The embryonic zebrafish brain is exclusively colonized by -dependent and lymphatic-independent population of microglia.

Microglia, the crucial immune cells inhabiting the central nervous system (CNS), perform a range of vital functions, encompassing immune defense and neuronal regulation. Microglia subsets with diverse functions and distinct developmental regulations have been identified recently. It is generally accepted that all microglia originate from hematopoiesis and depend on the myeloid transcription factor PU.1. However, a recent study reported the existence of microglia in zebrafish embryos, which are seemingly independent of Pu.1 and reliant on lymphatic vessels, sparking great interest in the possibility of lymphatic-originated microglia. To address this, we took advantage of a knock-in zebrafish allele for a detailed investigation. Our results conclusively showed that almost all zebrafish embryonic microglia (~95% on average) express . Further, lineage tracing and mutant analysis revealed that these microglia neither emerged from nor depended on lymphatic vessels. In essence, our study refutes the presence of -independent but lymphatic-dependent microglia.