In Toto Imaging and Reconstruction of Post-Implantation Mouse Development at the Single-Cell Level.

The mouse embryo has long been central to the study of mammalian development; however, elucidating the cell behaviors governing gastrulation and the formation of tissues and organs remains a fundamental challenge. A major obstacle is the lack of live imaging and image analysis technologies capable of systematically following cellular dynamics across the developing embryo. We developed a light-sheet microscope that adapts itself to the dramatic changes in size, shape, and optical properties of the post-implantation mouse embryo and captures its development from gastrulation to early organogenesis at the cellular level. We furthermore developed a computational framework for reconstructing long-term cell tracks, cell divisions, dynamic fate maps, and maps of tissue morphogenesis across the entire embryo. By jointly analyzing cellular dynamics in multiple embryos registered in space and time, we built a dynamic atlas of post-implantation mouse development that, together with our microscopy and computational methods, is provided as a resource. VIDEO ABSTRACT.

A Liquid-to-Solid Phase Transition of the ALS Protein FUS Accelerated by Disease Mutation.

Many proteins contain disordered regions of low-sequence complexity, which cause aging-associated diseases because they are prone to aggregate. Here, we study FUS, a prion-like protein containing intrinsically disordered domains associated with the neurodegenerative disease ALS. We show that, in cells, FUS forms liquid compartments at sites of DNA damage and in the cytoplasm upon stress. We confirm this by reconstituting liquid FUS compartments in vitro. Using an in vitro "aging" experiment, we demonstrate that liquid droplets of FUS protein convert with time from a liquid to an aggregated state, and this conversion is accelerated by patient-derived mutations. We conclude that the physiological role of FUS requires forming dynamic liquid-like compartments. We propose that liquid-like compartments carry the trade-off between functionality and risk of aggregation and that aberrant phase transitions within liquid-like compartments lie at the heart of ALS and, presumably, other age-related diseases.

Pluripotency of a founding field: rebranding developmental biology.

The field of developmental biology has declined in prominence in recent decades, with off-shoots from the field becoming more fashionable and highly funded. This has created inequity in discovery and opportunity, partly due to the perception that the field is antiquated or not cutting edge. A 'think tank' of scientists from multiple developmental biology-related disciplines came together to define specific challenges in the field that may have inhibited innovation, and to provide tangible solutions to some of the issues facing developmental biology. The community suggestions include a call to the community to help 'rebrand' the field, alongside proposals for additional funding apparatuses, frameworks for interdisciplinary innovative collaborations, pedagogical access, improved science communication, increased diversity and inclusion, and equity of resources to provide maximal impact to the community.

Self-supervised deep learning encodes high-resolution features of protein subcellular localization.

Explaining the diversity and complexity of protein localization is essential to fully understand cellular architecture. Here we present cytoself, a deep-learning approach for fully self-supervised protein localization profiling and clustering. Cytoself leverages a self-supervised training scheme that does not require preexisting knowledge, categories or annotations. Training cytoself on images of 1,311 endogenously labeled proteins from the OpenCell database reveals a highly resolved protein localization atlas that recapitulates major scales of cellular organization, from coarse classes, such as nuclear and cytoplasmic, to the subtle localization signatures of individual protein complexes. We quantitatively validate cytoself's ability to cluster proteins into organelles and protein complexes, showing that cytoself outperforms previous self-supervised approaches. Moreover, to better understand the inner workings of our model, we dissect the emergent features from which our clustering is derived, interpret them in the context of the fluorescence images, and analyze the performance contributions of each component of our approach.

DaXi-high-resolution, large imaging volume and multi-view single-objective light-sheet microscopy.

