Single-cell transcriptomics of the Drosophila wing disc reveals instructive epithelium-to-myoblast interactions

Abstract

In both vertebrates and invertebrates, generating a functional appendage requires interactions between ectoderm-derived epithelia and mesoderm-derived cells. To investigate such interactions, we used single-cell transcriptomics to generate a temporal cell atlas of the Drosophila wing disc from two developmental time points. Using these data, we visualized gene expression using a multi-layered model of the wing disc and catalogued ligand-receptor pairs that could mediate signaling between epithelial cells and adult muscle precursors (AMPs). We found that localized expression of the FGF ligands, Thisbe and Pyramus, in the disc epithelium regulates the number and location of the AMPs. In addition, Hedgehog ligand from the epithelium activates a specific transcriptional program within adjacent AMP cells, defined by AMP-specific targets Neurotactin and midline, that is critical for proper formation of direct flight muscles. More generally, our annotated temporal cell atlas provides an organ-wide view of potential cell-cell interactions between epithelial and myogenic cells.

Critical genetic program forDrosophilaimaginal disc regeneration revealed by single-cell analysis

“…Regenerating discs were dissected within 1 hour in Supplemented Schneider’s Medium. The samples were then processed according to the protocol outlined in (Everetts et al ., 2021). Briefly, we used a mixture of trypsin and collagenase to enzymatically dissociate the tissues.…”

“…Before cell filtering, we used scvi-tools to harmonize our single-cell data from regenerating wing discs with the single-cell data from developing wild-type wing discs presented in our previous study (accession number GSE155543) (Everetts et al ., 2021). We used Seurat’s variance-stabilizing transformation method to select 1000 variable genes for each batch, and the scVI VAE model was trained on the union of these genes with the following parameters: n_latent = 15, n_layers = 2, gene_likelihood = “nb”, max_epochs = 400, and train_size = 0.8.…”

“…For each identity combination (hinge-pouch, pouch-notum, and notum-hinge), gene signatures were constructed as follows: First, differential expression was performed between wild-type (non-regenerating) cells of each identity pair (e.g., for the hinge-pouch signature, differential expression was performed between cells from (Everetts et al ., 2021) classified as hinge vs. cells classified as pouch). This was conducted using a Wilcoxon test via Seurat’s FindMarkers function, selecting genes with a natural-log fold-change of greater than 0.25 (logfc.threshold = 0.25) and a Bonferroni-corrected p-value of < 0.05.…”

“…Data colored by sample of origin and labeled. Samples were derived from developing discs at the middle at late stages of the third larval instar (L3), as described previously (Everetts et al ., 2021), and from regenerating discs 24h after the downshift to 18° C. Two biological replicates were obtained for each sample (see Materials and Methods). The three major cell types identified were epithelial cells, myoblasts and hemocytes.…”

Necrosis-induced apoptosis promotes regeneration in Drosophila wing imaginal discs

“…DUAL Control experiments utilized the DVE>>GAL4 driver that drives expression in the majority of the wing pouch (Figure S1A-B). In the experiment to examine egr (Figure S4H-J), egr-GAL4 was utilized instead of DVE>>GAL4 , which drives in the adult muscle precursor (AMP) cells of the disc (Everetts et al 2021).…”