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. 2021 Feb 8;17(2):e1009371. doi: 10.1371/journal.pgen.1009371

Fig 4. Six4 inhibits PntP1 expression in type II NB lineages.

Fig 4

In all images, type II NB lineages were labeled with mCD8-RFP or mCD8-GFP driven by pntP1-GAL4 and counterstained with a combination of various antibodies as indicated. Some type II NB lineages are highlighted with dashed lines as an example. Scale bars equal 10μm in (A-D’, E1, F1, G-H”) or 50μm in (E, F). (A-A”) In wild type type II NB lineages, PntP1 is expressed in type II NBs (white arrows) and imINPs (E(spl)mγ-, yellow arrows), but is absent in newly generated E(spl)mγ + mINPs (white arrowheads). (B-B”) PntP1 is expressed in newly generated E(spl)mγ+ mINPs (white arrowheads) in addition to type II NBs (white arrows) and imINPs (yellow arrows) in Six4 knockdown type II NB lineages. (C-C’) Erm is expressed in Ase- (open white arrows) and Ase+ (yellow arrows) imINPs in wild type type II NB lineages (dashed lines). (D-D’) Erm remains expressed in Ase- (open white arrows) and Ase+ (yellow arrows) imINPs in Six4 knockdown type II NB lineages. (E-E1”) Ase is absent in wild type type II NBs (arrows) and there are multiple mINPs (arrowheads) in individual lineages. (E1-E1”) show an enlarged view of the lineage highlighted with a dashed square in (E). (F-F1”) Six4 overexpression leads to ectopic Ase expression in the NBs (open yellow arrows) and loss of mINPs in a subset of type II NB lineages. Instead, only GMCs (yellow arrowheads) are generated in these lineages as shown in the enlarged view (F1-F1”) of the lineage highlighted with a dashed square in (F). (G-H”) Six4 overexpression (H-H”) leads to a significant reduction in PntP1 expression in Ase- type II NBs (white arrows), Ase- (open white arrows) and Ase+ (yellow arrows) imINPs compared to the wild type control (G-G”). PntP1 is almost abolished in Ase+ type II NBs (open yellow arrows) and their associated Ase+ progeny (open yellow arrowheads) in the Six4 overexpressing brain (H-H”). (I-M) Quantifications of PntP1 (I) or Erm (J) staining intensity, the number of Ase- type II NBs (K), the number of type II NB lineages with mINPs (L), and the number of mINPs/lineage (M) in wild type, Six4 knockdown or Six4 overexpressing brains. The number on top of each bar indicates the number of cells or lineages examined. ***P<0.001. NS, no significant. (N-O) Quantifications of PntP1 intensity in type II NBs (N) and imINPs (O) in wild type and Six4 overexpressing type II NB lineages with or without ectopic Ase expression in the NBs. The number on top of each bar indicates the number of cells or lineages examined. ***, p < 0.001.