What can earthworms, salamanders, some fish, and some frogs do that mammals (such as humans) cannot? If you said that they can all regenerate extensive parts of their bodies after amputation, you are correct! Scientists have been studying these animals to try to discover why they can “re-grow” amputated limbs, whereas humans cannot. If researchers can understand how these animals regenerate damaged tissues, perhaps treatments could be developed to help humans regenerate lost limbs, repair damaged organs, and reverse the effects of spinal cord injury. In the March 15th issue of Genes and Development, Dr. Kenneth Poss and colleagues at Duke University and the University of North Carolina at Chapel Hill, report a breakthrough in the understanding of how zebrafish can regrow fins following amputation. These researchers show that during fin regeneration, complex changes in the regulation of limb growth programs occur. These changes can be controlled, at least in part, by changing levels of a single microRNA, miR-133.

MicroRNA: Small RNAs with a Big Job

In the traditional scientific view, the main function of RNA is to carry a copy of the genetic information on the DNA from the nucleus (where the DNA is located) out into the cell, where the information on the RNA is used as the instructions for making proteins. Although that role is obviously important, scientists are now beginning to discover that RNA can play other, equally important, roles in the cell. MicroRNAs are very short RNA sequences that do not encode proteins but instead bind to matching sequences on full-length RNAs that do encode proteins. When a microRNA binds to a full-length RNA, it stops that RNA from making its encoded protein. Because microRNAs often have many different targets, microRNAs can stop, or “silence”, groups of genes in a tightly controlled, regulated manner. MicroRNAs have been shown to be involved in early developmental processes, such as the formation of organs and the maintenance of stem cells, as well as having important roles in adult organs, such as protection from disease. Because microRNAs play a role during both early and later stages of development, scientists suspected that they might be important for limb regeneration, in which older organisms must grow a new limb in much the same way that embryos originally develop limbs.

MiR-133 and Fin Regeneration

In order to model complex limb regeneration, Dr. Poss and colleagues turned to the zebrafish, a common aquarium fish that has the ability to regrow fins following amputation. Although a fin might look simple, regeneration involves the coordination of many different types of cells: bone, epidermis (skin), blood vessels, nerves, and connective tissue. Despite this complexity, zebrafish can regenerate up to 95% of their missing fin within two weeks of amputation. Because microRNAs look like good candidates to play a role in limb regeneration, this study examined whether or not any microRNAs were changed during fin regeneration, and whether any of these microRNAs were also affected by a molecule called fibroblast growth factor (Fgf) which is known to be crucial for fin regeneration. Scientists found that the levels of one microRNA, miR-133, were dramatically decreased during fin regeneration. Blocking Fgf signaling, which also blocks fin regeneration, resulted in higher levels of miR-133. These results suggest that during regeneration, Fgf signaling decreases levels of miR-133.

To confirm the effects of miR-133, the researchers manipulated levels of miR-133 experimentally, either by adding extra miR-133 or by blocking the effects of miR-133. As predicted, increasing levels of miR-133 blocked fin regeneration, while decreasing levels of miR-133 accelerated fin regeneration. Next, scientists looked to see which gene or genes miR-133 targeted by looking for RNAs that “matched” the sequence of miR-133, and were also involved in regenerative processes. One of these matches was for a gene called mps1, a protein known to be present in regenerating fin tissue and that is critical for fin regeneration. When miR-133 was present, levels of the mps1 protein decreased; when miR-133 was absent, levels of mps1 increased.

Taken together, these experiments suggest that during fin regeneration, Fgf signaling works to decrease the amount of miR-133. Because miR-133 normally represses genes that play a role in fin growth, the Fgf-mediated miR-133 decrease results in higher levels of these genes that promote regrowth, particularly mps1. In this way, the decrease in miR-133 activates genes that promote fin regeneration.

Earthworm bodies, fish fins, frog legs… and human limbs?

Many scientists believe that one of the reasons why some animals can extensively regenerate tissues is that these animals can “reactivate” a developmental program that originally was responsible for the growth of limbs in the embryo. Once embryos grow limbs, these growth programs are repressed, and one potential mechanism for this repression could be the silencing action of one or more microRNAs. Humans, and other mammals, have these developmental programs for growing embryonic limbs, but do not seem to be able to reactivate them to regenerate amputated limbs. The discovery that reduction of miR-133 is a critical step in re-activating this limb growth process has scientists excited that regulation of human microRNAs might lead to a similar effect on regeneration. Normally, amputation does not reactivate a human limb growth program, but if treatments could be developed to manipulate a key regulator of the growth program, such as a microRNA, then maybe we could “kick-start” a regeneration process in humans. Similar programs could potentially be used to regenerate damaged organs, such as hearts, spinal cords, and retinas. Such treatments are a long way off, but discoveries such as miR-133 and its role in fin regeneration are pointing us in promising new directions.

–Stephanie Courchesne, Harvard Medical School

For More Information:

MicroRNA and the path to limb regeneration:
< http://www.scientificblogging.com/news_releases/microrna_and_the_pathway_to_limb_regeneration >

MicroRNAs help zebrafish regenerate fins:
< http://www.sciencedaily.com/releases/2008/03/080314202107.htm >

Primary Literature:

Yin, V.P., Thomson, M., Thummel, R., Hyde, D.R., Hammond, S.M., and Poss, K.D. (2008). Fgf-dependent depletion of microRNA-133 promotes appendage regeneration in zebrafish. Genes and Development, 22, 728-733.