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. 2018 Feb 5;7(1):11–31.

Figure 8.

Figure 8

Overexpression of wild-type ter94 restored nuclear TBPH signal intensities in the larval CNS with neuron-specific TBPH knockdown. (A-F) Are representative images of corresponding genotypes. (A1-A3) Are immunofluorescent images of the larval CNS taken from driver control larvae carrying yw/Y; UAS-GFP-IR/+; elav-GAL4/+ (elav>UAS-GFP-IR/+). The larval CNS comprises the brain-ventral ganglia complex (BVGC). The BVGC of driver control larvae carrying elav>UAS-GFP-IR/+ showed nuclear signals from endogenous TBPH (A1). Anti-TBPH antibody immunoreactivity was evident in the nucleus of neuronal cells (B1). The TBPH signal did not colocalize with phalloidin-stained actin filaments (B2, B1+B2). (C1-C3) Are the BVGCs of TBPH knockdown larvae carrying yw/Y; UAS-TBPH-IR517-531/UAS-GFP; elav-GAL4/+ (elav>UAS-TBPH-IR/UAS-GFP). (E1-E3) Are the BVGCs of larvae that overexpress wild-type ter94 on the background of TBPH knockdown carrying yw/Y; UAS-TBPH-IR517-531/UAS-ter94; elav-GAL4/+ (elav>UAS-TBPH-IR/UAS-ter94). Panels (B1) to (B3), (D1) to (D3), and (F1) to (F3) are higher magnification images of the boxed areas in (A1), (C1), and (E1), respectively. (B1+B2, B1+B3, D1+D2, D1+D3, F1+F2, and F1+F3) Are merged images. The indirect immunofluorescence in (A1, B1, C1, D1, E1, and F1) is the signal from the polyclonal anti-TBPH antibody (based on Alexa Fulor 546 channel). The fluorescence in (A2, B2, C2, D2, E2, and F2) is from phalloidin (based on Alexa Fulor 488 channel), which labels actin. The fluorescence in (A3, B3, C3, D3, E3, and F3) is from DAPI (based on DAPI channel). (G) This graph plots the mean (± SE) of the intensity of the nuclear TBPH signal in BVGCs from third instar larvae as fluorescence emission in arbitrary units for each genotype. Columns and horizontal bars show the mean and SE of 15 nuclei, respectively. ***P < 0.001. Compared to the signal intensity of nuclear TBPH in the BVGCs of the control larvae (A1), the TBPH signal intensity in the BVGCs of larvae carrying elav>UAS-TBPH-IR/UAS-GFP (C1) was decreased (P < 0.001, G). The signal intensity of nuclear TBPH was significantly higher in larvae carrying elav>UAS-TBPH-IR/UAS-ter94 (F1) than in larvae carrying elav>UAS-TBPH-IR/UAS-GFP (D1) (P < 0.001, G). Neuron-specific TBPH knockdown reduces nuclear TBPH signal intensities in the larval CNS. And overexpression of wild-type ter94 restored nuclear TBPH signal intensities in the larval CNS with neuron-specific TBPH knockdown. The scale bars indicate 100 µm (A1 to A3, C1 to C3, and E1 to E3) and 2 µm (B1 to B3, C1 to C3, and F1 to F3).