Fig. 3.

Shared regulatory inputs of the wing blade and the spot activities. (A) A map of the yellow locus 5′ region highlighting the position of the spot¹⁹⁶ core. (B–E) The wing blade and the spot activities are strongly affected by discrete mutations in D2. (Left) Construct schematics. (Center) Average phenotype. (Right) Comparison with construct D5 phenotype (difference). The positions of mutated sites as well as those of blocks 4 and 5 (SI Appendix, Fig. S2) are depicted on blown-up schematics of the spot¹⁹⁶ core in E. (B) RR is a negative control, the same randomized fragment as in Fig. 1 B and C. (C) D2 is identical to Fig. 1 B and C. (D) Mutating all four characterized Dll binding sites (16) of spot¹⁹⁶ in the context of D2 (D2^Dll-KO) reduces the spot activity strongly and the wing blade activity moderately, as seen when comparing this mutant construct with D5. (E) Mutating a newly identified activator site (28) of spot¹⁹⁶ (spot^{196 [6]}, 12 bp mutated) in the context of D2 (D2^[6]-KO) reduces both spot and wing blade activities strongly, as seen when comparing this mutant construct with D5. (F) Chromatin accessibility measured with ATAC-seq at the D2 and D2^[6]-KO transgenes at the onset of spot activity (SI Appendix, Fig. S3) (66-h pupal wings) differs significantly in a 500-bp region overlapping spot^{196 [6]} (dotted black and magenta line). This is the only region in the entire locus identified as a differentiated site using diffBind (50, 51) and DEseq2 (52) analyses (Materials and Methods) (adjusted P value from the DESeq2 analysis: 7.21E-08). ATAC-seq traces represent the pooled signal of three replicates for each transgenic line (SI Appendix, Fig. S4). The discrepancy between the enhancer boundaries defined in Fig. 1 and the accessible region of F may stem in part from the different stages at which these properties were assessed. Average activity phenotypes of each construct also shown in C and E are indicated in Insets under each construct diagram as a reminder.