Faculty Opinions recommendation of Tissue-resident macrophages originate from yolk-sac-derived erythro-myeloid progenitors.

Abstract Abstract 1599 The ontogeny of hematopoiesis in mammalian embryos is complicated by the requirement for functional blood cells prior to the emergence of hematopoietic stem cells or the bone marrow microenvironment. In the murine embryo, transplantable HSC are first evident at embryonic day (E) 10.5 and the first few HSC are found in the fetal liver hematopoietic environment by E12.5. However, two overlapping waves of hematopoietic potential arise in the yolk sac before E10.5. The first “primitive” wave produces progenitors from E7.25 to E8.5 with primitive erythroid, megakaryocyte and macrophage potentials. The resulting primitive erythroid cells mature within the circulation and support embryonic growth past E9.5. At E8.5, a second wave of hematopoiesis begins in the yolk sac and generates definitive erythroid and multiple myeloid progenitors that are the proposed source of the hematopoietic progenitors seeding the fetal liver before HSC colonization. We have identified a cell population displaying a unique cell surface immunophenotype in the E9.5 yolk sac that contains the potential to form definitive erythroid cells, megakaryocytes, macrophages and all forms of granulocytes within days of in vitro culture. Furthermore, all definitive hematopoietic colony-forming cells (BFU-E, CFC-myeloid and HPP-CFC) in the E9.5 yolk sac have this immunophenotype. These erythro-myeloid progenitors (EMP) are lineage-negative and co-express ckit, CD41, CD16/32 and Endoglin. Interestingly, this is not an immunophenotype evident in the adult bone marrow. Other markers that have been associated with HSC formation (AA4.1, ScaI) or with lymphoid potential (IL7R, Flt3) are not present on these cells at E9.5. Consistent with the lack of lymphoid markers, we also do not observe short-term development of B-cells (CD19+B220+ expressing Rag2 RNA) in cultures of the E9.5 sorted EMP, while bone marrow Lin-/ckit+/ScaI- cells do form B-cells under the same conditions. Clonal analysis of sorted EMP cells revealed single cells with both erythroid and granulocyte potential, similar to the common myeloid progenitors in adult bone marrow. Though these EMP are enriched at E9.5 in the yolk sac, they are also found at low levels in the fetal blood, embryo proper and placenta, consistent with their entrance into the circulation. By E10.5, EMP were most highly enriched in the newly formed fetal liver. Additionally by E12.5, a time when the first few HSCs are detected in the fetal liver, we find active erythropoiesis and granulopoiesis in the liver and the first definitive red blood cells and neutrophils in the bloodstream. Therefore, we believe the yolk sac definitive progenitors' fate is to populate the fetal liver and thus provide the first definitive erythrocytes and granulocytes for the embryo. The differentiation of embryonic stem cells (ES) and induced pluripotent stem cells (iPS) cells into mature cells types offers the hope of cell-based therapies. Analysis of differentiating murine ES cells reveals overlapping waves of primitive and definitive hematopoietic colony forming potential. We demonstrate the appearance of an EMP-like (ckit+/CD41+/FcGR+) population coincident with the emergence of definitive hematopoietic progenitors during murine ES cell differentiation as embryoid bodies. We have confirmed with colony forming assays that definitive hematopoietic potential is associated with this immunophenotypic group. Our studies support the concept that blood cell emergence during ES cell differentiation closely mimics pre-HSC hematopoiesis in the yolk sac. Disclosures: No relevant conflicts of interest to declare.

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Definitive Erythro-Myeloid Progenitors (EMP) Emerge in the Yolk Sac From Hemogenic Endothelium and Share Transcriptional Regulators with Adult Hematopoiesis

Blood ◽

10.1182/blood.v118.21.910.910 ◽

2011 ◽

Vol 118 (21) ◽

pp. 910-910

Author(s):

Kathleen E McGrath ◽

Katherine H Fegan ◽

Jenna M Frame ◽

Paul D Kingsley ◽

James Palis

Keyword(s):

