The first two blastomeres contribute unequally to the human embryo.

Retrospective lineage reconstruction of humans predicts that dramatic clonal imbalances in the body can be traced to the 2-cell stage embryo. However, whether and how such clonal asymmetries arise in the embryo is unclear. Here, we performed prospective lineage tracing of human embryos using live imaging, non-invasive cell labeling, and computational predictions to determine the contribution of each 2-cell stage blastomere to the epiblast (body), hypoblast (yolk sac), and trophectoderm (placenta). We show that the majority of epiblast cells originate from only one blastomere of the 2-cell stage embryo. We observe that only one to three cells become internalized at the 8-to-16-cell stage transition. Moreover, these internalized cells are more frequently derived from the first cell to divide at the 2-cell stage. We propose that cell division dynamics and a cell internalization bottleneck in the early embryo establish asymmetry in the clonal composition of the future human body.

Mechanics of human embryo compaction.

The shaping of human embryos begins with compaction, during which cells come into close contact1,2. Assisted reproductive technology studies indicate that human embryos fail compaction primarily because of defective adhesion3,4. On the basis of our current understanding of animal morphogenesis5,6, other morphogenetic engines, such as cell contractility, could be involved in shaping human embryos. However, the molecular, cellular and physical mechanisms driving human embryo morphogenesis remain uncharacterized. Using micropipette aspiration on human embryos donated to research, we have mapped cell surface tensions during compaction. This shows a fourfold increase of tension at the cell-medium interface whereas cell-cell contacts keep a steady tension. Therefore, increased tension at the cell-medium interface drives human embryo compaction, which is qualitatively similar to compaction in mouse embryos7. Further comparison between human and mouse shows qualitatively similar but quantitively different mechanical strategies, with human embryos being mechanically least efficient. Inhibition of cell contractility and cell-cell adhesion in human embryos shows that, whereas both cellular processes are required for compaction, only contractility controls the surface tensions responsible for compaction. Cell contractility and cell-cell adhesion exhibit distinct mechanical signatures when faulty. Analysing the mechanical signature of naturally failing embryos, we find evidence that non-compacting or partially compacting embryos containing excluded cells have defective contractility. Together, our study shows that an evolutionarily conserved increase in cell contractility is required to generate the forces driving the first morphogenetic movement shaping the human body.

Basal delamination during mouse gastrulation primes pluripotent cells for differentiation.

The blueprint of the mammalian body plan is laid out during gastrulation, when a trilaminar embryo is formed. This process entails a burst of proliferation, the ingression of embryonic epiblast cells at the primitive streak, and their priming toward primitive streak fates. How these different events are coordinated remains unknown. Here, we developed and characterized a 3D culture of self-renewing mouse embryonic cells that captures the main transcriptional and architectural features of the early gastrulating mouse epiblast. Using this system in combination with microfabrication and in vivo experiments, we found that proliferation-induced crowding triggers delamination of cells that express high levels of the apical polarity protein aPKC. Upon delamination, cells become more sensitive to Wnt signaling and upregulate the expression of primitive streak markers such as Brachyury. This mechanistic coupling between ingression and differentiation ensures that the right cell types become specified at the right place during embryonic development.

CD32 captures committed haemogenic endothelial cells during human embryonic development.

During embryonic development, blood cells emerge from specialized endothelial cells, named haemogenic endothelial cells (HECs). As HECs are rare and only transiently found in early developing embryos, it remains difficult to distinguish them from endothelial cells. Here we performed transcriptomic analysis of 28- to 32-day human embryos and observed that the expression of Fc receptor CD32 (FCGR2B) is highly enriched in the endothelial cell population that contains HECs. Functional analyses using human embryonic and human pluripotent stem cell-derived endothelial cells revealed that robust multilineage haematopoietic potential is harboured within CD32+ endothelial cells and showed that 90% of CD32+ endothelial cells are bona fide HECs. Remarkably, these analyses indicated that HECs progress through different states, culminating in FCGR2B expression, at which point cells are irreversibly committed to a haematopoietic fate. These findings provide a precise method for isolating HECs from human embryos and human pluripotent stem cell cultures, thus allowing the efficient generation of haematopoietic cells in vitro.

