Explore the vast range of titles available.

Zebrafish, a popular organism for studying embryonic development and for modeling human diseases, has so far lacked a systematic functional annotation program akin to those in other animal models. To address this, we formed the international DANIO-CODE consortium and created a central repository to store and process zebrafish developmental functional genomic data. Our data coordination center ( https://danio-code.zfin.org ) combines a total of 1,802 sets of unpublished and re-analyzed published genomic data, which we used to improve existing annotations and show its utility in experimental design. We identified over 140,000 cis -regulatory elements throughout development, including classes with distinct features dependent on their activity in time and space. We delineated the distinct distance topology and chromatin features between regulatory elements active during zygotic genome activation and those active during organogenesis. Finally, we matched regulatory elements and epigenomic landscapes between zebrafish and mouse and predicted functional relationships between them beyond sequence similarity, thus extending the utility of zebrafish developmental genomics to mammals. The DANIO-CODE consortium leverages a large-scale multiomic dataset to improve zebrafish genome annotation. They identify ~140,000 cis -regulatory elements throughout development and perform a comparison with the mouse regulatory landscape.

Many questions in developmental biology depend on measuring the position and movement of individual cells within developing embryos. Yet, tools that provide this data are often challenged by high cell density and their accuracy is difficult to measure. Here, we present a three-step procedure to address this problem. Step one is a novel segmentation algorithm based on image derivatives that, in combination with selective post-processing, reliably and automatically segments cell nuclei from images of densely packed tissue. Step two is a quantitative validation using synthetic images to ascertain the efficiency of the algorithm with respect to signal-to-noise ratio and object density. Finally, we propose an original method to generate reliable and experimentally faithful ground truth datasets: Sparse-dense dual-labeled embryo chimeras are used to unambiguously measure segmentation errors within experimental data. Together, the three steps outlined here establish a robust, iterative procedure to fine-tune image analysis algorithms and microscopy settings associated with embryonic 3D image data sets.

In eukaryotes, DNA is packed inside the cell nucleus in the form of chromatin, which consists of DNA, proteins such as histones, and RNA. Euchromatin, which is permissive for transcription, is spatially organized into transcriptionally inactive domains interspersed with pockets of transcriptional activity. While transcription and RNA have been implicated in euchromatin organization, it remains unclear how their interplay forms and maintains transcription pockets. Here we combine theory and experiment to analyze the dynamics of euchromatin organization as pluripotent zebrafish cells exit mitosis and begin transcription. We show that accumulation of RNA induces formation of transcription pockets which displace transcriptionally inactive chromatin. We propose that the accumulating RNA recruits RNA-binding proteins that together tend to separate from transcriptionally inactive euchromatin. Full phase separation is prevented because RNA remains tethered to transcribed euchromatin through RNA polymerases. Instead, smaller scale microphases emerge that do not grow further and form the typical pattern of euchromatin organization. How euchromatin organisation and transcription are related is unclear. Here, the authors observe the dynamics of euchromatin organization showing that accumulating RNA recruits RNA-binding proteins that together with transcribed euchromatin separate from non-transcribed euchromatin, forming microphases.

To expand the toolbox of imaging in living cells, we have engineered a single-chain variable fragment binding the linear HA epitope with high affinity and specificity in vivo. The resulting probe, called the HA frankenbody, can light up in multiple colors HA-tagged nuclear, cytoplasmic, membrane, and mitochondrial proteins in diverse cell types. The HA frankenbody also enables state-of-the-art single-molecule experiments in living cells, which we demonstrate by tracking single HA-tagged histones in U2OS cells and single mRNA translation dynamics in both U2OS cells and neurons. Together with the SunTag, we also track two mRNA species simultaneously to demonstrate comparative single-molecule studies of translation can now be done with genetically encoded tools alone. Finally, we use the HA frankenbody to precisely quantify the expression of HA-tagged proteins in developing zebrafish embryos. The versatility of the HA frankenbody makes it a powerful tool for imaging protein dynamics in vivo. Expression of genetically encoded antibodies for cell labelling is often limited by folding issues. Here, the authors engineer an anti-HA scFv antibody that works in the cellular environment and use it to track mRNA translation dynamics in living cells and to label proteins in live zebrafish embryos.

