Interleukin-17-Producing γδ T Cells Originate from SOX13+ Progenitors that Are Independent of γδTCR Signaling.

Lineage-committed αß and γδ T cells are thought to originate from common intrathymic multipotent progenitors following instructive T cell receptor (TCR) signals. A subset of lymph node and mucosal Vγ2+ γδ T cells is programmed intrathymically to produce IL-17 (Tγδ17 cells), however the role of the γδTCR in development of these cells remains controversial. Here we generated reporter mice for the Tγδ17 lineage-defining transcription factor SOX13 and identified fetal-origin, intrathymic Sox13+ progenitors. In organ culture developmental assays, Tγδ17 cells derived primarily from Sox13+ progenitors, and not from other known lymphoid progenitors. Single cell transcriptome assays of the progenitors found in TCR-deficient mice demonstrated that Tγδ17 lineage programming was independent of γδTCR. Instead, generation of the lineage committed progenitors and Tγδ17 cells was controlled by TCF1 and SOX13. Thus, T lymphocyte lineage fate can be prewired cell-intrinsically and is not necessarily specified by clonal antigen receptor signals.

Multiple waves of fetal-derived immune cells constitute adult immune system.

It has been over three decades since Drs. Herzenberg and Herzenberg proposed the layered immune system hypothesis, suggesting that different types of stem cells with distinct hematopoietic potential produce specific immune cells. This layering of immune system development is now supported by recent studies showing the presence of fetal-derived immune cells that function in adults. It has been shown that various immune cells arise at different embryonic ages via multiple waves of hematopoiesis from special endothelial cells (ECs), referred to as hemogenic ECs. However, it remains unknown whether these fetal-derived immune cells are produced by hematopoietic stem cells (HSCs) during the fetal to neonatal period. To address this question, many advanced tools have been used, including lineage-tracing mouse models, cellular barcoding techniques, clonal assays, and transplantation assays at the single-cell level. In this review, we will review the history of the search for the origins of HSCs, B-1a progenitors, and mast cells in the mouse embryo. HSCs can produce both B-1a and mast cells within a very limited time window, and this ability declines after embryonic day (E) 14.5. Furthermore, the latest data have revealed that HSC-independent adaptive immune cells exist in adult mice, which implies more complicated developmental pathways of immune cells. We propose revised road maps of immune cell development.

ABCG2-Expressing Clonal Repopulating Endothelial Cells Serve to Form and Maintain Blood Vessels.

BACKGROUND: Most organs are maintained lifelong by resident stem/progenitor cells. During development and regeneration, lineage-specific stem/progenitor cells can contribute to the growth or maintenance of different organs, whereas fully differentiated mature cells have less regenerative potential. However, it is unclear whether vascular endothelial cells (ECs) are also replenished by stem/progenitor cells with EC-repopulating potential residing in blood vessels. It has been reported recently that some EC populations possess higher clonal proliferative potential and vessel-forming capacity compared with mature ECs. Nevertheless, a marker to identify vascular clonal repopulating ECs (CRECs) in murine and human individuals is lacking, and, hence, the mechanism for the proliferative, self-renewal, and vessel-forming potential of CRECs is elusive. METHODS: We analyzed colony-forming, self-renewal, and vessel-forming potential of ABCG2 (ATP binding cassette subfamily G member 2)-expressing ECs in human umbilical vessels. To study the contribution of Abcg2-expressing ECs to vessel development and regeneration, we developed Abcg2CreErt2;ROSA TdTomato mice and performed lineage tracing during mouse development and during tissue regeneration after myocardial infarction injury. RNA sequencing and chromatin methylation chromatin immunoprecipitation followed by sequencing were conducted to study the gene regulation in Abcg2-expressing ECs. RESULTS: In human and mouse vessels, ECs with higher ABCG2 expression (ABCECs) possess higher clonal proliferative potential and in vivo vessel-forming potential compared with mature ECs. These cells could clonally contribute to vessel formation in primary and secondary recipients after transplantation. These features of ABCECs meet the criteria of CRECs. Results from lineage tracing experiments confirm that Abcg2-expressing CRECs (AbcCRECs) contribute to arteries, veins, and capillaries in cardiac tissue development and vascular tissue regeneration after myocardial infarction. Transcriptome and epigenetic analyses reveal that a gene expression signature involved in angiogenesis and vessel development is enriched in AbcCRECs. In addition, various angiogenic genes, such as Notch2 and Hey2, are bivalently modified by trimethylation at the 4th and 27th lysine residue of histone H3 (H3K4me3 and H3K27me3) in AbcCRECs. CONCLUSIONS: These results are the first to establish that a single prospective marker identifies CRECs in mice and human individuals, which holds promise to provide new cell therapies for repair of damaged vessels in patients with endothelial dysfunction.