Bone Marrow ◽

Mast Cell ◽

Mast Cells ◽

Fetal Liver ◽

Transcriptional Regulators ◽

Yolk Sac ◽

Erythroid Cells ◽

Hemogenic Endothelium ◽

Cell Potential ◽

Myeloid Progenitors

Abstract Abstract 910 In the mammalian embryo, hematopoietic stem cells (HSC) emerge from vascular beds and colonize the fetal liver. The first HSC are found in the murine fetal liver by embryonic day 12.5 (E12.5). However blood function is required before HSC have formed, and two earlier waves of hematopoietic potential arise to sustain the embryo. The first wave of hematopoietic progenitors are formed in the yolk sac between E7.25 and E8.5 and are termed “primitive” because, along with megakaryocyte and macrophage potential, they differentiate into primitive erythroid cells that mature in the circulation and express embryonic globins. A second lineage of hematopoietic potential has been characterized in the murine and human yolk sac as well as in zebrafish. These cells have been termed “EMP”, erythro-myeloid progenitors, that generate definitive erythroid and myeloid lineages, including granulocytes. We find that EMP emerging in the mouse embryo express many of the markers associated with HSC emergence from hemogenic endothelium. At early stages of their emergence (E8.5), EMP express not only kit and CD41, but also VE-cadherin, CD31 and CD34. A day later, EMP constitute a robust population of over 1,000 cells in the yolk sac and display diminished expression of VE-cadherin and increased expression of CD45. However, unlike HSC, EMP do not express Sca1, but do express the myeloid progenitor marker CD16/32 (low affinity FCgamma receptor II/III). Like CD45, CD16/32 is expressed on a subset of the CD41+/kit+ cells at E8.5. Colony forming assays confirm that EMP potential is found in both CD16/32 positive and negative CD41+/kit+ cells. By E9.5, over 90% of CD41+/kit+ cells also express CD16/32 and all of the hematopoietic colony-forming potential at E9.5 is found in this triple-positive population. The transition from endothelial-associated to hematopoietic-associated genes suggests that EMP may emerge from hemogenic endothelial intermediates. Between E9.5 and E11.5, cells with the EMP immunophenotype are found in the bloodstream and become concentrated in the liver, where evidence of very robust erythro-myeloid differentiation precedes HSC colonization. In culture, EMP rapidly expand, dividing twice daily, and within 6 days generate predominately erythroid cells as well as smaller numbers of megakaryocyte, macrophage and mast cells, but only rare granulocytes. This is in contrast to lin-/kit+ ScaI- bone marrow progenitors grown in the same culture conditions that generate predominately myeloid cells, particularly granulocytes, and rarely mast cells. In agreement with data in the zebrafish, we also do not see evidence of lymphoid potential or RAG2 expression in EMP cultures or expression of the lymphoid markers Flt3 and IL7 receptor on the cell surface of EMP. In order to better understand the lineage potential of EMP, we examined the expression of known transcriptional regulators of bone marrow hematopoiesis. In the adult, relative levels of GATA1 versus Pu.1 are proposed to determine fate between megakaryocyte/erythroid progenitors (MEP- GATA1 hi) and granulocyte/macrophage progenitors (GMP-Pu.1 hi). Consistent with their erythro-myeloid potential, EMP expressed both GATA1 and Pu.1 at intermediate levels compared to adult marrow-derived MEP and GMP. In the adult, Gfi-1 and C/EBPalpha are both proposed to upregulate granulocyte versus macrophage differentiation. We found lower levels of these regulators in EMP compared to GMP, consistent with EMP cultures generating small numbers of granulocytes versus macrophages. In addition, the GATA1 expression within the Pu.1+ GMP is found to increase mast cell potential and, thus, the high GATA1 and Pu.1 expression in EMP may account for their high mast cell potential. Taken together, these data suggest that, like HSC, EMP emerge from hemogenic endothelium and their erythro-myeloid potential is governed by the action of shared regulatory networks. However, the transcription factors and markers are present in the EMP in unique combinations consistent with their specific role in providing a transient initial wave of definitive hematopoiesis in the embryo. Disclosures: No relevant conflicts of interest to declare.

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Tissue-resident macrophages originate from yolk-sac-derived erythro-myeloid progenitors

Nature ◽

10.1038/nature13989 ◽

2014 ◽

Vol 518 (7540) ◽

pp. 547-551 ◽

Cited By ~ 897

Author(s):

Elisa Gomez Perdiguero ◽

Kay Klapproth ◽

Christian Schulz ◽

Katrin Busch ◽

Emanuele Azzoni ◽

...

