EF co-misexpression overrides the Type I->0 switch and NB exit.

(A–F) Whole CNS, AFT stage, stained for PH3 (A–C) or DAPI (D–F). (A) In control, a small number of dividing cells is observed in the brain and an occasional division in the nerve cord. (B) In insc-Gal4/UAS-4xCC, aberrant cell divisions are observed in both the brain and nerve cord. (C) In insc-Gal4/UAS-6xEF, aberrant divisions are even more frequent. (A′–C′) Close-up of dividing NBs and daughter cells, identified by PH3, Dpn, and Pros. (G) Quantification of proliferation in the brain and nerve cord. Both UAS-4xCC and UAS-6xEF CNSs show significantly more proliferation than control, and UAS-6xEF significantly more than UAS-4xCC (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, Student two-tailed t test; n = 10 CNSs; ±SD). (H–J) Quantification of DAPI cell volume, in the entire CNS (H), the nerve cord (I), and the brain (J). In the CNS and brain, UAS-6xEF shows significantly larger cell volume than both control and UAS-4xCC. In the nerve cord, UAS-6xEF is only significantly larger than control. UAS-4xCC is not significantly larger than control in any measurement (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, Student two-tailed t test; n = 10 CNSs; ±SD). (K–L) NB5-6T lineages at larvae stage L1 in control and lbe(K)-Gal4/UAS-6xEF dissected CNSs. Boxed regions are magnified to the right. In control, no divisions are observed in NB5-6, neither in T nor A segments. In 6xEF co-misexpression, dividing NBs and daughter cells can be observed in both T and A segments, and the lineage is larger. (M) Quantification of the number of cells in NB5-6T at stage L1 (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, Student two-tailed t test; n = 40 lineages; ±SD). (N) Cartoon depicting the NB5-6T lineage in control and in lbe(K)-Gal4/UAS-6xEF. The numerical data underlying this figure are included in S1 Data. Genotypes: (A, D, G–J) insc-Gal4/+. (B, E, G–J) insc-Gal4/UAS-4xCC. (C, F, G–J) insc-Gal4/UAS-6xEF. (K, M) lbe(K)-Gal4, UAS-nls-myc-EGFP/+. (L–M) lbe(K)-Gal4, UAS-nls-myc-EGFP/UAS-6xEF. A, abdominal; AFT, air-filled trachea; CNS, central nervous system; DAPI, 4′,6-diamidino-2-phenylindole; DC, daughter cell; Dpn, Deadpan; EF, Early Factor; EGFP, Enhanced Green Fluorescent Protein; Gal4, Galactose4; insc, inscuteable; lbe(K), ladybird early gene fragment K; myc, C-myc epitope tag; NB, neuroblast; nls, nuclear localization signal; PH3, phosphorylated Ser10 on Histone-H3; Pros, Prospero; T, thoracic; UAS, upstream activating sequence.