Axolotl Limb Regeneration Research Articles

Non-invasive cell tracking of SPIO labeled cells in an intrinsic regenerative environment: The axolotl limb.

Non-invasive methods to track the progress of stem cell therapies are important in the development of future regenerative therapies. Super-paramagnetic iron oxide particles (SPIOs) have previously been applied to track cells using magnetic resonance imaging (MRI) in vivo in non-regenerative animal models. To the best of the author's knowledge, the present study investigated for the first time, the feasibility of tracking SPIO labeled cells in an intrinsic regenerative environment, the regenerating limb of the axolotl, and investigated the homing of stem cell-like blastema cells to the regenerative zone. Viability and labeling success of labeled axolotl blastema cells was tested in vitro using cell culture and histology. SPIO labeling was performed in situ by intramuscular injections and mapped using MRI. Enhanced permeability and retention (EPR) effects were evaluated in the blastema, liver, heart, kidney and a back muscle. Finally, SPIO/Fluorophore-labeled blastema cells were injected intravascularly and tracked using MRI and fluorescence imaging. It was demonstrated that SPIO labeling had no effect on axolotl cell viability in vitro. In situ labeling resulted in an MRI signal alteration during 48 days of regeneration. EPR effect of unbound SPIO was observed only in the liver. MRI tracking revealed increased concentrations of SPIO labeled blastema cells in the liver, kidney and heart, however not the blastema of intravascularly injected axolotls. In conclusion, the results demonstrated that SPIO labeling facilitated non-invasive tracking of injected cells in the regenerating axolotl limb. An early homing mechanism of injected blastema cells to an injury site was not observed.

Pseudotyped baculovirus is an effective gene expression tool for studying molecular function during axolotl limb regeneration

Axolotls can regenerate complex structures through recruitment and remodeling of cells within mature tissues. Accessing the underlying mechanisms at a molecular resolution is crucial to understand how injury triggers regeneration and how it proceeds. However, gene transformation in adult tissues can be challenging. Here we characterize the use of pseudotyped baculovirus (BV) as an effective gene transfer method both for cells within mature limb tissue and within the blastema. These cells remain competent to participate in regeneration after transduction. We further characterize the effectiveness of BV for gene overexpression studies by overexpressing Shh in the blastema, which yields a high penetrance of classic polydactyly phenotypes. Overall, our work establishes BV as a powerful tool to access gene function in axolotl limb regeneration.

Identification of regenerative roadblocks via repeat deployment of limb regeneration in axolotls

Axolotl salamanders are powerful models for understanding how regeneration of complex body parts can be achieved, whereas mammals are severely limited in this ability. Factors that promote normal axolotl regeneration can be examined in mammals to determine if they exhibit altered activity in this context. Furthermore, factors prohibiting axolotl regeneration can offer key insight into the mechanisms present in regeneration-incompetent species. We sought to determine if we could experimentally compromise the axolotl’s ability to regenerate limbs and, if so, discover the molecular changes that might underlie their inability to regenerate. We found that repeated limb amputation severely compromised axolotls’ ability to initiate limb regeneration. Using RNA-seq, we observed that a majority of differentially expressed transcripts were hyperactivated in limbs compromised by repeated amputation, suggesting that mis-regulation of these genes antagonizes regeneration. To confirm our findings, we additionally assayed the role of amphiregulin, an EGF-like ligand, which is aberrantly upregulated in compromised animals. During normal limb regeneration, amphiregulin is expressed by the early wound epidermis, and mis-expressing this factor lead to thickened wound epithelium, delayed initiation of regeneration, and severe regenerative defects. Collectively, our results suggest that repeatedly amputated limbs may undergo a persistent wound healing response, which interferes with their ability to initiate the regenerative program. These findings have important implications for human regenerative medicine.