Keyword(s):

Yolk Sac ◽

Myeloid Progenitors

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Ezh2 is essential for the generation of functional yolk sac derived erythro-myeloid progenitors

Nature Communications ◽

10.1038/s41467-021-27140-8 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Wen Hao Neo ◽

Yiran Meng ◽

Alba Rodriguez-Meira ◽

Muhammad Z. H. Fadlullah ◽

Christopher A. G. Booth ◽

...

Keyword(s):

Wnt Signaling ◽

Epigenetic Regulation ◽

Yolk Sac ◽

Specific Role ◽

Primary Reason ◽

Erythroid Cells ◽

Hemogenic Endothelium ◽

Epigenetic Regulator ◽

Myeloid Progenitors

AbstractYolk sac (YS) hematopoiesis is critical for the survival of the embryo and a major source of tissue-resident macrophages that persist into adulthood. Yet, the transcriptional and epigenetic regulation of YS hematopoiesis remains poorly characterized. Here we report that the epigenetic regulator Ezh2 is essential for YS hematopoiesis but dispensable for subsequent aorta–gonad–mesonephros (AGM) blood development. Loss of EZH2 activity in hemogenic endothelium (HE) leads to the generation of phenotypically intact but functionally deficient erythro-myeloid progenitors (EMPs), while the generation of primitive erythroid cells is not affected. EZH2 activity is critical for the generation of functional EMPs at the onset of the endothelial-to-hematopoietic transition but subsequently dispensable. We identify a lack of Wnt signaling downregulation as the primary reason for the production of non-functional EMPs. Together, our findings demonstrate a critical and stage-specific role of Ezh2 in modulating Wnt signaling during the generation of EMPs from YS HE.

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Faculty Opinions recommendation of Tissue-resident macrophages originate from yolk-sac-derived erythro-myeloid progenitors.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.725255026.793553574 ◽

2018 ◽

Author(s):

John Grainger

Keyword(s):

Yolk Sac ◽

Myeloid Progenitors

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HSC-Independent Yolk Sac Progenitors Bear Hallmarks of JMML in a PTPN11D61Y Mouse Model

Blood ◽

10.1182/blood.v124.21.3236.3236 ◽

2014 ◽

Vol 124 (21) ◽

pp. 3236-3236

Author(s):

Stefan Pasichnyk Tarnawsky ◽

Momoko Yoshimoto ◽

Gordon Chan ◽

Benjamin Neel ◽

Rebecca J. Chan ◽

...

Keyword(s):

