Antigen presentation plays positive roles in the regenerative response to cardiac injury in zebrafish.

In contrast to adult mammals, adult zebrafish can fully regenerate injured cardiac tissue, and this regeneration process requires an adequate and tightly controlled immune response. However, which components of the immune response are required during regeneration is unclear. Here, we report positive roles for the antigen presentation-adaptive immunity axis during zebrafish cardiac regeneration. We find that following the initial innate immune response, activated endocardial cells (EdCs), as well as immune cells, start expressing antigen presentation genes. We also observe that T helper cells, a.k.a. Cd4+ T cells, lie in close physical proximity to these antigen-presenting EdCs. We targeted Major Histocompatibility Complex (MHC) class II antigen presentation by generating cd74a; cd74b mutants, which display a defective immune response. In these mutants, Cd4+ T cells and activated EdCs fail to efficiently populate the injured tissue and EdC proliferation is significantly decreased. cd74a; cd74b mutants exhibit additional defects in cardiac regeneration including reduced cardiomyocyte dedifferentiation and proliferation. Notably, Cd74 also becomes activated in neonatal mouse EdCs following cardiac injury. Altogether, these findings point to positive roles for antigen presentation during cardiac regeneration, potentially involving interactions between activated EdCs, classical antigen-presenting cells, and Cd4+ T cells.

Dissecting the spatiotemporal diversity of adult neural stem cells.

Adult stem cells are important for tissue turnover and regeneration. However, in most adult systems it remains elusive how stem cells assume different functional states and support spatially patterned tissue architecture. Here, we dissected the diversity of neural stem cells in the adult zebrafish brain, an organ that is characterized by pronounced zonation and high regenerative capacity. We combined single-cell transcriptomics of dissected brain regions with massively parallel lineage tracing and in vivo RNA metabolic labeling to analyze the regulation of neural stem cells in space and time. We detected a large diversity of neural stem cells, with some subtypes being restricted to a single brain region, while others were found globally across the brain. Global stem cell states are linked to neurogenic differentiation, with different states being involved in proliferative and non-proliferative differentiation. Our work reveals principles of adult stem cell organization and establishes a resource for the functional manipulation of neural stem cell subtypes.

Inference of differentiation trajectories by transfer learning across biological processes.

Stem cells differentiate into distinct fates by transitioning through a series of transcriptional states. Current computational approaches allow reconstruction of differentiation trajectories from single-cell transcriptomics data, but it remains unknown to what degree differentiation can be predicted across biological processes. Here, we use transfer learning to infer differentiation processes and quantify predictability in early embryonic development and adult hematopoiesis. Overall, we find that non-linear methods outperform linear approaches, and we achieved the best predictions with a custom variational autoencoder that explicitly models changes in transcriptional variance. We observed a high accuracy of predictions in embryonic development, but we found somewhat lower agreement with the real data in adult hematopoiesis. We demonstrate that this discrepancy can be explained by a higher degree of concordant transcriptional processes along embryonic differentiation compared with adult homeostasis. In summary, we establish a framework for quantifying and exploiting predictability of cellular differentiation trajectories.

Spatial transcriptomics unveils ZBTB11 as a regulator of cardiomyocyte degeneration in arrhythmogenic cardiomyopathy.

