The EnR-Ets chimeric repressor antagonizes endogenous PntP1’s function.

NB lineages are labeled with mCD8-GFP (in green) driven by insc-GAL4 for type I NB lineages (B-F) or by pntP1-GAL4 for type II NB lineages (G-L), and counterstained with anti-Dpn (in red) or anti-Ase (in blue) antibodies. White arrows point to some Ase⁺ Dpn⁺ type I NBs or Ase^- Dpn⁺ type II NBs as examples. White arrowheads point to Ase⁺ Dpn⁺ mINPs or mINP-like cells. Yellow arrowheads point to Ase⁺ Dpn^- GMCs. Dashed lines highlight representative NB lineages enlarged in insets. In this and all the following figures, only images from one brain lobe or the thoracic segments of VNCs are shown. The brain lobes are oriented so that the midline is to the right and the anterior side of the brain up. Scale bars equal 50μm. (A) Schematic diagrams of constructs used to express PntP1, NLS-EnR-Ets, NLS-Ets, or NLS-EnR. (B-B’) VNCs contain only Ase⁺ Dpn⁺ type I NBs, which directly produce Ase⁺ Dpn^- GMCs. (C-C’) Misexpressing UAS-pntP1 in type I NBs represses Ase expression and induces Ase⁺ Dpn⁺ mINP-like cells. (D-F’) Misexpressing UAS-NLS-EnR-Ets (D-D’), UAS-NLS-Ets (E-E’) or UAS-NLS-EnR (F-F’) in type I NBs does not repress Ase expression or induce any mINP-like cells. (G-H’) A wild type (G-G’) or UAS-pntP1 overexpressing brain lobe (H-H’) has eight type II NB lineages, each of which contains an Ase^- Dpn⁺ type II NB and multiple Ase⁺ Dpn⁺ mINPs. (I-I’) Type II NBs with UAS-NLS-EnR-Ets misexpression ectopically expresses Ase and directly produces Ase⁺ Dpn^- GMCs instead of INPs. Note that UAS-NLS-EnR-Ets misexpression also leads to the generation of supernumerary type II NB (see white arrows) lineages. (J-K’) Misexpressing UAS-NLS-Ets (J-J’) or UAS-NLS-EnR (K-K’) does not affect the normal development of type II NB lineages, including the repression of Ase, the number of type II NBs, or the composition of cell types. (L-L’) Knocking down PntP1 by expressing UAS-pnt RNAi results in ectopic Ase expression in type II NBs, loss of INPs and generation of supernumerary type II NBs. (M-N) Quantifications of the percentage of Ase^- type I NBs and the percentage of type I NB lineages with mINPs in VNCs (M), and the percentage of Ase⁺ type II NBs, the percentage of type II NB lineages with mINPs, and the number of total GFP⁺ type II NBs per brain lobe (N) with indicated genotypes. ***, P < 0.001. Values of the bars are mean ± SD. The numbers on top of, or within each bar in graphs in this and all following figures indicate the number of samples examined.

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