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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–D) Live confocal images of class III and IV dorsal cluster da neurons at the third larval instar labeled with the F-actin reporter UAS-GMA driven by the ppk-GAL4 transgene. Class III ddaA and ddaF neuron cell bodies are indicated by arrows and for clarity the class III neuron cell bodies and dendrites have been highlighted by a magenta pseudo-color trace overlay. (A) Representative image of wild-type class III da neuron displaying regularly distributed, unbranched, actin-rich dendritic filopodia (dashed line circles and high magnification image (A′)) projecting from the primary dendritic branches. (B) Full length Trio overexpression dramatically altered dendritic filopodia producing a hyper-branched, splintered morphology (dashed line circles and high magnification image (B′)). In addition, the filopodia displayed a more clustered distribution as compared to control. (C) Overexpression of the Trio-GEF1 domain increased dendritic branching overall and produced a highly similar splintered morphology on dendritic filopodia. (D) Overexpression of the Trio-GEF2 domain produces the opposite effect by reducing dendritic branching overall and leading to a dramatic decrease in the number of dendritic filopodia. (E) Quantitative analyses of the number of dendritic terminals as a measure of dendritic branching reveals no significant change with full length Trio overexpression, whereas upregulation of Trio-GEF1 dramatically leads to a significant increase in terminals both proximal and distal to the cell body, while upregulation of Trio-GEF2 leads to a significant decrease in terminals both proximal and distal to the cell body. The total n value for each neuron and genotype quantified is reported on the bar graph. Statistically significant p values are reported on the graph as follows (n.s. = not significant; *** = p<0.001). Genotypes: WT: UAS-GMA/+;ppk-GAL4/+;+. TRIO: UAS-GMA/UAS-trio;ppk-GAL4/+;+. GEF1: UAS-GMA/+;ppk-GAL4/UAS-trio-GEF1-myc;+. GEF2: UAS-GMA/+;ppk-GAL4/+;UAS-trio-GEF2-myc/+.