In silico-designed vitrification protocols: an approach to improve survival of in vitro produced-bovine embryos.

In brief: In silico predictions validated in this study demonstrate the potential for designing shorter equilibration protocols that improve post-warming re-expansion and hatching rates of D7 and D8 in vitro-produced bovine embryos. Our results benefit the livestock industry by providing a refined and reproducible approach to cryopreserving bovine embryos, which, in addition, could be useful for other mammalian species. Abstract: The cryopreservation of in vitro-produced (IVP) embryos is vital in the cattle industry for genetic selection and crossbreeding programs. Despite its importance, there is no standardized protocol yielding pregnancy rates comparable to fresh embryos. Current approaches often neglect the osmotic tolerance responses to cryoprotectants based on temperature and time. Hereby, we propose improved vitrification methods using shorter dehydration-based protocols. Blastocysts cultured for 7 (D7) or 8 days (D8) were exposed to standard equilibration solution (ES) at 25ºC and 38.5ºC. Optimized exposure times for each temperature and their impact on post-warming re-expansion, hatching rates, cell counts, and apoptosis rate were determined. In silico predictions aligned with in vitro observations, showing original volume recovery within 8 min 30 s at 25ºC or 3 min 40 s at 38.5ºC (D7 blastocysts) and 4 min 25 s at 25ºC and 3 min 15 s at 38.5ºC (D8 blastocysts) after exposure to ES. Vitrification at 38.5ºC resulted in D7 blastocysts re-expansion and hatching rates (93.1% and 38.1%, respectively) comparable to fresh embryos (100.0% and 32.4%, respectively), outperforming the 25ºC protocol (86.2% and 24.4%, respectively; P < 0.05). No differences were observed between D7 and D8 blastocysts using the 38.5ºC protocol. Total cell number was maintained for D7 and D8 blastocysts vitrified at 38.5ºC but decreased at 25ºC (P < 0.05). Apoptosis rates increased post-warming (P < 0.05), except for D8 blastocysts vitrified at 38.5ºC, resembling fresh controls. In conclusion, based on biophysical permeability data, new ES incubation times of 3 min 40 s for D7 blastocysts and 3 min 15 s for D8 blastocysts at 38.5ºC were validated for optimizing vitrification/warming methods for bovine IVP blastocysts.

Mouse SAS-6 is required for centriole formation in embryos and integrity in embryonic stem cells.

SAS-6 (SASS6) is essential for centriole formation in human cells and other organisms but its functions in the mouse are unclear. Here, we report that Sass6-mutant mouse embryos lack centrioles, activate the mitotic surveillance cell death pathway, and arrest at mid-gestation. In contrast, SAS-6 is not required for centriole formation in mouse embryonic stem cells (mESCs), but is essential to maintain centriole architecture. Of note, centrioles appeared after just one day of culture of Sass6-mutant blastocysts, from which mESCs are derived. Conversely, the number of cells with centrosomes is drastically decreased upon the exit from a mESC pluripotent state. At the mechanistic level, the activity of the master kinase in centriole formation, PLK4, associated with increased centriolar and centrosomal protein levels, endow mESCs with the robustness in using a SAS-6-independent centriole-biogenesis pathway. Collectively, our data suggest a differential requirement for mouse SAS-6 in centriole formation or integrity depending on PLK4 activity and centrosome composition.

A single-cell time-lapse of mouse prenatal development from gastrula to birth.

