CRISPR/Cas9 Deletions in a Conserved Exon of Distal-Less Generates Gains and Losses in a Recently Acquired Morphological Novelty in Flies

Abstract

Distal-less has been repeatedly co-opted for the development of many novel traits. Here, we document its curious role in the development of a novel abdominal appendage (“sternite brushes”) in sepsid flies. CRISPR/Cas9 deletions in the homeodomain result in losses of sternite brushes, demonstrating that Distal-less is necessary for their development. However, deletions in the coding exon upstream of the homeodomain (Exon 2) produces both losses and gains of brushes. A dissection of Exon 2 reveals that the likely mechanism for gains involves a deletion in an exon-splicing enhancer site that leads to exon-skipping. Such contradictory phenotypes are also observed in butterflies which suggests that deletions in the conserved upstream regions have the potential to generate phenotypic variability in insects that diverged 300 million years ago. Our results demonstrate the importance of Distal-less for the development of a novel abdominal appendage in insects and highlight how site-specific mutations in the same exon can produce contradictory phenotypes.