AIMS: Arrhythmogenic cardiomyopathy (ACM) is an inherited cardiac disorder that is characterized by progressive loss of myocardium that is replaced by fibro-fatty cells, arrhythmias, and sudden cardiac death. While myocardial degeneration and fibro-fatty replacement occur in specific locations, the underlying molecular changes remain poorly characterized. Here, we aim to delineate local changes in gene expression to identify new genes and pathways that are relevant for specific remodelling processes occurring during ACM. METHODS AND RESULTS: Using Tomo-Seq, genome-wide transcriptional profiling with high spatial resolution, we created transmural epicardial-to-endocardial gene expression atlases of explanted ACM hearts to gain molecular insights into disease-driving processes. This enabled us to link gene expression profiles to the different regional remodelling responses and allowed us to identify genes that are potentially relevant for disease progression. In doing so, we identified distinct gene expression profiles marking regions of cardiomyocyte degeneration and fibro-fatty remodelling and revealed Zinc finger and BTB domain-containing protein 11 (ZBTB11) to be specifically enriched at sites of active fibro-fatty replacement of myocardium. Immunohistochemistry indicated ZBTB11 to be induced in cardiomyocytes flanking fibro-fatty areas, which could be confirmed in multiple cardiomyopathy patients. Forced overexpression of ZBTB11 induced autophagy and cell death-related gene programmes in human cardiomyocytes, leading to increased apoptosis. CONCLUSION: Our study shows the power of Tomo-Seq to unveil new molecular mechanisms in human cardiomyopathy and uncovers ZBTB11 as a novel driver of cardiomyocyte loss.

A single-cell RNA labeling strategy for measuring stress response upon tissue dissociation.

Tissue dissociation, a crucial step in single-cell sample preparation, can alter the transcriptional state of a sample through the intrinsic cellular stress response. Here we demonstrate a general approach for measuring transcriptional response during sample preparation. In our method, transcripts made during dissociation are labeled for later identification upon sequencing. We found general as well as cell-type-specific dissociation response programs in zebrafish larvae, and we observed sample-to-sample variation in the dissociation response of mouse cardiomyocytes despite well-controlled experimental conditions. Finally, we showed that dissociation of the mouse hippocampus can lead to the artificial activation of microglia. In summary, our approach facilitates experimental optimization of dissociation procedures as well as computational removal of transcriptional perturbation response.

Long-term imaging reveals behavioral plasticity during C. elegans dauer exit.

BACKGROUND: During their lifetime, animals must adapt their behavior to survive in changing environments. This ability requires the nervous system to undergo adjustments at distinct temporal scales, from short-term dynamic changes in expression of neurotransmitters and receptors to longer-term growth, spatial and connectivity reorganization, while integrating external stimuli. The nematode Caenorhabditis elegans provides a model of nervous system plasticity, in particular its dauer exit decision. Under unfavorable conditions, larvae will enter the non-feeding and non-reproductive stress-resistant dauer stage and adapt their behavior to cope with the harsh new environment, with active reversal under improved conditions leading to resumption of reproductive development. However, how different environmental stimuli regulate the exit decision mechanism and thereby drive the larva's behavioral change is unknown. To fill this gap and provide insights on behavioral changes over extended periods of time, we developed a new open hardware method for long-term imaging (12h) of C. elegans larvae. RESULTS: Our WormObserver platform comprises open hardware and software components for video acquisition, automated processing of large image data (> 80k images/experiment) and data analysis. We identified dauer-specific behavioral motifs and characterized the behavioral trajectory of dauer exit in different environments and genetic backgrounds to identify key decision points and stimuli promoting dauer exit. Combining long-term behavioral imaging with transcriptomics data, we find that bacterial ingestion triggers a change in neuropeptide gene expression to establish post-dauer behavior. CONCLUSIONS: Taken together, we show how a developing nervous system can robustly integrate environmental changes activate a developmental switch and adapt the organism's behavior to a new environment. WormObserver is generally applicable to other research questions within and beyond the C. elegans field, having a modular and customizable character and allowing assessment of behavioral plasticity over longer periods.

Origin and function of activated fibroblast states during zebrafish heart regeneration.

The adult zebrafish heart has a high capacity for regeneration following injury. However, the composition of the regenerative niche has remained largely elusive. Here, we dissected the diversity of activated cell states in the regenerating zebrafish heart based on single-cell transcriptomics and spatiotemporal analysis. We observed the emergence of several transient cell states with fibroblast characteristics following injury, and we outlined the proregenerative function of collagen-12-expressing fibroblasts. To understand the cascade of events leading to heart regeneration, we determined the origin of these cell states by high-throughput lineage tracing. We found that activated fibroblasts were derived from two separate sources: the epicardium and the endocardium. Mechanistically, we determined Wnt signalling as a regulator of the endocardial fibroblast response. In summary, our work identifies specialized activated fibroblast cell states that contribute to heart regeneration, thereby opening up possible approaches to modulating the regenerative capacity of the vertebrate heart.

