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As embryos develop, numerous cell types with distinct functions and morphologies arise from pluripotent cells. Three research groups have used single-cell RNA sequencing to analyze the transcriptional changes accompanying development of vertebrate embryos (see the Perspective by Harland). Wagner et al. sequenced the transcriptomes of more than 90,000 cells throughout zebrafish development to reveal how cells differentiate during axis patterning, germ layer formation, and early organogenesis. Farrell et al. profiled the transcriptomes of tens of thousands of embryonic cells and applied a computational approach to construct a branching tree describing the transcriptional trajectories that lead to 25 distinct zebrafish cell types. The branching tree revealed how cells change their gene expression as they become more and more specialized. Briggs et al. examined whole frog embryos, spanning zygotic genome activation through early organogenesis, to map cell states and differentiation across all cell lineages over time. These data and approaches pave the way for the comprehensive reconstruction of transcriptional trajectories during development. Science , this issue p. 981 , p. eaar3131 , p. eaar5780 ; see also p. 967 Single-cell RNA sequencing and a computational technique reveal cell trajectories that form the complex body plan of the zebrafish embryo. During embryogenesis, cells acquire distinct fates by transitioning through transcriptional states. To uncover these transcriptional trajectories during zebrafish embryogenesis, we sequenced 38,731 cells and developed URD, a simulated diffusion-based computational reconstruction method. URD identified the trajectories of 25 cell types through early somitogenesis, gene expression along them, and their spatial origin in the blastula. Analysis of Nodal signaling mutants revealed that their transcriptomes were canalized into a subset of wild-type transcriptional trajectories. Some wild-type developmental branch points contained cells that express genes characteristic of multiple fates. These cells appeared to trans-specify from one fate to another. These findings reconstruct the transcriptional trajectories of a vertebrate embryo, highlight the concurrent canalization and plasticity of embryonic specification, and provide a framework with which to reconstruct complex developmental trees from single-cell transcriptomes.

D’Arcy Thompson predicted a century ago that animal body shape is conditioned by gravity, but there has been no animal model to study how cellular forces are coordinated to generate body shapes that withstand gravity; the hirame medaka fish mutant, with pronounced body flattening, reveals how the hirame /YAP gene controls gravity-resisting cellular forces to produce complex 3D organs and body shapes. YAP protein gives tissue shape How is tissue tension controlled at the organismal level to maintain body shape and complex three-dimensional structures? Makoto Furutani-Seiki and colleagues describe a medaka fish mutant, hirame ( hir ), with a flattened body. They show that the phenotype is due to reduction of internal forces caused by the absence of YAP protein, part of the Hippo signalling pathway. This striking effect is linked to the control exerted by YAP on actomyosin-mediated tension through the regulation of the RhoGAP GTPase activating protein ARHGAP18. YAP also controls the 3D structure of human cell spheres in this manner in a human cell culture system. Vertebrates have a unique 3D body shape in which correct tissue and organ shape and alignment are essential for function. For example, vision requires the lens to be centred in the eye cup which must in turn be correctly positioned in the head 1 . Tissue morphogenesis depends on force generation, force transmission through the tissue, and response of tissues and extracellular matrix to force 2 , 3 . Although a century ago D’Arcy Thompson postulated that terrestrial animal body shapes are conditioned by gravity 4 , there has been no animal model directly demonstrating how the aforementioned mechano-morphogenetic processes are coordinated to generate a body shape that withstands gravity. Here we report a unique medaka fish ( Oryzias latipes ) mutant, hirame ( hir ), which is sensitive to deformation by gravity. hir embryos display a markedly flattened body caused by mutation of YAP, a nuclear executor of Hippo signalling that regulates organ size. We show that actomyosin-mediated tissue tension is reduced in hir embryos, leading to tissue flattening and tissue misalignment, both of which contribute to body flattening. By analysing YAP function in 3D spheroids of human cells, we identify the Rho GTPase activating protein ARHGAP18 as an effector of YAP in controlling tissue tension. Together, these findings reveal a previously unrecognised function of YAP in regulating tissue shape and alignment required for proper 3D body shape. Understanding this morphogenetic function of YAP could facilitate the use of embryonic stem cells to generate complex organs requiring correct alignment of multiple tissues.