Efficient gene knockin in axolotl and its use to test the role of satellite cells in limb regeneration

Salamanders exhibit extensive regenerative capacities and serve as a unique model in regeneration research. However, due to the lack of targeted gene knockin approaches, it has been difficult to label and manipulate some of the cell populations that are crucial for understanding the mechanisms underlying regeneration. Here we have established highly efficient gene knockin approaches in the axolotl (Ambystoma mexicanum) based on the CRISPR/Cas9 technology. Using a homology-independent method, we successfully inserted both the Cherry reporter gene and a larger membrane-tagged Cherry-ERT2-Cre-ERT2 (∼5-kb) cassette into axolotl Sox2 and Pax7 genomic loci. Depending on the size of the DNA fragments for integration, 5-15% of the F0 transgenic axolotl are positive for the transgene. Using these techniques, we have labeled and traced the PAX7-positive satellite cells as a major source contributing to myogenesis during axolotl limb regeneration. Our work brings a key genetic tool to molecular and cellular studies of axolotl regeneration.

Transcriptional correlates of proximal-distal identify and regeneration timing in axolotl limbs

Cells within salamander limbs retain memories that inform the correct replacement of amputated tissues at different positions along the length of the arm, with proximal and distal amputations completing regeneration at similar times. We investigated the possibility that positional memory is associated with variation in transcript abundances along the proximal-distal limb axis. Transcripts were deeply sampled from Ambystoma mexicanum limbs at the time they were administered fore arm vs upper arm amputations, and at 19 post-amputation time points. After amputation and prior to regenerative outgrowth, genes typically expressed by differentiated muscle cells declined more rapidly in upper arms while cell cycle transcripts were expressed more highly. These and other expression patterns suggest upper arms undergo more robust tissue remodeling and cell proliferation responses after amputation, and thus provide an explanation for why the overall time to complete regeneration is similar for proximal and distal amputations. Additionally, we identified candidate positional memory genes that were expressed differently between fore and upper arms that encode a surprising number of epithelial proteins and a variety of cell surface, cell adhesion, and extracellular matrix molecules. Also, genes were discovered that exhibited different, bivariate patterns of gene expression between fore and upper arms, implicating dynamic transcriptional regulation for the first time in limb regeneration. Finally, 43 genes expressed differently between fore and upper arm samples showed similar transcriptional patterns during retinoic acid-induced reprogramming of fore arm blastema cells into upper arm cells. Our study provides new insights about the basis of positional information in regenerating axolotl limbs.

A new and improved algorithm for the quantification of chromatin condensation from microscopic data shows decreased chromatin condensation in regenerating axolotl limb cells.

The nuclear landscape plays an important role in the regulation of tissue and positional specific genes in embryonic and developing cells. Changes in this landscape can be dynamic, and are associated with the differentiation of cells during embryogenesis, and the de-differentiation of cells during induced pluripotent stem cell (iPSC) formation and in many cancers. However, tools to quantitatively characterize these changes are limited, especially in the in vivo context, where numerous tissue types are present and cells are arranged in multiple layers. Previous tools have been optimized for the monolayer nature of cultured cells. Therefore, we present a new algorithm to quantify the condensation of chromatin in two in vivo systems. We first developed this algorithm to quantify changes in chromatin compaction and validated it in differentiating spermatids in zebrafish testes. Our algorithm successfully detected the typical increase in chromatin compaction as these cells differentiate. We then employed the algorithm to quantify the changes that occur in amphibian limb cells as they participate in a regenerative response. We observed that the chromatin in the limb cells de-compacts as they contribute to the regenerating organ. We present this new tool as an open sourced software that can be readily accessed and optimized to quantify chromatin compaction in complex multi-layered samples.

Lineage tracing of genome-edited alleles reveals high fidelity axolotl limb regeneration

Salamanders are unparalleled among tetrapods in their ability to regenerate many structures, including entire limbs, and the study of this ability may provide insights into human regenerative therapies. The complex structure of the limb poses challenges to the investigation of the cellular and molecular basis of its regeneration. Using CRISPR/Cas, we genetically labelled unique cell lineages within the developing axolotl embryo and tracked the frequency of each lineage within amputated and fully regenerated limbs. This allowed us, for the first time, to assess the contributions of multiple low frequency cell lineages to the regenerating limb at once. Our comparisons reveal that regenerated limbs are high fidelity replicas of the originals even after repeated amputations.

