Extended Data Fig. 9: Live tissue staining assays to test direct binding between CSP pairs.

a, Confocal images of pupal brains (48 h APF) showing neuropil staining by N-cadherin antibody (blue), and binding of Sns-Fc-V5 in live pupa brains followed by fixing and staining against Fc (red). Control kirre conditionally tagged brains incubated with medium alone showed minimal background signal for Fc staining (left column). kirre conditionally tagged brains incubated with Sns-Fc-V5 proteins in medium have detectable signal for Fc staining in multiple brain regions and have differential signal in different glomeruli (middle column). These signals are substantially diminished in kirre mutant brains incubated with Sns-Fc-V5 proteins (right column), especially in the glomeruli where signal is high in the kirre conditionally tagged brains (arrowheads in the magnified images in the bottom rows). These results suggest Sns-Fc-V5 binds to endogenous Kirre, which serve as a positive control for this binding assay.b, Confocal images of pupal brains (48 h APF) showing neuropil staining by N-cadherin antibody (blue), and binding of Fili-Fc-V5 in live pupal brains followed by fixing and staining against Fc (red). kek1 conditionally tagged brains (middle column) incubated with Fili-Fc-V5 proteins in medium do not exhibit an increase of Fc signal in the antennal lobe (magnified in the bottom row) comparing to the control without the addition of Fili-Fc-V5 (left column) or Fili-Fc-V5 in kek1 homozygous mutant brain (right column). These results suggest that Kek1 does not bind to Fili in this assay. c, Confocal images of pupal brains (48 h APF) showing neuropil staining by N-cadherin antibody (blue), and binding of Toll2-Fc-V5 in live pupa brains followed by fixing and staining against Fc (red). Control Ptp10D conditionally tagged brains incubated with medium alone showed minimal background signal for Fc staining (left column). Ptp10D conditionally tagged brains incubated with Toll2-Fc-V5 proteins in medium have detectable signal for Fc staining in multiple brain regions (middle column). However, these signals are still present in Ptp10D mutant brains incubated with Toll2-Fc-V5 proteins (right column). These results suggest that Toll2-Fc-V5 binds to other proteins expressed in the brain, masking the detection of its potential binding to Ptp10D. d, Confocal images of wing discs dissected from 3rd instar larvae showing ectopic expression of UAS-kirre-HA in the posterior compartment driven by engrailed (en)-GAL4 (HA staining, red), and binding of Sns-Fc-V5 in live wing discs followed by fixing and staining against Fc (magenta). Control Kirre-overexpressed wing disc incubated with medium alone shows minimal background signal for Fc staining (left column). Kirre-overexpressed wing disc incubated with Sns-Fc-V5 proteins in medium has specific binding signal in the posterior compartment where Kirre was overexpressed from en-GAL4 (middle column). Interestingly, Sns binds strongest to regions with intermediate but not highest levels of Kirre overexpression. Without Kirre overexpression, no specific signal in the posterior compartment is detected (right column). These results suggest Sns-Fc-V5 binds to Kirre, which serves as a positive control for this binding assay. e, Confocal images of wing discs dissected from 3rd instar larvae showing ectopic expression of UAS-kek1-HA in the posterior compartment driven by en-GAL4 (HA staining, red), and binding of Fili-Fc-V5 in live wing discs followed by fixing and staining against Fc (magenta). Fc signal is not detectable in Kek1-overexpressed wing disc incubated with medium alone, Kek1-overexpressed wing disc incubated with Fili-Fc-V5, or wild-type wing disc incubated with Fili-Fc-V5. These results suggest that Kek1 does not bind to Fili in this assay. f, Confocal images of wing discs dissected from 3rd instar larvae showing ectopic expression of UAS-Ptp10D-HA in the posterior compartment driven by en-GAL4 (HA staining, red), and binding of Toll2-Fc-V5 in live wing discs followed by fixing and staining against Fc (magenta). Control Ptp10D-overexpressed wing disc incubated with medium alone showed minimal background signal for Fc staining (left column). Ptp10D-overexpressed wing disc incubated with Toll2-Fc-V5 proteins in medium has binding signal throughout the wing disc, not restricted to the posterior compartment where Toll2 was overexpressed (middle column). These signals are still present in wild-type wing disc incubated with Toll2-Fc-V5 proteins (right column). These results suggest Toll2-Fc-V5 binds to other proteins expressed in the wing disc, masking the detection of its potential binding to Ptp10D. The Fc-V5-tagged proteins used in this figure are visualized on Western blots in Extended Data Fig. 8e. Scale bar = 20 µm (a–c); Scale bar = 50 µm (d–f).