Development and regeneration of Drosophila wing imaginal discs: the role of IncRNAs and the stress sensor D-GADD45

Abstract

Long non-coding RNAs (lncRNAs) are defined as transcripts longer than 200 nucleotides that lack protein-coding potential. Although multiple examples of functional lncRNAs have been described, particularly regulating gene expression at different levels, the function of the vast majority of them remains to be elucidated. Here, we use the Drosophila wing imaginal discs as a model system to study the involvement of lncRNAs in development and regeneration. Additionally, we also studied the role of the stress sensor protein Drosophila Growth Arrest and DNA Damage 45 (D-GADD45) in the wing disc. For the study of lncRNAs, we used transcriptomic data from developing and regenerating wing discs. We identified a set of ~200 lncRNAs expressed in development, as well as 131 differentially-expressed (DE) lncRNAs in regeneration. Among them, we focused on the study of lncRNAs CR40469 and CR34335, which share 99.1% sequence identity, however, their expression pattern is far from similar: while CR40469 is upregulated in regeneration, the expression of CR34335 is inhibited upon damage. We generated a CR40469 knock-out mutant, for which no phenotypes were detected in normal conditions. Nevertheless, upon the induction of cell death, these mutants lost their regeneration capacity, suggesting a putative function of CR40469 in regeneration. Moreover, we characterised the molecular changes occurring in the mutant, revealing a set of 95 DE genes compared to controls. On the other hand, no phenotypes were detected for CR34335 mutants, neither in development nor in regeneration, suggesting that this lncRNA is dispensable for both processes. Regarding the study of D-GADD45, we activated its expression ectopically in the wing discs, resulting in increased apoptosis. Through genetic interaction experiments, we described the D-GADD45-induced cell death as dependent on the activation of the JNK signalling pathway. Additionally, we described a JNK-independent decrease in cell proliferation upon sustained activation of D-GADD45. Finally, we identified D-GADD45 as an essential gene for the regeneration of wing discs, as the use of RNAi constructs against D-GADD45 severely impairs the recovery process after the induction of cell death.