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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.

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.

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.

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.

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.

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.

SUMMARY Combinatorial transcription factor (TF) interactions control cellular phenotypes and, therefore, underpin stem cell formation, maintenance, and differentiation. Here, we report the genome-wide binding patterns and combinatorial interactions for ten key regulators of blood stem/progenitor cells (SCL/ TAL1, LYL1, LMO2, GATA2, RUNX1, MEIS1, PU.1, ERG, FLI-1, and GFI1B), thus providing the most comprehensive TF data set for any adult stem/ progenitor cell type to date. Genome-wide computational analysis of complex binding patterns, followed by functional validation, revealed the following: first, a previously unrecognized combinatorial interaction between a heptad of TFs (SCL, LYL1, LMO2, GATA2, RUNX1, ERG, and FLI-1). Second, we implicate direct protein-protein interactions between four key regulators (RUNX1, GATA2, SCL, and ERG) in stabilizing complex binding to DNA. Third, Runx1 +/� ::Gata2 +/� compound heterozygous mice are not viable with severe hematopoietic defects at midgestation. Taken together, this study demonstrates the power of genome-wide analysis in generating novel functional insights into the transcriptional control of stem and progenitor cells.

The placenta is a large, highly vascularised hematopoietic tissue that functions during the embryonic and foetal development of eutherian mammals. Although recognised as the interface tissue important in the exchange of oxygen, nutrients and waste products between the foetus and mother, the placenta has increasingly become a focus of research concerning the ontogeny of the blood system. Here, we describe recent data showing the intrinsic hematopoietic potential and appearance of hematopoietic cells in the mouse and human placenta and probe the biological rationale behind its hematopoietic function. As a rest tissue that contains potent hematopoietic stem cells (HSCs), the human placenta could represent (in addition to umbilical cord blood cells) an accessible supplemental source of cells for therapeutic strategies.

SummaryCytokines are important in adult hematopoiesis, yet their function in embryonic hematopoiesis has been largely unexplored. During development, hematopoietic stem cells (HSCs) are found in the aorta-gonad-mesonephros (AGM) region, yolk sac (YS), and placenta and require the Runx1 transcription factor for their normal generation. Since IL-3 is a Runx1 target and this cytokine acts on adult hematopoietic cells, we examined whether IL-3 affects HSCs in the mouse embryo. Using Runx1 haploinsufficient mice, we show that IL-3 amplifies HSCs from E11 AGM, YS, and placenta. Moreover, we show that IL-3 mutant embryos are deficient in HSCs and that IL-3 reveals the presence of HSCs in the AGM and YS prior to the stage at which HSCs are normally detected. Thus, our studies support an unexpected role for IL-3 during development and strongly suggest that IL-3 functions as a proliferation and/or survival factor for the earliest HSCs in the embryo.

Recent research on the ontogeny of the hematopoietic system in mammals has shown that a simple textbook steady-state hematopoietic hierarchy can not be strictly applied to the hematopoietic cells found within the embryo. During embryonic development, hematopoietic cells originate, migrate and differentiate in a number of distinct anatomical sites such as the yolk sac, AGM region and liver and thus represent various classes of cells within diverse microenvironments. In this manuscript we review both cellular and molecular aspects of developmental hematopoiesis and present our current views on the numerous complex mechanisms underlying the establishment of definitive hematopoiesis.

Hematopoiesis is an extensive developmental program and differentiation process beginning at approximately day 7 during mouse embryogenesis. Totipotential mouse embryonic stem cells have been used as a model system to study the earliest hematopoietic events in mammalian development. Analysis of hematopoietic colony formation and gene expression have been performed to describe the in vitro hematopoietic potential of differentiating ES cells. Transplantations of differentiated ES cells into recipient mice have been done to assess in vivo hematopoietic repopulating potential. Also, ES cells have been used to study the expression of hematopoietic specific genes and their transcriptional control elements. Here we describe the hematopoietic characteristics of differentiating ES cells and their potentials and limitations in blood cell development.

Adult-type hematopoietic stem cells (HSCs) emerge in the aorta-gonad-mesonephros (AGM) region in the mid-gestation mouse embryo. Thereafter, HSCs migrate to the fetal liver (FL) and at birth, colonize the adult bone marrow. The molecular signaling cascades controlling HSCs in the AGM region is not yet known. Exogenous Hedgehog (Hh) added to AGM explants prior to HSC generation induces AGM HSCs and BMP pathway inhibition abolishes AGM HSC activity. In the FL, the BMP signaling pathway is not required for hematopoiesis. A role for the MAPK signaling pathway has been demonstrated in the adult hematopoiesis, but whether this pathway also controls AGM HSCs is not known. We hypothesize that Hh and BMP control the induction/expansion of AGM HSCs and cooperate with the MAPK pathway to control their maintenance and differentiation. To test whether HSCs are directly activated by BMP, we used transgenic BMP-response element (BRE)-gfp mice, in which GFP expression is an indicator of BMP pathway activation. When BRE-gfp E11 AGM cells were sorted for GFP expression and injected into adult irradiated mice, all long term repopulating HSCs were found in the BMP activated (GFP+) fraction. We next asked whether HSCs in the FL are activated by BMP. Surprisingly, we found high level, multilineage, self-renewing HSCs in both GFP+ and GFPfractions. This suggests that subsets of HSCs shift from an exclusively BMP-activated state, to a state of non-activation. This shift may be involved in the expansion of HSCs in the FL. Since HSCs expand in explant cultures of E11 AGMs, we examined whether after explant, AGM HSCs remain BMP-activated. Interestingly, after explant, both GFP+ and GFPfractions contained multilineage, self-renewing HSCs. In such explants when Hh or MAPK pathways are inhibited, only the non-BMP-activated HSC activity is abolished. The Hh inhibition effect on AGM HSCs could be rescued by exogeneous VEGF addition, but VEGF could not rescue the HSC defects caused by MAPK inhibition. These results suggest that VEGF acts downstream of Hh and further activates the MAPK pathway to control AGM HSCs. In conclusion, our data provide clear evidence of an interactive molecular cascade involving the BMP, Hh and MAPK signaling pathways in the control of AGM HSC development. P1087 TWO ADULT-TYPE HSC SUBSETS COEXISTENT IN MOUSE Mihaela Crisan, Chris Vink, Tomoko Yamada-Inagawa, Alex Neagu, Caterina Purini, Sofia Karkanpouna, Reinir van der Linden, and Elaine Dzierzak Cell Biology, Erasmus MC Stem Cell Institute, Rotterdam, Netherlands; Dept. of Molecular and Cell Biology, Centre of Biomedical Genetics, Leiden, Netherlands

