Using a micro-device with a deformable ceiling to probe stiffness heterogeneities within 3D cell aggregates.

Recent advances in the field of mechanobiology have led to the development of methods to characterise 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 (3D) 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 polydimethylsiloxane piece cast on a 3D-printed mould 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 fewµm 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 labelling 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 neighbourhoods within the aggregate. The complete experimental and imaging platform can now be used to provide multi-scale measurements that link single-cell behaviour with the global mechanical response of the aggregates.

Mechanical feedback defines organizing centers to drive digit emergence.

During embryonic development, digits gradually emerge in a periodic pattern. Although genetic evidence indicates that digit formation results from a self-organizing process, the underlying mechanisms are still unclear. Here, we find that convergent-extension tissue flows driven by active stresses underlie digit formation. These active stresses simultaneously shape cartilage condensations and lead to the emergence of a compressive stress region that promotes high activin/p-SMAD/SOX9 expression, thereby defining digit-organizing centers via a mechanical feedback. In Wnt5a mutants, such mechanical feedback is disrupted due to the loss of active stresses, organizing centers do not emerge, and digit formation is precluded. Thus, digit emergence does not result solely from molecular interactions, as was previously thought, but requires a mechanical feedback that ensures continuous coupling between phalanx specification and elongation. Our work, which links mechanical and molecular signals, provides a mechanistic context for the emergence of organizing centers that may underlie various developmental processes.

Force-generating apoptotic cells orchestrate avian neural tube bending.

Apoptosis plays an important role in morphogenesis, and the notion that apoptotic cells can impact their surroundings came to light recently. However, how this applies to vertebrate morphogenesis remains unknown. Here, we use the formation of the neural tube to determine how apoptosis contributes to morphogenesis in vertebrates. Neural tube closure defects have been reported when apoptosis is impaired in vertebrates, although the cellular mechanisms involved are unknown. Using avian embryos, we found that apoptotic cells generate an apico-basal force before being extruded from the neuro-epithelium. This force, which relies on a contractile actomyosin cable that extends along the apico-basal axis of the cell, drives nuclear fragmentation and influences the neighboring tissue. Together with the morphological defects observed when apoptosis is prevented, these data strongly suggest that the neuroepithelium keeps track of the mechanical impact of apoptotic cells and that the apoptotic forces, cumulatively, contribute actively to neural tube bending.

LocalZProjector and DeProj: a toolbox for local 2D projection and accurate morphometrics of large 3D microscopy images.

BACKGROUND: Quantitative imaging of epithelial tissues requires bioimage analysis tools that are widely applicable and accurate. In the case of imaging 3D tissues, a common preprocessing step consists of projecting the acquired 3D volume on a 2D plane mapping the tissue surface. While segmenting the tissue cells is amenable on 2D projections, it is still very difficult and cumbersome in 3D. However, for many specimen and models used in developmental and cell biology, the complex content of the image volume surrounding the epithelium in a tissue often reduces the visibility of the biological object in the projection, compromising its subsequent analysis. In addition, the projection may distort the geometry of the tissue and can lead to strong artifacts in the morphology measurement. RESULTS: Here we introduce a user-friendly toolbox built to robustly project epithelia on their 2D surface from 3D volumes and to produce accurate morphology measurement corrected for the projection distortion, even for very curved tissues. Our toolbox is built upon two components. LocalZProjector is a configurable Fiji plugin that generates 2D projections and height-maps from potentially large 3D stacks (larger than 40 GB per time-point) by only incorporating signal of the planes with local highest variance/mean intensity, despite a possibly complex image content. DeProj is a MATLAB tool that generates correct morphology measurements by combining the height-map output (such as the one offered by LocalZProjector) and the results of a cell segmentation on the 2D projection, hence effectively deprojecting the 2D segmentation in 3D. In this paper, we demonstrate their effectiveness over a wide range of different biological samples. We then compare its performance and accuracy against similar existing tools. CONCLUSIONS: We find that LocalZProjector performs well even in situations where the volume to project also contains unwanted signal in other layers. We show that it can process large images without a pre-processing step. We study the impact of geometrical distortions on morphological measurements induced by the projection. We measured very large distortions which are then corrected by DeProj, providing accurate outputs.

Roadmap for the multiscale coupling of biochemical and mechanical signals during development.

