Adult Axolotls Research Articles

Radiographic anatomical description of the skeletal system of adult and subadult axolotls (Ambystoma mexicanum): a pilot study

In this study, the use of radiographic imaging to examine the skeletal structures of living axolotls (Ambystoma mexicanum) is described. Dorsoventral and left lateral radiographs were taken of both healthy adult and subadult axolotls without restraint, directly on the X-ray table. The aim was to comprehensively describe their skeletal anatomy using radiographic assessment. The method accurately depicted bone shapes and structures, identifying bone names based on comparisons with similar salamanders. However, internal organs within the coelomic cavity were not clearly visible except for the lungs. Interpreting subadult axolotl radiographs was harder, likely due to less developed skeletal ossification or X-ray parameter variations. Variations were observed in the number of certain bones and vertebrae types among individuals. This method offers a non-invasive way to understand healthy animal skeletal appearance, eliminating the need for autopsies. The radiographic approach contributes to better understand axolotl skeletal morphology, benefiting comparative anatomy, veterinary medicine and conservation efforts.

GROSS AND ULTRASONOGRAPHIC ANATOMY OF THE COELOMIC ORGANS OF HEALTHY AXOLOTLS (AMBYSTOMA MEXICANUM).

The objectives of this study were to describe the gross anatomy and ultrasonographic appearance of coelomic organs in subadult and adult axolotls (Ambystoma mexicanum), to describe an ultrasound technique, and to test correlations of ultrasonographic measurement with body length, width, and weight. Necropsies of coelomic organs were conducted on 10 axolotls (females = 5; males = 5) and ultrasound on 11 (males = 5; females = 6). Animals were kept in water and maintained conscious during ultrasound. The heart, caudal vena cava, liver, gallbladder, spleen, esophagus, stomach, colon, kidneys, ovaries, and fat bodies were identified in all study subjects, although testicles were identified in only 6/7 subjects. The pancreas and adrenal glands could not be identified in any animals, either during necropsy or ultrasonography. Coelomic and pericardial effusion was present in all animals. Ultrasonographic measurements of the liver, spleen, myocardial thickness, and right and left kidney length were highly repeatable (correlation value [CV] < 5%) and the esophagus, spleen, caudal vena cava, fat bodies, gallbladder, colon thickness, right kidney height and width, and right testicle diameter were statistically repeatable (CV < 10%).

Effects of Water Temperature on Gonads Growth in Ambystoma mexicanum Axolotl Salamanders.

The thermal environment is a major factor influencing amphibians. For example, amphibian reproduction occurs in specific temperatures, and minor changes in this aspect could have negative impacts on this biological process. Understanding the potential effects of temperature on reproductive output is important from both an ecological and captive breeding colony point of view. I examined temperature effects on reproduction in axolotl reared from egg to adult at 4 temperatures (15 °C, 19 °C, 23 °C, and 27 °C) These adult axolotls (n = 174) were measured and weighed, dissected, and their gonads were removed and weighed to obtain an individual's reproductive allocation. Female axolotls reared at 23 °C had a greater Gonadosomatic Index (GSI) compared to axolotl reared at each of the other temperatures with axolotls reared at 27 °C having the lowest reproductive output. Moreover, all GSI pair-wise comparisons in the four temperature treatments were significantly different from each other (ANOVA, F (3, 66) = 61.681, p < 0.0001). Additionally male rearing temperature significantly influenced GSI (ANOVA, F (3, 89) = 10.441, p < 0.0001). Male axolotls reared at 19 °C had significantly greater GSI compared to males reared at the three other temperatures. There were no statistical differences among each of the other pair-wise comparisons. As seen in this experiment, axolotls may be especially sensitive to climate-driven warming due to their highly permeable skin and paedomorphic life history. Understanding how axolotls and other amphibian species adjust to the challenges of climate change is important in the management of this imperiled taxa.