The Axolotl Limb Regeneration Model as a Discovery Tool for Engineering the Stem Cell Niche.

Recent advances in genomics and gene editing have expanded the range of model organisms to include those with interesting biological capabilities such as regeneration. Among these are the classic models of regeneration biology, the salamander. Although stimulating endogenous regeneration in humans likely is many years away, with advances in stem cell biology and biomedical engineering (e.g. bio-inspired materials), it is evident that there is great potential to enhance regenerative outcomes by approaching the problem from an engineering perspective. The question at this point is what do we need to engineer? The value of regeneration models is that they show us how regeneration works, which then can guide efforts to mimic these developmental processes therapeutically. Among these models, the Accessory Limb Model (ALM) was developed in the axolotl as a gain-of-function assay for the sequential steps that are required for successful regeneration. To date, this model has identified a number of proregenerative signals, including growth factor signaling associated with nerves, and signals associated with the extracellular matrix (ECM) that induce pattern formation. Identification of these signals through the use of models in highly regenerative vertebrates (e.g. the axolotl) offers a wide range of possible modifications for engineering bio-inspired, biomimetic materials to create a dynamic stem cell niche for regeneration and scar-free repair.

Tissue regeneration in dentistry: Can salamanders provide insight?

The ability to regenerate damaged tissues would be of tremendous benefit for medicine and dentistry. Unfortunately, humans are unable to regenerate tissues such as teeth and fingers or to repair injured spinal cord. With an aging population, health problems are more prominent and dentistry is no exception as loss of bone tissue in the orofacial sphere from periodontal disease is on the rise. Humans can repair oral soft tissues exceptionally well; however, hard tissues, such as bone and teeth, are devoid of the ability to repair well or at all. Fortunately, Mother Nature has solved nearly every problem that we would like to solve for our own benefit and tissue regeneration is no exception. By studying animals that can regenerate, like Axolotls (Mexican salamander), we hope to find ways to stimulate regeneration in humans. We will discuss the role of the transforming growth factor beta cytokines as they are central to wound healing in humans and regeneration in Axolotls. We will also compare wound healing in humans (skin and oral mucosa) to Axolotl skin wound healing and limb regeneration. Finally, we will address the problem of bone regeneration and present results in salamanders which indicate that in order to regenerate bone you need to recruit non-bone cells. Fundamental research, such as the work being performed in animals that can regenerate, offers insight to help understand why some treatments are successful while others fail when it comes to specific tissues such as bones.

Diffusion tensor tractography reveals muscle reconnection during axolotl limb regeneration.

Axolotls have amazing ability to regenerate their lost limbs. Our previous works showed that after amputation the remnant muscle ends remained at their original location whilst sending satellite cells into the regenerating parts to develop into early muscle fibers in the late differentiation stage. The parental and the newly formed muscle fibers were not connected until very late stage. The present study used non-invasive diffusion tensor imaging (DTI) to monitor weekly axolotl upper arm muscles after amputation of their upper arms. DTI tractography showed that the regenerating muscle fibers became visible at 9-wpa (weeks post amputation), but a gap was observed between the regenerating and parental muscles. The gap was filled at 10-wpa, indicating reconnection of the fibers of both muscles. This was confirmed by histology. The DTI results indicate that 23% of the muscle fibers were reconnected at 10-wpa. In conclusion, DTI can be used to visualize axolotls’ skeletal muscles and the results of muscle reconnection were in accordance with our previous findings. This non-invasive technique will allow researchers to identify the timeframe in which muscle fiber reconnection takes place and thus enable the study of the mechanisms underlying this reconnection.

Neuregulin-1 signaling is essential for nerve-dependent axolotl limb regeneration.