An ERK5-NRF2 Axis Mediates Senescence-Associated Stemness and Atherosclerosis.

BACKGROUND: ERK5 (extracellular signal-regulated kinase 5) is a dual kinase transcription factor containing an N-terminal kinase domain and a C-terminal transcriptional activation domain. Many ERK5 kinase inhibitors have been developed and tested to treat cancer and inflammatory diseases. However, recent data have raised questions about the role of the catalytic activity of ERK5 in proliferation and inflammation. We aimed to investigate how ERK5 reprograms myeloid cells to the proinflammatory senescent phenotype, subsequently leading to atherosclerosis. METHODS: A ERK5 S496A (dephosphorylation mimic) knock in (KI) mouse model was generated using CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeat-associated 9), and atherosclerosis was characterized by hypercholesterolemia induction. The plaque phenotyping in homozygous ERK5 S496A KI and wild type (WT) mice was studied using imaging mass cytometry. Bone marrow-derived macrophages were isolated from hypercholesterolemic mice and characterized using RNA sequencing and functional in vitro approaches, including senescence, mitochondria reactive oxygen species, and inflammation assays, as well as by metabolic extracellular flux analysis. RESULTS: We show that atherosclerosis was inhibited in ERK5 S496A KI mice. Furthermore, ERK5 S496 phosphorylation mediates both senescence-associated secretory phenotype and senescence-associated stemness by upregulating AHR (aryl hydrocarbon receptor) in plaque and bone marrow-derived macrophages isolated from hypercholesterolemic mice. We also discovered that ERK5 S496 phosphorylation could induce NRF2 (NFE2-related factor 2) SUMOylation at a novel K518 site to inhibit NRF2 transcriptional activity without altering ERK5 catalytic activity and mediates oxidized LDL (low-density lipoprotein)-induced senescence-associated secretory phenotype. Specific ERK5 kinase inhibitors (AX15836 and XMD8-92) also inhibited ERK5 S496 phosphorylation, suggesting the involvement of ERK5 S496 phosphorylation in the anti-inflammatory effects of these ERK5 kinase inhibitors. CONCLUSIONS: We discovered a novel mechanism by which the macrophage ERK5-NRF2 axis develops a unique senescence-associated secretory phenotype/stemness phenotype by upregulating AHR to engender atherogenesis. The finding of senescence-associated stemness phenotype provides a molecular explanation to resolve the paradox of senescence in proliferative plaque by permitting myeloid cells to escape the senescence-induced cell cycle arrest during atherosclerosis formation.

B1 lymphocytes develop independently of Notch signaling during mouse embryonic development.

