Explore the vast range of titles available.

Formation of the body of vertebrate embryos proceeds sequentially by posterior addition of tissues from the tail bud. Cells of the tail bud and the posterior presomitic mesoderm, which control posterior elongation 1 , exhibit a high level of aerobic glycolysis that is reminiscent of the metabolic status of cancer cells experiencing the Warburg effect 2 , 3 . Glycolytic activity downstream of fibroblast growth factor controls WNT signalling in the tail bud 3 . In the neuromesodermal precursors of the tail bud 4 , WNT signalling promotes the mesodermal fate that is required for sustained axial elongation, at the expense of the neural fate 3 , 5 . How glycolysis regulates WNT signalling in the tail bud is currently unknown. Here we used chicken embryos and human tail bud-like cells differentiated in vitro from induced pluripotent stem cells to show that these cells exhibit an inverted pH gradient, with the extracellular pH lower than the intracellular pH, as observed in cancer cells 6 . Our data suggest that glycolysis increases extrusion of lactate coupled to protons via the monocarboxylate symporters. This contributes to elevating the intracellular pH in these cells, which creates a favourable chemical environment for non-enzymatic β-catenin acetylation downstream of WNT signalling. As acetylated β-catenin promotes mesodermal rather than neural fate 7 , this ultimately leads to activation of mesodermal transcriptional WNT targets and specification of the paraxial mesoderm in tail bud precursors. Our work supports the notion that some tumour cells reactivate a developmental metabolic programme. The authors show that metabolic activity leads to an increase in the intracellular pH of neuromesodermal precursors, and that this increase in pH, by allowing post-translational modification of β-catenin, is required for the activation of WNT signalling and mesodermal fate acquisition.

In classical descriptions of vertebrate development, the segregation of the three embryonic germ layers completes by the end of gastrulation. Body formation then proceeds in a head to tail fashion by progressive deposition of lineage-committed progenitors during regression of the primitive streak (PS) and tail bud (TB). The identification by retrospective clonal analysis of a population of neuromesodermal progenitors (NMPs) contributing to both musculoskeletal precursors (paraxial mesoderm) and spinal cord during axis formation challenged these notions. However, classical fate mapping studies of the PS region in amniotes have so far failed to provide direct evidence for such bipotential cells at the single-cell level. Here, using lineage tracing and single-cell RNA sequencing in the chicken embryo, we identify a resident cell population of the anterior PS epiblast, which contributes to neural and mesodermal lineages in trunk and tail. These cells initially behave as monopotent progenitors as classically described and only acquire a bipotential fate later, in more posterior regions. We show that NMPs exhibit a conserved transcriptomic signature during axis elongation but lose their epithelial characteristicsin the TB. Posterior to anterior gradients of convergence speed and ingression along the PS lead to asymmetric exhaustion of PS mesodermal precursor territories. Through limited ingression and increased proliferation, NMPs are maintained and amplified as a cell population which constitute the main progenitors in the TB. Together, our studies provide a novel understanding of the PS and TB contribution through the NMPs to the formation of the body of amniote embryos.

The segmental organization of the vertebral column is established early in embryogenesis, when pairs of somites are rhythmically produced by the presomitic mesoderm (PSM). The tempo of somite formation is controlled by a molecular oscillator known as the segmentation clock 1 , 2 . Although this oscillator has been well-characterized in model organisms 1 , 2 , whether a similar oscillator exists in humans remains unknown. Genetic analyses of patients with severe spine segmentation defects have implicated several human orthologues of cyclic genes that are associated with the mouse segmentation clock, suggesting that this oscillator might be conserved in humans 3 . Here we show that human PSM cells derived in vitro—as well as those of the mouse 4 —recapitulate the oscillations of the segmentation clock. Human PSM cells oscillate with a period two times longer than that of mouse cells (5 h versus 2.5 h), but are similarly regulated by FGF, WNT, Notch and YAP signalling 5 . Single-cell RNA sequencing reveals that mouse and human PSM cells in vitro follow a developmental trajectory similar to that of mouse PSM in vivo. Furthermore, we demonstrate that FGF signalling controls the phase and period of oscillations, expanding the role of this pathway beyond its classical interpretation in ‘clock and wavefront’ models 1 . Our work identifying the human segmentation clock represents an important milestone in understanding human developmental biology. Human presomitic mesoderm cells derived in vitro demonstrate oscillations of the segmentation clock, thus providing a window into an otherwise inaccessible stage of human development.

