A csf1rb mutation uncouples two waves of microglia development in zebrafish.

In vertebrates, the ontogeny of microglia, the resident macrophages of the central nervous system, initiates early during development from primitive macrophages. Although murine embryonic microglia then persist through life, in zebrafish these cells are transient, as they are fully replaced by an adult population originating from larval hematopoietic stem cell (HSC)-derived progenitors. Colony-stimulating factor 1 receptor (Csf1r) is a fundamental regulator of microglia ontogeny in vertebrates, including zebrafish, which possess two paralogous genes: csf1ra and csf1rb Although previous work has shown that mutation in both genes completely abrogates microglia development, the specific contribution of each paralog remains largely unknown. Here, using a fate-mapping strategy to discriminate between the two microglial waves, we uncover non-overlapping roles for csf1ra and csf1rb in hematopoiesis, and identified csf1rb as an essential regulator of adult microglia development. Notably, we demonstrate that csf1rb positively regulates HSC-derived myelopoiesis, resulting in macrophage deficiency, including microglia, in adult mutant animals. Overall, this study contributes to new insights into evolutionary aspects of Csf1r signaling and provides an unprecedented framework for the functional dissection of embryonic versus adult microglia in vivo.

Zebrafish Galectin 3 binding protein is the target antigen of the microglial 4C4 monoclonal antibody.

BACKGROUND: Two decades ago, the fish-specific monoclonal antibody 4C4 was found to be highly reactive to zebrafish microglia, the macrophages of the central nervous system. This has resulted in 4C4 being widely used, in combination with available fluorescent transgenic reporters to identify and isolate microglia. However, the target protein of 4C4 remains unidentified, which represents a major caveat. In addition, whether the 4C4 expression pattern is strictly restricted to microglial cells in zebrafish has never been investigated. RESULTS: Having demonstrated that 4C4 is able to capture its native antigen from adult brain lysates, we used immunoprecipitation/mass-spectrometry, coupled to recombinant expression analyses, to identify its target. The cognate antigen was found to be a paralog of Galectin 3 binding protein (Lgals3bpb), known as MAC2-binding protein in mammals. Notably, 4C4 did not recognize other paralogs, demonstrating specificity. Moreover, our data show that Lgals3bpb expression, while ubiquitous in microglia, also identifies leukocytes in the periphery, including populations of gut and liver macrophages. CONCLUSIONS: The 4C4 monoclonal antibody recognizes Lgals3bpb, a predicted highly glycosylated protein whose function in the microglial lineage is currently unknown. Identification of Lgals3bpb as a new pan-microglia marker will be fundamental in forthcoming studies using the zebrafish model.

Isolation of Tissue Macrophages in Adult Zebrafish.

Tissue macrophages are essential components of the immune system that also play key roles in vertebrate development and homeostasis, including in zebrafish, which has gained popularity over the years as a translational model for human disease. Commonly, zebrafish macrophages are identified based on expression of fluorescent transgenic reporters, allowing for real-time imaging in living animals. Several of these lines have also proven instrumental to isolate pure populations of macrophages in the developing embryo and larvae using fluorescence-activated cell sorting (FACS). However, the identification of tissue macrophages in adult fish is not as clear, and robust protocols are needed that would take into account changes in reporter specificity as well as the heterogeneity of mononuclear phagocytes as fish reach adulthood. In this chapter, we describe the methodology for analyzing macrophages in various tissues in the adult zebrafish by flow cytometry. Coupled with FACS, these protocols further allow for the prospective isolation of enriched populations of tissue-specific mononuclear phagocytes that can be used in downstream transcriptomic and/or epigenomic analyses. Overall, we aim at providing a guide for the zebrafish community based on our expertise investigating the adult mononuclear phagocyte system.

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

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.

Embryonic Microglia Derive from Primitive Macrophages and Are Replaced by cmyb-Dependent Definitive Microglia in Zebrafish.

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.