The ablation/regeneration system that we have developed has several advantages over traditional methods that use surgical ablation and transplantation. First and foremost, this approach is far less labor-intensive and therefore allows for the examination of large numbers of regenerating discs in any experiment. Second, since the extent of ablation is defined by the expression of the pro-apoptotic gene under the control of tissue-specific regulatory elements, rather than a surgical instrument, the extent of ablation is relatively reproducible. Furthermore, by using different GAL4 driver lines, this method can be adapted to ablate tissue in other imaginal discs or in other organs during development. Finally, this process occurs entirely in vivo and hence allows an examination of disc-specific as well as systemic factors that regulate regeneration. Taken together, the advantages of this method will facilitate screens for genes that regulate regenerative growth.
The data presented here suggest that regeneration in response to surgical or apoptotic tissue damage occurs via at least one common mechanism, the upregulation of Wg and Myc. In addition, JNK activity is normally upregulated after injury and plays an important role in wound healing and regeneration in response to surgical damage (Bosch et al., 2008
; Bosch et al., 2005
; Mattila et al., 2005
). While Eiger activity has been shown to induce JNK signaling (Igaki et al., 2002
), we have also observed elevated JNK signaling in discs damaged by expression of UASreaper
(data not shown). Therefore activation of JNK in response to tissue damage is also likely a universal characteristic of regenerating discs and not unique to discs damaged by expression of UASeiger
. However, by activating JNK directly, Eiger may facilitate the initiation of tissue repair. In addition to activating JNK, surgical damage to discs leads to the upregulation of other molecules involved in wound healing such as Mmp1 (McClure et al., 2008
). We have observed that Mmp1 is similarly expressed in discs regenerating in response to tissue ablation by Eiger (data not shown). Thus regeneration after tissue damage may involve several common mechanisms irrespective of the method by which tissue ablation occurs.
The expression of Wg is markedly elevated during regeneration, with the highest levels observed in proliferating cells that are close to the site of ablation. In addition to Drosophila
imaginal discs, the upregulation of specific Wnt-family genes is observed in regenerating tissue in a wide variety of organisms including planaria (Gurley et al., 2008
; Petersen and Reddien, 2008
), Xenopus and zebrafish, as well as mammalian liver and skeletal muscle (Stoick-Cooper et al., 2007
). Thus Wnts may have an important function in regenerative growth that is evolutionarily conserved. Recent studies have shown that Wnt-family proteins can maintain several types of stem cells in a self-renewing state (Nusse, 2008
). Thus, increased expression of Wg may also antagonize differentiation signals in imaginal disc cells and allow the regenerating tissue to maintain a capacity for proliferation that is normally observed in even younger discs.
Our studies have revealed a likely role for Myc in driving regenerative growth. Others have observed that overexpression of CyclinD and Cdk4 is more effective than Myc at increasing wing size during normal development (de la Cova et al., 2004
). Thus the regulation of growth during regeneration may differ from the regulation of growth during normal development. Consistent with this idea is the observation that in surgically cut discs, cell size and the proliferation kinetics of the regenerating tissue are different from those that are observed at any time during normal disc development (Sustar and Schubiger, 2005
). The ability of Myc to potentiate regenerative growth may relate to Myc’s role in promoting cell plasticity, which was observed when the overexpression of c-myc
together with three other genes (oct3/4
) converted cultured adult fibroblasts into pluripotent stem cells (Takahashi and Yamanaka, 2006
). Indeed, our observations that some cell fate commitment and stage-appropriate patterning are abrogated in regenerating discs are consistent with such a role for Myc. These findings also raise the possibility that the ability of Wnt proteins to promote self-renewal in mammalian stem cells may be mediated, at least in part, by Myc.
A fundamental and unsolved question that underlies much of the research on regeneration is why the same tissues from different species or from different stages of development in the same species show a markedly different capacity for regenerative growth. In a similar vein, we have demonstrated that ablation of the wing pouch elicits a robust regenerative response until the middle of the third larval instar. The loss of the ability to regenerate likely reflects developmentally regulated changes in the disc epithelium that interfere with the activation of the gene expression programs that are required for regenerative growth. This block in regeneration may be the result of changes in the properties of the disc cells themselves or the result of changes in humoral factors that occur prior to the onset of metamorphosis. Indeed, these scenarios are not mutually exclusive as recent work has shown that Wg expression can be repressed by Ecdysone signaling (Mitchell et al., 2008
). The inability to elicit regeneration in older discs either by overexpressing Myc or by activating Wg signaling indicates that additional growth regulators may have an important role in permitting or preventing regeneration. Indeed, some of these regulators may be unique to mature discs and would not have been identified in previous studies of the growth that occurs during normal development. Thus the development of the system described here can facilitate large-scale unbiased genetic screens for novel regulators of regenerative growth.