The promise of single-objective light-sheet microscopy is to combine the convenience of standard single-objective microscopes with the speed, coverage, resolution and gentleness of light-sheet microscopes. We present DaXi, a single-objective light-sheet microscope design based on oblique plane illumination that achieves: (1) a wider field of view and high-resolution imaging via a custom remote focusing objective; (2) fast volumetric imaging over larger volumes without compromising image quality or necessitating tiled acquisition; (3) fuller image coverage for large samples via multi-view imaging and (4) higher throughput multi-well imaging via remote coverslip placement. Our instrument achieves a resolution of 450 nm laterally and 2 µm axially over an imaging volume of 3,000 × 800 × 300 µm. We demonstrate the speed, field of view, resolution and versatility of our instrument by imaging various systems, including Drosophila egg chamber development, zebrafish whole-brain activity and zebrafish embryonic development - up to nine embryos at a time.

Applications, promises, and pitfalls of deep learning for fluorescence image reconstruction.

Deep learning is becoming an increasingly important tool for image reconstruction in fluorescence microscopy. We review state-of-the-art applications such as image restoration and super-resolution imaging, and discuss how the latest deep learning research could be applied to other image reconstruction tasks. Despite its successes, deep learning also poses substantial challenges and has limits. We discuss key questions, including how to obtain training data, whether discovery of unknown structures is possible, and the danger of inferring unsubstantiated image details.

Content-aware image restoration: pushing the limits of fluorescence microscopy.

Fluorescence microscopy is a key driver of discoveries in the life sciences, with observable phenomena being limited by the optics of the microscope, the chemistry of the fluorophores, and the maximum photon exposure tolerated by the sample. These limits necessitate trade-offs between imaging speed, spatial resolution, light exposure, and imaging depth. In this work we show how content-aware image restoration based on deep learning extends the range of biological phenomena observable by microscopy. We demonstrate on eight concrete examples how microscopy images can be restored even if 60-fold fewer photons are used during acquisition, how near isotropic resolution can be achieved with up to tenfold under-sampling along the axial direction, and how tubular and granular structures smaller than the diffraction limit can be resolved at 20-times-higher frame rates compared to state-of-the-art methods. All developed image restoration methods are freely available as open source software in Python, FIJI, and KNIME.

PCM1 conveys centrosome asymmetry to polarized endosome dynamics in regulating daughter cell fate.

Vertebrate radial glia progenitors (RGPs), the principal neural stem cells, balance self-renewal and differentiation through asymmetric cell division (ACD), during which unequal inheritance of centrosomes is observed. Mechanistically, how centrosome asymmetry leads to distinct daughter cell fate remains largely unknown. Here we find that the centrosome protein Pericentriolar Material 1 (Pcm1), asymmetrically distributed at the centrosomes, regulates polarized endosome dynamics and RGP fate. In vivo time-lapse imaging and nanoscale-resolution expansion microscopy of zebrafish embryonic RGPs detect Pcm1 on Notch ligand-containing endosomes, in a complex with the polarity regulator Par-3 and dynein motor. Loss of pcm1 disrupts endosome dynamics, with clonal analysis uncovering increased neuronal production at the expense of progenitors. Pcm1 facilitates an exchange of Rab5b (early) for Rab11a (recycling) endosome markers and promotes the formation of Par-3 and dynein macromolecular complexes on recycling endosomes. Finally, in human-induced pluripotent stem cell-derived brain organoids, PCM1 shows asymmetry and co-localization with PARD3 and RAB11A in mitotic neural progenitors. Our data reveal a new mechanism by which centrosome asymmetry is conveyed by Pcm1 to polarize endosome dynamics and Notch signaling in regulating ACD and progenitor fate.

Single-cell transcriptional dynamics in a living vertebrate.

The ability to quantify transcriptional dynamics in individual cells via live imaging has revolutionized our understanding of gene regulation. However, such measurements are lacking in the context of vertebrate embryos. We addressed this deficit by applying MS2-MCP mRNA labeling to the quantification of transcription in zebrafish, a model vertebrate. We developed a platform of transgenic organisms, light sheet fluorescence microscopy, and optimized image analysis that enables visualization and quantification of MS2 reporters. We used these tools to obtain the first single-cell, real-time measurements of transcriptional dynamics of the segmentation clock. Our measurements challenge the traditional view of smooth clock oscillations and instead suggest a model of discrete transcriptional bursts that are organized in space and time. Together, these results highlight how measuring single-cell transcriptional activity can reveal unexpected features of gene regulation and how this data can fuel the dialogue between theory and experiment.

