Anthony L. Mescher

Regeneration or scarring: an immunologic perspective.

Complete regeneration of complex tissues and organs is usually precluded by fibrotic reactions that lead to scarring. Fish, salamanders, and larval anurans are among the few vertebrates capable of regenerating lost appendages, and this process seems to recapitulate ontogenic development of the structure in most respects. Recent work has revealed a capacity for excellent regeneration in certain mammalian tissues: embryonic or fetal skin and the ear of the MRL mouse. Analyses of these two systems suggest that processes of regenerative growth and patterning for the formation of new structures such as hair follicles may involve modulation of the inflammatory response to the injury in a way that reduces fibrosis and formation of scar tissue. We review evidence that this modulation includes changes in cytokine signaling and may involve properties of the extracellular matrix mediated by factors that include hyaluronic acid and “anti‐adhesive substrates” such as tenascin‐C. New studies and classic work on the capacity for limb regeneration in amphibians are then reviewed, focusing on the loss of this ability in prometamorphic anuran hindlimbs and the view that changing properties of the immune system may also underlie the declining regenerative potential in this system. Finally, we review recent work in comparative and developmental immunology, which raises the possibility that phylogenetic changes in regenerative capacity may be the result of evolutionary changes in cellular activities of the immune system. Developmental Dynamics 226:268–279, 2003.© 2003 Wiley‐Liss, Inc.

Limb Regeneration in Amphibians: Immunological Considerations

We review key aspects of what is known about limb regeneration in urodele and anuran amphibians, with a focus on the early events of the process that lead to formation of the regeneration blastema. This includes the role of the nerves and wound epithelium, but also covers the inflammatory effects of the amputation trauma and their importance for regenerative growth. We propose that immunotolerance is important for limb regeneration and changes in its regulation may underlie the loss of regenerative capacity during anuran metamorphosis.

Transferrin is necessary and sufficient for the neural effect on growth in amphibian limb regeneration blastemas.

Cell proliferation during the early phase of growth in regenerating amphibian limbs requires a permissive influence of nerves. Based on analyses of proliferative activity in denervated blastemas, it was proposed that nerves provide factors important for cells to complete the proliferative cycle rather than for mitogenesis itself. One such factor, the iron‐transport protein transferrin (Tf), is abundant in regenerating peripheral nerves where it is axonally transported and released at growth cones. Using blastemas in organ culture, which have been widely used in previous investigations of the neural effect on growth, it was shown here that the growth‐promoting activity of neural extract was completely removed by immuno‐absorption with antiserum against Tf and restored by addition of Tf. Purified Tf or a low molecular weight ferric ionophore were as active as the neural extract in this assay, indicating that the trophic effect of Tf involves its capacity for iron delivery. Both Tf and ferric ionophore also maintained DNA synthesis in denervated blastemas in vivo. A dose‐response assay indicated that purified axolotl Tf stimulates growth of cultured blastemal cells at concentrations as low as 100 ng/mL. The Tf mRNA in axolotl nervous tissue was shown by northern analysis to be similar in size to that of liver. These results are discussed together with those from previous in vitro studies of blastemal growth and support the hypothesis that cell division in the blastema depends on axonally released Tf during the early, nerve‐dependent phase of limb regeneration.

The Developing Xenopus Limb as a Model for Studies on the Balance between Inflammation and Regeneration

The roles of inflammation and immune cell reactivity triggered by amputation have only recently begun to be addressed in investigations of epimorphic regeneration, although studies of tissue repair in mammals clearly show the importance of the immune system in determining the quality of the repair process. Here, we first review inflammation‐related work in non‐mammalian systems of epimorphic regeneration which suggests that regeneration of an amputated appendage requires continuous modulation of the local immune response, from the first hours after amputation through the period of blastema patterning. We then present data on the effects of anti‐inflammatory and proinflammatory agents on regeneration of larval Xenopus hindlimbs. Treatment with the glucocorticoid beclomethasone immediately after amputation inhibits regeneration in regeneration‐complete stage 53 limbs. Other anti‐inflammatory agents, including the inhibitors of cyclooxygenase‐2 (COX‐2) activity celecoxib and diclofenac, applied similarly to larvae amputated at stage 55, when the capacity for limb regeneration is normally being lost, restore regenerative capacity. This suggests that although injury‐related events sensitive to glucocorticoids are necessary for regeneration, resolution of the inflammatory response may also be required to allow the complete regenerative response and normal blastema patterning. Conversely, if resolution of inflammation is prevented by local treatment of amputated limbs with beryllium, a strong immunoadjuvant, regeneration is inhibited, and gene expression data suggest that this inhibition results from a failure of normal blastema patterning. Both positive and negative effects of immune‐ or inflammation‐related activities occur during anuran limb regeneration and this underscores the importance of considering immune cells in studies of epimorphic regeneration. Anat Rec, 2012. ©2012 Wiley Periodicals, Inc.

