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Regeneration, the ability to heal damaged or lost tissues, is both an everyday and a real-life superpower. Health research inspired by the remarkable regeneration of animals like axolotls or starfish asks how future therapeutics could promote the regrowth of hard-to-heal tissues, limbs, or even entire organs.

A new study in Science Advances is a step toward clarifying a less visible—yet vital—corollary: how regeneration comes to a successful close when a tissue has healed.

"Many people have asked the question 'How does regeneration begin, and why does it begin in some animals and tissues and not in others?'" said Rachel Smith-Bolton, an associate professor of cell and developmental biology. "Those are really important questions and things we've been looking at, but not a lot of people have asked, at the end of the process . . . 'How does it end and rebuild the structure it's supposed to rebuild?'"

Smith-Bolton, the principal investigator of the study, worked with graduate student Anish Bose and their colleagues to identify mechanisms supporting the end of regeneration in an unlikely animal: the larval fruit fly, Drosophila. Fruit flies lack the dramatic regenerative capabilities that a few other animals are known for, but decades of biological research make them a powerful model for studying gene functions. Their development from larva to pupa to adult also presents an unexpected opportunity to study regeneration.

Fruit fly larvae, like other insects that undergo metamorphosis, contain tissues called imaginal disks. Imaginal disks are like epithelial building blocks that, during pupation, will transform to form adult anatomical structures such as eyes, antennae, legs, and wings. If the imaginal disks are damaged before pupation, they will regenerate. Researchers can cause this damage in a precise way and track how the imaginal disks regrow.

Bose, Smith-Bolton, and their co-authors hypothesized that particular genes are responsible for making sure that regeneration ends with the right number and types of cells. One of the genes that Smith-Bolton's lab was interested in looking more closely at was a gene whose whimsical name, Zelda, contrasts with its important known roles in development. Using a special form of the gene that is inactivated when the larva is exposed to blue light, they tracked how well the larval wing disk could recover when Zelda was inactivated at different points of the regeneration process.

In larvae with inactivated Zelda, undamaged wing disks developed normally, and damaged disks could begin regrowth. But without Zelda, the new cells didn't know how to finish the job. When the flies emerged after pupating, their wings had grown incorrectly, with missing or misplaced veins and bristles, as well as confused boundaries between different sections of the wing.

"One of the most exciting findings from the study is Zelda's surprising specificity during regeneration," said Bose, who was first author on the publication. "During the mid-stages of regeneration, Zelda becomes crucial for the wing disk to properly recover. This reveals a striking distinction between how tissues grow during development and how they repair themselves after injury."

The researchers considered how to fit this surprising finding with Zelda's already known function in development. Using this gene, cells produce a protein that is a transcription factor—its job is to help activate other genes. In regenerating disks, further experiments showed that Zelda is helping to control the activity of genes that help guide proper tissue development.

Even among transcription factors, Zelda is in a special category.

"This class of transcription factor is called a pioneer transcription factor. And what that means is that if a region of the genome is tightly closed down to not express a gene, it can go in and open that region up in order to allow expression of a gene," Smith-Bolton said. "Our hypothesis now is that pioneer transcription factors may play important roles in these shifts."

Although Zelda itself is an arthropod-specific gene, its identity as a pioneer transcription factor points back to what studies like this one, which was supported by the NIH and the University of Illinois, help us understand about healing and disease. In addition, inappropriate tissue growth is a hallmark of cancer, and study of other pioneer transcription factors has led to the identification of new cancer therapeutics.

"If the idea behind regenerative therapy is that you're going to add factors that drive the process, you have to know how to control those factors appropriately and also understand what mistakes they might be causing and how you can prevent those mistakes," Smith-Bolton said. "How would you control and prevent changes in pattern and cell fate? As a lab, we've been identifying some things that help prevent those errors."

Bose is also looking forward to the potential to better understand Zelda's differing functions, and by extension, the dynamics of regeneration.

"Could artificially increasing Zelda levels during normal development be harmful? And what molecular mechanisms are in place to keep Zelda in check when regeneration isn't occurring?" Bose said. "These are the exciting directions our lab is eager to explore next."