Yolk Sac Erythro-myeloid Progenitors Research Articles

Single-cell RNA sequencing-guided fate-mapping toolkit delineates the contribution of yolk sac erythro-myeloid progenitors

Erythro-myeloid progenitors of the yolk sac that originates during early embryo development has been suggested to generate tissue-resident macrophage, mast cell, and even endothelial cell populations from fetal to adult stages. However, the heterogeneity of erythro-myeloid progenitors (EMPs) is not well characterized. Here, we adapt single-cell RNA sequencing to dissect the heterogeneity of EMPs and establish several fate-mapping tools for each EMP subset to trace the contributions of different EMP subsets. We identify two primitive and one definitive EMP subsets from the yolk sac. In addition, we find that primitive EMPs are decoupled from definitive EMPs. Furthermore, we confirm that primitive and definitive EMPs give rise to microglia and other tissue-resident macrophages, respectively. In contrast, only Kit+ Csf1r- primitive EMPs generate endothelial cells transiently during early embryo development. Overall, our results delineate the contribution of yolk sac EMPs more clearly based on the single-cell RNA sequencing (scRNA-seq)-guided fate-mapping toolkit.

Megakaryocyte production is sustained by direct differentiation from erythromyeloid progenitors in the yolk sac until midgestation

SummaryThe extra-embryonic yolk sac contains the first definitive multipotent hematopoietic cells, denominated erythromyeloid progenitors. They originate in situ prior to the emergence of hematopoietic stem cells and give rise to erythroid, monocytes, granulocytes, mast cells and macrophages, the latter in a Myb transcription factor-independent manner. We uncovered here the heterogeneity of yolk sac erythromyeloid progenitors, at the single cell level, and discriminated multipotent from committed progenitors, prior to fetal liver colonization. We identified two temporally distinct megakaryocyte differentiation pathways. The first occurs in the yolk sac, bypasses intermediate bipotent megakaryocyte-erythroid progenitors and, similar to the differentiation of macrophages, is Myb-independent. By contrast, the second originates later, from Myb-dependent bipotent progenitors expressing Csf2rb and colonize the fetal liver, where they give rise to megakaryocytes and to large numbers of erythrocytes. Understanding megakaryocyte development is crucial as they play key functions during vascular development, in particular in separating blood and lymphatic networks.

Microglia Development and Maturation and Its Implications for Induction of Microglia-Like Cells from Human iPSCs.

Microglia are resident immune cells of the central nervous system and play critical roles during the development, homeostasis, and pathologies of the brain. Originated from yolk sac erythromyeloid progenitors, microglia immigrate into the embryonic brain parenchyma to undergo final postnatal differentiation and maturation driven by distinct chemokines, cytokines, and growth factors. Among them, TGFβ1 is an important regulator of microglial functions, mediating homeostasis, anti-inflammation, and triggering the expression of microglial homeostatic signature genes. Since microglia studies are mainly based on rodent cells and the isolation of homeostatic microglia from human tissue is challenging, human-induced pluripotent stem cells have been successfully differentiated into microglia-like cells recently. However, employed differentiation protocols strongly vary regarding used cytokines and growth factors, culture conditions, time span, and cell yield. Moreover, the incomplete differentiation of human microglia can hamper the similarity to primary human microglia and dramatically influence the outcome of follow-up studies with these differentiated cells. This review summarizes the current knowledge of the molecular mechanisms driving rodent microglia differentiation in vivo, further compares published differentiation protocols, and highlights the potential of TGFβ as an essential maturation factor.

Yolk sac, but not hematopoietic stem cell-derived progenitors, sustain erythropoiesis throughout murine embryonic life.

