Abstract
ABSTRACTMicroglia 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 (YSMs) into the embryonic brain, or regulate their acquisition of specialized properties, are currently known. Here, we present a CRISPR/Cas9-based in vivo reverse genetic screening pipeline to identify new microglia regulators using zebrafish. Zebrafish larvae are particularly suitable due to their external development, transparency and conserved microglia features. We targeted putative microglia regulators, 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 zebrafish colony-stimulating factor 1 receptor (Csf1r) ligand, Il34, caused reduced microglia numbers. Previous studies on the role of IL34 in 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. Our data also indicate that Il34 is required for YSM distribution to other organs. Disruption of the other Csf1r ligand, Csf1, did not reduce microglia numbers in mutants, whereas overexpression increased the number of microglia. This shows that Csf1 can influence microglia numbers, but might not be essential for the early seeding of the brain. In all, we identified il34 as a modifier of microglia colonization, by affecting distribution of YSMs to target organs, validating our reverse genetic screening pipeline in zebrafish.This article has an associated First Person interview with the joint first authors of the paper.
Highlights
Tissue macrophages, in addition to their immunological roles, modulate organogenesis and exhibit organ-specific regulatory properties that are thought to affect virtually all organs in vertebrates (Gordon and Martinez-Pomares, 2017; Pollard, 2009)
Similar to csf1ra−/− mutants, csf1ra CRISPants showed an 80% reduction in the number of Neutral Red (NR)+ microglia compared with controls, suggesting highly effective targeting in F0 injected embryos (Fig. 1A)
This shows that CRISPR/ Cas9-based mutagenesis can be used to reproduce mutant microglia phenotypes in Cas9-guide RNAs (gRNAs)-ribonucleoprotein complexes (RNPs)-injected zebrafish larvae
Summary
In addition to their immunological roles, modulate organogenesis and exhibit organ-specific regulatory properties that are thought to affect virtually all organs in vertebrates (Gordon and Martinez-Pomares, 2017; Pollard, 2009). Microglia are the brain’s resident macrophages, and have roles in brain development and homeostasis. Described functions of microglia include the removal of dead cells and debris, modulation of neuronal connectivity by synaptic pruning and maintenance of myelinproducing cells (Colonna and Butovsky, 2017; Hagemeyer et al, 2017; Salter and Stevens, 2017; Thion and Garel, 2017). The exact genes and mechanisms underlying the emergence of microglia in the brain and acquisition of their functional properties are still poorly understood
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