C4da neuron dendrites along the dorsal midline (ppk-CD4-tdTomato) and the extracellular matrix (trol-GFP or vkg-GFP) were co-visualized in third instar larvae. High resolution two-color confocal z-stacks were analyzed for dendrite-ECM interaction and detached dendrite segments are indicated in magenta as indicated schematically. (A) In wildtype animals, very few dendrite segments were not in contact with the ECM. Magnified regions of (A) and cross-sections illustrate dendrite-ECM proximity with few dendrite segments not contacting the ECM (A′, A′′, ECM in green, dendrites in magenta, see schematic for color code). (B) A strong increase of detached dendrites could be observed in Ret mutant animals, highlighted in the magnified sections displaying severe displacement of dendrites from the ECM (B′ and B′′). Scale bar: 25 μm. (C) Quantitative analysis of dendrite-ECM interaction in wildtype and Ret mutant C4da neurons revealed a strong increase in dendrite detachment in Ret deficient animals (mean ± SD, n = 5, p < 0.001, Student's two-tailed t-test). (D–E) Genetic interaction analysis of dendrite-ECM adhesion in (D) RetC168, (E) mewM6, (F) mys1 heterozygous and (G) mewM6/RetC168 (H) mys1/Ret C168 trans-heterozygous third instar larvae. The combination of either integrin mutant with the RetC168 allele showed highly increased loss of dendrite-ECM interaction (G and H) compared to wildtype or the heterozygous alleles alone. Scale bar: 25 μm. (I) Quantitative analysis of dendrite detachment for the individual genotypes as indicated. mewM6/RetC168 and mys1/RetC168 trans-heterozygotes showed significantly impaired dendrite-ECM adhesion (mean ± SD, p < 0.05, n = 4, Mann–Whitney U-test).