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Evolutionarily conserved, “natural” (n)IgM is broadly reactive to both self and foreign antigens. Its selective deficiency leads to increases in autoimmune diseases and infections. In mice, nIgM is secreted independent of microbial exposure to bone marrow (BM) and spleen B-1 cell–derived plasma cells (B-1PC), generating the majority of nIgM, or by B-1 cells that remain non-terminally differentiated (B-1sec). Thus, it has been assumed that the nIgM repertoire is broadly reflective of the repertoire of body cavity B-1 cells. Studies here reveal, however, that B-1PC generate a distinct, oligoclonal nIgM repertoire, characterized by short CDR3 variable immunoglobulin heavy chain regions, 7–8 amino acids in length, some public, many arising from convergent rearrangements, while specificities previously associated with nIgM were generated by a population of IgM-secreting B-1 (B-1sec). BM, but not spleen B-1PC, or B-1sec also required the presence of TCRαβ CD4 T cells for their development from fetal precursors. Together, the studies identify important previously unknown characteristics of the nIgM pool.

Infection directly influences adult hematopoietic stem cell (HSC) function and differentiation, but much less is known about the fetal hematopoietic response to infection during pregnancy. Here, we investigated the fetal hematopoietic response to maternal infection withToxoplasma gondii(T. gondii), an intracellular parasite that elicits Type II IFNγ-mediated maternal immunity. The effects of maternal IFNγ on developing HSCs and the signals that mediate these interactions have not been investigated. Our investigation reveals that the fetal HSCs respond toT. gondiiinfection with virulence-dependent changes in proliferation, self-renewal potential, and lineage output. We demonstrate that maternal IFNγ crosses the fetal-maternal interface and is perceived by fetal HSCs. By comparing the effects of maternal IFNγ injection with maternalT. gondiiinfection, our observations reveal that IFNγ mimics aspects of the fetal HSC response to infection. Moreover, our data disentangle the role of infection-induced inflammatory cytokines in driving the expansion of downstream hematopoietic progenitors. Our findings illuminate that the fetal HSC response to prenatal infection is distinct from the adult HSC response to IFNγ-induced inflammation.

Hematopoietic stem cells (HSCs) are responsible for the generation and maintenance of pools of multipotent precursors that ultimately give rise to all fully differentiated blood and immune cells. Proper identification and isolation of HSCs for functional analysis has greatly facilitated our understanding of both normal and abnormal adult hematopoiesis. Whereas adult hematopoiesis in mice and humans is driven by quiescent HSCs that reside almost exclusively within the bone marrow (BM), developmental hematopoiesis is characterized by a series of transient progenitors driving waves of increasingly mature hematopoietic cell production that occur across multiple anatomical sites. These waves of hematopoietic cell production are also responsible for the generation of distinct immune cell populations during development that persist into adulthood and contribute uniquely to adult immunity. Therefore, methods to properly isolate and characterize fetal progenitors with high purity across development become increasingly important not only for defining developmental hematopoietic pathways, but also for understanding the contribution of developmental hematopoiesis to the immune system. Here, we describe and discuss methods and considerations for the isolation and characterization of HSCs from the fetal liver, the primary hematopoietic organ during fetal development.

Leukocytes and endothelial cells frequently cooperate to resolve inflammatory events. In most cases, these interactions are transient in nature and triggered by immunological insults. Here, we report that, in areas of disturbed blood flow, aortic endothelial cells permanently and intimately associate with a population of specialized macrophages. These macrophages are recruited at birth from the closing ductus arteriosus and share the luminal surface with the endothelium, becoming interwoven in the tunica intima. Anatomical changes that affect hemodynamics, such as in patent ductus arteriosus, alter macrophage seeding to coincide with regions of disturbed flow. Aortic resident macrophages expand in situ via direct cell renewal. Induced depletion of intimal macrophages leads to thrombin-mediated endothelial cell contraction, progressive fibrin accumulation and formation of microthrombi that, once dislodged, cause blockade of vessels in several organs. Together the findings reveal that intravascular resident macrophages are essential to regulate thrombin activity and clear fibrin deposits in regions of disturbed blood flow.

Adult hematopoietic stem and progenitor cells (HSPCs) respond directly to inflammation and infection, resulting in both acute and persistent changes to quiescence, mobilization, and differentiation. Here we show that fetal HSPCs respond to prenatal inflammation in utero, and that the fetal response shapes postnatal hematopoiesis and immunity. Heterogenous fetal HSPCs showed divergent responses to maternal immune activation (MIA), including changes in quiescence, expansion, and lineage-biased output. Single cell transcriptomic analysis of fetal HSPCs in response to MIA revealed specific upregulation of inflammatory gene profiles in discrete, transient HSC populations, that propagated expansion of lymphoid-biased progenitors. Beyond fetal development, MIA caused the inappropriate expansion and persistence of fetal lymphoid-biased progenitors postnatally, concomitant with increased cellularity and hyperresponsiveness of fetal-derived innate-like lymphocytes. Our investigation demonstrates how inflammation in utero can direct the trajectory of output and function of fetal-derived immune cells by reshaping fetal HSC establishment.