In the embryo, the first hematopoietic cells derive from the yolk sac and are thought to be rapidly replaced by the progeny of hematopoietic stem cells. We used three lineage-tracing mouse models to show that, contrary to what was previously assumed, hematopoietic stem cells do not contribute significantly to erythrocyte production up until birth. Lineage tracing of yolk sac erythromyeloid progenitors, which generate tissue resident macrophages, identified highly proliferative erythroid progenitors that rapidly differentiate after intra-embryonic injection, persisting as the major contributors to the embryonic erythroid compartment. We show that erythrocyte progenitors of yolk sac origin require 10-fold lower concentrations of erythropoietin than their hematopoietic stem cell-derived counterparts for efficient erythrocyte production. We propose that, in a low erythropoietin environment in the fetal liver, yolk sac-derived erythrocyte progenitors efficiently outcompete hematopoietic stem cell progeny, which fails to generate megakaryocyte and erythrocyte progenitors.

Two populations of self-maintaining monocyte-independent macrophages exist in adult epididymis and testis

Macrophages are the principal immune cells of the epididymis and testis, but their origins, heterogeneity, development, and maintenance are not well understood. Here, we describe distinct populations of epididymal and testicular macrophages that display an organ-specific cellular identity. Combining in vivo fate-mapping, chimeric and parabiotic mouse models with in-depth cellular analyses, we found that CD64hiMHCIIlo and CD64loMHCIIhi macrophage populations of epididymis and testis arise sequentially from yolk sac erythro-myeloid progenitors, embryonic hematopoiesis, and nascent neonatal monocytes. While monocytes were the major developmental source of both epididymal and testicular macrophages, both populations self-maintain in the steady-state independent of bone marrow hematopoietic precursors. However, after radiation-induced macrophage ablation or during infection, bone marrow-derived circulating monocytes are recruited to the epididymis and testis, giving rise to inflammatory macrophages that promote tissue damage. These results define the layered ontogeny, maintenance and inflammatory response of macrophage populations in the male reproductive organs.

The human bone marrow harbors a CD45- CD11B+ cell progenitor permitting rapid microglia-like cell derivative approaches.

Microglia, the immune sentinel of the central nervous system (CNS), are generated from yolk sac erythromyeloid progenitors that populate the developing CNS. Interestingly, a specific type of bone marrow‐derived monocyte is able to express a yolk sac microglial signature and populate CNS in disease. Here we have examined human bone marrow (hBM) in an attempt to identify novel cell sources for generating microglia‐like cells to use in cell‐based therapies and in vitro modeling. We demonstrate that hBM stroma harbors a progenitor cell that we name stromal microglial progenitor (STR‐MP). STR‐MP single‐cell gene analysis revealed the expression of the consensus genetic microglial signature and microglial‐specific genes present in development and CNS pathologies. STR‐MPs can be expanded and generate microglia‐like cells in vitro, which we name stromal microglia (STR‐M). STR‐M cells show phagocytic ability, classically activate, and survive and phagocyte in human brain tissue. Thus, our results reveal that hBM harbors a source of microglia‐like precursors that can be used in patient‐centered fast derivative approaches.

Two Sequential and Independent Pathways of Erythromyeloid Progenitor Commitment in Their Niche of Emergence

The extra-embryonic yolk sac contains the first definitive multipotent hematopoietic cells, denominated erythromyeloid progenitors. They originate in situ prior to the emergence of hematopoietic stem cells and give rise to erythroid, monocyte/granulocytes, mast cells and macrophages, the latter in a Myb-independent manner. We uncovered here the heterogeneity of yolk sac erythromyeloid progenitors, at the single cell level, and discriminated multipotent from committed progenitors, prior to fetal liver colonization. We identified two temporally distinct megakaryocyte differentiation pathways. The first occurs in the yolk sac, bypasses intermediate bipotent megakaryocyte-erythroid progenitors and, similar to the differentiation of macrophages, is Myb-independent. By contrast, the second originates later, from bipotent progenitors and is absent in Myb-deficient embryos. We identified Csf2rb as a novel marker of these Myb-dependent megakaryocyte-erythroid bipotent progenitors that colonize the fetal liver, where they give rise to megakaryocytes and to large numbers of erythrocytes.

