The Greatest Regeneration

Figure 1

In the regeneration game, mammals pale before such creatures as salamanders. Humans barely avert complete disappointment, regrowing digit tips if they are severed past the last joint, while salamanders are superheroes, regrowing any structure that can be cut off without killing them. Though the salamander is a stalwart regeneration model, research is expanding into model organisms such as the zebrafish and mouse, which offer broader tools and possibly better translation to clinical medicine. "We want to take advantage of the strength of genetics," says Ken Muneoka of Tulane University in New Orleans, who switched from working on salamanders to digit-tip regeneration in the mouse.Developmental studies in the salamander have built a solid framework for studying regeneration. Researchers have described, for example, the organization of the cone-shaped blastema from which newly grown structures emerge, and they have implicated molecules such as fibroblast growth factor and retinoic acid in the process."Something like the axolotl [Ambystoma mexicanum] is a very powerful system because it demonstrates that regeneration is a biological reality," says David Gardiner, University of California, Irvine. Yet, there's only so much researchers can do in a system that lacks good genetics and genome sequence data. Muneoka says that along with having more powerful tools, mammalian research also may be more directly applicable to problems such as regenerating human limbs or treating spinal cord injuries, even if the phenotypes aren't as dramatic as those seen in the salamander. " [The digit tip] is really a minor injury compared to the [salamander] limb, but it is a bona fide regeneration response," he says.The different regeneration systems have enough similarities that researchers believe findings in one system will translate to others (see 5-Prime, p. 11). "The most clear commonality is the formation of the blastema," says Ken Poss, who studies zebrafish fin regeneration at Duke University. Once this bump of cells coalesces, regrowth of the missing structures seems to go on autopilot. For example, mouse digit tips will regenerate in vitro after forming a blastema, and grafting a salamander blastema onto a frog amazingly still yields a salamander limb, suggesting that endogenous signals are superfluous after blastema formation. Were it not for technical difficulties such as body temperature differences, Gardiner says, a salamander blastema could conceivably grow a salamander arm on a human. Figure 2

RESTARTING REGENERATIONThe regenerative failure of MSX1 mutant digit tips can be restored by application of Bone Morphogenetic Protein 4. In the mouse digit, the expression of BMP(4)is controlled in part by MSX1. BMP(4)transcripts are localized to the digit tips as shown in this whole-mount in situ hybridization preparation of a fetal limb.Results from other animal models often support hypotheses created using prolific regenerators like salamanders. While working in Mark Keating's lab at Harvard, Poss and Steve Johnson, now at Washington University in St. Louis, conducted zebrafish genetic screens to identify fin- and/or heart-regeneration mutants. The first of these mutants studied in detail had defects in genes that reflect the importance of cell division in the blastema: a mitotic checkpoint kinase, MPS1, and a protein trafficking gene, SLY1.Mouse work has shown that genes involved in digit development are also important for digit-tip regeneration, supporting the hypothesis generated by salamander researchers that the later stages of regrowth mirror development. For example, Muneoka has tested candidate mouse mutants to identify those that interfere with digit-tip regeneration. MSX1 (a homeobox-containing transcriptional repressor) is expressed in the limb during development. When Muneoka tested MSX1 mutant mice, he found defects in digit-tip regeneration, and further found that MSX1 regulates bone morphogenetic protein 4 (BMP4), which is also expressed in the developing limb bud.1Using a mouse strain called MRL, which has the unique ability to regenerate its heart, Ellen Heber-Katz of the Wistar Institute, Philadelphia, has shown that the matrix metallo-proteinase enzymes MMP2 and MMP9 are important for regenerating heart and other tissues in this system,2 lending support to amphibian studies showing that regeneration requires extracellular matrix remodeling. For example, the reappearance of a basement membrane at a wound site is dramatically delayed when a blastema forms, as compared to the wound simply healing. This work also mirrors findings that MMPs may be involved in optic nerve regeneration in rats; Martin Berry of Kings College London presented this work in December at the 10th International Symposium on Neural Regeneration in Pacific Grove, Calif.But even with new models being explored, very few labs work on regeneration, and tantalizing questions remain. "We would love for the field to expand and for there to be more people asking questions," says Poss.Mignon Fogarty mignon@welltopia.com is a freelance writer in Santa Cruz, Calif.

The Greatest Regeneration

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