Single-cell transcriptomic characterization of a gastrulating human embryo.

Gastrulation is the fundamental process in all multicellular animals through which the basic body plan is first laid down1-4. 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 specimens5-8, experimental models9-12 or, more recently, in vitro cultured samples13-16. 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.

CIARA: a cluster-independent algorithm for identifying markers of rare cell types from single-cell sequencing data.

A powerful feature of single-cell genomics is the possibility of identifying cell types from their molecular profiles. In particular, identifying novel rare cell types and their marker genes is a key potential of single-cell RNA sequencing. Standard clustering approaches perform well in identifying relatively abundant cell types, but tend to miss rarer cell types. Here, we have developed CIARA (Cluster Independent Algorithm for the identification of markers of RAre cell types), a cluster-independent computational tool designed to select genes that are likely to be markers of rare cell types. Genes selected by CIARA are subsequently integrated with common clustering algorithms to single out groups of rare cell types. CIARA outperforms existing methods for rare cell type detection, and we use it to find previously uncharacterized rare populations of cells in a human gastrula and among mouse embryonic stem cells treated with retinoic acid. Moreover, CIARA can be applied more generally to any type of single-cell omic data, thus allowing the identification of rare cells across multiple data modalities. We provide implementations of CIARA in user-friendly packages available in R and Python.

A single-cell molecular map of mouse gastrulation and early organogenesis.

Across the animal kingdom, gastrulation represents a key developmental event during which embryonic pluripotent cells diversify into lineage-specific precursors that will generate the adult organism. Here we report the transcriptional profiles of 116,312 single cells from mouse embryos collected at nine sequential time points ranging from 6.5 to 8.5 days post-fertilization. We construct a molecular map of cellular differentiation from pluripotency towards all major embryonic lineages, and explore the complex events involved in the convergence of visceral and primitive streak-derived endoderm. Furthermore, we use single-cell profiling to show that Tal1-/- chimeric embryos display defects in early mesoderm diversification, and we thus demonstrate how combining temporal and transcriptional information can illuminate gene function. Together, this comprehensive delineation of mammalian cell differentiation trajectories in vivo represents a baseline for understanding the effects of gene mutations during development, as well as a roadmap for the optimization of in vitro differentiation protocols for regenerative medicine.

Recent advances in understanding cell types during human gastrulation.

Gastrulation is a fundamental process during embryonic development, conserved across all multicellular animals [1]. In the majority of metazoans, gastrulation is characterised by large scale morphogenetic remodeling, leading to the conversion of an early pluripotent embryonic cell layer into the three primary 'germ layers': an outer ectoderm, inner endoderm and intervening mesoderm layer. The morphogenesis of these three layers of cells is closely coordinated with cellular diversification, laying the foundation for the generation of the hundreds of distinct specialized cell types in the animal body. The process of gastrulation has for a long time attracted tremendous attention in a broad range of experimental systems ranging from sponges to mice. In humans the process of gastrulation starts approximately 14 days after fertilization and continues for slightly over a week. However our understanding of this important process, as it pertains to human, is limited. Donations of human fetal material at these early stages are exceptionally rare, making it nearly impossible to study human gastrulation directly. Therefore, our understanding of human gastrulation is predominantly derived from animal models such as the mouse [2,3] and from studies of limited collections of fixed whole samples and histological sections of human gastrulae [4-7], some of which date back to over a century ago. More recently we have been gaining valuable molecular insights into human gastrulation using in vitro models of hESCs [8-12] and increasingly, in vitro cultured human and non-human primate embryos [13-16]. However, while methods have been developed to culture human embryos into this stage (and probably beyond), current ethical standards prohibit the culture of human embryos past 14 days again limiting our ability to experimentally probe human gastrulation. This review discusses recent molecular insights from the study of a rare CS 7 human gastrula obtained as a live sample and raises several questions arising from this recent study that it will be interesting to address in the future using emerging models of human gastrulation.