B1 lymphocytes are a small but unique component of the innate immune-like cells. However, their ontogenic origin is still a matter of debate. Although it is widely accepted that B1 cells originate early in fetal life, whether or not they arise from hematopoietic stem cells (HSCs) is still unclear. In order to shed light on the B1 cell origin, we set out to determine whether their lineage specification is dependent on Notch signaling, which is essential for the HSC generation and, therefore, all derivatives lineages. Using mouse embryonic stem cells (mESCs) to recapitulate murine embryonic development, we have studied the requirement for Notch signaling during the earliest B-cell lymphopoiesis and found that Rbpj-deficient mESCs are able to generate B1 cells. Their Notch independence was confirmed in ex vivo experiments using Rbpj-deficient embryos. In addition, we found that upregulation of Notch signaling induced the emergence of B2 lymphoid cells. Taken together, these findings indicate that control of Notch signaling dose is crucial for different B-cell lineage specification from endothelial cells and provides pivotal information for their in vitro generation from PSCs for therapeutic applications. This article has an associated 'The people behind the papers' interview.

Hematopoietic stem cell-independent hematopoiesis and the origins of innate-like B lymphocytes.

The current paradigm that a single long-term hematopoietic stem cell can regenerate all components of the mammalian immune system has been challenged by recent findings in mice. These findings show that adult tissue-resident macrophages and innate-like lymphocytes develop early in fetal hematopoiesis from progenitors that emerge prior to, and apparently independently of, conventional long-term hematopoietic stem cells. Here, we discuss these recent findings, which show that an early and distinct wave of hematopoiesis occurs for all major hematopoietic lineages. These data provide evidence that fetal hematopoietic progenitors not derived from the bona fide long-term hematopoietic stem cells give rise to tissue-resident immune cells that persist throughout adulthood. We also discuss recent insights into B lymphocyte development and attempt to synthesize seemingly contradictory recent findings on the origins of innate-like B-1a lymphocytes during fetal hematopoiesis.

Bmi1 Maintains the Self-Renewal Property of Innate-like B Lymphocytes.

The self-renewal ability is a unique property of fetal-derived innate-like B-1a lymphocytes, which survive and function without being replenished by bone marrow (BM) progenitors. However, the mechanism by which IgM-secreting mature B-1a lymphocytes self-renew is poorly understood. In this study, we showed that Bmi1 was critically involved in this process. Although Bmi1 is considered essential for lymphopoiesis, the number of mature conventional B cells was not altered when Bmi1 was deleted in the B cell lineage. In contrast, the number of peritoneal B-1a cells was significantly reduced. Peritoneal cell transfer assays revealed diminished self-renewal ability of Bmi1-deleted B-1a cells, which was restored by additional deletion of Ink4-Arf, the well-known target of Bmi1 Fetal liver cells with B cell-specific Bmi1 deletion failed to repopulate peritoneal B-1a cells, but not other B-2 lymphocytes after transplantation assays, suggesting that Bmi1 may be involved in the developmental process of B-1 progenitors to mature B-1a cells. Although Bmi1 deletion has also been shown to alter the microenvironment for hematopoietic stem cells, fat-associated lymphoid clusters, the reported niche for B-1a cells, were not impaired in Bmi1 -/- mice. RNA expression profiling suggested lysine demethylase 5B (Kdm5b) as another possible target of Bmi1, which was elevated in Bmi1-/- B-1a cells in a stress setting and might repress B-1a cell proliferation. Our work has indicated that Bmi1 plays pivotal roles in self-renewal and maintenance of fetal-derived B-1a cells.

Endotoxin Preconditioning Reprograms S1 Tubules and Macrophages to Protect the Kidney.

Preconditioning with a low dose of endotoxin confers unparalleled protection against otherwise lethal models of sepsis. The mechanisms of preconditioning have been investigated extensively in isolated immune cells such as macrophages. However, the role of tissue in mediating the protective response generated by preconditioning remains unknown. Here, using the kidney as a model organ, we investigated cell type-specific responses to preconditioning. Compared with preadministration of vehicle, endotoxin preconditioning in the cecal ligation and puncture mouse model of sepsis led to significantly enhanced survival and reduced bacterial load in several organs. Furthermore, endotoxin preconditioning reduced serum levels of proinflammatory cytokines, upregulated molecular pathways involved in phagocytosis, and prevented the renal function decline and injury induced in mice by a toxic dose of endotoxin. The protective phenotype involved the clustering of macrophages around S1 segments of proximal tubules, and full renal protection required both macrophages and renal tubular cells. Using unbiased S1 transcriptomic and tissue metabolomic approaches, we identified multiple protective molecules that were operative in preconditioned animals, including molecules involved in antibacterial defense, redox balance, and tissue healing. We conclude that preconditioning reprograms macrophages and tubules to generate a protective environment, in which tissue health is preserved and immunity is controlled yet effective. Endotoxin preconditioning can thus be used as a discovery platform, and understanding the role and participation of both tissue and macrophages will help refine targeted therapies for sepsis.

