seminars in CELL & DEVELOPMENTAL BIOLOGY, Vol. 13, 2002: pp. 345–352 doi:10.1016/S1084–9521(02)00090-3, available online at http://www.idealibrary.com on The molecular basis of amphibian limb regeneration: integrating the old with the new David M. Gardiner∗, Tetsuya Endo and Susan V. Bryant Is regeneration close to revealing its secrets? Rapid advances classical studies to guide the identification of the in technology and genomic information, coupled with several functions of this large set of genes. useful models to dissect regeneration, suggest that we soon The challenge of understanding the mechanisms may be in a position to encourage regeneration and enhanced controlling the biology of complex systems is hardly repair processes in humans. unique to regeneration biology. In recent years, techniques have become available to identify all the Key words: limb / regeneration / pattern formation / molecular components of a system, and to study the urodele / fibroblast / dedifferentiation / stem cells interactions between those components. Key to the success of such an approach is the ability to identify © 2002 Elsevier Science Ltd. All rights reserved. the molecules, while at the same time having an un- derstanding of the cell and tissue level properties of the system. The goal of this reviewis to discuss key insights from the classical literature as well as more recent molecular findings. We focus on three criti- Introduction cally important cell types: fibroblasts, epidermis and nerves. Each of these is necessary, and together they The study of amphibian limb regeneration has a rich are sufficient for the regeneration of a limb. Although experimental history. After many decades of research the final limb is composed of a variety of other cells at the tissue and cellular levels, much is known about and tissues, such as muscle, blood vessels and pigment the phenomenology and the basic organizing prin- cells, these other cell types do not appear to be neces- ciples of regeneration. In recent years, molecular sary for the control of growth and pattern formation analyses have begun to provide insights into the mech- during regeneration, but rather they respond to sig- anisms controlling regeneration. It is already clear that nals from nerves, fibroblasts and the epidermis. Thus regeneration involves complex molecular interactions at this time, these three cell types pose both great between multiple tissues, and thus we can expect that challenges and promising opportunities for research there will be many important genes involved, rather directed toward developing an integrated view of the than just a small number of ‘regeneration genes’. With molecular interactions controlling limb regeneration. the recent input from the axolotl EST project to the databases, nearly 500 non-redundant ‘regeneration’ genes have been cloned and identified,1 a number Key insights from pre-molecular studies that likely will increase dramatically over the next few years. Consequently, the availability of cloned regen- Fibroblasts eration genes will not be a limiting factor to progress in understanding limb regeneration. The challenge During regeneration, growth and pattern formation will entail drawing on the wealth of information from are coordinately regulated by interactions between cells that are derived from fibroblasts of the connec- tive tissues of the amputated stump (see References From the Department ofDevelopmental and Cell Biology, Developmental 2, 3). The function of connective tissue fibroblasts Biology Center, University ofCalifornia Irvine, Irvine, CA 92697, USA. is demonstrated qualitatively by grafting studies to *Corresponding author. E-mail: [email protected] © 2002 Elsevier Science Ltd. All rights reserved. induce newor altered pattern and quantitatively by 1084–9521 / 02 / $– see front matter cell contribution studies. As a generalized conclusion, 345 D.M. Gardiner et al. grafts of tissues that contain fibroblasts affect growth of specialized epidermis is critical for the success and pattern formation; whereas, grafts that do not of regeneration. Epidermal cells function to enable contain fibroblasts do not.4 Among these various tis- outgrowth, and may also function in the control of sues, the dermis plays a particularly dominant role pattern formation, as is the case during limb devel- during regeneration. opment. A specialized wound epidermis (WE) forms A direct demonstration of the importance of the der- during the initial healing of the wound surface by mis comes from studies of regeneration in X-irradiated the migration of an epidermal sheet of cells derived limbs. These limbs are inhibited from regenerating, from the basal cells of the mature skin epidermis but can be rescued by grafts of unirradiated skin.5–7 (see Reference 