Notch signalling in angiogenesis Notch signalling coordinates angiogenesis by controlling the specification of endothelial cells into tip cells that lead the way in growing blood vessels and the stalk cells that follow. Michael Potente and colleagues have identified a previously unknown component in Notch signalling regulation in endothelial cells that may provide a mechanism for fine-tuning angiogenesis in response to metabolic requirements and angiogenic stress. They show that the metabolism- and redox-sensing deacetylase SIRT1 deacetylates the Notch1 intracellular domain directly, thereby controlling its stability and turnover and negatively modulating Notch signalling. Inactivation of SIRT1 impairs angiogenesis in zebrafish and mice. Notch signalling is a key intercellular communication mechanism that is essential for cell specification and tissue patterning, and which coordinates critical steps of blood vessel growth 1 , 2 , 3 . Although subtle alterations in Notch activity suffice to elicit profound differences in endothelial behaviour and blood vessel formation 2 , 3 , little is known about the regulation and adaptation of endothelial Notch responses. Here we report that the NAD + -dependent deacetylase SIRT1 acts as an intrinsic negative modulator of Notch signalling in endothelial cells. We show that acetylation of the Notch1 intracellular domain (NICD) on conserved lysines controls the amplitude and duration of Notch responses by altering NICD protein turnover. SIRT1 associates with NICD and functions as a NICD deacetylase, which opposes the acetylation-induced NICD stabilization. Consequently, endothelial cells lacking SIRT1 activity are sensitized to Notch signalling, resulting in impaired growth, sprout elongation and enhanced Notch target gene expression in response to DLL4 stimulation, thereby promoting a non-sprouting, stalk-cell-like phenotype. In vivo , inactivation of Sirt1 in zebrafish and mice causes reduced vascular branching and density as a consequence of enhanced Notch signalling. Our findings identify reversible acetylation of the NICD as a molecular mechanism to adapt the dynamics of Notch signalling, and indicate that SIRT1 acts as rheostat to fine-tune endothelial Notch responses.

Tartrazine (E102) is a controversial coloring agent whose potential impacts on human health are not fully understood. Our study reveals the vascular disrupting effects of tartrazine (TTZ) on developing zebrafish embryos in vivo and on human umbilical vein endothelial cells in vitro. The dye was shown to cause dose-dependent hemorrhages in zebrafish embryos. Analyzing transgenic zebrafish harboring fluorescent endothelial cells revealed that TTZ treatment disrupted cell organization into vessels in both the sub-intestinal vein and the brain area. Assays on human umbilical vein endothelial cells demonstrated that TTZ inhibited endothelial proliferation, tube formation, and migration in a dose-dependent manner. Taken together, our results indicate for the first time that TTZ can affect endothelial cell properties, possibly by disrupting Rho family GTPase pathways which control the cytoskeleton. Our finding provides a credible explanation for many reported human health impacts and offers prospective applications for biomedicine.

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.

