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The first hematopoietic stem cells (HSCs) are formed through endothelial-to-hematopoietic transition (EHT) during embryonic development. The transcription factor GATA2 is a crucial regulator of EHT and HSC function throughout life. Because patients with GATA2 haploinsufficiency have inborn mutations, prenatal defects are likely to influence disease development. In mice, Gata2 haploinsufficiency (Gata2+/−) reduces the number and functionality of embryonic hematopoietic stem and progenitor cells (HSPCs) generated through EHT. However, the embryonic HSPC pool is heterogeneous and the mechanisms underlying this defect in Gata2+/− embryos remain unclear. Here, we investigated whether Gata2 haploinsufficiency selectively affects a cellular subset undergoing EHT. We showed that Gata2+/− HSPCs initiate, but cannot fully activate, hematopoietic programming during EHT. In addition, due to the reduced activity of the endothelial repressor Gfi1b, Gata2+/− HSPCs cannot repress endothelial identity to complete maturation. Finally, we showed that hematopoietic-specific induction of gfi1b could restore HSC production in gata2b-null (gata2b−/−) zebrafish embryos. This study illustrates the pivotal role of Gata2 in the regulation of the transcriptional network governing HSPC identity throughout the EHT.

The adult-definitive hematopoietic hierarchy and hematopoietic stem cells (HSCs) residing in the bone marrow are established during embryonic development. In mouse, human, and many other mammals, it is the sudden formation of so-called intra-aortic/arterial hematopoietic clusters (IAHCs) that best signifies and visualizes this de novo generation of HSCs and hematopoietic progenitor cells (HPCs). Cluster cells arise through an endothelial-to-hematopoietic transition and, for some time, express markers/genes of both tissue types, although acquiring more hematopoietic features and losing endothelial ones. Among several hundreds of IAHC cells, the midgestation mouse embryo contains only very few bona fide adult-repopulating HSCs, suggestive of a challenging cell fate to achieve. Most others are HPCs of various types, some of which have the potential to mature into HSCs in vitro. Based on the number of cells that reveal hematopoietic function, a fraction of IAHC cells is uncharacterized. This review aims to explore the current state of knowledge on IAHC cells. We will describe markers useful for isolation and characterization of these fleetingly produced, yet vitally important, cells and for the refined enrichment of the HSCs they contain and speculate on the role of some IAHC cells that are as-yet functionally uncharacterized.

Integrated molecular signals regulate cell fate decisions in the embryonic aortic endothelium to drive hematopoietic stem cell (HSC) generation during development. The G-protein–coupled receptor 56 (Gpr56, also called Adgrg1) is the most highly upregulated receptor gene in cells that take on hematopoietic fate and is expressed by adult bone marrow HSCs. Despite the requirement for Gpr56 in hematopoietic stem/progenitor cell (HS/PC) generation in zebrafish embryos and the highly upregulated expression of GPR56 in treatment-resistant leukemic patients, its function in normal mammalian hematopoiesis remains unclear. Here, we examine the role of Gpr56 in HS/PC development in Gpr56 conditional knockout (cKO) mouse embryos and Gpr knockout (KO) embryonic stem cell (ESC) hematopoietic differentiation cultures. Our results show a bias toward myeloid differentiation of Gpr56 cKO fetal liver HSCs and an increased definitive myeloid progenitor cell frequency in Gpr56KO ESC differentiation cultures. Surprisingly, we find that mouse Gpr97 can rescue Gpr56 morphant zebrafish hematopoietic generation, and that Gpr97 expression is upregulated in mouse Gpr56 deletion models. When both Gpr56 and Gpr97 are deleted in ESCs, no or few hematopoietic PCs (HPCs) are generated upon ESC differentiation. Together, our results reveal novel and redundant functions for these 2 G-protein coupled receptors in normal mammalian hematopoietic cell development and differentiation.

The first hematopoietic stem cells (HSCs) are formed through endothelial-to-hematopoietic transition (EHT) events during embryonic development. The transcription factor GATA2 is a crucial regulator of EHT and HSC function throughout life. Because GATA2 haploinsufficiency patients have inborn mutations, prenatal defects are likely to have an influence on disease development. In mice, Gata2 haploinsufficiency (Gata2+/-) reduces the number and the functionality of embryonic hematopoietic stem and progenitor cells (HSPCs) generated through EHT. However, the embryonic HSPC pool is heterogeneous and the mechanisms underlying this defect in Gata2+/- embryos are unclear. Here, we investigated whether Gata2 haploinsufficiency selectively affects a cellular subset undergoing EHT. We show that Gata2+/- HSPCs initiate but cannot fully activate hematopoietic programming during EHT. In addition, due to reduced activity of the endothelial repressor Gfi1b, Gata2+/- HSPCs cannot repress the endothelial identity to complete maturation. Finally, we show that hematopoietic-specific induction of gfi1b can restore HSC production in gata2b-null (gata2b-/-) zebrafish embryos. This study illustrates pivotal roles of Gata2 on the regulation of transcriptional network governing HSPC identity throughout EHT.HighlightsMaturation of embryonic Gata2+/- HSPCs is disturbed due to aberrant endothelial gene expression and incomplete activation of hematopoietic transcriptional programming.Gata2 activates Gfi1b to repress endothelial identity of embryonic HSPCs during maturation.Hematopoietic-specific induction of gfi1b restores the number of embryonic HSCs in gata2b-/- zebrafish.

