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• W2008982471 abstract "•Embryonic lymphoid lineage restriction initiates prior to emergence of definitive HSC•Embryonic lymphoid-primed progenitors have immune-restricted lymphomyeloid potential•Embryonic lymphoid-primed progenitors contribute to myelo- and lymphopoiesis in utero•Lympho-myelo immune restriction is an early-fate decision in embryonic hematopoiesis In jawed vertebrates, development of an adaptive immune-system is essential for protection of the born organism against otherwise life-threatening pathogens. Myeloid cells of the innate immune system are formed early in development, whereas lymphopoiesis has been suggested to initiate much later, following emergence of definitive hematopoietic stem cells (HSCs). Herein, we demonstrate that the embryonic lymphoid commitment process initiates earlier than previously appreciated, prior to emergence of definitive HSCs, through establishment of a previously unrecognized entirely immune-restricted and lymphoid-primed progenitor. Notably, this immune-restricted progenitor appears to first emerge in the yolk sac and contributes physiologically to the establishment of lymphoid and some myeloid components of the immune-system, establishing the lymphomyeloid lineage restriction process as an early and physiologically important lineage-commitment step in mammalian hematopoiesis. In jawed vertebrates, development of an adaptive immune-system is essential for protection of the born organism against otherwise life-threatening pathogens. Myeloid cells of the innate immune system are formed early in development, whereas lymphopoiesis has been suggested to initiate much later, following emergence of definitive hematopoietic stem cells (HSCs). Herein, we demonstrate that the embryonic lymphoid commitment process initiates earlier than previously appreciated, prior to emergence of definitive HSCs, through establishment of a previously unrecognized entirely immune-restricted and lymphoid-primed progenitor. Notably, this immune-restricted progenitor appears to first emerge in the yolk sac and contributes physiologically to the establishment of lymphoid and some myeloid components of the immune-system, establishing the lymphomyeloid lineage restriction process as an early and physiologically important lineage-commitment step in mammalian hematopoiesis. The mammalian hematopoietic system is considered the paradigmatic and best-understood model for how multilineage diversity is achieved by stepwise lineage restriction of a multipotent stem cell (Orkin and Zon, 2008aOrkin S.H. Zon L.I. Hematopoiesis: an evolving paradigm for stem cell biology.Cell. 2008; 132: 631-644Abstract Full Text Full Text PDF PubMed Scopus (670) Google Scholar). Although multipotent hematopoietic stem cells (HSCs) possessing all blood-lineage potentials have been unequivocally established to reside at the top of the hematopoietic hierarchy (Osawa et al., 1996Osawa M. Hanada K.-I. Hamada H. Nakauchi H. Long-term lymphohematopoietic reconstitution by a single CD34-low/negative hematopoietic stem cell.Science. 1996; 273: 242-245Crossref PubMed Scopus (1718) Google Scholar), the exact roadmap for hematopoietic lineage restriction remains disputed, in particular with regard to the first lineage-restriction steps of HSCs (Orkin and Zon, 2008aOrkin S.H. Zon L.I. Hematopoiesis: an evolving paradigm for stem cell biology.Cell. 2008; 132: 631-644Abstract Full Text Full Text PDF PubMed Scopus (670) Google Scholar, Schlenner and Rodewald, 2010Schlenner S.M. Rodewald H.R. Early T cell development and the pitfalls of potential.Trends Immunol. 2010; 31: 303-310Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, Ye and Graf, 2007Ye M. Graf T. Early decisions in lymphoid development.Curr. Opin. Immunol. 