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.

Regenerating tissue must initiate the signaling that drives regenerative growth, and sustain that signaling long enough for regeneration to complete. How these key signals are sustained is unclear. To gain a comprehensive view of the changes in gene expression that occur during regeneration, we performed whole-genome mRNAseq of actively regenerating tissue from damaged Drosophila wing imaginal discs. We used genetic tools to ablate the wing primordium to induce regeneration, and carried out transcriptional profiling of the regeneration blastema by fluorescently labeling and sorting the blastema cells, thus identifying differentially expressed genes. Importantly, by using genetic mutants of several of these differentially expressed genes we have confirmed that they have roles in regeneration. Using this approach, we show that high expression of the gene moladietz (mol), which encodes the Duox-maturation factor NIP, is required during regeneration to produce reactive oxygen species (ROS), which in turn sustain JNK signaling during regeneration. We also show that JNK signaling upregulates mol expression, thereby activating a positive feedback signal that ensures the prolonged JNK activation required for regenerative growth. Thus, by whole-genome transcriptional profiling of regenerating tissue we have identified a positive feedback loop that regulates the extent of regenerative growth.

Regenerating tissue must replace lost structures with cells of the proper identity and function. How regenerating tissue establishes or maintains correct cell fates during regrowth is an open question. We have identified a gene, taranis, that is essential for maintaining proper cell fate in damaged and regenerating Drosophila wing imaginal discs but that is dispensable for these fates in normal wing development. In regenerating tissue with reduced levels of Taranis, expression of the posterior selector gene engrailed is silenced through an autoregulatory silencing mechanism that requires the PRC1 component polyhomeotic, resulting in a transformation of posterior tissue into anterior tissue late in regeneration. An essential component of the wound response, JNK signaling, induces this misregulation of engrailed expression. Taranis can suppress these JNK-induced cell fate changes without interfering with JNK signaling activity. Thus, taranis protects regenerating tissue from deleterious side effects of wound healing and regeneration.

, §Drosophila Corkscrew protein and its vertebrate ortholog SHP-2 (now known as Ptpn11) positively modulate receptor tyrosine kinase (RTK) signaling during development, but how these tyrosine phosphatases promote tyrosine kinase signaling is not well understood. Sprouty proteins are tyrosine-phosphorylated RTK feedback inhibitors, but their regulation and mechanism of action are also poorly understood. Here, we show that Corkscrew/SHP-2 proteins control Sprouty phosphorylation and function. Genetic experiments demonstrate that Corkscrew/SHP-2 and Sprouty proteins have opposite effects on RTK-mediated developmental events in Drosophila and an RTK signaling process in cultured mammalian cells, and the genes display dose-sensitive genetic interactions. In cultured cells, inactivation of SHP-2 increases phosphorylation on the critical tyrosine of Sprouty 1. SHP-2 associates in a complex with Sprouty 1 in cultured cells and in vitro, and a purified SHP-2 protein dephosphorylates the critical tyrosine of Sprouty 1. Substrate-trapping forms of Corkscrew bind Sprouty in cultured Drosophila cells and the developing eye. These results identify Sprouty proteins as in vivo targets of Corkscrew/SHP-2 tyrosine phosphatases and show how Corkscrew/SHP-2 proteins can promote RTK signaling by inactivating a feedback inhibitor. We propose that this double-negative feedback circuit shapes the output profile of RTK signaling events.

Regeneration is a complex process that requires an organism to recognize and repair tissue damage, as well as grow and pattern new tissue. Here, we describe a genetic screen to identify novel regulators of regeneration. We ablated the larval wing primordium by inducing apoptosis in a spatially and temporally controlled manner and allowed the tissue to regenerate and repattern. To identify genes that regulate regeneration, we carried out a dominant-modifier screen by assessing the amount and quality of regeneration in adult wings heterozygous for isogenic deficiencies. We have identified 31 regions on the right arm of the third chromosome that modify the regenerative response. Interestingly, we observed several distinct phenotypes: mutants that regenerated poorly, mutants that regenerated faster or better than wild-type, and mutants that regenerated imperfectly and had patterning defects. We mapped one deficiency region to (), the Nrf2 ortholog, which is required for regeneration. Cnc regulates reactive oxygen species levels in the regenerating epithelium, and affects c-Jun N-terminal protein kinase (JNK) signaling, growth, debris localization, and pupariation timing. Here, we present the results of our screen and propose a model wherein Cnc regulates regeneration by maintaining an optimal level of reactive oxygen species to promote JNK signaling.