Multicellular systems possess an intrinsic capacity to autonomously generate nonrandom state distributions or morphologies in a process termed self-organization. Facets of self-organization, such as pattern formation, pattern elaboration, and symmetry breaking, are frequently observed in developing embryos. Artificial stem cell-derived structures including embryoid bodies (EBs), gastruloids, and organoids also demonstrate self-organization, but with a limited capacity compared to their in vivo developmental counterparts. There is a pressing need for better tools to allow user-defined control over self-organization in these stem cell-derived structures. Here, we employ synthetic biology to establish an efficient platform for the generation of self-organizing coaggregates, in which HEK-293 cells overexpressing P-cadherin (Cdh3) spontaneously form cell clusters attached mostly to one or two locations on the exterior of EBs. These Cdh3-expressing HEK cells, when further engineered to produce functional mouse WNT3A, evoke polarized and gradual Wnt/β-catenin pathway activation in EBs during coaggregation cultures. The localized WNT3A provision induces nascent mesoderm specification within regions of the EB close to the Cdh3-Wnt3a-expressing HEK source, resulting in pattern elaboration and symmetry breaking within EBs. This synthetic biology-based approach puts us closer toward engineering synthetic organizers to improve the realism in stem cell-derived structures.

E-cadherin is a calcium-dependent cell-cell adhesion molecule extensively studied for its involvement in tissue formation, epithelial cell behavior, and suppression of cancer. However, E-cadherin expression in the hematopoietic system has not been fully elucidated. Combining single-cell RNA-sequencing analyses and immunophenotyping, we revealed that progenitors expressing high levels of E-cadherin and contained within the granulocyte-monocyte progenitors (GMPs) fraction have an enriched capacity to differentiate into basophils and mast cells. We detected E-cadherin expression on committed progenitors before the expression of other reported markers of these lineages. We named such progenitors pro-BMPs (pro-basophil and mast cell progenitors). Using RNA sequencing, we observed transcriptional priming of pro-BMPs to the basophil and mast cell lineages. We also showed that GATA-2 directly regulates E-cadherin expression in the basophil and mast cell lineages, thus providing a mechanistic connection between the expression of this cell surface marker and the basophil and mast cell fate specification. ISSN:2470-9468

Summary Whereas hundreds of cells in the mouse embryonic aorta transdifferentiate to hematopoietic cells, only very few establish hematopoietic stem cell (HSC) identity at a single time point. The Gata2 transcription factor is essential for HSC generation and function. In contrast to surface-marker-based cell isolation, Gata2-based enrichment provides a direct link to the internal HSC regulatory network. Here, we use iterations of index-sorting of Gata2-expressing intra-aortic hematopoietic cluster (IAHC) cells, single-cell transcriptomics, and functional analyses to connect HSC identity to specific gene expression. Gata2-expressing IAHC cells separate into 5 major transcriptomic clusters. Iterative analyses reveal refined CD31, cKit, and CD27 phenotypic parameters that associate specific molecular profiles in one cluster with distinct HSC and multipotent progenitor function. Thus, by iterations of single-cell approaches, we identify the transcriptome of the first functional HSCs as they emerge in the mouse embryo and localize them to aortic clusters containing 1–2 cells., Graphical Abstract, Highlights • Single-cell iterations capture the transcriptome of the first functional HSCs in mouse • Gata2 connects the “inner” HSC regulatory network with single-cell function • Specific Gata2, cKit, and CD27 levels define all HSCs in embryonic aortic clusters • HSCs emerge from endothelium as single cells within aortic clusters of 1–2 cells, Vink and colleagues capture the transcriptome of the first functional HSCs in mouse by single-cell RNA-seq, index-sorting, and in vivo and invitro hematopoietic analyses. The HSC transcriptome is unique compared to HPCs, and heterogeneous expression of pivotal genes suggests that establishment of functional hematopoietic fate during cell emergence from embryonic aortic endothelium is stochastic.

Hematopoietic stem cells (HSCs) develop from the hemogenic endothelium in cluster structures that protrude into the embryonic aortic lumen. Although much is known about the molecular characteristics of the developing hematopoietic cells, we lack a complete understanding of their origin and the three-dimensional organization of the niche. Here, we use advanced live imaging techniques of organotypic slice cultures, clonal analysis, and mathematical modeling to show the two-step process of intra-aortic hematopoietic cluster (IACH) formation. First, a hemogenic progenitor buds up from the endothelium and undergoes division forming the monoclonal core of the IAHC. Next, surrounding hemogenic cells are recruited into the IAHC, increasing their size and heterogeneity. We identified the Notch ligand Dll4 as a negative regulator of the recruitment phase of IAHC. Blocking of Dll4 promotes the entrance of new hemogenic Gfi1+ cells into the IAHC and increases the number of cells that acquire HSC activity. Mathematical modeling based on our data provides estimation of the cluster lifetime and the average recruitment time of hemogenic cells to the cluster under physiologic and Dll4-inhibited conditions. We thank Thomas Graf (CRG), Minhong Yan (Genentech), Georges Lacaud (CRUK Manchester Institut) and Ralf Adams (Max Planck Institute for Molecular Medicine, Münster) for important mice models and reagents. Thanks to Catherine Robin, Timo Zimmermann and, Raul Gómez-Riera for imaging advice. Thanks to the PRBB facilities: animal facility, flow cytometry, CNAGCRG genomics, confocal microscopy for critical technical assistance. Thanks to Marta Garrido and Luis Galán for experimental support and all the members of the lab for helpful critical discussions. This research was funded by the Ministerio de Economía y Competitividad (SAF2016 -75613-R) and Generalitat de Catalunya, Agència de Gestió d’Ajuds Universitaris i de Recerca (AGAUR) (2017 SGR 135) and PERIS-SLT002/16/00299 to AB and ERC AdG 341096 to ED. CP was a recipient of Juan de la Cierva fellowship (FJCI2014-19870), RT is a recipient of Beatriu de Pinos (2016 BP 00021), JG is a recipient of PERIS (SLT002/16/00070).

