Notch信号通路调控斑马鱼小神经胶质细胞分化的分子机制研究

Microglia, as colonized macrophages of the brain tissue, is the first line of defense of the brain's immune system. In addition to playing the role of scavenger to clean up cell debris and pathogens, it also plays a role in the physiological activities of neurons. Important functions such as support, nutrition, protection and repair of microglia are essential for maintaining the homeostasis of the brain environment. But the molecular mechanism of microglial differentiation in the brain is still unclear. Through in vivo imaging and 3D reconstruction based on transgenic zebrafish strains that simultaneously labeled neurons and microglia, we found that the differentiation process of microglia relies on close contact with neurons. Later, by observing transgenic lines labeling microglia and Notch signals, the results suggest that microglia expresses activated Notch signals during differentiation, and the ligands and receptors of Notch such as dla, dlb, dlc, dld, jag1a, jag2b, notch1a, notch1b and notch3 is expressed in the midbrain, suggesting that microglia differentiation is related to Notch signal activation. A closer inspection of 3dpf zebrafish Notch-deficient mutant mibta52b showed that the number of myeloid cells in the midbrain did not change but the number of microglia decreased significantly. At the same time, activation of the Notch signaling pathway in myeloid precursor cells can promote the early maturation of microglia. These data suggest that microglia differentiation requires activation of the Notch signaling pathway mediated by neurons. This is a candidate signaling pathway for neuron to regulate microglia differentiation which has not been reported yet. It is of great significance to further analyze the molecular regulation mechanism of microglia development. Later, we will focus on 1) elucidating specific ligands of Notch signaling pathway that regulate microglia differentiation in neurons and their corresponding receptors , 2) exploring important downstream molecular networks of Notch signaling pathway in regulating microglia differentiation, 3) revealing important molecules and mechanisms that mediate close interaction between neurons and microglia.

小神经胶质细胞（microglia）对神经系统的构建及稳态维持至关重要。虽然已有研究表明microglia起源于原始髓系细胞，但它们在脑中分化的分子机理仍不清楚。在前期工作中，我们发现1）microglia在分化过程中与神经元紧密接触；2）且Notch信号会被激活；3）在神经元中过表达Notch配体或在髓系前体细胞中激活Notch信号均可使microglia提前分化。这些结果表明microglia的分化可能依赖神经元介导的Notch信号通路激活，这是目前未见报道的可能调控机制，对于进一步解析microglia发育的分子调控机理意义重大。在本研究中将：1）继续明确神经元细胞中调控microglia前体分化的特异Notch配体及对应受体，2）探究Notch信号通路调控microglia分化的重要下游分子信号网络，3）揭示介导神经元和microglia紧密互作的重要分子及其机制。

小神经质细胞（microglia）作为定植于脑组织的巨噬细胞，是大脑免疫系统的第一道防线。除发挥清除细胞碎片和病原体的清道夫作用外，还在神经元的生理活动中发挥作用。microglia的支持、营养、保护和修复等重要功能对维持脑环境的稳态至关重要。但脑内microglia分化的分子机制仍不清楚。研究基于斑马鱼胚胎发育早期microglia前体分化成熟的主要特征（特异分子标志物的表达；细胞形态的改变；吞噬功能的增加；细胞运动速率的减弱），共聚焦活体实时成像和3D重构发现microglia在分化过程中与神经元互相接触，深入解析揭示了发育神经元凋亡通过核苷酸信号介导microglia的迁移以及与神经元互作，且单细胞转录组测序明确microglia中Notch途径被激活。进一步深入分析揭示神经元通过调控Notch配体DeltaB，进而结合microglia细胞膜上的Notch1a，最终调控与其密切接触的microglia前体的Notch活性，使其成熟。进一步实验数据证实脑微环境的DLL3-Notch信号轴调控microglia成熟在高等哺乳类动物中是保守的。深入解析表明Notch通路通过溶酶体途径调控microglia的吞噬功能。综上所述，该研究提示了神经元通过与microglia前体互作来激活后者的Notch，从而促进microglia分化。我们的研究解析的一个保守的脑微环境孵育microglia特性的分子机制有助于我们理解神经系统和免疫系统的互作对于microglia发育和功能的影响，进而为实现有效诱导microglia成熟并将其作为潜在靶点来缓解和治疗神经系统疾病提供指导（Nature Communications，2022）。此外，通过硝基还原酶/甲硝唑系统(NTR/MTZ)诱导斑马鱼幼鱼胸腺淋巴细胞损伤模型，揭示胸腺淋巴细胞再生主要来源于肾脏迁移而来的前体细胞，以及Cxcl12b/Cxcr4b趋化因子信号通路被激活以促进前体细胞的迁移，进而恢复受损的胸腺淋巴细胞（Journal of Genetics and Genomics，2022）。该研究构建的斑马鱼体内胸腺淋巴细胞损伤模型以及相关分子机制，有助于为胸腺淋巴细胞受损后提供治疗和药物筛选价值。