2007; 19: 123-128Crossref PubMed Scopus (60) Google Scholar). The classical hematopoietic hierarchy still prevailing in textbooks predicts that the first lineage-restriction decision made by adult HSCs results in strictly separated pathways for the lymphoid and myeloerythroid lineages (Orkin and Zon, 2008bOrkin S.H. Zon L.I. SnapShot: hematopoiesis.Cell. 2008; 132: 712Abstract Full Text Full Text PDF PubMed Scopus (1672) Google Scholar, Seita and Weissman, 2010Seita J. Weissman I.L. Hematopoietic stem cell: self-renewal versus differentiation.Wiley Interdiscip Rev Syst Biol Med. 2010; 2: 640-653Crossref PubMed Scopus (506) Google Scholar) as supported by the identification of common lymphoid (Kondo et al., 1997Kondo M. Weissman I.L. Akashi K. Identification of clonogenic common lymphoid progenitors in mouse bone marrow.Cell. 1997; 91: 661-672Abstract Full Text Full Text PDF PubMed Scopus (1655) Google Scholar) and common myeloid (Akashi et al., 2000Akashi K. Traver D. Miyamoto T. Weissman I.L. A clonogenic common myeloid progenitor that gives rise to all myeloid lineages.Nature. 2000; 404: 193-197Crossref PubMed Scopus (1906) Google Scholar, Arinobu et al., 2007Arinobu Y. Mizuno S. Chong Y. Shigematsu H. Iino T. Iwasaki H. Graf T. Mayfield R. Chan S. Kastner P. Akashi K. Reciprocal activation of GATA-1 and PU.1 marks initial specification of hematopoietic stem cells into myeloerythroid and myelolymphoid lineages.Cell Stem Cell. 2007; 1: 416-427Abstract Full Text Full Text PDF PubMed Scopus (234) Google Scholar) progenitors (CLPs and CMPs, respectively). However, earlier in vitro findings demonstrating that clonal lineage outputs of heterogeneous fetal hematopoietic progenitors excluded combined B and T cell generation in the absence of myeloid cells, challenged the classical hematopoietic hierarchy, and led to the proposal that the first lineage decisions of HSCs might rather result in a separation into a CMP and a lymphomyeloid progenitor pathway (Katsura and Kawamoto, 2001Katsura Y. Kawamoto H. Stepwise lineage restriction of progenitors in lympho-myelopoiesis.Int. Rev. Immunol. 2001; 20: 1-20Crossref PubMed Scopus (41) Google Scholar), a hypothesis supported by additional studies (Cumano et al., 1992Cumano A. Paige C.J. Iscove N.N. Brady G. Bipotential precursors of B cells and macrophages in murine fetal liver.Nature. 1992; 356: 612-615Crossref PubMed Scopus (303) Google Scholar, Lacaud et al., 1998Lacaud G. Carlsson L. Keller G. Identification of a fetal hematopoietic precursor with B cell, T cell, and macrophage potential.Immunity. 1998; 9: 827-838Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). More recently, lymphoid-primed multipotent progenitors (LMPPs) were prospectively isolated and shown at the single-cell level to possess combined granulocyte-macrophage (GM) and B and T lymphocyte potential but little or no megakaryocyte-erythroid (MkE) potential (Adolfsson et al., 2005Adolfsson J. Månsson R. Buza-Vidas N. Hultquist A. Liuba K. Jensen C.T. Bryder D. Yang L. Borge O.J. Thoren L.A. et al.Identification of Flt3+ lympho-myeloid stem cells lacking erythro-megakaryocytic potential a revised road map for adult blood lineage commitment.Cell. 2005; 121: 295-306Abstract Full Text Full Text PDF PubMed Scopus (916) Google Scholar, Månsson et al., 2007Månsson R. Hultquist A. Luc S. Yang L. Anderson K. Kharazi S. Al-Hashmi S. Liuba K. Thorén L. Adolfsson J. et al.Molecular evidence for hierarchical transcriptional lineage priming in fetal and adult stem cells and multipotent progenitors.Immunity. 