Advances in live imaging early mouse development: exploring the researcher's interdisciplinary toolkit.

Live imaging is an important part of the developmental biologist's armoury of methods. In the case of the mouse embryo, recent advances in several disciplines including embryo culture, microscopy hardware and computational analysis have all contributed to our ability to probe dynamic events during early development. Together, these advances have provided us with a versatile and powerful 'toolkit', enabling us not only to image events during mouse embryogenesis, but also to intervene with them. In this short Spotlight article, we summarise advances and challenges in using live imaging specifically for understanding early mouse embryogenesis.

Establishment of a relationship between blastomere geometry and YAP localisation during compaction.

Precise patterning within the three-dimensional context of tissues, organs and embryos implies that cells can sense their relative position. During preimplantation development, outside and inside cells rely on apicobasal polarity and the Hippo pathway to choose their fate. Despite recent findings suggesting that mechanosensing might be central to this process, the relationship between blastomere geometry (i.e. shape and position) and the Hippo pathway effector YAP remains unknown. We used a highly quantitative approach to analyse information on the geometry and YAP localisation of individual blastomeres of mouse and human embryos. We identified the proportion of exposed cell surface area as most closely correlating with the nuclear localisation of YAP. To test this relationship, we developed several hydrogel-based approaches to alter blastomere geometry in cultured embryos. Unbiased clustering analyses of blastomeres from such embryos revealed that this relationship emerged during compaction. Our results therefore pinpoint the time during early embryogenesis when cells acquire the ability to sense changes in geometry and provide a new framework for how cells might integrate signals from different membrane domains to assess their relative position within the embryo.

Asymmetry in the frequency and position of mitosis in the mouse embryo epiblast at gastrulation.

At gastrulation, a subpopulation of epiblast cells constitutes a transient posteriorly located structure called the primitive streak, where cells that undergo epithelial-mesenchymal transition make up the mesoderm and endoderm lineages. Mouse embryo epiblast cells were labelled ubiquitously or in a mosaic fashion. Cell shape, packing, organization and division were recorded through live imaging during primitive streak formation. Posterior epiblast displays a higher frequency of rosettes, some of which associate with a central cell undergoing mitosis. Cells at the primitive streak, in particular delaminating cells, undergo mitosis more frequently than other epiblast cells. In pseudostratified epithelia, mitosis takes place at the apical side of the epithelium. However, mitosis is not restricted to the apical side of the epiblast, particularly on its posterior side. Non-apical mitosis occurs specifically in the streak even when ectopically located. Posterior non-apical mitosis results in one or two daughter cells leaving the epiblast layer. Cell rearrangement associated with mitotic cell rounding in posterior epiblast, in particular when non-apical, might thus facilitate cell ingression and transition to a mesenchymal phenotype.

The value of administering intravenous antibiotics during haemodialysis in the treatment of diabetic foot infections.

OBJECTIVE: People with diabetes who are on haemodialysis (HD) are at a high risk of diabetic foot infections (DFI) and related complications. We explored the value of treating DFI with intravenous (IV) antibiotics during HD. METHOD: This was an observational study of consecutively treated patients with DFIs with IV antibiotics during HD. Data collected included baseline characteristics, IV antibiotics used, details of multidisciplinary interventions and DFI treatment outcome. RESULTS: A cohort of 11 patients, mean (±standard deviation) age 62.4±12.7 years, had 15 episodes of treatment with IV antibiotics during HD. Of the patients, six (54.5%) were male and nine (81.8%) had type 2 diabetes. The estimated mean glomerular filtration rate (eGFR) was 11.4±3.9ml/minute. All patients had infected foot ulceration, soft tissue infection, six (54.5%) patients had osteomyelitis, and two (18.2%) had wet gangrene. The commonest IV antibiotic used was vancomycin (10/15 episodes, 66.7%). Other IV antibiotics used were daptomycin and meropenem. In three episodes, oral ciprofloxacin was used with IV antibiotics. The mean duration of antibiotic treatment was 9.2±4.9 weeks. Of the episodes, 11 (73.3%) were treated successfully with IV antibiotics alone and two (13.3%) episodes required minor surgical debridement/amputation. Some 10 (90.9%) members of the cohort had peripheral arterial disease and of those, five (50%) underwent angioplasty during IV antibiotic treatment. CONCLUSION: HD provides a good opportunity for treatment with IV antibiotics in DFI. This mode of administration of IV antibiotics, along with multidisciplinary intervention, is associated with ulcer healing and resolution of infection in over three-quarters of patients with DFI. DECLARATION OF INTEREST: The authors have no conflicts of interest to declare.

