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. 2012 Mar 19;7(3):e33634. doi: 10.1371/journal.pone.0033634

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Iyer et al.

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.

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(A–F) Live confocal images of third instar larval dorsal ddaC class IV da neurons labeled with GAL4⁴⁷⁷,UAS-mCD8::GFP. As compared to control (A), Trio overexpression results in decreased dendritic branching (B). (C) In contrast, Trio-GEF1 overexpression leads to an increase in dendritic branching, whereas Trio-GEF2 overexpression results in a reduction in dendritic branching (D). (E) RNAi knockdown of Rac1 in a Trio-GEF1 overexpression background suppresses defects in dendritic development as compared to Trio-GEF1 overexpression alone. (F) RNAi knockdown of Rho1 in a Trio-GEF2 overexpression background suppresses defects in dendrite morphogenesis as compared to Trio-GEF2 overexpression alone. (G) Analyses of the number of dendritic terminals reveals a decrease in dendritic branching with Trio and Trio-GEF2 overexpression whereas Trio-GEF1 overexpression leads to an increase in dendritic branching relative to wild-type controls. Knockdown of Rac1 via RNAi or by co-expression of the dominant negative Rac1.N17 suppresses defects in dendritic branching relative to Trio-GEF1 overexpression alone, whereas knockdown of Rho1 via RNAi suppresses defects in branching as compared to Trio-GEF2 overexpression alone. (H) Quantitation of total dendritic length reveals a mild to moderate reduction with Trio, Trio-GEF1 and Trio-GEF2 overexpression as compared to wild-type controls. Consistent with dendritic branching, disrupting Rac1 or Rho1 function suppresses defects in dendritic length as compared to Trio-GEF1 or Trio-GEF2 overexpression, respectively. (I) Relative to control (n = 6), dendritic branch order analyses reveal a proximal shift in the percentage of lower order branching with Trio (n = 8) and Trio-GEF2 (n = 8) overexpression, whereas Trio-GEF1 (n = 8) overexpression results in a distal shift towards higher order branching in class IV ddaC neurons. Images were taken at 20× magnification and size bar represents 50 microns. The total n value for each neuron and genotype quantified is reported on the bar graph. Statistically significant p values are reported on the graphs as follows (* = p<0.05; ** = p<0.01; *** = p<0.001). Genotypes: WT: GAL4⁴⁷⁷,UAS-mCD8::GFP/+. TRIO: UAS-trio/+;GAL4⁴⁷⁷,UAS-mCD8::GFP/+. GEF1: UAS-trio-GEF1-myc/GAL4⁴⁷⁷,UAS-mCD8::GFP. GEF2: GAL4⁴⁷⁷,UAS-mCD8::GFP/+;UAS-trio-GEF2-myc/+. GEF1+Rac1-RNAi: UAS-trio-GEF1-myc/GAL4⁴⁷⁷,UAS-mCD8::GFP;UAS-Rac1^JF02813/+. GEF1+Rac1.N17: UAS-trio-GEF1-myc/GAL4⁴⁷⁷,UAS-mCD8::GFP;UAS-Rac1.N17/+ GEF2+Rho1-RNAi: GAL4⁴⁷⁷,UAS-mCD8::GFP/+;UAS-trio-GEF2-myc/UAS-Rho1-dsRNA.