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. Author manuscript; available in PMC: 2016 May 1.
Published in final edited form as: Dev Neurobiol. 2014 Oct 20;75(5):485–493. doi: 10.1002/dneu.22237

Figure 2. Reducing Bj1 levels leads to loss of larval neuroblasts by an apoptosis-independent mechanism.

Figure 2

(A) The number of neuroblasts present per brain lobe at four time points after larval hatching (ALH) were quantified for wild type control RNAi and Bj1 RNAi larvae (at 26°C). There were ~100 Dpn+ neuroblasts per brain lobe in the control at each time point, whereas the number of Dpn+ neuroblasts per lobe steadily decreased over time in the Bj1 LOF background.

(B–E) Wild type control and Bj1 LOF brain lobes are shown stained for Mira, Dpn, and Pros (which is weakly expressed in the cytoplasm of wild type neuroblasts and strongly expressed in wild type neurons). (B, D) The neuroblasts shown in the wild type control brain and the Bj1 LOF brain at 24h ALH are equally as large, suggesting the neuroblasts in both genotypes have exited quiescence and have grown in order to re-enter the cell cycle. (C, E) By 48h ALH, the number of neuroblasts has decreased in the Bj1 LOF background compared to wild type, though the neuroblasts present are still as large as the wild type neuroblasts, showing they are not entering quiescence.

(F) Neuroblasts in the Bj1 RNAi background are not undergoing apoptosis because the addition of baculovirus p35 (a potent inhibitor of apoptosis) does not rescue neuroblast number. The first genotype is Wor-Gal4, UAS-Dicer2; UAS-NLS:GFP/TRiP mCherry RNAi (denoted as WT, for wild type) and it is a control that yields wild type larvae; the second genotype is Wor-Gal4, UAS-Dicer2; UAS-p35/TRiP mCherry RNAi (denoted WT, p35) and it is a control using p35; the third genotype is Wor-Gal4, UAS-Dicer2/Bj1 TRiP RNAi; UAS-NLS:GFP (denoted Bj1−) and is a knockdown of Bj1; and the fourth genotype is Wor-Gal4, UAS-Dicer2/Bj1 TRiP RNAi; UAS-p35 (denoted Bj1−, p35) and it is an attempted rescue of Bj1 RNAi neuroblast number by the addition of p35. Each genotype assessed had a total of 4 transgenes, with 2 total transgenes per genotype driven by the neuroblast specific Wor-Gal4 promoter. The UAS-NLS:GFP controls for the UAS-p35, while the TRiP mCherry RNAi is a nonsense RNAi that controls for the Bj1 TRiP RNAi. Scale bars in (B-E) are 50μm. For (A), n = 3, 5, 5, and 3 for WT and n = 5, 5, 4, and 4 for Bj1 RNAi, at 24, 48, 72, and 96 h ALH respectively. For (F), n = 10 for all four genotypes. Error bars are +/- one standard deviation.