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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. Whole-genome sequencing of haematopoietic colonies from human fetuses reveals the somatic mutations acquired by individual progenitors, which are used as barcodes to construct a phylogenetic tree of blood development.

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.

The blood and immune system are characterised by utmost diversity in its cellular components. This heterogeneity can solely be resolved with the application of single-cell technologies that enable precise examination of cell-to-cell variation. Single-cell transcriptomics is continuously pushing forward our understanding of processes driving haematopoiesis and immune responses in physiological settings as well as in disease. Remarkably, in the last five years, a number of studies involving single-cell RNA sequencing (scRNA-seq) allowed the discovery of new immune cell types and revealed that haematopoiesis is a continuous rather than a stepwise process, thus challenging the classical haematopoietic lineage tree model. This review summarises the most recent studies which applied scRNA-seq to answer outstanding questions in the fields of haematology and immunology and discusses the present challenges and future directions.

Spatial transcriptomics is revolutionising the study of single-cell RNA and tissue-wide cell heterogeneity, but few robust methods connecting spatially resolved cells to so-called marker genes from single-cell RNA sequencing, which generate significant insight gleaned from spatial methods, exist. Here we present SMaSH, a general computational framework for extracting key marker genes from single-cell RNA sequencing data for spatial transcriptomics approaches. SMaSH extracts robust and biologically well-motivated marker genes, which characterise the given data-set better than existing and limited computational approaches for global marker gene calculation.

Summary 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., Graphical abstract, Highlights • The epigenetic and transcriptional landscape of human fetal hematopoiesis • Blood stem cells differentiate into three distinct oligopotent progenitor populations • Changes in motif accessibility in blood stem cells precede transcriptional priming • Refined sorting strategy to isolate and enrich for human fetal blood stem cells, Ranzoni et al. provide a detailed transcriptional and chromatin accessibility map of fetal liver and bone marrow hematopoietic stem cells (HSCs). Within HSCs, they revealed extensive epigenetic but not transcriptional priming. They identified transcriptional and functional differences between HSCs from liver and bone marrow.

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).

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).

To date, ontogeny of the human haematopoietic system during foetal development has been characterized mainly through careful microscopic observations. Here we used whole-genome sequencing (WGS) of 511 single-cell derived haematopoietic colonies from healthy human foetuses of 8 and 18 post-conception weeks (pcw) coupled with deep targeted sequencing of tissues of known embryonic origin to reconstruct a phylogenetic tree of blood development. We found that in healthy foetuses, individual haematopoietic progenitors acquire tens of somatic mutations by 18 pcw. Using these mutations as barcodes, we 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 analysis has shown that ectoderm originates from a smaller set of blood antecedents compared to endoderm and mesoderm. Finally, our data support a hypoblast origin of the extra-embryonic mesoderm and primitive blood in humans.

The ontogeny of the human haematopoietic system during fetal development has previously been characterized mainly through careful microscopic observations

Regulation of haematopoiesis during human development remains poorly defined. Here, we applied single-cell (sc)RNA-Seq and scATAC-Seq analysis to over 8,000 human immunophenotypic blood cells from foetal liver and bone marrow. We inferred their differentiation trajectory and identified three highly proliferative oligopotent progenitor populations downstream from haematopoietic stem cell/multipotent progenitors (HSC/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 HSC/MPPs prior to their lineage commitment. Finally, we refined and functionally validated the sorting strategy for the HSC/MPPs and achieved around 90% enrichment. Our study provides a useful framework for future investigation of human developmental haematopoiesis in the context of blood pathologies and regenerative medicine.

Human lungs enable efficient gas exchange and form an interface with the environment, which depends on mucosal immunity for protection against infectious agents. Tightly controlled interactions between structural and immune cells are required to maintain lung homeostasis. Here, we use single-cell transcriptomics to chart the cellular landscape of upper and lower airways and lung parenchyma in healthy lungs, and lower airways in asthmatic lungs. We report location-dependent airway epithelial cell states and a novel subset of tissue-resident memory T cells. In the lower airways of patients with asthma, mucous cell hyperplasia is shown to stem from a novel mucous ciliated cell state, as well as goblet cell hyperplasia. We report the presence of pathogenic effector type 2 helper T cells (TH2) in asthmatic lungs and find evidence for type 2 cytokines in maintaining the altered epithelial cell states. Unbiased analysis of cell-cell interactions identifies a shift from airway structural cell communication in healthy lungs to a TH2-dominated interactome in asthmatic lungs.