Summary In vivo hematopoietic generation occurs in waves of primitive and definitive cell emergence. Differentiation cultures of pluripotent embryonic stem cells (ESCs) offer an accessible source of hematopoietic cells for blood-related research and therapeutic strategies. However, despite many approaches, it remains a goal to robustly generate hematopoietic progenitor and stem cells (HP/SCs) in vitro from ESCs. This is partly due to the inability to efficiently promote, enrich, and/or molecularly direct hematopoietic emergence. Here, we use Gata2Venus (G2V) and Ly6a(SCA1)GFP (LG) reporter ESCs, derived from well-characterized mouse models of HP/SC emergence, to show that during in vitro differentiation they report emergent waves of primitive hematopoietic progenitor cells (HPCs), definitive HPCs, and B-lymphoid cell potential. These results, facilitated by enrichment of single and double reporter cells with HPC properties, demonstrate that in vitro ESC differentiation approximates the waves of hematopoietic cell generation found in vivo, thus raising possibilities for enrichment of rare ESC-derived HP/SCs., Highlights • Gata2 reports waves of hematopoietic cell potential during ESC differentiation • Ly6aGFP expression distinguishes a late wave of ESC hematopoietic differentiation • Fluorescent reporters enrich ESC-derived cells with hematopoietic potential • Double reporter ESCs verify waves of hematopoietic progenitor generation, Kauts et al. demonstrate that Gata2Venus, Ly6aGFP, and double reporter expression in differentiating mouse embryonic stem cells (ESCs) discriminates and facilitates enrichment of most hematopoietic progenitors generated in vitro. Sequential waves of ESC-derived reporter cell emergence show increasing hematopoietic lineage potency and approximate the in vivo waves of hematopoietic cell generation found in the mouse embryo.

The Runx family of transcription factors (Runx1, Runx2, and Runx3) are highly conserved and encode proteins involved in a variety of cell lineages, including blood and blood-related cell lineages, during developmental and adult stages of life. They perform activation and repressive functions in the regulation of gene expression. The requirement for Runx1 in the normal hematopoietic development and its dysregulation through chromosomal translocations and loss-of-function mutations as found in acute myeloid leukemias highlight the importance of this transcription factor in the healthy blood system. Whereas another review will focus on the role of Runx factors in leukemias, this review will provide an overview of the normal regulation and function of Runx factors in hematopoiesis and focus particularly on the biological effects of Runx1 in the generation of hematopoietic stem cells. We will present the current knowledge of the structure and regulatory features directing lineage-specific expression of Runx genes, the models of embryonic and adult hematopoietic development that provide information on their function, and some of the mechanisms by which they affect hematopoietic function.

Summary Estrogen stimulates breast development during puberty and mammary tumors in adulthood through estrogen receptor-α (ERα). These effects are proposed to occur via ERα+ luminal cells and not the mammary stem cells (MaSCs) that are ERαneg. Since ERα+ luminal cells express stem cell antigen-1 (SCA-1), we sought to determine if SCA-1 could define an ERα+ subset of EpCAM+/CD24+/CD49fhi MaSCs. We show that the MaSC population has a distinct SCA-1+ population that is abundant in pre-pubertal mammary glands. The SCA-1+ MaSCs have less stem cell markers and less in vivo repopulating activity than their SCA-1neg counterparts. However, they express ERα and specifically enter the cell cycle at puberty. Using estrogen-deficient aromatase knockouts (ArKO), we showed that the SCA-1+ MaSC could be directly modulated by estrogen supplementation. Thus, SCA-1 enriches for an ERα+, estrogen-sensitive subpopulation within the CD24+/CD49fhi MaSC population that may be responsible for the hormonal sensitivity of the developing mammary gland., Highlights • SCA-1+ delineates ER-positive cells in the CD24+ CD49fhi mammary stem population • SCA-1+ cells have lower repopulation activity • SCA-1+ cells are estrogen responsive, Mouse mammary stem cells are thought to be estrogen-receptor negative and receive hormonal influence via estrogen-receptor-positive luminal neighbors. In this article, Britt and colleagues describe a population within the mammary stem cell-enriched compartment that is estrogen-receptor positive and directly responsive to estrogens. This has implications for understanding how aberrant hormone exposure affects breast cancer risk.