The house mouse (Mus musculus) is an exceptional model system, combining genetic tractability with close evolutionary affinity to humans1,2. Mouse gestation lasts only 3 weeks, during which the genome orchestrates the astonishing transformation of a single-cell zygote into a free-living pup composed of more than 500 million cells. Here, to establish a global framework for exploring mammalian development, we applied optimized single-cell combinatorial indexing3 to profile the transcriptional states of 12.4 million nuclei from 83 embryos, precisely staged at 2- to 6-hour intervals spanning late gastrulation (embryonic day 8) to birth (postnatal day 0). From these data, we annotate hundreds of cell types and explore the ontogenesis of the posterior embryo during somitogenesis and of kidney, mesenchyme, retina and early neurons. We leverage the temporal resolution and sampling depth of these whole-embryo snapshots, together with published data4-8 from earlier timepoints, to construct a rooted tree of cell-type relationships that spans the entirety of prenatal development, from zygote to birth. Throughout this tree, we systematically nominate genes encoding transcription factors and other proteins as candidate drivers of the in vivo differentiation of hundreds of cell types. Remarkably, the most marked temporal shifts in cell states are observed within one hour of birth and presumably underlie the massive physiological adaptations that must accompany the successful transition of a mammalian fetus to life outside the womb.

Hypoblast from human pluripotent stem cells regulates epiblast development.

Recently, several studies using cultures of human embryos together with single-cell RNA-seq analyses have revealed differences between humans and mice, necessitating the study of human embryos1-8. Despite the importance of human embryology, ethical and legal restrictions have limited post-implantation-stage studies. Thus, recent efforts have focused on developing in vitro self-organizing models using human stem cells9-17. Here, we report genetic and non-genetic approaches to generate authentic hypoblast cells (naive hPSC-derived hypoblast-like cells (nHyCs))-known to give rise to one of the two extraembryonic tissues essential for embryonic development-from naive human pluripotent stem cells (hPSCs). Our nHyCs spontaneously assemble with naive hPSCs to form a three-dimensional bilaminar structure (bilaminoids) with a pro-amniotic-like cavity. In the presence of additional naive hPSC-derived analogues of the second extraembryonic tissue, the trophectoderm, the efficiency of bilaminoid formation increases from 20% to 40%, and the epiblast within the bilaminoids continues to develop in response to trophectoderm-secreted IL-6. Furthermore, we show that bilaminoids robustly recapitulate the patterning of the anterior-posterior axis and the formation of cells reflecting the pregastrula stage, the emergence of which can be shaped by genetically manipulating the DKK1/OTX2 hypoblast-like domain. We have therefore successfully modelled and identified the mechanisms by which the two extraembryonic tissues efficiently guide the stage-specific growth and progression of the epiblast as it establishes the post-implantation landmarks of human embryogenesis.

Emergence of replication timing during early mammalian development.

DNA replication enables genetic inheritance across the kingdoms of life. Replication occurs with a defined temporal order known as the replication timing (RT) programme, leading to organization of the genome into early- or late-replicating regions. RT is cell-type specific, is tightly linked to the three-dimensional nuclear organization of the genome1,2 and is considered an epigenetic fingerprint3. In spite of its importance in maintaining the epigenome4, the developmental regulation of RT in mammals in vivo has not been explored. Here, using single-cell Repli-seq5, we generated genome-wide RT maps of mouse embryos from the zygote to the blastocyst stage. Our data show that RT is initially not well defined but becomes defined progressively from the 4-cell stage, coinciding with strengthening of the A and B compartments. We show that transcription contributes to the precision of the RT programme and that the difference in RT between the A and B compartments depends on RNA polymerase II at zygotic genome activation. Our data indicate that the establishment of nuclear organization precedes the acquisition of defined RT features and primes the partitioning of the genome into early- and late-replicating domains. Our work sheds light on the establishment of the epigenome at the beginning of mammalian development and reveals the organizing principles of genome organization.

Complete human day 14 post-implantation embryo models from naive ES cells.

