Pu.1和Spi-b在斑马鱼小胶质细胞维持中的功能和机制研究

Microglia are a group of specified macrophages residing in central nervous system (CNS) and play pivotal roles in the regulation of neuronal development and maintaining the homeostasis of CNS in physiological condition. Furthermore, in pathological conditions, microglia are also highly associated with the onset and progression of many neuronal disorders, such as Alzheimer’s disease, Parkinson disease and multiple sclerosis (MS). In mice and zebrafish, it was documented that early development of microglia is tightly controlled by the ETS family transcription factor PU.1. However, due to the complete loss of microglia/macrophages in PU.1-deficient mouse or zebrafish embryos, our understanding of the role of Pu.1 in the maintenance of microglia pool after its establishment is still lacking. In our previous work, we generated a pu.1 conditional knockout allele pu.1KI via Non-homologous end jointing (NHEJ) mediated knockin method. Phenotypical analysis revealed that conditional depletion of Pu.1 in microglia had no effect on their maintenance. However, when we inactivated Pu.1 in the microglia of spi-b mutant, their number rapidly declined. Hence, in this project, we are planning to figure out the cellular basis underlying the reduction of microglia number in this process and further investigate the genetic interactions and molecular mechanism of Pu.1 and Spi-b in the regulation of microglia maintenance. Successful fulfillment of this project will not only broaden our knowledge on the functions of Pu.1 and Spi-b, but also provide new insights into the understanding of molecular mechanisms underlying microglia maintenance, which could hopefully contribute to the intervention and treatment of microglia-related neuronal disorders.

小胶质细胞是驻扎在中枢神经系统的巨噬细胞，它们不仅在生理状态下对中枢神经系统的发育和稳态的维持具有重要的作用，而且在病理状态下还与多种神经系统疾病，比如阿尔兹海默综合症等的诱发与恶化有着紧密的联系。研究人员发现，无论小鼠和斑马鱼，早期小胶质细胞的发育都受到ETS家族转录因子PU.1的严格调控。然而由于PU.1缺陷导致小胶质细胞的完全缺失，因此我们对PU.1在小胶质细胞稳态维持中的作用却不得而知。在我们的前期工作中，我们通过CRISPER/Cas9介导的非同源末端连接的基因敲入手段建立了pu.1条件性敲除斑马鱼品系，并发现小胶质细胞中单独敲除pu.1对其后续的数量维持没有影响。于此同时，当在spi-b突变体中对pu.1进行条件性敲除后，小胶质细胞的数量则在短时间内发生了明显的减少。因此，本项目中，我们将针对这一现象进一步探究Pu.1和Spi-b缺陷后小胶质细胞数量减少的细胞学基础和分子机制。

小胶质细胞是驻扎在中枢神经系统的巨噬细胞，它们对于中枢神经系统的稳态维持至关重要。小胶质细胞的发育或功能异常通常由于多种神经退行性疾病的发生和发展联系紧密。因而探究小胶质细胞群体的建立和维持对于小胶质细胞的功能行使，以及对于相关疾病的理解和干预都有着重要的意义。Pu.1是ETS家族转录因子的一员，它对于小胶质细胞的发育至为重要。在小鼠和斑马鱼中Pu.1功能缺陷导致小胶质细胞不能正常发育，然而Pu.1在小胶质细胞维持中的功能却不得而知。本项目中，我们利用 CRISPR/Cas9 介导的非同源末端连接的基因敲入手段建立了 pu.1 条件性敲除斑马鱼品系。研究发现当在 spi-b突变体的小胶质细胞中将 pu.1 条件性敲除后，无论RBI起源的胚胎小胶质细胞还是VDA起源的成年小胶质细胞均在短时间内发生细胞凋亡。 进一步的研究表明，转录因子Mafba， Mafbb位于Pu.1和Spi-b下游。在mafba，mafbb双突变体中，其大脑的小胶质细胞也不能存活，表明Pu.1和Spi-b缺失导致小胶质细胞凋亡的其中一个机制是下游Mafba， Mafbb的缺失。此外，我们还发现单独敲除Pu.1导致小胶质细胞的长期维持发生障碍，具体在细胞水平表现为其增殖能力下降，并且凋亡增加。综上所述，该研究中，我们通过构建pu.1条件性敲除斑马鱼品系，揭示了转录因子Pu.1在小胶质细胞短期存活以及长期维持中的不同功能。该项目加深了对小胶质细胞种群维持分子机制的理解，对于相关神经退行性疾病的致病机理和干预提供了一定的理论基础。