Summary The study of regeneration would be aided greatly by systems that support large-scale genetic screens. Here we describe a non-surgical method for inducing tissue damage and regeneration in Drosophila larvae by inducing apoptosis in the wing imaginal disc in a spatially and temporally regulated manner. Tissue damage results in localized regenerative proliferation characterized by altered expression of patterning genes and growth regulators as well as a temporary loss of markers of cell fate commitment. Wingless and Myc are induced by tissue damage and are important for regenerative growth. Furthermore, ectopic Myc enhances regeneration when other growth drivers tested do not. As the animal matures, the ability to regenerate is lost and cannot be restored by activation of Wg or Myc. This system is conducive to forward genetic screens, enabling an unbiased search for genes that regulate both the extent of and the capacity for regeneration.

The study of regeneration in Drosophila imaginal discs provides an opportunity to use powerful genetic tools to address fundamental problems pertaining to tissue regeneration and cell plasticity. We present a historical overview of the field and describe how the application of modern methods has made the study of disc regeneration amenable to genetic analysis. Discs respond to tissue damage in several ways: (a) Removal of part of the disc elicits localized cell proliferation and regeneration of the missing tissue. (b) Damage at specific locations in the disc can cause cells to generate disc-inappropriate structures (e.g., wing instead of leg), a phenomenon known as transdetermination. (c) Diffuse damage to imaginal discs, results in compensatory proliferation of surviving cells. Candidate-gene approaches have implicated the JNK, Wingless, and Hippo pathways in regeneration. Recently developed systems will enable extensive genetic screens that could provide new insights into tissue regeneration, transdetermination and compensatory proliferation.

SUMMARYTwo types of information are particularly valuable in understanding the development of a tissue or an organ from a small population of founder cells. First, it is useful to know the composition of the final structure in terms the contribution of individual founder cells. Second, it is important to understand cell-cell interactions. To facilitate the study of both of these aspects of organ development at a tissue-wide level, we have developed a method, TIE-DYE, that allows simultaneous lineage tracing of multiple cell populations as well as the genetic manipulation of a subset of these populations. Seven uniquely marked categories of cells are produced by sitedirected recombination of three independent cassettes. We have used the TIE-DYE method to estimate the number of founder cells that give rise to the wing-imaginal disc during normal development and following compensatory growth caused by X-ray irradiation of the founder cells. We also show that four out of the seven types of marked clones can be genetically manipulated by gene overexpression or RNAi knockdown, allowing an assessment of the consequences of these manipulations on the entire wing disc. We demonstrate the utility of this system in studying the consequences of alterations in growth, patterning and cell-cell affinity.

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.

Regeneration following tissue damage often necessitates a mechanism for cellular re-programming, so that surviving cells can give rise to all cell types originally found in the damaged tissue. This process, if unchecked, can also generate cell types that are inappropriate for a given location. We conducted a screen for genes that negatively regulate the frequency of notum-to-wing transformations following genetic ablation and regeneration of the wing pouch, from which we identified mutations in the transcriptional co-repressor C-terminal Binding Protein (CtBP). When CtBP function is reduced, ablation of the pouch can activate the JNK/AP-1 and JAK/STAT pathways in the notum to destabilize cell fates. Ectopic expression of Wingless and Dilp8 precede the formation of the ectopic pouch, which is subsequently generated by recruitment of both anterior and posterior cells near the compartment boundary. Thus, CtBP stabilizes cell fates following damage by opposing the destabilizing effects of the JNK/AP-1 and JAK/STAT pathways.