Hematopoietic stem cells (HSCs) develop from the hemogenic endothelium in cluster structures that protrude into the embryonic aortic lumen. Although much is known about the molecular characteristics of the developing hematopoietic cells, we lack a complete understanding of their origin and the three-dimensional organization of the niche. Here we use advanced live imaging techniques of organotypic slice cultures, clonal analysis, and mathematical modelling to show the two-step process of intra-aortic hematopoietic cluster (IACH) formation. First, a hemogenic progenitor buds up from the endothelium and undergoes division forming the monoclonal core of the IAHC. Next, surrounding hemogenic cells are recruited into the IAHC, increasing their size and heterogeneity. We identified the Notch ligand Dll4 as a negative regulator of the recruitment phase of IAHC. Blocking of Dll4 promotes the entrance of new hemogenic Gfi1+ cells into the IAHC and increases the number of cells that acquire HSC activity. Mathematical modelling based on our data provides estimation of the cluster lifetime and the average recruitment time of hemogenic cells to the cluster under physiologic and Dll4-inhibited conditions.

Along with the aorta-gonad-mesonephros region, the head is a site of hematopoietic stem and progenitor cell (HS/PC) development in the mouse embryo. Macrophages are present in both these embryonic hemogenic sites, and recent studies indicate a functional interaction of macrophages with hematopoietic cells as they are generated in the aorta. Whereas brain macrophages or “microglia” are known to affect neuronal patterning and vascular circuitry in the embryonic brain, it is unknown whether macrophages play a role in head hematopoiesis. Here, we characterize head macrophages and examine whether they affect the HS/PC output of the hindbrain-branchial arch (HBA) region of the mouse embryo. We show that HBA macrophages are CD45(+)F4/80(+)CD11b(+)Gr1(−) and express the macrophage-specific Csf1r-GFP reporter. In the HBA of chemokine receptor-deficient (Cx3cr1(−/−)) embryos, a reduction in erythropoiesis is concomitant with a decrease in HBA macrophage percentages. In cocultures, we show that head macrophages boost hematopoietic progenitor cell numbers from HBA endothelial cells > twofold, and that the proinflammatory factor tumor necrosis factor-α is produced by head macrophages and influences HBA hematopoiesis in vitro. Taken together, head macrophages play a positive role in HBA erythropoiesis and HS/PC expansion and/or maturation, acting as microenvironmental cellular regulators in hematopoietic development.

Hematopoietic stem cells (HSCs) are generated from specialized endothelial cells of the embryonic aorta. Inflammatory factors are implicated in regulating mouse HSC development, but which cells in the aorta-gonad-mesonephros (AGM) microenvironment produce these factors is unknown. In the adult, macrophages play both pro- and anti-inflammatory roles. We sought to examine whether macrophages or other hematopoietic cells found in the embryo prior to HSC generation were involved in the AGM HSC-generative microenvironment. CyTOF analysis of CD45+ AGM cells revealed predominance of two hematopoieticcell types, mannose-receptor positive macrophages and mannose-receptor negative myeloid cells. We show here that macrophage appearance in the AGM was dependent on the chemokine receptor Cx3cr1. These macrophages expressed a pro-inflammatory signature, localized to the aorta, and dynamically interacted with nascent and emerging intra-aortic hematopoietic cells (IAHCs). Importantly, upon macrophage depletion, no adult-repopulating HSCs were detected, thus implicating a role for pro-inflammatory AGM-associated macrophages in regulating the development of HSCs.

The Gata2 transcription factor is a pivotal regulator of hematopoietic cell development and maintenance, highlighted by the fact that Gata2 haploinsufficiency has been identified as the cause of some familial cases of acute myelogenous leukemia/myelodysplastic syndrome and in MonoMac syndrome. Genetic deletion in mice has shown that Gata2 is pivotal to the embryonic generation of hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs). It functions in the embryo during endothelial cell to hematopoietic cell transition to affect hematopoietic cluster, HPC, and HSC formation. Gata2 conditional deletion and overexpression studies show the importance of Gata2 levels in hematopoiesis, during all developmental stages. Although previous studies of cell populations phenotypically enriched in HPCs and HSCs show expression of Gata2, there has been no direct study of Gata2 expressing cells during normal hematopoiesis. In this study, we generate a Gata2Venus reporter mouse model with unperturbed Gata2 expression to examine the hematopoietic function and transcriptome of Gata2 expressing and nonexpressing cells. We show that all the HSCs are Gata2 expressing. However, not all HPCs in the aorta, vitelline and umbilical arteries, and fetal liver require or express Gata2. These Gata2-independent HPCs exhibit a different functional output and genetic program, including Ras and cyclic AMP response element-binding protein pathways and other Gata factors, compared with Gata2-dependent HPCs. Our results, indicating that Gata2 is of major importance in programming toward HSC fate but not in all cells with HPC fate, have implications for current reprogramming strategies.

