roles for the TET3 hydroxylase and passive demethylation, together with base excision repair (BER) and the elongator complex, in methylation erasure from the zygote. Deamination by AID, BER and passive demethylation have been implicated in reprogramming in PGCs, but the process in its entirety is still poorly understood. In this review, we discuss the dynamics of DNA methylation reprogramming in PGCs and the zygote, the mechanisms involved and the biological significance of these events. Advances in our understanding of such natural epigenetic reprogramming are beginning to aid enhancement of experimental reprogramming in which the role of potential mechanisms can be investigated in vitro. Conversely, insights into in vitro reprogramming techniques may aid our understanding of epigenetic reprogramming in the germline and supply important clues in reprogramming for therapies in regenerative medicine.

Gastrulation is the fundamental process in all multicellular animals through which the basic body plan is first laid down 1 – 4 . It is pivotal in generating cellular diversity coordinated with spatial patterning. In humans, gastrulation occurs in the third week after fertilization. Our understanding of this process in humans is relatively limited and based primarily on historical specimens 5 – 8 , experimental models 9 – 12 or, more recently, in vitro cultured samples 13 – 16 . Here we characterize in a spatially resolved manner the single-cell transcriptional profile of an entire gastrulating human embryo, staged to be between 16 and 19 days after fertilization. We use these data to analyse the cell types present and to make comparisons with other model systems. In addition to pluripotent epiblast, we identified primordial germ cells, red blood cells and various mesodermal and endodermal cell types. This dataset offers a unique glimpse into a central but inaccessible stage of our development. This characterization provides new context for interpreting experiments in other model systems and represents a valuable resource for guiding directed differentiation of human cells in vitro. The single-cell transcriptional profile of a human embryo between 16 and 19 days after fertilization reveals parallels and differences in gastrulation in humans as compared with mouse and non-human primate models.

Mammalian organogenesis is a remarkable process. Within a short timeframe, the cells of the three germ layers transform into an embryo that includes most of the major internal and external organs. Here we investigate the transcriptional dynamics of mouse organogenesis at single-cell resolution. Using single-cell combinatorial indexing, we profiled the transcriptomes of around 2 million cells derived from 61 embryos staged between 9.5 and 13.5 days of gestation, in a single experiment. The resulting ‘mouse organogenesis cell atlas’ (MOCA) provides a global view of developmental processes during this critical window. We use Monocle 3 to identify hundreds of cell types and 56 trajectories, many of which are detected only because of the depth of cellular coverage, and collectively define thousands of corresponding marker genes. We explore the dynamics of gene expression within cell types and trajectories over time, including focused analyses of the apical ectodermal ridge, limb mesenchyme and skeletal muscle. Data from single-cell combinatorial-indexing RNA-sequencing analysis of 2 million cells from mouse embryos between embryonic days 9.5 and 13.5 are compiled in a cell atlas of mouse organogenesis, which provides a global view of developmental processes occurring during this critical period.

Embryogenesis requires the precise activation and repression of many transcriptional regulators. The Polycomb group proteins and the associated H3K27me3 histone mark are essential to maintain the inactive state of many of these genes. Mammalian Hox genes are targets of Polycomb proteins and form local 3D clusters centered on the H3K27me3 mark. More distal contacts have also been described, yet their selectivity, dynamics, and relation to other layers of chromatin organization remained elusive. We report that repressed Hox genes form mutual intra- and interchromosomal interactions with other genes located in strong domains labeled by H3K27me3. These interactions occur in a central and active nuclear environment that consists of the HiC compartment A, away from peripheral lamina-associated domains. Interactions are independent of nearby H3K27me3-marked loci and determined by chromosomal distance and cell-type–specific scaling factors, thus inducing a moderate reorganization during embryogenesis. These results provide a simplified view of nuclear organization whereby Polycomb proteins may have evolved to repress genes located in gene-dense regions whose position is restricted to central, active, nuclear environments. Significance The development of an embryo from a single fertilized cell is orchestrated by a large set of key regulatory genes whose activities need to be precisely controlled. Proteins from the Polycomb group (PcG) family maintain the inactive state of these genes by modifying the surrounding histone H3 tails. Here we report that these inactive genes contact other PcG-rich regions within otherwise active environments in the cell nucleus, suggesting the presence of repressive microenvironments. PcG positive genes interact independently from neighboring genes, depending on their linear distance and more global chromosome folding characteristics, with only moderate changes during embryonic development.

