Cheng Siao Muk, Ho Chi-Lin, Chen Shiou-Lan, Huang Yi-Te, Mao Pin-Cheng, Lin Tzu-Chia, Chen Jia-Shing, Sun H Sunny, Hwang Daw-Yang, Chu Chun-Hsien
National Institute of Cancer Research, National Health Research Institutes, Tainan, Taiwan.
Institute of Molecular Medicine, College of Medicine, National Cheng Kung University, Tainan, Taiwan.
Glia. 2025 Nov;73(11):2236-2252. doi: 10.1002/glia.70064. Epub 2025 Jul 25.
As the primary brain-resident macrophages, embryonic microglia (EM) display functional diversity and significant heterogeneity, which are essential for normal brain development and growth. However, the heterogeneous nature of EM and their developmental trajectory remain contentious. This study isolated individual cells from the brains of embryonic day 14 (E14) mice without using a microglial cell sorting method and subsequently performed single-cell RNA sequencing (scRNA-seq) analysis. Unsupervised subclustering of the microglial population based on gene expression profiles revealed two novel EM subclusters: approximately 60% EM1 (CD68-negative and Iba-1-positive) and about 40% EM2 (CD68- and Iba-1-double-positive). Additionally, bioinformatics analyses indicated that the EM1 cluster represents relatively early and immature microglia with high proliferative capacity. In contrast, the EM2 cluster exhibits a higher expression of genes involved in the stepwise program of microglial development, synaptic phagocytosis, regulation of neuron differentiation and projection, and interaction with other brain cells. To further confirm these findings, double or triple immunofluorescence staining of Iba-1, CD68, or the presynaptic marker synaptophysin demonstrated the presence of the EM1 and EM2 clusters in E14 mouse brains, as well as increased synaptic phagocytosis in the EM2 cluster. Moreover, by monitoring their proportional changes in the brains on postnatal days 1, 14, and 90, our data disclosed the developmental trajectory of the EMs as they transition from CD68-negative to CD68-positive after the postnatal period stages. Overall, this study opens new avenues for exploring the functional diversity and developmental trajectory of EMs during embryonic brain development and growth.
作为主要的脑内常驻巨噬细胞，胚胎小胶质细胞（EM）表现出功能多样性和显著的异质性，这对正常脑发育和生长至关重要。然而，EM的异质性本质及其发育轨迹仍存在争议。本研究未使用小胶质细胞分选方法，从胚胎第14天（E14）小鼠的大脑中分离出单个细胞，随后进行单细胞RNA测序（scRNA-seq）分析。基于基因表达谱对小胶质细胞群体进行无监督亚聚类，发现了两个新的EM亚群：约60%的EM1（CD68阴性且Iba-1阳性）和约40%的EM2（CD68和Iba-1双阳性）。此外，生物信息学分析表明，EM1亚群代表相对早期和不成熟的小胶质细胞，具有较高的增殖能力。相比之下，EM2亚群在小胶质细胞发育的逐步程序、突触吞噬、神经元分化和投射的调节以及与其他脑细胞的相互作用等方面，表现出更高的基因表达。为进一步证实这些发现，对Iba-1、CD68或突触前标记物突触素进行双重或三重免疫荧光染色，证明E14小鼠大脑中存在EM1和EM2亚群，且EM2亚群的突触吞噬增加。此外，通过监测出生后第1天、第14天和第90天大脑中它们的比例变化，我们的数据揭示了EM在出生后阶段从CD68阴性转变为CD68阳性的发育轨迹。总体而言，本研究为探索胚胎脑发育和生长过程中EM的功能多样性和发育轨迹开辟了新途径。