Construction of the axolotl cell landscape using combinatorial hybridization sequencing at single-cell resolution

The Mexican axolotl (Ambystoma mexicanum) is a well-established tetrapod model for regeneration and developmental studies. Remarkably, neotenic axolotls may undergo metamorphosis, a process that triggers many dramatic changes in diverse organs, accompanied by gradually decline of their regeneration capacity and lifespan. However, the molecular regulation and cellular changes in neotenic and metamorphosed axolotls are still poorly investigated. Here, we develop a single-cell sequencing method based on combinatorial hybridization to generate a tissue-based transcriptomic landscape of the neotenic and metamorphosed axolotls. We perform gene expression profiling of over 1 million single cells across 19 tissues to construct the first adult axolotl cell landscape. Comparison of single-cell transcriptomes between the tissues of neotenic and metamorphosed axolotls reveal the heterogeneity of non-immune parenchymal cells in different tissues and established their regulatory network. Furthermore, we describe dynamic gene expression patterns during limb development in neotenic axolotls. This system-level single-cell analysis of molecular characteristics in neotenic and metamorphosed axolotls, serves as a resource to explore the molecular identity of the axolotl and facilitates better understanding of metamorphosis.

An Identification and Characterization of the Axolotl (Ambystoma mexicanum, Amex) Telomerase Reverse Transcriptase (Amex TERT).

The Mexican axolotl is one of the few vertebrates that is able to replace its lost body parts during lifespan. Due to its remarkable regenerative abilities, the axolotl emerged as a model organism especially for limb regeneration. Telomeres and the telomerase enzyme are crucial for regeneration and protection against aging processes and degenerating diseases. Despite its relevance for regeneration, the axolotl telomerase and telomere length have not yet been investigated. Therefore, in the present paper, we reveal the sequence of the axolotl telomerase reverse transcriptase gene (Tert) and protein (TERT). Multiple sequence alignment (MSA) showed the known conserved RT- and TERT-specific motifs and residues found in other TERTs. In addition, we establish methods to determine the Tert expression (RT-PCR) and telomerase activity (Q-TRAP) of adult axolotl and blastema tissues. We found that both differentiated forelimb tissue and regenerating blastema tissue express Tert and show telomerase activity. Furthermore, blastema tissue appears to exhibit a higher Tert expression and telomerase activity. The presence of active telomerase in adult somatic cells is a decisive difference to somatic cells of non-regenerating vertebrates, such as humans. These findings indicate that telomere biology may play a key role in the regenerative abilities of cells.

Identification of the Adult Hematopoietic Liver as the Primary Reservoir for the Recruitment of Pro-regenerative Macrophages Required for Salamander Limb Regeneration.

The lack of scar-free healing and regeneration in many adult human tissues imposes severe limitations on the recovery of function after injury. In stark contrast, salamanders can functionally repair a range of clinically relevant tissues throughout adult life. The impressive ability to regenerate whole limbs after amputation, or regenerate following cardiac injury, is critically dependent on the recruitment of (myeloid) macrophage white blood cells to the site of injury. Amputation in the absence of macrophages results in regeneration failure and scar tissue induction. Identifying the exact hematopoietic source or reservoir of myeloid cells supporting regeneration is a necessary step in characterizing differences in macrophage phenotypes regulating scarring or regeneration across species. Mammalian wounds are dominated by splenic-derived monocytes that originate in the bone marrow and differentiate into macrophages within the wound. Unlike mammals, adult axolotls do not have functional bone marrow but instead utilize liver and spleen tissues as major sites for adult hematopoiesis. To interrogate leukocyte identity, tissue origins, and modes of recruitment, we established several transgenic axolotl hematopoietic tissue transplant models and flow cytometry protocols to study cell migration and identify the source of pro-regenerative macrophages. We identified that although bidirectional trafficking of leukocytes can occur between spleen and liver tissues, the liver is the major source of leukocytes recruited to regenerating limbs. Recruitment of leukocytes and limb regeneration occurs in the absence of the spleen, thus confirming the dependence of liver-derived myeloid cells in regeneration and that splenic maturation is dispensable for the education of pro-regenerative macrophages. This work provides an important foundation for understanding the hematopoietic origins and education of myeloid cells recruited to, and essential for, adult tissue regeneration.