Here it is shown that epithelia extrude live but not dying cells at sites of high strain, elucidating a mechanism for maintaining homeostatic cell numbers. Crowd control in epithelia For an epithelial-cell layer to retain its structure and provide a protective barrier, it needs to maintain a balance between the number of cells dividing and the number dying. Buzz Baum and colleagues study this process in Drosophila tissues and demonstrate a direct link between physical forces in a tissue and the rates of cell loss. In regions of tissue that are overcrowded, some of the cells undergo a loss of cell-adhesive junctions and are squeezed out by neighbouring cells. This process of live-cell delamination buffers epithelial cells against variations in growth and contributes to normal tissue homeostasis. As a link between epithelial hyperplasia and cell invasion, it may have relevance to the early stages of cancer development. In a second paper, Jody Rosenblatt and colleagues study epithelial-cell monolayers and find that epithelia extrude live but not dying cells at sites of high strain. The extruded cells undergo cell death owing to loss of survival factors. Hence, extrusion could provide a tumour-suppressive mechanism that could be used to eliminate excess cells. In carcinomas with high levels of survival signalling pathways, extrusion may promote tumour-cell invasion. For an epithelium to provide a protective barrier, it must maintain homeostatic cell numbers by matching the number of dividing cells with the number of dying cells. Although compensatory cell division can be triggered by dying cells 1 , 2 , 3 , it is unknown how cell death might relieve overcrowding due to proliferation. When we trigger apoptosis in epithelia, dying cells are extruded to preserve a functional barrier 4 . Extrusion occurs by cells destined to die signalling to surrounding epithelial cells to contract an actomyosin ring that squeezes the dying cell out 4 , 5 , 6 . However, it is not clear what drives cell death during normal homeostasis. Here we show in human, canine and zebrafish cells that overcrowding due to proliferation and migration induces extrusion of live cells to control epithelial cell numbers. Extrusion of live cells occurs at sites where the highest crowding occurs in vivo and can be induced by experimentally overcrowding monolayers in vitro . Like apoptotic cell extrusion, live cell extrusion resulting from overcrowding also requires sphingosine 1-phosphate signalling and Rho-kinase-dependent myosin contraction, but is distinguished by signalling through stretch-activated channels. Moreover, disruption of a stretch-activated channel, Piezo1, in zebrafish prevents extrusion and leads to the formation of epithelial cell masses. Our findings reveal that during homeostatic turnover, growth and division of epithelial cells on a confined substratum cause overcrowding that leads to their extrusion and consequent death owing to the loss of survival factors. These results suggest that live cell extrusion could be a tumour-suppressive mechanism that prevents the accumulation of excess epithelial cells.

Cell identity is governed by the complex regulation of gene expression, represented as gene-regulatory networks 1 . Here we use gene-regulatory networks inferred from single-cell multi-omics data to perform in silico transcription factor perturbations, simulating the consequent changes in cell identity using only unperturbed wild-type data. We apply this machine-learning-based approach, CellOracle, to well-established paradigms—mouse and human haematopoiesis, and zebrafish embryogenesis—and we correctly model reported changes in phenotype that occur as a result of transcription factor perturbation. Through systematic in silico transcription factor perturbation in the developing zebrafish, we simulate and experimentally validate a previously unreported phenotype that results from the loss of noto , an established notochord regulator. Furthermore, we identify an axial mesoderm regulator, lhx1a . Together, these results show that CellOracle can be used to analyse the regulation of cell identity by transcription factors, and can provide mechanistic insights into development and differentiation. A machine-learning-based strategy called CellOracle combines computational perturbation with modelling of gene-regulatory networks to analyse how cell identity is regulated by transcription factors, and correctly predicts phenotypic changes after transcription factor perturbation in the developing zebrafish.

The limited per-pixel bandwidth of most microscopy methods requires compromises between field of view, sampling density and imaging speed. This limitation constrains studies involving complex motion or fast cellular signaling, and presents a major bottleneck for high-throughput structural imaging. Here, we combine high-speed intensified camera technology with a versatile, reconfigurable and dramatically improved Swept, Confocally Aligned Planar Excitation (SCAPE) microscope design that can achieve high-resolution volumetric imaging at over 300 volumes per second and over 1.2 GHz pixel rates. We demonstrate near-isotropic sampling in freely moving Caenorhabditis elegans, and analyze real-time blood flow and calcium dynamics in the beating zebrafish heart. The same system also permits high-throughput structural imaging of mounted, intact, cleared and expanded samples. SCAPE 2.0’s significantly lower photodamage compared to point-scanning techniques is also confirmed. Our results demonstrate that SCAPE 2.0 is a powerful, yet accessible imaging platform for myriad emerging high-speed dynamic and high-throughput volumetric microscopy applications.