The past 20 years of comparative developmental genetic studies have shown that blueprints of animal forms are encoded in complex gene regulatory and signaling networks made of evolutionary conserved components. Morphological evolution is thought to largely result from modifications in the interactions among components of genetic networks, rather than from the diversification of gene repertoires or gene functions. However it remains unclear how novel morphological traits were gained, and how the underlying complex genetic regulatory network appeared. The evolution of a pigmentation spot located on the wing of some Drosophila males is a great model to tackle this question, notably because it is genetically tractable and has evolved several times in the melanogaster group. Using a comparative and integrative approach our goal is to map on the drosophila tree the genomic changes that caused changes in the genetic regulatory network of wing pigmentation, which in turn led to the evolution of this morphological trait. In order to identify genes whose changes in spatio-temporal expression underlie the evolution of wing pigmentation, we have compared gene expression using RNA-seq in 3 different species and between sexes, at 11 time points spanning wing pupal development and in a spatially resolved manner. In order to understand the genetic determinism of these gene expression changes we have employed functional genomics methods such as ChIP-seq and FAIRE. We are testing the involvement of a dozen of candidates in the evolution of wing spot formation by using a combination of CRISPR/Cas9-targeted mutagenesis, RNAi knockdown and overexpression assays in pupae of the spotted species D. biarmipes.