Genetic DISC-section of regeneration in Drosophila

Abstract

Although regeneration has long fascinated biologists, it remains a challenging field of study with much yet to learn at the molecular level. In this issue of Developmental Cell, Bolton-Smith et al. introduce a genetic ablation system in Drosophila melanogaster with the potential for large-scale identification of new regulators of regeneration.

Many animals are capable of regenerating lost body parts, and there has historically been great interest among biologists in understanding how and why regeneration occurs. Through its study, we learn 1) how injury provokes growth, not scarring; 2) how stem and progenitor cells are directed successfully toward morphogenesis; 3) how newly created cells integrate into an existing, patterned organ; and 4) how identity of position is maintained and recalled by adult tissues. Yet, the study of regeneration has been hampered over the years by inconvenient model systems and the inaccessibility of adult developmental events to genetic manipulation. In particular, there exist few unbiased, large-scale approaches to regeneration. Current methods include RNAi-mediated knockdown screens in planarians, which provide a spectacular, stem cell-based prototype of animal regeneration and homeostatic maintenance, and mutagenesis screens in zebrafish, which offer the advantages of studying elevated regenerative capacity in the contexts of vertebrate organs (Poss et al., 2002; Reddien et al., 2005). These model organisms have generated excitement of late, as both the understanding of regenerative events and the available techniques continue to expand and mature. In this issue of Developmental Cell, Bolton-Smith and colleagues describe a new method for identifying regulators of regeneration in a particularly established and tractable genetic model system, Drosophila melanogaster (Bolton-Smith et al., 2009).

While adult Drosphila appendages lack regenerative capacity, it has been known for decades that larval imaginal discs regenerate following injury and transplantation into the adult female abdomen (Hadorn, 1963). A related phenomenon displayed by discs is transdetermination, referring to a process during which disc cells switch identity; e.g. wing cells to leg cells. Mechanisms of imaginal disc regeneration and transdetermination have been vigorously pursued (McClure and Schubiger, 2007). However, transdetermination is a distinct process from regeneration, and difficulties associated with transplantation techniques have hindered high-throughput examination of imaginal disc regeneration. The method described by Bolton-Smith et al. incorporates powerful genetic tools to achieve a reproducible ablation of a large region of the third instar imaginal wing disc, and offers new potential for unbiased searches for regulators of tissue regeneration.

The authors employ the GAL4-UAS system to ectopically express either Eiger (the Drosophila TNFα ortholog) or Reaper, resulting in apoptosis of most expressing cells. Ablation is restricted to the larval wing pouch by the use of the rnGAL4 enhancer trap line, while temporal control is achieved through inclusion of a temperature-sensitive GAL80 (tubGAL80^ts). Consequently, injury is controlled both spatially and temporally by a simple temperature shift (Figure 1). As is the case in other examples of regeneration across phyla, the capacity for complete regeneration of a new ablation injury is gradually lost as development proceeds. However, the authors find a period during development when injury to the presumptive wing results in high-frequency restoration of a properly sized and patterned wing in the adult fly. Expression and lineage-tracing experiments indicate that the regenerate originates from cells inside the lesioned wing pouch that expressed Eiger, as well as from cells adjacent to the pouch that have potentially switched fates. As part of this regenerative response, wing compartment markers like engrailed and cubitus interruptus maintain their expression domains after ablation. At the same time, growth regulators like dpp and vestigial display temporary expansion and reduction in expression, respectively, although with largely intact regional localization. By contrast, the wingless gene loses its typical localization after injury, and is induced throughout the disc and highest in the pouch. Additional organ-wide and systemic responses occur after ablation, including the restriction of cellular proliferation from undamaged regions, and delays in pupariation and eclosion. Thus, the authors establish a large number of cellular and molecular features of this injury/regeneration system (Figure 1).

Figure 1.

Genetic ablation system to provoke injury and regeneration in the Drosophila wing disc.

After characterizing the injury response, Bolton-Smith et al. complete several functional studies, first focusing on Wingless. A role for Wingless in regeneration might have been predicted from studies of embryonic wing development and in regeneration in other organisms (Couso et al., 1993; Stoick-Cooper et al., 2007; Lengfeld et al., 2009). Following ablation, Wingless is induced in non-apoptotic regenerating cells of the wing pouch, which also induce the growth-promoting transcription factor Myc. Using available loss- and gain-of-function tools, the authors describe a mechanism in which Wingless inhibits Notch activity, enabling Myc expression and regeneration (Figure 1). Myc appears to have specificity in this system. Transgenic Myc overexpression increased the frequency of full wing regeneration, unlike other drivers of cell growth, but was not sufficient to extend the developmental period during which when the disc can regenerate after ablation. Perhaps most importantly, Bolton-Smith et al. perform additional experiments that demonstrate great potential for identifying new modifiers of regeneration. First, they show that heterozygosity in the capicua gene, which was previously described as a negative regulator of growth control, can enhance the average size of regenerates. Second, the authors test a series of chromosomal deletion lines, from which they identify deficiencies that can either negatively or positively modify regeneration. There is thus considerable promise for discovering many more regulators by a comprehensive deficiency screening or mutagenesis, and then elegantly manipulating them during regeneration.

The system described here is the most comprehensive package to date of genetic tools necessary for large-scale dissection of an injury and regeneration paradigm, including ablation injury, transgenesis, and forward genetics. Modifications of technical aspects of the screen will further increase its impact. For instance, the use of the GAL4-UAS system for ablation limits the efficacy of UAS-RNAi and overexpressor transgenes, which currently are localized to cells also expressing death signals. To uncover the widest range of regulators specific to the regenerative process itself, manipulation of knockdown or overexpressor cassettes should be targeted post-injury in the absence of apoptotic influences. Even so, the strengths of the system are clear and provide an excellent glimpse into the future of regeneration research. Non-surgical ablation systems that employ spatiotemporal control of cytotoxins for provoking injury and examining regeneration are also available for use in vertebrates. For example, recent ablation studies in zebrafish larvae have employed the bacterial enzyme nitroreductase, which can be expressed from a tissue-restricted promoter and converts an experimentally applied prodrug metronidazole into a cytotoxic compound (Pisharath et al., 2007; Curado et al., 2007). Thus, there are high hopes and expectations for large-scale regeneration screens across the phylogenetic spectrum, and for the collective light that genes identified by these approaches will shed on our understanding of regenerative events.