Single-cell transcriptional profiling: a window into embryonic cell-type specification.

During mammalian embryonic development, a single fertilized egg cell will proliferate and differentiate into all the cell lineages and cell types that eventually form the adult organism. Cell lineage diversification involves repeated cell fate choices that ultimately occur at the level of the individual cell rather than at the cell-population level. Improvements in single-cell technologies are transforming our understanding of mammalian development, not only by overcoming the limitations presented by the extremely low cell numbers of early embryos but also by enabling the study of cell fate specification in greater detail. In this Review, we first discuss recent advances in single-cell transcriptomics and imaging and provide a brief outline of current bioinformatics methods available to analyse the resulting data. We then discuss how these techniques have contributed to our understanding of pre-implantation and early post-implantation development and of in vitro pluripotency. Finally, we overview the current challenges facing single-cell research and highlight the latest advances and potential future avenues

A single-cell resolution map of mouse hematopoietic stem and progenitor cell differentiation.

Maintenance of the blood system requires balanced cell fate decisions by hematopoietic stem and progenitor cells (HSPCs). Because cell fate choices are executed at the individual cell level, new single-cell profiling technologies offer exciting possibilities for mapping the dynamic molecular changes underlying HSPC differentiation. Here, we have used single-cell RNA sequencing to profile more than 1600 single HSPCs, and deep sequencing has enabled detection of an average of 6558 protein-coding genes per cell. Index sorting, in combination with broad sorting gates, allowed us to retrospectively assign cells to 12 commonly sorted HSPC phenotypes while also capturing intermediate cells typically excluded by conventional gating. We further show that independently generated single-cell data sets can be projected onto the single-cell resolution expression map to directly compare data from multiple groups and to build and refine new hypotheses. Reconstruction of differentiation trajectories reveals dynamic expression changes associated with early lymphoid, erythroid, and granulocyte-macrophage differentiation. The latter two trajectories were characterized by common upregulation of cell cycle and oxidative phosphorylation transcriptional programs. By using external spike-in controls, we estimate absolute messenger RNA (mRNA) levels per cell, showing for the first time that despite a general reduction in total mRNA, a subset of genes shows higher expression levels in immature stem cells consistent with active maintenance of the stem-cell state. Finally, we report the development of an intuitive Web interface as a new community resource to permit visualization of gene expression in HSPCs at single-cell resolution for any gene of choice.This work was supported by grants from Bloodwise, Cancer Research UK, Biotechnology and Biological Sciences Research Council, Leukemia Lymphoma Society, the National Institute for Health Research Cambridge Biomedical Research Centre, and core support grants by Wellcome Trust to the Cambridge Institute for Medical Research and Wellcome Trust-Medical Research Council Cambridge Stem Cell Institute. S.N. and F.K.H. are recipients of Medical Research Council PhD studentships. D.G.K. is the recipient of a Bennett Fellowship from Bloodwise, and E.L. is the recipient of a Sir Henry Dale Fellowship from the Wellcome Trust.This is the author accepted manuscript. The final version is available from the American Society of Hematology via http://dx.doi.org/10.1182/blood-2016-05-71648

Defining murine organogenesis at single-cell resolution reveals a role for the leukotriene pathway in regulating blood progenitor formation.

During gastrulation, cell types from all three germ layers are specified and the basic body plan is established 1 . However, molecular analysis of this key developmental stage has been hampered by limited cell numbers and a paucity of markers. Single-cell RNA sequencing circumvents these problems, but has so far been limited to specific organ systems 2 . Here, we report single-cell transcriptomic characterization of >20,000 cells immediately following gastrulation at E8.25 of mouse development. We identify 20 major cell types, which frequently contain substructure, including three distinct signatures in early foregut cells. Pseudo-space ordering of somitic progenitor cells identifies dynamic waves of transcription and candidate regulators, which are validated by molecular characterization of spatially resolved regions of the embryo. Within the endothelial population, cells that transition from haemogenic endothelial to erythro-myeloid progenitors specifically express Alox5 and its co-factor Alox5ap, which control leukotriene production. Functional assays using mouse embryonic stem cells demonstrate that leukotrienes promote haematopoietic progenitor cell generation. Thus, this comprehensive single-cell map can be exploited to reveal previously unrecognized pathways that contribute to tissue development

