Protein sequence evolution underlies interspecies incompatibility of a cell fate determinant

Novel and rapidly evolving genes can integrate into conserved gene networks and play critical roles in development. Understanding how sequence variation across the orthologs of such genes influences functional interactions with the molecular products of older, more conserved genes requires investigation at the level of protein function. Here, we elucidate how protein-coding sequence evolution in oskar, a gene required for primordial germ cell specification and embryonic patterning in fruit flies, has led to functional incompatibility between orthologs from Drosophila melanogaster and Drosophila virilis. We generated chimeric versions of oskar comprising different combinations of Oskar protein domains from each species, expressed these chimeric oskar sequences in D. melanogaster, and quantified their ability to assemble functional germ line and abdominal patterning determinants (germ plasm). We found that a specific portion of D. virilis Oskar, namely the OSK domain, was primarily responsible for the cross-species incompatibility of Oskar. In the absence of endogenous D. melanogaster Oskar, chimeras containing the D. virilis OSK domain could not localize posterior germ plasm well enough to generate primordial germ cells, but were sufficient to specify the anteroposterior axis. We also found that the D. virilis OSK domain had dominant-negative effects on D. melanogaster Oskar's ability to localize germ plasm mRNA, resulting in severe axial patterning defects. We propose that evolved changes in the biophysical properties of the OSK domain between species are linked to distinct molecular interactions with conserved germ plasm molecules. Under this hypothesis, an essential germ line determinant evolved to be incompatible across species of the same genus in less than 50 million years, while retaining functional within-species molecular interactions. This case study illustrates how investigating in vivo protein function can bridge genomic and molecular evolution with phenotypic variation and fitness at higher scales.