Protein Tyrosine Phosphatase PRL2 Mediates Notch and Kit Signals in Early T Cell Progenitors.

The molecular pathways regulating lymphoid priming, fate, and development of multipotent bone marrow hematopoietic stem and progenitor cells (HSPCs) that continuously feed thymic progenitors remain largely unknown. While Notch signal is indispensable for T cell specification and differentiation, the downstream effectors are not well understood. PRL2, a protein tyrosine phosphatase that regulates hematopoietic stem cell proliferation and self-renewal, is highly expressed in murine thymocyte progenitors. Here we demonstrate that protein tyrosine phosphatase PRL2 and receptor tyrosine kinase c-Kit are critical downstream targets and effectors of the canonical Notch/RBPJ pathway in early T cell progenitors. While PRL2 deficiency resulted in moderate defects of thymopoiesis in the steady state, de novo generation of T cells from Prl2 null hematopoietic stem cells was significantly reduced following transplantation. Prl2 null HSPCs also showed impaired T cell differentiation in vitro. We found that Notch/RBPJ signaling upregulated PRL2 as well as c-Kit expression in T cell progenitors. Further, PRL2 sustains Notch-mediated c-Kit expression and enhances stem cell factor/c-Kit signaling in T cell progenitors, promoting effective DN1-DN2 transition. Thus, we have identified a critical role for PRL2 phosphatase in mediating Notch and c-Kit signals in early T cell progenitors. Stem Cells 2017;35:1053-1064.

Yolk sac erythromyeloid progenitors expressing gain of function PTPN11 have functional features of JMML but are not sufficient to cause disease in mice.

BACKGROUND: Accumulating evidence suggests the origin of juvenile myelomonocytic leukemia (JMML) is closely associated with fetal development. Nevertheless, the contribution of embryonic progenitors to JMML pathogenesis remains unexplored. We hypothesized that expression of JMML-initiating PTPN11 mutations in HSC-independent yolk sac erythromyeloid progenitors (YS EMPs) would result in a mouse model of pediatric myeloproliferative neoplasm (MPN). RESULTS: E9.5 YS EMPs from VavCre+;PTPN11D61Y embryos demonstrated growth hypersensitivity to granulocyte-macrophage colony-stimulating factor (GM-CSF) and hyperactive RAS-ERK signaling. Mutant EMPs engrafted the spleens of neonatal recipients, but did not cause disease. To assess MPN development during unperturbed hematopoiesis we generated CSF1R-MCM+;PTPN11E76K ;ROSAYFP mice in which oncogene expression was restricted to EMPs. Yellow fluorescent protein-positive progeny of mutant EMPs persisted in tissues one year after birth and demonstrated hyperactive RAS-ERK signaling. Nevertheless, these mice had normal survival and did not demonstrate features of MPN. CONCLUSIONS: YS EMPs expressing mutant PTPN11 demonstrate functional and molecular features of JMML but do not cause disease following transplantation nor following unperturbed development. Developmental Dynamics 246:1001-1014, 2017. © 2017 Wiley Periodicals, Inc.

[Hematopoietic stem cell emergence and stem cell-independent hematopoiesis in the mouse embryo].

