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

Abstract

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...

Csf1rbmutation uncouples two waves of microglia development in zebrafish

“…3A,B, the complete loss of csf1r had no consequence on primitive macrophage ontogeny, as the number of mfap4 + cells in double mutants were similar to that of wildtype and single homozygous mutant embryos. This is consistent with our recent findings using the macrophage mpeg1:EGFP reporter line (Kuil et al, 2020). We next investigated the requirement of the different csf1r paralogs for the establishment of embryonic microglia, which differentiate in the brain parenchyma from primitive macrophages starting at 60 hpf (Ferrero et al, 2018; Herbomel et al, 2001).…”

“…Also, since the very few microglia present at 3 dpf in csf1ra or il34 -deficient embryos retain their proliferative capacity and can be increased by csf1a overexpression (Kuil et al, 2019; Oosterhof et al, 2018), it is tempting to speculate that csf1a and/or csf1b signalling through csf1rb triggers the proliferation of embryonic microglia in csf1ra mutants. In line with this interpretation, we recently showed that primitive macrophages in csf1r DM embryos, which lose interaction with all Csf1r ligands, exhibit both migration and proliferation defects (Kuil et al, 2020). Further investigation in zebrafish mutants combining Csf1 ligands and receptors knockout will be instrumental in testing this hypothesis.…”

“…Tissue macrophages constitute a highly heterogeneous compartment, based on their origin and the niche they inhabit (Bennett and Bennett, 2019; Guilliams et al, 2020). CSF1R signalling is a common pathway regulating the development of most macrophages in vertebrates, as demonstrated by their dramatic loss (including microglia) in Csf1r -deficient mice and csf1r -deficient zebrafish (Dai, 2002; Kuil et al, 2020; Oosterhof et al, 2018; Rojo et al, 2019). However, while CSF1R is represented only once in mammalian genomes, zebrafish possess two copies of the gene and their relative contribution to myelopoiesis has remained unknown.…”

A Population of Injury-Responsive Lymphoid Cells Expresses mpeg1.1 in the Adult Zebrafish Heart

“…mpeg1.1 transgenics may therefore aid the characterization of these elusive cells. Our data also support the recent description of mpeg1.1 + epidermal metaphocytes (37,38), suggesting additional markers will be required to stratify these mpeg1.1 + csf1ra 2 populations in studies in the skin.…”

“…2C, 2F), this may suggest that mpeg1.1 is expressed in an additional csf1ra-negative population unique to the skin/fin. Indeed, the morphology of these cells is similar to that of the recently described nonmyeloid cell type, metaphocytes, which perform Ag processing functions in the skin of adult zebrafish (37,38). Therefore, our data suggest that two populations of mpeg1.1 + cells that do not express csf1ra exist in adult skin.…”