Adult hematopoietic stem and progenitor cells (HSPCs) respond directly to inflammation and infection, causing both acute and persistent changes to quiescence, mobilization, and differentiation. Here we show that murine fetal HSPCs respond to prenatal inflammation in utero and that the fetal response shapes postnatal hematopoiesis and immune cell function. Heterogeneous fetal HSPCs show divergent responses to maternal immune activation (MIA), including changes in quiescence, expansion, and lineage-biased output. Single-cell transcriptomic analysis of fetal HSPCs in response to MIA reveals specific upregulation of inflammatory gene profiles in discrete, transient hematopoietic stem cell (HSC) populations that propagate expansion of lymphoid-biased progenitors. Beyond fetal development, MIA causes the inappropriate expansion and persistence of fetal lymphoid-biased progenitors postnatally, concomitant with increased cellularity and hyperresponsiveness of fetal-derived innate-like lymphocytes. Our investigation demonstrates how inflammation in utero can direct the output and function of fetal-derived immune cells by reshaping fetal HSC establishment.

Therapeutic discovery for mantle cell lymphoma (MCL) has been hindered by a lack of preclinical mouse models that recapitulate human disease. In this issue, Pieters and colleagues (2021. J. Exp. Med.https://doi.org/10.1084/jem.20202280) establish a novel mouse model of MCL driven by overexpression of cyclin D2 and identify fetal-derived B1a cells as putative cell of origin for MCL.

Tissue-resident lymphoid cells (TLCs) span the spectrum of innate-to-adaptive immune function. Unlike traditional, circulating lymphocytes that are continuously generated from hematopoietic stem cells (HSCs), many TLCs are of fetal origin and poorly generated from adult HSCs. Here, we sought to further understand murine TLC development and the roles of Flk2 and IL7Rα, two cytokine receptors with known function in traditional lymphopoiesis. Using Flk2- and Il7r-Cre lineage tracing, we found that peritoneal B1a cells, splenic marginal zone B (MZB) cells, lung ILC2s and regulatory T cells (Tregs) were highly labeled. Despite high labeling, loss of Flk2 minimally affected the generation of these cells. In contrast, loss of IL7Rα, or combined deletion of Flk2 and IL7Rα, dramatically reduced the number of B1a cells, MZBs, ILC2s and Tregs, both in situ and upon transplantation, indicating an intrinsic and essential role for IL7Rα. Surprisingly, reciprocal transplants of wild-type HSCs showed that an IL7Rα−/− environment selectively impaired reconstitution of TLCs when compared with TLC numbers in situ. Taken together, our data defined Flk2- and IL7Rα-positive TLC differentiation paths, and revealed functional roles of Flk2 and IL7Rα in TLC establishment.

Traditional, adult-derived lymphocytes that circulate provide adaptive immunity to infection and pathogens. However, subsets of lymphoid cells are also found in non-lymphoid tissues and are called tissue-resident lymphoid cells (TLCs). TLCs encompass a wide array of cell types that span the spectrum of innate-to-adaptive immune function. Unlike traditional lymphocytes that are continuously generated from hematopoietic stem cells (HSCs), many TLCs are of fetal origin and poorly generated from adult HSCs. Here, we sought to understand the development of murine TLCs across multiple tissues and therefore probed the roles of Flk2 and IL7Rα, two cytokine receptors with known roles in traditional lymphopoiesis. Using Flk2- and Il7r-Cre lineage tracing models, we found that peritoneal B1a cells, splenic marginal zone B (MZB) cells, lung ILC2s and regulatory T cells (Tregs) were highly labeled in both models. Despite this high labeling, highly quantitative, in vivo functional approaches showed that the loss of Flk2 minimally affected the generation of these cells in situ. In contrast, the loss of IL7Rα, or combined deletion of Flk2 and IL7Rα, dramatically reduced the cell numbers of B1a cells, MZBs, ILC2s, and Tregs both in situ and upon transplantation, indicating an intrinsic and more essential role for IL7Rα. Surprisingly, reciprocal transplants of WT HSCs showed that an IL7Rα-/- environment selectively impaired reconstitution of TLCs when compared to TLC numbers in situ. Taken together, our data revealed functional roles of Flk2 and IL7Rα in the establishment of tissue-resident lymphoid cells.

Fetal and adult hematopoietic stem cells (HSCs) have distinct proliferation rates, lineage biases, gene expression profiles and gene dependencies. While these differences are widely recognized, it is not clear how the transition from fetal to adult identity is coordinated. Here we show that murine HSCs and committed hematopoietic progenitors (HPCs) undergo a gradual, rather than precipitous, transition from fetal to adult transcriptional states. The transition begins prior to birth and is punctuated by a late prenatal spike in type I interferon signaling that promotes perinatal HPC expansion and sensitizes progenitors to the leukemogenic FLT3(ITD) mutation. Most other changes in gene expression and enhancer activation are imprecisely timed and poorly coordinated. Thus, heterochronic enhancer elements, and their associated transcripts, are activated independently of one another rather than as part of a robust network. This simplifies the regulatory programs that guide neonatal HSC/HPC ontogeny, but it creates heterogeneity within these populations.

Summary Hematopoiesis is arguably one of the best understood stem cell systems; however, significant challenges remain to reach a consensus understanding of the lineage potential, heterogeneity, and relationships of hematopoietic stem and progenitor cell populations. To gain new insights, we performed quantitative analyses of mature cell production from hematopoietic stem cells (HSCs) and multiple hematopoietic progenitor populations. Assessment of the absolute numbers of mature cell types produced by each progenitor cell revealed a striking erythroid dominance of all myeloid-competent progenitors assessed, accompanied by strong platelet reconstitution. All populations with myeloid potential also produced robust numbers of red blood cells and platelets in vivo. Clonal analysis by single-cell transplantation and by spleen colony assays revealed that a significant fraction of HSCs and multipotent progenitors have multilineage potential at the single-cell level. These new insights prompt an erythroid-focused model of hematopoietic differentiation., Graphical Abstract, Highlights • RBCs are the predominant cell type produced by multipotent hematopoietic progenitors • All cell types with myeloid potential also produced RBCs and platelets in vivo • Single HSCs and MPPF cells are capable of multilineage hematopoietic reconstitution • Erythroid cell production emerges as a default hematopoietic fate, In this article, Forsberg and colleagues use quantitative reconstitution assays to demonstrate that all myeloid-competent progenitor cells also contribute to erythroid and platelet production. Single-cell in vivo analyses showed that hematopoietic stem cells and multipotent progenitors are clonally multipotent. These results suggest that erythropoiesis is a default hematopoietic fate and challenges current views on hematopoietic lineage potential and fate decisions.

