Embryonic microglia derive from primitive macrophages and are replaced by cmyb-dependent definitive microglia in zebrafish

Ferrero, Giuliano; Mahony, Christopher B.; Dupuis, Eleonore; Yvernogeau, Laurent; Ruggiero, Elodie Di; Miserocchi, Magali; Caron, Marianne; Robin, Catherine; Traver, David; Bertrand, Julien; Wittamer, Valérie

doi:10.3389/conf.fnins.2019.96.00078

Cited by 26 publications

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“…In mpeg1.1 : GFP or mpeg1.1 : mCherry transgenic adult zebrafish, parenchymal microglia can be isolated from other CNS‐associated macrophages by flow cytometry based on fluorophore expression levels . We therefore sought to investigate whether mpeg1.1 reporters could similarly discriminate distinct macrophage subsets in other tissues.…”

Section: Resultsmentioning

confidence: 99%

“…Guts were dissected from adult fish, fixed in 4% paraformaldehyde (PFA) overnight (O/N) and incubated in 30% sucrose:PBS O/N before snap‐freezing in OCT. Scales were manually detached from euthanized fish and pretreated with 100 mM DTT before O/N fixation in 4% PFA. Immunostaining on gut cryosections or floating scales was performed as described, using the following primary and secondary antibodies: chicken anti‐GFP polyclonal antibody (1:500; Abcam, Cambridge, UK), rabbit anti‐DsRed polyclonal antibody (1:500; Clontech, Mountain view, CA, USA), Alexa Fluor 488‐conjugated anti‐chicken IgG antibody (1:500; Invitrogen, Carlsbad, CA, USA), Alexa Fluor 594‐conjugated anti‐rabbit IgG (1:500; Abcam). Images were taken with a Zeiss LSM 780 inverted microscope (Zeiss, Oberkochen, Germany), using a Plan Apochromat 20× objective.…”

Section: Methodsmentioning

confidence: 99%

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Themacrophage-expressed gene(mpeg)1identifies a subpopulation of B cells in the adult zebrafish

Ferrero

Gomez

lyer

et al. 2020

Journal of Leukocyte Biology

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The mononuclear phagocytic system consists of many cells, in particular macrophages, scattered throughout the body. However, there is increasing evidence for the heterogeneity of tissue‐resident macrophages, leading to a pressing need for new tools to discriminate mononuclear phagocytic system subsets from other hematopoietic lineages. Macrophage‐expressed gene (Mpeg)1.1 is an evolutionary conserved gene encoding perforin‐2, a pore‐forming protein associated with host defense against pathogens. Zebrafish mpeg1.1:GFP and mpeg1.1:mCherry reporters were originally established to specifically label macrophages. Since then more than 100 peer‐reviewed publications have made use of mpeg1.1‐driven transgenics for in vivo studies, providing new insights into key aspects of macrophage ontogeny, activation, and function. Whereas the macrophage‐specific expression pattern of the mpeg1.1 promoter has been firmly established in the zebrafish embryo, it is currently not known whether this specificity is maintained through adulthood. Here we report direct evidence that beside macrophages, a subpopulation of B‐lymphocytes is marked by mpeg1.1 reporters in most adult zebrafish organs. These mpeg1.1+ lymphoid cells endogenously express mpeg1.1 and can be separated from mpeg1.1+ macrophages by virtue of their light‐scatter characteristics using FACS. Remarkably, our analyses also revealed that B‐lymphocytes, rather than mononuclear phagocytes, constitute the main mpeg1.1‐positive population in irf8null myeloid‐defective mutants, which were previously reported to recover tissue‐resident macrophages in adulthood. One notable exception is skin macrophages, whose development and maintenance appear to be independent from irf8, similar to mammals. Collectively, our findings demonstrate that irf8 functions in myelopoiesis are evolutionary conserved and highlight the need for alternative macrophage‐specific markers to study the mononuclear phagocytic system in adult zebrafish.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Themacrophage-expressed gene(mpeg)1identifies a subpopulation of B cells in the adult zebrafish

Ferrero

Gomez

lyer

et al. 2020

Journal of Leukocyte Biology

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“…Unlike macrophages, metaphocytes are of non-hematopoietic, likely ectodermal origin (Lin et al, 2019). We recently proposed that skin macrophages and metaphocytes, based on these different ontogenies, could be discriminated in the adult zebrafish using the Tg( kdrl : Cre ; ßactin2 :loxP-STOP-loxP- DsRed ) fate-mapping model that labels EMPs, HSCs and their progenies (Bertrand et al, 2010a; Ferrero et al, 2018). Genetic, permanent labeling with DsRed of adult leukocytes, including branched skin macrophages is induced by constitutive expression of Cre recombinase in endothelial cells and hemogenic endothelium (Bertrand et al, 2010a).…”

Section: Resultsmentioning

confidence: 99%

Zebrafish macrophage developmental arrest underlies depletion of microglia and reveals Csf1r-independent metaphocytes

