Functional Diversity of Two Novel Embryonic Microglial Subpopulations and Their Developmental Trajectories in Developing Mouse Brains

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.