Dev. Biol. Oct 3. pii: S0012-1606(12)00541-6. doi: 10.1016/j.ydbio.2012.09.021.

Yu, L, Han, M, Yan, M, Lee, J, and Muneoka, K. 2012.

PMID: 23041115

An intense interest in regenerative medicine has driven studies aimed at unraveling the molecular mechanisms underpinning natural regeneration.  Building on research examining digit regeneration in neonatal and adult mice, this study demonstrates the potential of a single molecule, Bmp2, to induce bone regeneration in digital elements or long bones of the limb following amputation.

Bmp2-directed long bone regeneration

In a straightforward and elegant presentation, Yu and colleagues show that bead-soaked implantation of recombinant human Bmp2 protein can direct bone regeneration from the second element of the digit (P2) by induction of a de novo ossification center.  Although not structurally perfect, the result is a continuation of the marrow cavity and re-growth of the bone up to, but not including the joint distal to this element or the distal (P3) element itself.  This same pattern was dramatically presented using µCT data to show the regeneration and fusion of the tibia and fibia following mid-level amputation through these long bones (pictured at right). Given the ability of Bmp2 to also induce regeneration of the more distal P3 element, the authors note the perplexing finding that regeneration of either, but not both occurs following Bmp2 treatment. This suggests the importance of additional signaling pathways to coordinate and direct a complete regenerative response.

Salamanders and newts regenerate amputated limbs through formation of a blastema, a lineage-restricted mass of progenitor cells.  Although the authors found that a blastema formed only transiently, this work reinforces the likelihood that different molecular pathways direct different components of the regenerative response that are subsequently integrated during complete regeneration. Further work in this system will compliment future studies designed at dissecting the molecular control of blastema formation during regeneration of ear punches in rabbits and spiny mice.

Gene expression patterns specific to the regenerating limb of the Mexican axolotl

Monaghan, J. R., Athippozhy, A., Seifert, A.W., Putta, S., Stromberg, A., Maden, M., Gardiner, D. M., and Voss, S. R. (2012). Denervation quantitatively and systemically alters transcription within the wound epithelium of a regeneration-competent salamander limb. (in press, Open Biology) (Click here for PDF)

Among vertebrates, experimental studies have established the absolute requirement for an adequate nerve supply during regeneration of pectoral fins, froglet limbs, urodele limbs, and lizard tails (see further reading below).  For instance, severing the nerve supply to the limb will prevent a salamander from regenerating that limb.  Salamander limb regeneration is also dependent upon tissue interactions that are local to the amputation site.  Communication among limb epidermis, peripheral nerves, and mesenchyme coordinate cell migration, cell proliferation, and tissue patterning to generate a blastema, a mass of lineage-restricted progenitor cells that forms the missing limb structures.  An outstanding question is how molecular cross-talk between these tissues gives rise to this regeneration blastema.

In collaboration with James Monaghan, Randal Voss and others, our study has identified genes associated specifically with nerve-dependent regeneration.  To accomplish this, we examined histological and transcriptional changes during the first week of regeneration in the wound epidermis and subjacent cells between three injury types; 1) a flank wound on the side of the animal that will not regenerate a limb, 2) a denervated limb that will not regenerate a limb, and 3) an innervated limb that will regenerate a limb. Early, histological and transcriptional changes were highly similar between the three injury types, likely because a common wound-healing program is employed across anatomical locations.  However, we identified transcripts that were enriched in the limb compared to the flank and are associated with vertebrate limb development.  Many of these genes were activated before blastema outgrowth and in situ hybridization showed that some of these genes were expressed in specific tissue types including the epidermis, peripheral nerve, and mesenchyme.

Summary of differentially expressed genes between innervated limb (NL) denervated limb (DL) and flank wound (FW).

We also identified a relatively small group of transcripts that were more highly expressed in innervated limbs versus denervated limbs. These transcripts encode for proteins that are associated with myelination of peripheral nerves, epidermal maintenance, and cell proliferation, suggesting that denervation affects myelinating Schwann cells, epidermal cell function, and proliferation of mesenchymal cells.  Overall, our study identifies limb-specific and nerve-dependent genes that are upstream of regenerative growth, and thus promising candidates for the regulation of blastema formation.

Further Reading:

Geraudie, J. & Singer, M. (1985). Necessity of an adequate nerve supply for regeneration of the amputated pectoral fin in the teleost Fundulus. Journal of Experimental Zoology 234, 367-74

Suzuki, M., Satoh, A., Ide, H. & Tamura, K. (2005). Nerve-dependent and -independent events in blastema formation during Xenopus froglet limb regeneration. Developmental Biology 286, 361-75.

Singer, M. (1952). The influence of the nerve in regeneration of the amphibian extremity. Quarterly Review of Biology 27, 169-200.

