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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.

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.

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.

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.

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 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.

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.

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.

GATA2 function is essential for the generation of HSCs during the stage of endothelial-to-hematopoietic cell transition and thereafter for HSC survival, Knowledge of the key transcription factors that drive hematopoietic stem cell (HSC) generation is of particular importance for current hematopoietic regenerative approaches and reprogramming strategies. Whereas GATA2 has long been implicated as a hematopoietic transcription factor and its dysregulated expression is associated with human immunodeficiency syndromes and vascular integrity, it is as yet unknown how GATA2 functions in the generation of HSCs. HSCs are generated from endothelial cells of the major embryonic vasculature (aorta, vitelline, and umbilical arteries) and are found in intra-aortic hematopoietic clusters. In this study, we find that GATA2 function is essential for the generation of HSCs during the stage of endothelial-to-hematopoietic cell transition. Specific deletion of Gata2 in Vec (Vascular Endothelial Cadherin)-expressing endothelial cells results in a deficiency of long-term repopulating HSCs and intra-aortic cluster cells. By specific deletion of Gata2 in Vav-expressing hematopoietic cells (after HSC generation), we further show that GATA2 is essential for HSC survival. This is in contrast to the known activity of the RUNX1 transcription factor, which functions only in the generation of HSCs, and highlights the unique requirement for GATA2 function in HSCs throughout all developmental stages.

Hes1 is required for the development of hematopoietic stem cells in the mouse embryo through repression of Gata2., Previous studies have identified Notch as a key regulator of hematopoietic stem cell (HSC) development, but the underlying downstream mechanisms remain unknown. The Notch target Hes1 is widely expressed in the aortic endothelium and hematopoietic clusters, though Hes1-deficient mice show no overt hematopoietic abnormalities. We now demonstrate that Hes is required for the development of HSC in the mouse embryo, a function previously undetected as the result of functional compensation by de novo expression of Hes5 in the aorta/gonad/mesonephros (AGM) region of Hes1 mutants. Analysis of embryos deficient for Hes1 and Hes5 reveals an intact arterial program with overproduction of nonfunctional hematopoietic precursors and total absence of HSC activity. These alterations were associated with increased expression of the hematopoietic regulators Runx1, c-myb, and the previously identified Notch target Gata2. By analyzing the Gata2 locus, we have identified functional RBPJ-binding sites, which mutation results in loss of Gata2 reporter expression in transgenic embryos, and functional Hes-binding sites, which mutation leads to specific Gata2 up-regulation in the hematopoietic precursors. Together, our findings show that Notch activation in the AGM triggers Gata2 and Hes1 transcription, and next HES-1 protein represses Gata2, creating an incoherent feed-forward loop required to restrict Gata2 expression in the emerging HSCs.

© 2016 by The American Society of Hematology. Enhancers are the primary determinants of cell identity, and specific promoter/enhancer combinations of Endoglin (ENG) have been shown to target blood and endothelium in the embryo. Here, we generated a series of embryonic stem cell lines, each targeted with reporter constructs driven by specific promoter/enhancer combinations of ENG, to evaluate their discriminative potential and value as molecular probes of the corresponding transcriptome. The Eng promoter (P) in combination with the 28/17/19-kb enhancers, targeted cells in FLK1 mesoderm that were enriched for blast colony forming potential, whereas the P/28-kb enhancer targeted TIE21/c-KIT1/CD412 endothelial cells that were enriched for hematopoieticpotential. These fractions were isolated using reporter expression and their transcriptomes profiled by RNA-seq. There was high concordance between our signatures and those from embryos with defects at corresponding stages of hematopoiesis. Of the 6 genes that were upregulated in both hemogenic mesoderm and hemogenic endothelial fractions targeted by the reporters,LRP2, amultiligandreceptor,wasthe only gene that hadnot previously been associated with hematopoiesis.WeshowthatLRP2is indeedinvolved indefinitivehematopoiesisandbydoingsovalidatetheuseof reportergene-coupled enhancers as probes to gain insights into transcriptional changes that facilitate cell fate transitions.

