Annotation of embryonic glial clusters in the VNC.

(A) UMAP of the 11 embryonic glial clusters labelled with both the cluster number (left) and our annotation of the specific glial class (and subtype; right) based on marker gene validation. (B) Expression plot of all marker genes selected for annotation, which were validated in vivo (except for Lobe). The size of the dot represents the percentage of cells with expression in each cluster, while the colour of the dot represents the level of average expression in the cluster. (C–H) MCFO clones (0 h after larval hatching) generated with the Gal4 lines indicated belonged to the main embryonic glial subtypes as accessed by their morphology. (C) Surface-only perineurial (N = 269 clones from N = 11 brains); (D) the newly defined perineurial glial subtype, termed channel-associated perineurial glia (N = 51 clones from N = 13 brains); (E) subperineurial, all morphological categories (N = 95 clones from N = 12 brains); (F) cortex (N = 295 clones from N = 13 brains); (G) ensheathing, both tract (t) and neuropil (n) (N = 190 clones from N = 11 brains); and (H) astrocyte (N = 117 clones from N = 36 brains). All clones labelled in cyan, with Repo in green and Prospero in magenta. Insets in (F–H) show Prospero and Repo in glial nuclei in alrm>MCFO clones in (H) were positive for Prospero. See S11–S13 Figs for additional in vivo marker gene validation. Dashed lines outline the neuropils. Prime panels show MCFO clones alone. Scale bars are 10 μm. The data underlying (A, B) can be found in S2 and S6 Data files. MCFO, multicolour flip-out; VNC, ventral nerve cord; UMAP, uniform manifold approximation and projection.