Single-cell RNA-seq reveals novel mitochondria-related musculoskeletal cell populations during adult axolotl limb regeneration process.

While the capacity to regenerate tissues or limbs is limited in mammals, including humans, axolotls are able to regrow entire limbs and major organs after incurring a wound. The wound blastema has been extensively studied in limb regeneration. However, due to the inadequate characterization of ECM and cell subpopulations involved in the regeneration process, the discovery of the key drivers for human limb regeneration remains unknown. In this study, we applied large-scale single-cell RNA sequencing to classify cells throughout the adult axolotl limb regeneration process, uncovering a novel regeneration-specific mitochondria-related cluster supporting regeneration through energy providing and the ECM secretion (COL2+) cluster contributing to regeneration through cell-cell interactions signals. We also discovered the dedifferentiation and re-differentiation of the COL1+/COL2+ cellular subpopulation and exposed a COL2-mitochondria subcluster supporting the musculoskeletal system regeneration. On the basis of these findings, we reconstructed the dynamic single-cell transcriptome of adult axolotl limb regenerative process, and identified the novel regenerative mitochondria-related musculoskeletal populations, which yielded deeper insights into the crucial interactions between cell clusters within the regenerative microenvironment.

Inhibition of Wound Epidermis Formation via Full Skin Flap Surgery During Axolotl Limb Regeneration.

Classical experiments in salamander regenerative biology over the last century have long established that the wound epidermis is a crucial signaling structure that forms rapidly post-amputation and is required for limb regeneration. However, methods to study its precise function at the molecular level over the last decades have been limited due to a paucity of precise functional techniques and genomic information available in salamander model systems. Excitingly, the recent plethora of sequencing technologies coupled with the release of various salamander genomes and the advent of functional genetic testing methods, including CRISPR, makes it possible to re-visit these foundational experiments at unprecedented molecular resolution. Here, I describe how to perform the classically developed full skin flap (FSF) surgery in adult axolotls in order to inhibit wound epidermis formation immediately following amputation. The wound epidermis normally forms via distal migration of epithelial cells in the skin proximal to the amputation plane to seal off the wound from the outside environment. The surgery entails immediately suturing full thickness skin (which includes both epidermal and dermal layers) over the amputation plane to hinder epithelial cell migration and contact with the underlying damaged mesenchymal tissues. Successful surgeries result in the inhibition of blastema formation and limb regeneration. By combining this surgery method with contemporary downstream molecular and functional analyses, researchers can begin to uncover the molecular underpinnings of wound epidermis function and biology during limb regeneration.

Meningeal Foam Cells and Ependymal Cells in Axolotl Spinal Cord Regeneration.