Characterization of embryonic surface ectoderm cell protrusions.

BACKGROUND: During embryonic development, complex changes in cell behavior generate the final form of the tissues. Extension of cell protrusions have been described as an important component in this process. Cellular protrusions have been associated with generation of traction, intercellular communication or establishment of signaling gradients. Here, we describe and compare in detail from live imaging data the dynamics of protrusions in the surface ectoderm of chick and mouse embryos. In particular, we explore the differences between cells surrounding the lens placode and other regions of the head. RESULTS: Our results showed that protrusions from the eye region in mouse embryos are longer than those in chick embryos. In addition, protrusions from regions where there are no significant changes in tissue shape are longer and more stable than protrusions that surround the invaginating lens placode. We did not find a clear directionality to the protrusions in any region. Finally, we observed intercellular trafficking of membrane puncta in the protrusions of both embryos in all the regions analyzed. CONCLUSIONS: In summary, the results presented here suggest that the dynamics of these protrusions adapt to their surroundings and possibly contribute to intercellular communication in embryonic cephalic epithelia.

Mechanics of mouse blastocyst hatching revealed by a hydrogel-based microdeformation assay.

Mammalian embryos are surrounded by an acellular shell, the zona pellucida. Hatching out of the zona is crucial for implantation and continued development of the embryo. Clinically, problems in hatching can contribute to failure in assisted reproductive intervention. Although hatching is fundamentally a mechanical process, due to limitations in methodology most studies focus on its biochemical properties. To understand the role of mechanical forces in hatching, we developed a hydrogel deformation-based method and analytical approach for measuring pressure in cyst-like tissues. Using this approach, we found that, in cultured blastocysts, pressure increased linearly, with intermittent falls. Inhibition of Na/K-ATPase led to a dosage-dependent reduction in blastocyst cavity pressure, consistent with its requirement for cavity formation. Reducing blastocyst pressure reduced the probability of hatching, highlighting the importance of mechanical forces in hatching. These measurements allowed us to infer details of microphysiology such as osmolarity, ion and water transport kinetics across the trophectoderm, and zona stiffness, allowing us to model the embryo as a thin-shell pressure vessel. We applied this technique to test whether cryopreservation, a process commonly used in assisted reproductive technology (ART), leads to alteration of the embryo and found that thawed embryos generated significantly lower pressure than fresh embryos, a previously unknown effect of cryopreservation. We show that reduced pressure is linked to delayed hatching. Our approach can be used to optimize in vitro fertilization (IVF) using precise measurement of embryo microphysiology. It is also applicable to other biological systems involving cavity formation, providing an approach for measuring forces in diverse contexts.

Peristaltic Elastic Instability in an Inflated Cylindrical Channel.

A long cylindrical cavity through a soft solid forms a soft microfluidic channel, or models a vascular capillary. We observe experimentally that, when such a channel bears a pressurized fluid, it first dilates homogeneously, but then becomes unstable to a peristaltic elastic instability. We combine theory and numerics to fully characterize the instability in a channel with initial radius a through an incompressible bulk neo-Hookean solid with shear modulus µ. We show instability occurs supercritically with wavelength 12.278…a when the cavity pressure exceeds 2.052…µ. In finite solids, the wavelength for peristalsis lengthens, with peristalsis ultimately being replaced by a long-wavelength bulging instability in thin-walled cylinders. Peristalsis persists in Gent strain-stiffening materials, provided the material can sustain extension by more than a factor of 6. Although naively a pressure driven failure mode of soft channels, the instability also offers a route to fabricate periodically undulating channels, producing, e.g., waveguides with photonic or phononic stop bands.