A single-cell atlas of de novo ß-cell regeneration reveals the contribution of hybrid ß/δ-cells to diabetes recovery in zebrafish.

Regeneration-competent species possess the ability to reverse the progression of severe diseases by restoring the function of the damaged tissue. However, the cellular dynamics underlying this capability remain unexplored. Here, we have used single-cell transcriptomics to map de novo ß-cell regeneration during induction and recovery from diabetes in zebrafish. We show that the zebrafish has evolved two distinct types of somatostatin-producing δ-cells, which we term δ1- and δ2-cells. Moreover, we characterize a small population of glucose-responsive islet cells, which share the hormones and fate-determinants of both ß- and δ1-cells. The transcriptomic analysis of ß-cell regeneration reveals that ß/δ hybrid cells provide a prominent source of insulin expression during diabetes recovery. Using in vivo calcium imaging and cell tracking, we further show that the hybrid cells form de novo and acquire glucose-responsiveness in the course of regeneration. The overexpression of dkk3, a gene enriched in hybrid cells, increases their formation in the absence of ß-cell injury. Finally, interspecies comparison shows that plastic δ1-cells are partially related to PP cells in the human pancreas. Our work provides an atlas of ß-cell regeneration and indicates that the rapid formation of glucose-responsive hybrid cells contributes to the resolution of diabetes in zebrafish.

Single-cell-resolved dynamics of chromatin architecture delineate cell and regulatory states in zebrafish embryos.

DNA accessibility of cis-regulatory elements (CREs) dictates transcriptional activity and drives cell differentiation during development. While many genes regulating embryonic development have been identified, the underlying CRE dynamics controlling their expression remain largely uncharacterized. To address this, we produced a multimodal resource and genomic regulatory map for the zebrafish community, which integrates single-cell combinatorial indexing assay for transposase-accessible chromatin with high-throughput sequencing (sci-ATAC-seq) with bulk histone PTMs and Hi-C data to achieve a genome-wide classification of the regulatory architecture determining transcriptional activity in the 24-h post-fertilization (hpf) embryo. We characterized the genome-wide chromatin architecture at bulk and single-cell resolution, applying sci-ATAC-seq on whole 24-hpf stage zebrafish embryos, generating accessibility profiles for ∼23,000 single nuclei. We developed a genome segmentation method, ScregSeg (single-cell regulatory landscape segmentation), for defining regulatory programs, and candidate CREs, specific to one or more cell types. We integrated the ScregSeg output with bulk measurements for histone post-translational modifications and 3D genome organization and identified new regulatory principles between chromatin modalities prevalent during zebrafish development. Sci-ATAC-seq profiling of npas4l/cloche mutant embryos identified novel cellular roles for this hematovascular transcriptional master regulator and suggests an intricate mechanism regulating its expression. Our work defines regulatory architecture and principles in the zebrafish embryo and establishes a resource of cell-type-specific genome-wide regulatory annotations and candidate CREs, providing a valuable open resource for genomics, developmental, molecular, and computational biology.

Single cell biology-a Keystone Symposia report.

Single cell biology has the potential to elucidate many critical biological processes and diseases, from development and regeneration to cancer. Single cell analyses are uncovering the molecular diversity of cells, revealing a clearer picture of the variation among and between different cell types. New techniques are beginning to unravel how differences in cell state-transcriptional, epigenetic, and other characteristics-can lead to different cell fates among genetically identical cells, which underlies complex processes such as embryonic development, drug resistance, response to injury, and cellular reprogramming. Single cell technologies also pose significant challenges relating to processing and analyzing vast amounts of data collected. To realize the potential of single cell technologies, new computational approaches are needed. On March 17-19, 2021, experts in single cell biology met virtually for the Keystone eSymposium "Single Cell Biology" to discuss advances both in single cell applications and technologies.