Kuil

Oosterhof

Ferrero

et al. 2020

Preprint

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20Macrophages derive from multiple sources of hematopoietic progenitors. Most macrophages 21 require colony-stimulating factor 1 receptor (CSF1R), but some macrophages persist in the 22 absence of CSF1R. Here, we analyzed mpeg1:GFP-expressing macrophages in csf1r-23 deficient zebrafish and report that embryonic macrophages emerge followed by their 24 developmental arrest. In larvae, mpeg1+ cell numbers then increased showing two distinct 25 types in the skin: branched, putative Langerhans cells, and amoeboid cells. In contrast, 26 although numbers also increased in csf1r-mutants, exclusively amoeboid mpeg1+ cells were 27 present, which we showed by genetic lineage tracing to have a non-hematopoietic origin. 28They expressed macrophage-associated genes, but also showed decreased phagocytic 29 gene expression and increased epithelial-associated gene expression, characteristic of 30 metaphocytes, recently discovered ectoderm-derived cells. We further demonstrated that 31 juvenile csf1r-deficient zebrafish exhibit systemic macrophage depletion. Thus, Csf1r 32 deficiency disrupts embryonic to adult macrophage development. Csf1r-deficient zebrafish 33 are viable and permit analyzing the consequences of macrophage loss throughout life. 34 35 36 106 fate mapping and gene expression profiling, we identified csf1r DM mpeg1+ cells as 107 metaphocytes, a population of ectoderm-derived macrophage-like cells recently reported in 108 zebrafish (Alemany et al., 2018a; Lin et al., 2019). Extending our analyses, we further 109 demonstrated that adult csf1r DM fish exhibit a global defect in macrophage generation. In 110 5 conclusion, our study highlights distinct requirements for Csf1r during macrophage 111 generation and metaphocyte ontogeny, resolving part of the presumed macrophage 112 heterogeneity and their sensitivity to loss of Csf1r. 113 Results 114Zebrafish embryonic macrophages are formed independently of csf1r but display 115 migration and proliferation defects 207 both csf1r DM primitive macrophages and early microglia. 208Next, we assessed the presence of macrophages in developing csf1r DM animals by in 209 vivo fluorescence imaging of one lateral side of entire, individual larvae on 4 consecutive 210 days, starting at 5 dpf. We visualized ~450 macrophages in control animals, whereas csf1r DM 211 animals contained > 4-fold fewer (~100) ( Figure 3D). Over the next 4 days, macrophage 212 numbers in both groups remained stable ( Figure 3D). This suggests that, at this stage, there 213 is neither proliferative expansion of embryonic macrophages nor supply of macrophages 214 from an alternative source, causing macrophage numbers in csf1r DM larvae to remain much 215 lower than those in controls up to 9 dpf. Together these data indicate that, onwards from the 216 initiation of embryonic tissue colonization, proliferative expansion of macrophages remains 217 halted in csf1r DM animals.218 219 csf1r DM skin lacks highly branched putative Langerhans cells 220 8Given that macrophages are produced by consecutive waves of primiti...

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“…Importantly, the mechanisms underlying these functions are the same as those employed by mammalian microglia, suggesting a high degree of conservation across species (Sieger & Peri, ). Interestingly, in zebrafish, these larval microglia seem to be replaced by a second wave of definitive microglia that persist throughout adulthood and are derived from cmyb‐dependent hematopoietic stem cells (Ferrero et al, ; Xu et al, ). The transcriptome of these adult zebrafish microglia was recently analyzed and a comparison with available data sets from mouse showed that a large fraction of the mouse microglia specific gene expression signature is conserved in the zebrafish (Oosterhof et al, ).…”

Section: Introductionmentioning

confidence: 99%

Gene expression profiling reveals a conserved microglia signature in larval zebrafish

Mazzolini

Clerc

Morisse

et al. 2019

Glia

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Microglia are the resident macrophages of the brain. Over the past decade, our understanding of the function of these cells has significantly improved. Microglia do not only play important roles in the healthy brain but are involved in almost every brain pathology. Gene expression profiling allowed to distinguish microglia from other macrophages and revealed that the full microglia signature can only be observed in vivo. Thus, animal models are irreplaceable to understand the function of these cells. One of the popular models to study microglia is the zebrafish larva. Due to their optical transparency and genetic accessibility, zebrafish larvae have been employed to understand a variety of microglia functions in the living brain. Here, we performed RNA sequencing of larval zebrafish microglia at different developmental time points: 3, 5, and 7 days post fertilization (dpf). Our analysis reveals that larval zebrafish microglia rapidly acquire the core microglia signature and many typical microglia genes are expressed from 3 dpf onwards. The majority of changes in gene expression happened between 3 and 5 dpf, suggesting that differentiation mainly takes place during these days. Furthermore, we compared the larval microglia transcriptome to published data sets of adult zebrafish microglia, mouse microglia, and human microglia. Larval microglia shared a significant number of expressed genes with their adult counterparts in zebrafish as well as with mouse and human microglia. In conclusion, our results show that larval zebrafish microglia mature rapidly and express the core microglia gene signature that seems to be conserved across species.

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Embryonic microglia derive from primitive macrophages and are replaced by cmyb-dependent definitive microglia in zebrafish

Cited by 26 publications

References 0 publications

Themacrophage-expressed gene(mpeg)1identifies a subpopulation of B cells in the adult zebrafish

Themacrophage-expressed gene(mpeg)1identifies a subpopulation of B cells in the adult zebrafish

Zebrafish macrophage developmental arrest underlies depletion of microglia and reveals Csf1r-independent metaphocytes

Gene expression profiling reveals a conserved microglia signature in larval zebrafish

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