Hematopoiesis is initiated in several distinct tissues in the mouse conceptus. The aorta-gonad-mesonephros (AGM) region is of particular interest,as it autonomously generates the first adult type hematopoietic stem cells(HSCs). The ventral position of hematopoietic clusters closely associated with the aorta of most vertebrate embryos suggests a polarity in the specification of AGM HSCs. Since positional information plays an important role in the embryonic development of several tissue systems, we tested whether AGM HSC induction is influenced by the surrounding dorsal and ventral tissues. Our explant culture results at early and late embryonic day 10 show that ventral tissues induce and increase AGM HSC activity, whereas dorsal tissues decrease it. Chimeric explant cultures with genetically distinguishable AGM and ventral tissues show that the increase in HSC activity is not from ventral tissue-derived HSCs, precursors or primordial germ cells (as was previously suggested). Rather, it is due to instructive signaling from ventral tissues. Furthermore, we identify Hedgehog protein(s) as an HSC inducing signal.

Summary Hematopoietic stem cells (HSC), the self-renewing cells of the adult blood differentiation hierarchy, are generated during embryonic stages. The first HSCs are produced in the aorta-gonad-mesonephros (AGM) region of the embryo through endothelial to a hematopoietic transition. BMP4 and Hedgehog affect their production and expansion, but it is unknown whether they act to affect the same HSCs. In this study using the BRE GFP reporter mouse strain that identifies BMP/Smad-activated cells, we find that the AGM harbors two types of adult-repopulating HSCs upon explant culture: One type is BMP-activated and the other is a non-BMP-activated HSC type that is indirectly controlled by Hedgehog signaling through the VEGF pathway. Transcriptomic analyses demonstrate that the two HSC types express distinct but overlapping genetic programs. These results revealing the bifurcation in HSC types at early embryonic stages in the AGM explant model suggest that their development is dependent upon the signaling molecules in the microenvironment., Graphical Abstract, Highlights • AGM explants contain two HSC types, BMP-activated and non-BMP-activated • Non-BMP-activated HSCs are dependent on Hedgehog/VEGF • Changes in the microenvironment ex vivo contribute to novel HSC composition, In this study, Dzierzak and colleagues demonstrate that the AGM contains two distinct HSC types when cultured ex vivo. Manipulation of the culture reveals that the HSC types are dependent upon distinct signaling molecules in the microenvironment. This could explain the persistence of HSCs in signaling pathway disruption mouse models.

Summary The first adult-repopulating hematopoietic stem cells (HSCs) emerge in the aorta-gonads-mesonephros (AGM) region of the embryo. We have recently identified the transcription factor Gata3 as being upregulated in this tissue specifically at the time of HSC emergence. We now demonstrate that the production of functional and phenotypic HSCs in the AGM is impaired in the absence of Gata3. Furthermore, we show that this effect on HSC generation is secondary to the role of Gata3 in the production of catecholamines, the mediators of the sympathetic nervous system (SNS), thus making these molecules key components of the AGM HSC niche. These findings demonstrate that the recently described functional interplay between the hematopoietic system and the SNS extends to the earliest stages of their codevelopment and highlight the fact that HSC development needs to be viewed in the context of the development of other organs., Graphical Abstract Highlights ► Gata3-deficient AGMs have reduced hematopoietic stem cell (HSC) numbers ► Gata3-dependent catecholamine production regulates HSC emergence ► Catecholamines regulate HSC emergence independent of blood flow ► Nascent HSCs express catecholamine receptors, Catecholamines produced by the sympathetic nervous system regulate hematopoietic stem cell emergence during embryonic development.

Deletion of β-catenin from mouse embryonic endothelium, but not embryonic hematopoietic cells, prevents hematopoietic differentiation; thus Wnt/β-catenin signaling is needed for emergence but not maintenance of HSCs., Understanding how hematopoietic stem cells (HSCs) are generated and the signals that control this process is a crucial issue for regenerative medicine applications that require in vitro production of HSC. HSCs emerge during embryonic life from an endothelial-like cell population that resides in the aorta-gonad-mesonephros (AGM) region. We show here that β-catenin is nuclear and active in few endothelial nonhematopoietic cells closely associated with the emerging hematopoietic clusters of the embryonic aorta during mouse development. Importantly, Wnt/β-catenin activity is transiently required in the AGM to generate long-term HSCs and to produce hematopoietic cells in vitro from AGM endothelial precursors. Genetic deletion of β-catenin from the embryonic endothelium stage (using VE-cadherin–Cre recombinase), but not from embryonic hematopoietic cells (using Vav1-Cre), precludes progression of mutant cells toward the hematopoietic lineage; however, these mutant cells still contribute to the adult endothelium. Together, those findings indicate that Wnt/β-catenin activity is needed for the emergence but not the maintenance of HSCs in mouse embryos.

