Spatial distribution of Scabrous protein during bristle patterning stages.

(A) Example of apicobasal distribution of Scabrous protein (arrowheads, white puncta, anti-Scabrous) in the notum, with bristle precursor cell marked by red nuclei (anti-senseless). Phalloidin is used to stain filamentous actin (green). Scale bar, 5 μm. Apical (+ μm) and basal (- μm) are relative to the middle of the sensless positive nuclei (widest diameter). Genotype: w1118. (B) Apicobasal distribution of Scabrous protein (red) in a bristle precursor cell labeled with anti-GFP (green). a, apical; b, basal. Scale bars, 5 μm. Genotype: neur-GAL4, UAS-GMCA/+. (C) Anti-scabrous staining for extracellular only (Sca-E) or total (Sca-T) Scabrous protein near a bristle precursor cell (green in merge). Pink arrowhead = double positive Sca puncta, indicative of extracellular protein. Yellow arrowheads = single positive Sca puncta, stained after permeabilization. Genotype: neur-GAL4, UAS-GMCA/+. Scale bar, 10 μm. (D) Demonstration of Scabrous and Rab7 colocalization patterns. Sca = scabrous, anti-GFP staining; sens = senseless staining to mark bristle cell precursor nuclei. DNA = DAPI stain to mark all nuclei. White arrowhead = puncta double positive for anti-GFP and anti-rab7. Pink arrowhead = puncta single positive for anti-GFP. Yellow arrowhead = puncta single positive for rab7. Note most puncta closely associated with senseless nuclei are single positive for anti-GFP. Genotype: scabrousGSFTF. Scale bar, 5 μm. (E) Pairwise quantification of Scabrous puncta at apical or basal z-planes for n = 9 nota. *, p = 0.04, r = 0.83 by paired student’s t-test. (C) XY distribution of Scabrous puncta relative to the center of bristle precursor cells (0 μm). The number of puncta are area adjusted, across n = 9 nota. See also S1 Fig and S1 Movie.