Simpson, S. B., Jr. (1970). Studies on regeneration of the lizard’s tail. American Zoologist 10, 157-65.

Nature. 2011 Aug 24;476(7361):409-13. doi: 10.1038/nature10346

Yuval Rinkevich, Paul Lindau, Hiroo Ueno, Michael T. Longaker & Irving L. Weissman

from Rinkevich et al., 2011

The source and identity of cells used during regeneration has taken on new importance as stem cell biologists tinker with how to manipulate the potency of cells for regenerative medicine. Recent work using current molecular tools has confirmed that, while planarians can regenerate an entire replacement from one pluripotent reserve cell (see Wagner et al., 2011), regeneration in larval vertebrates is dependent on lineage-restricted progenitor cells (see Gargioli and Slack 2004, Kragl et al., 2009, Tu and Johnson, 2011). Delving deep into the transgenic mouse toolbox, this study now extends these findings to mammals and demonstrates that partially regenerating structures in young mice digit tips rely on lineage-restricted progenitor cells sourced from the digit itself (e.g. using En1cre transgenic mice, red arrows pictured at left show contribution of ventral ectoderm to regenerated sweat glands) . Additionally, their analysis shows that circulating hematopoietic cells do not contribute to new vasculature or regenerating tissues. Together, these studies strongly suggest that when regeneration in vertebrates occurs, it utilizes an evolutionarily conserved strategy to replace missing tissue.  It will be of great interest to examine regeneration in adult animals to in order to extend these findings and ask whether a progressive reduction in resident progenitor cell populations might partly underlie a failure to regenerate.

For those interested in the original (and detailed) account of mouse digit tip regeneration through the distal phalange, please see this paper by Richard Borgens (Science. 1982 Aug 20;217(4561):747-50). Borgens’ account sets the stage for this paper by stating “…that the regrowth of distal structure only occurred if a counterpart was left in the stump.“  Is is also worth noting that he makes clear in his paper that digit tip regeneration in these juvenile mice is not structurally perfect, although it is cosmetically so. This extends to human digit tip regeneration where although the tip appears to regenerate, the bony elements within the distal phalange are not regenerated. This can be visualized in a nice review by Ken Muneoka (Birth Defects Res C Embryo Today. 2008 Dec;84(4):265-80.).

{1} Wagner et al. Science 2011, 332:811-6 [PMID:21566185].
{2} Gargioli and Slack, Development 2004, 131:2669-79 [PMID:15148301].
{3} Tu and Johnson, Dev Cell 2011, 20:725-732 [PMID:21571228].
{4} Kragl et al. Nature 2009, 460:60-65 [PMID:19571878]

Nat Commun. 2011 Jul 12;2:384. doi: 10.1038/ncomms1389.

Goro Eguchi, Yukiko Eguchi, Kenta Nakamura, Manisha C. Yadav, José Luis Millán & Panagiotis A. Tsonis

from Eguchi et al. 2011

A nagging question in the field of regeneration biology is how ageing affects regenerative capacity in urodeles.  Examining repeated lens regeneration in 16 year-old newts (continuing till age 30), Eguchi et al. show that iris pigmented retinal epithelial cells (PECs) retain the ability to fully regenerate a lens after multiple removals. Regardless of age, the rate of regeneration and quality of the regenerated lenses were identical (new lens pictured at right showing normal fiber orientation in regenerated lens).  In the face of repeated lentectomies this finding suggests that newts maintain a progenitor population of PECs that last throughout life.  Alternatively, it is possible that the act of regenerating a lens induced progenitor cell maintenance and raises the possibility that reparative regeneration might help newt PECs escape cellular senescence. This interesting study will hopefully stimulate researchers to test regenerative capacity in other organ systems in aged salamanders.

link to F1000 Review

Some of the major findings are discussed below in the context of mammalian wound healing.

Skin regeneration in adult axolotls: a blueprint for scar-free healing in vertebrates

Authors: Ashley W. Seifert^1†, James R. Monaghan¹, S. Randal Voss^2,3, and Malcolm Maden¹