GATA2 is the most frequently mutated gene in childhood AML. These patients have germline mutations in one allele of GATA2 and have a high predisposition for MDS/AML. Also, in mouse, Gata2 is required for embryonic HSC generation and survival and Gata2 haploinsufficiency results in a severe reduction in the formation of HSCs. The first HSCs are formed at embryonic day (E)10.5 through a process called endothelial-to-hematopoietic transition (EHT) from specialized hemogenic endothelial cells of the dorsal aorta in the aorta-gonad-mesonephros (AGM) region. As all patients suffering from GATA2 haploinsufficiency syndromes have innate GATA2 mutations we want to understand how Gata2 haploinsufficiency affects embryonic HSC generation. Therefore, we sorted phenotypic HSPCs (CD31+cKit+ cells) from WT and Gata2+/- E11 AGMs and performed transcriptome analysis. Surprisingly, we found that the hematopoietic transcriptional program is not abrogated in Gata2+/- mouse E11 CD31+cKit+ cells. However, Gata2+/- HSPCs still express the endothelial program indicating that the hematopoietic cells are stuck in the endothelium and cannot complete EHT. We found that Gfi1b is downregulated in Gata2+/- HSPCs. As Gfi1b is known to be required for downregulation of the endothelial program for EHT during primitive hematopoiesis, we hypothesize that Gata2 directly regulates Gfi1b and that this regulation is required for normal EHT. We are currently studying if a specific HSPC subtype can overcome Gata2 haploinsufficiency and will still undergo EHT as this may have implications for the hematopoietic cells that eventually populate the hematopoietic tissues.

Cell fate and behavior are regulated by dynamic intrinsic and extrinsic processes. These processes affect single cells to produce a fully integrated organism with distinct functional cell types and tissues. Experimental hematology provides insight into the independent iterations of blood cell development (primitive, definitive progenitor and definitive stem) in the different embryonic sites/times prior to adult hematopoietic system formation. Hematopoietic stem cell (HSC) generation occurs in a short developmental time window from a natural transdifferentiation of aortic endothelial-to-hematopoietic cells (EHT). The Gata2 transcription factor is a dosage-dependent intrinsic regulator of EHT controlled by signals from the extrinsic developmental niche. Hematopoietic progenitor cells (HPC) are also generated by EHT, although at an earlier stage. So, what mechanistically determines HPC or HSC fate during EHT? Vital time-lapse imaging reveals highly dynamic Gata2 expression changes in cells emerging during EHT. We used Gata2 levels to examine the “inner” HSC transcriptional regulatory network. Single cell iterative RNAseq, index-sorting and in vivo/vitro hematopoietic analyses show that the transcriptome of the first aortic HSCs is unique compared to HPCs. The observed heterogeneity and quantitative expression differences of pivotal EHT/HSC genes suggest that establishment of functional hematopoietic fate is a stochastic process. Additionally, dynamic changes occur in the aortic niche. Transient cell-cell interactions occur between motile yolk sac HPC-derived macrophages and emerging aortic hematopoietic cells to affect hematopoietic output/function. The extrinsic signals from this new pro-inflammatory macrophage subset, together with intrinsic dynamic Gata2 expression changes in endothelial cells could assist strategies for ex vivo HSC generation.

Vascular smooth muscle cells (VSMCs) show pronounced heterogeneity across and within vascular beds, with direct implications for their function in injury response and atherosclerosis. Here we combine single-cell transcriptomics with lineage tracing to examine VSMC heterogeneity in healthy mouse vessels. The transcriptional profiles of single VSMCs consistently reflect their region-specific developmental history and show heterogeneous expression of vascular disease-associated genes involved in inflammation, adhesion and migration. We detect a rare population of VSMC-lineage cells that express the multipotent progenitor marker Sca1, progressively downregulate contractile VSMC genes and upregulate genes associated with VSMC response to inflammation and growth factors. We find that Sca1 upregulation is a hallmark of VSMCs undergoing phenotypic switching in vitro and in vivo, and reveal an equivalent population of Sca1-positive VSMC-lineage cells in atherosclerotic plaques. Together, our analyses identify disease-relevant transcriptional signatures in VSMC-lineage cells in healthy blood vessels, with implications for disease susceptibility, diagnosis and prevention.

Hematopoietic stem cells (HSCs) develop from the hemogenic endothelium in the embryonic aorta. In most vertebrates, hemogenic cells protrude into the aortic lumen forming clusters where the hematopoietic features are acquired. Here we used advanced live imaging techniques to visualize the origin and 3D-organization of intraortic hematopoietic clusters (IAHC) in the mouse embryo and how they evolve over time. Using organotypic slice cultures, clonal analysis and mathematical modelling we show that IAHC formation is a two-step process. First, a hemogenic progenitor buds up from the endothelium and undergoes division forming the monoclonal core of the cluster. Next, cells from the surrounding hemogenic endothelium are also recruited into the IAHCs, increasing their size and heterogeneity. We found that the recruitment phase of IAHC formation is negatively controlled by a Notch and Delta-like-4 (Dll4) and its blockage promotes the entrance of new hemogenic Gfi1+ cells into the IAHC thus increasing the number of cells that acquire HSC activity. Mathematical modelling based on our data estimates the cluster lifetime and the average recruitment time of hemogenic cells to the cluster under physiologic and Dll4-inhibited conditions. Upon blockage of Dll4 the recruitment time was dramatically decreased, with changes in the number of cells composing a cluster. Transcriptome analysis at single cell indicates that cell identities are preserved after Dll4 blockage. Our data provides for the first time a detailed characterization of the aortic hematopoietic stem cell niche and describe its process of maturation, identifying the Notch ligand Dll4 as a major molecular player governing cluster formation and definitive HSCs production.