2007; 26: 407-419Abstract Full Text Full Text PDF PubMed Scopus (276) Google Scholar), providing direct evidence for the existence of a lymphomyeloid-restricted pathway in mammalian hematopoiesis. However, the revised hematopoietic hierarchy has been challenged by studies implying that LMPPs possess considerable MkE potential in vivo (Boyer et al., 2011Boyer S.W. Schroeder A.V. Smith-Berdan S. Forsberg E.C. All hematopoietic cells develop from hematopoietic stem cells through Flk2/Flt3-positive progenitor cells.Cell Stem Cell. 2011; 9: 64-73Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar, Forsberg et al., 2006Forsberg E.C. Serwold T. Kogan S. Weissman I.L. Passegué E. New evidence supporting megakaryocyte-erythrocyte potential of flk2/flt3+ multipotent hematopoietic progenitors.Cell. 2006; 126: 415-426Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar). Thus, whether the first lineage fate decision of multipotent stem-progenitor cells results in establishment of completely separated pathways of the myeloid and lymphoid components of the immune system or a common lymphomyeloid immune pathway remains unresolved (Boyer et al., 2012Boyer S.W. Beaudin A.E. Forsberg E.C. Mapping differentiation pathways from hematopoietic stem cells using Flk2/Flt3 lineage tracing.Cell Cycle. 2012; 11: 3180-3188Crossref PubMed Scopus (26) Google Scholar, Orkin and Zon, 2008aOrkin S.H. Zon L.I. Hematopoiesis: an evolving paradigm for stem cell biology.Cell. 2008; 132: 631-644Abstract Full Text Full Text PDF PubMed Scopus (670) Google Scholar, Schlenner and Rodewald, 2010Schlenner S.M. Rodewald H.R. Early T cell development and the pitfalls of potential.Trends Immunol. 2010; 31: 303-310Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, Seita and Weissman, 2010Seita J. Weissman I.L. Hematopoietic stem cell: self-renewal versus differentiation.Wiley Interdiscip Rev Syst Biol Med. 2010; 2: 640-653Crossref PubMed Scopus (506) Google Scholar, Ye and Graf, 2007Ye M. Graf T. Early decisions in lymphoid development.Curr. Opin. Immunol. 2007; 19: 123-128Crossref PubMed Scopus (60) Google Scholar). Moreover, the in vivo physiological relevance of the lymphomyeloid pathway has been challenged based on in vivo fate mapping studies concluding that adult lymphomyeloid-restricted progenitors contribute almost exclusively to lymphopoiesis and not myelopoiesis (Boyer et al., 2011Boyer S.W. Schroeder A.V. Smith-Berdan S. Forsberg E.C. All hematopoietic cells develop from hematopoietic stem cells through Flk2/Flt3-positive progenitor cells.Cell Stem Cell. 2011; 9: 64-73Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar, Schlenner et al., 2010Schlenner S.M. Madan V. Busch K. Tietz A. Läufle C. Costa C. Blum C. Fehling H.J. Rodewald H.R. Fate mapping reveals separate origins of T cells and myeloid lineages in the thymus.Immunity. 2010; 32: 426-436Abstract Full Text Full Text PDF PubMed Scopus (225) Google Scholar, Welner et al., 2009Welner R.S. Esplin B.L. Garrett K.P. Pelayo R. Luche H. Fehling H.J. Kincade P.W. Asynchronous RAG-1 expression during B lymphopoiesis.J. Immunol. 2009; 183: 7768-7777Crossref PubMed Scopus (38) Google Scholar). Lineage-fate decisions of multipotent stem and progenitor cells take place for the first time during early embryonic development. Blood cells appear in the mouse embryo at embryonic day (E) 7 in the yolk sac (YS). This first wave of primitive hematopoiesis is myeloerythroid-restricted and transient (Medvinsky et al., 2011Medvinsky A. Rybtsov S. Taoudi S. Embryonic origin of the adult hematopoietic system: advances and questions.Development. 2011; 138: 1017-1031Crossref PubMed Scopus (277) Google Scholar). In addition, resident tissue macrophages, such as brain microglia, liver Kupffer cells, and epidermal Langerhans cells, develop between E8.5 and E9.5 independently of definitive HSCs (Schulz et al., 2012Schulz C. Gomez Perdiguero E. Chorro L. Szabo-Rogers H. Cagnard N. Kierdorf K. Prinz M. Wu B. Jacobsen S.E. Pollard J.W. et al.A lineage of myeloid cells independent of Myb and hematopoietic stem cells.Science. 