Axolotl Limb Regeneration Research Articles

Cells keep a memory of their tissue origin during axolotl limb regeneration

During limb regeneration adult tissue is converted into a zone of undifferentiated progenitors called the blastema that reforms the diverse tissues of the limb. Previous experiments have led to wide acceptance that limb tissues dedifferentiate to form pluripotent cells. Here we have reexamined this question using an integrated GFP transgene to track the major limb tissues during limb regeneration in the salamander Ambystoma mexicanum (the axolotl). Surprisingly, we find that each tissue produces progenitor cells with restricted potential. Therefore, the blastema is a heterogeneous collection of restricted progenitor cells. On the basis of these findings, we further demonstrate that positional identity is a cell-type-specific property of blastema cells, in which cartilage-derived blastema cells harbour positional identity but Schwann-derived cells do not. Our results show that the complex phenomenon of limb regeneration can be achieved without complete dedifferentiation to a pluripotent state, a conclusion with important implications for regenerative medicine.

Expression of matrix metalloproteinases (MMPs) during axolotl limb regeneration

Expression of matrix metalloproteinases (MMPs) during axolotl limb regeneration

Pitx1 expression in developing and regenerating axolotl limbs

The expression of the homeobox transcription factor Pitx1 was investigated in the Mexican axolotl (Ambystoma mexicanum) during limb development and regeneration by whole-mount mRNA in situ hybridizations. This clone shares high amino acid identity with Pitx1 from other vertebrates (92% Xenopus; 87% chick; 75% human and mouse) within the region isolated. In the developing limbs, Pitx1 was expressed in hindlimb mesenchyme, as has been reported in other species. The expression pattern in the hindlimb might have been conserved during evolution. In the regenerating limbs, Pitx1 was expressed in both fore- and hindlimb blastemas. Our observations suggest two roles of Pitx1 in the axolotl: one is to determine the hindlimb pattern during development, and the other that relates to proliferation of regenerating tissues without regard to fore- or hindlimb.

Transforming Growth Factor: β Signaling Is Essential for Limb Regeneration in Axolotls

Axolotls (urodele amphibians) have the unique ability, among vertebrates, to perfectly regenerate many parts of their body including limbs, tail, jaw and spinal cord following injury or amputation. The axolotl limb is the most widely used structure as an experimental model to study tissue regeneration. The process is well characterized, requiring multiple cellular and molecular mechanisms. The preparation phase represents the first part of the regeneration process which includes wound healing, cellular migration, dedifferentiation and proliferation. The redevelopment phase represents the second part when dedifferentiated cells stop proliferating and redifferentiate to give rise to all missing structures. In the axolotl, when a limb is amputated, the missing or wounded part is regenerated perfectly without scar formation between the stump and the regenerated structure. Multiple authors have recently highlighted the similarities between the early phases of mammalian wound healing and urodele limb regeneration. In mammals, one very important family of growth factors implicated in the control of almost all aspects of wound healing is the transforming growth factor-beta family (TGF-β). In the present study, the full length sequence of the axolotl TGF-β1 cDNA was isolated. The spatio-temporal expression pattern of TGF-β1 in regenerating limbs shows that this gene is up-regulated during the preparation phase of regeneration. Our results also demonstrate the presence of multiple components of the TGF-β signaling machinery in axolotl cells. By using a specific pharmacological inhibitor of TGF-β type I receptor, SB-431542, we show that TGF-β signaling is required for axolotl limb regeneration. Treatment of regenerating limbs with SB-431542 reveals that cellular proliferation during limb regeneration as well as the expression of genes directly dependent on TGF-β signaling are down-regulated. These data directly implicate TGF-β signaling in the initiation and control of the regeneration process in axolotls.

The short toes mutation of the axolotl

The axolotl mutant strain, short toes (s/s), can regenerate spinal cord and tail, but not limbs. This makes s/s potentially very useful for limb regeneration studies. This mutant merits a new examination that integrates the original description of the mutant, existing experimental studies, new data and current thinking about stem cells and regeneration. There are still major gaps in information about this mutant; the gene(s) causing the defects has not yet been discovered, and even the histological description is incomplete, especially regarding muscle abnormalities. In the short toes limb, MyHC (myosin heavy chain)-1, MyHC-2b and pax7 are down-regulated. In particular, all three MyHC genes and pax7 are highly expressed in the normal limb, but almost lost in the s/s limb. MyHC genes are one of the main components of skeletal muscle, and Pax7 is the skeletal muscle satellite cell marker. Histological experiments confirm that severe s/s has lost most skeletal muscle and myosin. These results suggest that skeletal muscle, which includes satellite cells, could play an important role in axolotl limb regeneration.

