Mapping Ds functional domains. (A) Structure of the Ds ICD and the deletions generated for the S2 cell recruitment assay and CRISPR mutations in vivo. Full-length Ds ICD or Ds ICD deletions (ΔA, ΔB, ΔC, and ΔD) were fused to the Ed ECD for S2 cell experiments. The same domains are deleted with CRISPR-Cas9 in vivo. Abbreviations: Conserved motif 1–3 (CM1–3, red boxes): transmembrane domain (TM, sky blue box). (B) Ed:Ds:Flag recruits Dachs:GFP, App, and Dlish:HA to cell contacts (yellow arrows). Ed homophilic interactions promote the accumulation of the Ed fusion proteins at cell–cell contacts. (C–F) Recruitment of Dachs:GFP by Ds ICD and its deletions. Ed:Ds:Flag and Ed:DsΔA:Flag recruit Dachs:GFP to cell contacts (yellow arrows), but Ed:DsΔB:Flag and Ed:DsΔD:Flag fail to recruit Dachs:GFP. (G–L) Adult wings of the indicated genotypes. (M) Quantification of wing size in wild-type and ds ICD deletion mutants. ****P < 0.0001 (two-tailed unpaired t test between each genotype). Wild-type (n = 55), ds^:Flag (n = 62), ds^ΔICD:GFP (n = 31), ds^ΔA:Flag (n = 28), ds^ΔB:Flag (n = 23), ds^ΔC:Flag (n = 94), ds^ΔD:Flag (n = 22). (N–S) Tarsal segments in legs from flies of the indicated genotypes. Numbers in the top right corner indicate the percentage of animals with normal tarsal segments. Fused tarsal segments are marked with yellow arrow heads. (T–W) Dachs localization in wing discs of the indicated genotypes. Dachs colocalizes with Ds in ds^:Flag (T) and ds^ΔA:Flag (U), but they colocalize less in ds^ΔB:Flag (V) and ds^ΔD:Flag (W). Yellow circles indicate Ds puncta. (X) App puncta, stained extracellularly with a pulse-chase protocol (see Materials and methods) are lost in ds^ΔD:Flag clones. Scale bars (B, C–F, and X) 5 µm, (G–L and N–S) 100 µm, (T–W) 1 µm. In this and subsequent figures where epitope tags (Flag, HA, V5) are indicated, antibodies were used that recognize those tags.