Zeb2 (Sip1/Zfhx1b) is a member of the zinc-finger E-box-binding (ZEB) family of transcriptional repressors previously demonstrated to regulate epithelial-to-mesenchymal transition (EMT) processes during embryogenesis and tumor progression. We found high Zeb2 mRNA expression levels in HSCs and hematopoietic progenitor cells (HPCs), and examined Zeb2 function in hematopoiesis through a conditional deletion approach using the Tie2-Cre and Vav-iCre recombination mouse lines. Detailed cellular analysis demonstrated that Zeb2 is dispensable for hematopoietic cluster and HSC formation in the aorta-gonadomesonephros region of the embryo, but is essential for normal HSC/HPC differentiation. In addition, Zeb2-deficient HSCs/HPCs fail to properly colonize the fetal liver and/or bone marrow and show enhanced adhesive properties associated with increased β1 integrin and Cxcr4 expression. Moreover, deletion of Zeb2 resulted in embryonic (Tie2-Cre) and perinatal (Vav-icre) lethality due to severe cephalic hemorrhaging and decreased levels of angiopoietin-1 and, subsequently, improper pericyte coverage of the cephalic vasculature. These results reveal essential roles for Zeb2 in embryonic hematopoiesis and are suggestive of a role for Zeb2 in hematopoietic-related pathologies in the adult. ispartof: Blood vol:117 issue:21 pages:5620-5630 ispartof: location:United States status: published

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.

The recent description of the placenta as a tissue rich in haematopoietic stem and progenitor cells has not only opened up a whole new line of investigation into how haematopoiesis is regulated in this unique mammalian tissue, but has also resulted in the revisiting of long-standing and yet unanswered questions about the significance of having multiple haematopoietic organs during development. Due to its remarkable capacity for haematopoietic stem/progenitor cell expansion, the study of placental haematopoiesis is also of obvious clinical interest. In the following pages, we summarise what is currently known about the haematopoietic regulatory processes in the murine placenta and describe our most recent data demonstrating that the human placenta, like its murine counterpart, is also a source of haematopoietic stem and progenitor cells throughout development.

The first adult-repopulating hematopoietic stem cells (HSCs) are detected starting at day 10.5 of gestation in the aorta-gonads-mesonephros (AGM) region of the mouse embryo. Despite the importance of the AGM in initiating HSC production, very little is currently known about the regulators that control HSC emergence in this region. We have therefore further defined the location of HSCs in the AGM and incorporated this information into a spatial and temporal comparative gene expression analysis of the AGM. The comparisons included gene expression profiling (1) in the newly identified HSC-containing region compared with the region devoid of HSCs, (2) before and after HSC emergence in the AGM microenvironment, and (3) on populations enriched for HSCs and their putative precursors. Two genes found to be up-regulated at the time and place where HSCs are first detected, the cyclin-dependent kinase inhibitor p57Kip2/Cdkn1c and the insulin-like growth factor 2, were chosen for further analysis. We demonstrate here that they play a novel role in AGM hematopoiesis. Interestingly, many genes involved in the development of the tissues surrounding the dorsal aorta are also up-regulated during HSC emergence, suggesting that the regulation of HSC generation occurs in coordination with the development of other organs.

SummaryHematopoietic stem cells (HSCs) are responsible for the life-long production of the blood system and are pivotal cells in hematologic transplantation therapies. During mouse and human development, the first HSCs are produced in the aorta-gonad-mesonephros region. Subsequent to this emergence, HSCs are found in other anatomical sites of the mouse conceptus. While the mouse placenta contains abundant HSCs at midgestation, little is known concerning whether HSCs or hematopoietic progenitors are present and supported in the human placenta during development. In this study we show, over a range of developmental times including term, that the human placenta contains hematopoietic progenitors and HSCs. Moreover, stromal cell lines generated from human placenta at several developmental time points are pericyte-like cells and support human hematopoiesis. Immunostaining of placenta sections during development localizes hematopoietic cells in close contact with pericytes/perivascular cells. Thus, the human placenta is a potent hematopoietic niche throughout development.