We have recently reported that IL7R signaling regulates fetal macrophage development, in addition to lymphocyte lineage commitment and development. Specifically, IL7R deletion and pharmacological blockade during a defined fetal developmental window impairs the establishment of fetal macrophage populations in the liver, lung, brain, and epidermis. We next hypothesized that IL-7 is also required for fetal macrophage establishment and that IL-7 signaling activates developmental programs that facilitate macrophage development via proliferation and survival. Immunofluorescent staining of E14.5 murine embryos revealed abundant co-localized IL7R and F4/80 expression in fetal liver, lung, brain, and skin. IL7R+ F4/80-expressing cells were also found in close proximity to IL-7+ cells, suggesting they participate in IL-7 signaling. Moreover, germline deletion of IL-7 also resulted in postnatal depletion of fetal-derived macrophage populations. Antibody blockade of IL-7R receptor during late gestation revealed tissue-specific deficits in survival and proliferation the day before birth. Specifically, liver and lung macrophages showed increased apoptosis, as determined by Annexin V staining, while brain and epidermal macrophages were less proliferative, as determined by Ki67 staining. Here we have shown that IL-7, in addition to IL7R, regulates establishment of tissue-resident macrophages and that fetal blockade of IL7R signaling regulates establishment by impairing survival and proliferation during late gestation. Ongoing work seeks to define IL7/IL7R signaling pathways in developing macrophages, as well as use genetic models to pinpoint the requirement for IL7R in fetal myeloid specification. Supported by grants from NIH (R01 10057561).

Cytomegalovirus (CMV) is the most common congenital viral infection in the developed world and the leading cause of non-genetic sensorineural hearing loss (SNHL) in children. Congenital CMV causes progressive SNHL long after acute infection has resolved, suggesting a lasting effect on the developing host immune response within the cochlea and surrounding temporal bone marrow (TB). Using fate-mapping models, we have investigated the contribution of fetal-derived tissue resident macrophages (TRMs) to normal cochlear development and function, and their role in mediating CMV-associated SNHL. Our analysis defined two distinct myeloid population within the inner ear: 1) classical tissue resident macrophages (F4/80hiCD11blo) that highly express CD64 and are found exclusively in cochlear tissue; and 2) transitional monocytes (F4/80midCD11bhi) that express both CD64 and Ly6C and are found in cochlea and TB. Fate mapping of these populations uncovered dual contribution of fetal-derived cochlear TRMs from yolk sac (YS) and fetal liver (FL) hematopoiesis. Transitional monocytes increase during acute CMV infection, acting as drivers of expanded lymphoid populations after CMV resolves. Importantly, analysis of Flt3-Cre labeling within myeloid populations suggests that TRMs are replaced by BM-derived populations at later postnatal stages following CMV infection, coinciding with SNHL. Ongoing work examines spatial distribution of distinct fetal-derived TRMs along cochlear architecture, defining transcriptional regulation differences between YS-, FL-, and adult-derived TRMs in response to CMV, and examining how replacement of fetal-derived TRMs with adult-derived TRMs affects tissue function and response to infection. Supported by grants from University of Utah Molecular Medicine and Immunology, Inflammation and Infectious Disease (3i) Initiative and NIAID (T32 5T32AI138945)

CD8 T cells can play both a protective and pathogenic role in inflammation and autoimmune development. Recent studies have highlighted the ability of CD8 T cells to function as T follicular helper (Tfh) cells in the germinal center in the context of infection. However, whether this phenomenon occurs in autoimmunity and contributes to autoimmune pathogenesis is largely unexplored. In this study, we show that CD8 T cells acquire a CD4 Tfh profile in the absence of functional regulatory T cells in both the IL-2–deficient and scurfy mouse models. Depletion of CD8 T cells mitigates autoimmune pathogenesis in IL-2–deficient mice. CD8 T cells express the B cell follicle–localizing chemokine receptor CXCR5, a principal Tfh transcription factor Bcl6, and the Tfh effector cytokine IL-21. CD8 T cells localize to the B cell follicle, express B cell costimulatory proteins, and promote B cell differentiation and Ab isotype class switching. These data reveal a novel contribution of autoreactive CD8 T cells to autoimmune disease, in part, through CD4 follicular-like differentiation and functionality.

During embryonic development, multiple waves of hematopoietic progenitors with distinct lineage potential are differentially regulated in time and space. Two different waves of thymic progenitors colonize the fetal thymus where they contribute to thymic organogenesis and homeostasis. The origin, the lineage differentiation potential of the first wave, and their relative contribution in shaping the thymus architecture, remained, however, unclear. Here, we show that the first wave of thymic progenitors comprises a unique population of bipotent T and innatel lymphoid cells (T/ILC), generating a lymphoid tissue inducer cells (LTi's), in addition to invariant Vγ5(+) T cells. Transcriptional analysis revealed that innate lymphoid gene signatures and, more precisely, the LTi-associated transcripts were expressed in the first, but not in the second, wave of thymic progenitors. Depletion of early thymic progenitors in a temporally controlled manner showed that the progeny of the first wave is indispensable for the differentiation of autoimmune regulator–expressing medullary thymic epithelial cells (mTECs). We further show that these progenitors are of strict hematopoietic stem cell origin, despite the overlap between lymphopoiesis initiation and the transient expression of lymphoid-associated transcripts in yolk sac (YS) erythromyeloid-restricted precursors. Our work highlights the relevance of the developmental timing on the emergence of different lymphoid subsets, required for the establishment of a functionally diverse immune system.

