Studies of Limb Regeneration in Larval Xenopus.

A basic protocol is given for animal maintenance and surgery in studies of hindlimb regeneration in larval Xenopus laevis Unlike urodele limbs, those of larval frogs typically show much more variation in the extent of regeneration after amputation. Such variation can be reduced by optimizing the conditions of larval maintenance to regulate the rates of growth and development, by selecting only larvae with normal rates of growth and morphological development for experimental use, and by attention to precision and consistency in the proximo-distal level of surgical amputation.

Macrophages and fibroblasts during inflammation and tissue repair in models of organ regeneration.

This review provides a concise summary of the changing phenotypes of macrophages and fibroblastic cells during the local inflammatory response, the onset of tissue repair, and the resolution of inflammation which follow injury to an organ. Both cell populations respond directly to damage and present coordinated sequences of activation states which determine the reparative outcome, ranging from true regeneration of the organ to fibrosis and variable functional deficits. Recent work with mammalian models of organ regeneration, including regeneration of full-thickness skin, hair follicles, ear punch tissues, and digit tips, is summarized and the roles of local immune cells in these systems are discussed. New investigations of the early phase of amphibian limb and tail regeneration, including the effects of pro-inflammatory and anti-inflammatory agents, are then briefly discussed, focusing on the transition from the normally covert inflammatory response to the initiation of the regeneration blastema by migrating fibroblasts and the expression of genes for limb patterning.

Inflammation and immunity in organ regeneration.

The ability of vertebrates to regenerate amputated appendages is increasingly well-understood at the cellular level. Cells mediating an innate immune response and inflammation in the injured tissues are a prominent feature of the limb prior to formation of a regeneration blastema, with macrophage activity necessary for blastema growth and successful development of the new limb. Studies involving either anti-inflammatory or pro-inflammatory agents suggest that the local inflammation produced by injury and its timely resolution are both important for regeneration, with blastema patterning inhibited in the presence of unresolved inflammation. Various experiments with Xenopus larvae at stages where regenerative competence is declining show improved digit formation after treatment with certain immunosuppressive, anti-inflammatory, or antioxidant agents. Similar work with the larval Xenopus tail has implicated adaptive immunity with regenerative competence and suggests a requirement for regulatory T cells in regeneration, which also occurs in many systems of tissue regeneration. Recent analyses of the human nail organ indicate a capacity for local immune tolerance, suggesting roles for adaptive immunity in the capacity for mammalian appendage regeneration. New information and better understanding regarding the neuroendocrine-immune axis in the response to stressors, including amputation, suggest additional approaches useful for investigating effects of the immune system during repair and regeneration.

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-1ß 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-1ß 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.

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.

Dedifferentiation and the role of sall4 in reprogramming and patterning during amphibian limb regeneration.

A central feature of epimorphic regeneration during amphibian limb regeneration is cellular dedifferentiation. Two questions are discussed. First, what is the origin and nature of the soluble factors involved in triggering local cellular and tissue dedifferentiation? Secondly, what role does the key stem cell transcription factor Sall4 play in reprogramming gene expression during dedifferentiation? The pattern of Sall4 expression during Xenopus hindlimb regeneration is consistent with the hypothesis that Sall4 plays a role in dedifferentiation (reprogramming) and in maintaining limb blastema cells in an undifferentiated state. Sall4 is involved in maintenance of ESC pluripotency, is a major repressor of differentiation, plays a major role in reprogramming differentiated cells into iPSCs, and is a component of the stemness regulatory circuit of pluripotent ESCs and iPSCs. These functions suggest Sall4 as an excellent candidate to regulate reprogramming events that produce and maintain dedifferentiated blastema cells required for epimorphic regeneration.

RNA helicase Ddx39 is expressed in the developing central nervous system, limb, otic vesicle, branchial arches and facial mesenchyme of Xenopus laevis.

Ddx39, a DEAD-box RNA helicase, is a part of the homeostatic machinery that regulates the switch between cellular proliferation and differentiation. Ddx39 was shown to be differentially regulated in Xenopus laevis using a differential screen of mRNAs from regenerating limbs (King et al., 2003). Here, the expression patterns of Ddx39 in developing limb and nervous system are reported. Ddx39 was detected by RT-PCR in the Xenopus embryo, the earliest stage examined. Localization of the message by whole-mount in situ hybridization at stage 17 showed it to be localized primarily to the developing nervous system. Ddx39 was present in the ventricular region of the developing neural tube up to and including stage 48, and was also localized to the head mesenchyme, pharyngeal arches, and paraxial mesoderm. Strong label was also present in the developing limb buds at stages 48-55. Analysis of expression patterns in cryosections of the developing eye at stage 38 and 47 showed Ddx39 in the ciliary marginal zone (CMZ) adjacent to the neural retina and within the lens epithelium. Ddx39 was also present in the anterior eye during fibroblast growth factor 2 (FGF2)-mediated retinal regeneration. BrDU incorporation analyses and double-label studies with proliferating cell nuclear antigen showed that Ddx39 message was restricted to a subpopulation of proliferating cells in the developing and regenerating optic cup.

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.

Neural MMP-28 expression precedes myelination during development and peripheral nerve repair.