Living systems are more robust, diverse, complex, and supportive of human life than any technology yet created. However, our ability to create novel lifeforms is currently limited to varying existing organisms or bioengineering organoids in vitro. Here we show a scalable pipeline for creating functional novel lifeforms: AI methods automatically design diverse candidate lifeforms in silico to perform some desired function, and transferable designs are then created using a cell-based construction toolkit to realize living systems with the predicted behaviors. Although some steps in this pipeline still require manual intervention, complete automation in future would pave the way to designing and deploying unique, bespoke living systems for a wide range of functions.

We assayed the toxicity of polystyrene nanoparticles (PS-NP, 52 nm) to Daphnia galeata . Survival and reproduction were significantly decreased in individuals exposed to 5 mg/L of PS-NP for 5 days, and embryos showed abnormal development, including a low hatching rate. Using fluorescence confocal microscopy, we recorded the transfer of PS-NP from the external surface of the body to the internal organs, including the thoracic appendices, ovaries, caudal appendices, and brood chamber, as well as PS-NP storage in lipid droplets. Although embryos were exposed to PS-NP in the brood chamber, they did not internalize PS-NP. Exposed D . galeata adults that were not pregnant stored significantly fewer lipid droplets than did the control group, and the lipid droplets that they did store were smaller; meanwhile, there were no significant changes in lipid storage in exposed pregnant individuals. Some embryos showed a high level of lipid storage, a response that occurs when embryos experience an abnormal state, and these embryos showed a very low hatching rate. However, the offspring of exposed adults showed normal survival and lipid storage. This study provides visual evidence that confirms the transfer and effects of PS-NP in Daphnia species, and suggests a relationship between toxicity and lipid storage.

Morphogen signalling forms an activity gradient and instructs cell identities in a signalling strength-dependent manner to pattern developing tissues. However, developing tissues also undergo dynamic morphogenesis, which may produce cells with unfit morphogen signalling and consequent noisy morphogen gradients. Here we show that a cell competition-related system corrects such noisy morphogen gradients. Zebrafish imaging analyses of the Wnt/β-catenin signalling gradient, which acts as a morphogen to establish embryonic anterior-posterior patterning, identify that unfit cells with abnormal Wnt/β-catenin activity spontaneously appear and produce noise in the gradient. Communication between unfit and neighbouring fit cells via cadherin proteins stimulates apoptosis of the unfit cells by activating Smad signalling and reactive oxygen species production. This unfit cell elimination is required for proper Wnt/β-catenin gradient formation and consequent anterior-posterior patterning. Because this gradient controls patterning not only in the embryo but also in adult tissues, this system may support tissue robustness and disease prevention. Gradients of morphogens such as Wnt provide instructive cues for cell identities during development. Here, the authors report that in the developing zebrafish embryo, cell competition and elimination of unfit cells are required for proper Wnt gradient formation.

Early amniote development is highly self-organized, capable of adapting to interference through local and long-range cell–cell interactions. This process, called embryonic regulation 1 , has been well illustrated in experiments on avian embryos, in which subdividing the epiblast disk into different parts not only redirects cell fates to eventually form a complete and well-proportioned embryo at its original location, but also leads to the self-organization of additional, fully formed embryos 2 , 3 in the other separated parts. The cellular interactions underlying embryonic self-organization are widely believed to be mediated by molecular signals, yet the identity of such signals is unclear. Here, by analysing intact and mechanically perturbed quail embryos, we show that the mechanical forces that drive embryogenesis self-organize, with contractility locally self-activating and the ensuing tension acting as a long-range inhibitor. This mechanical feedback governs the persistent pattern of tissue flows that shape the embryo 4 – 6 and also steers the concomitant emergence of embryonic territories by modulating gene expression, ensuring the formation of a single embryo under normal conditions, yet allowing the emergence of multiple, well-proportioned embryos after perturbations. Thus, mechanical forces act at the core of embryonic self-organization, shaping both tissues and gene expression to robustly yet plastically canalize early development. Mechanical forces act at the core of bird embryonic self-organization, shaping both tissues and gene expression to robustly yet plastically canalize early development.