Mesoderm diversification during mouse embryonic development

During early mouse embryonic development, a single cell, the fertilised egg, will give rise to a wide range of cell types that become specialised at a functional and molecular level. Gastrulation and early organogenesis are two of the most critical events during these early stages, when pluripotent cells, able to generate any cell in the embryo, proliferate and become lineage-restricted into the progenitors of the major organs. The vast amount of cell fate decisions taking place in this 48-hour window makes these stages a suitable paradigm to study cell type diversification. Nevertheless, the low cell numbers in early mouse embryos and the limited strategies to isolate homogenous cell populations have restricted the study of the transcriptional programs and regulatory processes that underlie these processes $\textit{in vivo}$. With the advent of high-throughput single-cell genome-wide technologies, it is now possible to obtain the molecular profiles of hundreds of individual cells at once, thus opening a new window for the study of early embryogenesis. To delineate the molecular events underlying gastrulation and early organogenesis, we have therefore generated a comprehensive single-cell transcriptomic atlas of these stages. In Chapter 3, I introduce this atlas and give a general overview of the lineages that have been captured. Due to the importance of the haemato-endothelial lineages to establish the circulatory system in the embryo for appropriate oxygenation, in Chapter 4, I characterise their emergence using the atlas. My analyses uncover a rapid formation of primitive erythrocytes that do not transition through mature endothelium. Furthermore, I report the transcriptomes of megakaryocytic and myeloid progenitors as well as show that endothelial cells from different embryonic locations present distinctive transcriptional signatures. Getting a better characterisation of embryogenesis gives us a solid baseline to understand the consequences of genetic mutations. In Chapter 5, I explore the effects of disrupting the blood regulator $\textit{Tal1}$ using mouse embryonic chimaeras and reveal that endothelial cells are transcriptionally aberrant at early organogenesis and express genes characteristic of other mesodermal lineages. Although single-cell transcriptomics unveils the molecular programs defining each cell type, studying gene expression is not enough if we want to highlight the regulatory events behind cell type diversification. Therefore, in Chapter 5, I examine the use of single-cell transcriptomics to detect RNAs at enhancers, which may represent a surrogate for enhancer activity. Due to the limitations encountered, in Chapter 7, I perform single-nucleus ATAC-seq in cells at early organogenesis, a time-point by which all major progenitors are established. Analysing the resulting chromatin accessibility maps together with subsequent $\textit{in vivo}$ validation experiments have allowed the discovery of two novel endothelial enhancers as well as a previously unrecognised role for the ETS transcription factor FEV in the establishment of haemato-endothelial lineages. In conclusion, single-cell genome-wide technologies have permitted the comprehensive characterisation of the molecular programs and regulatory events underlying gastrulation and the start of organogenesis in the early mouse embryo. Having acquired this information has not only contributed to our understanding of embryonic development, but it will also help the optimisation of $\textit{in vitro}$ differentiation protocols in the future.Wellcome Trust 4-Year PhD Programme in Stem Cell Biology and Medicine and the University of Cambridge, United Kingdom

Single-cell chromatin accessibility maps reveal regulatory programs driving early mouse organogenesis.

During mouse embryonic development, pluripotent cells rapidly divide and diversify, yet the regulatory programs that define the cell repertoire for each organ remain ill-defined. To delineate comprehensive chromatin landscapes during early organogenesis, we mapped chromatin accessibility in 19,453 single nuclei from mouse embryos at 8.25 days post-fertilization. Identification of cell-type-specific regions of open chromatin pinpointed two TAL1-bound endothelial enhancers, which we validated using transgenic mouse assays. Integrated gene expression and transcription factor motif enrichment analyses highlighted cell-type-specific transcriptional regulators. Subsequent in vivo experiments in zebrafish revealed a role for the ETS factor FEV in endothelial identity downstream of ETV2 (Etsrp in zebrafish). Concerted in vivo validation experiments in mouse and zebrafish thus illustrate how single-cell open chromatin maps, representative of a mammalian embryo, provide access to the regulatory blueprint for mammalian organogenesis.Wellcome Trust, Bloodwise, MRC, CRUK, NIH NIDD

Myelo-lymphoid lineage restriction occurs in the human haematopoietic stem cell compartment before lymphoid-primed multipotent progenitors.