Not all hematopoietic stem cells (HSCs) are alike. They differ in their physical characteristics such as cell cycle status and cell surface marker phenotype, they respond to different extrinsic signals, and they have different lineage outputs following transplantation. The growing body of evidence that supports heterogeneity within HSCs, which constitute the most robust cell fraction at the foundation of the adult hematopoietic system, is currently of great interest and raises questions as to why HSC subtypes exist, how they are generated and whether HSC heterogeneity affects leukemogenesis or treatment options. This Review provides a developmental overview of HSC subtypes during embryonic, fetal and adult stages of hematopoiesis and discusses the possible origins and consequences of HSC heterogeneity., Summary: This Review takes a close look at hematopoietic stem cell heterogeneity during development and in the adult, and discusses several different ways in which this heterogeneity may arise.

Hematopoietic cell generation in the midgestation mouse embryo occurs through the natural transdifferentiation of temporally and spatially restricted set of hemogenic endothelial cells. These cells take on hematopoietic fate in the aorta, vitelline and umbilical arteries and appear as hematopoietic cell clusters that emerge from the vascular wall. Genetic and live imaging data have supported this. Recently, the embryonic head has been shown to contain fully functional hematopoietic stem cells (HSC). By lineage tracing, cerebrovascular specific endothelial cells were shown to contribute to the postnatal mouse hematopoietic system. Since Ly6aGFP is a marker of all HSCs, some hematopoietic cluster cells and hemogenic endothelial cells in the midgestation mouse aorta, we examine here whether embryonic head HSCs and vascular endothelial cells are positive for this marker. Whereas some head vasculature, single hematopoietic cells and all HSCs are Ly6aGFP expressing, we do not find clusters of hematopoietic cells emerging from the cerebrovasculature that are characteristic of endothelial-to-hematopoietic transition., Highlights • All midgestation mouse head HSCs and some HPCs are Ly6aGFP expressing. • Ly6aGFP is expressed in the major head vasculature, particularly in the hindbrain. • No hematopoietic cell clusters are found within the lumen of the head vasculature.

Haemopoietic stem cells (HSCs) are the foundation of the adult blood system and sustain the lifelong production of all blood lineages. These rare cells are generally defined by their ability to self-renew through a process of asymmetric cell division, the outcome of which is an HSC and a differentiating cell. In health, HSCs provide homeostatic maintenance of the system through their ability to differentiate and generate the hundreds of millions of erythrocytes and leukocytes needed each day. In trauma and physiological stress, HSCs ensure the replacement of the lost or damaged blood cells. The tight regulation of HSC self-renewal ensures the appropriate balance of blood cell production. Perturbation of this regulation and unchecked growth of HSCs and/or immature blood cells results in leukaemia. Over the last 50 years, great success has been achieved with bone marrow transplantation as a stem cell regenerative therapy. However, insufficient numbers of HSCs are still a major constraint in clinical applications. As the pivotal cells in this essential tissue, HSCs are the focus of intense research to 1) further our understanding of their normal behaviour and the basis of their dysfunction in haemopoietic disease and leukaemia and, 2) to provide insights for new strategies for improved and patient-specific stem cell therapies. This chapter provides current and historical information on the organization of the adult haemopoietic cell differentiation hierarchy, the ontogeny of HSCs, the stromal microenvironment supporting these cells, and the molecular mechanisms involved in the regulation of HSCs.

Eich et al. reveal the dynamic expression of the Gata2 transcription factor in single aortic cells transitioning to hematopoietic fate by vital imaging of Gata2Venus mouse embryos. Pulsatile expression level changes highlight an unstable genetic state during hematopoietic cell generation., Cell fate is established through coordinated gene expression programs in individual cells. Regulatory networks that include the Gata2 transcription factor play central roles in hematopoietic fate establishment. Although Gata2 is essential to the embryonic development and function of hematopoietic stem cells that form the adult hierarchy, little is known about the in vivo expression dynamics of Gata2 in single cells. Here, we examine Gata2 expression in single aortic cells as they establish hematopoietic fate in Gata2Venus mouse embryos. Time-lapse imaging reveals rapid pulsatile level changes in Gata2 reporter expression in cells undergoing endothelial-to-hematopoietic transition. Moreover, Gata2 reporter pulsatile expression is dramatically altered in Gata2+/− aortic cells, which undergo fewer transitions and are reduced in hematopoietic potential. Our novel finding of dynamic pulsatile expression of Gata2 suggests a highly unstable genetic state in single cells concomitant with their transition to hematopoietic fate. This reinforces the notion that threshold levels of Gata2 influence fate establishment and has implications for transcription factor–related hematologic dysfunctions.

Evidence of the diversity and multi-layered organization of the hematopoietic system is leading to new insights that may inform ex vivo production of blood cells. Interestingly, not all long-lived hematopoietic cells derive from hematopoietic stem cells (HSCs). Here we review the current knowledge on HSC-dependent cell lineages and HSC-independent tissue-resident hematopoietic cells and how they arise during embryonic development. Classical embryological and genetic experiments, cell fate tracing data, single-cell imaging, and transcriptomics studies provide information on the molecular/cell trajectories that form the complete hematopoietic system. We also discuss the current developmentally informed efforts toward generating engraftable and multilineage blood cells. The authors thank Lluis Espinosa for critical reading of the review and lab members for helpful discussions. We acknowledge the support of the ERC Advanced Grant (341096 DENOVOHSC) to E.D. and SAF2016-75613-R from MINECO and PERIS- SLT002/16/00299 and 2017SGR135 from Generalitat de Catalunya to A.B.

