Fig 2: Notch signalling is reduced in Snr1 knockdown cells. (A) Combined UMAP of optic lobe neuroepithelial cells and neuroblasts from control brains and brains with knockdown of Snr1 in neuroepithelial cells. (B) UMAP showing distribution of optic lobe cells from the control brains (CTRL, red) and the Snr1 knockdown brains (KD, blue). (C,D) UMAPs showing expression of Notch targets E(spl)m 4-BFM and E(spl)m?-HLH in neuroepithelial cells and neuroblasts represented in A and B. (E) Average expression of E(spl) genes in control and the Snr1 knockdown in neuroepithelial cells.

Fig 3: Cells expressing the neuroblast marker Deadpan are present in Snr1R3 clones in the adult optic lobe. Clones generated at 31 h after larval hatching (ALH). (A-E) Control MARCM clones marked with GFP. (A′-E′) Higher magnification images of region outlined in E. (F-J) Snr1R3 MARCM clone marked with GFP. (F′-J′) Higher magnification images of region outlined in J. In the merge, DAPI is in blue, GFP is in green, Dpn is in white and Snr1 is in red. Scale bars: 50 µm in E,J; 20 µm in E′,J′.

Fig 4: Snr1 is required for the transition from neuroepithelial cells to neuroblasts. (A) Timeline of larval development showing timing of clonal induction and dissections after larval hatching (ALH). (B-C′) Snr1 mutant clones in the optic lobe have altered location and morphology. (B,B′) Control MARCM clone at surface of brain imaged in cross-section. (C,C′) Snr1R3 MARCM clone located deeper inside the brain showing ectopic expression of Deadpan (Dpn; arrowheads). Cell junctions are marked by DE-cad, neuroblasts are marked by Dpn, MARCM clones are marked by GFP and GMCs are marked by Pros. (D-E″) Snr1 mutant clones deep in the medulla express markers for neuroepithelial cells and neuroblasts. (D-D″) Control optic lobe MARCM clone marked by GFP. Clone is outlined by a white dashed line. (E-E″) Snr1R3 optic lobe MARCM clone marked by GFP. Clone is outlined by a white dashed line. Neuroepithelial cells marked by PatJ (red) and neuroblasts marked by Dpn (white). (F-F′) Snr1R3 clone cells on the surface of the brain are smaller (arrowheads) and express neuroepithelial (NE) cell marker. Neuroepithelial cell junctions are marked by PatJ (magenta) and MARCM clones are marked by GFP (green). (G-H‴) MARCM clones imaged in cross-section at mid third instar stage. Brackets indicate transition zone. Arrows indicate ectopic Dpn+ cells. Scale bars: 20 µm.

Fig 5: Expression of Broad (br) and Eip93F is reduced in Snr1 knockdown cells. (A-D) Violin plots showing expression of Snr1 (log2FC=-1.17, P=6e-11), br (log2FC=-1.94, P=1e-8), Eip93F (log2FC=-2.34, P=2e-11) and inscuteable (insc) (P=0.10) in neuroblasts from control brains and from brains expressing Snr1RNAi in neuroblasts. ***P<0.001; ns, not significant. (E) FLP-out control clones. (E′) Higher magnification images of the region outlined in E. Clone is outlined by a yellow dashed line. (F) FLP-out clones expressing Snr1RNAi. (F′) Higher magnification image of the region outlined in F. Clone is outlined by a yellow dashed line. In merged images in E-F′, Snr1 is shown in green, Broad-Core (Br-C) is in white, RFP is in red and DAPI is in blue. (G) FLP-out control clones. (G′) Higher magnification image of region outlined in G. Clone is outlined by a yellow dashed line. (H) FLP-out clones expressing Snr1RNAi. (H′) Higher magnification image of region outlined in H. Clone is outlined by a yellow dashed line. In merged images in G-H′, Snr1 is shown in green, Eip93F (E93) is in white, RFP is in red and DAPI is in blue. Scale bars: 50 µm in E,F,G,H; 20 µm in E′,F′,G′,H′.