The haemopoietic system is a dynamic and selfrenewing hierarchy of cells. The constant production of blood cells occurs through a series of differentiation steps, with terminally differentiated blood cells and their intermediates arising from haemopoietic stem cells that possess multilineage differentiation potential, as well as enormous proliferative potential. There is much interest in cell replacement therapy using stem cells. Haemopoietic stem cells derived from adult bone marrow have been used for many years to replace abnormal or deficient blood cells, such as sickle or thalassaemic cells. Successful use of HLA-mismatched umbilical cord blood cells in clinical transplantation for life-threatening haematological disorders suggests that these ontogenically earlier stem cells might provide therapeutic advantage. However, little is known about the qualitative differences between haemopoietic stem cells from various sources, or about the mechanisms that determine the fate of haemopoietic cells, the expansion of a clone, or how these cells home. Our first insight into the developmental origins of the adult haemopoietic system came from studies of bird and frog embryos. In these embryos, the adult blood system originates in an intraembryonic site encompassing the dorsal aorta, pronephros, and the surrounding mesenchyme. The yolk sac is responsible only for transient embryonic haemopoiesis. In mouse embryos, however, adult blood seemed to be derived from an extraembryonic yolk sac (figure). Analysis of the intraembryonic aorta-gonadmesonephros (AGM) region of the mouse embryo has since shown that the first adult-type haemopoietic stem cells are generated in the AGM region. These studies indicate that the dorsal aorta and underlying mesenchyme are responsible for the emergence of the first transplantable haemopoietic stem cells, and that high concentrations of such cells are associated with major vasculature of mouse and human embryos. What are the precursors of these adult-type haemopoietic stem cells? The haemopoietic system is derived from the mesodermal germ layer of the embryo and develops in association with the vasculature. Indeed, the idea of the haemangioblast, a common mesodermal precursor cell for the haemopoietic and endothelial lineages, was proposed nearly 100 years ago. The morphology of the yolk sac blood islands and work on embryonic stem cells, which mimic the in-vivo haemopoietic events that occur in the early gestation yolk sac, suggest the existence of such a precursor. The notion of an intraembryonic haemangioblast is less well accepted, since the vasculature is formed earlier than adult-type haemopoietic stem cells and, intraembryonic blood islands are not apparent. Nonetheless, gene targeting studies in mice have implicated common genetic requirements by cells of the endothelial and adult haemopoietic lineages. Will vascular endothelial cells or mesenchymal cells be found to be the precursors of haemopoietic stem cells? If so, the signals required for fate determination might be a means by which such precursors can be grown and induced to differentiate into haemopoietic cells for cell therapy and transplantation. How do AGM haemopoietic stem cells differ from the early gestation yolk sac cells that produce the embryonic haemopoietic system? The first morphologically recognisable and earliest blood cells to appear in the embryo are primitive yolk sac erythrocytes. Primitive erythroid cells are different to those found in the adult; embryonic globin genes are expressed in nucleated primitive erythrocytes whereas adult globin genes are expressed in small enucleated erythrocytes. In further contrast to the adult, early haemopoietic cell production in the yolk sac is biased towards the embryonic erythroid lineage with no production of adult-type haemopoietic stem cells. Although the microenvironment might account for these differences, the findings in mammals of at least two independent sites of haemopoietic emergence (yolk sac and AGM) indicate separate lineages of cells are responsible. Gene targeting studies in mice support this suggestion. These studies suggest that the genetic requirements for the production of the adult haemopoietic system beginning in the midgestation mouse embryo are overlapping with, but also distinct from, those of the primitive yolk sac haemopoietic system. Indeed, the development of the adult haemopoietic system requires the expression of many additional genes including transcription factors, growth factor receptors, adhesion molecules, and other genes that lead to the establishment of a stem cell phenotype. The key to the initiation of the adult haemopoietic stem cell programme will probably be found in the differential gene expression profiles obtained by comparison of expression patterns of precursor cells from the early embryo with those first haemopoietic stem cells generated in the midgestation AGM. With such information, manipulation of haemopoietic stem cells or precursors from adult bone marrow might result in production of unlimited numbers of cells. Embryonic sites of haemopoietic activity