Using highly sensitive RNAseq to examine the whole transcriptome of enriched aortic hematopoietic stem cells and endothelial cells, the authors find G-protein–coupled receptor, Gpr56, is required to generate the first HSCs during endothelial to hematopoietic cell transition., Hematopoietic stem cells (HSCs) are generated via a natural transdifferentiation process known as endothelial to hematopoietic cell transition (EHT). Because of small numbers of embryonal arterial cells undergoing EHT and the paucity of markers to enrich for hemogenic endothelial cells (ECs [HECs]), the genetic program driving HSC emergence is largely unknown. Here, we use a highly sensitive RNAseq method to examine the whole transcriptome of small numbers of enriched aortic HSCs, HECs, and ECs. Gpr56, a G-coupled protein receptor, is one of the most highly up-regulated of the 530 differentially expressed genes. Also, highly up-regulated are hematopoietic transcription factors, including the “heptad” complex of factors. We show that Gpr56 (mouse and human) is a target of the heptad complex and is required for hematopoietic cluster formation during EHT. Our results identify the processes and regulators involved in EHT and reveal the surprising requirement for Gpr56 in generating the first HSCs.

The bone morphogenetic protein (BMP)/SMAD signaling pathway is a critical regulator of angiogenic sprouting and is involved in vascular development in the embryo. SMAD1 and SMAD5, the core mediators of BMP signaling, are vital for this activity, yet little is known about their transcriptional regulation in endothelial cells. Here, we have integrated multispecies sequence conservation, tissue-specific chromatin, in vitro reporter assay, and in vivo transgenic data to identify and validate Smad1 + 63 and the Smad5 promoter as tissue-specific cis-regulatory elements that are active in the developing endothelium. The activity of these elements in the endothelium was dependent on highly conserved ETS, GATA, and E-box motifs, and chromatin immunoprecipitation showed high levels of enrichment of FLI1, GATA2, and SCL at these sites in endothelial cell lines and E11 dorsal aortas in vivo. Knockdown of FLI1 and GATA2 but not SCL reduced the expression of SMAD1 and SMAD5 in endothelial cells in vitro. In contrast, CD31(+) cKit(+) endothelial cells harvested from embryonic day 9 (E9) aorta-gonad-mesonephros (AGM) regions of GATA2 null embryos showed reduced Smad1 but not Smad5 transcript levels. This is suggestive of a degree of in vivo selection where, in the case of reduced SMAD1 levels, endothelial cells with more robust SMAD5 expression have a selective advantage.

Identifying signaling pathways that regulate hematopoietic stem and progenitor cell (HSPC) formation in the embryo will guide efforts to produce and expand HSPCs ex vivo. Here we show that sterile tonic inflammatory signaling regulates embryonic HSPC formation. Expression profiling of progenitors with lymphoid potential and hematopoietic stem cells (HSCs) from aorta/gonad/mesonephros (AGM) regions of midgestation mouse embryos revealed a robust innate immune/inflammatory signature. Mouse embryos lacking interferon γ (IFN-γ) or IFN-α signaling and zebrafish morphants lacking IFN-γ and IFN-ϕ activity had significantly fewer AGM HSPCs. Conversely, knockdown of IFN regulatory factor 2 (IRF2), a negative regulator of IFN signaling, increased expression of IFN target genes and HSPC production in zebrafish. Chromatin immunoprecipitation (ChIP) combined with sequencing (ChIP-seq) and expression analyses demonstrated that IRF2-occupied genes identified in human fetal liver CD34+ HSPCs are actively transcribed in human and mouse HSPCs. Furthermore, we demonstrate that the primitive myeloid population contributes to the local inflammatory response to impact the scale of HSPC production in the AGM region. Thus, sterile inflammatory signaling is an evolutionarily conserved pathway regulating the production of HSPCs during embryonic development.

Hematopoietic stem cells (HSC) are responsible for life-long maintenance and regeneration of the adult vertebrate blood system. The first HSCs arise from intra-aortic hematopoietic cluster cells (IAHC) in the mouse aorta at embryonic day (E)10.5 through a transdifferentiation process called endothelial-to-hematopoietic-transition (EHT). Previously we showed that the heptad factor Gata2, one of a group of 7 (heptad) transcription factors pivotal to definitive hematopoiesis, is required for HSC generation and survival. Gata2-/- embryos suffer lethality at E10.5 and Gata2+/- HSCs are qualitatively defective. Utilizing our Gata2Venus reporter mouse for time-lapse imaging of EHT in vivo, we found rapid pulsatile Gata2 expression level changes in single transitioning cells, implicating transcriptional instability during establishment of hematopoietic fate. Flow cytometric analysis of IAHCs revealed cells with varying levels of Venus (Vmedium, Vhigh). Hematopoietic progenitors (HPC) were highly enriched and HSCs exclusively found in the Vmed fraction. From these results, we hypothesized that fate choice is based on stochastic expression (levels and combinations) of the pivotal heptad factors. Here we test this by single cell transcriptomics and functional assays. Single-cell RNA sequencing of index-sorted Vmed cells shows surprising heterogeneity in heptad gene expression and has facilitated further HSC enrichment, defining intensity levels of known markers and providing additional markers. These new markers were functionally validated and their expression visualized by immunohistochemistry. Single-cell HPC assays also allowed for enrichment of immature HPCs. Further sorting and sequencing reiterations will refine the HSC transcriptomic profile and provide information on whether transcriptome heterogeneity correlates with and plays a role in generating both HSCs and HPCs.

