UC Berkeley

Why some tissues can regenerate, while other cannot is a fundamental question for regenerative medicine. The ability to regenerate at least some tissues is widespread across diverse animals. Some, such as Hydra and planaria can regenerate the majority of their body, while urodele amphibians such as salamanders can regenerate the spinal cord in their tails and also a complete limb. Humans can regenerate tissues such as the liver, muscle and skin. There are diverse mechanisms of regenerations, from stem cell based replacement to the local dedifferentiation of tissues adjacent to the site of injury. A better understanding of the genetic regulation of regenerative growth could potentially impact the design of therapeutic strategies to promote regeneration.

The genetically tractable model organism Drosophila melanogaster has the ability to regenerate imaginal disc tissues during larval development and is therefore a powerful system to study the molecular mechanisms that regulate regeneration. We have developed a system to study imaginal-disc regeneration on large scale. The new system is an improvement over methods used in the past in Drosophila, because it is non-surgical and therefore more suited to large-scale screens. Tissue damage is induced in the larval primordium of the adult wing (the wing imaginal disc), in a spatially and temporally controlled manner. Drosophila imaginal discs are capable of regenerating lost tissue. Following damage caused by inducing cell death in the central portion of the wing pouch in the wing imaginal disc, cells surrounding the damaged area undergo proliferation to replace the lost cells, a type of regenerative growth referred to as epimorphic regeneration. Salamander limb regeneration is also a form of epimorphic regeneration. The region of proliferating cells exhibits changed expression patterns of genes that are involved in patterning and growth regulation. In addition, the cells that are undergoing regenerative growth cease to express some markers associated with committed cell fates. The morphogen and Wnt-homolog, Wingless, along with the transcription-factor Myc are expressed at higher levels in the regenerating tissue. Wnt proteins have been found to play a role in vertebrate regeneration and Myc protein is involved in cell proliferation. Increasing the amount of Myc leads to improved regeneration in the wing imaginal disc, while increasing levels of other growth-promoting genes had no effect. This suggests that Myc levels may be specifically important for regenerative growth. In addition, the extent of regeneration of the imaginal discs differs based on the age of the larva. As the animal matures, less regeneration is observed. Using this decrease in regenerative capacity, we have screened for dominant enhancers of regenerative growth. However, the screen was unsuccessful in clearly identifying a negative regulator of regenerative growth, but we did uncover mutants that slow down developmental timing and suppressor of Eiger-induced cell death.

In a different type of genetic screen for modification of regeneration, I identified a mutant that increases the frequency of transdetermination following ablation. Transdetermination is a phenomenon where cells that have been determined to generate one type of structure swich fates to adopt a different determined state. This mutant, which we have named Chameleon, dominantly increases the frequency of notum-to-wing transdetermination following Eiger-induced ablation. We have characterized the formation of the ectopic wing pouch and the data suggest that multiple cells are adopting new fates, possibly switching compartment identity and that local proliferation generates the new structure. The molecular lesion that is responsible for the Chameleon mutant phenotype remains unknown, however I have candidate mutations identified using whole genome sequencing.

In order to better understand different changes in fate that might occur during the processes of regeneration and transdetermination, we have developed a new method, TIE-DYE, which allows simultaneous tracing of multiple cell lineages as well as the genetic manipulation of a subset of these populations. The method creates seven uniquely marked categories of cells and we have used the 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 demonstrate the utility of this system in studying the consequences of alterations in growth, patterning and cell-cell affinity.