Mouse Model ◽

Fetal Liver ◽

Yolk Sac ◽

T Test ◽

Erk Signaling ◽

Erk Pathway ◽

Gm Csf ◽

Student’S T ◽

Student’S T Test ◽

Myeloid Progenitors

Abstract Juvenile Myelomonocytic Leukemia is the most common pediatric myeloproliferative neoplasm (MPN). JMML is characterized by myeloid populations with mutually-exclusive mutations in Ras-Erk signaling genes, most commonly PTPN11, which confer growth hypersensitivity to GM-CSF. JMML is notable among pediatric MPNs as being refractory to chemotherapy and having a 50% relapse rate following allogeneic hematopoietic stem cell (HSC) transplantation. As such, there is an urgent need for novel JMML therapies. The recent discovery of yolk sac myeloid lineages that persist into adulthood independently of bone marrow HSC contributions suggests a mechanism for JMML relapse following HSC transplantation. In this study, we sought to determine whether yolk sac HSC-independent myeloid progenitors bear hallmarks of MPN in a mouse model of JMML. Using the Vav1 promoter-directed Cre recombinase, we generated a mouse model of JMML that expresses the PTPN11D61Y gain of function mutation in all waves of embryonic and adult hematopoiesis, including yolk sac myeloid progenitors that emerge prior to and independently from HSCs. PTPN11D61Y/+; VavCre+ mice are viable, born at expected Mendelian ratios, and develop peripheral blood monocytosis as early as 4 weeks of age. Given this early onset, we hypothesized MPN may develop in these mice during embryonic development. E14.5 fetal liver progenitors from PTPN11D61Y/+; VavCre+ embryos displayed marked GM-CSF hypersensitivity in methylcellulose colony forming assays (Figure-1A), possessed hyperactive Ras-Erk pathway signaling (Figure-2), and had a skewed progenitor distribution with a greater proportion of megakaryocyte-erythroid progenitors (63.5% vs. 50.1%, p<0.01) and fewer common myeloid progenitors (9.2% vs. 19.3%, p<0.01) than littermate controls. Since the E14.5 fetal liver contains both HSC-dependent and HSC-independent myeloid progenitors, we repeated these experiments using E9.5 yolk sac samples to restrict our analysis to HSC-independent hematopoiesis. Compared to littermate controls, PTPN11D61Y/+; VavCre+ E9.5 embryos had no difference in overall number of yolk sac myeloid progenitors (Ter119-; cKit+, CD41Dim), and in the number and distribution of CFU-E/GM/GEMM colonies in methylcellulose assay. However, PTPN11D61Y/+; VavCre+ yolk sac progenitors demonstrated marked GM-CSF hypersensitivity in colony forming assay (Figure-1B) as well as hyperactive Ras-Erk signalling by western blot analysis (Figure-2) and intracellular flow cytometry with antibodies against pSTAT5 and pERK (Figure-3). We have demonstrated that HSC-independent myeloid lineages from the murine yolk sac possess GM-CSF hypersensitivity and Ras-Erk pathway hyperactivation in a mouse model of JMML. These findings suggest that HSC-independent hematopoietic populations may be involved in the development of JMML. Our study highlights the need to further assess the role of bone marrow-independent myeloid lineages in pediatric MPN, and to identify innovative therapies that can specifically target HSC-independent hematopoietic lineages. Figure 1. Embryonic myeloid progenitors from PTPN11D61Y/+; VavCre+ embryos demonstrate GM-CSF growth hypersensitivity. A) E14.5 fetal liver mononuclear cells (n=7) or B) E9.5 yolk sac cells (n=8) were plated in methylcellulose colony forming assays and colonies were counted 7 days later. * p<0.05; ** p<0.001 by two-tailed Student’s t-test. Figure 1. Embryonic myeloid progenitors from PTPN11D61Y/+; VavCre+ embryos demonstrate GM-CSF growth hypersensitivity. A) E14.5 fetal liver mononuclear cells (n=7) or B) E9.5 yolk sac cells (n=8) were plated in methylcellulose colony forming assays and colonies were counted 7 days later. * p<0.05; ** p<0.001 by two-tailed Student’s t-test. Figure 2. Western blot analysis demonstrates hyperactive Ras-Erk signaling in E14.5 fetal liver and E9.5 yolk sac progenitors from PTPN11D61Y/+; VavCre+ embryos. Cultured progenitors were starved overnight and stimulated with GM-CSF for 60min prior to protein extraction. p, phosphorylated protein; t, total protein; WT, wild type. Figure 2. Western blot analysis demonstrates hyperactive Ras-Erk signaling in E14.5 fetal liver and E9.5 yolk sac progenitors from PTPN11D61Y/+; VavCre+ embryos. Cultured progenitors were starved overnight and stimulated with GM-CSF for 60min prior to protein extraction. p, phosphorylated protein; t, total protein; WT, wild type. Figure 3. Yolk Sac Myeloid Progenitors from PTPN11D61Y/+; VavCre+ embryos demonstrate Ras-Erk pathway hypersensitivity at baseline and following GM-CSF stimulation. Cultured yolk sac progenitors were stimulated for 30min with 5ng/ml of GM-CSF, processed for intracellular flow cytometry, and stained with the indicated fluorescent antibody. Histograms display representative median fluorescence intensity among CD45+ cells in each sample (n=3). * p<0.05 by two-tailed Student’s t-test. ADDIN EN.REFLIST Figure 3. Yolk Sac Myeloid Progenitors from PTPN11D61Y/+; VavCre+ embryos demonstrate Ras-Erk pathway hypersensitivity at baseline and following GM-CSF stimulation. Cultured yolk sac progenitors were stimulated for 30min with 5ng/ml of GM-CSF, processed for intracellular flow cytometry, and stained with the indicated fluorescent antibody. Histograms display representative median fluorescence intensity among CD45+ cells in each sample (n=3). * p<0.05 by two-tailed Student’s t-test. ADDIN EN.REFLIST Disclosures No relevant conflicts of interest to declare.

