Figure 1.
Trio is expressed in PNS sensory neurons.
Trio staining (arrows) in dorsal cluster sensory neurons from (a–c) 109(2)80 Gal4- UAS-GFP larvae highlighting all md-da neurons and (d–f) Gal4 2–21 UAS-GFP larvae highlighting Class I neurons. All the md-da sensory neurons express Trio. While it is readily detected in the cell body and proximal dendritic branches, the signal in small, higher order dendritic branches is difficult to discriminate from immunofluorescence deriving from Trio in associated epithelial cells. Scale bar 20 µm.
Figure 2.
trio mutations affect dendritic branching of Class I vpda neurons cell autonomously.
a–c) A vpda neuron from a wandering third instar larvae of (a) trio1 Gal42–21UAS-GFP/+ (control), (b) trio1Gal42–21UAS-GFP/trioM89 and (c) trio1Gal42–21UAS-GFP/UAS-trio RNAi; scale bar 50 µm. d–e) Quantification of dendritic branching in control, trio mutant and UAS-trio RNAi expressing vpda neurons. d) Dendritic branching of vpda neuron: trio knockdown reduces number of dendritic branches, e) average number of branches per branch order of vpda neuron: trio knockdown affects number of only higher order branches. f–g) Quantification of dendritic length in control and trio mutant vpda neuron. f) Total dendritic length of vpda neuron: trio mutations reduce total dendritic length g) average dendritic length per branch order of vpda neuron: trio mutations do not affect average dendritic length. The number of samples (n value) for each genotype is indicated by the number inside the respective bar, error bars represent standard error of the mean (SEM) and asterisks indicate p value<0.05.
Figure 3.
trio mutations affect dendritic branching of dorsal cluster Class IV ddaC neuron.
a–b) A ddaC neuron from a wandering third instar larvae of (a) Gal44–77-UAS-GFP/ppk-Gal4 control, and (b) Gal44–77-UAS-GFP/ppkGal4; trio1/trioM89; scale bar 50 µm. c–f) Quantification of different dendritic parameters in control and trio mutant ddaC neurons. c) Dendritic branching: trio mutations reduce dendritic branching. d) Dendritic arbor area: trio mutations do not affect dendritic arbor area, e) Total dendritic length: trio mutations reduce total dendritic length. f) Average dendritic length: trio mutations increase average dendritic length per branch. The number of samples (n value) for each genotype is indicated by the number inside the respective bar, error bars represent standard error of the mean (SEM) and asterisks indicate p value<0.05.
Figure 4.
trio over-expression in Class I neurons.
a–d) third instar larval Class I neurons in control Gal42–21 UAS-GFP/+ (a) vpda, (c) ddaE, and Gal42–21 UAS-GFP/UAS-trio (b) vpda, (d) ddaE; scale bar 50 µm. e–j) Quantitative analysis of dendritic parameters in control and UAS-trio expressing vpda and ddaE neuron. e,g) Dendritic branches: UAS-trio promotes dendritic branching of vpda (e) and ddaE (g) neuron. f,h) Average number of branches per branch order: UAS-trio affects only higher order branches in both vpda (f) and ddaE (h) neurons. i) Dendritic length: UAS-trio doesn't alter total dendritic length. j) Average dendritic length by branch order: UAS-trio reduces average length only of secondary branches. The number of samples (n value) for each genotype is indicated by the number inside the respective bar, error bars represent standard error of the mean (SEM) and asterisks indicate p value<0.05.
Figure 5.
trio over-expression in Class IV neuron.
a,b) third instar larval Class IV ddaC neurons in control Gal44–77UAS-GFP/ppk-eGFP and UAS-trio/+; Gal44–77UAS-GFP/ppk-eGFP; scale bar 50 µm. c–f) Quantitative analysis of dendritic parameters in control and UAS-trio expressing ddaC neuron. c) Dendritic branches: trio over-expression promotes dendritic branching of ddaC neuron. d) Dendritic arbor area: trio over-expression does not affect dendritic arbor area e) Dendritic length: trio over-expression doesn't alter total dendritic length. d) Average dendritic length: trio over-expression reduces average dendritic length per branch. The number of samples (n value) for each genotype is indicated by the number inside the respective bar, error bars represent standard error of the mean (SEM) and asterisks indicate p value<0.05.
Figure 6.
Trio acts via its GEF1 domain to regulate dendrogenesis.
a,b) Third instar larval vpda neuron expressing (a) UAS- trioGEF1mu and (b) UAS- trioGEF2mu; scale bar 50 µm c–d) magnified view of dendritic branching (c) and (d) are highlighted areas in (a) and (b). e) Quantitative analysis of dendritic branching: ectopic expression of UAS- trioGEF2mu but not UAS- trioGEF1mu promotes formation of extra dendritic branches. The number of samples (n value) for each genotype is indicated by the number inside the respective bar, error bars represent standard error of the mean (SEM) and asterisks indicate p value<0.05.
Figure 7.
Reducing Rac suppresses the effect of Trio overexpression.
Larvae of the indicated genotypes were dissected and vpda branch number was quantified. (A–C) Representative examples of vpda neurons labeled with mCD8-GFP and visualized by fluorescent microscopy. (A) rac1J11rac2ΔmtlΔ/GAL42–21UAS-mCD8-GFP, (B) UAS-Trio; GAL42–21UAS-mCD8-GFP, (C) UAS-Trio; rac1J11rac2ΔmtlΔ/GAL42–21UAS-mCD8-GFP. (D) quantification of vpda branch number. Vertical bars report number of branches for the indicated genotypes, lines indicate SEM. Number in each bar is the number of samples. Asterisks indicate p<0.05.
Figure 8.
Trio does not transform class identity of md-da neurons.
trio1/trioM89 mutant larvae were filleted and stained with the pan-neuronal antibody anti-HRP (green) and the indicated antibodies against class-specific markers (red). (A-A″) anti-Abrupt (Class I). Absence of Trio does not prevent expression of Abrupt in the Class I neurons ddaE or ddaF (arrows), nor does it induce expression of Abrupt in the Class IV neuron ddaC (arrowhead). (B-B″) anti-Knot (Class IV). Absence of Trio does not prevent expression of Knot in the Class IV neuron v' ada (arrow).