Over the last century, the alarming surge in allergy and autoimmune disease has led to the hypothesis that decreasing exposure to microbes, which has accompanied industrialization and modern life in the Western world, has fundamentally altered the immune response. In its current iteration, the “hygiene hypothesis” suggests that reduced microbial exposures during early life restricts the production and differentiation of immune cells suited for immune regulation. Although it is now well-appreciated that the increase in hypersensitivity disorders represents a “perfect storm” of many contributing factors, we argue here that two important considerations have rarely been explored. First, the window of microbial exposure that impacts immune development is not limited to early childhood, but likely extends into the womb. Second, restricted microbial interactions by an expectant mother will bias the fetal immune system toward hypersensitivity. Here, we extend this discussion to hypothesize that the cell types sensing microbial exposures include fetal hematopoietic stem cells, which drive long-lasting changes to immunity.

The discovery of a fetal origin for tissue-resident macrophages (trMacs) has inspired an intense search for the mechanisms underlying their development. Here, we performed in vivo lineage tracing of cells with an expression history of IL-7Rα, a marker exclusively associated with the lymphoid lineage in adult hematopoiesis. Surprisingly, we found that IL7R-Cre labeled fetal-derived, adult trMacs. Labeling was almost complete in some tissues and partial in other organs. The putative progenitors of trMacs, yolk sac (YS) erythromyeloid progenitors (EMPs), did not express IL-7R, and YS hematopoiesis was unperturbed in IL-7R-deficient mice. In contrast, tracking of IL-7Rα message levels, surface protein expression, and IL7R-Cre-mediated labeling across fetal development revealed dynamic regulation of IL-7Rα mRNA expression and rapid upregulation of IL-7Rα surface protein upon transition from monocyte to macrophage within fetal tissues. Fetal liver monocyte differentiation in vitro produced IL-7R+ macrophages, supporting a direct progenitor-progeny relationship. Additionally, blockade of IL-7R function during late gestation specifically impaired the establishment of fetal-derived tissue macrophages in vivo. These data provide evidence for a distinct function of IL-7Rα in fetal myelopoiesis and identify IL-7R as a novel regulator of tissue-resident macrophage development.

Hematopoietic stem cells (HSCs) have long been considered the continuous source of all hematopoietic cells for the life of an individual. Recent findings have questioned multiple aspects of this view, including the ability of lifelong HSCs to contribute to tissue-resident immune cells. Here we discuss the most recent findings on the source of B1a cells, innatelike lymphocytes that primarily reside in serous cavities. Powerful experimental approaches including bar coding, single cell transplantation, in vivo lineage tracing, and HSC-specific pulse-chase labeling have provided novel insights on B1a-cell generation during ontogeny. We evaluate the evidence for fetal vs adult B1a-cell production capacity and the identity of putative cells of origin. Integrating these most recent findings with previous work, we propose a working model that encapsulates our current understanding of waves of immune development.

The generation of distinct hematopoietic cell types, including tissue-resident immune cells, distinguishes fetal from adult hematopoiesis. However, the mechanisms underlying differential cell production to generate a layered immune system during hematopoietic development are unclear. Using an irreversible lineage-tracing model, we identify a definitive hematopoietic stem cell (HSC) that supports long-term multilineage reconstitution upon transplantation into adult recipients but does not persist into adulthood in situ. These HSCs are fully multipotent, yet they display both higher lymphoid cell production and greater capacity to generate innate-like B and T lymphocytes as compared to coexisting fetal HSCs and adult HSCs. Thus, these developmentally restricted HSCs (drHSCs) define the origin and generation of early lymphoid cells that play essential roles in establishing self-recognition and tolerance, with important implications for understanding autoimmune disease, allergy, and rejection of transplanted organs.

During development, transient fetal hematopoietic stem cells (HSCs) are responsible for the production of "unconventional" innate-like immune cells that contribute to adult immunity. Whereas dysregulation of fetal-derived immune cells contributes to pathogenesis in a variety of immune tolerance disorders, the cellular and molecular drivers of pathogenesis are unknown. We have previously identified a transient developmentally-restricted HSC (drHSC) that specifically gives rise to innate-like lymphocytes during the perinatal period1. Under homeostatic conditions, the drHSC disappears postnatally, but its innate-like lymphocyte progeny persist into adulthood. Our discovery of a transient cell-of-origin for a specialized component of adult immunity underscores a “critical window” of immune development, during which phenotype of the adult immune system can be shaped by extrinsic inputs in early life. Here, we tested how developmental perturbation induced in utero by a single low-dose injection of poly(I:C) at mid-gestation drives both immediate and lasting changes to hematopoiesis and immunity in offspring. In utero perturbation disrupted the entrance of fetal HSCs into quiescence, causing disproportionate expansion of drHSCs, and a resultant shift in multipotent progenitor output. Postnatally, we observed sustained expansion and inappropriate persistence of the drHSC population into adulthood. These fundamental changes to the postnatal HSC compartment resulted in parallel expansion and hyperactivation of innate-like lymphocytes, thereby altering immune landscape and function in offspring. Our work validates the existence of a critical window of immune development by revealing how early perturbation of distinct fetal HSCs can drive long-term changes to immunity and disease susceptibility in offspring.

