Microglia, the tissue-resident macrophages of the CNS, represent major targets for therapeutic intervention in a wide variety of neurological disorders. Efficient reprogramming protocols to generate ...microglia-like cells in vitro using patient-derived induced pluripotent stem cells will, however, require a precise understanding of the cellular and molecular events that instruct microglial cell fates. This remains a challenge since the developmental origin of microglia during embryogenesis is controversial. Here, using genetic tracing in zebrafish, we uncover primitive macrophages as the unique source of embryonic microglia. We also demonstrate that this initial population is transient, with primitive microglia later replaced by definitive microglia that persist throughout adulthood. The adult wave originates from cmyb-dependent hematopoietic stem cells. Collectively, our work challenges the prevailing model establishing erythro-myeloid progenitors as the sole and direct microglial precursor and provides further support for the existence of multiple waves of microglia, which originate from distinct hematopoietic precursors.
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•Microglia ontogeny occurs in two distinct waves in zebrafish•EMPs do not contribute to microglia development•Primitive macrophages give rise to primitive microglia•Embryonic HSCs give rise to adult definitive microglia
Using zebrafish to investigate microglia ontogeny during vertebrate development, Ferrero et al. find that embryonic “primitive” microglia exclusively derive from primitive macrophages, while adult “definitive” microglia originate from cmyb-dependent hematopoietic stem cells.
Abstract
Reactive oxygen species (ROS) represent a by-product of metabolism and their excess is toxic for hematopoietic stem and progenitor cells (HSPCs). During embryogenesis, a small number of ...HSPCs are produced from the hemogenic endothelium, before they colonize a transient organ where they expand, for example the fetal liver in mammals. In this study, we use zebrafish to understand the molecular mechanisms that are important in the caudal hematopoietic tissue (equivalent to the mammalian fetal liver) to promote HSPC expansion. High levels of ROS are deleterious for HSPCs in this niche, however this is rescued by addition of antioxidants. We show that
Cx41.8
is important to lower ROS levels in HSPCs. We also demonstrate a new role for
ifi30
, known to be involved in the immune response. In the hematopoietic niche,
Ifi30
can recycle oxidized glutathione to allow HSPCs to dampen their levels of ROS, a role that could be conserved in human fetal liver.
In mammals, embryonic hematopoiesis occurs in successive waves, culminating with the emergence of hematopoietic stem cells (HSCs) in the aorta. HSCs first migrate to the fetal liver (FL), where they ...expand, before they seed the bone marrow niche, where they will sustain hematopoiesis throughout adulthood. In zebrafish, HSCs emerge from the dorsal aorta and colonize the caudal hematopoietic tissue (CHT). Recent studies showed that they interact with endothelial cells (ECs), where they expand, before they reach their ultimate niche, the kidney marrow. We identified tfec, a transcription factor from the mitf family, which is highly enriched in caudal endothelial cells (cECs) at the time of HSC colonization in the CHT. Gain-of-function assays indicate that tfec is capable of expanding HSC-derived hematopoiesis in a non–cell-autonomous fashion. Furthermore, tfec mutants (generated by CRISPR/Cas9) showed reduced hematopoiesis in the CHT, leading to anemia. Tfec mediates these changes by increasing the expression of several cytokines in cECs from the CHT niche. Among these, we found kitlgb, which could rescue the loss of HSCs observed in tfec mutants. We conclude that tfec plays an important role in the niche to expand hematopoietic progenitors through the modulation of several cytokines. The full comprehension of the mechanisms induced by tfec will represent an important milestone toward the expansion of HSCs for regenerative purposes.
•tfec controls the expression of cytokines in the vascular niche.•tfec expands HSCs in a non–cell-autonomous fashion.
Abstract
Background
Synovial fibroblasts in rheumatoid arthritis (RAFLS) exhibit a pathological aberration of glycolysis and glutaminolysis. Henceforth, we aimed to investigate if dual inhibition of ...these pathways by phytobiological compound c28MS has the potential of synergistic therapy for arthritis by targeting both glucose and glutamine metabolism.
