Exploring the transcriptomic landscape of the Drosophila melanogaster embryonic salivary gland.
Many internal organs in all animals are composed of epithelial tubes that are formed during development. Despite much research into their formation over the years, the gene programmes and morphogenetic effectors responsible for controlling the morphogenesis of tubes and their order of action is yet to be comprehensively assessed. To investigate this, I utilised a simple model of tubulogenesis found in the Drosophila melanogaster embryonic salivary gland. Once specified, two mirror-symmetric placodes internalise from a flat epithelium in a coordinated manner, form two narrow-lumen epithelial tubes on the inside and eventually fuse to form a Y-shaped organ. Salivary gland cells do not undergo cell death or division, instead only through changing their shape, size, and neighbours. The short time window of specification, small cell population, continual lineage of cells and the established spatial patterning of genes within the salivary gland primordium prior to morphogenesis makes the Drosophila melanogaster embryonic salivary gland an excellent candidate for single cell RNA sequencing to identify novel genes involved in the process of tube budding and to delineate their order of expression. In my PhD, I used a combinatorial method of strictly staged embryos, fluorophore driven embryo cell sorting and 10x chromium single cell sequencing, to generate two novel stage 11 Drosophila melanogaster embryonic single cell sequencing data sets: a general epidermal dataset and a salivary gland primordium enriched dataset. Both datasets cover identical developmental time points of salivary gland specification and early invagination, and provide insights into a lineage of cells never previously identified in existing single cell sequencing datasets. Differential expression analysis between datasets successfully identified genes previously implicated in salivary gland development, alongside many novel genes with unknown roles in the process. The analysis also identified a subset of genes actively excluded from the salivary gland primordium, behaviour previously only reported for the genes Sex combs reduced and homothorax involved in the earliest specification of the salivary gland primordium. The salivary gland enriched dataset successfully clustered secretory cells and duct cells separately. Validation of novel genes identified from this dataset by in-situ hybridisation unveiled novel gene expression patterns within the salivary gland primordium, a new collection of genes not under the control of secretory cell fate maintenance transcription factor forkhead, and evidence of upregulation of the protein secretory machinery far earlier than previously reported. Pseudotime analysis enabled the identification of temporally controlled expression of transcription factors and adhesion molecules in time with morphogenetic movement of the tissue.
Drosophila, Drosophila embryo, Drosophila embryogenesis, Morphogenesis, Pseudotime, Salivary gland, scRNAseq, Single cell sequencing
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