Ontogeny of arterial macrophages defines their functions in homeostasis and inflammation

Arterial macrophages have different developmental origins, but the association of macrophage ontogeny with their phenotypes and functions in adulthood is still unclear. Here, we combine macrophage fate-mapping analysis with single-cell RNA sequencing to establish their cellular identity during homeostasis, and in response to angiotensin-II (AngII)-induced arterial inflammation. Yolk sac erythro-myeloid progenitors (EMP) contribute substantially to adventitial macrophages and give rise to a defined cluster of resident immune cells with homeostatic functions that is stable in adult mice, but declines in numbers during ageing and is not replenished by bone marrow (BM)-derived macrophages. In response to AngII inflammation, increase in adventitial macrophages is driven by recruitment of BM monocytes, while EMP-derived macrophages proliferate locally and provide a distinct transcriptional response that is linked to tissue regeneration. Our findings thus contribute to the understanding of macrophage heterogeneity, and associate macrophage ontogeny with distinct functions in health and disease.

3027 – HSC-INDEPENDENT EMP CONTAIN ERYTHROID/MEGAKARYOCYTE AND INNATE LYMPHOID/MYELOID LINEAGE HETEROGENEITY PRIOR TO SEEDING THE FETAL LIVER

Hematopoietic ontogeny unfolds in two broad programs- a complex hematopoietic stem cell (HSC)-independent program followed by an HSC-dependent program. We have previously determined that HSC-independent, multi-lineage "erythro-myeloid" progenitors (EMP) emerge from hemogenic endothelium in the yolk sac of the murine embryo and engraft the fetal liver to generate the first definitive erythroid, granulocyte, monocyte, and NK cells prior to the establishment of HSC-derived hematopoiesis. Analysis of single cell transcriptomic data from E9.5 EMP and from hematopoietic cells in the E12.5 fetal liver provides a unique opportunity to tie predictions of progenitor identity to subsequent differentiation states. We analyzed 2,959 kit+CD41+CD16/32+ yolk sac EMP and 7,495 fetal liver hematopoietic cells pooled from 30 E9.5 and 3 E12.5 embryos, respectively, using the 10X Genomics and inDrops platforms. Dimensionality reduction coupled with graph-based clustering and analysis of differentially expressed marker genes allowed us to presumptively identify erythroid-, megakaryocyte-, macrophage-, and myeloid-biased clusters both in EMP and in the E12.5 fetal liver. Functional colony-forming assays confirm the existence of primary myeloid- and erythroid-biased EMP in E9.5 embryos. E9.5 EMP also contain a cluster of innate lymphoid-biased progenitors, consistent with the recently identified NK cell lineage potential of EMP. Integration of EMP and fetal liver datasets further supported the concept that EMP consist of a surprising, early complexity of lineage-biased progenitors, whose differentiation continue in the fetal liver. Interestingly, EMP also contained a cluster of hemogenic endothelial-biased progenitors that were not found in the fetal liver, and which serve as a source of diverging erythroid/megakaryocyte and innate lymphoid/myeloid pseudotime trajectories. Taken together, our data support the concept that EMP emerge from hemogenic endothelium and begin to differentiate into multiple lineage-biased progenitors before engrafting the fetal liver to complete their differentiation along erythroid/megakaryocytic and innate immune cell paths to meet the needs of the developing embryo prior to the establishment of HSC-derived blood cell production.

Histone Deacetylase 3 regulates the ontogeny and maintenance of tissue-resident macrophages

