Reverse genetic screen reveals that Il34 facilitates yolk sac macrophage distribution and seeding of the brain

Abstract

Microglia are brain resident macrophages, which have specialized functions important in brain development and in disease. They colonize the brain in early embryonic stages, but few factors that drive the migration of yolk sac macrophages into the embryonic brain, or regulate their acquisition of specialized properties are currently known.\n\nHere, we present a CRISPR/Cas9-based in vivo reverse genetic screening pipeline to identify new regulators important for microglia development using zebrafish. Zebrafish larvae are particularly suitable due to their external development, transparency, high fecundity and conserved microglia features. We targeted putative microglia regulators, including signature genes and non-cell autonomous factors, by Cas9/gRNA-complex injections, followed by neutral red-based visualization of microglia. Microglia were quantified automatically in 3-day-old larvae using a software tool we called SpotNGlia. We identified that loss of function of the zebrafish homolog of the colony stimulating factor 1 receptor (CSF1R) ligand IL34, caused strongly reduced microglia numbers in early development. Previous studies on the role of the IL34 on microglia development in vivo were ambiguous. Our data, and a concurrent paper, show that in zebrafish, il34 is required during the earliest seeding of the brain by microglia progenitors. Our data also indicate that Il34 is required for distribution to other organs.\n\nPreviously, we showed that csf1ra and csf1rb double mutant zebrafish have no microglia. As there is a moderate effect of il34 on microglia development, relative to the effect csf1r, additional Csf1r-dependent signalling may be needed for establishment of the microglia network. In all, we identified il34 as a modifier of microglia colonization, by affecting distribution of yolk sac macrophages to target organs, validating our reverse genetic screening pipeline in zebrafish which can be used for the identification of additional regulators of microglia development.