Exploring Regenerative Potential in Zebrafish and Humans

Xray image of a Zebrafish Fin
Image of a zebrafish fin that has regenerated after amputation, with the bone forming osteoblast cells labeled green.

ISCRM faculty member Ron Kwon, PhD, is an Associate Professor of Orthopaedics and Sports Medicine, and the director of the Musculoskeletal Systems Biology Lab. There are two broad areas of interest for Kwon and his team. The first area of interest is skeletal genetics – the study of how genetic variation influences musculoskeletal tissue development, repair, and risk for  diseases like osteoporosis.

At the same time, the lab is also trying to answer a question that has tantalized scientists for centuries. Why are some animals, including zebrafish and salamanders, able to regrow bony appendages such as limbs after amputation – and could something in the biology of these animals teach us to someday regenerate human limbs?

Fortunately for us, zebrafish make a surprisingly good model. For starters, 70 percent of human genes are also found in zebrafish. Moreover, their tail fins contain bones that, when amputated, are able to regenerate in a process similar to limb regeneration in salamanders. If researchers could pinpoint the genes that drive fin regeneration in these aquatic creatures, the discovery could reveal useful information about similar genetic pathways in humans.

Head shot of Joyce Tang
Joyce Tang, MS is a Research Scientist in the Musculoskeletal Systems Biology Lab

Unfortunately for us, there are more than 30,000 genes to sort through. That is a lot of puzzle pieces. Propelled by advances in technology, the picture is coming together. Joyce Tang, MS, a Research Scientist in the Kwon Lab, is the lead author of a paper published this month in the journal iScience that sheds a light on the characteristics of zebrafish cells that are able to differentiate from progenitor cells into bone cells, and then revert back to a progenitor state.

While genetic similarities between zebrafish and humans offer hope, fundamental differences between the species also provide clues. “In mammals, differentiation usually only goes one way,” explains Kwon. “Stem cells turn into osteoblasts (bone cells) and that’s it. But in zebrafish and salamanders, those cells can de-differentiate and re-differentiate, seemingly in perpetuity.”

Faculty head shot of Ron Kwon
ISCRM Faculty Member Ron Kwon, PhD

The idea of converting mature cells into progenitor cells with potential for large-scale regeneration by activating specific genes has obvious allure for scientists, physicians, and patients. However, researchers first need to know which of the approximately 30,000 genes in the zebrafish genome are involved in regeneration. For this, the researchers turned to single-cell RNA sequencing, a technology that has allowed scientists to precisely identify the genes activated by cells as they transition from one type to the other.

In their study, Tang and her colleagues discovered that during zebrafish fin regeneration, osteoblasts activate a large number of genes involved in a process called EMT/MET, or epithelial-to-mesenchymal transition (EMT) – and vice-versa. Specifically, in EMT, epithelial cells such as those that line the outer surfaces of organs and blood vessels throughout the body, acquire characteristics of mesenchymal cells, which tend to reside within connective tissues and be highly motile. While this process is critical for development, in some types of cancer EMT/MET is a key process that allows solid tumors to metastasize and become more invasive.

“Our findings build on a study in 2014 by Stewart et al., who first showed that EMT markers are exhibited during fin regeneration. Our study provides support that, at the genome-wide level, there are many genes involved in EMT/MET being expressed by osteoblasts during the process of differentiation and de-differentiation,” says Kwon.  “This finding lends support to the idea that EMT and potentially MET might be important biological processes enabling reversible switching between de- and re-differentiated states.”

Does the reversible nature of zebrafish osteoblasts mean that human osteoblasts might also have an untapped regenerative potential? Our own biology may offer evidence that it does. “EMT is a normal part of development and occurs in diseases such as cancer,” says Kwon, adding that EMT and MET enable cancer cells to go back and forth between differentiated and de-differentiated states. “Thus, humans have the genes necessary for EMT/MET; if the activation of EMT/MET is sufficient to enable reversible switching between de- and re-differentiation in human osteoblasts becomes a compelling question.”

The sequencing work was performed in partnership with the Brotman Baty Institute and was supported by a grant Kwon shares with ISCRM faculty members Drs. Olivia Bermingham-McDonogh, Tom Reh, Andrea Wills, and Hannele Ruohola-Baker. The grand vision of that grant, says Kwon, is “to generate a single cell atlas of different models of regeneration and to find broadly conserved genes that could be activated across these systems.” In other words, the group, informally known as the Regeneration Club, wants to compare and understand how regeneration works across species, organs, and tissues.

Faculty headshot of Christopher Allan, MD
ISCRM Faculty Member Christopher Allan, MD

Christopher Allan, MD, an Orthopaedic Surgeon and an investigator in the Musculoskeletal Systems Biology Lab, provided key scientific expertise on the study. Like Kwon, Allan is fascinated by the possibility that humans have innate potential for appendage regeneration, with a specific interest in the comparison of human tissues with appendage regeneration in other species. Allan has also has helped to develop a therapeutic glove that is designed to regenerate digits. The fit and comfort of the glove are now being tested in clinical trials.

For now, Kwon is eager to build on the findings detailed in the new paper. “We now have an idea that EMT/MET as a biological process might be important for osteoblast de-and re-differentiation during appendage regeneration and have generated a list of genes that are known to be important for this process and which appear to be differentially regulated. What we want to know next is when and where those genes are being turned on and off, and what their functions might be.”