Colocalization Analysis for Cryosectioned and Immunostained Tissue Samples with or without Label Retention Expansion Microscopy (LR-ExM) by JACoP.

Asymmetric cell division (ACD) is fundamental for balancing cell proliferation and differentiation in metazoans. During active neurogenesis in the developing zebrafish forebrain, radial glia progenitors (RGPs) mainly undergo ACD to produce one daughter with high activity of Delta/Notch signaling (proliferative cell fate) and another daughter with low Delta/Notch signaling (differentiative cell fate). The cell polarity protein partitioning-defective 3 (Par-3) is critical for regulating this process. To understand how polarized Par-3 on the cell cortex can lead to differential Notch activity in the nuclei of daughter cells, we combined an anti-Delta D (Dld) -atto 647N antibody uptake assay with label retention expansion microscopy (LR-ExM), to obtain high resolution immunofluorescent images of Par-3, dynein light intermediate chain 1 (Dlic1), and Dld endosomes in mitotic RGPs. We then developed a protocol for analyzing the colocalization of Par-3, Dlic1, and endosomal DeltaD, using JACoP (Just Another Co-localization Plugin) in ImageJ software (Bolte and Cordelières, 2006). Through such analyses, we have shown that cytosolic Par-3 is associated with Dlic1 on Dld endosomes. Our work demonstrates a direct involvement of Par-3 in dynein-mediated polarized transport of Notch signaling endosomes. This bio-protocol may be generalizable for analysis of protein co-localization in any cryosectioned and immunostained tissue samples.

Neuropilin-1 Mediates SARS-CoV-2 Infection of Astrocytes in Brain Organoids, Inducing Inflammation Leading to Dysfunction and Death of Neurons.

Coronavirus disease 2019 (COVID-19) is frequently associated with neurological deficits, but how severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) induces these effects remains unclear. Here, we show that astrocytes are readily infected by SARS-CoV-2, but surprisingly, neuropilin-1, not angiotensin-converting enzyme 2 (ACE2), serves as the principal receptor mediating cell entry. Infection is further positively modulated by the two-pore segment channel 2 (TPC2) protein that regulates membrane trafficking and endocytosis. Astrocyte infection produces a pathological response closely resembling reactive astrogliosis characterized by elevated type I interferon (IFN) production, increased inflammation, and the decreased expression of transporters of water, ions, choline, and neurotransmitters. These combined events initiated within astrocytes produce a hostile microenvironment that promotes the dysfunction and death of uninfected bystander neurons. IMPORTANCE SARS-CoV-2 infection primarily targets the lung but may also damage other organs, including the brain, heart, kidney, and intestine. Central nervous system (CNS) pathologies include loss of smell and taste, headache, delirium, acute psychosis, seizures, and stroke. Pathological loss of gray matter occurs in SARS-CoV-2 infection, but it is unclear whether this is due to direct viral infection, indirect effects associated with systemic inflammation, or both. Here, we used induced pluripotent stem cell (iPSC)-derived brain organoids and primary human astrocytes from the cerebral cortex to study direct SARS-CoV-2 infection. Our findings support a model where SARS-CoV-2 infection of astrocytes produces a panoply of changes in the expression of genes regulating innate immune signaling and inflammatory responses. The deregulation of these genes in astrocytes produces a microenvironment within the CNS that ultimately disrupts normal neuron function, promoting neuronal cell death and CNS deficits.

OpenCell: Endogenous tagging for the cartography of human cellular organization.

Elucidating the wiring diagram of the human cell is a central goal of the postgenomic era. We combined genome engineering, confocal live-cell imaging, mass spectrometry, and data science to systematically map the localization and interactions of human proteins. Our approach provides a data-driven description of the molecular and spatial networks that organize the proteome. Unsupervised clustering of these networks delineates functional communities that facilitate biological discovery. We found that remarkably precise functional information can be derived from protein localization patterns, which often contain enough information to identify molecular interactions, and that RNA binding proteins form a specific subgroup defined by unique interaction and localization properties. Paired with a fully interactive website (opencell.czbiohub.org), our work constitutes a resource for the quantitative cartography of human cellular organization.