Towards understanding the roles of position and geometry on cell fate decisions during preimplantation development.

The first lineage segregation event in mouse embryos produces two separate cell populations: inner cell mass and trophectoderm. This is understood to be brought about by cells sensing their position within the embryo and differentiating accordingly. The cellular and molecular underpinnings of this process remain under investigation and have variously been considered to be completely stochastic or alternately, subject to some predisposition set up at fertilisation or before. Here, we consider these views in light of recent publications, discuss the possible role of cell geometry and mechanical forces in this process and describe how modelling could contribute in addressing this issue.

Limited predictive value of blastomere angle of division in trophectoderm and inner cell mass specification.

The formation of trophectoderm (TE) and pluripotent inner cell mass (ICM) is one of the earliest events during mammalian embryogenesis. It is believed that the orientation of division of polarised blastomeres in the 8- and 16-cell stage embryo determines the fate of daughter cells, based on how asymmetrically distributed lineage determinants are segregated. To investigate the relationship between angle of division and subsequent fate in unperturbed embryos, we constructed cellular resolution digital representations of the development of mouse embryos from the morula to early blastocyst stage, based on 4D confocal image volumes. We find that at the 16-cell stage, very few inside cells are initially produced as a result of cell division, but that the number increases due to cell movement. Contrary to expectations, outside cells at the 16-cell stage represent a heterogeneous population, with some fated to contributing exclusively to the TE and others capable of contributing to both the TE and ICM. Our data support the view that factors other than the angle of division, such as the position of a blastomere, play a major role in the specification of TE and ICM.

Bi-modal strategy of gastrulation in reptiles.

BACKGROUND: Amniote gastrulation is often described with respect to human, mouse and chick development by the presence of the primitive streak, a posterior-to-anterior midline morphological cell ingression feature that has come to define Amniote gastrulation. How this midline, ingression-based strategy of gastrulation evolved from the ancestral blastopore, a circumferential involution event in Anamniotes, is unknown. However, within the Amniote clade there exists a more diverse range of gastrulation strategies than just the primitive streak. Investigating gastrulation in a wider range of Amniotes provides a way to understand evolutionary transition from blastopore to the primitive streak. RESULTS: We analysed early to late gastrulation stages of Chamaeleo calyptratus, showing their unique morphology through confocal imaging of F-actin and laminin-stained embryos to visualise cell morphology and assess basal lamina integrity. We analysed the expression pattern of core mesodermal markers Brachyury and Fgf8 and complimented this analysis with that of the turtle, Trachemys scripta. CONCLUSIONS: Our analysis suggests that reptile gastrulation is bi-modal; primary internalization occurs anteriorly by means of an incomplete blastopore-like opening, while posteriorly the cells undergo ingression in the Brachyury-expressing blastoporal plate. This strategy stands mid-way between Anamniotes and Avians/Mammals, suggesting that blastoporal plate is a precursor of the avian primitive streak. Developmental Dynamics 244:1144-1157, 2015. © 2015 Wiley Periodicals, Inc.

Multi-cellular rosettes in the mouse visceral endoderm facilitate the ordered migration of anterior visceral endoderm cells.