Innate lymphoid cells (ILCs) are important mediators of the immune response and homeostasis in barrier tissues of mammals. However, the existence and function of ILCs in other vertebrates is poorly understood. Here, we use single-cell RNA sequencing to generate a comprehensive atlas of zebrafish lymphocytes during tissue homeostasis and following immune challenge. We profiled 14,080 individual cells from the gut of wild-type zebrafish, as well as of rag1-deficient zebrafish which lack T and B cells, and discovered populations of ILC-like cells. We uncovered a rorc-positive subset of innate lymphoid cells that could express cytokines associated with type 1, 2 and 3 responses upon immune challenge. Specifically, these ILC-like cells expressed il22 and tnfa following exposure to inactivated bacteria, or il13 following exposure to helminth extract. Cytokine-producing ILC-like cells express a specific repertoire of novel immune-type receptors (NITRs), likely involved in recognition of environmental cues. We identified additional novel markers of zebrafish ILCs and generated a cloud repository for their in-depth exploration.

Innate lymphoid cells (ILCs) are important mediators of the immune response and homeostasis in barrier tissues of mammals. However, the existence and function of ILCs in other vertebrates is poorly understood. Here, we use single-cell RNA sequencing to generate a comprehensive atlas of zebrafish lymphocytes during tissue homeostasis and following immune challenge. We profiled 14,080 individual cells from the gut of wild-type zebrafish, as well as of rag1-deficient fish which lack T and B cells, and discovered diverse populations of helper ILC-like cells. Unexpectedly, fish displayed a rorc-positive, naïve subset that established a Type 3 or Type 2 response only upon immune challenge. Specifically, naïve ILC-like cells expressed il22 and tnfa following exposure to inactivated bacteria, or il13 following exposure to helminth extract. Cytokine-producing ILC-like cells express a specific repertoire of novel immune-type receptors, likely involved in recognition of environmental cues. We identified additional novel markers of zebrafish ILCs and generated a cloud repository for their in-depth exploration.

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.

To access publisher's full text version of this article, please click on the hyperlink in Additional Links field or click on the hyperlink at the top of the page marked Files. This article is open access. Nearly three-quarters of the 143 genetic signals associated with platelet and erythrocyte phenotypes identified by meta-analyses of genome-wide association (GWA) studies are located at non-protein-coding regions. Here, we assessed the role of candidate regulatory variants associated with cell type-restricted, closely related hematological quantitative traits in biologically relevant hematopoietic cell types. We used formaldehyde-assisted isolation of regulatory elements followed by next-generation sequencing (FAIRE-seq) to map regions of open chromatin in three primary human blood cells of the myeloid lineage. In the precursors of platelets and erythrocytes, as well as in monocytes, we found that open chromatin signatures reflect the corresponding hematopoietic lineages of the studied cell types and associate with the cell type-specific gene expression patterns. Dependent on their signal strength, open chromatin regions showed correlation with promoter and enhancer histone marks, distance to the transcription start site, and ontology classes of nearby genes. Cell type-restricted regions of open chromatin were enriched in sequence variants associated with hematological indices. The majority (63.6%) of such candidate functional variants at platelet quantitative trait loci (QTLs) coincided with binding sites of five transcription factors key in regulating megakaryopoiesis. We experimentally tested 13 candidate regulatory variants at 10 platelet QTLs and found that 10 (76.9%) affected protein binding, suggesting that this is a frequent mechanism by which regulatory variants influence quantitative trait levels. Our findings demonstrate that combining large-scale GWA data with open chromatin profiles of relevant cell types can be a powerful means of dissecting the genetic architecture of closely related quantitative traits. Marie-Curie Initial Training Network NETSIM British Heart Foundation RG/09/12/28096 National Institutes for Health RP-PG-0310-1002 Wellcome Trust 098051 info:eu-repo/grantAgreement/EC/FP7/282510

The homologous recombination protein RAD51 has been extensively studied in prokaryotes and lower eukaryotes. However, there is a significant lack of knowledge of the role of this protein and its regulation in an in vivo context in vertebrates. Here we report the first viable vertebrate mutant model of rad51 in zebrafish. These mutant fish enabled us to confirm the recently discovered role of RAD51 in Fanconi anemia pathogenesis. We report that p53-linked embryonic stem cell defects directly lead to hematological impairments later in life. Comutation of rad51 with p53 rescues the observed hematological defects, but predisposes the fish to early tumor development. The application of this model opens new possibilities to advance Fanconi anemia drug discovery.