Methods
The presence of HK2 and GLS across various cell types and associated gene expression in human synovial cells and a murine model of arthritis was evaluated by scRNA-seq. The metabolic profiling of RAFLS cells was done using H
1
-nuclear magnetic resonance spectroscopy under glycolytic and glutaminolytic inhibitory conditions by incubating with 3-bromopyruvate, CB839, or dual inhibitor c28MS. FLS functional analysis was conducted under similar conditions. ELISA was employed for the quantification of IL-6, CCL2, and MMP3. K/BxN sera was administered to mice to induce arthritis for in vivo arthritis experiments.
Results
scRNA-seq analysis revealed that many fibroblasts expressed
Hk2
along with
Gls
with several genes including
Ptgs2
,
Hif1a
,
Timp1
,
Cxcl5
, and
Plod2
only associated with double-positive fibroblasts, suggesting that dual inhibition can be an attractive target for fibroblasts. Metabolomic and functional analysis revealed that c28MS decreased the aggressive behavior of RAFLS by targeting both upregulated glycolysis and glutaminolysis. c28MS administered in vivo significantly decreased the severity of arthritis in the K/BxN model.
Conclusion
Our findings imply that dual inhibition of glycolysis and glutaminolysis could be an effective approach for the treatment of RA. It also suggests that targeting more than one metabolic pathway can be a novel treatment approach in non-cancer diseases.
The whole kidney marrow (WKM) is the site for hematopoiesis in the adult zebrafish. Here, we present a protocol for analyzing hematopoietic lineages in the WKM of adult zebrafish. We describe steps ...for the isolation of hematopoietic cells from the WKM, the downstream analysis of total marrow cellularity, and analysis of cell populations by flow cytometry. We then detail procedures for May-Grünwald-Giemsa staining for analysis of cellular morphology and phenotyping.
For complete details on the use and execution of this protocol, please refer to Mahony et al.1
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•Isolation of hematopoietic cells from the adult zebrafish kidney marrow•Protocol enables total cell counts and analysis of cell populations by flow cytometry•Steps for preparation of cells for cytospins/cytosmears•May-Grünwald-Giemsa staining of cells for morphological analysis
Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.
The whole kidney marrow (WKM) is the site for hematopoiesis in the adult zebrafish. Here, we present a protocol for analyzing hematopoietic lineages in the WKM of adult zebrafish. We describe steps for the isolation of hematopoietic cells from the WKM, the downstream analysis of total marrow cellularity, and analysis of cell populations by flow cytometry. We then detail procedures for May-Grünwald-Giemsa staining for analysis of cellular morphology and phenotyping.
Inherited bone marrow failure associated with heterozygous mutations in GATA2 predisposes toward hematological malignancies, but the mechanisms remain poorly understood. Here, we investigate the ...mechanistic basis of marrow failure in a zebrafish model of GATA2 deficiency. Single-cell transcriptomics and chromatin accessibility assays reveal that loss of gata2a leads to skewing toward the erythroid lineage at the expense of myeloid cells, associated with loss of cebpa expression and decreased PU.1 and CEBPA transcription factor accessibility in hematopoietic stem and progenitor cells (HSPCs). Furthermore, gata2a mutants show impaired expression of npm1a, the zebrafish NPM1 ortholog. Progressive loss of npm1a in HSPCs is associated with elevated levels of DNA damage in gata2a mutants. Thus, Gata2a maintains myeloid lineage priming through cebpa and protects against genome instability and marrow failure by maintaining expression of npm1a. Our results establish a potential mechanism underlying bone marrow failure in GATA2 deficiency.
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•Zebrafish gata2a enhancer mutants recapitulate human GATA2 deficiency disease•A Gata2a/Cebpa axis maintains marrow cellularity and myeloid lineage output•Gata2a regulates expression of DNA damage repair and replication-associated genes•Progressive loss of npm1a in mutant HSPCs is associated with increased DNA damage
Mahony et al. use single-cell genomics to uncover the molecular defects and examine progression to marrow failure in a zebrafish model of GATA2 deficiency. They show that Gata2a/Cebpa regulates marrow cellularity and myeloid lineage output, whereas HSPC survival and genome stability are protected by maintaining expression of DNA damage repair and replication-associated genes.
