Microglia are predominant immune cells resident in central nervous system (CNS) and play numerous functions for maintaining CNS homeostasis and participating in immune responses locally. In zebrafish, there are at least two waves of hematopoiesis contributing to microglia. RBI derived primitive microglia colonize in brain at embryonic stage and they are gradually replaced by VDA derived definitive microglia during development until adulthood. Microglia are long been believed to be maintained by self-renew without contribution from circulation in physiological conditions. However, the mechanisms for microglia maintenance are still largely unknown. Here, we firstly established the visible conditional knockout method with high efficiency to achieve spatiotemporal inactivation of p...[ Read more ]

Microglia are predominant immune cells resident in central nervous system (CNS) and play numerous functions for maintaining CNS homeostasis and participating in immune responses locally. In zebrafish, there are at least two waves of hematopoiesis contributing to microglia. RBI derived primitive microglia colonize in brain at embryonic stage and they are gradually replaced by VDA derived definitive microglia during development until adulthood. Microglia are long been believed to be maintained by self-renew without contribution from circulation in physiological conditions. However, the mechanisms for microglia maintenance are still largely unknown. Here, we firstly established the visible conditional knockout method with high efficiency to achieve spatiotemporal inactivation of pu.1. This method was based on intron-targeting non homologous end joining (NHEJ)-mediated knockin of large fragment into genome and we generated the visible conditional knockout pu.1^KI allele. Next, we demonstrated that pu.1 was critical for definitive microglia long-term maintenance. Inactivation of pu.1 in microglia reduced their proliferation rate and led them gradually replaced by wild type microglia. Finally, we figured out that pu.1 and spi-b played redundant roles for microglia short-term survival. This mechanism was conserved in both primitive and definitive microglia. Taken together, our works provided a useful platform for zebrafish genetic study and gave new insights into functions of pu.1 and spi-b for microglia maintenance.