It is currently accepted that stem cells of the definitive blood cell lines originate from the yolk-sac blood islands. Experiments were devised to examine the validity of this theory in the avian embryo. These involved grafting two-day-old quail embryos on to chick yolk-sacs of comparable developmental stages, i.e. before or shortly after the establishment of vascularization. The conclusions of the experiments are based on the possibility of distinguishing chick cell nuclei from those of the quail. In the developing haemopoietic organs (spleen and thymus) of quail embryos grafted on to the chick and subsequently incubated for 6 – 11 days, all cells, whether belonging to the granulopoietic, erythropoietic or lymphopoietic series, are of quail type. Thus these organs have not been colonized by chick stem cells. On the other hand, coelomic graft experiments show that the development of these organs is indeed dependent on an extrinsic colonization by haemopoietic cells; quail spleen or thymus rudiment, developing in the coelom of a chick, is populated by chick cells. Thus no incompatibility which would prevent heterospecific colonization exists in this system. It is concluded that haemopoietic stem cells of the definitive blood cell series originate from some source other than the yolk-sac, and that this source must be intra-embryonic.

The hematopoietic system is one of the first complex tissues to develop in the mammalian conceptus. Of particular interest in the field of developmental hematopoiesis is the origin of adult bone marrow hematopoietic stem cells. Tracing their origin is complicated because blood is a mobile tissue and because hematopoietic cells emerge from many embryonic sites. The origin of the adult mammalian blood system remains a topic of lively discussion and intense research. Interest is also focused on developmental signals that induce the adult hematopoietic stem cell program, as these may prove useful for generating and expanding these clinically important cell populations ex vivo. This review presents a historical overview of and the most recent data on the developmental origins of hematopoiesis.

Hematopoiesis during development is a dynamic process, with many factors involved in the emergence and regulation of hematopoietic stem cells (HSCs) and progenitor cells. Whereas previous studies have focused on developmental signaling and transcription factors in embryonic hematopoiesis, the role of well-known adult hematopoietic cytokines in the embryonic hematopoietic system has been largely unexplored. The cytokine interleukin-1 (IL-1), best known for its proinflammatory properties, has radioprotective effects on adult bone marrow HSCs, induces HSC mobilization, and increases HSC proliferation and/or differentiation. Here we examine IL-1 and its possible role in regulating hematopoiesis in the midgestation mouse embryo. We show that IL-1, IL-1 receptors (IL-1Rs), and signaling mediators are expressed in the aorta-gonad-mesonephros (AGM) region during the time when HSCs emerge in this site. IL-1 signaling is functional in the AGM, and the IL-1RI is expressed ventrally in the aortic subregion by some hematopoietic, endothelial, and mesenchymal cells. In vivo analyses of IL-1RI–deficient embryos show an increased myeloid differentiation, concomitant with a slight decrease in AGM HSC activity. Our results suggest that IL-1 is an important homeostatic regulator at the earliest time of HSC development, acting to limit the differentiation of some HSCs along the myeloid lineage.

The development of the hematopoietic system during early embryonic stages occurs in spatially and temporally distinct waves. Hematopoietic stem cells (HSC), the most potent and self-renewing cells of this system, are produced in the final 'definitive' wave of hematopoietic cell generation. In contrast to HSCs in the adult, which differentiate via intermediate progenitor populations to produce functional blood cells, the generation of hematopoietic cells in the embryo prior to HSC generation occurs in the early waves by producing blood cells without intermediate progenitors (such as the 'primitive' hematopoietic cells). The lineage relationship between the early hematopoietic cells and the cells giving rise to HSCs, the genetic networks controlling their emergence, and the precise temporal determination of HSC fate remain topics of intense research and debate. This Review article discusses the current knowledge on the step-wise embryonic establishment of the adult hematopoietic system, examines the roles of pivotal intrinsic regulators in this process, and raises questions concerning the temporal onset of HSC fate determination.

Background: The Interleukin-1 (IL-1) signaling component TAK1 binding protein 2 (TAB2) plays a role in activating the NFκB and JNK signaling pathways. Additionally, TAB2 functions in the nucleus as a repressor of NFκB-mediated gene regulation. Objective: To obtain insight into the function of TAB2 in the adult mouse, we analyzed the in vivo TAB2 expression pattern. Materials and methods: Cell lines and adult mouse tissues were analyzed for TAB2 protein expression and localization. Results: Immunohistochemical staining for TAB2 protein revealed expression in the vascular endothelium of most tissues, hematopoietic cells and brain cells. While TAB2 is localized in both the nucleus and the cytoplasm in cell lines, cytoplasmic localization predominates in hematopoietic tissues in vivo. Conclusions: The TAB2 expression pattern shows striking similarities with previously reported IL-1 receptor expression and NFκB activation patterns, suggesting that TAB2 in vivo is playing a role in these signaling pathways.