In classical descriptions of vertebrate development, the segregation of the three embryonic germ layers is completed by the end of gastrulation. Body formation then proceeds in a head to tail fashion by progressive deposition of lineage committed progenitors during regression of the Primitive Streak (PS) and tail bud (Pasteels, 1937b; Stern, 2004). Identification of Neuro-Mesodermal Progenitors (NMPs) contributing to both musculo-skeletal precursors (paraxial mesoderm) and spinal cord during axis formation by retrospective clonal analysis challenged these notions (Henrique et al., 2015; Tzouanacou et al., 2009). However, in amniotes such as mouse and chicken, the precise identity and localization of these cells has remained unclear despite a wealth of fate mapping analyses of the PS region. Here, we use lineage tracing in the chicken embryo to show that single cells located in the SOX2/T positive anterior PS region contribute to both neural and mesodermal lineages in the trunk and tail, but only express this bipotential fate with some delay. We demonstrate that posterior to anterior gradients of convergence speed and ingression along the PS gradually lead to exhaustion of all mesodermal precursor territories except for NMPs where limited ingression and increased proliferation maintain and amplify this pool of axial progenitors. As a result, most of the remaining mesodermal precursors from the PS in the tail bud are bipotential NMPs. Together, our results provide a novel understanding of the contribution of the PS and tail bud to the formation of the body of amniote embryos.

Investigating single-cell dynamics and morphology in tissues and embryos requires highly accurate quantitative analysis of microscopy images. Despite significant advances in the field of bioimage analysis, even the most sophisticated segmentation and tracking algorithms inevitably produce errors (e.g. : over segmentation, missing objects, miss-connected objects). Although error rate may be small, their propagation throughout a time-lapse sequence has catastrophic effects on the accuracy of tracking and extraction of single cell parameters. Extracting single cell temporal information in the context of tissue/embryo requires thus expert curation to identify and correct segmentation errors. In the movies commonly used in developmental biology and stem cell research, both the number of imaged cells and the duration of recording are large, making this manual correction task extremely time-consuming. This has now become a major bottleneck in the fields of development, stem cell biology and bioimage analysis. We present here EpiCure (Epithelial Curation), a versatile tool designed to streamline and accelerate manual curation of segmentation and tracking in 2D movies of large epithelial tissues. EpiCure uses temporal information and morphometric parameters to automatically identify segmentation and tracking errors and provides user-friendly tools to correct them. It focuses on ergonomics and offers several visualization options to help navigating in movies of tissue covering a large number of cells, speeding up the detection of errors and their curation. EpiCure is highly interoperable and supports input from a wide range of segmentation tools. It also includes multiple export filters, enabling seamless integration with downstream analysis pipelines. In this paper, using movies from several animal models, we highlight the importance of curating cell segmentation and tracking for accurate downstream analysis, and demonstrate how EpiCure helps the curation process for extracting accurate single cell dynamics and cellular events detection, making it faster and amenable on large dataset.

Despite progress in probing tissue mechanics, direct long-term measurements in live embryonic epithelia are lacking. This limits our understanding of amniote embryonic morphogenesis, which takes place over hours or days. Here, we introduce a combined technological and analytical framework to directly measure active forces and material properties in developing quail embryos, in a minimally invasive way. We find that the embryonic epithelium behaves elastically on short timescales with a modulus ∼ 2 nN · μm−1 but flows over longer timescales with a viscosity ∼ 4 μN · s · μm−1, and that both properties are under active regulation. Notably, we demonstrate that cell division is an active epithelial fluidizer, with division rates correlating with tissue fluidity. This fluidization is crucial for the emergence of the primary embryonic axis, which we show is shaped by a force ∼ 2 μN. Altogether, our approach delivers a complete and quantitative view of early embryonic mechanics, and opens new avenues for biomechanical studies in live embryos and tissues.