All blood cells are produced by hematopoietic stem cells (HSCs) in the adult bone marrow. However, prior to the emergence of the first HSCs at embryonic day (E) 11, mouse embryos present several hematopoiesis waves, such as primitive hematopoietic cells, definitive erythro-myeloid progenitors, lymphoid potent cells, and multi-potent progenitors. Non-HSC-derived hematopoiesis is called HSC-independent hematopoiesis and has been considered a transient wave that is diminished after birth. Recent reports have shown that tissue-resident macrophages are derived from the yolk sac (YS) and are HSC-independent. Similarly, the presence of a developmental pathway for innate-like B-1a cells, independent of fetal liver HSCs, has been shown, although the question of whether B-1a cells are produced by fetal liver HSCs remains controversial. The present review focuses on the process of HSC development and introduces recent information regarding HSC-independent tissue macrophages and B-1 lymphocytes.

Functional B-1 progenitor cells are present in the hematopoietic stem cell-deficient embryo and depend on Cbfß for their development.

The fetal liver is a major hematopoietic site containing progenitor cells that give rise to nearly all blood cells, including B-1 cells. Because the fetal liver is not a de novo site of hematopoietic stem cell (HSC) or progenitor-cell emergence, it must be seeded by yolk sac (YS)-derived erythromyeloid progenitors at embryonic day (E) 8.5-E10 and aorta-gonado-mesonephros (AGM)-derived HSCs at E10.5-E11.5. Although the B-1 progenitor cell pool in the fetal liver is considered to be of HSC origin, we have previously proposed that YS-derived B-1 progenitors may also contribute to this pool. Until now, it has been impossible to determine whether HSC-independent B-1 progenitor cells exist in the fetal liver. Here, we demonstrate the presence of transplantable fetal-liver B-1 and marginal zone B progenitor cells in genetically engineered HSC-deficient embryos. HSC-deficient YS and AGM tissues produce B-1 progenitors in vitro and thus may serve as sites of origin for the B-1 progenitors that seed the fetal liver. Furthermore, we have found that core-binding factor beta (Cbfß) expression is required for fetal-liver B-1 progenitor cell maturation and expansion. Our data provide, to our knowledge, the first evidence for the presence of B-1 progenitor cells in the fetal liver that arise independently of HSCs and implicate Cbfß as a critical molecule in the development of this lineage.

Fkbp1a controls ventricular myocardium trabeculation and compaction by regulating endocardial Notch1 activity.

Trabeculation and compaction of the embryonic myocardium are morphogenetic events crucial for the formation and function of the ventricular walls. Fkbp1a (FKBP12) is a ubiquitously expressed cis-trans peptidyl-prolyl isomerase. Fkbp1a-deficient mice develop ventricular hypertrabeculation and noncompaction. To determine the physiological function of Fkbp1a in regulating the intercellular and intracellular signaling pathways involved in ventricular trabeculation and compaction, we generated a series of Fkbp1a conditional knockouts. Surprisingly, cardiomyocyte-restricted ablation of Fkbp1a did not give rise to the ventricular developmental defect, whereas endothelial cell-restricted ablation of Fkbp1a recapitulated the ventricular hypertrabeculation and noncompaction observed in Fkbp1a systemically deficient mice, suggesting an important contribution of Fkbp1a within the developing endocardia in regulating the morphogenesis of ventricular trabeculation and compaction. Further analysis demonstrated that Fkbp1a is a novel negative modulator of activated Notch1. Activated Notch1 (N1ICD) was significantly upregulated in Fkbp1a-ablated endothelial cells in vivo and in vitro. Overexpression of Fkbp1a significantly reduced the stability of N1ICD and direct inhibition of Notch signaling significantly reduced hypertrabeculation in Fkbp1a-deficient mice. Our findings suggest that Fkbp1a-mediated regulation of Notch1 plays an important role in intercellular communication between endocardium and myocardium, which is crucial in controlling the formation of the ventricular walls.

Plasma cell alloantigen ENPP1 is expressed by a subset of human B cells with potential regulatory functions.