The way in which interactions between mechanics and biochemistry lead to the emergence of complex cell and tissue organization is an old question that has recently attracted renewed interest from biologists, physicists, mathematicians and computer scientists. Rapid advances in optical physics, microscopy and computational image analysis have greatly enhanced our ability to observe and quantify spatiotemporal patterns of signalling, force generation, deformation, and flow in living cells and tissues. Powerful new tools for genetic, biophysical and optogenetic manipulation are allowing us to perturb the underlying machinery that generates these patterns in increasingly sophisticated ways. Rapid advances in theory and computing have made it possible to construct predictive models that describe how cell and tissue organization and dynamics emerge from the local coupling of biochemistry and mechanics. Together, these advances have opened up a wealth of new opportunities to explore how mechanochemical patterning shapes organismal development. In this roadmap, we present a series of forward-looking case studies on mechanochemical patterning in development, written by scientists working at the interface between the physical and biological sciences, and covering a wide range of spatial and temporal scales, organisms, and modes of development. Together, these contributions highlight the many ways in which the dynamic coupling of mechanics and biochemistry shapes biological dynamics: from mechanoenzymes that sense force to tune their activity and motor output, to collectives of cells in tissues that flow and redistribute biochemical signals during development.

Transgenesis and web resources in quail.

Due to its amenability to manipulations, to live observation and its striking similarities to mammals, the chicken embryo has been one of the major animal models in biomedical research. Although it is technically possible to genome-edit the chicken, its long generation time (6 months to sexual maturity) makes it an impractical lab model and has prevented it widespread use in research. The Japanese quail (Coturnix coturnix japonica) is an attractive alternative, very similar to the chicken, but with the decisive asset of a much shorter generation time (1.5 months). In recent years, transgenic quail lines have been described. Most of them were generated using replication-deficient lentiviruses, a technique that presents diverse limitations. Here, we introduce a novel technology to perform transgenesis in quail, based on the in vivo transfection of plasmids in circulating Primordial Germ Cells (PGCs). This technique is simple, efficient and allows using the infinite variety of genome engineering approaches developed in other models. Furthermore, we present a website centralizing quail genomic and technological information to facilitate the design of genome-editing strategies, showcase the past and future transgenic quail lines and foster collaborative work within the avian community.

The quail genome: insights into social behaviour, seasonal biology and infectious disease response.

BACKGROUND: The Japanese quail (Coturnix japonica) is a popular domestic poultry species and an increasingly significant model species in avian developmental, behavioural and disease research. RESULTS: We have produced a high-quality quail genome sequence, spanning 0.93 Gb assigned to 33 chromosomes. In terms of contiguity, assembly statistics, gene content and chromosomal organisation, the quail genome shows high similarity to the chicken genome. We demonstrate the utility of this genome through three diverse applications. First, we identify selection signatures and candidate genes associated with social behaviour in the quail genome, an important agricultural and domestication trait. Second, we investigate the effects and interaction of photoperiod and temperature on the transcriptome of the quail medial basal hypothalamus, revealing key mechanisms of photoperiodism. Finally, we investigate the response of quail to H5N1 influenza infection. In quail lung, many critical immune genes and pathways were downregulated after H5N1 infection, and this may be key to the susceptibility of quail to H5N1. CONCLUSIONS: We have produced a high-quality genome of the quail which will facilitate further studies into diverse research questions using the quail as a model avian species.

A tensile ring drives tissue flows to shape the gastrulating amniote embryo.

Tissue morphogenesis is driven by local cellular deformations that are powered by contractile actomyosin networks. How localized forces are transmitted across tissues to shape them at a mesoscopic scale is still unclear. Analyzing gastrulation in entire avian embryos, we show that it is driven by the graded contraction of a large-scale supracellular actomyosin ring at the margin between the embryonic and extraembryonic territories. The propagation of these forces is enabled by a fluid-like response of the epithelial embryonic disk, which depends on cell division. A simple model of fluid motion entrained by a tensile ring quantitatively captures the vortex-like "polonaise" movements that accompany the formation of the primitive streak. The geometry of the early embryo thus arises from the transmission of active forces generated along its boundary.

Timed Collinear Activation of Hox Genes during Gastrulation Controls the Avian Forelimb Position.

Limb position along the body is highly consistent within one species but very variable among vertebrates. Despite major advances in our understanding of limb patterning in three dimensions, how limbs reproducibly form along the antero-posterior axis remains largely unknown. Hox genes have long been suspected to control limb position; however, supporting evidences are mostly correlative and their role in this process is unclear. Here, we show that limb position is determined early in development through the action of Hox genes. Dynamic lineage analysis revealed that, during gastrulation, the forelimb, interlimb, and hindlimb fields are progressively generated and concomitantly patterned by the collinear activation of Hox genes in a two-step process. First, the sequential activation of Hoxb genes controls the relative position of their own collinear domains of expression in the forming lateral plate mesoderm, as demonstrated by functional perturbations during gastrulation. Then, within these collinear domains, we show that Hoxb4 anteriorly and Hox9 genes posteriorly, respectively, activate and repress the expression of the forelimb initiation gene Tbx5 and instruct the definitive position of the forelimb. Furthermore, by comparing the dynamics of Hoxb genes activation during zebra finch, chicken, and ostrich gastrulation, we provide evidences that changes in the timing of collinear Hox gene activation might underlie natural variation in forelimb position between different birds. Altogether, our results that characterize the cellular and molecular mechanisms underlying the regulation and natural variation of forelimb positioning in avians show a direct and early role for Hox genes in this process.

