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

Abstract

SENTENCEMpeg1 is not a restricted macrophage marker, but also labels B cells in the adult zebrafish. Therefore, previously identified irf8-independent macrophages likely consist of B lymphocytes. 2 ABSTRACTThe 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. Mpeg1.1 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, 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. However, while the macrophagespecific 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 Blymphocytes, rather than mononuclear phagocytes, constitute the main mpeg1.1positive population in irf8 null myeloid-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 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 MPS in adult zebrafish.

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

“…Of note, a possible caveat is that mpeg1 + primitive macrophages, which derive directly from kdrl -negative mesoderm, are also not marked by DsRed in this setting, which could complicate the interpretation of results. However, as we previously documented, there seems to be no contribution from primitive hematopoiesis to mpeg1 -expressing cells in the adult skin (Ferrero et al, 2020). In addition, primitive macrophages appear virtually absent in Csf1r-deficient zebrafish, thus making this approach suitable to address the identity of mpeg1+ cells in csf1r DM skin.…”

“…However, these GFP + DsRed + cells were lost and GFP + DsRed − cell numbers were increased in csf1r DM . As the presence of metaphocytes was reported in skin but also in the intestine (Ferrero et al, 2020; Lin et al, 2019), intestinal GFP + DsRed − cells are likely also csf1r- independent metaphocytes. In all, mpeg1+ macrophages are largely Csf1r-dependent, whereas mpeg1+ cells present in the skin and intestine are Csf1r-independent non-hematopoietic metaphocytes.…”

“…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). As suggested by restricted expression of the metaphocyte marker cldnh in mpeg1 -GFP + DsRed − cells, non-hematopoietic metaphocytes lack DsRed labeling (Ferrero et al 2020). The presence or absence of DsRed expression could thus be used to discriminate between metaphocytes (GFP + DsRed − ) and macrophages (GFP + DsRed + ).…”

“…It remains unclear whether these are true Langerhans cells, as there is no known zebrafish ortholog of langerin (CD207), the main marker of LCs in humans and mice. LCs are likely to exist in zebrafish, as Birbeck granules, the morphological markers of LCs, have been identified in zebrafish skin macrophages (Lugo-Villarino et al, 2010), and we recently demonstrated that zebrafish branched skin macrophages, develop independently of the transcription factor Irf8 (Ferrero et al, 2020), similar to mammalian LCs (Chopin et al, 2013; Hambleton et al, 2011). Their dependence on Il34 provides additional evidence for the conservation of LCs in zebrafish.…”

Live Imaging of Heart Injury in Larval Zebrafish Reveals a Multi-Stage Model of Neutrophil and Macrophage Migration

“…Imaging neutrophils and macrophages simultaneously using timelapse LSFM led to the unexpected discovery that a small population of motile immune cells are marked by both the neutrophil marker mpx and macrophage marker mpeg1. Although, mpeg1 is known to mark B-cells in the adult (Ferrero et al, 2020), in larvae these markers are considered mutually exclusive and a pan macrophage marker (Ellett et al, 2011). To our knowledge the only other evidence of such cells comes from the previously mentioned bulk RNAseq study characterizing larval zebrafish macrophage and neutrophil gene expression (Rougeot et al, 2019).…”