Comparison of activities of endogenously expressed cleaved and uncleaved Bnl. (A) Schematic map of the genomic bnl:gfp^endo locus and the products of its subsequent CRISPR/Cas9-based editing; orange box, coding exon; gray box, noncoding exon; line, introns; red star, M1 mutation. (B and C) Representative images of αHA-stained (red) ASP and wing disc from homozygous wt^endo (n = 85) and m1^endo (n = 79) larvae. (D and E) Representative images of αHA-immunostained (red) ASP and wing disc from wt^endo larvae after 5 h of ex vivo culture in the absence (control; n = 18) and presence (n = 29) of Furin inhibitors. In B–E, white dashed line, ASP; blue, phalloidin-Alexa Fluor 647; arrow, t-WT^endo; arrowhead, uncleaved WT^endo or M1^endo. (F–G′) Receptor-colocalized t-WT^endo and M1^endo puncta (arrow) in trans-heterozygous btl:Cherry^endo/wt^endo (F and F′) and btl:Cherry^endo/m1^endo (G and G′) ASP; split red channels (F′ and G′). (H–I′) Live images of CD8:Cherry-marked ASPs showing the long (>15 µm) oriented ASP cytonemes (arrows) containing t-WT^endo (H and H′) and M1^endo (I and I′) puncta (arrowheads). (J and J′) Surface αGFP^ex immunostaining (white) detecting M1^endo on the ASP cytoneme surfaces of btl:cherry^endo/m1^endo larvae; arrow and arrowhead, receptor-colocalized intracellular (bright green) and surface M1^endo, respectively. (K) Graph comparing the number of cytonemes (>15 µm long) counted from a 60-µm perimeter centering the ASP tip (Materials and methods) in wt^endo (n = 28) and m1^endo (n = 38). Scale bars: 20 µm (B–G′, J, and J′); 10 µm (H–I′).