Lineage tracing of human development through somatic mutations.

The ontogeny of the human haematopoietic system during fetal development has previously been characterized mainly through careful microscopic observations1. Here we reconstruct a phylogenetic tree of blood development using whole-genome sequencing of 511 single-cell-derived haematopoietic colonies from healthy human fetuses at 8 and 18 weeks after conception, coupled with deep targeted sequencing of tissues of known embryonic origin. We found that, in healthy fetuses, individual haematopoietic progenitors acquire tens of somatic mutations by 18 weeks after conception. We used these mutations as barcodes and timed the divergence of embryonic and extra-embryonic tissues during development, and estimated the number of blood antecedents at different stages of embryonic development. Our data support a hypoblast origin of the extra-embryonic mesoderm and primitive blood in humans.

Analysis of single-cell RNA sequencing data based on autoencoders.

BACKGROUND: Single-cell RNA sequencing (scRNA-Seq) experiments are gaining ground to study the molecular processes that drive normal development as well as the onset of different pathologies. Finding an effective and efficient low-dimensional representation of the data is one of the most important steps in the downstream analysis of scRNA-Seq data, as it could provide a better identification of known or putatively novel cell-types. Another step that still poses a challenge is the integration of different scRNA-Seq datasets. Though standard computational pipelines to gain knowledge from scRNA-Seq data exist, a further improvement could be achieved by means of machine learning approaches. RESULTS: Autoencoders (AEs) have been effectively used to capture the non-linearities among gene interactions of scRNA-Seq data, so that the deployment of AE-based tools might represent the way forward in this context. We introduce here scAEspy, a unifying tool that embodies: (1) four of the most advanced AEs, (2) two novel AEs that we developed on purpose, (3) different loss functions. We show that scAEspy can be coupled with various batch-effect removal tools to integrate data by different scRNA-Seq platforms, in order to better identify the cell-types. We benchmarked scAEspy against the most used batch-effect removal tools, showing that our AE-based strategies outperform the existing solutions. CONCLUSIONS: scAEspy is a user-friendly tool that enables using the most recent and promising AEs to analyse scRNA-Seq data by only setting up two user-defined parameters. Thanks to its modularity, scAEspy can be easily extended to accommodate new AEs to further improve the downstream analysis of scRNA-Seq data. Considering the relevant results we achieved, scAEspy can be considered as a starting point to build a more comprehensive toolkit designed to integrate multi single-cell omics.

Single-cell biology: resolving biological complexity, one cell at a time.

In March 2018, over 250 researchers came together at the Wellcome Genome Campus in Hinxton, Cambridge, UK, to present their latest research in the area of single-cell biology. A highly interdisciplinary meeting, the Single Cell Biology conference covered a variety of topics, ranging from cutting-edge technological innovation, developmental biology and stem cell research to evolution and cancer. This meeting report summarises the key findings presented and the major research themes that emerged during the conference.

Single-cell transcriptome analysis of fish immune cells provides insight into the evolution of vertebrate immune cell types.

The immune system of vertebrate species consists of many different cell types that have distinct functional roles and are subject to different evolutionary pressures. Here, we first analyzed conservation of genes specific for all major immune cell types in human and mouse. Our results revealed higher gene turnover and faster evolution of trans-membrane proteins in NK cells compared with other immune cell types, and especially T cells, but similar conservation of nuclear and cytoplasmic protein coding genes. To validate these findings in a distant vertebrate species, we used single-cell RNA sequencing of lck:GFP cells in zebrafish and obtained the first transcriptome of specific immune cell types in a nonmammalian species. Unsupervised clustering and single-cell TCR locus reconstruction identified three cell populations, T cells, a novel type of NK-like cells, and a smaller population of myeloid-like cells. Differential expression analysis uncovered new immune-cell-specific genes, including novel immunoglobulin-like receptors, and neofunctionalization of recently duplicated paralogs. Evolutionary analyses confirmed the higher gene turnover of trans-membrane proteins in NK cells compared with T cells in fish species, suggesting that this is a general property of immune cell types across all vertebrates.

Power analysis of single-cell RNA-sequencing experiments.

Single-cell RNA sequencing (scRNA-seq) has become an established and powerful method to investigate transcriptomic cell-to-cell variation, thereby revealing new cell types and providing insights into developmental processes and transcriptional stochasticity. A key question is how the variety of available protocols compare in terms of their ability to detect and accurately quantify gene expression. Here, we assessed the protocol sensitivity and accuracy of many published data sets, on the basis of spike-in standards and uniform data processing. For our workflow, we developed a flexible tool for counting the number of unique molecular identifiers (https://github.com/vals/umis/). We compared 15 protocols computationally and 4 protocols experimentally for batch-matched cell populations, in addition to investigating the effects of spike-in molecular degradation. Our analysis provides an integrated framework for comparing scRNA-seq protocols.