Abstract Tissue-resident macrophages (TRMs) are essential for tissue homeostasis, immune-surveillance, and resolution of inflammation, and contribute to the pathogenesis of cancer, autoimmune, and inflammatory diseases. Most adult TRMs are derived from embryonic yolk sac erythro-myeloid progenitors and self-maintain independently from adult hematopoiesis. A few genes have been recently identified to be required for TRM tissue-specific development. However, the master regulator that centrally controls TRM ontogeny remains a mystery. Using a fate-mapping strategy, here we report that conditional deletion of histone deacetylase 3 (HDAC3) in mice essentially abrogates epidermal Langerhans cells and TRMs in dermis, lung, kidney, and heart at embryonic stage and after birth. In contrast, HDAC3 deletion promotes liver TRM ontogeny, while without significant effects on brain TRM microglia. Using lung TRM alveolar macrophage (AM) as a model, we further indicate that HDAC3 controls metabolism and cell death, at least by directly targeting PPAR-γ during AM embryonic development. Moreover, single-cell RNA sequencing identifies four distinct AM sub-clusters and lack of HDAC3 dysregulates AM functions in a cluster-specific manner. Overall, this is the first study to demonstrate that HDAC3 serves as a key epigenetic regulator of embryonic TRM ontogeny and maintenance in a spatial- and tissue-specific manner. Not only do these findings provide a new conceptual framework for our understanding of epigenetic regulation in TRM ontogeny and homeostasis, they also shed light on HDAC3 as a potential therapeutic target for TRM-based intervention in cancer and autoimmune diseases.

Abstract A127: Role of macrophage subsets in liver metastasis

Abstract Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal disease and frequently metastasize to the liver. The presence of macrophages (F4/80+ cells or CD68+ cells) is reported to correlate with tumor progression and metastasis in pancreatic cancer. Emerging evidence has revised the traditional concept of macrophage ontogeny and made it increasingly apparent that there exists functional diversity between resident macrophages originating from yolk sac erythro-myeloid progenitors (EMPs) and hematopoietic stem cells (HSCs)-derived macrophages. Sophisticated therapeutic intervention requires in-depth understanding of these macrophage subsets in the metastasis microenvironment. However, the role and heterogeneity of macrophages in tumor development, in particular in PDAC liver metastasis, remains poorly understood. Here, we utilized murine PDAC model to inidcate that Kupffer cells and tumor associated macrophages (TAMs) are two developmentally and functionally distinct subsets of macrophages. Removal of Kupffer cells by depletion of Id3 in macrophage progenitors or CSF1R blockade increases tumor burden. These findings reveal that Kupffer cells play a key role in restraining PDAC liver metastasis and show the necessity to understand macrophage origin when developing therapeutics for cancer treatment. Citation Format: Zihou Deng, Frederic Geissmann. Role of macrophage subsets in liver metastasis [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A127.

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

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). 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. 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.

Neuroscience: From embryo mutation to adult degeneration.

Mutations in embryonic blood-cell precursors called erythro-myeloid progenitors cause abnormal activation of their descendants — immune cells called microglia — leading to neurodegeneration in mice. See Letter p.389 Microglia—immune cells in the brain—derive from yolk-sac erythro-myeloid progenitors (EMPs), which are distinct from haematopoietic stem cells (HSCs). Frederic Geissmann and colleagues show that mosaic expression of a mutant BRAF, which activates the RAS–MEK–ERK pathway and causes tumours when expressed in HSCs, results in expansion of tissue-resident macrophages and late-onset neurodegeneration when expressed in EMPs. They show in a mouse model that neurobehavioural abnormalities, astrogliosis, deposition of amyloid precursor protein, synaptic loss and neuronal death are driven by ERK-activated microglia and can be prevented by BRAF inhibition. These results show that, in mice, activation of the MAP kinase pathway in microglia can cause neurodegeneration. These findings may explain the neurodegeneration observed in patients with histiocytosis—disorders of myeloid cell expansion associated with somatic mutations in the RAS–MEK–ERK pathway, such as the BRAF mutation studied here.

A somatic mutation in erythro-myeloid progenitors causes neurodegenerative disease.