Identification of genes expressed during Xenopus laevis limb regeneration by using subtractive hybridization

Suppression polymerase chain reaction–based subtractive hybridization was used to identify genes that are expressed during Xenopus laevis hindlimb regeneration. Subtractions were done by using RNAs extracted from the regeneration‐competent stage (stage 53) and regeneration‐incompetent stage (stage 59) of limb development. Forward and reverse subtractions were done between stage 53 7‐day blastema and stage 53 contralateral limb (competent stage), stage 59 7‐day pseudoblastema and stage 59 contralateral limb (incompetent stage), and stage 53 7‐day blastema and stage 59 7‐day pseudoblastema. Several thousand clones were analyzed from the various subtracted libraries, either by random selection and sequencing (1,920) or by screening subtracted cDNA clones (6,150), arrayed on nylon membranes, with tissue‐specific probes. Several hundred clones were identified from the array screens whose expression levels were at least twofold higher in experimental tissue vs. control tissue (e.g., blastema vs. limb) and selected for sequencing. In addition, primers were designed to assay several of the randomly selected clones and used to assess the level of expression of these genes during regeneration and normal limb development. Approximately half of the selected clones were differentially expressed, as expected, including several that demonstrate blastema‐specific enhancement of expression. Three distinct categories of expression were identified in our screens: (1) clones that are expressed in both regeneration‐competent blastemas and ‐incompetent pseudoblastemas, (2) clones that are expressed at highest levels in regeneration‐competent blastemas, and (3) clones that are expressed at highest levels in regeneration‐incompetent pseudoblastemas. Characterizing the role of each of these three categories of genes will be important in furthering our understanding of the process of tissue regeneration. Developmental Dynamics 226:398–409, 2003.

Global analysis of gene expression in Xenopus hindlimbs during stage-dependent complete and incomplete regeneration

Xenopus laevis tadpoles are capable of limb regeneration after amputation, in a process that initially involves the formation of a blastema. However, Xenopus has full regenerative capacity only through premetamorphic stages. We have used the Affymetrix Xenopus laevis Genome Genechip microarray to perform a large‐scale screen of gene expression in the regeneration‐complete, stage 53 (st53), and regeneration‐incomplete, stage 57 (st57), hindlimbs at 1 and 5 days postamputation. Through an exhaustive reannotation of the Genechip and a variety of comparative bioinformatic analyses, we have identified genes that are differentially expressed between the regeneration‐complete and ‐incomplete stages, detected the transcriptional changes associated with the regenerating blastema, and compared these results with those of other regeneration researchers. We focus particular attention on striking transcriptional activity observed in genes associated with patterning, stress response, and inflammation. Overall, this work provides the most comprehensive views yet of a regenerating limb and different transcriptional compositions of regeneration‐competent and deficient tissues. Developmental Dynamics 235:2667–2685, 2006.

Transferrin and the Growth-Promoting Effect of Nerves

In addition to its role in the activity of specialized proteins such as hemoglobin and myoglobin, iron is required as a cofactor in several important enzymes common to most animal cells. One such enzyme, ribonucleotide reductase, which regulates the production of deoxyribonucleotides during DNA synthesis, requires a continuous supply of iron to maintain its activity throughout the process of DNA replication. The mechanism by which animal cells normally acquire iron involves receptor-mediated uptake of iron-loaded transferrin, followed by release of apotransferrin. The density of transferrin receptors on the cell surface is greatly increased in rapidly dividing normal and neoplastic cells. Various mitogens and certain organogenic tissue interactions have been shown to induce the appearance of transferrin receptors, signalling the onset of DNA replication. Interference with this process of iron delivery causes the rapid arrest of cell cycling, frequently during the S phase itself, which underscores the importance of iron for DNA replication. Although most circulating transferrin is synthesized in the liver and embryonic yolk sac, smaller quantities are produced in several other embryonic organs and certain other adult tissues. It has been suggested that local synthesis and/or release of transferrin supplies the iron required by rapidly growing cells in situations where the cells do not have ready access to adequate amounts of plasma transferrin due to incomplete development of the vasculature or the presence of blood-tissue barriers (Ekblom and Thesleff, 1985; Meek and Adamson, 1985). Oligodendrocytes and Schwann cells have been shown to synthesize and/or contain high concentrations of transferrin and these cells therefore may constitute a local source of this factor for neurons, whose growth and survival in vitro require transferrin. Transferrin in central and peripheral nervous tissues may be significant for the trophic or growth-promoting effect neurons exert on cells of certain tissues. Transferrin duplicates the activity of neural tissue or neural extracts on growth and development of cultured skeletal myoblasts from chick embryos and on proliferation of mesenchymal cells in blastemas from regenerating amphibian limbs, two systems that have been widely used in investigations of the growth-promoting influence of nerves. Moreover, removal of active transferrin from neural extracts, either with antibodies to transferrin or chelation of the iron, inhibits reversibly the effect of the extract in these developing systems. While the physiological significance of the extract in these developing systems.(ABSTRACT TRUNCATED AT 400 WORDS)