Loss of the homologous recombination gene rad51 leads to Fanconi anemia-like symptoms in zebrafish.

RAD51 is an indispensable homologous recombination protein, necessary for strand invasion and crossing over. It has recently been designated as a Fanconi anemia (FA) gene, following the discovery of two patients carrying dominant-negative mutations. FA is a hereditary DNA-repair disorder characterized by various congenital abnormalities, progressive bone marrow failure, and cancer predisposition. In this report, we describe a viable vertebrate model of RAD51 loss. Zebrafish rad51 loss-of-function mutants developed key features of FA, including hypocellular kidney marrow, sensitivity to cross-linking agents, and decreased size. We show that some of these symptoms stem from both decreased proliferation and increased apoptosis of embryonic hematopoietic stem and progenitor cells. Comutation of p53 was able to rescue the hematopoietic defects seen in the single mutants, but led to tumor development. We further demonstrate that prolonged inflammatory stress can exacerbate the hematological impairment, leading to an additional decrease in kidney marrow cell numbers. These findings strengthen the assignment of RAD51 as a Fanconi gene and provide more evidence for the notion that aberrant p53 signaling during embryogenesis leads to the hematological defects seen later in life in FA. Further research on this zebrafish FA model will lead to a deeper understanding of the molecular basis of bone marrow failure in FA and the cellular role of RAD51.

CD4-Transgenic Zebrafish Reveal Tissue-Resident Th2- and Regulatory T Cell-like Populations and Diverse Mononuclear Phagocytes.

CD4+ T cells are at the nexus of the innate and adaptive arms of the immune system. However, little is known about the evolutionary history of CD4+ T cells, and it is unclear whether their differentiation into specialized subsets is conserved in early vertebrates. In this study, we have created transgenic zebrafish with vibrantly labeled CD4+ cells allowing us to scrutinize the development and specialization of teleost CD4+ leukocytes in vivo. We provide further evidence that CD4+ macrophages have an ancient origin and had already emerged in bony fish. We demonstrate the utility of this zebrafish resource for interrogating the complex behavior of immune cells at cellular resolution by the imaging of intimate contacts between teleost CD4+ T cells and mononuclear phagocytes. Most importantly, we reveal the conserved subspecialization of teleost CD4+ T cells in vivo. We demonstrate that the ancient and specialized tissues of the gills contain a resident population of il-4/13b-expressing Th2-like cells, which do not coexpress il-4/13a Additionally, we identify a contrasting population of regulatory T cell-like cells resident in the zebrafish gut mucosa, in marked similarity to that found in the intestine of mammals. Finally, we show that, as in mammals, zebrafish CD4+ T cells will infiltrate melanoma tumors and obtain a phenotype consistent with a type 2 immune microenvironment. We anticipate that this unique resource will prove invaluable for future investigation of T cell function in biomedical research, the development of vaccination and health management in aquaculture, and for further research into the evolution of adaptive immunity.

The Ribosome Biogenesis Protein Nol9 Is Essential for Definitive Hematopoiesis and Pancreas Morphogenesis in Zebrafish.

Ribosome biogenesis is a ubiquitous and essential process in cells. Defects in ribosome biogenesis and function result in a group of human disorders, collectively known as ribosomopathies. In this study, we describe a zebrafish mutant with a loss-of-function mutation in nol9, a gene that encodes a non-ribosomal protein involved in rRNA processing. nol9sa1022/sa1022 mutants have a defect in 28S rRNA processing. The nol9sa1022/sa1022 larvae display hypoplastic pancreas, liver and intestine and have decreased numbers of hematopoietic stem and progenitor cells (HSPCs), as well as definitive erythrocytes and lymphocytes. In addition, ultrastructural analysis revealed signs of pathological processes occurring in endothelial cells of the caudal vein, emphasizing the complexity of the phenotype observed in nol9sa1022/sa1022 larvae. We further show that both the pancreatic and hematopoietic deficiencies in nol9sa1022/sa1022 embryos were due to impaired cell proliferation of respective progenitor cells. Interestingly, genetic loss of Tp53 rescued the HSPCs but not the pancreatic defects. In contrast, activation of mRNA translation via the mTOR pathway by L-Leucine treatment did not revert the erythroid or pancreatic defects. Together, we present the nol9sa1022/sa1022 mutant, a novel zebrafish ribosomopathy model, which recapitulates key human disease characteristics. The use of this genetically tractable model will enhance our understanding of the tissue-specific mechanisms following impaired ribosome biogenesis in the context of an intact vertebrate.