A fundamental goal of wound healing research is to develop therapies that will replace imperfect healing and scarring with regeneration and perfect restoration of damaged tissue. The basic strategies of regenerative medicine involve engineering replacement parts (like fixing a car) or coaxing the natural regenerative potential of the damaged tissue.  Inducing natural regeneration from existing tissue offers tremendous promise towards tackling the medical burden of injury, loss, and degeneration of musculoskeletal and skin tissues.  This paper establishes a new model for perfect skin regeneration using adult axolotls to model full thickness excisional wounds. Using this model, our research demonstrates that aquatic axolotls are capable of regenerating their dermis and associated glands.  We also induced metamorphosis to examine the effects of terrestriality on skin regeneration. While these experiments demonstrated that terrestrial axolotls are similarly capable of scar-free healing (albeit more slowly), they exhibited delayed re-epithelialization, increased cellularity within the wound bed and a protracted fibrotic response. Compared to mammals, both axolotl morphs exhibited a substantial reduction in hemostasis, faster rate of re-epithelialization, lower neutrophil infiltration and a relatively long delay in production of new extracellular matrix (ECM).  Genetic and protein level analysis implicated matrix metalloproteinases in the control of fibrosis and tensacin-c in the facilitation of dermis regeneration.  This research firmly establishes the terrestrial axolotl as an excellent model of scar-free healing in adult vertebrates. Future research will continue to uncover the cellular and molecular mechanisms underlying scar-free healing in these animals towards comparing these findings to understand how these pathways behave during scarring in mammals.  The figure below summarizes these conclusions.

Check out the full paper: http://www.nature.com/nature/journal/v489/n7417/full/nature11499.html
And a nice commentary in Nature as well: http://www.nature.com/nature/journal/v489/n7417/full/489508a.html

Weak skin and tissue regeneration in African spiny mice (Acomys)

It is generally accepted that mammals have lost much of the regenerative capacity that lower vertebrates enjoy. Alternatively, the capacity to regenerate damaged tissue may be suppressed, locked away in favor of mechanisms that promote rapid and efficient wound healing. One of my research projects sent me to Kenya where I’ve been researching the ability that two species of African spiny mice possess to “come out of their skin” when attacked by a predator.  As it turns out, this is not far from the truth. Our study has found that these mice  have incredibly weak skin, so weak in fact that very small amounts of tension loaded rapidly induces tearing. In response to such tearing these mice display an ability to regenerate hair follicles in the wound bed, something most mammals fail to do in response to injury. Perhaps even more exciting is the ability of these mice to heal large circular ear holes by regenerating hair follicles, dermis and cartilage. While it has been known for some time that rabbits are capable of the same fantastic feat, the wealth of resources available for work in rodents makes this a tractable system for future study. As re-emergent interest in regenerative medicine seeks to isolate molecular pathways controlling tissue regeneration in mammals, Acomys may prove useful in identifying mechanisms to promote regeneration in lieu of fibrosis and scarring.

Check out the F1000 review as a “must read”; http://f1000.com/717958161#eval793462471

Below is a sampling of some nice press on the article.

Audio:
Nature Podcast:  http://www.nature.com/nature/podcast/index-2012-09-27.html
NPR All Things Considered:  http://www.npr.org/2012/09/26/161817096/mammalian-surprise-african-mouse-can-regrow-skin

Articles:
http://www.bbc.co.uk/news/health-19727622
http://www.nature.com/news/african-spiny-mice-can-regrow-lost-skin-1.11488
http://blogs.discovermagazine.com/notrocketscience/2012/09/26/spiny-mice-flaying-skin-healing-factor/
http://news.discovery.com/animals/spiny-mouse-skin-peels-and-reseals-120926.html
http://news.sciencemag.org/sciencenow/2012/09/african-spiny-mouse.html?ref=hp
http://www.newscientist.com/article/dn22310-zoologger-spiny-mice-save-their-skin-by-shedding-it.html

The influences of fundamental traits on mechanisms controlling appendage regeneration

Biol Rev Camb Philos Soc. 2012 May;87(2):330-45. doi: 10.1111/j.1469-185X.2011.00199.x

Authors: Ashley W. Seifert, James R. Monaghan, Matthew D. Smith, Bret Pasch, Adrian C. Stier, François Michonneau and Malcolm Maden

ABSTRACT

One of the most compelling questions in evolutionary biology is why some animals can regenerate injured structures while others cannot.  Appendage regeneration appears to be common when viewed across the metazoan phylogeny, yet this ability has been lost in many taxa to varying degrees.  Within species, the capacity for regeneration also can vary ontogenetically among individuals.  Here we argue that appendage regeneration along the secondary body axis may be constrained by fundamental traits such as body size, aging, life stage, and growth pattern.  Studies of the molecular mechanisms affecting regeneration have been conducted primarily with small organisms at early life stages.  Such investigations disregard the dramatic shifts in morphology and physiology that organisms undergo as they age, grow, and mature.  To help explain interspecific and intraspecific constraints on regeneration, we link particular fundamental traits to specific molecular mechanisms that control regeneration.  We present a new synthesis for how these fundamental traits may affect the molecular mechanisms of regeneration at the tissue, cellular, and genomic levels of biological organization.  Future studies that explore regeneration in organisms across a broad phylogenetic scale, and within an ontogenetic framework, will help elucidate the proximate mechanisms that modulate regeneration and may reveal new biomedical applications for use in regenerative medicine.

If you cannot access the paper please e-mail me for a pdf.