Capturing where and how multipotency is lost is crucial to understand how blood formation is controlled. Blood lineage specification is currently thought to occur downstream of multipotent haematopoietic stem cells (HSC). Here we show that, in human, the first lineage restriction events occur within the CD19-CD34+CD38-CD45RA-CD49f+CD90+ (49f+) HSC compartment to generate myelo-lymphoid committed cells with no erythroid differentiation capacity. At single-cell resolution, we observe a continuous but polarised organisation of the 49f+ compartment, where transcriptional programmes and lineage potential progressively change along a gradient of opposing cell surface expression of CLEC9A and CD34. CLEC9AhiCD34lo cells contain long-term repopulating multipotent HSCs with slow quiescence exit kinetics, whereas CLEC9AloCD34hi cells are restricted to myelo-lymphoid differentiation and display infrequent but durable repopulation capacity. We thus propose that human HSCs gradually transition to a discrete lymphoid-primed state, distinct from lymphoid-primed multipotent progenitors, representing the earliest entry point into lymphoid commitment.We thank the Cambridge NIHR BRC Cell Phenotyping Hub, particularly Anna Petrunkina-Harrison and Esther Perez for their flow cytometry advice; the Cambridge Blood and Stem Cell Biobank, specifically Joanna Baxter and the team of nurses consenting and collecting cord blood samples; David Kent for critical reading of the manuscript. E.L. is supported by a Sir Henry Dale fellowship from the Wellcome Trust (WT)/Royal Society. S.B. is supported by a CRUK Cambridge Cancer Center PhD fellowship. Research in the E.L. and B.G. laboratories is supported by the WT, EHA, CRUK, Bloodwise, MRC, BBSRC, NIH-NIDDK, and core support grants by the WT and MRC to the WT-MRC Cambridge Stem Cell Institute

A single-cell hematopoietic landscape resolves 8 lineage trajectories and defects in Kit mutant mice.

Hematopoietic stem and progenitor cells (HSPCs) maintain the adult blood system, and their dysregulation causes a multitude of diseases. However, the differentiation journeys toward specific hematopoietic lineages remain ill defined, and system-wide disease interpretation remains challenging. Here, we have profiled 44 802 mouse bone marrow HSPCs using single-cell RNA sequencing to provide a comprehensive transcriptional landscape with entry points to 8 different blood lineages (lymphoid, megakaryocyte, erythroid, neutrophil, monocyte, eosinophil, mast cell, and basophil progenitors). We identified a common basophil/mast cell bone marrow progenitor and characterized its molecular profile at the single-cell level. Transcriptional profiling of 13 815 HSPCs from the c-Kit mutant (W41/W41) mouse model revealed the absence of a distinct mast cell lineage entry point, together with global shifts in cell type abundance. Proliferative defects were accompanied by reduced Myc expression. Potential compensatory processes included upregulation of the integrated stress response pathway and downregulation of proapoptotic gene expression in erythroid progenitors, thus providing a template of how large-scale single-cell transcriptomic studies can bridge between molecular phenotypes and quantitative population changes

An atlas of rabbit development as a model for single-cell comparative genomics.

Traditionally, the mouse has been the favoured vertebrate model for biomedical research, due to its experimental and genetic tractability. However, non-rodent embryological studies highlight that many aspects of early mouse development, such as its egg-cylinder gastrulation and method of implantation, diverge from other mammals, thus complicating inferences about human development. Like the human embryo, rabbits develop as a flat-bilaminar disc. Here we constructed a morphological and molecular atlas of rabbit development. We report transcriptional and chromatin accessibility profiles for over 180,000 single cells and high-resolution histology sections from embryos spanning gastrulation, implantation, amniogenesis and early organogenesis. Using a neighbourhood comparison pipeline, we compare the transcriptional landscape of rabbit and mouse at the scale of the entire organism. We characterize the gene regulatory programmes underlying trophoblast differentiation and identify signalling interactions involving the yolk sac mesothelium during haematopoiesis. We demonstrate how the combination of both rabbit and mouse atlases can be leveraged to extract new biological insights from sparse macaque and human data. The datasets and computational pipelines reported here set a framework for a broader cross-species approach to decipher early mammalian development, and are readily adaptable to deploy single-cell comparative genomics more broadly across biomedical research