Proteomics analysis of regenerating amphibian limbs: changes during the onset of regeneration

During amphibian epimorphic limb regeneration, local injury produces metabolic changes that lead to cellular dedifferentiation and formation of a blastema, but few details of these changes have been elucidated. Here we report the first global proteomic analysis of epimorphic regeneration comparing the profiles of abundant proteins in larval limbs of the anuran Xenopus laevis (stage 53) at the time of amputation (0dPA) and 3 days post-amputation when the regeneration blastema is developing (3dPA). We identified and quantified 1517 peptides, of which 1067 were identified with high peptide ID confidence. Of these 1067 proteins, 489 showed significant changes in quantity between the two groups. Taking into account identical peptides whose fold changes were within 20%, and not including peptides whose fold changes were below the observed fold changes of peptides for the internal standard (chicken lysozyme), we were able to identify 145 peptides elevated in 3dPA relative to 0dPA and 220 peptides in 0dPA relative to 3dPA. In this report, we focus on those proteins that were elevated in the 3dPA tissue relative to 0dPA. In this class were members of the annexin family (e.g. ANXA1, ANXA2, ANXA5) and the ANXA2-binding partner S100A10, which have important immunoregulatory roles in other systems and were also shown to be differentially expressed in stage 53 and 57 3dPA and 5dPA blastemas in our previous microarray studies. Besides elucidating the possible modulation of inflammation during amphibian limb regeneration, our proteomic study also provides insight into dedifferentiation by revealing up-regulation of proteins known to characterize many stem cells.

Changes in the Inflammatory Response to Injury and Its Resolution during the Loss of Regenerative Capacity in Developing Xenopus Limbs

Tissue and organ regeneration, unlike development, involves an injury that in postembryonic animals triggers inflammation followed by resolution. How inflammation affects epimorphic regeneration is largely uninvestigated. Here we examine inflammation and its resolution in Xenopus laevis hindlimb regeneration, which declines during larval development. During the first 5 days postamputation, both regeneration-competent stage 53 and regeneration-deficient stage 57 hindlimbs showed very rapid accumulation of leukocytes and cells expressing interleukin-1β and matrix metalloproteinase 9. Expression of genes for factors mediating inflammatory resolution appeared more persistent at stages 55 and 57 than at stage 53, suggesting changes in this process during development. FoxP3, a marker for regulatory T cells, was upregulated by amputation in limbs at all three stages but only persisted at stage 57, when it was also detected before amputation. Expression of genes for cellular reprogramming, such as SALL4, was upregulated in limbs at all 3 stages, but markers of limb patterning, such as Shh, were expressed later and less actively after amputation in regeneration-deficient limbs. Topical application of specific proinflammatory agents to freshly amputated limbs increased interleukin-1β expression locally. With aqueous solutions of the proinflammatory metal beryllium sulfate, this effect persisted through 7 days postamputation and was accompanied by inhibition of regeneration. In BeSO4-treated limbs expression of markers for both inflammation and resolution, including FoxP3, was prolonged, while genes for cellular reprogramming were relatively unaffected and those for limb patterning failed to be expressed normally. These data imply that in Xenopus hindlimbs postamputation inflammation and its resolution change during development, with little effect on cellular dedifferentiation or reprogramming, but potentially interfering with the expression of genes required for blastema patterning. The results suggest that developmental changes in the larval anuran immune system may be involved in the ontogenetic loss of epimorphic regeneration in this system.

Apoptosis in regenerating and denervated, nonregenerating urodele forelimbs

Denervated limbs of larval salamanders fail to regenerate if amputated and, unlike adult limbs, undergo regression. The cellular basis of the tissue loss is poorly understood. We used TUNEL staining of larval axolotl limbs fixed and sectioned at intervals after bilateral amputation and unilateral denervation to investigate the role of apoptosis during normal limb regeneration and denervated limb regression. In the first week after amputation a small percentage of apoptotic cells was found in both innervated and denervated limbs. During the second week the apoptotic index remained low in the mitotically active mesenchymal cells of the regenerating limbs, but increased twofold in the nondividing, dedifferentiated cells of the regressing limbs. TUNEL‐positive cells resembling apoptotic bodies were restricted primarily to the dedifferentiated area beneath the wound epithelium, but were also present within the wound epithelium itself. Macrophages were identified immunohistochemically and were also found in increased numbers in distal areas of the denervated regressing limbs. The results suggest a role for apoptosis in the early phase of normal regeneration and indicate that denervated limb regression involves an increased rate of apoptosis and removal of apoptotic bodies by macrophages and the wound epithelium.