2012; 336: 86-90Crossref PubMed Scopus (1725) Google Scholar). The first definitive multipotent HSCs, defined as cells capable of long-term repopulation of all myeloerythroid and lymphoid lineages in adult recipient mice are not found until after E10.5 (Medvinsky and Dzierzak, 1996Medvinsky A. Dzierzak E. Definitive hematopoiesis is autonomously initiated by the AGM region.Cell. 1996; 86: 897-906Abstract Full Text Full Text PDF PubMed Scopus (1161) Google Scholar). These HSCs, first generated in the aorta-gonad-mesonephros (AGM) region, subsequently at E11 seed the fetal liver (FL), the main fetal hematopoietic site (Kumaravelu et al., 2002Kumaravelu P. Hook L. Morrison A.M. Ure J. Zhao S. Zuyev S. Ansell J. Medvinsky A. Quantitative developmental anatomy of definitive haematopoietic stem cells/long-term repopulating units (HSC/RUs): role of the aorta-gonad-mesonephros (AGM) region and the yolk sac in colonisation of the mouse embryonic liver.Development. 2002; 129: 4891-4899PubMed Google Scholar, Medvinsky and Dzierzak, 1996Medvinsky A. Dzierzak E. Definitive hematopoiesis is autonomously initiated by the AGM region.Cell. 1996; 86: 897-906Abstract Full Text Full Text PDF PubMed Scopus (1161) Google Scholar). Because the first lymphoid-restricted embryonic progenitors have been identified after definitive HSCs seed the FL (Douagi et al., 2002Douagi I. Vieira P. Cumano A. Lymphocyte commitment during embryonic development, in the mouse.Semin. Immunol. 2002; 14: 361-369Crossref PubMed Scopus (37) Google Scholar), it has been assumed that lymphoid lineage restriction does not initiate before the FL is seeded by definitive HSCs. However, there are several findings compatible with lymphoid lineage commitment potentially initiating earlier. Progenitors possessing all myeloerythroid and lymphoid lineage potentials have been identified before emergence of definitive HSCs and FL hematopoiesis (Kieusseian et al., 2012Kieusseian A. Brunet de la Grange P. Burlen-Defranoux O. Godin I. Cumano A. Immature hematopoietic stem cells undergo maturation in the fetal liver.Development. 2012; 139: 3521-3530Crossref PubMed Scopus (66) Google Scholar, Rybtsov et al., 2011Rybtsov S. Sobiesiak M. Taoudi S. Souilhol C. Senserrich J. Liakhovitskaia A. Ivanovs A. Frampton J. Zhao S. Medvinsky A. Hierarchical organization and early hematopoietic specification of the developing HSC lineage in the AGM region.J. Exp. Med. 2011; 208: 1305-1315Crossref PubMed Scopus (172) Google Scholar, Yoshimoto et al., 2012Yoshimoto M. Porayette P. Glosson N.L. Conway S.J. Carlesso N. Cardoso A.A. Kaplan M.H. Yoder M.C. Autonomous murine T-cell progenitor production in the extra-embryonic yolk sac before HSC emergence.Blood. 2012; 119: 5706-5714Crossref PubMed Scopus (114) Google Scholar) and at E10.5–E11, low-level expression of interleukin-7 receptor alpha (IL7Rα) and the recombination activating gene 1 (Rag1), regulators of early lymphoid development, have been described in the embryo (Kawamoto et al., 2000Kawamoto H. Ikawa T. Ohmura K. Fujimoto S. Katsura Y. T cell progenitors emerge earlier than B cell progenitors in the murine fetal liver.Immunity. 2000; 12: 441-450Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar, Yokota et al., 2006Yokota T. Huang J. Tavian M. Nagai Y. Hirose J. Zúñiga-Pflücker J.C. Péault B. Kincade P.W. Tracing the first waves of lymphopoiesis in mice.Development. 