The pathophysiology of neurodegenerative diseases is poorly understood, and therapeutic options are few. Neurodegenerative diseases are hallmarked by progressive neuronal dysfunction and loss, associated with chronic glial activation1. Whether microglial activation, which is viewed in general as a secondary process, is detrimental or protective in neurodegeneration remains unclear1–8. Late-onset neurodegenerative disease observed in patients with histiocytoses9–12, which are clonal myeloid diseases associated with somatic mutations in the RAS/MEK/ERK pathway such as BRAFV600E 13–17, suggests a possible role of somatic mutations in myeloid cells in neurodegeneration. Yet expression of BRAFV600E in the hematopoietic stem cell (HSC) lineage causes leukemic and tumoral diseases but not neurodegenerative disease18,19. Microglia belong to a lineage of adult tissue-resident myeloid cells that develop during organogenesis from yolk sac erythro-myeloid progenitors (EMP) distinct from HSC20–23. We thus hypothesized that a somatic BRAFV600E mutation in the EMP lineage may cause neurodegeneration. Here we show that mosaic expression of BRAFV600E in EMP results in clonal expansion of tissue-resident macrophages and a severe late-onset neurodegenerative disorder, associated with accumulation of ERK-activated amoeboid microglia in mice, also observed in human histiocytoses patients. In the murine model, neurobehavioral signs, astrogliosis, amyloid precursor protein deposition, synaptic loss and neuronal death were driven by ERK-activated microglia and were preventable by BRAF inhibition. These results identify the fetal precursors of tissue-resident macrophages as a potential cell-of-origin for histiocytoses, and demonstrate in mice that a somatic mutation in the EMP lineage can drive late-onset neurodegeneration. Moreover, these data identify activation of the MAP kinase pathway in microglia as a cause of neurodegeneration, and provide opportunities for therapeutic intervention aimed at preventing neuronal death in neurodegenerative diseases.

Hemangioblast, hemogenic endothelium, and primitive versus definitive hematopoiesis.

The types of progenitors generated during the successive stages of embryonic blood development are now fairly well characterized. The terminology used to describe these waves, however,can still be confusing. What is truly primitive? What is uniquely definitive? These questions become even more challenging to answer when blood progenitors are derived invitro upon the differentiation of embryonic stem cells or induced pluripotent stem cells. Similarly, the cellular origin of these blood progenitors can be controversial. Are all blood cells, including the primitive wave, derived from hemogenic endothelium? Is the hemangioblast an invitro artifact or is this mesoderm entity also present in the developing embryo? Here, we discuss the latest findings and propose some consensus relating to these controversial issues.

The Ron Receptor Tyrosine Kinase Regulates Macrophage Heterogeneity and Plays a Protective Role in Diet-Induced Obesity, Atherosclerosis, and Hepatosteatosis.

Obesity is a chronic inflammatory disease mediated in large part by the activation of inflammatory macrophages. This chronic inflammation underlies a whole host of diseases including atherosclerosis, hepatic steatosis, insulin resistance, type 2 diabetes, and cancer, among others. Macrophages are generally classified as either inflammatory or alternatively activated. Some tissue-resident macrophages are derived from yolk sac erythromyeloid progenitors and fetal liver progenitors that seed tissues during embryogenesis and have the ability to repopulate through local proliferation. These macrophages tend to be anti-inflammatory in nature and are generally involved in tissue remodeling, repair, and homeostasis. Alternatively, during chronic inflammation induced by obesity, bone marrow monocyte-derived macrophages are recruited to inflamed tissues, where they produce proinflammatory cytokines and exacerbate inflammation. The extent to which these two populations of macrophages are plastic in their phenotype remains controversial. We have demonstrated previously that the Ron receptor tyrosine kinase is expressed on tissue-resident macrophages, where it limits inflammatory macrophage activation and promotes a repair phenotype. In this study, we demonstrate that Ron is expressed in a subpopulation of macrophages during chronic inflammation induced by obesity that exhibit a repair phenotype as determined by the expression of arginase 1. In addition, we demonstrate that the Ron receptor plays a protective role in the progression of diet-induced obesity, hepatosteatosis, and atherosclerosis. These results suggest that altering macrophage heterogeneity in vivo could have the potential to alleviate obesity-associated diseases.