Mammalian matrix metalloproteinase 28 (MMP-28) is expressed in several normal adult tissues, and during cutaneous wound healing. We show that, in frog and mouse embryos, MMP-28 is expressed predominantly throughout the nervous system. Xenopus expression increases during neurulation and remains elevated through early limb development where it is expressed in nerves. In the mouse, neural expression peaks at embryonic day (E) 14 but remains detectable through E17. During frog hindlimb regeneration XMMP-28 is not initially expressed in the regenerating nerves but is detectable before myelination. Following hindlimb denervation, XMMP-28 expression is detectable along regenerating nerves before myelination. In embryonic rat neuron-glial co-cultures, MMP-28 decreases after the initiation of myelination. Incubation of embryonic brain tissue with purified MMP-28 leads to the degradation of multiple myelin proteins. These results suggest that MMP-28 plays an evolutionarily conserved role in neural development and is likely to modulate the axonal-glial extracellular microenvironment.

Cells of cutaneous immunity in Xenopus: studies during larval development and limb regeneration.

The anuran Xenopus laevis is an experimental model for vertebrate development, immunology, and regenerative biology. Using histochemistry and immunohistochemistry (IHC) we examined embryonic, larval, and postmetamorphic Xenopus skin for the presence of dendritic cells (DCs), Langerhans cells (LCs), and dendritic epidermal T cells (DETCs), all components of cutaneous immunity that have been implicated in skin repair and regeneration. Cells expressing three markers for dendritic and Langerhans cells (formalin-resistant ATPase activity, major histocompatibility complex [MHC] class II antigens, and vimentin) and having morphology like that of these cells first appeared during late embryonic stages, becoming abundant by prometamorphosis. Cells positive for these markers were also numerous in the wound epithelia of regenerating hindlimbs at both early and late larval stages. Cells tentatively identified as DETCs were found, beginning at early larval stages, using IHC with antibodies against heterologous CD3epsilon chain and T-cell receptor delta. Further characterization and work with the putative DCs, LCs, and DETCs demonstrated here will allow not only greater understanding of the amphibian immune system, but also further elucidation of regenerative growth and scarring.

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.

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.

Expression of Xenopus XlSALL4 during limb development and regeneration.

The multi-C2H2 zinc-finger domain containing transcriptional regulators of the spalt (SAL) family plays important developmental regulatory roles. In a competitive subtractive hybridization screen of genes expressed in Xenopus laevis hindlimb regeneration blastemas, we identified a SAL family member that, by phylogenetic analysis, falls in the same clade as human SALL4 and have designated it as XlSALL4. Mutations of human SALL4 have been linked to Okihiro syndrome, which includes preaxial (anterior) limb defects. The expression pattern of XlSALL4 transcripts during normal forelimb and hindlimb development and during hindlimb regeneration at the regeneration-competent and regeneration-incompetent stages is temporally and regionally dynamic. We show for the first time that a SAL family member (XlSALL4) is expressed at the right place and time to play a role regulating both digit identity along the anterior/posterior axis and epimorphic limb regeneration.

Regenerative capacity and the developing immune system.

Many components of the vertebrate immune system have evolved with dual, interrelated functions of both protecting injured tissues from infection and providing for tissue maintenance and repair of injuries. The capacity for organ regeneration, prominent among invertebrates and certain phylogenically primitive vertebrates, is poorly developed in mammals. We have proposed that evolution of the mammalian immune system has produced inflammatory cellular interactions at sites of injury which have optimized tissue defense and facilitated tissue repair, but that these improvements included concomitant loss of regenerative capacity. This chapter briefly reviews work in two regenerating systems: scar-free repair of fetal mammalian skin and regeneration of amputated limbs in larval frogs. In both organs the potential to regenerate anatomically and functionally complete new structures is lost gradually during ontogeny and this loss coincides with development of an immune system producing an inflammatory response in injured tissues. Failure of organ regeneration has long been associated with scarring or fibrosis and this phenomenon is a direct result of inflammatory interactions of immune cells and fibroblasts at sites of injury. Several aspects of immunity related to repair are reviewed, including the importance of antigen-presenting cells and lymphocytes, relevant cytokines and growth factors released by these and other cells, immune functions of extracellular matrix components, and immunological functions of fibroblasts. Skin repair in various transgenic mouse models has been especially informative. Further study of immune mechanisms associated with the loss of regenerative capacity in the skin and amphibian limb will be useful for efforts to promote mammalian organ regeneration.

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.

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.

Vasculature in pre-blastema and nerve-dependent blastema stages of regenerating forelimbs of the adult newt, Notophthalmus viridescens.

Immunocytochemistry utilizing a monoclonal antibody (BV1; blood vessel 1) highly reactive to the vasculature of the adult newt showed that a developing vasculature was present during early, pre-blastema, and early-bud blastema stages of forelimb regeneration in this species. Infusion of Prussian Blue and DiI into the brachial artery further delineated the intactness of this early vasculature. Finally, macroscopic observations of vascular flow underneath the apical epithelial cap (AEC) and microsurgical removal of the AEC and observation of subsequent bleeding buttressed the conclusion that an intact vasculature exists during early nerve-dependent stages of newt forelimb regeneration. The results suggest that this process of neovascular formation is angiogenesis, i.e., the formation of new vessels from pre-existing vessels in the stump. Furthermore, angiogenesis is an ongoing process initiated early after amputation. Blastema cells and the AEC are likely sourcesof factors that stimulate neovascularization.