The visceral endoderm (VE) is a simple epithelium that forms the outer layer of the egg-cylinder stage mouse embryo. The anterior visceral endoderm (AVE), a specialised subset of VE cells, is responsible for specifying anterior pattern. AVE cells show a stereotypic migratory behaviour within the VE, which is responsible for correctly orientating the anterior-posterior axis. The epithelial integrity of the VE is maintained during the course of AVE migration, which takes place by intercalation of AVE and other VE cells. Though a continuous epithelial sheet, the VE is characterised by two regions of dramatically different behaviour, one showing robust cell movement and intercalation (in which the AVE migrates) and one that is static, with relatively little cell movement and mixing. Little is known about the cellular rearrangements that accommodate and influence the sustained directional movement of subsets of cells (such as the AVE) within epithelia like the VE. This study uses an interdisciplinary approach to further our understanding of cell movement in epithelia. Using both wild-type embryos as well as mutants in which AVE migration is abnormal or arrested, we show that AVE migration is specifically linked to changes in cell packing in the VE and an increase in multi-cellular rosette arrangements (five or more cells meeting at a point). To probe the role of rosettes during AVE migration, we develop a mathematical model of cell movement in the VE. To do this, we use a vertex-based model, implemented on an ellipsoidal surface to represent a realistic geometry for the mouse egg-cylinder. The potential for rosette formation is included, along with various junctional rearrangements. Simulations suggest that while rosettes are not essential for AVE migration, they are crucial for the orderliness of this migration observed in embryos. Our simulations are similar to results from transgenic embryos in which Planar Cell Polarity (PCP) signalling is disrupted. Such embryos have significantly reduced rosette numbers, altered epithelial packing, and show abnormalities in AVE migration. Our results show that the formation of multi-cellular rosettes in the mouse VE is dependent on normal PCP signalling. Taken together, our model and experimental observations suggest that rosettes in the VE epithelium do not form passively in response to AVE migration. Instead, they are a PCP-dependent arrangement of cells that acts to buffer the disequilibrium in cell packing generated in the VE by AVE migration, enabling AVE cells to migrate in an orderly manner.

The fibronectin leucine-rich repeat transmembrane protein Flrt2 is required in the epicardium to promote heart morphogenesis.

The epicardium, the outermost tissue layer that envelops the developing heart and provides essential trophic signals for the myocardium, derives from the pro-epicardial organ (PEO). Two of the three members of the Flrt family of transmembrane glycoproteins, Flrt2 and Flrt3, are strongly co-expressed in the PEO. However, beginning at around day 10 of mouse development, following attachment and outgrowth, Flrt3 is selectively downregulated, and only Flrt2 is exclusively expressed in the fully delaminated epicardium. The present gene-targeting experiments demonstrate that mouse embryos lacking Flrt2 expression arrest at mid-gestation owing to cardiac insufficiency. The defects in integrity of the epicardial sheet and disturbed organization of the underlying basement membrane closely resemble those described in Flrt3-deficient embryos that fail to maintain cell-cell contacts in the anterior visceral endoderm (AVE) signalling centre that normally establishes the A-P axis. Using in vitro and in vivo reconstitution assays, we demonstrate that Flrt2 and Flrt3 are functionally interchangeable. When acting alone, either of these proteins is sufficient to rescue functional activities in the AVE and the developing epicardium.

Coordination of cell proliferation and anterior-posterior axis establishment in the mouse embryo.

During development, the growth of the embryo must be coupled to its patterning to ensure correct and timely morphogenesis. In the mouse embryo, migration of the anterior visceral endoderm (AVE) to the prospective anterior establishes the anterior-posterior (A-P) axis. By analysing the distribution of cells in S phase, M phase and G2 from the time just prior to the migration of the AVE until 18 hours after its movement, we show that there is no evidence for differential proliferation along the A-P axis of the mouse embryo. Rather, we have identified that as AVE movements are being initiated, the epiblast proliferates at a much higher rate than the visceral endoderm. We show that these high levels of proliferation in the epiblast are dependent on Nodal signalling and are required for A-P establishment, as blocking cell division in the epiblast inhibits AVE migration. Interestingly, inhibition of migration by blocking proliferation can be rescued by Dkk1. This suggests that the high levels of epiblast proliferation function to move the prospective AVE away from signals that are inhibitory to its migration. The finding that initiation of AVE movements requires a certain level of proliferation in the epiblast provides a mechanism whereby A-P axis development is coordinated with embryonic growth.