Stem/progenitor cells in amphibian limb and spinal cord regeneration

Amphibian regeneration may replace tissue via homotypic cells, dedifferentiation, transdifferentiation or stem cells. The mechanism may vary with life cycle stage. Expression of Notch signaling components suggests a role for stem cells. We examine the role of stem cells in regeneration-competent and incompetent stages of Xenopus laevis (clawed frog) tadpoles, juvenile and adult axolotls (the Mexican salamander Ambystoma mexicanum) and the regeneration-deficient axolotl mutant short-toes. Notch1 signaling is a key pathway in maintaining stem cell populations. The stem cell marker Musashi (Msi) is an RNA-binding protein inhibiting translation of Numb to maintain Notch1 signaling. Isoforms of Msi1 and Msi2 are expressed in temporal and spatial patterns that correlate with regeneration behavior. Msi-1 is expressed in specific tissue compartments in regeneration-competent Xenopus limb and spinal cord and decreases in incompetent tadpole and adult Xenopus tissues. Msi2 isoforms change between competent and incompetent Xenopus stages. Rapidly growing juvenile axolotls show high levels of Msi1 in both cord and limb, while intact adult axolotl tissues shows low levels of Msi1 that are upregulated following injury. Msi expression is also lost in limbs of the axolotl limb regeneration mutant short-toes, but maintained in the regeneration competent mutant spinal cord. Support: Indiana 21st Century Research and Technology Fund ; W.M. Keck Foundation.

Publication of a Special Issue: Limb and Fin Regeneration

• Limb regeneration in amphibians: Immunological considerations • Limb regeneration in Axolotl: Is it superhealing? • Limb regeneration in Xenopus laevis froglet • Transcriptional profiling of caudal fin regeneration in Zebrafish • Newt opportunities for understanding the dedifferentiation process • Comparative experimental analysis of fin lepidotrichia in fishes and several skeletal tissues in vertebrates including humans

Limb Regeneration in Axolotl: Is It Superhealing?

Limb Regeneration in Axolotl: Is It Superhealing?

Identification of the non-specific cytotoxic cell receptor protein 1 (NCCRP1) in regenerating axolotl limbs

The teleost non-specific cytotoxic cells (NCC) are evolutionary precursors of the mammalian natural killer (NK) cells and an important element of innate immunity. The non-specific cytotoxic cell receptor protein (NCCRP1) is a characteristic cell surface protein with main functions in target cell recognition and cytotoxicity with sequence information available for many species of fish. We have isolated a cDNA encoding the Axolotl homologue of fish NCCRP1 out of limb regeneration blastema and analysed its expression by RT-PCR. Sequence analysis revealed a high degree of homology with teleost NCCRP1 on nucleotide and deduced amino acid levels. NCCRP1 contains a conserved C-terminal F-box-associated domain (FBA) and proline-rich motifs (PRM) characteristic for this protein family. NCCRP1 is expressed in multiple tissues with high levels in limb regeneration blastema. The present work describes for the first time the cloning of the NCCRP1 gene in a tetrapod vertebrate providing a valuable link between fish and higher vertebrates. Our findings suggest the existence of NCC in axolotl and a role of the innate immune system in the processes of limb regeneration.

Limb Regeneration in Axolotl: Is It Superhealing?

The ability of axolotls to regenerate their limbs is almost legendary. In fact, urodeles such as the axolotl are the only vertebrates that can regenerate multiple structures like their limbs, jaws, tail, spinal cord, and skin (the list goes on) throughout their lives. It is therefore surprising to realize, although we have known of their regenerative potential for over 200 years, how little we understand the mechanisms behind this achievement of adult tissue morphogenesis. Many observations can be drawn between regeneration and other disciplines such as development and wound healing. In this review, we present new developments in functional analysis that will help to address the role of specific genes during the process of regeneration. We also present an analysis of the resemblance between wound healing and regeneration, and discuss whether axolotls are superhealers. A better understanding of these animals' regenerative capacity could lead to major benefits by providing regenerative medicine with directions on how to develop therapeutic approaches leading to regeneration in humans.