New gene functions in megakaryopoiesis and platelet formation.

Platelets are the second most abundant cell type in blood and are essential for maintaining haemostasis. Their count and volume are tightly controlled within narrow physiological ranges, but there is only limited understanding of the molecular processes controlling both traits. Here we carried out a high-powered meta-analysis of genome-wide association studies (GWAS) in up to 66,867 individuals of European ancestry, followed by extensive biological and functional assessment. We identified 68 genomic loci reliably associated with platelet count and volume mapping to established and putative novel regulators of megakaryopoiesis and platelet formation. These genes show megakaryocyte-specific gene expression patterns and extensive network connectivity. Using gene silencing in Danio rerio and Drosophila melanogaster, we identified 11 of the genes as novel regulators of blood cell formation. Taken together, our findings advance understanding of novel gene functions controlling fate-determining events during megakaryopoiesis and platelet formation, providing a new example of successful translation of GWAS to function.

A loss of function screen of identified genome-wide association study Loci reveals new genes controlling hematopoiesis.

The formation of mature cells by blood stem cells is very well understood at the cellular level and we know many of the key transcription factors that control fate decisions. However, many upstream signalling and downstream effector processes are only partially understood. Genome wide association studies (GWAS) have been particularly useful in providing new directions to dissect these pathways. A GWAS meta-analysis identified 68 genetic loci controlling platelet size and number. Only a quarter of those genes, however, are known regulators of hematopoiesis. To determine function of the remaining genes we performed a medium-throughput genetic screen in zebrafish using antisense morpholino oligonucleotides (MOs) to knock down protein expression, followed by histological analysis of selected genes using a wide panel of different hematopoietic markers. The information generated by the initial knockdown was used to profile phenotypes and to position candidate genes hierarchically in hematopoiesis. Further analysis of brd3a revealed its essential role in differentiation but not maintenance and survival of thrombocytes. Using the from-GWAS-to-function strategy we have not only identified a series of genes that represent novel regulators of thrombopoiesis and hematopoiesis, but this work also represents, to our knowledge, the first example of a functional genetic screening strategy that is a critical step toward obtaining biologically relevant functional data from GWA study for blood cell traits.

Mechanisms of fate decision and lineage commitment during haematopoiesis.

Blood stem cells need to both perpetuate themselves (self-renew) and differentiate into all mature blood cells to maintain blood formation throughout life. However, it is unclear how the underlying gene regulatory network maintains this population of self-renewing and differentiating stem cells and how it accommodates the transition from a stem cell to a mature blood cell. Our current knowledge of transcriptomes of various blood cell types has mainly been advanced by population-level analysis. However, a population of seemingly homogenous blood cells may include many distinct cell types with substantially different transcriptomes and abilities to make diverse fate decisions. Therefore, understanding the cell-intrinsic differences between individual cells is necessary for a deeper understanding of the molecular basis of their behaviour. Here we review recent single-cell studies in the haematopoietic system and their contribution to our understanding of the mechanisms governing cell fate choices and lineage commitment.

Single-cell and spatial transcriptomics analysis of non-small cell lung cancer.

Lung cancer is the second most frequently diagnosed cancer and the leading cause of cancer-related mortality worldwide. Tumour ecosystems feature diverse immune cell types. Myeloid cells, in particular, are prevalent and have a well-established role in promoting the disease. In our study, we profile approximately 900,000 cells from 25 treatment-naive patients with adenocarcinoma and squamous-cell carcinoma by single-cell and spatial transcriptomics. We note an inverse relationship between anti-inflammatory macrophages and NK cells/T cells, and with reduced NK cell cytotoxicity within the tumour. While we observe a similar cell type composition in both adenocarcinoma and squamous-cell carcinoma, we detect significant differences in the co-expression of various immune checkpoint inhibitors. Moreover, we reveal evidence of a transcriptional "reprogramming" of macrophages in tumours, shifting them towards cholesterol export and adopting a foetal-like transcriptional signature which promotes iron efflux. Our multi-omic resource offers a high-resolution molecular map of tumour-associated macrophages, enhancing our understanding of their role within the tumour microenvironment.

Silencing of RhoA nucleotide exchange factor, ARHGEF3, reveals its unexpected role in iron uptake.