Cell Division Drives Epithelial Cell Rearrangements during Gastrulation in Chick.

During early embryonic development, cells are organized as cohesive epithelial sheets that are continuously growing and remodeled without losing their integrity, giving rise to a wide array of tissue shapes. Here, using live imaging in chick embryo, we investigate how epithelial cells rearrange during gastrulation. We find that cell division is a major rearrangement driver that powers dramatic epithelial cell intercalation events. We show that these cell division-mediated intercalations, which represent the majority of epithelial rearrangements within the early embryo, are absolutely necessary for the spatial patterning of gastrulation movements. Furthermore, we demonstrate that these intercalation events result from overall low cortical actomyosin accumulation within the epithelial cells of the embryo, which enables dividing cells to remodel junctions in their vicinity. These findings uncover a role for cell division as coordinator of epithelial growth and remodeling that might underlie various developmental, homeostatic, or pathological processes in amniotes.

A relative shift in cloacal location repositions external genitalia in amniote evolution.

The move of vertebrates to a terrestrial lifestyle required major adaptations in their locomotory apparatus and reproductive organs. While the fin-to-limb transition has received considerable attention, little is known about the developmental and evolutionary origins of external genitalia. Similarities in gene expression have been interpreted as a potential evolutionary link between the limb and genitals; however, no underlying developmental mechanism has been identified. We re-examined this question using micro-computed tomography, lineage tracing in three amniote clades, and RNA-sequencing-based transcriptional profiling. Here we show that the developmental origin of external genitalia has shifted through evolution, and in some taxa limbs and genitals share a common primordium. In squamates, the genitalia develop directly from the budding hindlimbs, or the remnants thereof, whereas in mice the genital tubercle originates from the ventral and tail bud mesenchyme. The recruitment of different cell populations for genital outgrowth follows a change in the relative position of the cloaca, the genitalia organizing centre. Ectopic grafting of the cloaca demonstrates the conserved ability of different mesenchymal cells to respond to these genitalia-inducing signals. Our results support a limb-like developmental origin of external genitalia as the ancestral condition. Moreover, they suggest that a change in the relative position of the cloacal signalling centre during evolution has led to an altered developmental route for external genitalia in mammals, while preserving parts of the ancestral limb molecular circuitry owing to a common evolutionary origin.

Vertebrate limb bud formation is initiated by localized epithelial-to-mesenchymal transition.

Vertebrate limbs first emerge as small buds at specific locations along the trunk. Although a fair amount is known about the molecular regulation of limb initiation and outgrowth, the cellular events underlying these processes have remained less clear. We show that the mesenchymal limb progenitors arise through localized epithelial-to-mesenchymal transition (EMT) of the coelomic epithelium specifically within the presumptive limb fields. This EMT is regulated at least in part by Tbx5 and Fgf10, two genes known to control limb initiation. This work shows that limb buds initiate earlier than previously thought, as a result of localized EMT rather than differential proliferation rates.

WNT5A/JNK and FGF/MAPK pathways regulate the cellular events shaping the vertebrate limb bud.

BACKGROUND: The vertebrate limb is a classical model for understanding patterning of three-dimensional structures during embryonic development. Although decades of research have elucidated the tissue and molecular interactions within the limb bud required for patterning and morphogenesis of the limb, the cellular and molecular events that shape the limb bud itself have remained largely unknown. RESULTS: We show that the mesenchymal cells of the early limb bud are not disorganized within the ectoderm as previously thought but are instead highly organized and polarized. Using time-lapse video microscopy, we demonstrate that cells move and divide according to this orientation. The combination of oriented cell divisions and movements drives the proximal-distal elongation of the limb bud necessary to set the stage for subsequent morphogenesis. These cellular events are regulated by the combined activities of the WNT and FGF pathways. We show that WNT5A/JNK is necessary for the proper orientation of cell movements and cell division. In contrast, the FGF/MAPK signaling pathway, emanating from the apical ectodermal ridge, does not regulate cell orientation in the limb bud but instead establishes a gradient of cell velocity enabling continuous rearrangement of the cells at the distal tip of the limb. CONCLUSIONS: Together, these data shed light on the cellular basis of vertebrate limb bud morphogenesis and uncover new layers to the sequential signaling pathways acting during vertebrate limb development.