Expression of heat‐shock protein 70 during limb development and regeneration in the axolotl

Urodele amphibians (e.g., axolotls) have the unique ability, among vertebrates, to regenerate perfectly many parts of their body after amputation. The limb has been the most widely studied regenerating structure in these organisms and provides an ideal model in which to understand how vertebrates can regenerate complex tissues. The present study focuses on Hsp-70, a member of the stress-related heat-shock protein family. This protein is normally induced after a stress or trauma such as heat-shock, ultraviolet irradiation, or wounding. Thus, studying its expression during axolotl limb regeneration, a response to an important traumatic event (amputation), is of great interest to further understand how the regenerative process is mediated. Using molecular biology and biochemical techniques, we have characterized both the spatiotemporal and quantitative expression patterns of Hsp-70 in axolotl development and regeneration. Our results show that Hsp-70 is expressed and regulated during axolotl development as in other vertebrates. Our data also demonstrate an up-regulation of the RNA transcript for Hsp-70 during limb regeneration as early as 24 hr after amputation that is maintained up to early differentiation. We also demonstrate a similar pattern of expression for the protein during regeneration. Finally, we show that axolotl Hsp-70 is induced threefold after heat-shock as observed in other vertebrates.

Expression of complement 3 and complement 5 in newt limb and lens regeneration.

Some urodele amphibians possess the capacity to regenerate their body parts, including the limbs and the lens of the eye. The molecular pathway(s) involved in urodele regeneration are largely unknown. We have previously suggested that complement may participate in limb regeneration in axolotls. To further define its role in the regenerative process, we have examined the pattern of distribution and spatiotemporal expression of two key components, C3 and C5, during limb and lens regeneration in the newt Notophthalmus viridescens. First, we have cloned newt cDNAs encoding C3 and C5 and have generated Abs specifically recognizing these molecules. Using these newt-specific probes, we have found by in situ hybridization and immunohistochemical analysis that these molecules are expressed during both limb and lens regeneration, but not in the normal limb and lens. The C3 and C5 proteins were expressed in a complementary fashion during limb regeneration, with C3 being expressed mainly in the blastema and C5 exclusively in the wound epithelium. Similarly, during the process of lens regeneration, C3 was detected in the iris and cornea, while C5 was present in the regenerating lens vesicle as well as the cornea. The distinct expression profile of complement proteins in regenerative tissues of the urodele lens and limb supports a nonimmunologic function of complement in tissue regeneration and constitutes the first systematic effort to dissect its involvement in regenerative processes of lower vertebrate species.

Retinoid antagonists inhibit normal patterning during limb regeneration in the axolotl, Ambystoma mexicanum.

Retinoic acid (RA) has been detected in the regenerating limb of the axolotl, and exogenous RA can proximalize, posteriorize, and ventralize blastemal cells. Thus, RA may be an endogenous regulatory factor during limb regeneration. We have investigated whether endogenous retinoids are essential for patterning during axolotl (Ambystoma mexicanum) limb regeneration by using retinoid antagonists that bind to specific RAR (retinoic acid receptor) or RXR (retinoid X receptor) retinoid receptor subtypes. Retinoid antagonists (Ro41-5253, Ro61-8431, LE135, and LE540) were administered to regenerating limbs using implanted silastin blocks loaded with each antagonist. The skeletal pattern of regenerated limbs treated with Ro41-5253 or Ro61-8431 differed only slightly from control limbs. Treatment with LE135 inhibited limb regeneration, while treatment with LE540 allowed relatively normal limb regeneration. When LE135 and LE540 were implanted together, regeneration was not completely inhibited and a hand-like process regenerated. These results demonstrate that interfering with retinoid receptors can modify pattern in the regenerating limb indicating that endogenous retinoids are important during patterning of the regenerating limb.

Expression of fibroblast growth factors 4, 8, and 10 in limbs, flanks, and blastemas of Ambystoma.