Genomewide association meta-analysis studies have identified > 100 independent genetic loci associated with blood cell indices, including volume and count of platelets and erythrocytes. Although several of these loci encode known regulators of hematopoiesis, the mechanism by which most sequence variants exert their effect on blood cell formation remains elusive. An example is the Rho guanine nucleotide exchange factor, ARHGEF3, which was previously implicated by genomewide association meta-analysis studies in bone cell biology. Here, we report on the unexpected role of ARHGEF3 in regulation of iron uptake and erythroid cell maturation. Although early erythroid differentiation progressed normally, silencing of arhgef3 in Danio rerio resulted in microcytic and hypochromic anemia. This was rescued by intracellular supplementation of iron, showing that arhgef3-depleted erythroid cells are fully capable of hemoglobinization. Disruption of the arhgef3 target, RhoA, also produced severe anemia, which was, again, corrected by iron injection. Moreover, silencing of ARHGEF3 in erythromyeloblastoid cells K562 showed that the uptake of transferrin was severely impaired. Taken together, this is the first study to provide evidence for ARHGEF3 being a regulator of transferrin uptake in erythroid cells, through activation of RHOA.

Exploiting somatic mutations to decipher human blood production: a natural lineage-tracing strategy.

Lineage tracing using fluorescent proteins, genetic barcodes, and various other strategies has provided critical insights into the dynamics of both fetal and adult hematopoiesis in model organisms. However, these technologies cannot be readily used to study hematopoiesis in human beings. Therefore, there is a critical need to develop strategies to assess cellular dynamics within human hematopoietic tissues in vivo. Recently, researchers have used naturally acquired somatic mutations, coupled with other single-cell technologies, to retrospectively analyze clonal cellular dynamics. In summer 2022, the International Society for Experimental Hematology's New Investigator Committee hosted a webinar focused on novel approaches to dissect fetal and adult hematopoiesis, with presentations from Drs. Ana Cvejic and Vijay Sankaran. Here, we provide an overview of these exciting technological advances and some of the novel insights they have already provided in studying human hematopoiesis.

Functional genomics in zebrafish permits rapid characterization of novel platelet membrane proteins.

In this study, we demonstrate the suitability of the vertebrate Danio rerio (zebrafish) for functional screening of novel platelet genes in vivo by reverse genetics. Comparative transcript analysis of platelets and their precursor cell, the megakaryocyte, together with nucleated blood cell elements, endothelial cells, and erythroblasts, identified novel platelet membrane proteins with hitherto unknown roles in thrombus formation. We determined the phenotype induced by antisense morpholino oligonucleotide (MO)-based knockdown of 5 of these genes in a laser-induced arterial thrombosis model. To validate the model, the genes for platelet glycoprotein (GP) IIb and the coagulation protein factor VIII were targeted. MO-injected fish showed normal thrombus initiation but severely impaired thrombus growth, consistent with the mouse knockout phenotypes, and concomitant knockdown of both resulted in spontaneous bleeding. Knockdown of 4 of the 5 novel platelet proteins altered arterial thrombosis, as demonstrated by modified kinetics of thrombus initiation and/or development. We identified a putative role for BAMBI and LRRC32 in promotion and DCBLD2 and ESAM in inhibition of thrombus formation. We conclude that phenotypic analysis of MO-injected zebrafish is a fast and powerful method for initial screening of novel platelet proteins for function in thrombosis.

The role of meis1 in primitive and definitive hematopoiesis during zebrafish development.

BACKGROUND: The Meis1 protein represents an important cofactor for Hox and Pbx1 and is implicated in human and murine leukemias. Though much is known about the role of meis1 in leukemogenesis, its function in normal hematopoiesis remains largely unclear. Here we characterized the role of the proto-oncogene, meis1, during zebrafish primitive and definitive hematopoiesis. DESIGN AND METHODS: Zebrafish embryos were stained with o-dianisidine to detect hemoglobin-containing cells and Sudan black to quantify neutrophils. The numbers of other cells (scl-, gata1- and alas2-positive cells) were also quantified by measuring the corresponding stained areas of the embryos. We used anti-Meis1 antibody and whole mount immunohistochemistry to determine the pattern of expression of Meis1 during zebrafish development and then analyzed the functional role of Meis1 by knocking-down the meis1 gene. RESULTS: Using antisense morpholino oligomers to interrupt meis1 expression we found that, although primitive macrophage development could occur unhampered, posterior erythroid differentiation required meis1, and its absence resulted in a severe decrease in the number of mature erythrocytes. Furthermore a picture emerged that meis1 exerts important effects on later stages of erythrocyte maturation and that these effects are independent of gata1, but under the control of scl. In addition, meis1 morpholino knock-down led to dramatic single arteriovenous tube formation. We also found that knock-down of pbx1 resulted in a phenotype that was strikingly similar to that of meis1 knock-down zebrafish. CONCLUSIONS: These results imply that meis1, jointly with pbx1, regulates primitive hematopoiesis as well as vascular development.

