Quantification of LNvs defasciculation (ventral projections) and branching (dorsal projections).

<p>(A-B) The <i>cyc</i><sup><i>01</i></sup> mutant has disrupted sLN<sub>v</sub> morphology. (A) Representative confocal images of anti-PDF staining in Canton-S control and <i>cyc</i><sup><i>01</i></sup> adult male brains. The sLN<sub>v</sub>s and optic tract (OT) are indicated. Scale bar = 25 μm. The inserts on the right show the sLN<sub>v</sub> projections with the signal intensity adjusted for visibility in the <i>cyc</i><sup><i>01</i></sup> mutants. The top insert shows the distal (dorsal) area and the bottom insert shows the proximal (ventral) area of the sLN<sub>v</sub> projections. Scale bar = 10 μm. (B) Representative images of eight brains of<i>; Pdf-RFP;cyc</i><sup><i>01</i></sup> experimental flies stained with anti-RFP (magenta). Flies were raised at 28°C. Most of <i>cyc</i><sup><i>01</i></sup> mutant flies (~78.5%, 11 out of 14 brains) exhibit severe phenotypes in their sLNv morphology compared to the effects of <i>cyc</i> downregulation in <i>Pdf</i>+ neurons. (C-E) Representative confocal images of adult (C, E) and L3 larvae (D) control brains stained with anti-RFP (magenta). (C) To determine the degree of defasciculation of the sLN<sub>v</sub> ventral projections in adult brains, 6 concentric circles separated by is 25 μm were centered at the point of intersection (POI), where the projections of the sLN<sub>v</sub>s and those of the lLN<sub>v</sub>s intersect. The most distant circle does not reach the main branching point (BP) in control brains; therefore, the dorsal termini are not included. The number of intersections between either the sLNvs or the lLN<sub>v</sub>s and each concentric circle were quantified. (D) Dorsal projection branching in the larval sLN<sub>v</sub>s was measured by counting the number of intersections the sLN<sub>v</sub>s had at each of the 6 concentric circles separated by 12.5 μm. (E) Adult sLN<sub>v</sub> dorsal projection branching was measured by counting the number of intersections the sLN<sub>v</sub>s had at each of 8 concentric circles separated by 12.5 μm. This was adapted from a previous study [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1011441#pgen.1011441.ref027" target="_blank">27</a>] to capture the hyperextended projection phenotype of <i>Pdf</i> > <i>Δ-Clk</i> flies. (F-I) Quantification of the LN<sub>v</sub> morphology phenotypes of experimental flies in which a <i>cyc</i><sup><i>RNAi</i></sup> transgene was driven by a; <i>Pdf-RFP</i>,<i>Pdf</i>-<i>Gal4</i>; driver compared to the parental controls. Flies were raised at 25°C. The sLN<sub>v</sub> projection length until the branching point (BP) (F), the total number of intersections of the sLN<sub>v</sub> ventral projections (G), the total sLN<sub>v</sub> projection length (H), and the total number of intersections of the lLN<sub>v</sub> projections along the optic tract (OT) (I) are shown. Graphs are representative of two independent experiments, with each dot representing one brain. For each genotype, n falls in the range: 18 ≤ n ≤ 21. (I-J). **p < 0.01, *** p < 0.001. Error bars indicate SEM. See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1011441#pgen.1011441.s006" target="_blank">S1 Table</a> for additional quantifications.</p> <p>(PDF)</p>