Abstract Revealing the mechanisms underlying the breathtaking morphological diversity observed in nature is a major challenge in Biology. It has been established that recurrent mutations in hotspot genes cause the repeated evolution of morphological traits, such as body pigmentation or the gain and loss of structures. To date, however, it remains elusive whether hotspot genes contribute to natural variation in the size and shape of organs. As natural variation in head morphology is pervasive in Drosophila, we studied the molecular and developmental basis of differences in compound eye size and head shape in two closely related Drosophila species. We show differences in the progression of retinal differentiation between species and we applied comparative transcriptomics and chromatin accessibility data to identify the GATA transcription factor Pannier (Pnr) as central factor associated with these differences. Although the genetic manipulation of Pnr affected multiple aspects of dorsal head development, the effect of natural variation is restricted to a subset of the phenotypic space. We present data suggesting that this developmental constraint is caused by the coevolution of expression of pnr and its cofactor u-shaped (ush). We propose that natural variation in expression or function of highly connected developmental regulators with pleiotropic functions is a major driver for morphological evolution and we discuss implications on gene regulatory network evolution. In comparison to previous findings, our data strongly suggest that evolutionary hotspots are not the only contributors to the repeated evolution of eye size and head shape in Drosophila.

Revealing the mechanisms underlying the breath-taking morphological diversity observed in nature is a major challenge in Biology. It has been established that recurrent mutations in hotspot genes cause the repeated evolution of rather simple morphological traits, such as body pigmentation or the gain and loss of structures. To date, however, it remains elusive whether hotspot genes contribute to natural variation in complex morphological traits, such as the size and shape of organs. Since natural variation in head morphology is pervasive in Drosophila, we studied the molecular and developmental basis of differences in compound eye size and head shape in two closely related Drosophila species. We show that differences in both traits are established late during head development and we applied comparative transcriptomics and chromatin accessibility data to identify the GATA transcription factor Pannier (Pnr) as central factor regulating these differences. Although the genetic manipulation of Pnr affected multiple aspects of dorsal head development, the effect of natural variation is restricted to a subset of the phenotypic space. We present data suggesting that this developmental constraint is caused by the co-evolution of expression of pnr and its co-factor u-shaped (ush). We propose that natural variation in highly connected developmental regulators with pleiotropic functions is a major driver for morphological evolution and we discuss implications on gene regulatory network evolution. In comparison to previous findings, our data strongly suggests that evolutionary hotspots do not contribute to the repeated evolution of eye size and head shape in Drosophila.

The size and shape of an organism is tightly controlled during embryonic and postembryonic development to ensure proper functionality. However, in the light of the breath-taking diversity of body forms observed in nature, developmental processes must have evolved to allow evolutionary changes in adult morphology. Therefore, gene regulatory networks (GRNs) that orchestrate organ development are mostly constrained, but nodes and edges within such networks must change to give rise to morphological divergence. Identifying such tuning nodes remains a major challenge in evolutionary developmental biology. Here, we combined comparative transcriptomics and chromatin accessibility data to study developmental differences leading to natural variation in compound eye size and head shape in the two closely related Drosophila species D. melanogaster and D. mauritiana. We show that variation in expression of the GATA transcription factor Pannier (Pnr) is associated with extensive remodeling of the transcriptomic landscape during head development. Since U-shaped (Ush), a co-factor of Pnr, is involved in the same regulatory context, we argue that variation in expression of both factors may be a driver of divergence in head morphology. Applying functional genetics and geometric morphometrics we confirmed that manipulation of pnr expression in D. melanogaster largely phenocopies D. mauritiana dorsal head shape and ommatidia number. Therefore, we propose that the regulatory module composed of Pnr and Ush represents a tuning node within the otherwise highly conserved GRN underlying head development in Drosophila.