Genome-wide expression profiling and functional network analysis upon neuroectodermal conversion of human mesenchymal stem cells suggest HIF-1 and miR-124a as important regulators.

Tissue-specific stem cells, such as bone-marrow-derived human mesenchymal stem cells (hMSCs), are thought to be lineage restricted and therefore, could only be differentiated into cell types of the tissue of origin. Several recent studies however have suggested that these types of stem cells might be able to break barriers of germ layer commitment and differentiate in vitro into cells with neuroectodermal properties. We reported earlier about efficient conversion of adult hMSCs into a neural stem cell (NSC)-like population (hmNSCs, for human marrow-derived NSC-like cells) with all major properties of NSCs including functional neuronal differentiation capacity. Here we compared the transcriptomes from hMSCs and hmNSCs using a novel strategy by combining classic Affymetrix oligonucleotide microarray profiling with regulatory and protein interaction network analyses to shed light on regulatory protein networks involved in this neuroectodermal conversion process. We found differential regulation of extracellular matrix protein transcripts, up-regulation of distinct neuroectodermal and NSCs marker genes and local chromosomal transcriptional up-regulation at chromosome 4q13.3. In comparison to hMSCs and primary adult hippocampal NSCs, the transcriptome of hmNSCs displayed minor overlap with both other cell populations. Advanced bioinformatics of regulated genes upon neuroectodermal conversion identified transcription factor networks with HIF-1 and microRNA miR-124a as potential major regulators. Together, transgerminal neuroectodermal conversion of hMSCs into NSC-like cells is accompanied by extensive changes of their global gene expression profile, which might be controlled in part by transcription factor networks related to HIF-1 and miR-124a.

GoGene: gene annotation in the fast lane.

High-throughput screens such as microarrays and RNAi screens produce huge amounts of data. They typically result in hundreds of genes, which are often further explored and clustered via enriched GeneOntology terms. The strength of such analyses is that they build on high-quality manual annotations provided with the GeneOntology. However, the weakness is that annotations are restricted to process, function and location and that they do not cover all known genes in model organisms. GoGene addresses this weakness by complementing high-quality manual annotation with high-throughput text mining extracting co-occurrences of genes and ontology terms from literature. GoGene contains over 4,000,000 associations between genes and gene-related terms for 10 model organisms extracted from more than 18,000,000 PubMed entries. It does not cover only process, function and location of genes, but also biomedical categories such as diseases, compounds, techniques and mutations. By bringing it all together, GoGene provides the most recent and most complete facts about genes and can rank them according to novelty and importance. GoGene accepts keywords, gene lists, gene sequences and protein sequences as input and supports search for genes in PubMed, EntrezGene and via BLAST. Since all associations of genes to terms are supported by evidence in the literature, the results are transparent and can be verified by the user. GoGene is available at http://gopubmed.org/gogene.

ZAF, the first open source fully automated feeder for aquatic facilities.

In the past few decades, aquatic animals have become popular model organisms in biology, spurring a growing need for establishing aquatic facilities. Zebrafish are widely studied and relatively easy to culture using commercial systems. However, a challenging aspect of maintaining aquatic facilities is animal feeding, which is both time- and resource-consuming. We have developed an open-source fully automatic daily feeding system, Zebrafish Automatic Feeder (ZAF). ZAF is reliable, provides a standardized amount of food to every tank, is cost-efficient and easy to build. The advanced version, ZAF+, allows for the precise control of food distribution as a function of fish density per tank, and has a user-friendly interface. Both ZAF and ZAF+ are adaptable to any laboratory environment and facilitate the implementation of aquatic colonies. Here, we provide all blueprints and instructions for building the mechanics, electronics, fluidics, as well as to setup the control software and its user-friendly graphical interface. Importantly, the design is modular and can be scaled to meet different user needs. Furthermore, our results show that ZAF and ZAF+ do not adversely affect zebrafish culture, enabling fully automatic feeding for any aquatic facility.