Our lab previously identified a developmentally-restricted hematopoietic stem cell (drHSC) that only exists during fetal development and specifically gives rise to innate-like lymphocytes that persist across the lifespan. The identification of a developmentally-limited cell of origin for innate-like immune cells defines a “critical window” for immune development, in which the phenotype of the developing immune system can be shaped via extrinsic inputs. To test how perturbation during this critical window drives immune dysfunction, we examined underlying changes to innate lymphoid cells (ILCs) in the lung. ILCs are a recently identified family of fetal-derived innate-like lymphocytes that mimic the adaptive T-helper arm of our immune system. In the lung, type-2 innate lymphoid cells (ILC2s) produce IL5 and IL13, cytokines important for eosinophil recruitment, activation and goblet cell hyperplasia during allergic airway inflammation. Surprisingly, a single low-dose injection of poly (i:c) at mid-gestation robustly increased proliferative capacity and cellularity of lung ILC2s in offspring at postnatal day (P)9 and P14, respectively, concomitant with drHSC and common-helper innate lymphoid progenitor (ChILP) cell expansion during fetal development. Additionally, in poly (i:c) perturbed offspring, lung ILC2s exhibited heightened IL5 and IL13 production upon in-vitro stimulation with PMA and ionomycin. Ongoing experiments will examine how perturbation of transient progenitors during fetal development may impact the ChILP cell immune trajectory, altering the establishment and function of neonatal lung ILC2s, and ultimately contributing to allergic asthma susceptibility into adulthood.

RNA-seq is a powerful technique to investigate and quantify entire transcriptomes. Recent advances in the field have made it possible to explore the transcriptomes of single cells. However, most widely used RNA-seq protocols fail to provide crucial information regarding transcription start sites. Here we present a protocol, Tn5Prime, that takes advantage of the Tn5 transposase based Smartseq2 protocol to create RNA-seq libraries that capture the 5’ end of transcripts. The Tn5Prime method dramatically streamlines the 5’ capture process and is both cost effective and reliable. By applying Tn5Prime to bulk RNA and single cell samples we were able to define transcription start sites as well as quantify transcriptomes at high accuracy and reproducibility. Additionally, similar to 3’ end based high-throughput methods like Drop-Seq and 10X Genomics Chromium, the 5’ capture Tn5Prime method allows the introduction of cellular identifiers during reverse transcription, simplifying the analysis of large numbers of single cells. In contrast to 3’ end based methods, Tn5Prime also enables the assembly of the variable 5’ ends of antibody sequences present in single B-cell data. Therefore, Tn5Prime presents a robust tool for both basic and applied research into the adaptive immune system and beyond.

SummaryAlthough classified as hematopoietic cells, tissue-resident macrophages (MFs) arise from embryonic precursors that seed the tissues prior to birth to generate a self-renewing population, which is maintained independently of adult hematopoiesis. Here we reveal the identity of these embryonic precursors using an in utero MF-depletion strategy and fate-mapping of yolk sac (YS) and fetal liver (FL) hematopoiesis. We show that YS MFs are the main precursors of microglia, while most other MFs derive from fetal monocytes (MOs). Both YS MFs and fetal MOs arise from erythro-myeloid progenitors (EMPs) generated in the YS. In the YS, EMPs gave rise to MFs without monocytic intermediates, while EMP seeding the FL upon the establishment of blood circulation acquired c-Myb expression and gave rise to fetal MOs that then seeded embryonic tissues and differentiated into MFs. Thus, adult tissue-resident MFs established from hematopoietic stem cell-independent embryonic precursors arise from two distinct developmental programs.

Understanding gene regulation and function requires a genome-wide method capable of capturing both gene expression levels and isoform diversity at the single-cell level. Short-read RNAseq is limited in its ability to resolve complex isoforms because it fails to sequence full-length cDNA copies of RNA molecules. Here, we investigate whether RNAseq using the long-read single-molecule Oxford Nanopore MinION sequencer is able to identify and quantify complex isoforms without sacrificing accurate gene expression quantification. After benchmarking our approach, we analyse individual murine B1a cells using a custom multiplexing strategy. We identify thousands of unannotated transcription start and end sites, as well as hundreds of alternative splicing events in these B1a cells. We also identify hundreds of genes expressed across B1a cells that display multiple complex isoforms, including several B cell-specific surface receptors. Our results show that we can identify and quantify complex isoforms at the single cell level., Short-read RNA-seq is limited in its ability to resolve complex transcript isoforms since it cannot sequence full-length cDNA. Here the authors use Oxford Nanopore MinION and their Mandalorion analysis pipeline to measure complex isoforms in B1a cells.

Our lab has previously identified a developmentally-restricted hematopoietic stem cell (drHSC) that only exists in the perinatal period and specifically gives rise to innate-like lymphocytes. The identification of a developmentally-limited cell of origin for innate-like immune cells that persist across the lifespan defines a “critical window” for immune development, in which the phenotype of the adult immune system can be shaped by extrinsic inputs. To test how developmental perturbation during this critical window drives immune dysfunction, we examined underlying changes to innate-like immune cells in the lung and susceptibility to airway dysfunction following maternal immune stimulation. Lung type-2 innate lymphoid cells (ILC2s) are recognized as potent producers of IL5 and IL13, cytokines important for eosinophil recruitment, activation and goblet cell hyperplasia during allergic airway inflammation. Surprisingly, maternal immune stimulation via a single low-dose injection of poly (i:c) at mid-gestation robustly increased the cellularity of lung ILC2s in offspring at postnatal day 14, concomitant with drHSC and ILC2 progenitor expansion during fetal development. Additionally, lung ILC2s exhibited heightened IL5 and IL13 production upon in-vitro stimulation with PMA and ionomycin in poly (i:c) perturbed offspring. Ongoing experiments examine how altered ILC2 functionality, as a result of developmental perturbation, underlie susceptibility to allergic asthma in response to secondary house-dust mite immune stimulation. Together, our data suggest perinatal immune perturbation may contribute to allergic asthma susceptibility by altering drHSC establishment and training lung ILC2 “innate immune memory”.

