Single cell RNA Sequencing of Autofluorescent Cluster Complexes in Mouse Neonatal Retina

During early postnatal retinal development in mice, tightly coordinated interactions between neurones, blood vessels, and microglia shape the neurovascular unit. Spontaneous retinal waves—progressing through gap-junction (stage 1), cholinergic (stage 2), and glutamatergic (stage 3) signalling phases, coincide with vascular expansion. During stage 2 waves, autofluorescent cluster complexes (ACCs) appear beneath the growing vascular plexus. ACCs were isolated via FACS and then sequenced using 10x Genomics and examined via cluster analysis. Clusters were sequenced, achieving a depth of approximately 50,000 reads per cell. Raw sequencing output (BCL files) was processed with CellRanger, which converted them into FASTQ files, aligned reads to the GRCh38 reference genome, and quantified gene expression. Quality control was performed in R, where low-quality cells were removed if they contained fewer than 1000 reads, fewer than 500 detected genes, or more than 10% mitochondrial reads. Differential expression analysis was conducted to identify genes enriched in each cluster, retaining only those with adjusted p-values (p_val_adj) of 0.05 or lower. These gene lists were uploaded into Ingenuity Pathway Analysis (IPA), which integrates data from curated experimental studies to identify significant pathways, upstream regulators, and biological networks. Cluster identities were further refined through manual annotation, informed by existing retinal single-cell datasets and relevant literature.In the gene expression outputs, several variables are important to interpret. The p_val represents the probability that a result is due to chance, while the p_val_adj is corrected for multiple comparisons and therefore more reliable. The avg_log2FC indicates the average log₂ fold-change in gene expression between groups, with positive values representing upregulation and negative values downregulation. The pct.1 and pct.2 values denote the percentage of cells in each group that express a given gene. Each cluster represents a computationally defined group of cells with similar gene expression patterns, and the function field refers to the predicted or known biological role of a gene, inferred from pathway databases or published literature. Together, these methods and variables provide a comprehensive picture of retinal single-cell gene expression and the biological pathways underlying developmental processes.Cluster 0, the predominant cluster and corresponds to microglia, the retina’s resident immune cells. This identification is supported by the strong expression of markers such as <i>Aif1</i>, <i>Sparc</i>, <i>C1qb</i>, <i>Sall1</i>, <i>P2ry12</i>, <i>Cd68</i>, <i>Dock10</i>, and <i>Cx3cr1. </i>In this cluster, <i>Rbpms</i>, a well-known RGC marker, is present as well as apoptotic markers <i>Bax</i> and <i>Bbc3</i>.<br>Cluster 1 is most likely made up of endothelial cells, as indicated by strong expression of genetic markers <i>Dabd2</i> and <i>F13a1</i>.<br>Cluster 2 represents proliferative precursor cells, demonstrating notably high expression of <i>Stmn1</i> and <i>Tubb3</i>.Cluster 3 likely represents retinal progenitor cells (RPCs), as evidenced by elevated expression of proliferation-associated genes such as <i>Top2a</i>.<br><i>Neurod1</i> and <i>Rorb</i> are the most highly expressed genes in cluster 4, suggesting this cluster represents progenitor cells.This scRNA-seq data reveals microglia as key regulators of retinal development, they express genes involved in phagocytosis, apoptotic clearance, immune and neurovascular signalling, and synaptic refinement (e.g., <i>Aif1</i>, <i>P2ry12</i>, <i>Sall1</i>, <i>C1qb</i>). Microglia actively clear dying RGCs, contributing to developmental pruning and neural circuit refinement. Overall, the study demonstrates that microglial clearance of ACCs during retinal waves links neuronal activity, metabolic demand, and vascular development, highlighting microglia’s integral role in orchestrating neurovascular maturation.