2006; 133: 2041-2051Crossref PubMed Scopus (74) Google Scholar) Although the identity of these early IL7Rα and Rag1-expressing cells has not been investigated in detail, they could potentially reflect multilineage transcriptional-primed multipotent progenitors (Hu et al., 1997Hu M. Krause D. Greaves M. Sharkis S. Dexter M. Heyworth C. Enver T. Multilineage gene expression precedes commitment in the hemopoietic system.Genes Dev. 1997; 11: 774-785Crossref PubMed Scopus (616) Google Scholar, Månsson et al., 2007Månsson R. Hultquist A. Luc S. Yang L. Anderson K. Kharazi S. Al-Hashmi S. Liuba K. Thorén L. Adolfsson J. et al.Molecular evidence for hierarchical transcriptional lineage priming in fetal and adult stem cells and multipotent progenitors.Immunity. 2007; 26: 407-419Abstract Full Text Full Text PDF PubMed Scopus (276) Google Scholar). However, the earliest IL7Rα-expressing progenitors have rather been suggested to be fully B or T lymphocyte-restricted and to lack myeloid lineage potential (Kawamoto et al., 2000Kawamoto H. Ikawa T. Ohmura K. Fujimoto S. Katsura Y. T cell progenitors emerge earlier than B cell progenitors in the murine fetal liver.Immunity. 2000; 12: 441-450Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar), coinciding with or even preceding evidence of lymphomyeloid lineage restriction, suggesting that B and T lymphoid lineage restriction might occur independently of a lymphomyeloid lineage restriction step. Herein we prospectively identified an IL7Rα-expressing lymphomyeloid-restricted embryonic progenitor as early as E11.5 that sustains combined lymphoid and GM transcriptional lineage priming and potential at the single-cell level but lacks MkE potential. We tracked this lymphomyeloid progenitor back to E9.5 YS, preceding both hematopoietic colonization of the FL and the establishment of definitive HSCs. Through in vivo fate mapping, we confirmed the inability of Rag1-expressing early embryonic progenitors to significantly contribute to the MkE lineage while unequivocally and robustly contributing to the myeloid innate, as well as lymphoid adaptive, immune systems of the mammalian embryo. These findings identify the developmentally earliest immune-restricted progenitor and establish the lymphomyeloid restriction as a physiologically important lineage-commitment step in embryonic mammalian hematopoiesis, preceding the emergence of definitive HSCs. Expression of IL7Rα has been suggested to be restricted to lymphoid progenitors. We investigated IL7Rα expression in FL at E11.5 and in agreement with previous findings (Kawamoto et al., 2000Kawamoto H. Ikawa T. Ohmura K. Fujimoto S. Katsura Y. T cell progenitors emerge earlier than B cell progenitors in the murine fetal liver.Immunity. 2000; 12: 441-450Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar) we found IL7Rα+ cells coexpressing the pan-hematopoietic marker CD45 and the progenitor cell receptor c-Kit (Kit) but negative for mature lineage markers (Lin−) (Figure 1A). A large fraction of Lin−Kit+IL7Rα+ cells also expressed the myeloid colony-stimulating factor 1 receptor (Csf1r) and the thrombopoietin receptor (ThpoR) (Figure 1A), also expressed by HSCs (Solar et al., 1998Solar G.P. Kerr W.G. Zeigler F.C. Hess D. Donahue C. de Sauvage F.J. Eaton D.L. Role of c-mpl in early hematopoiesis.Blood. 1998; 92: 4-10Crossref PubMed Google Scholar). Most IL7Rα+ cells (>70%) coexpressed c-fms-like tyrosine kinase 3 receptor (Flt3) (0.20% of FL cells; Figure 1B), highly expressed on early lymphomyeloid progenitors in adult bone marrow (Adolfsson et al., 2005Adolfsson J. Månsson R. Buza-Vidas N. Hultquist A. Liuba K. Jensen C.T. Bryder D. Yang L. Borge O.J. Thoren L.A. et al.Identification of Flt3+ lympho-myeloid stem cells lacking erythro-megakaryocytic potential a revised road map for adult blood lineage commitment.Cell. 2005; 121: 295-306Abstract Full Text Full Text PDF PubMed Scopus (916) Google Scholar). Single-cell assays demonstrated that Lin−Kit+Flt3+IL7Rα+ E11.5 FL cells possess considerable GM potential with as many as 43% of single cells forming GM