The blood group Vel was discovered 60 years ago(1), but the underlying gene is unknown. Individuals negative for the Vel antigen are rare and are required for the safe transfusion of patients with antibodies to Vel(2). To identify the responsible gene, we sequenced the exomes of five individuals negative for the Vel antigen and found that four were homozygous and one was heterozygous for a low-frequency 17-nucleotide frameshift deletion in the gene encoding the 78-amino-acid transmembrane protein SMIM1. A follow-up study showing that 59 of 64 Vel-negative individuals were homozygous for the same deletion and expression of the Vel antigen on SMIM1-transfected cells confirm SMIM1 as the gene underlying the Vel blood group. An expression quantitative trait locus (eQTL), the common SNP rs1175550 contributes to variable expression of the Vel antigen (P = 0.003) and influences the mean hemoglobin concentration of red blood cells (RBCs; P = 8.6 x 10(-15))(3). In vivo, zebrafish with smim1 knockdown showed a mild reduction in the number of RBCs, identifying SMIM1 as a new regulator of RBC formation. Our findings are of immediate relevance, as the homozygous presence of the deletion allows the unequivocal identification of Vel-negative blood donors.

The success of marker-based approaches for dissecting haematopoiesis in mouse and human is reliant on the presence of well-defined cell surface markers specific for diverse progenitor populations. An inherent problem with this approach is that the presence of specific cell surface markers does not directly reflect the transcriptional state of a cell. Here, we used a marker-free approach to computationally reconstruct the blood lineage tree in zebrafish and order cells along their differentiation trajectory, based on their global transcriptional differences. Within the population of transcriptionally similar stem and progenitor cells, our analysis reveals considerable cell-to-cell differences in their probability to transition to another committed state. Once fate decision is executed, the suppression of transcription of ribosomal genes and upregulation of lineage-specific factors coordinately controls lineage differentiation. Evolutionary analysis further demonstrates that this haematopoietic programme is highly conserved between zebrafish and higher vertebrates., Traditionally marker-based approaches are used to define haematopoietic cell type or state. Here, the authors use single-cell RNA-seq to establish a cellular hierarchy of lineage development in zebrafish haematopoiesis, and propose a refined model of developmental progression of haematopoietic cells.

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.

All data generated in this study have been deposited in the ArrayExpress database under accession codes E-MTAB-5480, E-MTAB-5481, E-MTAB-5482, E-MTAB-5483, E-MTAB-5484, E-MTAB-5485, and E-MTAB-5486. Summary tables are provided as supplementary files., 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., We are grateful to O. Stegle and J.K. Kim for helpful discussions and comments on the manuscript. We thank M. Lynch for support with the C1 experiments, X. Chen for discussions on spike-ins, and M. Quail for help with 10× Chromium experiments. We extend our gratitude to S. Linnarsson and A. Zeisel for invaluable support in implementing STRT-seq in our laboratory and for help with sequencing the STRT library. We also thank D. Grün for sharing smFISH molecule counts. Finally we thank R. Kirchner for many improvements to the umis tool. This study was supported by Cancer Research UK grant C45041/A14953 to A.C. and C.L.; European Research Council project 677501–ZF_Blood to A.C.; a core support grant from the Wellcome Trust and MRC to the Wellcome Trust–Medical Research Council Cambridge Stem Cell Institute; ERC grant ThSWITCH to S.A.T. (grant 260507); and a Lister Institute Research Prize to S.A.T. K.N.N. was supported by the Wellcome Trust Strategic Award ‘Single cell genomics of mouse gastrulation’. We thank P. Liu (Wellcome Trust Sanger Institute) for providing cells., info:eu-repo/semantics/publishedVersion