Spatio-temporal mRNA tracking in the early zebrafish embryo.

Early stages of embryogenesis depend on subcellular localization and transport of maternal mRNA. However, systematic analysis of these processes is hindered by a lack of spatio-temporal information in single-cell RNA sequencing. Here, we combine spatially-resolved transcriptomics and single-cell RNA labeling to perform a spatio-temporal analysis of the transcriptome during early zebrafish development. We measure spatial localization of mRNA molecules within the one-cell stage embryo, which allows us to identify a class of mRNAs that are specifically localized at an extraembryonic position, the vegetal pole. Furthermore, we establish a method for high-throughput single-cell RNA labeling in early zebrafish embryos, which enables us to follow the fate of individual maternal transcripts until gastrulation. This approach reveals that many localized transcripts are specifically transported to the primordial germ cells. Finally, we acquire spatial transcriptomes of two xenopus species and compare evolutionary conservation of localized genes as well as enriched sequence motifs.

Systematic identification of A-to-I RNA editing in zebrafish development and adult organs.

A-to-I RNA editing is a common post transcriptional mechanism, mediated by the Adenosine deaminase that acts on RNA (ADAR) enzymes, that increases transcript and protein diversity. The study of RNA editing is limited by the absence of editing maps for most model organisms, hindering the understanding of its impact on various physiological conditions. Here, we mapped the vertebrate developmental landscape of A-to-I RNA editing, and generated the first comprehensive atlas of editing sites in zebrafish. Tens of thousands unique editing events and 149 coding sites were identified with high-accuracy. Some of these edited sites are conserved between zebrafish and humans. Sequence analysis of RNA over seven developmental stages revealed high levels of editing activity in early stages of embryogenesis, when embryos rely on maternal mRNAs and proteins. In contrast to the other organisms studied so far, the highest levels of editing were detected in the zebrafish ovary and testes. This resource can serve as the basis for understanding of the role of editing during zebrafish development and maturity.

Single-cell genomics to study developmental cell fate decisions in zebrafish.

New developments in single-cell genomics have transformed developmental biology in recent years by enabling systematic analysis of embryonic cell types and differentiation trajectories. Ongoing efforts in experimental and computational method development aim to reveal gene-regulatory mechanisms and to provide additional spatio-temporal information about developmental cell fate decisions. Here, we discuss recent technological developments as well as biological applications of single-cell genomics, with a particular focus on analysis of developmental cell fate decisions. Although the approaches described here are generally applicable to a broad range of model systems, we focus our discussion on applications in zebrafish, which has proven to be a particularly powerful model organism for establishing novel methods in single-cell genomics.

Variability of an Early Developmental Cell Population Underlies Stochastic Laterality Defects.

Embryonic development seemingly proceeds with almost perfect precision. However, it is largely unknown how much underlying microscopic variability is compatible with normal development. Here, we quantify embryo-to-embryo variability in vertebrate development by studying cell number variation in the zebrafish endoderm. We notice that the size of a sub-population of the endoderm, the dorsal forerunner cells (DFCs, which later form the left-right organizer), exhibits significantly more embryo-to-embryo variation than the rest of the endoderm. We find that, with incubation of the embryos at elevated temperature, the frequency of left-right laterality defects is increased drastically in embryos with a low number of DFCs. Furthermore, we observe that these fluctuations have a large stochastic component among fish of the same genetic background. Hence, a stochastic variation in early development leads to a remarkably strong macroscopic phenotype. These fluctuations appear to be associated with maternal effects in the specification of the DFCs.

The transcriptome dynamics of single cells during the cell cycle.