The Mexican axolotl (Ambystoma mexicanum) is capable of fully regenerating amputated limbs, but denervation of the limb inhibits the formation of the post-injury proliferative mass called the blastema. The molecular basis behind this phenomenon remains poorly understood, but previous studies have suggested that nerves support regeneration via the secretion of essential growth-promoting factors. An essential nerve-derived factor must be found in the blastema, capable of rescuing regeneration in denervated limbs, and its inhibition must prevent regeneration. Here, we show that the neuronally secreted protein Neuregulin-1 (NRG1) fulfills all these criteria in the axolotl. Immunohistochemistry and in situ hybridization of NRG1 and its active receptor ErbB2 revealed that they are expressed in regenerating blastemas but lost upon denervation. NRG1 was localized to the wound epithelium prior to blastema formation and was later strongly expressed in proliferating blastemal cells. Supplementation by implantation of NRG1-soaked beads rescued regeneration to digits in denervated limbs, and pharmacological inhibition of NRG1 signaling reduced cell proliferation, blocked blastema formation and induced aberrant collagen deposition in fully innervated limbs. Taken together, our results show that nerve-dependent NRG1/ErbB2 signaling promotes blastemal proliferation in the regenerating limb and may play an essential role in blastema formation, thus providing insight into the longstanding question of why nerves are required for axolotl limb regeneration.

Cartilage and bone cells do not participate in skeletal regeneration in Ambystoma mexicanum limbs

The Mexican Axolotl is one of the few tetrapod species that is capable of regenerating complete skeletal elements in injured adult limbs. Whether the skeleton (bone and cartilage) plays a role in the patterning and contribution to the skeletal regenerate is currently unresolved. We tested the induction of pattern formation, the effect on cell proliferation, and contributions of skeletal tissues (cartilage, bone, and periosteum) to the regenerating axolotl limb. We found that bone tissue grafts from transgenic donors expressing GFP fail to induce pattern formation and do not contribute to the newly regenerated skeleton. Periosteum tissue grafts, on the other hand, have both of these activities. These observations reveal that skeletal tissue does not contribute to the regeneration of skeletal elements; rather, these structures are patterned by and derived from cells of non-skeletal connective tissue origin.

FGF8 and SHH substitute for anterior-posterior tissue interactions to induce limb regeneration.

In salamanders, grafting of a left limb blastema onto a right limb stump yields regeneration of three limbs, the normal limb and two 'supernumerary' limbs. This experiment and other research have shown that the juxtaposition of anterior and posterior limb tissue plus innervation are necessary and sufficient to induce complete limb regeneration in salamanders. However, the cellular and molecular basis of the requirement for anterior-posterior tissue interactions were unknown. Here we have clarified the molecular basis of the requirement for both anterior and posterior tissue during limb regeneration and supernumerary limb formation in axolotls (Ambystoma mexicanum). We show that the two tissues provide complementary cross-inductive signals that are required for limb outgrowth. A blastema composed solely of anterior tissue normally regresses rather than forming a limb, but activation of hedgehog (HH) signalling was sufficient to drive regeneration of an anterior blastema to completion owing to its ability to maintain fibroblast growth factor (FGF) expression, the key signalling activity responsible for blastema outgrowth. In blastemas composed solely of posterior tissue, HH signalling was not sufficient to drive regeneration; however, ectopic expression of FGF8 together with endogenous HH signalling was sufficient. In axolotls, FGF8 is expressed only in the anterior mesenchyme and maintenance of its expression depends on sonic hedgehog (SHH) signalling from posterior tissue. Together, our findings identify key anteriorly and posteriorly localized signals that promote limb regeneration and show that these single factors are sufficient to drive non-regenerating blastemas to complete regeneration with full elaboration of skeletal elements.

Activation of Smad2 but not Smad3 is required to mediate TGF-β signaling during axolotl limb regeneration.