Single-Cell Transcriptomic Analysis of Hematopoietic Cells.

Single-cell RNA sequencing (scRNA-Seq) allows the complete and unbiased analysis of the transcriptional state of an individual cell. In the past 5 years, scRNA-Seq contributed to the progress of the hematology field, advancing our knowledge of both normal and malignant hematopoiesis. Different scRNA-Seq methods are available, all relying on the conversion of RNA to cDNA, followed by amplification of cDNA in order to obtain a sufficient amount of genetic material for sequencing. Currently available scRNA-Seq platforms can be broadly divided into two categories: droplet-based and plate-based. Each of these approaches has advantages and disadvantages that need to be considered when designing the experiment. Here, we describe detailed protocols of two of the most used methods for scRNA-Seq of hematopoietic cells: Smart-Seq2 (plate-based) and 10× Genomics (droplet-based).

Integrative Single-Cell RNA-Seq and ATAC-Seq Analysis of Human Developmental Hematopoiesis.

Regulation of hematopoiesis during human development remains poorly defined. Here we applied single-cell RNA sequencing (scRNA-seq) and single-cell assay for transposase-accessible chromatin sequencing (scATAC-seq) to over 8,000 human immunophenotypic blood cells from fetal liver and bone marrow. We inferred their differentiation trajectory and identified three highly proliferative oligopotent progenitor populations downstream of hematopoietic stem cells (HSCs)/multipotent progenitors (MPPs). Along this trajectory, we observed opposing patterns of chromatin accessibility and differentiation that coincided with dynamic changes in the activity of distinct lineage-specific transcription factors. Integrative analysis of chromatin accessibility and gene expression revealed extensive epigenetic but not transcriptional priming of HSCs/MPPs prior to their lineage commitment. Finally, we refined and functionally validated the sorting strategy for the HSCs/MPPs and achieved around 90% enrichment. Our study provides a useful framework for future investigation of human developmental hematopoiesis in the context of blood pathologies and regenerative medicine.

Unsupervised generative and graph representation learning for modelling cell differentiation.

Using machine learning techniques to build representations from biomedical data can help us understand the latent biological mechanism of action and lead to important discoveries. Recent developments in single-cell RNA-sequencing protocols have allowed measuring gene expression for individual cells in a population, thus opening up the possibility of finding answers to biomedical questions about cell differentiation. In this paper, we explore unsupervised generative neural methods, based on the variational autoencoder, that can model cell differentiation by building meaningful representations from the high dimensional and complex gene expression data. We use disentanglement methods based on information theory to improve the data representation and achieve better separation of the biological factors of variation in the gene expression data. In addition, we use a graph autoencoder consisting of graph convolutional layers to predict relationships between single-cells. Based on these models, we develop a computational framework that consists of methods for identifying the cell types in the dataset, finding driver genes for the differentiation process and obtaining a better understanding of relationships between cells. We illustrate our methods on datasets from multiple species and also from different sequencing technologies.

Analysis of endothelial-to-haematopoietic transition at the single cell level identifies cell cycle regulation as a driver of differentiation.

BACKGROUND: Haematopoietic stem cells (HSCs) first arise during development in the aorta-gonad-mesonephros (AGM) region of the embryo from a population of haemogenic endothelial cells which undergo endothelial-to-haematopoietic transition (EHT). Despite the progress achieved in recent years, the molecular mechanisms driving EHT are still poorly understood, especially in human where the AGM region is not easily accessible. RESULTS: In this study, we take advantage of a human pluripotent stem cell (hPSC) differentiation system and single-cell transcriptomics to recapitulate EHT in vitro and uncover mechanisms by which the haemogenic endothelium generates early haematopoietic cells. We show that most of the endothelial cells reside in a quiescent state and progress to the haematopoietic fate within a defined time window, within which they need to re-enter into the cell cycle. If cell cycle is blocked, haemogenic endothelial cells lose their EHT potential and adopt a non-haemogenic identity. Furthermore, we demonstrate that CDK4/6 and CDK1 play a key role not only in the transition but also in allowing haematopoietic progenitors to establish their full differentiation potential. CONCLUSION: We propose a direct link between the molecular machineries that control cell cycle progression and EHT.