RAD51is 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 paper, we describe the first viable vertebrate model ofRAD51loss. Zebrafishrad51loss-of-function mutants developed key features of FA, including hypocellular kidney marrow, sensitivity to crosslinking 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. Co-mutation ofp53was 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 ofRAD51as 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 novel 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.Significance statementThe homologous recombination protein RAD51 has been extensively studied in prokaryotes and lower eukaryotes. However, there is a significant lack of knowledge of the role of this protein and its regulation in anin-vivocontext in vertebrates. Here we report the first viable vertebrate mutant model ofrad51in zebrafish. These mutant fish enabled us to confirm for the first time the recently discovered role ofRAD51in Fanconi anemia pathogenesis. We report that p53 linked embryonic stem cell defects directly lead to hematological impairments later in life. Co-mutation ofrad51withp53rescues the observed hematological defects, but predisposes the fish to early tumor development. The application of this model opens new possibilities to advance Fanconi anemia drug discovery.

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 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 analysed gene conservation of 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 to other immune cell populations, 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 to obtain the first transcriptome of specific immune cell types in a non-mammalian 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 a higher gene turnover and lower conservation of trans-membrane proteins in NK cells compared to T cells in fish species, suggesting that this is a general property of immune cell types across all vertebrates.

High-throughput single cell RNA sequencing (scRNA-seq) has become an established and powerful method to investigate transcriptomic cell-to-cell variation, and has revealed new cell types, and new insights into developmental process and stochasticity in gene expression. There are now several published scRNA-seq protocols, which all sequence transcriptomes from a minute amount of starting material. Therefore, a key question is how these methods compare in terms of sensitivity of detection of mRNA molecules, and accuracy of quantification of gene expression. Here, we assessed the sensitivity and accuracy of many published data sets based on standardized spike-ins with a uniform raw data processing pipeline. We developed a flexible and fast UMI counting tool (https://github.com/vals/umis) which is compatible with all UMI based protocols. This allowed us to relate these parameters to sequencing depth, and discuss the trade offs between the different methods. To confirm our results, we performed experiments on cells from the same population using three different protocols. We also investigated the effect of RNA degradation on spike-in molecules, and the average efficiency of scRNA-seq on spike-in molecules versus endogenous RNAs.

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.

Despite significant improvements in our knowledge of the mechanisms of normal and pathological hematopoiesis, our current understanding is most likely an oversimplification of the complexity of regulatory networks at play. Thus, considerable efforts have been made to catalogue the total sum of germline alterations in individual genomes affecting human hematopoiesis. These efforts ultimately led to the discovery of a large number of new genes not previously implicated in blood formation. Although identification of novel genes is important in revealing the profiles of genetic variations associated with normal hematopoiesis, further functional studies are necessary to improve our understanding of the mechanism(s) involved in these processes. In this review, we summarize the knowledge gained from genome-wide association studies to elucidate the relationship between genetics and blood cell traits. We discuss the most important recent advances, with an emphasis on functional follow-up studies that have been particularly useful in providing an insight into novel regulatory processes that influence blood cell formation and function. We also discuss potential future directions and challenges in the field.

Blood cells derive from hematopoietic stem cells through stepwise fating events. To characterize gene expression programs driving lineage choice, we sequenced RNA from eight primary human hematopoietic progenitor populations representing the major myeloid commitment stages and the main lymphoid stage. We identified extensive cell type-specific expression changes: 6711 genes and 10,724 transcripts, enriched in non-protein-coding elements at early stages of differentiation. In addition, we found 7881 novel splice junctions and 2301 differentially used alternative splicing events, enriched in genes involved in regulatory processes. We demonstrated experimentally cell-specific isoform usage, identifying nuclear factor I/B (NFIB) as a regulator of megakaryocyte maturation-the platelet precursor. Our data highlight the complexity of fating events in closely related progenitor populations, the understanding of which is essential for the advancement of transplantation and regenerative medicine. The work described in this manuscript was primarily supported by the European Commission Seventh Framework Program through the BLUEPRINT grant with code HEALTH-F5-2011-282510 (DH, FB, GC, JHAM, KD, LC, MF, SC, SF and SPG). Research in the Ouwehand laboratory is further supported by program grants from the National Institute for Health Research (NIHR, http://www.nihr.ac.uk; to AA, MK, PP, SBGJ, SN, and WHO); and the British Heart Foundation under numbers RP-PG-0310-1002 and RG/09/12/28096 (http://www.bhf.org.uk; to AR and WJA). KF and MK were supported by Marie Curie funding from the NETSIM FP7 program funded by the European Commission. The Cambridge BioResource (http://www.cambridgebioresource.org.uk), the Cell Phenotyping Hub, and the Cambridge Translational GenOmics laboratory (http://www.catgo.org.uk) are supported by an NIHR grant to the Cambridge NIHR Biomedical Research Centre (BRC). Research in the Soranzo laboratory (LV, NS and SW) is further supported by the Wellcome Trust (Grant Codes WT098051 and WT091310) and the EU FP7 EPIGENESYS initiative (Grant Code 257082). Research in the Cvejic laboratory (AC and CL) is funded by the Cancer Research UK under grant number C45041/A14953. SJS is funded by NIHR. MEF is supported by a British Heart Foundation Clinical Research Training Fellowship, number FS/12/27/29405. EBM is supported by a Wellcome Trust grant, number 084183/Z/07/Z. FAC, CL and SW are supported by MRC Clinical Training Fellowships and TB by a British Society of Haematology/NHS Blood and Transplant grant. RJR is a Principal Research Fellow of the Wellcome Trust, grant No. 082961/Z/07/Z. Research in the Flicek laboratory is also supported by the Wellcome Trust (grant number 095908) and EMBL. Research in the Bertone laboratory is supported by EMBL. KF and CvG are supported by FWO-Vlaanderen through grant G.0B17.13N

