Figure 6.

Mutations in the RNA-binding and DAZ domains reduce the ability of Boule to block axon pruning. A, Schematic of UAS-Boule-A::FLAG transgenes. The wt transgene expresses Boule-A with three FLAG epitopes (gray box) at the C terminus (Bol-A). Boule contains a conserved RNA-binding domain (black box) composed of type 1 and type 2 RRMs (lines), and a DAZ repeat domain (blue box). The BolPM1 transgene has a point mutation (K73L) in the RRM1 domain of Bol-A, and the BolΔDAZ transgene is truncated at amino acid 159, thereby deleting the DAZ domain. B, Western blot of brain extracts from larvae coexpressing mCD8::GFP and Boule-FLAG transgenic proteins in MB neurons driven by OK107-GAL4. The top panel shows staining with a monoclonal antibody against FLAG. The bottom panel shows the same blot after stripping and reprobing with anti-GFP as a control for protein loading. All larvae express mCD8::GFP. Larvae with two insertions of the BolPM1 transgene (2× PM1) or one insertion of BolΔDAZ have higher protein expression levels compared with larvae with one copy of the wt BolA transgene. C–F, MB γ neuron axon pruning at 24 h APF, in which γ neurons are labeled with mCD8::GFP driven by 201Y-GAL4. At this time point, axons have completely degenerated in wt γ neurons (C), with few axon fragments remaining at the tips of the former dorsal lobe (open arrowhead). Transgenic expression of wt Bol-A in γ neurons (D) partially inhibits axon pruning, as evident by the GFP-positive axons in the dorsal lobe (arrowhead). MB γ neurons expressing 2× PM1 (E) or BolΔDAZ (F) mutant protein prune normally. G–J, Axon pruning defects in adult MB neurons expressing Boule transgenes driven by OK107-GAL4. Transgenic protein expression levels were increased by using OK107-GAL4, which strongly expresses in all classes of MB neurons but has little expression outside of the CNS. The panels below G–J show enlarged images of the dorsal lobe, with FasII staining on the left and OK107-GAL4 driven mCD8::GFP on the right. MB γ neurons expressing transgenic wt Bol-A (H) or BolPM1 mutant protein (I) have a thick dorsal lobe with ectopic FasII-positive axons in the dorsal lobe (arrowheads), indicating a failure to prune. MB neurons expressing BolΔDAZ mutant protein (J) are similar to wt MBs (G). C–J show confocal Z projections of the MB axon lobes. n > 8 brains for each experiment. Scale bars, 50 μm.