The cell cycle is among the most basic phenomena in biology. Despite advances in single-cell analysis, dynamics and topology of the cell cycle in high-dimensional gene expression space remain largely unknown. We developed a linear analysis of transcriptome data which reveals that cells move along a planar circular trajectory in transcriptome space during the cycle. Non-cycling gene expression adds a third dimension causing helical motion on a cylinder. We find in immortalized cell lines that cell cycle transcriptome dynamics occur largely independently from other cellular processes. We offer a simple method ("Revelio") to order unsynchronized cells in time. Precise removal of cell cycle effects from the data becomes a straightforward operation. The shape of the trajectory implies that each gene is upregulated only once during the cycle, and only two dynamic components represented by groups of genes drive transcriptome dynamics. It indicates that the cell cycle has evolved to minimize changes of transcriptional activity and the related regulatory effort. This design principle of the cell cycle may be of relevance to many other cellular differentiation processes.

Multispecies RNA tomography reveals regulators of hematopoietic stem cell birth in the embryonic aorta.

The defined location of a stem cell within a niche regulates its fate, behavior, and molecular identity via a complex extrinsic regulation that is far from being fully elucidated. To explore the molecular characteristics and key components of the aortic microenvironment, where the first hematopoietic stem cells are generated during development, we performed genome-wide RNA tomography sequencing on zebrafish, chicken, mouse, and human embryos. The resulting anterior-posterior and dorsal-ventral transcriptional maps provided a powerful resource for exploring genes and regulatory pathways active in the aortic microenvironment. By performing interspecies comparative RNA sequencing analyses and functional assays, we explored the complexity of the aortic microenvironment landscape and the fine-tuning of various factors interacting to control hematopoietic stem cell generation, both in time and space in vivo, including the ligand-receptor couple ADM-RAMP2 and SVEP1. Understanding the regulatory function of the local environment will pave the way for improved stem cell production in vitro and clinical cell therapy.

Inclusion of temporal information in single cell transcriptomics.

Single cell transcriptomics has emerged as a powerful method for dissecting cell type diversity and for understanding mechanisms of cell fate decisions. However, inclusion of temporal information remains challenging, since each cell can be measured only once by sequencing analysis. Here, we discuss recent progress and current efforts towards inclusion of temporal information in single cell transcriptomics. Even from snapshot data, temporal dynamics can be computationally inferred via pseudo-temporal ordering of single cell transcriptomes. Temporal information can also come from analysis of intronic reads or from RNA metabolic labeling, which can provide additional evidence for pseudo-time trajectories and enable more fine-grained analysis of gene regulatory interactions. These approaches measure dynamics on short timescales of hours. Emerging methods for high-throughput lineage tracing now enable information storage over long timescales by using CRISPR/Cas9 to record information in the genome, which can later be read out by sequencing.

RNA Tomography for Spatially Resolved Transcriptomics (Tomo-Seq).

Embryonic development is heavily dependent on temporally and spatially restricted gene expression. Spatially resolved measurements of gene expression are therefore crucial for identifying novel regulators and the understanding of their function. However, in situ methods do not resolve global gene expression, and sequencing-based methods usually do not provide spatial information. Here, we describe tomo-seq, a method that combines classical histological sectioning of embryos or tissues with a highly sensitive RNA-sequencing technique. Application of tomo-seq to zebrafish embryos allows reconstructing the spatial gene expression of thousands of genes.

Spatial Transcriptomics of C. elegans Males and Hermaphrodites Identifies Sex-Specific Differences in Gene Expression Patterns.

To advance our understanding of the genetic programs that drive cell and tissue specialization, it is necessary to obtain a comprehensive overview of gene expression patterns. Here, we have used spatial transcriptomics to generate high-resolution, anteroposterior gene expression maps of C. elegans males and hermaphrodites. To explore these maps, we have developed computational methods for discovering region- and tissue-specific genes. We have found extensive sex-specific gene expression differences in the germline and sperm and discovered genes that are specifically expressed in the male reproductive tract. These include a group of uncharacterized genes that encode small secreted proteins that are required for male fertility. We conclude that spatial gene expression maps provide a powerful resource for identifying tissue-specific gene functions in C. elegans. Importantly, we found that expression maps from different animals can be precisely aligned, enabling transcriptome-wide comparisons of gene expression patterns.