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To understand glial development and function, we comprehensively annotated the glial cells from two large single-cell mRNA-sequencing (scRNA-seq) atlases of the developing\n                    <jats:italic>Drosophila<\/jats:italic>\n                    visual system. This allowed us to identify all glial cell types from larval to adult stages and follow their developmental trajectories to understand how the diversity of glial types is generated during development. We show that whereas most glial types, such as chiasm glia, gradually change their transcriptome as they mature during development, neuropil glia that transcriptionally appear as a single-cell class in larvae, splits into ensheathing (EG) and astrocyte-like (ALG) glia types during pupal stages. We have experimentally validated these developmental trajectories and identified the genetic markers expressed through the differentiation between EG and ALG classes. Unexpectedly, our analysis of scRNA-seq datasets allowed us to discover that the transcriptome of glial cell bodies can be distinguished from that of their processes. We have identified that processes are enriched for distinct mRNAs that were validated in vivo. This work provides the most detailed transcriptomic analysis of optic lobe glia during development and helps explain the expansion of glial diversity observed in the adult visual system. We also present an innovative computational approach to identify mRNA species that are differentially localized to cell bodies or cellular processes.\n                  <\/jats:p>","DOI":"10.1371\/journal.pbio.3004015","type":"journal-article","created":{"date-parts":[[2026,9,28]],"date-time":"2026-09-28T17:52:09Z","timestamp":1790617929000},"page":"e3004015","update-policy":"https:\/\/doi.org\/10.1371\/journal.pbio.corrections_policy","source":"Crossref","is-referenced-by-count":0,"title":["A developmental single-cell atlas of the Drosophila visual system glia reveals cell type diversification and subcellular mRNA compartmentalization"],"prefix":"10.1371","volume":"24","author":[{"given":"Amanda A. 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