A lack of models that recapitulate the complexity of human bone marrow has hampered mechanistic studies of normal and malignant hematopoiesis and the validation of novel therapies. Here, we describe ...a step-wise, directed-differentiation protocol in which organoids are generated from induced pluripotent stem cells committed to mesenchymal, endothelial, and hematopoietic lineages. These 3D structures capture key features of human bone marrow-stroma, lumen-forming sinusoids, and myeloid cells including proplatelet-forming megakaryocytes. The organoids supported the engraftment and survival of cells from patients with blood malignancies, including cancer types notoriously difficult to maintain ex vivo. Fibrosis of the organoid occurred following TGFβ stimulation and engraftment with myelofibrosis but not healthy donor-derived cells, validating this platform as a powerful tool for studies of malignant cells and their interactions within a human bone marrow-like milieu. This enabling technology is likely to accelerate the discovery and prioritization of novel targets for bone marrow disorders and blood cancers.
We present a human bone marrow organoid that supports the growth of primary cells from patients with myeloid and lymphoid blood cancers. This model allows for mechanistic studies of blood cancers in the context of their microenvironment and provides a much-needed ex vivo tool for the prioritization of new therapeutics. See related commentary by Derecka and Crispino, p. 263. This article is highlighted in the In This Issue feature, p. 247.
Glucose metabolism, specifically, hexokinase 2 (HK2), has a critical role in rheumatoid arthritis (RA) fibroblast-like synoviocyte (FLS) phenotype. HK2 localizes not only in the cytosol but also in ...the mitochondria, where it protects mitochondria against stress. We hypothesize that mitochondria-bound HK2 is a key regulator of RA FLS phenotype.
HK2 localization was evaluated by confocal microscopy after FLS stimulation. RA FLSs were infected with Green fluorescent protein (GFP), full-length (FL)-HK2, or HK2 lacking its mitochondrial binding motif (HK2ΔN) expressing adenovirus (Ad). RA FLS was also incubated with methyl jasmonate (MJ; 2.5 mM), tofacitinib (1 µM), or methotrexate (1 µM). RA FLS was tested for migration and invasion and gene expression. Gene associations with HK2 expression were identified by examining single-cell RNA sequencing (scRNA-seq) data from murine models of arthritis. Mice were injected with K/BxN serum and given MJ. Ad-FLHK2 or Ad-HK2ΔN was injected into the knee of wild-type mice.
Cobalt chloride (CoCl
) and platelet-derived growth factor (PDGF) stimulation induced HK2 mitochondrial translocation. Overexpression of the HK2 mutant and MJ incubation reversed the invasive and migrative phenotype induced by FL-HK2 after PDGF stimulation, and MJ also decreased the expression of C-X-C Motif Chemokine Ligand 1 (CXCL1) and Collagen Type I Alpha 1 Chain (COL1A1). Of interest, tofacitinib but not methotrexate had an effect on HK2 dissociation from the mitochondria. In murine models, MJ treatment significantly decreased arthritis severity, whereas HK2FL was able to induce synovial hypertrophy as opposed to HK2ΔN.
Our results suggest that mitochondrial HK2 regulates the aggressive phenotype of RA FLS. New therapeutic approaches to dissociate HK2 from mitochondria offer a safer approach than global glycolysis inhibition.
Hematopoiesis maintains the entire blood system, and dysregulation of this process can lead to malignancies (leukemia), immunodeficiencies or red blood cell diseases (anemia, polycythemia vera). We ...took advantage of the zebrafish model that shares most of the genetic program involved in hematopoiesis with mammals to characterize a new gene of unknown function,
, which is expressed in the erythroid lineage during primitive, definitive and adult hematopoiesis. This gene, required during primitive and definitive erythropoiesis, encodes a C2H2 zinc-finger protein that inhibits BMP signaling. We therefore named this gene blood-inducing factor 1 and BMP inhibitory factor 1 (
). We identified a
ortholog in
, another fish, and in the mouse genome. Both genes also inhibit BMP signaling when overexpressed in zebrafish. In conclusion, we have deorphanized a new zebrafish gene of unknown function:
codes for a zinc-finger protein that inhibits BMP signaling and also regulates primitive erythropoiesis and definitive hematopoiesis.
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