In adulthood, infection drives cytokine-mediated inflammation that can directly influence hematopoietic stem and progenitor cell (HSPC) function and differentiation, but the effects of maternal infection on the trajectory of fetal hematopoietic and immune cells have not been elucidated. Toxoplasma gondii is a TORCH pathogen that can be vertically transmitted upon acute infection during pregnancy, with dire consequences for the developing fetus. Maternal IFNγ production during congenital infection is required to prevent vertical transmission and promote parasite clearance, but at the cost of lowered birth weights and premature abortion. Here, we investigated the effects of acute congenital toxoplasmosis on fetal hematopoiesis. Our examination of fetal hematopoiesis in response to maternal T. gondii infection of varying virulence revealed that discrete HSPC populations, including subsets of fetal hematopoietic stem cells, B1 precursors, and myeloid progenitors are fundamentally altered in response to congenital toxoplasmosis. To assess the ability of IFNγ to mediate these observed changes and directly cross the placental barrier, we performed epistatic crosses of mice deficient for IFNγ receptor. Our results indicate that maternal IFNy produced in response to T. gondii infection may drive observed changes to the fetal HSPC compartment and that fetal HSPCs respond directly to maternal sources of IFNγ in a manner distinct from adult HSPCs. Ongoing experiments attempt to resolve the mechanisms by which vertical transmission and maternal inflammation perturb the establishment of the fetal immune system, and the direct role of IFNγ in mediating these phenomena.

Tissue-resident macrophages (TRMs) play critical roles in tissue homeostasis and disease. Many populations of TRMs derive from fetal progenitors and independently self-maintain across the lifespan through in situ proliferation. Here, we have identified the interleukin-7 receptor (IL7R) as a novel regulator of TRM development. Using an IL7R-Cre lineage tracing model, we observed that adult TRMs in the brain, epidermis, liver, and lung were highly labeled by IL7R-cre, in the absence of IL7Ra mRNA or protein expression. To gain insight into developmental expression of IL7Ra, we profiled surface expression, mRNA expression, and IL7R-cre driven labeling across fetal development. Erythromyeloid progenitors, putative TRM precursors, were barely labeled by IL7Ra-cre, and IL7R deletion did not affect YS hematopoiesis. In contrast, we observed IL7Ra mRNA expression in fetal monocytes, and robust IL7Ra surface expression on developing TRMs during late gestation. Sorted Ly6chi fetal liver monocytes cultured ex vivo with M-CSF differentiated into macrophages expressing IL7R, suggesting a precursor-product relationship. Blockade of the IL7R with a monoclonal antibody during gestation impaired liver, lung, and epidermal TRM cellularity at birth, with a concomitant increase in cellularity of liver monocytes, suggesting that IL7Ra regulates TRM differentiation from fetal monocytes during fetal development. These data reveal dynamic regulation of IL7Ra expression in TRMs and TRM precursors during late gestation, and provide evidence that IL7R signaling regulates fetal TRM development. Ongoing work addresses downstream signaling and the specific developmental processes regulated by IL7R signaling during fetal TRM development.

Impaired folate-mediated one-carbon metabolism (OCM) has emerged as a risk factor for several diseases associated with age-related cognitive decline, but the underlying mechanisms remain unknown and thus hinder the identification of subpopulations most vulnerable to OCM disruption. Here we investigated the role of serine hydroxymethyltransferase 1 (SHMT1), a folate-dependent enzyme regulating de novo thymidylate biosynthesis, in influencing neuronal and cognitive function in the adult mouse. We observed Shmt1 expression in the hippocampus, including the granule cell layer of the dentate gyrus (DG), and examined hippocampal neurogenesis and hippocampal-dependent fear conditioning in mice deficient for Shmt1. We used a 3 × 3 design in which adult male Shmt1(+/+), Shmt1(+/-), and Shmt1(-/-) mice were fed folic acid control (2 mg/kg), folic acid-deficient (0 mg/kg), or folic acid-supplemented (8 mg/kg) diets from weaning through the duration of the study. Proliferation within the DG was elevated by 70% in Shmt1(+/-) mice, yet the number of newborn mature neurons was reduced by 98% compared with that in Shmt1(+/+) mice. Concomitant with these alterations, Shmt1(+/-) mice showed a 45% reduction in mnemonic recall during trace fear conditioning. Dietary folate manipulations alone did not influence neural outcomes. Together, these data identify SHMT1 as one of the first enzymes within the OCM pathway to regulate neuronal and cognitive profiles and implicate impaired thymidylate biosynthesis in the etiology of folate-related neuropathogenesis.

