Clean-up of contamination of glial clusters with cells with typical neuronal profile.

(A)Frq1 (yellow) and Nckx30C (magenta) expression were detected by in situ HCR in the cortex area of the optic lobe. GFP labelled all glial cells in green. Single focal plane. Scale bar is 20 μm. (B, C) Single focal planes showing glial somas (green) with Frq1 (yellow) and Nckx30C (magenta) expression. DAPI marks all nuclei in white. Dashed lines outline glial somas. Scale bars are 5 μm. (D) Single focal planes of MCFO clones (cyan) generated with Rdl-Gal4. Repo in green and Prospero in magenta. Dashed lines outline the neuropil. Scale bar is 7 μm. (E) The distribution of Rdl expression levels in each glial cluster of the young adult optic lobe integrated dataset, before neuronal clean-up. (F) The distribution of Rdl expression levels in all cells of the 3-day-old adult optic lobe UMAP, from [57], except glial clusters. (G) UMAP of the 20 glial clusters obtained from the first integration of optic lobe datasets. (H) UMAP of 19 glial clusters after clean-up of potential neurons by excluding cells with normalised expression of Rdl, Frq1, and Nckx30C >1, and cells with Hml expression (>0 normalised expression) as potential hemocytes. (I) Cluster #3, #6, and #8 were excluded from the UMAP in (H) since less than 1% of the cells contained in them originated from the Kurmangaliyev dataset (see Materials and methods for details). (J) The distribution of Rdl expression in each glial cluster of the embryonic dataset, before neuronal clean-up. (K) The distribution of Rdl expression in all cells of the embryonic nervous system, excluding cells belonging to the 19 glial clusters. (L) UMAP of the 19 initial glial clusters of the embryonic nervous system, before neuronal clean-up. (M) Same UMAP as in (L) after exclusion of potential neurons (normalised expression >1 of Rdl, Frq1, and Nckx30C) and potential hemocytes (normalised expression >0 of Hml expression). (N) UMAP of the remaining cells in (M) after reanalysis and reclustering (see Materials and methods for details). The data underlying this figure can be found at NCBI GEO accessions GSE208324, GSE142787 and GSE156455, GitHubs: https://github.com/AustinSeroka/2022_stage17_glia and https://github.com/VilFernandesLab/2022_DrosophilaGlialAtlas, and in S6 and S7 Data files. (TIF)