Enhancer architecture and chromatin accessibility constrain phenotypic space during Drosophila development

Developmental enhancers bind transcription factors and dictate patterns of gene expression during development. Their molecular evolution can underlie phenotypical evolution, but the contributions of the evolutionary pathways involved remain little understood. Here, using mutation libraries in Drosophila melanogaster embryos, we observed that most point mutations in developmental enhancers led to changes in gene expression levels but rarely resulted in novel expression outside of the native pattern. In contrast, random sequences, often acting as developmental enhancers, drove expression across a range of cell types; random sequences including motifs for transcription factors with pioneer activity acted as enhancers even more frequently. Our findings suggest that the phenotypic landscapes of developmental enhancers are constrained by enhancer architecture and chromatin accessibility. We propose that the evolution of existing enhancers is limited in its capacity to generate novel phenotypes, whereas the activity of de novo elements is a primary source of phenotypic novelty.