The ability to study human post-implantation development remains limited owing to ethical and technical challenges associated with intrauterine development after implantation1. Embryo-like models with spatially organized morphogenesis and structure of all defining embryonic and extra-embryonic tissues of the post-implantation human conceptus (that is, the embryonic disc, the bilaminar disc, the yolk sac, the chorionic sac and the surrounding trophoblast layer) remain lacking1,2. Mouse naive embryonic stem cells have recently been shown to give rise to embryonic and extra-embryonic stem cells capable of self-assembling into post-gastrulation structured stem-cell-based embryo models with spatially organized morphogenesis (called SEMs)3. Here we extend those findings to humans using only genetically unmodified human naive embryonic stem cells (cultured in human enhanced naive stem cell medium conditions)4. Such human fully integrated and complete SEMs recapitulate the organization of nearly all known lineages and compartments of post-implantation human embryos, including the epiblast, the hypoblast, the extra-embryonic mesoderm and the trophoblast layer surrounding the latter compartments. These human complete SEMs demonstrated developmental growth dynamics that resemble key hallmarks of post-implantation stage embryogenesis up to 13-14 days after fertilization (Carnegie stage 6a). These include embryonic disc and bilaminar disc formation, epiblast lumenogenesis, polarized amniogenesis, anterior-posterior symmetry breaking, primordial germ-cell specification, polarized yolk sac with visceral and parietal endoderm formation, extra-embryonic mesoderm expansion that defines a chorionic cavity and a connecting stalk, and a trophoblast-surrounding compartment demonstrating syncytium and lacunae formation. This SEM platform will probably enable the experimental investigation of previously inaccessible windows of human early post implantation up to peri-gastrulation development.

LIS1 RNA-binding orchestrates the mechanosensitive properties of embryonic stem cells in AGO2-dependent and independent ways.

Lissencephaly-1 (LIS1) is associated with neurodevelopmental diseases and is known to regulate the molecular motor cytoplasmic dynein activity. Here we show that LIS1 is essential for the viability of mouse embryonic stem cells (mESCs), and it governs the physical properties of these cells. LIS1 dosage substantially affects gene expression, and we uncovered an unexpected interaction of LIS1 with RNA and RNA-binding proteins, most prominently the Argonaute complex. We demonstrate that LIS1 overexpression partially rescued the extracellular matrix (ECM) expression and mechanosensitive genes conferring stiffness to Argonaute null mESCs. Collectively, our data transforms the current perspective on the roles of LIS1 in post-transcriptional regulation underlying development and mechanosensitive processes.

Psychedelics promote plasticity by directly binding to BDNF receptor TrkB.

Psychedelics produce fast and persistent antidepressant effects and induce neuroplasticity resembling the effects of clinically approved antidepressants. We recently reported that pharmacologically diverse antidepressants, including fluoxetine and ketamine, act by binding to TrkB, the receptor for BDNF. Here we show that lysergic acid diethylamide (LSD) and psilocin directly bind to TrkB with affinities 1,000-fold higher than those for other antidepressants, and that psychedelics and antidepressants bind to distinct but partially overlapping sites within the transmembrane domain of TrkB dimers. The effects of psychedelics on neurotrophic signaling, plasticity and antidepressant-like behavior in mice depend on TrkB binding and promotion of endogenous BDNF signaling but are independent of serotonin 2A receptor (5-HT2A) activation, whereas LSD-induced head twitching is dependent on 5-HT2A and independent of TrkB binding. Our data confirm TrkB as a common primary target for antidepressants and suggest that high-affinity TrkB positive allosteric modulators lacking 5-HT2A activity may retain the antidepressant potential of psychedelics without hallucinogenic effects.

Pluripotent stem cell-derived model of the post-implantation human embryo.