Plasma cell alloantigen 1 (PC1), also known as ENPP1 (ectonucleotide pyrophosphatase/phosphodiesterase 1), is an enzyme involved primarily in hydrolysis of adenosine triphosphate at the cell surface. Although the expression pattern of PC1 is relatively broad, its expression in B cells is found at significant levels only in terminally differentiated germinal center B cells, plasma cells and a subset of B-1a cells in mice. Here we describe studies designed to determine whether expression of PC1 might define novel populations of human B cells with similarities to mouse B cells. We found that PC1 is expressed in small populations of human B lineage cells in peripheral blood, cord blood, tonsils, bone marrow and pediatric peritoneal fluid, with the highest levels in plasma cells. The characteristics of human PC1(+) B cells differ from mouse peritoneal B-1a subsets and from features of the human CD20(+)CD27(+)CD43(+)CD70(-) B-cell subset proposed to be human B-1 cells. Expression of PC1 was greatly increased in B cells stimulated with the combination of CD40 ligand, interleukin (IL)-4 and IL-21. In addition, PC1(+) B cells activated CD4(+) T regulatory cells. ENPP1 thus defines a subset of human B cells that differs significantly from mouse peritoneal B-1a and proposed human B-1 cells.

The macrophage mediates the renoprotective effects of endotoxin preconditioning.

Preconditioning is a preventative approach, whereby minimized insults generate protection against subsequent larger exposures to the same or even different insults. In immune cells, endotoxin preconditioning downregulates the inflammatory response and yet, preserves the ability to contain infections. However, the protective mechanisms of preconditioning at the tissue level in organs such as the kidney remain poorly understood. Here, we show that endotoxin preconditioning confers renal epithelial protection in various models of sepsis in vivo. We also tested the hypothesis that this protection results from direct interactions between the preconditioning dose of endotoxin and the renal tubules. This hypothesis is on the basis of our previous findings that endotoxin toxicity to nonpreconditioned renal tubules was direct and independent of immune cells. Notably, we found that tubular protection after preconditioning has an absolute requirement for CD14-expressing myeloid cells and particularly, macrophages. Additionally, an intact macrophage CD14-TRIF signaling pathway was essential for tubular protection. The preconditioned state was characterized by increased macrophage number and trafficking within the kidney as well as clustering of macrophages around S1 proximal tubules. These macrophages exhibited increased M2 polarization and upregulation of redox and iron-handling molecules. In renal tubules, preconditioning prevented peroxisomal damage and abolished oxidative stress and injury to S2 and S3 tubules. In summary, these data suggest that macrophages are essential mediators of endotoxin preconditioning and required for renal tissue protection. Preconditioning is, therefore, an attractive model to investigate novel protective pathways for the prevention and treatment of sepsis.

Human platelet lysate improves human cord blood derived ECFC survival and vasculogenesis in three dimensional (3D) collagen matrices.

Human cord blood (CB) is enriched in circulating endothelial colony forming cells (ECFCs) that display high proliferative potential and in vivo vessel forming ability. Since diminished ECFC survival is known to dampen the vasculogenic response in vivo, we tested how long implanted ECFC survive and generate vessels in three-dimensional (3D) type I collagen matrices in vitro and in vivo. We hypothesized that human platelet lysate (HPL) would promote cell survival and enhance vasculogenesis in the 3D collagen matrices. We report that the percentage of ECFC co-cultured with HPL that were alive was significantly enhanced on days 1 and 3 post-matrix formation, compared to ECFC alone containing matrices. Also, co-culture of ECFC with HPL displayed significantly more vasculogenic activity compared to ECFC alone and expressed significantly more pro-survival molecules (pAkt, p-Bad and Bcl-xL) in the 3D collagen matrices in vitro. Treatment with Akt1 inhibitor (A-674563), Akt2 inhibitor (CCT128930) and Bcl-xL inhibitor (ABT-263/Navitoclax) significantly decreased the cell survival and vasculogenesis of ECFC co-cultured with or without HPL and implicated activation of the Akt1 pathway as the critical mediator of the HPL effect on ECFC in vitro. A significantly greater average vessel number and total vascular area of human CD31(+) vessels were present in implants containing ECFC and HPL, compared to the ECFC alone implants in vivo. We conclude that implantation of ECFC with HPL in vivo promotes vasculogenesis and augments blood vessel formation via diminishing apoptosis of the implanted ECFC.