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Definitive Erythro-Myeloid Progenitors (EMPs) Emerge in the Myb-/- Embryo and Retain the Capacity to Differentiate into Macrophages

Blood ◽

10.1182/blood.v126.23.2372.2372 ◽

2015 ◽

Vol 126 (23) ◽

pp. 2372-2372

Author(s):

Jenna M. Frame ◽

Katherine H. Fegan ◽

Seana C. Catherman ◽

Joanna Tober ◽

Anne D. Koniski ◽

...

Keyword(s):

Conflicts Of Interest ◽

Fetal Liver ◽

Yolk Sac ◽

Mouse Embryos ◽

Zebrafish Embryos ◽

Normal Numbers ◽

Hematopoietic Progenitors ◽

Hematopoietic Stem ◽

Myeloid Progenitors

Abstract In the adult, the proto-oncogene Myb critically regulates both the maintenance of hematopoiesis and the differentiation of several hematopoietic lineages. Myb-/- mouse embryos die by embryonic day (E) 15 with severe anemia due to the absence of definitive erythropoiesis (Mucenski et al., Cell, 1991). Similarly, zebrafish embryos lacking myb do not express adult globin genes, have a reduction in other mature hematopoietic lineages by 48 hours post fertilization, and maintain a bloodless phenotype through adulthood (Soza-Ried et al., PNAS, 2010). These and other data have led to the concept that Myb-/- embryos entirely lack definitive hematopoiesis. In both mouse and zebrafish embryos, the first definitive hematopoietic potential arises as a hematopoietic stem cell (HSC)-independent wave of erythro-myeloid progenitors (EMPs). EMPs emerge in the murine yolk sac beginning at E8.25, partially overlapping with an earlier wave of primitive hematopoietic progenitors. We previously demonstrated that EMPs are multipotent progenitors, and are the major source of definitive erythroid potential in the early fetal liver, prior to the colonization of adult-repopulating HSCs (McGrath et al., Blood, 2011). Recently, we identified a unique cell surface phenotype that facilitates the prospective isolation of murine definitive EMPs, distinguishing them from primitive hematopoietic progenitors and maturing populations of megakaryocytes and macrophages in the yolk sac (McGrath et al., Cell Reports, 2015). We detected expression of Myb in sorted EMPs, suggesting that Myb may regulate the emergence and/or differentiation of EMPs. We tested this hypothesis by assessing the emergence, hematopoietic potential and expansion capacity of EMPs, compared with other maturing primitive hematopoietic lineages, in Myb-/- mouse embryos. Consistent with the proposed Myb-independence of the earlier wave of primitive progenitors, we observed normal numbers of maturing macrophages in E9.5 Myb-/- yolk sacs. Interestingly, E9.5 Myb-/- yolk sacs also contained normal numbers of immunophenotypic EMPs. These EMPs were present in hemogenic endothelial-derived clusters expressing Runx1, similar to littermate controls, suggesting that Myb is dispensable for EMP emergence from hemogenic endothelium. We next assessed the differentiation capability of Myb-/- EMPs in vitro. E9.5 Myb-/- yolk sacs lacked high proliferative colony-forming potential (HPP-CFC), a hallmark of immature definitive hematopoietic progenitors. In addition, both definitive erythroid and granulocyte colony-forming potential were absent in methylcellulose cultures of sorted Myb-/- EMPs, in contrast to littermate controls. Surprisingly, however, sorted Myb-/- EMPs gave rise to macrophage progenitors in colony-forming assays, and CD11b+ F4/80+ macrophages in differentiation cultures. These data indicate that Myb is not required for the differentiation of primary definitive EMPs into macrophages. Analysis of Myb-/- fetal liversalso confirmed the presence of F4/80+ macrophages. While these fetal liver macrophages have been previously proposed to be of primitive hematopoietic origin, our data raise the possibility that they may also be derived from EMPs. Further analysis of in vitro differentiation cultures demonstrated an inability of sorted Myb-/- EMPs to proliferate when compared with normal littermates, although these cultures still generated small numbers of macrophages. It is not yet clear whether this reduction in proliferation is due solely to the loss of differentiation of multiple hematopoietic lineages, or is also due to defective maintenance or expansion of EMPs. However, consistent with a role for Myb in continued emergence and/or expansion of EMPs, we observed a reduction in the total number of EMPs by E10.5 in yolk sacs of Myb-/- embryos compared with normal littermates. Taken together, these data indicate that Myb is a critical regulator not only of HSCs, but also of HSC-independent definitive hematopoietic progenitors (EMPs). While Myb is dispensable for the initial emergence of EMPs, it is required for their subsequent differentiation into erythroid and granulocyte lineages. Surprisingly, the persistence of EMPs, while reduced, may provide a source of definitive macrophages in Myb-/- embryos. Disclosures No relevant conflicts of interest to declare.