Members of the fibroblast growth factor (FGF) family of molecules are critical to limb outgrowth. Here, we examine the expression of Fgfs in three types of limbs-embryonic (developing), mature (differentiated), and regenerating-as well as in the surrounding non-limb tissues in the Mexican axolotl, Ambystoma mexicanum. We have previously cloned partial cDNAs of Fgf4, 8, and 10 from the axolotl (Christensen et al., 2001); the complete Fgf10 cDNA sequence is presented here. Axolotl Fgf10 showed deduced amino acid sequence identity with all other vertebrate Fgf10 coding sequences of >62%, and also included conserved 5' and 3' untranslated regions in nucleotide sequence comparisons. Semiquantitative reverse transcriptase-polymerase chain reaction showed that fibroblast growth factors are differentially expressed in axolotl limbs. Only Fgf8 and 10 were highly expressed during axolotl limb development, although Fgf4, 8, and 10 are all highly expressed during limb development of other vertebrates. Fgf4 expression, however, was highly expressed in the differentiated salamander limb, whereas expression levels of Fgf8 and 10 decreased. Expression levels of Fgf8 and 10 then increased during limb regeneration, whereas Fgf4 expression was completely absent. In addition, axolotl limb regeneration contrasted to limb development of other vertebrates in that Fgf8 did not seem to be as highly expressed in the distal epithelium; rather, its highest expression was found in the blastema mesenchyme. Finally, we investigated the expression of these Fgfs in non-limb tissues. The Fgfs were clearly expressed in developing flank tissue and then severely downregulated in mature flank tissue. Differential Fgf expression levels in the limb and shoulder (limb field) versus in the flank (non-limb field) suggest that FGFs may be instrumental during limb field specification as well as instrumental in maintaining the salamander limb in a state of preparation for regeneration.

Effect of retinoic acid onFgf‐8expression in regenerating urodele amphibian limbs

In our previous study, we have shown that Fgf‐8 is expressed in the basal layer of the apical epithelial cap (AEC) and in the underlying thin layer of mesenchymal tissue of the regenerating limbs of Mexican axolotl, Amby‐stoma mexicanum. Our present RT‐PCR data also demonstrate that Fgf‐8 transcript is localized both in the mesenchymal and epidermal tissues. To understand the effect of retinoic acid (RA) on the expression of Fgf‐8 in the regenerating axolotl limbs, RA was injected intraperitoneally at the dediffer‐entiation stage of limb regeneration. The RA treatment caused a change in the Fgf‐8 expression profile of the regenerating limbs. In RA‐treated limbs, duration of Fgf‐8 expression was prolonged and a high level of expression was maintained during dedifferentiation and blastema formation stages. These results suggest that Fgf‐8 is an important molecule in the process of pattern duplication of regenerating salamander limbs evoked by RA treatment.

Fibroblast growth factors in regenerating limbs of Ambystoma: cloning and semi-quantitative RT-PCR expression studies.

Urodele amphibians (newts and salamanders) have the ability to regenerate amputated limbs throughout their life span. Because fibroblast growth factors (Fgfs) play important roles in developing limbs, we initiated studies to investigate these growth factors in regenerating limbs. Partial cDNAs of Fgf4, 8, and 10 were cloned from both the Mexican axolotl, Ambystoma mexicanum, and locally collected spotted salamander, Ambystoma maculatum, two salamanders well recognized for their regenerative capabilities. cDNAs from the two Ambystoma species were virtually identical, ranging from 97-100% nucleotide identity. Axolotl Fgf4, 8, and 10 showed nucleotide sequence identity with chick Fgf4, 8, and 10 of 79%, 83%, and 72%, respectively. RT-PCR showed that these growth factors are expressed in regenerating axolotl limbs as well as in developing salamander larvae at the three-digit forelimb stage. Fgf8 and 10 are upregulated during regeneration and thus may be involved in distal signaling similar to that of the developing chick limb. Fgf4, however, was undetectable by RT-PCR in the distal tips of regenerates, suggesting that it does not play the same role in limb regeneration that it does in limb development. We also investigated the role these Fgfs may have in the nerve-dependence of regeneration. They were expressed similarly in aneurogenic and innervated limbs, suggesting that they are not the neurotrophic factors responsible for nerve-dependence. Denervation prevented Fgf8 and 10 upregulation, suggesting Fgf pathways are downstream of nerve-dependence. These data highlight important similarities and differences in Fgf expression between limb development and limb regeneration. J. Exp. Zool. 290:529-540, 2001.

Expression of Hoxb13 and Hoxc10 in Developing and Regenerating Axolotl Limbs and Tails

The expression of Hox complex genes in correct spatial and temporal order is critical to patterning of the body axis and limbs during embryonic development. In order to understand the role such genes play in appendage regeneration, we have compared the expression of two 5′ Hox complex genes: Hoxb13 and Hoxc10 during development and regeneration of the body axis and the limbs of axolotls. In contrast to higher vertebrates, Hoxb13 is expressed not only in the tip of the developing tail, but also in the distal mesenchyme of developing hind limbs, and at low levels in developing forelimbs. Hoxc10 is expressed as two transcripts during both development and regeneration. The short transcript (Hoxc10S) is expressed in the tip of the developing tail, in developing hind limbs, and at low levels in developing forelimbs. The long transcript (Hoxc10L) is expressed in a similar pattern, with the exception that no expression in developing forelimbs could be detected. Hoxb13 and both transcripts of Hoxc10 are expressed at high levels in the regenerating spinal cord during tail regeneration, and in both regenerating hind limbs and forelimbs. The up-regulation of expression of these genes during forelimb regeneration, relative to the very low levels of expression during forelimb development, suggests that they play a critical and perhaps unique role in regeneration. This is particularly true for Hoxc10L, which is not expressed during forelimb development, but is expressed during forelimb regeneration; thus making it the first truly “regeneration-specific” gene transcript identified to date.