Crystalline biominerals do not resemble faceted crystals. Current explanations for this property involve formation via amorphous phases. Using X-ray absorption near-edge structure (XANES) spectroscopy and photoelectron emission microscopy (PEEM), here we examine forming spicules in embryos of Strongylocentrotus purpuratus sea urchins, and observe a sequence of three mineral phases: hydrated amorphous calcium carbonate (ACC · H₂O)→ dehydrated amorphous calcium carbonate (ACC) → calcite. Unexpectedly, we find ACC · H₂O-rich nanoparticles that persist after the surrounding mineral has dehydrated and crystallized. Protein matrix components occluded within the mineral must inhibit ACC · H₂O dehydration. We devised an in vitro, also using XANES-PEEM, assay to identify spicule proteins that may play a role in stabilizing various mineral phases, and found that the most abundant occluded matrix protein in the sea urchin spicules, SM50, stabilizes ACC · H₂O in vitro.

During gastrulation, physical forces reshape the simple embryonic tissue to form the complex body plans of multicellular organisms 1 . These forces often cause large-scale asymmetric movements of the embryonic tissue 2 , 3 . In many embryos, the gastrulating tissue is surrounded by a rigid protective shell 4 . Although it is well-recognized that gastrulation movements depend on forces that are generated by tissue-intrinsic contractility 5 , 6 , it is not known whether interactions between the tissue and the protective shell provide additional forces that affect gastrulation. Here we show that a particular part of the blastoderm tissue of the red flour beetle ( Tribolium castaneum ) tightly adheres in a temporally coordinated manner to the vitelline envelope that surrounds the embryo. This attachment generates an additional force that counteracts tissue-intrinsic contractile forces to create asymmetric tissue movements. This localized attachment depends on an αPS2 integrin (inflated), and the knockdown of this integrin leads to a gastrulation phenotype that is consistent with complete loss of attachment. Furthermore, analysis of another integrin (the αPS3 integrin, scab) in the fruit fly ( Drosophila melanogaster ) suggests that gastrulation in this organism also relies on adhesion between the blastoderm and the vitelline envelope. Our findings reveal a conserved mechanism through which the spatiotemporal pattern of tissue adhesion to the vitelline envelope provides controllable, counteracting forces that shape gastrulation movements in insects. In the red flour beetle ( Tribolium castaneum ) and fruit fly ( Drosophila melanogaster ), spatiotemporally coordinated integrin-dependent attachments between the blastoderm and vitelline envelope counteract tissue-intrinsic contractile forces to create asymmetric movements of embryonic tissue.

Mammalian sex determination is controlled by the antagonistic interactions of two genetic pathways: The SRY-SOX9-FGF9 network promotes testis determination partly by opposing proovarian pathways, while RSPO1/WNT-β-catenin/FOXL2 signals control ovary development by inhibiting SRY-SOX9-FGF9. The molecular basis of this mutual antagonism is unclear. Here we show that ZNRF3, a WNT signaling antagonist and direct target of RSPO1-mediated inhibition, is required for sex determination in mice. XY mice lacking ZNRF3 exhibit complete or partial gonadal sex reversal, or related defects. These abnormalities are associated with ectopic WNT/β-catenin activity and reduced Sox9 expression during fetal sex determination. Using exome sequencing of individuals with 46, XY disorders of sex development, we identified three human ZNRF3 variants in very rare cases of XY female presentation. We tested two missense variants and show that these disrupt ZNRF3 activity in both human cell lines and zebrafish embryo assays. Our data identify a testis-determining function for ZNRF3 and indicate a mechanism of direct molecular interaction between two mutually antagonistic organogenetic pathways.

Single-cell RNA-seq quantifies biological heterogeneity across both discrete cell types and continuous cell transitions. Partition-based graph abstraction (PAGA) provides an interpretable graph-like map of the arising data manifold, based on estimating connectivity of manifold partitions ( https://github.com/theislab/paga ). PAGA maps preserve the global topology of data, allow analyzing data at different resolutions, and result in much higher computational efficiency of the typical exploratory data analysis workflow. We demonstrate the method by inferring structure-rich cell maps with consistent topology across four hematopoietic datasets, adult planaria and the zebrafish embryo and benchmark computational performance on one million neurons.