Item does not contain fulltext The exon-junction complex (EJC) performs essential RNA processing tasks. Here, we describe the first human disorder, thrombocytopenia with absent radii (TAR), caused by deficiency in one of the four EJC subunits. Compound inheritance of a rare null allele and one of two low-frequency SNPs in the regulatory regions of RBM8A, encoding the Y14 subunit of EJC, causes TAR. We found that this inheritance mechanism explained 53 of 55 cases (P < 5 x 10(-228)) of the rare congenital malformation syndrome. Of the 53 cases with this inheritance pattern, 51 carried a submicroscopic deletion of 1q21.1 that has previously been associated with TAR, and two carried a truncation or frameshift null mutation in RBM8A. We show that the two regulatory SNPs result in diminished RBM8A transcription in vitro and that Y14 expression is reduced in platelets from individuals with TAR. Our data implicate Y14 insufficiency and, presumably, an EJC defect as the cause of TAR syndrome.

The segmentation of thrombus and vessel in microscopic image sequences is of high interest for identifying genes linked to cardiovascular diseases. This task is however challenging because of the low contrast and the highly dynamic conditions observed in time-lapse DIC in-vivo microscopic scenes. In this work, we introduce a probabilistic framework for the joint segmentation of thrombus and vessel regions. Modeling the scene with dynamic textures, we derive two likelihood functions to account for both spatial and temporal discrepancies of the motion patterns. A tubular shape prior is moreover introduced to constrain the aortic region. Extensive experiments on microscopic sequences quantitatively show the good performance of our approach.