Adult haematopoiesis is the outcome of distinct haematopoietic stem cell (HSC) subtypes with self-renewable repopulating ability, but with different haematopoietic cell lineage outputs. The molecular basis for this heterogeneity is largely unknown. BMP signalling regulates HSCs as they are first generated in the aorta-gonad-mesonephros region, but at later developmental stages, its role in HSCs is controversial. Here we show that HSCs in murine fetal liver and the bone marrow are of two types that can be prospectively isolated--BMP activated and non-BMP activated. Clonal transplantation demonstrates that they have distinct haematopoietic lineage outputs. Moreover, the two HSC types differ in intrinsic genetic programs, thus supporting a role for the BMP signalling axis in the regulation of HSC heterogeneity and lineage output. Our findings provide insight into the molecular control mechanisms that define HSC types and have important implications for reprogramming cells to HSC fate and treatments targeting distinct HSC types.

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.

We investigated whether the in vitro differentiation of ES cells into haematopoietic progenitors could be enhanced by exposure to the aorta-gonadal-mesonephros (AGM) microenvironment that is involved in the generation of haematopoietic stem cells (HSC) during embryonic development. We established a co-culture system that combines the requirements for primary organ culture and differentiating ES cells and showed that exposure of differentiating ES cells to the primary AGM region results in a significant increase in the number of ES-derived haematopoietic progenitors. Co-culture of ES cells on the AM20-1B4 stromal cell line derived from the AGM region also increases haematopoietic activity. We conclude that factors promoting the haematopoietic activity of differentiating ES cells present in primary AGM explants are partially retained in the AM20.1B4 stromal cell line and that these factors are likely to be different to those required for adult HSC maintenance.

We recently established that two midgestation-derived stromal clones--UG26-1B6, urogenital ridge-derived, and EL08-1D2, embryonic liver-derived--support the maintenance of murine adult bone marrow and human cord blood hematopoietic repopulating stem cells (HSCs). In this study, we investigate whether direct HSC-stroma contact is required for this stem cell maintenance. Adult bone marrow ckit+ Ly-6C- side population (K6-SP) cells and stromal cells were cocultured under contact or noncontact conditions. These experiments showed that HSCs were maintained for at least 4 weeks in culture and that direct contact between HSCs and stromal cells was not required. To find out which factors might be involved in HSC maintenance, we compared the gene expression profile of EL08-1D2 and UG26-1B6 with four HSC-nonsupportive clones. We found that EL08-1D2 and UG26-1B6 both expressed 21 genes at a higher level, including the putative secreted factors fibroblast growth factor-7, insulin-like growth factor-binding proteins 3 and 4, pleiotrophin, pentaxin-related, and thrombospondin 2, whereas 11 genes, including GPX-3 and HSP27, were expressed at a lower level. In summary, we show for the first time long-term maintenance of adult bone marrow HSCs in stroma noncontact cultures and identify some secreted molecules that may be involved in this support.

SummaryIn the midgestation murine embryo, several major vascular tissues contain hematopoietic stem cell (HSC) activity. These include the aorta-gonad-mesonephros region (AGM), yolk sac, and fetal liver. Recently, the placenta was demonstrated to harbor hematopoietic progenitors, but it was not examined for HSC activity. We demonstrate here that the placenta also harbors adult-repopulating HSCs. Placental HSCs begin to be detected at embryonic day (E) 11, and HSC numbers increase dramatically between E11 and E12, exceeding the numbers in the circulating embryonic blood. Furthermore, all placental HSC activity is restricted to the GFP+ fraction of cells in Ly-6A (Sca-1) GFP transgenic embryos. Cells coexpressing GFP and endothelial markers CD34 and CD31 are found in the embryonic vasculature of the placental labyrinth. Moreover, placental cell expression of other HSC markers and transcription factors suggests that HSC emergence may occur in the placenta, as has been proposed for other embryonic hematopoietic sites.

Mesenchymal stem cells (MSCs) have great clinical potential for the replacement and regeneration of diseased or damaged tissue. They are especially important in the production of the hematopoietic microenvironment,which regulates the maintenance and differentiation of hematopoietic stem cells (HSCs). In the adult, MSCs and their differentiating progeny are found predominantly in the bone marrow (BM). However, it is as yet unknown in which embryonic tissues MSCs reside and whether there is a localized association of these cells within hematopoietic sites during development. To investigate the embryonic origins of these cells, we performed anatomical mapping and frequency analysis of mesenchymal progenitors at several stages of mouse ontogeny. We report here the presence of mesenchymal progenitors, with the potential to differentiate into cells of the osteogenic, adipogenic and chondrogenic lineages, in most of the sites harboring hematopoietic cells. They first appear in the aorta-gonad-mesonephros (AGM) region at the time of HSC emergence. However, at this developmental stage, their presence is independent of HSC activity. They increase numerically during development to a plateau level found in adult BM. Additionally, mesenchymal progenitors are found in the embryonic circulation. Taken together, these data show a co-localization of mesenchymal progenitor/stem cells to the major hematopoietic territories, suggesting that, as development proceeds,mesenchymal progenitors expand within these potent hematopoietic sites.

