Microglia, the pivotal players in maintaining the tissue homeostasis of the central nervous system (CNS), are specialized macrophages originating from the peripheral hematopoietic system. In mouse, production of macrophages comprises two successive waves, namely the transient primitive myelopoiesis occurring in the extra-embryo yolk sac (YS) and hematopoietic stem cell (HSC)-dependent definitive myelopoiesis initiating in the aorta-gonad-mesonephros (AGM) region. Similarly, macrophages in zebrafish are also generated by two successive waves of myelopoiesis. Rostral Blood Island (RBI), the zebrafish equivalent of mouse YS, gives rise to primitive macrophages that will colonize the developing brain and differentiate as microglia; whereas ventral wall of dorsal aorta (VDA), corres...[ Read more ]

Microglia, the pivotal players in maintaining the tissue homeostasis of the central nervous system (CNS), are specialized macrophages originating from the peripheral hematopoietic system. In mouse, production of macrophages comprises two successive waves, namely the transient primitive myelopoiesis occurring in the extra-embryo yolk sac (YS) and hematopoietic stem cell (HSC)-dependent definitive myelopoiesis initiating in the aorta-gonad-mesonephros (AGM) region. Similarly, macrophages in zebrafish are also generated by two successive waves of myelopoiesis. Rostral Blood Island (RBI), the zebrafish equivalent of mouse YS, gives rise to primitive macrophages that will colonize the developing brain and differentiate as microglia; whereas ventral wall of dorsal aorta (VDA), corresponding to mouse AGM, is responsible for the emergence of HSCs that produce definitive macrophages, which could potentially invade CNS in the late developmental stages. In this thesis, we took advantages of the tiny tractable zebrafish as a model and utilized both forward and reverse genetic methods to uncover molecular mechanisms underlying the development of microglia.

In the first part, we isolated a permanent micrgolia-defective zebrafish mutant, named 310, from an Ethylnitrosourea (ENU) mediated forward genetic screening. Phenotype characterization results indicated that before colonizing the brain to differentiate as microglia, macrophages in periphery are already defective, as elucidated by their reduced number as well as less branched morphology. Positional cloning and further mRNA rescue experiment confirmed that trim35-28, which codes a potential E3 ubiquitin ligase of the tripartite motif (RING-Bbox-Coiled coil) containing protein family, was responsible for the phenotypes observed in 310^-/- mutants. Ectopic expression of trim35-28 specific in myeloid cells restored microglia in 310^-/- mutants, demonstrating that this gene is cell-autonomously required for normal microglia and microphage development. Further cellular studies showed that loss of microglia and reduction of macrophages in 310^-/- mutants could solely be attributed to enhancement of p53-independent apoptosis. In summary, this study uncovered an essential factor trim35-28 that is cell-autonomously required for the development of microglia/macrophages.

In the second part, we determined that microglia arising from different origins (primitive myelopoiesis versus definitive myelopoiesis) were controlled by distinct genetic networks of pu.1 and pu.2, two essential transcription factors in the ETS family. Here, we showed that loss of pu.1, but not pu.2 was able to block the generation of primitive microglia, whereas for definitive microglia, they were only inhibited in pu.1 and pu.2 double null mutants. Further epistatic studies reveled that during the generation of primitive microglia, pu.1 acts upstream of pu.2 to induce its expression. Then expressed pu.1 and pu.2 function synergistically to control the downstream genes expression and sustain normal microglia development. Their status, i.e. more important roles of pu.1 than pu.2 in primitive microglia development relies on their endogenous expression levels and genetic hierarchies, since artificial expression of pu.2 to the level of pu.1 apparently rescued the microglia defect in pu.1 null mutants. By contrast, in the definitive wave, expression of pu.1 and pu.2 are reciprocally independent and both of them are equally important and function parallelly to sustain microglia development.

Taken together, our results uncovered the universal factor trim35-28, as well as distinct pu.1 and pu.2 genetic networks as essential molecular basis in controlling the development of primitive and definitive microglia.