A team of researchers at Duke University last week showed that inhibiting a specific microRNA, miR-133, can promote fin regeneration in injured zebrafish even when a growth factor-signaling pathway essential for tissue regeneration was inhibited.
The investigators, who published their data in Genes & Development, also found a number of other miRNAs that were regulated during the regeneration process, adding weight to the theory that miRNAs may have therapeutic applications.
“It looks like microRNAs are being regulated in different ways to affect the process of fin regeneration,” Kenneth Poss, an assistant professor of cell biology at Duke University and senior author on the paper, told RNAi News this week. “The implication is that, perhaps, some of these could be regulated to affect regeneration, in general, [as well as the repair of] normally non-regenerative [organs] like the mammalian heart.
“Everyone is looking for targets that can help wounds heal or tissue regenerate after organ damage,” he added. MicroRNAs “are great targets potentially for manipulating an entire program of genes [and] could be an elegant way to enhance or impact a complex process like tissue regeneration.”
Poss said his lab largely focuses on the mechanisms by which zebrafish are able to regenerate various tissues including heart muscle, the retina, the spinal cord, sensory hair cells, and appendages such as the fin.
Given the growing body of evidence linking miRNAs to developmental processes like organogenesis and apoptosis, “we were pretty optimistic about being able to find differential regulation of different microRNAs during different stages of fin regeneration,” Poss said.
After amputating the caudal fins of zebrafish, the research team, which included investigators from the University of North Carolina, Chapel Hill, and the University of Notre Dame, conducted miRNA microarray experiments using RNA collected from tissue at the site of injury at various stages of the regeneration process.
“Everyone is looking for targets that can help wounds heal or tissue regenerate after organ damage. [MicroRNAs] are great targets potentially for manipulating an entire program of genes [and] could be an elegant way to enhance or impact a complex process like tissue regeneration.”
They identified miRNAs that are both up-regulated and down-regulated during fin regeneration, but opted to focus on “microRNAs that have lower expression during regeneration as compared to a normal, uninjured adult fin,” Poss explained.
The team then set out to test the hypothesis that signaling by fibroblast growth factors, which has been shown to be essential for fin regeneration, is responsible for the changes in miRNA expression during the regeneration process.
To do so, they used “a transgenic zebrafish strain that carries a heat-inducible dominant-negative Fgf receptor [that] … effectively disrupts regeneration of fin and cardiac tissues in animals given daily heat shocks,” the investigators wrote in Genes & Development.
Of the 22 miRNAs that were found to be influenced by Fgf signaling during regeneration, one, miR-133, was especially regulated. Poss noted that this miRNA was also of particular interest because it has been linked to skeletal muscle proliferation and differentiation.
After further experimentation, “we found that if we could introduce more of that microRNA [in the form of RNA duplexes], we could slow the process of fin regeneration,” he said. “If we antagonized it [using antisense morpholinos], we could accelerate the process.”
The investigators wrote in the paper that miR-133 antagonism appears to rescue regeneration that has been inhibited by Fgfr blockade by “increasing the expression of regeneration genes and enabling cellular proliferation.
“That was the most surprising result: We could actually cause regeneration to occur more rapidly, even in the face of inhibiting a pathway that we know is important for the process,” Poss added.
A bioinformatics search led the researchers to a number of possible targets for miR-133, including mps1, a gene containing a single predicted binding site for the miRNA that also encodes a kinase that “regulates multiple aspects of cell proliferation during morphogenesis,” they wrote.
“Most importantly, mps1 is one of only four genes to date that have been shown by forward genetic approaches to be essential for fin regeneration,” they added. Experiments using zebrafish embryo sensor assays led them to conclude that the gene is indeed an in vivo target of miR-133.
“In conclusion, our experiments implicate post-transcriptional regulation by miRNAs in the process of complex tissue regeneration,” the researchers wrote. “We identified many miRNAs in zebrafish appendages that showed sharp increases or decreases in expression during the transition from uninjured to regenerating tissue. Through both gain-of-function and loss-of-function experiments, our data reveal that miR-133 is a regenerative brake whose regulated depletion ensures optimal fin regeneration,” they noted.
“With respect to regenerative medicine, our findings suggest that tactical modulation of key miRNAs and their target populations may be sufficient to revise the regenerative capacity of vertebrate organs,” they added.
Poss said that his lab plans to follow up on its findings by looking more closely at other miRNAs that may be important in fin regeneration, as well as those that might be at work during cardiac repair in zebrafish.
Noting that other labs are also exploring the role of miRNAs in animals capable of significant regeneration such as planarians and amphibians, he said that “we’ll be really excited to compare what we find with what they’re finding.”