Apoptosis is an essential process in embryonic tissue remodeling and adult tissue homeostasis. Within the adult hematopoietic system, it allows for tight regulation of hematopoietic cell subsets. Previously, it was shown that B-cell leukemia 2 (Bcl-2) overexpression in the adult increases the viability and activity of hematopoietic cells under normal and/or stressful conditions. However, a role for apoptosis in the embryonic hematopoietic system has not yet been established. Since the first hematopoietic stem cells (HSCs) are generated within the aortagonad-mesonephros (AGM; an actively remodeling tissue) region beginning at embryonic day 10.5, we examined this tissue for expression of apoptosis-related genes and ongoing apoptosis. Here, we show expression of several proapoptotic and antiapoptotic genes in the AGM. We also generated transgenic mice overexpressing Bcl-2 under the control of the transcriptional regulatory elements of the HSC marker stem cell antigen-1 (Sca-1), to test for the role of cell survival in the regulation of AGM HSCs. We provide evidence for increased numbers and viability of Sca-1+ cells in the AGM and subdissected midgestation aortas, the site where HSCs are localized. Most important, our in vivo transplantation data show that Bcl-2 overexpression increases AGM and fetal liver HSC activity, strongly suggesting that apoptosis plays a role in HSC development.

As the zinc-finger transcription factor specificity protein 3 (Sp3) has been implicated in the regulation of many hematopoietic-specific genes, we analyzed the role of Sp3 in hematopoiesis. At embryonic day 18.5 (E18.5), Sp3-/- mice exhibit a partial arrest of T-cell development in the thymus and B-cell numbers are reduced in liver and spleen. However, pre-B-cell proliferation and differentiation into immunoglobulin M-positive (IgM+) B cells in vitro are not affected. At E14.5 and E16.5, Sp3-/- mice exhibit a significant delay in the appearance of definitive erythrocytes in the blood, paralleled by a defect in the progression of differentiation of definitive erythroid cells in vitro. Perinatal death of the null mutants precludes the analysis of adult hematopoiesis in Sp3-/- mice. We therefore investigated the ability of E12.5 Sp3-/- liver cells to contribute to the hematopoietic compartment in an in vivo transplantation assay. Sp3-/- cells were able to repopulate the B- and T-lymphoid compartment, albeit with reduced efficiency. In contrast, Sp3-/- cells showed no significant engraftment in the erythroid and myeloid lineages. Thus, the absence of Sp3 results in cell-autonomous hematopoietic defects, affecting in particular the erythroid and myeloid cell lineages.

The first adult-repopulating hematopoietic stem cells (HSCs) emerge in the mouse aorta-gonad-mesonephros (AGM) region at embryonic day 10.5 prior to their appearance in the yolk sac and fetal liver. Although several genes are implicated in the regulation of HSCs, there are gaps in our understanding of the processes taking place in the AGM at the time of HSC emergence. To identify genes involved in AGM HSC emergence, we performed differential display reverse transcriptase–polymerase chain reaction (DD RT-PCR). Differentially expressed genes included β-catenin and homologs of human TM9SF2 and TAB2. We characterized the expression pattern of Wnt/β-catenin signaling,mTM9SF2, and mTAB2 in the embryo and adult. Interestingly, the expression of mouse TAB2 (mTAB2) in the E11 dorsal aorta endothelium suggests a role for mTAB2 in HSC emergence and/or regulation. The identification of differentially expressed genes in the AGM region should yield further insights into the development of this tissue and into the emergence and regulation of HSCs.