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Fetal Hematopoiesis is Driven by Privileged Expansion and Differentiation of Yolk Sac-Derived Erythro-Myeloid Progenitors

10.21203/rs.3.rs-1002760/v1 ◽

2021 ◽

Author(s):

Elisa Gomez Perdiguero ◽

Laina Freyer ◽

Lorea Iturri ◽

Anne Biton ◽

Alina Sommer ◽

...

Keyword(s):

Fetal Liver ◽

Perinatal Period ◽

Yolk Sac ◽

Cell Of Origin ◽

Blood Disorders ◽

Hematopoietic Stem ◽

Molecular Features ◽

Neonatal Immunity ◽

Fetal Hematopoiesis ◽

Myeloid Progenitors

Abstract Most blood and immune cells are produced by Hematopoietic Stem Cells (HSC) throughout life. However, several tissue resident immune populations can only be generated from developmentally restricted progenitors. This questions to what extent fetal HSC differentiate in utero, implicating an essential role for HSC-independent progenitors in supporting embryonic viability and innate immunity in the perinatal period. Among them, Erythro-Myeloid Progenitors (EMP) emerge from the extraembryonic yolk sac prior to HSC and their progeny (resident macrophages and skin mast cells) persist in adulthood. Here, we showed that HSC contributed minimally to fetal myelopoiesis as we exposed a developmentally-restricted privilege for erythro-myeloid differentiation from EMP in the fetal liver. EMP-derived myeloid progenitors displayed distinct molecular features and were functionally inequivalent to fetal HSC-derived counterparts. These findings inform future studies of HSC-dependent and HSC-independent hematopoiesis in view of neonatal immunity and pediatric blood disorders for which the cell of origin is poorly understood.

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Origins and unconventional behavior of neutrophils in developing zebrafish

Blood ◽

10.1182/blood-2007-06-095398 ◽

2008 ◽

Vol 111 (1) ◽

pp. 132-141 ◽

Cited By ~ 220

Author(s):

Dorothée Le Guyader ◽

Michael J. Redd ◽

Emma Colucci-Guyon ◽

Emi Murayama ◽

Karima Kissa ◽

...

Keyword(s):

Danio Rerio ◽

Yolk Sac ◽

Neutrophilic Granulocytes ◽

Cell Type ◽

Antimicrobial Defense ◽

Embryonic Tissues ◽

Vertebrate Embryos ◽

Myeloid Progenitors

The first leukocytes that arise in the development of vertebrate embryos are the primitive macrophages, which differentiate in the yolk sac and then quickly invade embryonic tissues. These macrophages have been considered to constitute a separate lineage, giving rise to no other cell type. Using an in vivo photoactivatable cell tracer in the transparent zebrafish (Danio rerio) embryo, we demonstrated that this lineage also gave rise to an equal or higher number of neutrophilic granulocytes. We were surprised to find that the differentiation of these primitive neutrophils occurs only after primitive myeloid progenitors have dispersed in the tissues. By 2 days after fertilization, these neutrophils have become the major leukocyte type found wandering in the epidermis and mesenchyme. Like the primitive macrophages, all primitive and larval neutrophils express PU.1 and L-plastin and they are highly attracted to local infections, yet only a small fraction of them phagocytose microbes, and to a much lesser extent per cell than the macrophages. They are also attracted to variously stressed or malformed tissues, suggesting a wider role than antimicrobial defense.

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