Genetic fate-mapping approaches provide a unique opportunity to assess differentiation pathways under physiological conditions. We have recently employed a lineage tracing approach to define hematopoietic differentiation pathways in relation to expression of the tyrosine kinase receptor Flk2.1 Based on our examination of reporter activity across all stem, progenitor and mature populations in our Flk2-Cre lineage model, we concluded that all mature blood lineages are derived through a Flk2+ intermediate, both at steady-state and under stress conditions. Here, we re-examine in depth our initial conclusions and perform additional experiments to test alternative options of lineage specification. Our data unequivocally support the conclusion that onset of Flk2 expression results in loss of self-renewal but preservation of multilineage differentiation potential. We discuss the implications of these data for defining stem cell identity and lineage potential among hematopoietic populations.

Background: Low dietary choline intake has been proposed to increase the risk of neural tube defects (NTDs) in human populations. Mice with reduced Shmt1 expression exhibit a higher frequency of NTDs when placed on a folate- and choline-deficient diet and may represent a model of human NTDs. The individual contribution of dietary folate and choline deficiency to NTD incidence in this mouse model is not known. Objective: To dissociate the effects of dietary folate and choline deficiency on Shmt1-related NTD sensitivity, we determined NTD incidence in embryos from Shmt1-null dams fed diets deficient in either folate or choline. Design: Shmt1+/+ and Shmt1−/− dams were maintained on a standard AIN93G diet (Dyets), an AIN93G diet lacking folate (FD), or an AIN93G diet lacking choline (CD). Virgin Shmt1+/+ and Shmt1−/− dams were crossed with Shmt1+/− males, and embryos were examined for the presence of NTDs at embryonic day (E) 11.5 or E12.5. Results: Exencephaly was observed only in Shmt1−/− embryos isolated from dams maintained on the FD diet (P = 0.004). Approximately 33% of Shmt1−/−embryos (n = 18) isolated from dams maintained on the FD diet exhibited exencephaly. NTDs were not observed in any embryos isolated from dams maintained on the CD (n = 100) or control (n = 152) diets or in any Shmt1+/+ (n = 78) or Shmt1+/− embryos (n = 182). Conclusion: Maternal folate deficiency alone is sufficient to induce NTDs in response to embryonic Shmt1 disruption.

Tissue-resident macrophages play critical roles in tissue homeostasis and disease. Many populations of tissue-resident macrophages derive from fetal progenitors and self-maintain across the lifespan through in situ proliferation, independent of bone marrow hematopoiesis. However, the developmental mechanisms that specify fetal-derived tissue-resident macrophages are poorly understood. Here, we have identified a novel cytokine regulating tissue-resident macrophage development using an IL7Ra-cre lineage tracing model. Adult tissue resident macrophages in the brain, skin, liver, and lung were extensively labeled by IL7ra-cre, in the absence of IL7ra message or protein expression. To gain insight into developmental expression of IL7ra, we profiled IL7Ra surface expression, mRNA expression, and IL7ra-cre-driven labeling across fetal myeloid development. We observed rapid upregulation of IL7ra surface expression during a limited window of tissue macrophage establishment, and dynamic regulation of IL7ra message and protein levels in monocyte precursors, suggesting that IL7ra regulated the transition from fetal liver monocytes into tissue macrophages. Blockade of the IL7R using a monoclonal antibody decreased cellularity of liver, lung and skin tissue resident macrophages at birth, and increased cellularity of fetal liver monocytes. These data suggest that late in gestation, IL7Ra is upregulated as fetal monocytes exit the fetal liver and differentiate into macrophages, and provides evidence that IL-7R signaling regulates fetal tissue-resident macrophage development. Ongoing work addresses the function of IL-7 signaling in myeloid development, and the function of IL-7R marked macrophages in adult immunity.

Neural tube defects (NTDs), including anencephaly and spina bifida, arise from the failure of neurulation during early embryonic development. Neural tube defects are common birth defects with a heterogenous and multifactorial etiology with interacting genetic and environmental risk factors. Although the mechanisms resulting in failure of neural tube closure are unknown, up to 70% of NTDs can be prevented by maternal folic acid supplementation. However, the metabolic mechanisms underlying the association between folic acid and NTD pathogenesis have not been identified. This review summarizes our current understanding of the mechanisms by which impairments in folate metabolism might ultimately lead to failure of neural tube closure, with an emphasis on untangling the relative contributions of nutritional deficiency and genetic risk factors to NTD pathogenesis.

On a series of attention tasks, male mice with a mutation targeted to the fragile X mental retardation 1 (Fmrl) gene (Fmrl knockout [KO] mice) committed a higher rate of premature responses than wild-type littermates, with the largest differences seen when task contingencies changed. This finding indicates impaired inhibitory control, particularly during times of stress or arousal. The KO mice also committed a higher rate of inaccurate responses than controls, particularly during the final third of each daily test session, indicating impaired sustained attention. In the selective attention task, the unpredictable presentation of potent olfactory distractors produced a generalized disruption in the performance of the KO mice, whereas for controls, the disruption produced by the distractors was temporally limited. Finally, the attentional disruption seen following an error was more pronounced for the KO mice than for controls, further implicating impaired regulation of arousal and/or negative affect. The present study provides the first evidence that the Fmrl KO mouse is impaired in inhibitory control, attention, and arousal regulation, hallmark areas of dysfunction in fragile X syndrome. The resistance to change also seen in these mice provides a behavioral index for studying the autistic features of this disorder.