The segregation of the extra-embryonic lineage is one of the earliest events and a key step in amniote development. Whereas the regulation of extra-embryonic cell fate specification has been extensively studied, little is known about the morphogenetic events underlying the formation of this lineage. Here, taking advantage of the amenability of avian embryos to live and quantitative imaging, we investigate the cell- and tissue-scale dynamics of epiboly, the process during which the epiblast expands to engulf the entire yolk. We show that tension arising from the outward migration of the epiblast border on the vitelline membrane stretches extra-embryonic cells, which reversibly transition from a columnar to squamous morphology. The propagation of this tension is strongly attenuated in the embryonic territory, which concomitantly undergoes fluid-like motion, culminating in the formation of the primitive streak. We formulate a simple viscoelastic model in which the tissue responds elastically to isotropic stress but flows in response to shear stress, and show that it recapitulates the flows and deformation of both embryonic and extra-embryonic tissues. Together, our results clarify the mechanical basis of early avian embryogenesis and provide a framework unifying the divergent mechanical behaviors observed in the contiguous embryonic and extra-embryonic territories that make up the epiblast.Competing Interest StatementThe authors have declared no competing interest.

Recent advances in the field of mechanobiology have led to the development of methods to characterize single-cell or monolayer mechanical properties and link them to their functional behaviour. However, there remains a strong need to establish this link for three-dimensional multicellular aggregates, which better mimic tissue function. Here we present a platform to actuate and observe many such aggregates within one deformable micro-device. The platform consists of a single PDMS piece cast on a 3D-printed mold and bonded to a glass slide or coverslip. It consists of a chamber containing cell spheroids, which is adjacent to air cavities that are fluidically independent. Controlling the air pressure in these air cavities leads to a vertical displacement of the chamber’s ceiling. The device can be used in static or dynamic modes over time-scales of seconds to hours, with displacement amplitudes from a few μm to several tens of microns. Further, we show how the compression protocols can be used to obtain measurements of stiffness heterogeneities within individual co-culture spheroids, by comparing image correlations of spheroids at different levels of compression with finite element simulations. The labeling of the cells and their cytoskeleton is combined with image correlation methods to relate the structure of the co-culture spheroid with its mechanical properties at different locations. The device is compatible with various microscopy techniques, including confocal microscopy, which can be used to observe the displacements and rearrangements of single cells and neighborhoods within the aggregate. The complete experimental and imaging platform can now be used to provide multi-scale measurements that link single-cell behavior with the global mechanical response of the aggregates.

Developmental morphogenesis is driven by tissue stresses acting on tissue rheology. Direct measurements of forces in small tissues (0.1-1mm) in situ such as in early embryos require high spatial precision and minimal invasiveness. Here we report tissue force microscopy (TiFM) integrating a vertical cantilever probe and live imaging to enable close-loop control of mechanical loading in early chicken embryos. By testing previously qualitatively characterized force-producing tissues in the elongating body axis, we show that TiFM quantitatively captures stress dynamics with high sensitivity. TiFM also provides the capacity of applying a stable, minimally-invasive and physiologically relevant load to drive tissue deformation, which alters morphogenetic progression and cell movements. Together, TiFM addresses a key technological gap in tissue force measurement and manipulation in small developing embryos, and promises to contribute to the quantitative understanding of complex multi-tissue mechanics during development.Competing Interest StatementThe authors have declared no competing interest.Footnotes* New results added to show deformation of different tissues under mechanical load (Figure 3) New discussion and technical notes added regarding probe depth and tissue contact area estimation, and sources and considerations of measurement errors (method sections 4.3, 4.4) New control experiments added for foil experiments (Figure S3, movies)

The vertebrate bauplan is primarily established via the formation of embryonic tissues in a head-to-tail progression. The biomechanics of this elongation, which requires the presomitic mesoderm (PSM), remains poorly understood. Here, we find that avian PSM explants can elongate autonomously when physically confined in vitro, producing a pushing force that can largely account for the posterior elongation of the embryo. Tissue elongation results from volumetric expansion that is driven by cellular activity and accompanied by inhomogeneous increase of the extracellular fraction along the AP axis. We show that FGF signaling promotes glycolysis-dependent production of Hyaluronic Acid (HA), which is required for expansion of the posterior PSM. Our findings link body axis elongation to tissue expansion through the metabolic control of extracellular matrix production downstream of FGF signaling. Active tissue expansion propels body elongation independent of cell proliferation-driven growth