The timing of emergence of muscle progenitors is controlled by an FGF/ERK/SNAIL1 pathway.

In amniotes, the dermomyotome is the source of all skeletal muscles of the trunk and the limbs. Trunk skeletal muscles form in two sequential stages: in the first stage, cells located at the four borders of the epithelial dermomyotome delaminate to generate the primary myotome, composed of post-mitotic, mononucleated myocytes. The epithelio-mesenchymal transition (EMT) of the central dermomyotome initiates the second stage of muscle formation, characterised by a massive entry of mitotic muscle progenitors from the central region of the dermomyotome into the primary myotome. The signals that regulate the timing of the dermomyotome EMT are unknown. Here, we propose that this process is regulated by an FGF signal emanating from the primary myotome, a known source of FGF. The over-expression of FGF results in a precocious EMT of the dermomyotome, while on the contrary, the inhibition of FGF signalling by the electoporation of a dominant-negative form of FGFR4 delays this process. Within the dermomyotome, FGF signalling triggers a MAPK/ERK pathway that leads to the activation of the transcription factor Snail1, a known regulator of EMT in a number of cellular contexts. The activation or the inhibition of the MAPK/ERK pathway and of Snail1 mimics that of FGF signalling and leads to an early or delayed EMT of the dermomyotome, respectively. Altogether, our results indicate that in amniotes, the primary myotome is an organizing center that regulates the timely entry of embryonic muscle progenitors within the muscle masses, thus initiating the growth phase of the trunk skeletal muscles.

Cell movements at Hensen's node establish left/right asymmetric gene expression in the chick.

In vertebrates, the readily apparent left/right (L/R) anatomical asymmetries of the internal organs can be traced to molecular events initiated at or near the time of gastrulation. However, the earliest steps of this process do not seem to be universally conserved. In particular, how this axis is first defined in chicks has remained problematic. Here we show that asymmetric cell rearrangements take place within chick embryos, creating a leftward movement of cells around the node. It is the relative displacement of cells expressing sonic hedgehog (Shh) and fibroblast growth factor 8 (Fgf8) that is responsible for establishing their asymmetric expression patterns. The creation of asymmetric expression domains as a passive effect of cell movements represents an alternative strategy for breaking L/R symmetry in gene activity.

WNT11 acts as a directional cue to organize the elongation of early muscle fibres.

The early vertebrate skeletal muscle is a well-organized tissue in which the primitive muscle fibres, the myocytes, are all parallel and aligned along the antero-posterior axis of the embryo. How myofibres acquire their orientation during development is unknown. Here we show that during early chick myogenesis WNT11 has an essential role in the oriented elongation of the myocytes. We find that the neural tube, known to drive WNT11 expression in the medial border of somites, is necessary and sufficient to orient myocyte elongation. We then show that the specific inhibition of WNT11 function in somites leads to the disorganization of myocytes. We establish that WNT11 mediates this effect through the evolutionary conserved planar cell polarity (PCP) pathway, downstream of the WNT/beta-catenin-dependent pathway, required to initiate the myogenic program of myocytes and WNT11 expression. Finally, we demonstrate that a localized ectopic source of WNT11 can markedly change the orientation of myocytes, indicating that WNT11 acts as a directional cue in this process. All together, these data show that the sequential action of the WNT/PCP and the WNT/beta-catenin pathways is necessary for the formation of fully functional embryonic muscle fibres. This study also provides evidence that WNTs can act as instructive cues to regulate the PCP pathway in vertebrates.

The chirality of gut rotation derives from left-right asymmetric changes in the architecture of the dorsal mesentery.

We have investigated the structural basis by which the counterclockwise direction of the amniote gut is established. The chirality of midgut looping is determined by left-right asymmetries in the cellular architecture of the dorsal mesentery, the structure that connects the primitive gut tube to the body wall. The mesenchymal cells of the dorsal mesentery are more condensed on the left side than on the right and, additionally, the overlying epithelium on the left side exhibits a columnar morphology, in contrast to a cuboidal morphology on the right. These properties are instructed by a set of transcription factors: Pitx2 and Isl1 specifically expressed on the left side, and Tbx18 expressed on the right, regulated downstream of the secreted protein Nodal which is present exclusively on the left side. The resultant differences in cellular organization cause the mesentery to assume a trapezoidal shape, tilting the primitive gut tube leftward.