Single-cell analysis of the early Drosophila salivary gland reveals that morphogenetic control involves both the induction and exclusion of gene expression programs.

How tissue shape and therefore function is encoded by the genome remains in many cases unresolved. The tubes of the salivary glands in the Drosophila embryo start from simple epithelial placodes, specified through the homeotic factors Scr/Hth/Exd. Previous work indicated that early morphogenetic changes are prepatterned by transcriptional changes, but an exhaustive transcriptional blueprint driving physical changes was lacking. We performed single-cell-RNAseq-analysis of FACS-isolated early placodal cells, making up less than 0.4% of cells within the embryo. Differential expression analysis in comparison to epidermal cells analyzed in parallel generated a repertoire of genes highly upregulated within placodal cells prior to morphogenetic changes. Furthermore, clustering and pseudotime analysis of single-cell-sequencing data identified dynamic expression changes along the morphogenetic timeline. Our dataset provides a comprehensive resource for future studies of a simple but highly conserved morphogenetic process of tube morphogenesis. Unexpectedly, we identified a subset of genes that, although initially expressed in the very early placode, then became selectively excluded from the placode but not the surrounding epidermis, including hth, grainyhead and tollo/toll-8. We show that maintaining tollo expression severely compromised the tube morphogenesis. We propose tollo is switched off to not interfere with key Tolls/LRRs that are expressed and function in the tube morphogenesis.