3). This epidermal layer subsequently The regenerated limbs are formed from graft-derived thickens, as a result of continued cell migration, to dermal fibroblasts, and have a normal pattern of skele- form the apical epithelial cap (AEC), and acquires tal and connective tissues, blood vessels and nerves, unique functions associated with regeneration. A though they lack muscles. Since the stump muscles newbasal lamina is not reformed until relatively late are irradiated, precluding muscle precursor cells from during regeneration, which presumably is critical migrating distally into the regenerate, it follows that in allowing for epithelial–mesenchymal interactions myogenic cells are not required to build a normal limb (see Reference 3). pattern during regeneration, in common with similar Treatments that affect the formation of the WE or findings in developing limbs.8 We conclude that the the AEC alter the course of regeneration (see Refer- fibroblast-derived mesenchymal blastema that even- ence 13). Formation of the WE is inhibited by a graft tually reforms the cartilaginous skeleton and associ- of mature skin over the amputation surface, in which ated connective tissues, forms a blueprint that guides case regeneration is inhibited. Removal of the WE or the migration and growth of nerves, blood vessels and AEC after it has formed also inhibits regeneration. myogenic cells. Conversely, experiments that relocate the position of Descriptive histological studies suggested that each the AEC induce limb outgrowth at the new position. tissue of the mature limb contributed cells in propor- tion to its availability in the stump,9, 10 but subsequent Nerves lineage analysis does not support this interpretation.11 The progeny of dermal fibroblasts account for be- The importance of nerves in regeneration has long tween 19 and 78% of the cells of the early blastema been recognized, but the molecular mechanisms me- (42% on average), even though dermal fibroblasts diating neuronal influences are still largely unknown. represent less than 20% of the cells of the stump, Nerves are severed during amputation, begin to re- suggesting that the fibroblast population is subject generate rapidly into the stump tissues at the amputa- to selective expansion in the blastema. Since dermal tion plane, and subsequently innervate the blastema fibroblasts account for about half of all fibroblasts and overlying epidermis (see Reference 3). If the limb in the limb,12 it is possible that essentially all of the is denervated during the early stages of regeneration, early blastemal cells are derived from fibroblasts, the limb fails to regenerate. In addition to axons, even though these cells account for less than half of nerves contain connective tissue cells and Schwann the cells of the mature limb. At present it is unclear cells. The function of nerves during limb regener- whether there is a population of quiescent stem cells ation is not considered to be associated with these in the dermis that are activated during regeneration, non-neuronal cells since they are present whether or or whether dermal fibroblasts become dedifferen- not the limb has been denervated. Consequently, the tiated by losing their differentiated phenotype and critical cell type is considered to be the neuron, which reversing their cell fate to become stem cells. Never- is hypothesized to produce a ‘neurotrophic factor’ theless, an important consideration for future exper- required for the initiation and progression of the imental work on the biology of stem cells for limb early stages of regeneration. Denervation of regener- regeneration is that such a population of cells likely ating limbs at later stages inhibits further growth of will be isolated from the dermis. the regenerate, but not redifferentiation of normally patterned limb tissues (see Reference 3). Epidermis The production of factors required for regenera- tion may be a normal function of nerves. Alternatively, Although epidermal cells do not contribute directly this function of nerves may be acquired in response to the blastema (see References 11, 13), a covering to amputation, as is the case for fibroblasts and the 346 Amphibian limb regeneration epidermis. Presumably this function would be induced to rescue regeneration is again basically negative in and maintained by interactions with blastema cells and design, and subject to experimental artifacts. Fortu- epidermal cells, and would be coupled with the inter- nately, as discussed next, there are a variety of exper- actions that stimulate growth and pattern formation. imental models that hold great potential to identify Since the behavior of nerves during regeneration has key signals controlling growth and pattern formation not been well characterized at the molecular level,