The human embryo undergoes morphogenetic transformations following implantation into the uterus, but our knowledge of this crucial stage is limited by the inability to observe the embryo in vivo. Models of the embryo derived from stem cells are important tools for interrogating developmental events and tissue-tissue crosstalk during these stages1. Here we establish a model of the human post-implantation embryo, a human embryoid, comprising embryonic and extraembryonic tissues. We combine two types of extraembryonic-like cell generated by overexpression of transcription factors with wild-type embryonic stem cells and promote their self-organization into structures that mimic several aspects of the post-implantation human embryo. These self-organized aggregates contain a pluripotent epiblast-like domain surrounded by extraembryonic-like tissues. Our functional studies demonstrate that the epiblast-like domain robustly differentiates into amnion, extraembryonic mesenchyme and primordial germ cell-like cells in response to bone morphogenetic protein cues. In addition, we identify an inhibitory role for SOX17 in the specification of anterior hypoblast-like cells2. Modulation of the subpopulations in the hypoblast-like compartment demonstrates that extraembryonic-like cells influence epiblast-like domain differentiation, highlighting functional tissue-tissue crosstalk. In conclusion, we present a modular, tractable, integrated3 model of the human embryo that will enable us to probe key questions of human post-implantation development, a critical window during which substantial numbers of pregnancies fail.

Multiple waves of fetal-derived immune cells constitute adult immune system.

It has been over three decades since Drs. Herzenberg and Herzenberg proposed the layered immune system hypothesis, suggesting that different types of stem cells with distinct hematopoietic potential produce specific immune cells. This layering of immune system development is now supported by recent studies showing the presence of fetal-derived immune cells that function in adults. It has been shown that various immune cells arise at different embryonic ages via multiple waves of hematopoiesis from special endothelial cells (ECs), referred to as hemogenic ECs. However, it remains unknown whether these fetal-derived immune cells are produced by hematopoietic stem cells (HSCs) during the fetal to neonatal period. To address this question, many advanced tools have been used, including lineage-tracing mouse models, cellular barcoding techniques, clonal assays, and transplantation assays at the single-cell level. In this review, we will review the history of the search for the origins of HSCs, B-1a progenitors, and mast cells in the mouse embryo. HSCs can produce both B-1a and mast cells within a very limited time window, and this ability declines after embryonic day (E) 14.5. Furthermore, the latest data have revealed that HSC-independent adaptive immune cells exist in adult mice, which implies more complicated developmental pathways of immune cells. We propose revised road maps of immune cell development.

NFATc1 marks articular cartilage progenitors and negatively determines articular chondrocyte differentiation.

The origin and differentiation mechanism of articular chondrocytes remain poorly understood. Broadly, the difference in developmental mechanisms of articular and growth-plate cartilage is still less elucidated. Here, we identified that the nuclear factor of activated T-cells cytoplasmic 1 (NFATc1) is a crucial regulator of articular, but not growth-plate, chondrocyte differentiation during development. At the early stage of mouse knee development (embryonic day 13.5), NFATc1-expressing cells were mainly located in the flanking region of the joint interzone. With development, NFATc1-expressing cells generated almost all articular chondrocytes but not chondrocytes in limb growth-plate primordium. NFATc1-expressing cells displayed prominent capacities for colony formation and multipotent differentiation. Transcriptome analyses revealed a set of characteristic genes in NFATc1-enriched articular cartilage progenitors. Strikingly, the expression of NFATc1 was diminished with articular chondrocyte differentiation, and suppressing NFATc1 expression in articular cartilage progenitors was sufficient to induce spontaneous chondrogenesis while overexpressing NFATc1 suppresses chondrogenesis. Mechanistically, NFATc1 negatively regulated the transcriptional activity of the Col2a1 gene. Thus, our results reveal that NFATc1 characterizes articular, but not growth-plate, cartilage progenitors during development and negatively determines articular chondrocyte differentiation at least partly through regulating COL2A1 gene transcription.

