A new Nature Physics study shows that zebrafish regrow fins to their original size using Erk gradients. Researchers found that a burst of growth signal at the fin tip guides this process. "Understanding how positional memory is orchestrated at the cellular level inspired the recently published work," the researchers said.
In a new Nature Physics study, researchers found that fin regrowth in zebrafish is driven by a brief burst of growth signal at the fin tip, sized to the amount lost, which sets up gradients of Erk activity that guide the regrowth.
Unlike mammals, animals such as zebrafish and salamanders can regrow major appendages like limbs and fins after amputation. The regrown structure matches the original in shape and size, with no stunting or overgrowth. This is known as positional memory.
Size control is a fundamental biological aspect. During normal development, limbs and organs are proportional to the body, and errors in size control can severely impair their function. Regeneration poses the same challenge. Researchers have identified many of the proteins needed to regrow a fin, but how they work together to restore the right size has remained a missing piece of the puzzle.
A leading idea has been that size information is stored in the fin before injury, for example, as a protein whose levels decrease along its length. While there is evidence for this in axolotl limbs, no such stored signal has been found in zebrafish.
Phys.org spoke with co-authors Ashley Rich and Stefano Di Talia, both at Duke University Medical Center, and Ken Poss at the Morgridge Institute for Research and the University of Wisconsin–Madison.
"Understanding how positional memory is orchestrated at the cellular level inspired the recently published work," the researchers explained.
Fins, rays and Erk activity
A zebrafish's tail fin consists of 16 to 18 thin bony structures, called rays, separated by soft tissue. Each ray contains different cell types, including osteoblasts, the bone-building cells. Each ray in an amputated tail fin grows independently. The rays that lose more tissue grow faster so that all of them return to their original length at roughly the same time.
Regrowth begins with a layer of wound skin forming over the cut. This skin releases growth factors that promote regeneration, including Fgf (fibroblast growth factor). A growth zone then forms at the tip of each injured ray.
The researchers focused on a protein called extracellular signal-regulated kinase, or Erk, which is activated inside cells by external signals such as Fgf. Erk promotes cell division, growth and movement. Both Fgf and Erk have previously been linked to fin regeneration.
"We focused on Erk for two reasons," the researchers explained. "First, Erk is a known regulator of cell proliferation, and prior research established that regenerating caudal fin rays exhibit large amounts of cell proliferation. Second, prior research from the Di Talia and Poss labs showed that Erk is a key regulator of bone regeneration in zebrafish scales."
To see how Erk guides regrowth, the team tracked its activity across thousands of cells in regenerating fins.
Tracking cells
To monitor regrowth at the cellular level, the researchers used genetically engineered zebrafish whose cells fluoresce under a microscope. Different fluorescent markers lit up osteoblasts, dividing cells, Erk activity and Fgf-producing skin cells.
The researchers amputated different amounts of fin tissue ranging from 0.7 to about 4 millimeters per ray. Since the tail is forked, a straight cut removes more from the outer rays than the inner ones. These rays were observed in living fish for nearly two weeks using high-magnification microscopy. Each ray was repeatedly photographed, and overlapping images were stitched together to capture the full regrowth in each ray.
Soon after the amputation, wound skin forms at the cut site. This skin marks the beginning of the regeneration process. It releases growth factors including Fgf. A "growth zone" then forms at the tip of every ray. The osteoblasts in the growth zone come mainly from existing bone cells near the cut.
These cells revert to a less specialized state and migrate into the growth zone. Some also come from reserve bone-forming cells at the ray's joints. The number of Fgf-producing skin cells peaks around day 3, while bone cells divide throughout the new tissue. The ray starts to lengthen past the cut.
By day 4, there's enough new tissue that researchers can distinctly identify a base, a middle and a tip section. This is when the marking begins.
The microscope laser is used to switch the color of a group of cells in each section so they are easy to follow. The researchers then followed these groups for 24 hours to see which section was growing. They also compared Erk activity with cell division in the same cells and mapped how Erk varied from the tip of each ray to the cut site over time.
To probe where the Erk signal comes from, they used drugs to block Erk and the Fgf receptor, measured where and when Fgf genes switched on, and counted the Fgf-producing skin cells at the fin tip. They then repeated key measurements in longfin mutants, whose fins overgrow, for comparison.
Finally, the team built mathematical and physics models to test whether the Erk patterns could predict how each ray grows and explain how those patterns form.
The marking experiment showed that only the cells at the tip of the new tissue expanded and gained cells, while those at the base and middle stayed put. Cell division accounted for about 83% of the bone's lengthening.
Cells with higher Erk activity were more likely to divide, regardless of how much of the fin had been removed, and blocking Erk stopped both division and growth. Along each ray, Erk activity formed a gradient, highest at the tip and lowest at the cut site. As the ray grew, the gradient stretched and faded while keeping the same shape.
"We found that gradients of Erk activity act as rulers during fin regeneration, keeping track of how much regenerative outgrowth has occurred and instructing the outgrowth that still needs to occur," the researchers said.
These gradients traced back to the Fgf-producing skin cells at the fin tip. Fgf was made in a brief burst, peaking around day 3, and the number of producing cells matched the amount of fin removed. Blocking the Fgf receptor shut down Erk activity, cell division and growth.
The team's model suggests the signal does not spread by diffusion. Instead, it is carried along by the growing tissue, a process called advection, and slowly breaks down. For this to work, the signal must last at least four days.
As the researchers put it, a given cell "need not 'know' how much tissue was cut off. Rather, a given cell simply needs to 'follow' the instructions already encoded during the early stage of regeneration."
In longfin mutants, the number of Fgf-producing cells did not match the amount removed, but it still predicted how much the fin actually regrew. Erk activity also faded more slowly in these fish, which may explain their overgrowth.
Looking ahead
The findings suggest that in zebrafish, size memory may be set by an early response to the injury, rather than stored in the fin beforehand.
The researchers believe their approach to studying cell behavior during regeneration could also inform human tissue repair.
"It will not be sufficient to simply turn human tissue regeneration on. Successful regenerative therapies will need to understand how to titrate pro-regenerative signals over time to ensure that appropriate size, and ultimately function, are achieved," they said.
While the study explains how regrowing bone cells read size information, it does not explain how that information is first set. How the patch of Fgf-producing skin cells comes to match the amount of fin removed remains unknown.
"A key future direction is understanding how positional (or size) memory is encoded in the earliest stages of fin regeneration," the researchers said.
"Another key future direction is expanding our studies to other tissues. The fin is composed of several tissues, including epidermis and mesenchymal fibroblasts. Each of these tissues has a unique organization and geometry, and we believe understanding how size is controlled during the regeneration of each of these tissues is a fascinating next step."