Nodal dependent differential localisation of dishevelled-2 demarcates regions of differing cell behaviour in the visceral endoderm.

The anterior visceral endoderm (AVE), a signalling centre within the simple epithelium of the visceral endoderm (VE), is required for anterior-posterior axis specification in the mouse embryo. AVE cells migrate directionally within the VE, thereby properly positioning the future anterior of the embryo and orientating the primary body axis. AVE cells consistently come to an abrupt stop at the border between the anterior epiblast and extra-embryonic ectoderm, which represents an end-point to their proximal migration. Little is known about the underlying basis for this barrier and how surrounding cells in the VE respond to or influence AVE migration. We use high-resolution 3D reconstructions of protein localisation patterns and time-lapse microscopy to show that AVE cells move by exchanging neighbours within an intact epithelium. Cell movement and mixing is restricted to the VE overlying the epiblast, characterised by the enrichment of Dishevelled-2 (Dvl2) to the lateral plasma membrane, a hallmark of Planar Cell Polarity (PCP) signalling. AVE cells halt upon reaching the adjoining region of VE overlying the extra-embryonic ectoderm, which displays reduced neighbour exchange and in which Dvl2 is excluded specifically from the plasma membrane. Though a single continuous sheet, these two regions of VE show distinct patterns of F-actin localisation, in cortical rings and an apical shroud, respectively. We genetically perturb PCP signalling and show that this disrupts the localisation pattern of Dvl2 and F-actin and the normal migration of AVE cells. In Nodal null embryos, membrane localisation of Dvl2 is reduced, while in mutants for the Nodal inhibitor Lefty1, Dvl2 is ectopically membrane localised, establishing a role for Nodal in modulating PCP signalling. These results show that the limits of AVE migration are determined by regional differences in cell behaviour and protein localisation within an otherwise apparently uniform VE. In addition to coordinating global cell movements across epithelia (such as during convergence extension), PCP signalling in interplay with TGFß signalling can demarcate regions of differing behaviour within epithelia, thereby modulating the movement of cells within them.

An integrated approach identifies the molecular underpinnings of murine anterior visceral endoderm migration.

The anterior visceral endoderm (AVE) differs from the surrounding visceral endoderm (VE) in its migratory behavior and ability to restrict primitive streak formation to the opposite side of the mouse embryo. To characterize the molecular bases for the unique properties of the AVE, we combined single-cell RNA sequencing of the VE prior to and during AVE migration with phosphoproteomics, high-resolution live-imaging, and short-term lineage labeling and intervention. This identified the transient nature of the AVE with attenuation of "anteriorizing" gene expression as cells migrate and the emergence of heterogeneities in transcriptional states relative to the AVE's position. Using cell communication analysis, we identified the requirement of semaphorin signaling for normal AVE migration. Lattice light-sheet microscopy showed that Sema6D mutants have abnormalities in basal projections and migration speed. These findings point to a tight coupling between transcriptional state and position of the AVE and identify molecular controllers of AVE migration.

Embryonic and extraembryonic tissues during mammalian development: shifting boundaries in time and space.

The first few days of embryonic development in eutherian mammals are dedicated to the specification and elaboration of the extraembryonic tissues. However, where the fetus ends and its adnexa begins is not always as self-evident during the early stages of development, when the definitive body axes are still being laid down, the germ layers being specified and a discrete form or bodyplan is yet to emerge. Function, anatomy, histomorphology and molecular identities have been used through the history of embryology, to make this distinction. In this review, we explore them individually by using specific examples from the early embryo. While highlighting the challenges of drawing discrete boundaries between embryonic and extraembryonic tissues and the limitations of a binary categorization, we discuss how basing such identity on fate is the most universal and conceptually consistent. This article is part of the theme issue 'Extraembryonic tissues: exploring concepts, definitions and functions across the animal kingdom'.