Gene expression divergence during Drosophila head development on single cell resolution

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Understanding the processes involved in the origin of the diversity of life forms observed on earth is a major aim of biological research. Adult morphology is defined during embryonic and postembryonic development by the action of developmental gene products, which are organized in gene regulatory networks (GRNs). Natural variation in the expression and thus the regulation of developmental genes has been shown to be a main driver for morphological diversification. While the developmental underpinnings of differences in relatively simple traits are being revealed, a comprehensive understanding of the molecular changes responsible for morphological divergence of complex traits is missing to date. To close this gap, we study the evolution of head development in three closely related Drosophila species. The insect head is a complex structure that consists of the main sensory organs (e.g. the compound eye and antennae) to perceive environmental information and the brain to process information to trigger behavioral responses. The Drosophila head develops from a composite eye-antennal imaginal disc that undergoes major patterning, growth and differentiation processes during larval stages. We and others have previously quantified differences in adult head size and shape among the three Drosophila species D. melanogaster, D. simulans and D. mauritiana. To identify candidate genes involved in the observed variation, we generated a comprehensive comparative transcriptomic dataset by sequencing entire eye-antennal imaginal discs throughout development (i.e. bulk-RNAseq). Based on this data we revealed a high level of context dependent gene expression and regulation recapitulating the composite nature of the developing eye-antennal imaginal discs. The identification of clear candidate genes and developmental processes, however, is hampered because the bulk-RNAseq data does not provide sufficient tissue specific resolution. Therefore, we propose here to study gene expression dynamics and divergence on single cell level using single cell mRNA sequencing (scRNAseq).We will establish a single cell expression atlas for five stages of eye-antennal imaginal disc development for D. melanogaster, D. simulans and D. mauritiana. This data will be used to reveal key developmental genes and regulatory interactions in specific cell clusters representing different regions of the developing head. The interspecific comparison of observed cell clusters with respect to gene expression and GRN architecture will allow identifying diverging cell populations. Two of these cell clusters will be functionally validated applying CRISPR/Cas9 mediated genome editing and re-sequencing of cluster specific cells. With the proposed project we will gain major insights into the development and evolution of the Drosophila head on an unprecedented resolution. Additionally, we will contribute to a broader mechanistic understanding of context dependent gene expression and regulation.