Chromosomes in proliferating metazoan cells undergo marked structural metamorphoses every cell cycle, alternating between highly condensed mitotic structures that facilitate chromosome segregation, and decondensed interphase structures that accommodate transcription, gene silencing and DNA replication. Here we use single-cell Hi-C (high-resolution chromosome conformation capture) analysis to study chromosome conformations in thousands of individual cells, and discover a continuum of cis -interaction profiles that finely position individual cells along the cell cycle. We show that chromosomal compartments, topological-associated domains (TADs), contact insulation and long-range loops, all defined by bulk Hi-C maps, are governed by distinct cell-cycle dynamics. In particular, DNA replication correlates with a build-up of compartments and a reduction in TAD insulation, while loops are generally stable from G1 to S and G2 phase. Whole-genome three-dimensional structural models reveal a radial architecture of chromosomal compartments with distinct epigenomic signatures. Our single-cell data therefore allow re-interpretation of chromosome conformation maps through the prism of the cell cycle. Single-cell Hi-C analysis in thousands of mouse embryonic stem cells shows that chromosomal compartments, topological-associated domains and long-range loops all have distinct cell-cycle dynamics. Chromosomal organization dynamics Eukaryotic chromosomes undergo a cycle of compaction and decondensation during the cell cycle. Here, Peter Fraser and colleagues have developed an improved single-cell Hi-C method to characterize the 3D organization of chromosomes through the cell cycle in thousands of individual mouse embryonic stem cells. They find that chromosomal compartments, topological-associated domains and loops are each governed by distinct dynamics and reveal a continuum of dynamic chromosomal structural features throughout the cell cycle. The results will be a new point of reference for interpreting chromosome conformation Hi-C maps.

DNA methylation is dynamically remodelled during the mammalian life cycle through distinct phases of reprogramming and de novo methylation. These events enable the acquisition of cellular potential followed by the maintenance of lineage-restricted cell identity, respectively, a process that defines the life cycle through successive generations. DNA methylation contributes to the epigenetic regulation of many key developmental processes including genomic imprinting, X-inactivation, genome stability and gene regulation. Emerging sequencing technologies have led to recent insights into the dynamic distribution of DNA methylation during development and the role of this epigenetic mark within distinct genomic contexts, such as at promoters, exons or imprinted control regions. Additionally, there is a better understanding of the mechanistic basis of DNA demethylation during epigenetic reprogramming in primordial germ cells and during pre-implantation development. Here, we discuss our current understanding of the developmental roles and dynamics of this key epigenetic system.

Gastruloids are three-dimensional aggregates of embryonic stem cells that display key features of mammalian development after implantation, including germ-layer specification and axial organization 1 – 3 . To date, the expression pattern of only a small number of genes in gastruloids has been explored with microscopy, and the extent to which genome-wide expression patterns in gastruloids mimic those in embryos is unclear. Here we compare mouse gastruloids with mouse embryos using single-cell RNA sequencing and spatial transcriptomics. We identify various embryonic cell types that were not previously known to be present in gastruloids, and show that key regulators of somitogenesis are expressed similarly between embryos and gastruloids. Using live imaging, we show that the somitogenesis clock is active in gastruloids and has dynamics that resemble those in vivo. Because gastruloids can be grown in large quantities, we performed a small screen that revealed how reduced FGF signalling induces a short-tail phenotype in embryos. Finally, we demonstrate that embedding in Matrigel induces gastruloids to generate somites with the correct rostral–caudal patterning, which appear sequentially in an anterior-to-posterior direction over time. This study thus shows the power of gastruloids as a model system for exploring development and somitogenesis in vitro in a high-throughput manner. Single-cell RNA sequencing and spatial transcriptomics reveal that the somitogenesis clock is active in mouse gastruloids, which can be induced to generate somites with the correct rostral–caudal patterning.