Defining differentiation pathways is central to understanding the pathogenesis of hematopoietic disorders, including leukemia. The function of the receptor tyrosine kinase Flk2 (Flt3) in promoting myeloid development remains poorly defined, despite being commonly mutated in acute myeloid leukemia. We investigated the effect of Flk2 deficiency on myelopoiesis, focusing on specification of progenitors between HSC and mature cells. We provide evidence that Flk2 is critical for proliferative expansion of multipotent progenitors that are common precursors for all lymphoid and myeloid lineages, including megakaryocyte/erythroid (MegE) cells. Flk2 deficiency impaired the generation of both lymphoid and myeloid progenitors by abrogating propagation of their common upstream precursor. At steady state, downstream compensatory mechanisms masked the effect of Flk2 deficiency on mature myeloid output, whereas transplantation of purified progenitors revealed impaired generation of all mature lineages. Flk2 deficiency did not affect lineage choice, thus dissociating the role of Flk2 in promoting cell expansion and regulating cell fate. Surprisingly, despite impairing myeloid development, Flk2 deficiency afforded protection against myeloablative insult. This survival advantage was attributed to reduced cell cycling and proliferation of progenitors in Flk2-deficient mice. Our data support the existence of a common Flk2(+) intermediate for all hematopoietic lineages and provide insight into how activating Flk2 mutations promote hematopoietic malignancy by non-Flk2-expressing myeloid cells.

Background: MTHFD1 encodes C1-tetrahydrofolate synthase, which is a folate-dependent enzyme that catalyzes the formation and interconversion of folate-activated one-carbon groups for nucleotide biosynthesis and cellular methylation. A polymorphism in MTHFD1 (1958G→A) impairs enzymatic activity and is associated with increased risk of adverse pregnancy outcomes, but the mechanisms are unknown. Objective: The objective of this study was to determine whether disruption of the embryonic or maternal Mthfd1 gene or both interacts with impaired folate and choline status to affect neural tube closure, fetal growth, and fertility in mice and to investigate the underlying metabolic disruptions. Design: Dams with a gene-trapped (gt) allele in Mthfd1 and wild-type dams were fed a control or folate- and choline-deficient AIN93G diet (Dyets Inc). Litters were examined for gross morphologic defects, crown-rump length, and resorptions. Folate status and amounts of folate-related metabolites were determined in pregnant dams. Results: Reduced folate and choline status resulted in severe fetal growth restriction (FGR) and impaired fertility in litters harvested from Mthfd1gt/+ dams, but embryonic Mthfd1gt/+ genotype did not affect fetal growth. Gestational supplementation of Mthfd1gt/+ dams with hypoxanthine increased FGR frequency and caused occasional neural tube defects (NTDs) in Mthfd1gt/+ embryos. Mthfd1gt/+ dams exhibited lower red blood cell folate and plasma methionine concentrations than did wild-type dams. Conclusions: Maternal Mthfd1gt/+ genotype impairs fetal growth but does not cause NTDs when dams are maintained on a folate- and choline-deficient diet. Mthfd1gt/+ mice exhibit a spectrum of adverse reproductive outcomes previously attributed to the human MTHFD1 1958G→A polymorphism. Mthfd1 heterozygosity impairs folate status in pregnant mice but does not significantly affect homocysteine metabolism.

Background: Folic acid supplementation prevents the occurrence and recurrence of neural tube defects (NTDs), but the causal metabolic pathways underlying folic acid–responsive NTDs have not been established. Serine hydroxymethyltransferase (SHMT1) partitions folate-derived one-carbon units to thymidylate biosynthesis at the expense of cellular methylation, and therefore SHMT1-deficient mice are a model to investigate the metabolic origin of folate-associated pathologies. Objectives: We examined whether genetic disruption of the Shmt1 gene in mice induces NTDs in response to maternal folate and choline deficiency and whether a corresponding disruption in de novo thymidylate biosynthesis underlies NTD pathogenesis. Design: Shmt1 wild-type, +/−, and −/− mice fed either folate- and choline-sufficient or folate- and choline-deficient diets were bred, and litters were examined for the presence of NTDs. Biomarkers of impaired folate metabolism were measured in the dams. In addition, the effect of Shmt1 disruption on NTD incidence was investigated in Sp mice, an established folate-responsive NTD mouse model. Results: +/− and −/− embryos exhibited exencephaly in response to maternal folate and choline deficiency. Shmt1 disruption on the Sp background exacerbated NTD frequency and severity. Pax3 disruption impaired de novo thymidylate and purine biosynthesis and altered amounts of SHMT1 and thymidylate synthase protein. Conclusions: SHMT1 is the only folate-metabolizing enzyme that has been shown to affect neural tube closure in mice by directly inhibiting folate metabolism. These results provide evidence that disruption of Shmt1 expression causes NTDs by impairing thymidylate biosynthesis and shows that changes in the expression of genes that encode folate-dependent enzymes may be key determinates of NTD risk.

Neural tube defects (NTDs) refer to a cluster of neurodevelopmental conditions associated with failure of neural tube closure during embryonic development. Worldwide prevalence of NTDs ranges from approximately 0.5 to 60 per 10,000 births, with regional and population-specific variation in prevalence. Numerous environmental and genetic influences contribute to NTD etiology; accumulating evidence from population-based studies has demonstrated that folate status is a significant determinant of NTD risk. Folate-mediated one-carbon metabolism (OCM) is essential for de novo nucleotide biosynthesis, methionine biosynthesis, and cellular methylation reactions. Periconceptional maternal supplementation with folic acid can prevent occurrence of NTDs in the general population by up to 70%; currently several countries fortify their food supply with folic acid for the prevention of NTDs. Despite the unambiguous impact of folate status on NTD risk, the mechanism by which folic acid protects against NTDs remains unknown. Identification of the mechanism by which folate status affects neural tube closure will assist in developing more efficacious and better targeted preventative measures. In this review, we summarize current research on the relationship between folate status and NTDs, with an emphasis on linking genetic variation, folate nutriture, and specific metabolic and/or genomic pathways that intersect to determine NTD outcomes.