Axolotls are unique among vertebrates in their ability to regenerate tissues, such as limbs, tail and skin. The axolotl limb is the most studied regenerating structure. The process is well characterized morphologically; however, it is not well understood at the molecular level. We demonstrate that TGF-β1 is highly upregulated during regeneration and that TGF-β signaling is necessary for the regenerative process. We show that the basement membrane is not prematurely formed in animals treated with the TGF-β antagonist SB-431542. More importantly, Smad2 and Smad3 are differentially regulated post-translationally during the preparation phase of limb regeneration. Using specific antagonists for Smad2 and Smad3 we demonstrate that Smad2 is responsible for the action of TGF-β during regeneration, whereas Smad3 is not required. Smad2 target genes (Mmp2 and Mmp9) are inhibited in SB-431542-treated limbs, whereas non-canonical TGF-β targets (e.g. Mmp13) are unaffected. This is the first study to show that Smad2 and Smad3 are differentially regulated during regeneration and places Smad2 at the heart of TGF-β signaling supporting the regenerative process.

DNA Methylation Dynamics Regulate the Formation of a Regenerative Wound Epithelium during Axolotl Limb Regeneration.

The formation of a blastema during regeneration of an axolotl limb involves important changes in the behavior and function of cells at the site of injury. One of the earliest events is the formation of the wound epithelium and subsequently the apical epidermal cap, which involves in vivo dedifferentiation that is controlled by signaling from the nerve. We have investigated the role of epigenetic modifications to the genome as a possible mechanism for regulating changes in gene expression patterns of keratinocytes of the wound and blastema epithelium that are involved in regeneration. We report a modulation of the expression DNMT3a, a de novo DNA methyltransferase, within the first 72 hours post injury that is dependent on nerve signaling. Treatment of skin wounds on the upper forelimb with decitabine, a DNA methyltransferase inhibitor, induced changes in gene expression and cellular behavior associated with a regenerative response. Furthermore, decitabine-treated wounds were able to participate in regeneration while untreated wounds inhibited a regenerative response. Elucidation of the specific epigenetic modifications that mediate cellular dedifferentiation likely will lead to insights for initiating a regenerative response in organisms that lack this ability.

Tissue specific reactions to positional discontinuities in the regenerating axolotl limb.

We investigated cellular contributions to intercalary regenerates and 180o supernumerary limbs during axolotl limb regeneration using the cell autonomous green fluorescent protein marker and exchanged blastemas between white and green fluorescent protein animals. After distal blastemas were grafted to proximal levels tissues of the intercalary regenerate behaved independently with regard to the law of distal transformation; graft epidermis was replaced by stump epidermis, muscle‐derived cells, blood vessels, and Schwann cells of the distal blastema moved proximally to the stylopodium and cartilage and dermal cells conformed to the law. After 180o rotation, blastemas showed contributions from stump tissues which failed to alter patterning of the blastema. Supernumerary limbs were composed of stump and graft tissues and extensive contributions of stump tissues generated inversions or duplications of polarity to produce limbs of mixed handedness. Tail skeletal muscle and cardiac muscle broke the law with cells derived from these tissues exhibiting an apparent anteroposterior polarity as they migrated to the anterior side of the blastema. We attribute this behavior to the possible presence of a chemotactic factor from the wound epidermis.

Gene expression during the first 28 days of axolotl limb regeneration I: Experimental design and global analysis of gene expression.

While it is appreciated that global gene expression analyses can provide novel insights about complex biological processes, experiments are generally insufficiently powered to achieve this goal. Here we report the results of a robust microarray experiment of axolotl forelimb regeneration. At each of 20 post‐amputation time points, we estimated gene expression for 10 replicate RNA samples that were isolated from 1 mm of heterogeneous tissue collected from the distal limb tip. We show that the limb transcription program diverges progressively with time from the non‐injured state, and divergence among time adjacent samples is mostly gradual. However, punctuated episodes of transcription were identified for five intervals of time, with four of these coinciding with well‐described stages of limb regeneration—amputation, early bud, late bud, and pallet. The results suggest that regeneration is highly temporally structured and regulated by mechanisms that function within narrow windows of time to coordinate transcription within and across cell types of the regenerating limb. Our results provide an integrative framework for hypothesis generation using this complex and highly informative data set.