Within the body are about 300 joints connecting bones together. Many factors ­ including trauma, inflammation, aging, and genetic changes ­ can affect the cushion tissue covering the end of the bones in these joints known as articular cartilage. This can lead to diseases such as osteoarthritis which cause chronic pain, and in some cases disability. To treat such conditions, it is essential to know how cells in the articular cartilage are formed during development. In the embryo, most cells come from groups of progenitor cells that are programmed to produce specific types of tissue. But which progenitor cells are responsible for producing the main cells in articular cartilage, chondrocytes, and the mechanisms that govern this transformation are poorly understood. In 2016, a group of researchers found that the gene for the protein NFATc1, which is important for building bone, is also expressed in a group of progenitor cells at the site where ligaments insert into bone in mice. Inactivation of NFATc1 in these progenitor cells has also been shown to cause abnormal cartilage to form, a condition termed osteochondromas. Building on this work, Zhang, Wang et al. ­ including some of the researchers involved in the 2016 study ­ set out to find whether NFATc1 is also involved in the normal development of articular chondrocytes. To investigate, the team used genetically modified mice in which any cells with NFATc1 also had a green fluorescent protein, and tracked these cells and their progeny over the course of joint development. This led them to discover a group of NFATc1-containing progenitor cells that gave rise to almost all articular chondrocytes in the knee joint. Further experiments revealed that when NFATc1 was removed, this made the progenitors become articular chondrocytes very quickly. In contrast, when the cells had excess amounts of the protein, the formation of articular chondrocytes was significantly reduced. This suggests that the level of NFATc1 governs when progenitors develop into articular chondrocytes. These findings have provided a way to track the progenitors of articular chondrocytes throughout development and study how articular cartilage is formed. In the future, this work could help researchers develop treatment strategies for osteoarthritis and other cartilage-based diseases. However, before this can happen, further work is needed to confirm that the effects observed in this study also relate to humans.

Cell lineage-specific mitochondrial resilience during mammalian organogenesis.

Mitochondrial activity differs markedly between organs, but it is not known how and when this arises. Here we show that cell lineage-specific expression profiles involving essential mitochondrial genes emerge at an early stage in mouse development, including tissue-specific isoforms present before organ formation. However, the nuclear transcriptional signatures were not independent of organelle function. Genetically disrupting intra-mitochondrial protein synthesis with two different mtDNA mutations induced cell lineage-specific compensatory responses, including molecular pathways not previously implicated in organellar maintenance. We saw downregulation of genes whose expression is known to exacerbate the effects of exogenous mitochondrial toxins, indicating a transcriptional adaptation to mitochondrial dysfunction during embryonic development. The compensatory pathways were both tissue and mutation specific and under the control of transcription factors which promote organelle resilience. These are likely to contribute to the tissue specificity which characterizes human mitochondrial diseases and are potential targets for organ-directed treatments.

Metabolic regulation of species-specific developmental rates.

Animals display substantial inter-species variation in the rate of embryonic development despite a broad conservation of the overall sequence of developmental events. Differences in biochemical reaction rates, including the rates of protein production and degradation, are thought to be responsible for species-specific rates of development1-3. However, the cause of differential biochemical reaction rates between species remains unknown. Here, using pluripotent stem cells, we have established an in vitro system that recapitulates the twofold difference in developmental rate between mouse and human embryos. This system provides a quantitative measure of developmental speed as revealed by the period of the segmentation clock, a molecular oscillator associated with the rhythmic production of vertebral precursors. Using this system, we show that mass-specific metabolic rates scale with the developmental rate and are therefore higher in mouse cells than in human cells. Reducing these metabolic rates by inhibiting the electron transport chain slowed down the segmentation clock by impairing the cellular NAD+/NADH redox balance and, further downstream, lowering the global rate of protein synthesis. Conversely, increasing the NAD+/NADH ratio in human cells by overexpression of the Lactobacillus brevis NADH oxidase LbNOX increased the translation rate and accelerated the segmentation clock. These findings represent a starting point for the manipulation of developmental rate, with multiple translational applications including accelerating the differentiation of human pluripotent stem cells for disease modelling and cell-based therapies.