Biomechanical forces promote embryonic haematopoiesis.

Biomechanical forces are emerging as critical regulators of embryogenesis, particularly in the developing cardiovascular system. After initiation of the heartbeat in vertebrates, cells lining the ventral aspect of the dorsal aorta, the placental vessels, and the umbilical and vitelline arteries initiate expression of the transcription factor Runx1 (refs 3-5), a master regulator of haematopoiesis, and give rise to haematopoietic cells. It remains unknown whether the biomechanical forces imposed on the vascular wall at this developmental stage act as a determinant of haematopoietic potential. Here, using mouse embryonic stem cells differentiated in vitro, we show that fluid shear stress increases the expression of Runx1 in CD41(+)c-Kit(+) haematopoietic progenitor cells, concomitantly augmenting their haematopoietic colony-forming potential. Moreover, we find that shear stress increases haematopoietic colony-forming potential and expression of haematopoietic markers in the para-aortic splanchnopleura/aorta-gonads-mesonephros of mouse embryos and that abrogation of nitric oxide, a mediator of shear-stress-induced signalling, compromises haematopoietic potential in vitro and in vivo. Collectively, these data reveal a critical role for biomechanical forces in haematopoietic development.

Autonomous murine T-cell progenitor production in the extra-embryonic yolk sac before HSC emergence.

The extra-embryonic yolk sac (YS) is the first hematopoietic site in the mouse embryo and is thought to generate only primitive erythroid and myeloerythroid progenitor cells before definitive HSC emergence within the embryo on E10.5. Here, we have shown the existence of T cell-restricted progenitors in the E9.5 YS that directly engraft in recipient immunodeficient mice. T-cell progenitors were also produced in vitro from both YS and para-aortic splanchnopleura hemogenic endothelial cells, and these T-cell progenitors repopulated the thymus and differentiated into mature T-cell subsets in vivo on transplantation. Our data confirm that the YS produces T-lineage-restricted progenitors that are available to colonize the thymus and provide new insight into the YS as a definitive hematopoietic site in the mouse embryo.

Embryonic day 9 yolk sac and intra-embryonic hemogenic endothelium independently generate a B-1 and marginal zone progenitor lacking B-2 potential.

The majority of B lymphocytes in the adult mouse are generated in the bone marrow from hematopoietic stem cells (HSCs) that first appear in the aorta-gonado-mesonephros region of the fetus on embryonic day (E) 10.5-11. Comparatively less is known about B-cell development during embryogenesis. For example, which specific embryonic tissues participate in B lymphopoiesis and whether hematopoietic differentiation is skewed toward specific B-cell subsets in the embryo are unanswered questions, because the systemic circulation is initiated early during embryogenesis, resulting in an admixture of cells potentially originating from multiple sites. We demonstrate, using Ncx1(-/-) mice that lack systemic blood circulation, that the E9 yolk sac (YS) and the intra-embryonic para-aortic splanchnopleura (P-Sp) tissues independently give rise to AA4.1(+)CD19(+)B220(lo-neg) B progenitor cells that preferentially differentiate into innate type B-1 and marginal zone (MZ) B cells but not into B-2 cells upon transplantation. We have further demonstrated that these B-1 progenitor cells arise directly from YS and P-Sp hemogenic endothelium. These results document the initial wave of innate B lymphopoietic progenitor cells available for seeding the fetal liver at E11. The results of these studies expand our knowledge of hemogenic endothelial sites specifying distinct B-1 and MZ cell fates apart from B-2 cells and independent of an HSC origin during development.