Regulation of Axolotl (Ambystoma mexicanum) Limb Blastema Cell Proliferation by Nerves and BMP2 in Organotypic Slice Culture.

We have modified and optimized the technique of organotypic slice culture in order to study the mechanisms regulating growth and pattern formation in regenerating axolotl limb blastemas. Blastema cells maintain many of the behaviors that are characteristic of blastemas in vivo when cultured as slices in vitro, including rates of proliferation that are comparable to what has been reported in vivo. Because the blastema slices can be cultured in basal medium without fetal bovine serum, it was possible to test the response of blastema cells to signaling molecules present in serum, as well as those produced by nerves. We also were able to investigate the response of blastema cells to experimentally regulated changes in BMP signaling. Blastema cells responded to all of these signals by increasing the rate of proliferation and the level of expression of the blastema marker gene, Prrx-1. The organotypic slice culture model provides the opportunity to identify and characterize the spatial and temporal co-regulation of pathways in order to induce and enhance a regenerative response.

Long-duration muscle dedifferentiation during limb regeneration in axolotls.

Although still debated, limb regeneration in salamanders is thought to depend on the dedifferentiation of remnant tissue occurring early after amputation and generating the progenitor cells that initiate regeneration. This dedifferentiation has been demonstrated previously by showing the fragmentation of muscle fibers into mononucleated cells and by revealing the contribution of mature muscle fibers to the regenerates by using lineage-tracing studies. Here, we provide additional evidence of dedifferentiation by showing that Pax7 (paired-box protein-7) transcripts are expressed at the ends of remnant muscle fibers in axolotls by using in situ hybridization and by demonstrating the presence of Pax7+ muscle-fiber nuclei in the early bud and mid-bud stages by means of immunohistochemical staining. During the course of regeneration, the remnant muscles did not progress; instead, muscle progenitors migrated out from the remnants and proliferated and differentiated in the new tissues at an early stage of differentiation. The regenerating muscles and remnant muscles were largely disconnected, and this left a gap between them until extremely late in the late stage of differentiation, at which point the new and old muscles connected together. Notably, Pax7 transcripts were detected in the regions of muscles that faced these gaps; thus, Pax7 expression might indicate dedifferentiation in the remnant-muscle ends and partial differentiation in the regenerating muscles. The roles of this long-duration dedifferentiation in the remnants remain unknown. However, the results presented here could support the hypothesis that long-duration muscle dedifferentiation facilitates the connection and fusion between the new and old muscles that are both in an immature state; this is because immature Pax7+ myoblasts readily fuse during developmental myogenesis.

Identification of reference genes and validation for gene expression studies in diverse axolotl (Ambystoma mexicanum) tissues

For the precise quantitative RT-PCR normalization a set of valid reference genes is obligatory. Moreover have to be taken into concern the experimental conditions as they bias the regulation of reference genes. Up till now, no reference targets have been described for the axolotl (Ambystoma mexicanum).In a search in the public database SalSite for genetic information of the axolotl we identified fourteen presumptive reference genes, eleven of which were further tested for their gene expression stability. This study characterizes the expressional patterns of 11 putative endogenous control genes during axolotl limb regeneration and in an axolotl tissue panel. All 11 reference genes showed variable expression. Strikingly, ACTB was to be found most stable expressed in all comparative tissue groups, so we reason it to be suitable for all different kinds of axolotl tissue-type investigations. Moreover do we suggest GAPDH and RPLP0 as suitable for certain axolotl tissue analysis. When it comes to axolotl limb regeneration, a validated pair of reference genes is ODC and RPLP0.With these findings, new insights into axolotl gene expression profiling might be gained.