The tal-1 gene encodes a basic helix-loop-helix (bHLH) transcription factor required for primitive and definitive hematopoiesis. Additionally, ectopic activation of thetal-1 gene during T lymphopoiesis occurs in numerous cases of human T-cell acute lymphoblastic leukemia. With the use of transgenic mice, we show that, in adult hematopoiesis, constitutive expression of TAL-1 protein causes disorders in the hematopoietic lineages that normally switch off tal-1 gene expression during their differentiation process. Myelopoiesis was characterized by a moderate increase of myeloid precursors and by Sca-1 antigen persistence. Although no lymphoid leukemia was observed, T lymphopoiesis and B lymphopoiesis were severely impaired. Transgenic mice showed reduced thymic cellularity together with a decrease in double-positive cells and a concurrent increase in the single-positive population. B cells exhibited a differentiation defect characterized by a reduction of the B-cell compartment most likely because of a differentiation block upstream of the intermediate pro-B progenitor. B cells escaping this defect developed normally, but transgenic splenocytes presented a defect in immunoglobulin class switch recombination. Altogether, these results enlighten the fine-tuning of TAL-1 expression during adult hematopoiesis and indicate why TAL-1 expression has to be switched off in the lymphoid lineages.

Throughout life, the hematopoietic system requires a supportive microenvironment that allows for the maintenance and differentiation of hematopoietic stem cells (HSC). To understand the cellular interactions and molecules that provide these functions, investigators have previously established stromal cell lines from the late gestational stage and adult murine hematopoietic microenvironments. However, the stromal cell microenvironment that supports the emergence, expansion and maintenance of HSCs during mid-gestational stages has been largely unexplored. Since several tissues within the mouse embryo are known to harbor HSCs (i.e. aortagonads-mesonephros, yolk sac, liver), we generated numerous stromal cell clones from these mid-gestational sites. Owing to the limited cell numbers, isolations were performed with tissues from transgenic embryos containing the ts SV40 Tag gene (tsA58) under the transcriptional control of constitutive and ubiquitously expressing promoters. We report here that the growth and cloning efficiency of embryonic cells (with the exception of the aorta) is increased in the presence of the tsA58 transgene. Furthermore, our results show that the large panel of stromal clones isolated from the different embryonal subregions exhibit heterogeneity in their ability to promote murine and human hematopoietic differentiation. Despite our findings of heterogeneity in hematopoietic growth factor gene expression profiles, high-level expression of some factors may influence hematopoietic differentiation. Interestingly, a few of these stromal clones express a recently described chordin-like protein, which is an inhibitor of bone morphogenic proteins and is preferentially expressed in cells of the mesenchymal lineage.

Objectives. To better understand the differentiation of stromal cells of the hematopoietic microenvironment, we set out to characterize stromal cells from the different developmental sites of hematopoiesis in the mouse (30 bone marrow, 7 spleen, 3 embryonic and 15 fetal liver, 6 yolk sac, and 6 aorta-gonad-mesonephros lines) for expression of 22 cytoskeletal, membrane, and extracellular matrix proteins. Materials and Methods. Western blotting, immunofluorescence, and flow cytometry were used. Statistical methods included principal components analysis and analysis of variance. Results. Stromal cells from 11 dpc mouse embryos express mesenchymal and vascular smooth muscle cell (VSMC) markers. Principal components analysis on the 70 stromal cell lines isolated from different anatomic sites and developmental stages allows classification of stromal lines along a mesenchymal to VSMC differentiation pathway. Stromal cells do not express endothelial and hematopoietic differentiation membrane antigens, but they do express integrin � 5 , � 6 , and � 1 subunits, vascular cell adhesion molecule-1, CD44, stem cell antigen-1, Thy-1, CD34, and endoglin. The intensity of expression of certain markers differs between lines according to the anatomic site of origin. Conclusions. This study indicates that stromal cells, whatever their anatomic site of origin, follow a VSMC differentiation pathway, suggesting a blood-forming tissue-specific differentiation of mesenchymal stem cells. Differential quantitative expression of distinct sets of markers appears to be correlated with the anatomic sites of origin of the stromal cells. © 2002 International Society for Experimental Hematology. Published by Elsevier Science Inc.

The aorta-gonads-mesonephros (AGM) region autonomously generates the first adult repopulating hematopoietic stem cells (HSCs) in the mouse embryo. HSC activity is initially localized to the dorsal aorta and mesenchyme (AM) and vitelline and umbilical arteries. Thereafter, HSC activity is found in the urogenital ridges (UGs), yolk sac, and liver. As increasing numbers of HSCs are generated, it is thought that these sites provide supportive microenvironments in which HSCs are harbored until the bone marrow microenvironment is established. However, little is known about the supportive cells within these midgestational sites, and particularly which microenvironment is most supportive for HSC growth and maintenance. Thus, to better understand the cells and molecules involved in hematopoietic support in the midgestation embryo, more than 100 stromal cell lines and clones were established from these sites. Numerous stromal clones were found to maintain hematopoietic progenitors and HSCs to a similar degree as, or better than, previously described murine stromal lines. Both the AM and UG subregions of the AGM produced many supportive clones, with the most highly HSC-supportive clone being derived from the UGs. Interestingly, the liver at this stage yielded only few supportive stromal clones. These results strongly suggest that during midgestation, not only the AM but also the UG subregion provides a potent microenvironment for growth and maintenance of the first HSCs.