Citral, an inhibitor of retinoic acid synthesis, modifies pattern formation during limb regeneration in the axolotlAmbystoma mexicanum

While the effects of exogenous retinoids on amphibian limb regeneration have been studied extensively, the role of endogenous retinoids is not clear. Hence, I wished to investigate the role of endogenous retinoic acid during axolotl limb regeneration. Citral is a known inhibitor of retinoic acid synthesis. Thus, I treated regenerating limbs of the larval axolotl Ambystoma mexicanum with citral. The result of this inhibition of retinoic acid synthesis was that limb regeneration became extremely irregular and hypomorphic, with serious pattern defects, or was inhibited altogether. I conclude that endogenous retinoic acid plays an important role in pattern formation during limb regeneration.

Expression of Mmp-9 and related matrix metalloproteinase genes during axolotl limb regeneration.

One of the earliest events in limb regeneration is the extensive remodeling of the extracellular matrix (ECM). Matrix metalloproteinases (MMPs) are a family of matrix degrading enzymes that have been identified in both normal and disease states. Using RT-PCR and cDNA library screening, we have isolated sequences homologous to four different Mmp genes. The spatial and temporal expression of one of these, Mmp-9, has been analyzed during axolotl limb regeneration. Northern blot analysis identifies a 3.8 kb transcript that is abundantly expressed during regeneration, and whole-mount in situ hybridization has uncovered an unusual bi-phasic expression pattern. The first phase begins at 2 hours after amputation, and expression is confined to the healed wound epithelium. This phase continues for 2 days, showing peak expression at 14 hours after amputation. This early phase may be needed to retard reformation of the basal lamina of the epidermis, and thereby facilitate the epidermal-mesenchymal interactions required for successful regeneration. The second phase begins a few days later when a small blastema has formed. During this phase, expression is in the mesenchyme, localized to cells around the tips of the cut skeletal elements. This expression is maintained through several stages until redifferentiation begins. The timing and position of the second phase of expression is consistent with a role for Mmp-9 in the removal of damaged cartilage matrix. We have also discovered that the time of onset of Mmp-9 expression is sensitive to denervation, which causes a delay of several hours. Finally, retinoids, known for their dramatic effects on the pattern of regenerating limbs, can cause a down regulation of Mmp-9 expression. Dev Dyn 1999;216:2-9.

Expression of Mmp9 and related matrix metalloproteinase genes during axolotl limb regeneration

One of the earliest events in limb regeneration is the extensive remodeling of the extracellular matrix (ECM). Matrix metalloproteinases (MMPs) are a family of matrix degrading enzymes that have been identified in both normal and disease states. Using RT-PCR and cDNA library screening, we have isolated sequences homologous to four different Mmp genes. The spatial and temporal expression of one of these, Mmp-9, has been analyzed during axolotl limb regeneration. Northern blot analysis identifies a 3.8 kb transcript that is abundantly expressed during regeneration, and whole-mount in situ hybridization has uncovered an unusual bi-phasic expression pattern. The first phase begins at 2 hours after amputation, and expression is confined to the healed wound epithelium. This phase continues for 2 days, showing peak expression at 14 hours after amputation. This early phase may be needed to retard reformation of the basal lamina of the epidermis, and thereby facilitate the epidermal-mesenchymal interactions required for successful regeneration. The second phase begins a few days later when a small blastema has formed. During this phase, expression is in the mesenchyme, localized to cells around the tips of the cut skeletal elements. This expression is maintained through several stages until redifferentiation begins. The timing and position of the second phase of expression is consistent with a role for Mmp-9 in the removal of damaged cartilage matrix. We have also discovered that the time of onset of Mmp-9 expression is sensitive to denervation, which causes a delay of several hours. Finally, retinoids, known for their dramatic effects on the pattern of regenerating limbs, can cause a down regulation of Mmp-9 expression. Dev Dyn 1999;216:2–9. © 1999 Wiley-Liss, Inc.