Gene expression divergence during Drosophila head development on single cell resolution
Developmental Biology
Final Report Abstract
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 is a main driver for morphological diversification. A comprehensive understanding of the molecular changes responsible for morphological divergence in organ size and shape is missing to date. 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. 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. The Drosophila head develops from a composite eye-antennal imaginal disc that undergoes major patterning, growth and differentiation processes during larval stages. In our previous work we employed extensive comparative transcriptomic datasets based on sequencing entire eye-antennal discs throughout development (i.e. bulk-RNAseq) to identify candidate genes that regulate observed differences in head morphology. Due to the composite nature of the disc, the identification of clear candidate genes and developmental processes had been hampered because the bulk-RNAseq data does not provide sufficient tissue specific resolution. Therefore, we studied gene expression dynamics and divergence on single cell level using single nucleus mRNA sequencing (snRNAseq). In this work, we established a highly efficient snRNAseq protocol for small tissue samples. Using this protocol, we generated single nucleus expression atlases for five stages of eye-antennal disc development for D. melanogaster, D. simulans and D. mauritiana. Clustering analyses followed by cell type annotation showed that our datasets contained all major cell types of the eye-antennal disc. Based on this high-quality data, we revealed significant differences in the contribution of retinal cell types, such as photoreceptors and their proliferative precursors between D. melanogaster and D. mauritiana, recapitulating observed differences in facet number in these two species. In contrast, the contribution of retinal cell types was much less variable between D. mauritiana and D. simulans, which is in line with consistent facet number in these two species. Cell type specific differential expression analyses in combination with the identification of central transcriptional regulators for each cell type revealed exciting candidate genes, such as members of the ecdysone signaling pathway, the dorsal-ventral regulator araucan and a cohesion subunit involved in gene regulation. In summary, we established a powerful comparative dataset that already resulted in the identification of novel candidate genes and that will allow multiple follow-up studies aiming at understanding Drosophila head development and evolution on an unprecedented resolution.
Publications
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Tissue dissociation for single-cell and single-nuclei RNA sequencing for low amounts of input material. Frontiers in Zoology, 19(1).
Wiegleb, Gordon; Reinhardt, Susanne; Dahl, Andreas & Posnien, Nico