The first definitive long-term repopulating hematopoietic stem cells (HSCs) emerge from and undergo rapid expansion in the embryonic aorta-gonad-mesonephros (AGM) region. To investigate the presumptive unique characteristics of the embryonic hematopoietic microenvironment and its surrounding tissues, we have generated stromal clones from subdissected day 10 and day 11 AGMs, embryonic livers (ELs) and gut mesentery. We here examine the ability of 19 of these clones to sustain extended long-term cultures (LTCs) of human CD34(+) umbilical cord blood (UCB) cells in vitro. The presence of in vitro repopulating cells was assessed by sustained production of progenitor cells (extended LTC-CFC) and cobblestone area-forming cells (CAFC). The embryonic stromal clones differed greatly in their support for human HSCs. Out of eight clones tested in the absence of exogenous cytokines, only one (EL-derived) clone was able to provide maintenance of HSCs. Addition of either Tpo or Flt3-L + Tpo improved the long-term support of about 50% of the tested clones. Cultures on four out of 19 clones, ie the EL-derived clone mentioned, two urogenital-ridge (UG)-derived clones and one gastrointestinal (GI)-derived clone, allowed a continuous expansion of primitive CAFC and CFU-GM with over several hundred-fold more CAFC(week6) produced in the 12th week of culture. This expansion was considerably higher than that found with the FBMD-1 cell line, which is appreciated by many investigators for its support of human HSCs, under comparable conditions. This stromal cell panel derived from the embryonic regions may be a powerful tool in dissecting the factors mediating stromal support for maintenance and expansion of HSCs.

During embryogenesis there is a sequential, temporal appearance of increasingly more-complex hematopoietic cells beginning with unipotential progenitors, proceeding to multipotential (myeloid, erythroid and lymphoid) progenitors and culminating with adult-repopulating hematopoietic stem cells. Current research has established an important role for the aorta-gonads-mesonephros region of the mouse embryo in the generation of multipotential progenitors and hematopoietic stem cells. Comparisons of normal and hematopoietic-cell-mutant mouse embryos have revealed several genes pivotal in hematopoietic stem cell generation/function. Other genes have been implicated in the critical generation of lymphoid lineage potential. Thus, an understanding of the cellular and molecular interactions within the midgestation aorta-gonads-mesonephros region offers insight into the mechanisms of hematopoietic lineage specification during ontogeny and perhaps will lead to a more complete knowledge of the adult hematopoietic system.

The aorta–gonad–mesonephros (AGM) region is a potent hematopoietic site within the mammalian embryo body, and the first place from which hematopoietic stem cells (HSCs) emerge. Within the complex embryonic vascular, excretory and reproductive tissues of the AGM region, the precise location of HSC development is unknown. To determine where HSCs develop, we subdissected the AGM into aorta and urogenital ridge segments and transplanted the cells into irradiated adult recipients. We demonstrate that HSCs first appear in the dorsal aorta area. Furthermore, we show that vitelline and umbilical arteries contain high frequencies of HSCs coincident with HSC appearance in the AGM. While later in development and after organ explant culture we find HSCs in the urogenital ridges, our results strongly suggest that the major arteries of the embryo are the most important sites from which definitive HSCs first emerge.

The ability of the many cell types within the adult blood system to be constantly replenished and renewed from hematopoietic stem cells is an interesting problem in development and differentiation and has led to questions concerning how, when and where thses stem cells for the adult hematopoietic system are generated within the embryo. During embryonic development many mature hematopoietic cells appear before adult-type hematopoietic stem cells thus the notion of a conventional hematopoietic hierarchy is challegned. Experiments probing the development of hematopoietic stem cells in the mouse embryo strongly suggest that at least two independent hematopoietic sites generate blood cells during development; the yolk sac, which produces the transient embryonic hematopoietic system, and the AGM (aorta-gonad-mesonephros) region, which initiates the long-lived adult hematopoietic system.