clones, whereas virtually no cells (<1%) possessed Mk or E potential (Figures 1C–1F). Single Lin−Kit+Flt3+IL7Rα+ cells generated 31% and 35% of B and T cell progeny, respectively (Figures 1G and 1H). In vitro cytokine response experiments with the ligands for Flt3 and IL7Rα demonstrated that Flt3 and IL7Rα are expressed at functionally relevant levels on Lin−Kit+Flt3+IL7Rα+ progenitors (see Figure S1A available online). However, in contrast to later emerging B and T lymphoid-restricted progenitors critically dependent on Flt3 and IL7Rα signaling (Sitnicka et al., 2003Sitnicka E. Brakebusch C. Martensson I.L. Svensson M. Agace W.W. Sigvardsson M. Buza-Vidas N. Bryder D. Cilio C.M. Ahlenius H. et al.Complementary signaling through flt3 and interleukin-7 receptor alpha is indispensable for fetal and adult B cell genesis.J. Exp. Med. 2003; 198: 1495-1506Crossref PubMed Scopus (130) Google Scholar), investigation of embryos genetically deleted for expression of both Flt3 ligand and the common gamma chain of the IL7R demonstrated that E11.5 Lin−Kit+Flt3+IL7Rα+ progenitors emerge independently of Flt3 and IL7R signaling (Figures S1B and S1C). Investigation of single Lin−Kit+Flt3+IL7Rα+ E11.5 FL cells demonstrated that more than 70% coexpressed lymphoid (Il7r, Rag1, and sIgh) and GM (Csf3r [Gcsfr] and Mpo), but not the MkE (Vwf, Epor, and Gata1) transcripts typically expressed by multipotent progenitors with MkE potential (Figures 2A and 2B )(Månsson et al., 2007Månsson R. Hultquist A. Luc S. Yang L. Anderson K. Kharazi S. Al-Hashmi S. Liuba K. Thorén L. Adolfsson J. et al.Molecular evidence for hierarchical transcriptional lineage priming in fetal and adult stem cells and multipotent progenitors.Immunity. 2007; 26: 407-419Abstract Full Text Full Text PDF PubMed Scopus (276) Google Scholar). When compared to Lin−Kit+Flt3−IL7Rα− cells, the downregulation of MkE-affiliated and upregulation of lymphoid genes expressed prior to B and T cell commitment was further verified through single-cell quantitative gene expression analysis (Figure 2C). Notably, this single-cell analysis demonstrated that every Lin−Kit+Flt3+IL7Rα+ progenitor cell expressing these lymphoid genes coexpressed myeloid lineage genes, whereas none of them expressed signature genes of the earliest B (Pax5) and T (Ptcra) cell restricted progenitors. Clones derived from single Lin−Kit+Flt3+IL7Rα+ E11.5 FL cells were analyzed for the combined potential for generation of GM, B, and T cells (Figures 2D and 2E). Of the single cell-derived clones that gave a lineage readout, as many as 45% produced mature cells of all three GM, B, and T cell lineages (GM/B/T; Figures 2F and 2G). Natural killer (NK) cells, a third lymphoid lineage and part of the innate immune system, were generated in the majority of the single cell-derived GM/B/T clones (Figure 2H). Virtually no erythroid cells were produced even upon inclusion of erythroid-promoting cytokines (Figure 2I), which stimulated erythropoiesis from the more heterogeneous Lin−Kit+Flt3−IL7Rα− progenitors (Figures S2A and S2B). Paired immunoglobulin like receptor (PIR) expression has been shown to separate T and B cell progenitors later in embryonic development. Specifically, in E13.5, FL T cell restricted progenitors were found to coexpress IL7Rα and PIR, whereas B cell progenitors were IL7Rα+PIR− (Masuda et al., 2005Masuda K. Kubagawa H. Ikawa T. Chen C.C. Kakugawa K. Hattori M. Kageyama R. Cooper M.D. Minato N. Katsura Y. Kawamoto H. Prethymic T-cell development defined by the expression of paired immunoglobulin-like receptors.EMBO J. 2005; 24: 4052-4060Crossref PubMed Scopus (61) Google Scholar). We found that only a small fraction (11%) of E11.5 FL Lin−Kit+Flt3+IL7Rα+ progenitors expressed cell surface PIR (Figure 2J). The PIR expression