Figure 1(A) Larval NSCs of control (ctrl; yw) and pr-set7716 (716) at 24h ALH were stained for Deadpan (Dpn) and Miranda (Mira). (B, C) Quantifications of cellular extensions (B) and NSC diameters (C) for genotypes in (A). In (B), n=1055, 12 brain lobes [BL] for control; n=1000, 15 BL for pr-set7716. In (C), n=101 for control; n=112 for pr-set7716. Data information: Quantification data are presented in as mean ± SD (B, C n numbers in (B, were total NSCs counted with total brain lobes measured, while each brain lobe considered as one biological replicate. White arrows point to NSCs and yellow arrowheads point to cellular extensions of NSCs. Statistical analyses were done comparing two different genotypes using Mann-Whitney test (B, C, ***P < 0.001. Scale bars, 5 μm (A, (D) A schematic representation of the pr-set7716 mutation. pr-set7716 contains a G to A point mutations at 831 nucleotides (nt). Drosophila Pr-set7 contains a SET domain (in yellow), which is required for its methyl transferase activity. (E) Larval NSCs of control (ctrl; yw) and pr-set7716/Df(3R) ED5644 (716/Df) at 96h ALH were labelled with Dpn and Mira. Data information: White arrows point to NSCs and yellow arrowheads point to cellular extensions of NSCs. Scale bars, 5 μm (F, G) Quantifications of cellular extensions (F) and NSC diameters (G) for genotypes in (E). In (F), n=1488, 15 BL for control; n=2014, 20 BL for pr-set7716/Df. In (G), n=92 for control; n=93 for pr-set7716/Df. (H) Quantifications of EdU incorporation for genotypes in (I). Control, n=1365, 16 BL; pr-set7716/Df, n=1006, 16 BL. Data information: Quantification data are presented in as mean ± SD F, G, H, n numbers in were total NSCs counted with total brain lobes measured, while each brain lobe considered as one biological replicate. Statistical analyses were done comparing two different genotypes using Mann-Whitney test F, G, H, ***P < 0.001. (I) Larval brains of control (ctrl; yw) and pr-set7716/Df at 96h ALH were labelled with Dpn and EdU. Data information: Scale bars, 30 μm (I). (J, K) Western blot and quantification analysis of larval brain extracts of control (ctrl; yw), pr-set7716/Df and UAS-pr-set7 induced with insc­-Gal4 at 72h ALH (n=2 as biological replicates). Blots were probed with anti-Pr-set7 antibody or anti-Actin antibody (loading control). Pr-set7 levels were normalized against actin intensity. (L) Larval NSCs of control (ctrl; yw), pr-set7716/Df, and UAS-pr-set7 induced with insc­-Gal4 at 72h ALH were stained for Pr-set7, Mira and DNA. (M, N) Quantifications of NSC diameters (M) at 24h ALH and EdU incorporation (N) for genotypes in (O). In (M), n=152 for control; n=129 for pr-set7716/Df. In (N), for control, n=601, 13 BL at 24h ALH; n=988, 14 BL at 48h ALH; n=813, 9 BL at 72h ALH and n=829, 10 BL at 96h ALH. For pr-set7716/Df, n=566, 12 BL at 24h ALH; n=808, 13 BL at 48h ALH; n=1032, 10 BL at 72h ALH and n=825, 9 BL at 96h ALH. Data information: Quantification data are presented in as mean ± SD M, N). n numbers in N) were total NSCs counted with total brain lobes measured, while each brain lobe considered as one biological replicate. White arrows point to NSCs Statistical analyses were done comparing two different genotypes using Mann-Whitney test M, N). ***P < 0.001. Scale bars, 5 μm L), (O) Larval brains of control (ctrl; yw) and pr-set7716/Df at 24h, 48h, 72h and 96h ALH were labelled with Dpn and EdU. Data information: Yellow dotted boxes indicate the region of zoomed-in images (O). White arrows point to NSCs The central brain is to the left of white dotted lines O). Scale bars, 30 μm (I).