Figure 7. Wnd restricts the expression level of presynaptic components Brp and VGlut in unc-104 mutants.

(A–D) Immunostaining for VGlut (green, (A and B) and Brp (magenta, (C and D) in motoneuron cell bodies (A and C) and NMJ terminals (B and D). In (A) motoneuron nuclei are indicated by Elav staining (blue). Note the increased intensity of VGlut and Brp in unc-104^bris/P350; wnd³ double mutants. (E) Quantification of VGlut and Brp intensity in cell bodies of motoneurons, corresponding to images in A and C. Note that total intensity measured at NMJ terminals is shown in Figure 2E–F. (F) Estimates generated for the total levels of VGlut and Brp proteins within motoneurons, accounting for the summed intensities measured in cell body, axonal, and synaptic compartments. Methods and assumptions used to calculate these estimates are described in Experimental Procedures. These proteins predominantly localize to the NMJ synaptic terminals (the proportion of the total protein localized to synapses is indicated with black shading). In unc-104-hypomorph mutants, a larger percentage is detected in cell bodies (medium gray shading), and the total summed intensity is reduced. In unc-104; wnd double mutants, the intensity increases in all of the compartments, cell body, axon (light gray shading) and synapse, compared to unc-104 mutants alone. Wt animals are Canton S. (G) Images of motoneuron nuclei, immunostained for DsRed in vglut promoter-DsRed reporter lines. (H) Quantification of vglut-DsRed intensity in (G), normalized to controlRNAi (moody RNAi). UAS-RNAi lines were driven by OK6-Gal4. For cell body analysis, at least 2 dorsal abdominal clusters of motoneurons per animal from at least 6 animals per genotype were examined. All data are represented as mean ±SEM; ****p<0.0001, ***p<0.001, **p<0.01, *p<0.05, Tukey test for multiple comparison; Scale bar, 20 μm. For additional data, see Figure 7—figure supplements 1–3.

Figure 7—figure supplement 1. In unc-104-null mutants, multiple synaptic proteins were down-regulated by Wnd, (related to Figure 7).

(A) Schematic cartoon showing the locations of motoneuron cell bodies (within one segment, denoted by dotted lines) and Neuropil (green) in the nerve cord at embryonic stage 17. (B) Brp, SytI, VGlut and CSP immunostaining in stage 17 embryonic nerve cord (20–21 AEL). Increased staining in unc-104^null;wnd³ mutants is noted in the cell body region. (C) Quantification of total intensity of SytI and VGlut from (B). At least 6 animals were examined per genotype. All data are represented as mean ±SEM; N.S., not significant, ****p<0.0001, ***p<0.001, *p<0.05, Tukey test for multiple comparison; Scale bar, 20 μm.

Figure 7—figure supplement 2. Wnd signaling components JNK/Bsk and Fos restrict the expression level of presynaptic components Brp and VGlut in cell bodies of unc-104 mutants, (related to Figure 7).

(A–B) Representative confocal images of motoneuron cell bodies immunostained for VGlut (Green) and Brp (magenta). Similarly to mutations in wnd (in Figure 7) inhibition of (A) bsk(JNK) or (B) fos in the unc-104-hypomorph mutant background via expression of dominant negative isoforms caused a dramatic enhancement of VGlut and Brp acccumulations in cell bodies. Two groups of motoneuron cell bodies are shown in each image. The genotypes are: wt (Canton-S), unc-104^bris/d11204, bsk^DN (elav-Gal4;; UAS-bsk^DN), unc-104^bris/d11204; bsk^DN (elav-Gal4; unc104^bris/d11204; UAS-bsk^DN), unc-104^bris/O3.1, unc-104^bris/O3.1; fos^DN (BG380-Gal4; unc104^bris/d11204; UAS-bsk^DN) and fos^DN (BG380-Gal4;; UAS-fos^DN). Scale bar, 20 μm. (C–D) Quantification of VGlut and Brp from (A–B). At least 6 animals were examined per genotype.

Figure 7—figure supplement 3. Total VGlut protein levels are reduced in unc-104-hypomorph mutants, in a Wnd-dependent manner (related to Figure 7).

Representative Western blot of 30 larval brains for VGlut and β-tubulin from wt (Canton S), unc-104^bris/P350, wnd³ and unc-104^bris/P350;wnd³.