Regeneration Discovery May One Day Inform Hearing Loss Treatment

Hair cells in the inner ear detect mechanical stimuli from sound waves and convert them into electrical signals that the brain can interpret. Some animals, such as zebrafish, can regenerate their inner ear hair cells upon damage, but their mammalian counterparts are much more limited in this capacity. To understand why, developmental biologist Tatjana Piotrowski at the Stowers Institute investigates the molecular mechanisms that allow zebrafish to regenerate their hair cells, in hopes of figuring out what parts of the process are missing in mice and humans.

“There’s really hope in the field that we will be able to trigger [hair cell] regeneration [in mammals] at some point,” Piotrowski said.

In a recent study, Piotrowski’s team found that two cyclin genes independently regulate two proliferating cell types in zebrafish during inner ear hair cell regeneration.¹ Their findings, published in Nature Communications, indicate that cell type-specific regulation, not specific genes, is the critical determinant for this regeneration.

“It was surprising that it’s not just one gene that makes all the cells divide, but you can have dividing cells that are driven by different genes,” Piotrowski said.

David Raible, a developmental biologist at the University of Washington who was not involved in the study, said, “It’s a really interesting study—a nice body of work.”

To circumvent the technical challenges of working with zebrafish inner ear hair cells, Piotrowski’s team used hair cells in neuromasts, which are garlic bulb-shaped sensory organs that line zebrafish’s body from head to tail. Like their counterparts in the inner ear, hair cells in neuromasts convert mechanical stimuli—in their case, from water movement—to electrical signals. Because neuromasts are located close to the skin surface, scientists can easily manipulate and image them, especially in the transparent zebrafish larvae.

Zebrafish neuromasts consist of two cell types: the differentiated hair cell progenitors at the organs’ center and the undifferentiated support cells that surround them. Hair cell progenitors divide to produce two more progenitors, while support cells divide asymmetrically, such that one daughter cell differentiates to become a progenitor hair cell, while the other remains undifferentiated like its parent.^2-6 The latter ensures that the pool of undifferentiated cells doesn’t get depleted. On the other hand, in newborn mice, inner ear hair cells can regenerate without proliferation through a process called transdifferentiation, in which support cells differentiate to replace the lost hair cells, but pups lose their capacity for regeneration within a week or so.⁷

“Cell proliferation is likely critical for regeneration,” Raible said.

To find the genes that regulate proliferation during regeneration, Piotrowski’s team induced hair cell death in zebrafish larvae using the antibiotic neomycin and then performed single-cell RNA-sequencing on neuromasts. They discovered that two cyclin D genes, ccndx and ccnd2a, were specifically expressed in progenitor and support cells, respectively. The researchers showed that upon damage, ccndx and ccnd2a expression levels were initially low, but they became significantly upregulated within several hours when cells began to proliferate.

To validate the functions of ccndx and ccnd2a, the researchers used CRISPR to delete these genes in zebrafish larvae and then observed how their loss affected hair cell regeneration upon neomycin-induced damage. They found that knockout of ccndx, which is present in zebrafish but not mice, eliminated proliferation in the hair cell progenitors, but its loss had no effect on either proliferation or differentiation in the support cells.

Raible didn’t expect that hair cell regeneration could occur in the absence of proliferation in zebrafish. He said, while other animals could regenerate hair cells without cell proliferation, researchers in the field widely thought that in zebrafish, proliferation was a critical aspect of hair cell regeneration.

“It seemed like there was a different mechanism for [zebrafish],” Raible said. “But this study showed that ccndx can uncouple proliferation from regeneration, and I think this commonality is interesting.”

In contrast to ccndx, Piotrowski’s team found that ccnd2a knockout only affected support cell proliferation during development, but not regeneration. They proposed that this was due to functional compensation by another cyclin D gene, ccnd1, which was also upregulated during regeneration. The researchers also showed, unexpectedly, that the expression of ccnd2a in progenitor cells could rescue hair cell regeneration in ccndx-knockout larvae. This indicated that it was the cell type-specific expression of ccndx, and not the gene itself, that was critical to induce regeneration.

“Of course, our first hope was that ccndx would be the key gene that allows regeneration,” Piotrowski said. “But we’re still, unfortunately, far away from making a functional hair cell.”

However, Piotrowski said, human organs that have regenerative capacity, like the skin, intestine, and blood, also have distinct cell populations that proliferate, and their regulatory mechanisms are still not well understood.

“To my knowledge, people have not really investigated if proliferation in different cell populations is regulated differently, so our findings could have implications for these other regenerating organ systems,” she said.