Chronic Myeloid Disorders Working Group of the International Cancer Genome Consortium.-- et al., The myelodysplastic syndromes are a heterogeneous group of hematologic cancers characterized by low blood counts, most commonly anemia, and a risk of progression to acute myeloid leukemia.1 These disorders have increased in prevalence and are expected to continue to do so. Blood films and bone marrow¿biopsy specimens from patients with myelodysplastic syndromes show dysplastic changes in myeloid cells, with abnormal proliferation and differentiation of one or more lineages. Target genes of recurrent chromosomal aberrations have been mapped,2,3 and several genes have been identified as recurrently mutated in these disorders, including NRAS (encoding neuroblastoma RAS viral oncogene homologue), TP53 (encoding tumor protein p53), RUNX1 (encoding runt-related transcription factor 1), CBL (encoding Cas-Br-M ecotropic retroviral transforming sequence),4,5 TET2 (encoding tet oncogene family member 2),6,7 ASXL1 (encoding additional sex combs¿like protein 1),8,9 and EZH2 (encoding enhancer of zeste homologue 2).10 With the exception of TET2, most of these genes are mutated in no more than 5 to 15% of cases, and generally the mutation rates are lower in the more benign subtypes of the disease. The myelodysplastic syndromes can be divided into several categories on the basis of bone marrow and peripheral-blood morphologic characteristics and cytogenetic changes.11 In low-risk disease, such as refractory anemia, cytopenias are the major clinical challenge, whereas high-risk disease, such as refractory anemia with excess blasts, is characterized by both cytopenias and a high rate of transformation to acute myeloid leukemia. More than a quarter of patients with myelodysplastic syndromes have large numbers of ring sideroblasts in the bone marrow,12 a sufficiently distinctive morphologic abnormality to warrant a separate designation. Ring sideroblasts are characteristically seen on iron staining of bone marrow aspirates as differentiating erythroid cells with a complete or partial ring of iron-laden mitochondria surrounding the nucleus. Several genetic lesions underpinning inherited sideroblastic anemias have been identified,13 including loss-of-function mutations in the genes ALAS2 (encoding delta aminolevulinate synthase 2), ABCB7 (encoding ATP-binding cassette, subfamily B, member 7), and SLC25A38 (solute carrier family 25, member 38). The pathogenesis of ring sideroblasts in myelodysplastic syndromes, however, remains obscure, although gene-expression studies have revealed up-regulation of genes involved in heme synthesis (including ALAS2) and down-regulation of ABCB7.14,15 We reasoned that the identification of recurrently mutated cancer genes in low-grade myelodysplastic syndromes could prove useful for the diagnosis of these disorders and provide new insights into the molecular pathogenesis of these syndromes, Supported by grants from the Wellcome Trust (077012/Z/05/Z, for the overall study, as well as WT088340MA, to Dr. Campbell), the Kay Kendall Leukaemia Fund, Leukemia Lymphoma Research (for the overall study and to Drs. Boultwood, Green, Vyas, and Wainscoat), the Adenoid Cystic Carcinoma Research Foundation, the Medical Research Council (MRC) (to Dr. Warren), the Oxford National Institutes for Health Research Biomedical Research Centre (to Drs. Boultwood, Vyas, and Wainscoat), the Swedish Cancer Society (to Dr. Hellstrom-Lindburg), the International Human Frontier Science Program Organization (to Dr. Varela), the Department of Veterans Affairs and the National Institutes of Health (R01-124929, P01-155249, P50- 100007, and P01-78378, to Drs. Munshi and Anderson), the Association for International Cancer Research and the Leukemia Lymphoma Society (to Drs. Warren and Green), Associazione Italiana per la Ricerca sul Cancro (to the University of Pavia, the University of Milan Bicocca, and Dr. Cazzola), and Fondazione Cariplo (to the University of Pavia and the University of Milan Bicocca).

Gray platelet syndrome (GPS) is a predominantly recessive platelet disorder that is characterized by mild thrombocytopenia with large platelets and a paucity of α-granules; these abnormalities cause mostly moderate but in rare cases severe bleeding. We sequenced the exomes of four unrelated individuals and identified NBEAL2 as the causative gene; it has no previously known function but is a member of a gene family that is involved in granule development. Silencing of nbeal2 in zebrafish abrogated thrombocyte formation.

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.

Summary Hematopoietic differentiation critically depends on combinations of transcriptional regulators controlling the development of individual lineages. Here, we report the genome-wide binding sites for the five key hematopoietic transcription factors—GATA1, GATA2, RUNX1, FLI1, and TAL1/SCL—in primary human megakaryocytes. Statistical analysis of the 17,263 regions bound by at least one factor demonstrated that simultaneous binding by all five factors was the most enriched pattern and often occurred near known hematopoietic regulators. Eight genes not previously appreciated to function in hematopoiesis that were bound by all five factors were shown to be essential for thrombocyte and/or erythroid development in zebrafish. Moreover, one of these genes encoding the PDZK1IP1 protein shared transcriptional enhancer elements with the blood stem cell regulator TAL1/SCL. Multifactor ChIP-Seq analysis in primary human cells coupled with a high-throughput in vivo perturbation screen therefore offers a powerful strategy to identify essential regulators of complex mammalian differentiation processes., Highlights ► Genome-wide TF binding analysis in primary human megakaryocyte ► Regulatory network reconstruction from TF, epigenetic, and expression data ► Simultaneous binding of five TFs marks hematopoietic regulators ► Five-factor binding reveals eight genes essential for zebrafish blood 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.

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. ispartof: BLOOD vol:113 issue:19 pages:4754-4762 ispartof: location:United States status: published