Hematopoietic stem cells (HSCs) are responsible for the lifelong production and maintenance of adult blood cell types. They arise from the aorta at the embryonic day (E) 10.5 from a subset of endothelial cells that undergo to endothelial-to-hematopoietic transition (EHT). Our lab discovered that a G-coupled receptor protein, Gpr56, is required for the generation of HSCs in zebrafish. However, its role in mammalian hematopoiesis remains controversial. We investigate the functional role of Gpr56 in vitro using our mouse Gata2Venus (G2V) embryonic stem cell (ESC) line which allows us to highly enrich for hematopoietic progenitor cells (HPCs). G2V.G56-/-. We tested the Gpr56 loss of function by generating a full knock out (KO) Gpr56 mouse G2V (G2V.G56-/-) ESC line. The in vitro examination of phenotypic hematopoietic cell production by FACS and colony forming unit-culture (CFU-C) assay reported an increase in hematopoietic cells (HC) and HPCs production from G2V.G56-/- ESC as compared to G2V ESC. Further analyses performed on cell-sorted HPC population showed that another GPR protein, Gpr97 (located in the same locus), is highly expressed when Gpr56 is deleted. This led us to generate a novel G2V Gpr56/Gpr97 double knockout (G2V.G56/97-/-) ESC line. The in vitro differentiation of the G2V.G56/97-/- revealed a significant decrease in HPCs, supporting the idea that a compensatory mechanism by Gpr97 is taking place when Gpr56 is absent. Preliminary in vivo data using a Gpr56 conditional KO mouse model corroborates our hypothesis. The data demonstrate that the Gpr97 performs a redundant function upon deletion of Gpr56 in the HS/PC population and the deletion of both Gpr56 and Gpr97 is sufficient to impair the production of HS/PC and HC in vitro. We conclude that GPR signalling through Gpr56 and/or Gpr97 is required for HPC production.

Hematopoietic stem cells (HSCs) are responsible for the life-long maintenance and regeneration of the adult vertebrate blood system. The first HSCs are generated from specialized endothelial cells of the embryonic aorta. Inflammatory factors have been implicated in regulating HSC development, but it is unclear what cells in the mouse embryonic aorta-gonad-mesonephros (AGM) microenvironment produce these factors. In the adult, macrophages play both pro- and anti-inflammatory roles and they are involved in bone marrow hematopoiesis. We sought to examine whether macrophages or other hematopoietic cells found in the embryo prior to HSC generation are involved in the AGM HSC-generative microenvironment. Our mass cytometry (CyTOF) results indicate the absence of lymphoid cells and mature neutrophils in E10.5 mouse AGM, while two abundant myeloid cell types are present – mannose-receptor positive AGM-associated macrophages (AGM-aM) and mannose-receptor negative macrophages/progenitors. We show that the appearance of macrophages in the AGM is dependent on CX3CR1 and it occurs just before the time of HSC generation. Differently from mannose-receptor positive adult macrophages, AGM-aM express a pro-inflammatory signature. They localize to the embryonic aorta and dynamically interact with nascent and emerging intra-aortic hematopoietic cells (IAHC). Importantly, upon macrophage depletion, no adult-repopulating HSCs are detected, thus implicating unique pro-inflammatory AGM-associated macrophages in regulating the embryonic development of HSCs.

Guiu et al. use ChIP-on-chip analysis for the Notch partner RBPj, using embryonic tissue from the aorta-gonad-mesonephros region to identify potential novel Notch target genes involved in HSC emergence. They show that c-MYC–responsive gene Cdca7 is expressed in different HSC and progenitor subpopulations and that CDCA7 is important for maintaining the undifferentiated phenotype. Cdca7 acts downstream of Notch in HSCs in zebrafish, mouse, and human, indicating a highly conserved Notch/RBPj/Cdca7 axis in hematopoietic development., Hematopoietic stem cell (HSC) specification occurs in the embryonic aorta and requires Notch activation; however, most of the Notch-regulated elements controlling de novo HSC generation are still unknown. Here, we identify putative direct Notch targets in the aorta-gonad-mesonephros (AGM) embryonic tissue by chromatin precipitation using antibodies against the Notch partner RBPj. By ChIP-on-chip analysis of the precipitated DNA, we identified 701 promoter regions that were candidates to be regulated by Notch in the AGM. One of the most enriched regions corresponded to the Cdca7 gene, which was subsequently confirmed to recruit the RBPj factor but also Notch1 in AGM cells. We found that during embryonic hematopoietic development, expression of Cdca7 is restricted to the hematopoietic clusters of the aorta, and it is strongly up-regulated in the hemogenic population during human embryonic stem cell hematopoietic differentiation in a Notch-dependent manner. Down-regulation of Cdca7 mRNA in cultured AGM cells significantly induces hematopoietic differentiation and loss of the progenitor population. Finally, using loss-of-function experiments in zebrafish, we demonstrate that CDCA7 contributes to HSC emergence in vivo during embryonic development. Thus, our study identifies Cdca7 as an evolutionary conserved Notch target involved in HSC emergence.