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

The mononuclear phagocytic system (MPS) 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 MPS subsets from other hematopoietic lineages.Mpeg1.1is an evolutionary conserved gene encodingperforin-2, a pore-forming protein associated with host defense against pathogens. Zebrafishmpeg1.1:GFPandmpeg1.1:mCherryreporters were originally established to specifically label macrophages. Since, more than 100 peer-reviewed publications have made use ofmpeg1.1-driven transgenics forin vivostudies, providing new insights into key aspects of macrophage ontogeny, activation and function. However, while the macrophage-specific expression pattern of thempeg1.1promoter 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 bympeg1.1reporters in most adult zebrafish organs. Thesempeg1.1+lymphoid cells endogenously expressmpeg1.1and can be separated frommpeg1.1+macrophages by virtue of their light-scatter characteristics using FACS. Remarkably, our analyses also revealed that B-lymphocytes, rather than mononuclear phagocytes, constitute the mainmpeg1.1-positive population inirf8nullmyeloid-defective mutants, which were previously reported to recover tissue-resident macrophages in adulthood. One notable exception are skin macrophages, whose development and maintenance appear to be independent fromirf8, similar to mammals. Collectively, our findings demonstrate thatirf8functions in myelopoiesis are evolutionary conserved and highlight the need for alternative macrophage-specific markers to study the MPS in adult zebrafish.SUMMARY SENTENCEMpeg1 is not a restricted macrophage marker, but also labels B cells in the adult zebrafish. Therefore, previously identifiedirf8-independent macrophages likely consist of B lymphocytes.Graphical Abstract