Single-cell RNA sequencing of Drosophila wing discs harboring the scrib mutant clones in combination with RasV12, YkiS168A or NAct signals.

It has long been proposed that cell competition functions to remove precancerous clones. A classical model is the removal of polarity-deficient clones such as the scribble (scrib) mutant clones in Drosophila imaginal discs. The activation of Ras, Yki or Notch signaling robustly reverses the scrib mutant clonal fate from elimination to tumorous growth. Using single-cell transcriptomics techniques to profile wing imaginal discs harboring the scrib mutant clones in combination with different signals, we found that a critical converging point downstream of Ras, Yki and Notch signals is the upregulation of Upd2, which is necessary to promote tumorous growth. Unexpectedly, while Upd2 is not required for cell survival per se, Upd2-deficient clones are efficiently wiped out from epithelia, indicating that Upd2 is a previously unrecognized cell competition factor. We generate the scrib mutant clones with activated Ras, Yki or Notch signals using MARCM system in Drosophila wing imaginal discs. The control MARCM GFP+ clones occupy around 30% of the whole disc area at 36 hours after clone induction (ACI) and around 40% of the disc area at 60 hours ACI. In contrast, the scrib mutant clones occupy around 20% of the whole disc area at 36 hours ACI and 10% of the whole disc area at the 60 hours ACI. When RasV12, YkiS168A or NAct is co-expressed in the scrib mutant clones, the GFP+ clones occupy around 30% of the whole disc area at 36 hours ACI. By 60 hours ACI, the scrib mutant clones co-expressing RasV12, YkiS168A or NAct start to exhibit tumor-like morphology and occupy around 30%-40% of the whole disc area. The clone size and morphology are comparable across all five experimental groups at 36 hours ACI, and cell competition process is an on-going process at this early stage. Thus, we choose to generate single-cell RNA transcriptomics datasets of wing discs carrying GFP+ clones of these genotypes: control, scrib-/-, scrib-/- RasV12, scrib-/- YkiS168A and scrib-/- NAct at 36 hours ACI. To generate MARCM clones for each genotype, around 50 embryos were collected within 3 hours and raised until 60 hours after egg laying (AEL). The larvae were then placed in water bath for 1 hour at 37°C for heat shock and dissected at 36 hours to perform single cell experiment.