was confirmed by quantitative PCR and shown to coincide with upregulation of other early lymphoid transcripts, whereas both PIR+ and PIR− Lin−Kit+Flt3+IL7Rα+ cells expressed myeloid genes (Figure 2K). In agreement with representing the E11.5 FL lymphomyeloid-restricted progenitors with combined GM, B, and T cell potential, Lin−Kit+Flt3+IL7Rα+PIR− cells showed robust readout of all three lineage potentials, whereas the small fraction of Lin−Kit+Flt3+IL7Rα+PIR+ cells showed reduced T and undetectable B cell potential (Figure 2L). Thus, at the time of its first seeding by definitive HSCs, the FL contains Lin−Kit+Flt3+IL7Rα+ progenitors with combined lymphomyeloid, but no MkE transcriptional priming or potential. A large number of Lin−Kit+Flt3+IL7Rα+ immune-restricted progenitors (1,200 ± 200 cells/FL) were already present by E11.5. In contrast, we did not detect any long-term multilineage reconstitution activity in E10.5 or E11.5 FL (whether transplanted intravenously or directly into the bones), whereas considerable HSC activity was found in E12.5 FL (Figure 3A; Figures S3A–S3D). These findings are in agreement with previous studies, which suggest that there is at most only one definitive HSC in the FL at E11.5 (Kumaravelu et al., 2002Kumaravelu P. Hook L. Morrison A.M. Ure J. Zhao S. Zuyev S. Ansell J. Medvinsky A. Quantitative developmental anatomy of definitive haematopoietic stem cells/long-term repopulating units (HSC/RUs): role of the aorta-gonad-mesonephros (AGM) region and the yolk sac in colonisation of the mouse embryonic liver.Development. 2002; 129: 4891-4899PubMed Google Scholar). To enhance the possibility to identify rare lymphoid-primed progenitors in the early FL, and because expression of Flt3 and Rag1-GFP highly overlap in Lin−Kit+Flt3+IL7Rα+ E11.5 progenitors (Figure 3B), we used a Rag1-GFP reporter to identify a distinct population of Lin−Kit+Rag1GFP+IL7Rα+ E10.5 FL progenitors (Figure 3C), which possessed GM, B, and T cell, but no MkE potential, as in the E11.5 FL (Figures 3D–3G; Figure S3E), establishing that immune-restricted progenitors are present in the FL already prior to and independently of definitive HSCs. We next explored how early Lin−Kit+Rag1GFP+IL7Rα+ lymphomyeloid-primed progenitors emerge in the embryo. In whole E8.5 concepti, virtually no Rag1GFP+IL7Rα+-positive cells were found, neither in the CD45− nor in the small CD45+ fraction (Figure 3H). Because the first hematopoietic cells to colonize the FL at E10 might come from the YS, we analyzed YS at E9.5 and observed a small but distinct Lin−Kit+Rag1GFP+IL7Rα+ cell population (30 ± 4 cells/YS; Figure 3I). Notably, whereas most hematopoietic cells at E9.5 have yet to upregulate expression of the pan-hematopoietic marker CD45, virtually all Lin−Kit+Rag1GFP+IL7Rα+ cells were CD45+ although most also expressed CD41, and a small fraction the endothelial marker VE-Cadherin (Figure 3J). The placenta and the para-aortic-splanchnopleura (PAS, to become the AGM) region from E9.5 embryos were also investigated, but no Rag1GFP+IL7Rα+ cells were detected above background level (Figure 3I). Almost 80% of the rare purified E9.5 YS Lin−Kit+Rag1GFP+IL7Rα+ cells expressed combined lymphoid GM but no MkE transcriptional priming at the single-cell level (Figures 3K and 3L), similar to E11.5 FL Lin−Kit+Flt3+IL7Rα+ cells (Figures 2A–2B), demonstrating that lymphomyeloid-primed progenitors emerge as early as E9.5. To further investigate the embryonic site of origin of the lymphoid-primed immune-restricted progenitors, whole-mount imaging of Rag1GFP+ embryos was performed. In agreement with the fluorescence-activated cell sorting (FACS) analysis, Rag1GFP+ cells coexpressing CD45 and the endothelial marker CD31 were localized in the YS at E9.5 in all embryos investigated (14/14; Figure 3M; Figure S3F), whereas in the PAS region Rag1GFP+ cells were rarely observed (Figure 3N). Notably, the majority of Rag1GFP+ cells in the YS were predominantly located in close proximity to the primitive vascular plexus, rather than in large vessels (Figures 3M–3N; Figure S3F). In line with these data, we also observed transcriptional expression of Rag2 and Il7r in E9.5 YS but little or no expression in E8.5 embryos or E9.5 PAS (Figure 3O). These data are most compatible with Lin−Kit+Rag1GFP+IL7Rα+ cells originating in E9.5 YS. However, because the connection between the YS and intraembryonic blood vessels occurs already at E8.5, we next dissected the YS and PAS regions from Rag1GFP+ embryos before circulation was established (≤6 somite pairs [sp]), short-term cultured the whole explants, and performed whole-mount immunolabeling (Cumano et al., 1996Cumano A. Dieterlen-Lievre F. Godin I. Lymphoid potential, probed before circulation in mouse, is restricted to caudal intraembryonic splanchnopleura.Cell. 1996; 86: 907-916Abstract Full Text Full Text PDF PubMed Scopus (475) Google Scholar). Strikingly, in six out of ten YS explants, Rag1GFP+ cells coexpressing CD45 and CD31 emerged after 48 hr, as did expression of Il7r and sIgh (Figures 3P and 3Q), whereas at the same time no Rag1GFP+ cells were generated by the precirculation PAS explants (seven embryos) (Figure S3G), supporting that Rag1GFP+IL7Rα+ lymphomyeloid-restricted progenitors arise de novo in the YS at E9.5. It is important to establish to what degree early lymphoid progenitors with sustained myeloid lineage potential in vitro contribute toward generation of myeloid cells under in vivo physiologically relevant conditions. Recent studies exploring this in adult hematopoiesis have questioned the physiological relevance of the residual myeloid potential of early lymphomyeloid progenitors (Boyer et al., 2011Boyer S.W. Schroeder A.V. Smith-Berdan S. Forsberg E.C. All hematopoietic cells develop from hematopoietic stem cells through Flk2/Flt3-positive progenitor cells.Cell Stem Cell. 2011; 9: 64-73Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar, Schlenner et al., 2010Schlenner S.M. Madan V. Busch K. Tietz A. Läufle C. Costa C. Blum C. Fehling H.J. Rodewald H.R. Fate mapping reveals separate origins of T cells and myeloid lineages in the thymus.Immunity. 2010; 32: 426-436Abstract Full Text Full Text PDF PubMed Scopus (225) Google Scholar, Welner et al., 2009Welner R.S. Esplin B.L. Garrett K.P. Pelayo R. Luche H. Fehling H.J. Kincade P.W. Asynchronous RAG-1 expression during B lymphopoiesis.J. Immunol. 2009; 183: 7768-7777Crossref PubMed Scopus (38) Google Scholar). Thus, although our in vitro experiments demonstrated that embryonic immune-restricted progenitors sustain lymphoid and GM but little or no MkE lineage potentials, they could not establish the contribution of these Rag1-expressing progenitors to different hematopoietic cell lineages during embryonic development. Thus, we applied a Rag1-Cre fate mapping in which only cells expressing Rag1 or their progeny will express a yellow fluorescent protein (YFP). As expected, virtually all B and T cells emerging later in the fetus were positive for YFP in E14.5 FL (Figures 4A–4B) as in adult BM (Figure S4A). Moreover, Rag1 expressin" @default.
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• W2008982471 date "2013-11-01" @default.
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• W2008982471 title "Lymphomyeloid Contribution of an Immune-Restricted Progenitor Emerging Prior to Definitive Hematopoietic Stem Cells" @default.
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• W2008982471 doi "https://doi.org/10.1016/j.stem.2013.08.012" @default.
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