A previously unreported population of foam cells (foamy macrophages) accumulates in the invasive fibrotic meninges during gap regeneration of transected adult Axolotl spinal cord (salamander Ambystoma mexicanum) and may act beneficially. Multinucleated giant cells (MNGCs) also occurred in the fibrotic meninges. Actin-label localization and transmission electron microscopy showed characteristic foam cell and MNGC podosome and ruffled border-containing sealing ring structures involved in substratum attachment, with characteristic intermediate filament accumulations surrounding nuclei. These cells co-localized with regenerating cord ependymal cell (ependymoglial) outgrowth. Phase contrast-bright droplets labeled with Oil Red O, DiI, and DyRect polar lipid live cell label showed accumulated foamy macrophages to be heavily lipid-laden, while reactive ependymoglia contained smaller lipid droplets. Both cell types contained both neutral and polar lipids in lipid droplets. Foamy macrophages and ependymoglia expressed the lipid scavenger receptor CD36 (fatty acid translocase) and the co-transporter toll-like receptor-4 (TLR4). Competitive inhibitor treatment using the modified fatty acid Sulfo-N-succinimidyl Oleate verified the role of the lipid scavenger receptor CD36 in lipid uptake studies in vitro. Fluoromyelin staining showed both cell types took up myelin fragments in situ during the regeneration process. Foam cells took up DiI-Ox-LDL and DiI-myelin fragments in vitro while ependymoglia took up only DiI-myelin in vitro. Both cell types expressed the cysteine proteinase cathepsin K, with foam cells sequestering cathepsin K within the sealing ring adjacent to the culture substratum. The two cell types act as sinks for Ox-LDL and myelin fragments within the lesion site, with foamy macrophages showing more Ox-LDL uptake activity. Cathepsin K activity and cellular localization suggested that foamy macrophages digest ECM within reactive meninges, while ependymal cells act from within the spinal cord tissue during outgrowth into the lesion site, acting in complementary fashion. Small MNGCs also expressed lipid transporters and showed cathepsin K activity. Comparison of 3H-glucosamine uptake in ependymal cells and foam cells showed that only ependymal cells produce glycosaminoglycan and proteoglycan-containing ECM, while the cathepsin studies showed both cell types remove ECM. Interaction of foam cells and ependymoglia in vitro supported the dispersion of ependymal outgrowth associated with tissue reconstruction in Axolotl spinal cord regeneration.

Transcriptomic landscape of the blastema niche in regenerating adult axolotl limbs at single-cell resolution

Regeneration of complex multi-tissue structures, such as limbs, requires the coordinated effort of multiple cell types. In axolotl limb regeneration, the wound epidermis and blastema have been extensively studied via histology, grafting, and bulk-tissue RNA-sequencing. However, defining the contributions of these tissues is hindered due to limited information regarding the molecular identity of the cell types in regenerating limbs. Here we report unbiased single-cell RNA-sequencing on over 25,000 cells from axolotl limbs and identify a plethora of cellular diversity within epidermal, mesenchymal, and hematopoietic lineages in homeostatic and regenerating limbs. We identify regeneration-induced genes, develop putative trajectories for blastema cell differentiation, and propose the molecular identity of fibroblast-like blastema progenitor cells. This work will enable application of molecular techniques to assess the contribution of these populations to limb regeneration. Overall, these data allow for establishment of a putative framework for adult axolotl limb regeneration.

Musashi and Plasticity of Xenopus and Axolotl Spinal Cord Ependymal Cells.

The differentiated state of spinal cord ependymal cells in regeneration-competent amphibians varies between a constitutively active state in what is essentially a developing organism, the tadpole of the frog Xenopus laevis, and a quiescent, activatable state in a slowly growing adult salamander Ambystoma mexicanum, the Axolotl. Ependymal cells are epithelial in intact spinal cord of all vertebrates. After transection, body region ependymal epithelium in both Xenopus and the Axolotl disorganizes for regenerative outgrowth (gap replacement). Injury-reactive ependymal cells serve as a stem/progenitor cell population in regeneration and reconstruct the central canal. Expression patterns of mRNA and protein for the stem/progenitor cell-maintenance Notch signaling pathway mRNA-binding protein Musashi (msi) change with life stage and regeneration competence. Msi-1 is missing (immunohistochemistry), or at very low levels (polymerase chain reaction, PCR), in both intact regeneration-competent adult Axolotl cord and intact non-regeneration-competent Xenopus tadpole (Nieuwkoop and Faber stage 62+, NF 62+). The critical correlation for successful regeneration is msi-1 expression/upregulation after injury in the ependymal outgrowth and stump-region ependymal cells. msi-1 and msi-2 isoforms were cloned for the Axolotl as well as previously unknown isoforms of Xenopus msi-2. Intact Xenopus spinal cord ependymal cells show a loss of msi-1 expression between regeneration-competent (NF 50–53) and non-regenerating stages (NF 62+) and in post-metamorphosis froglets, while msi-2 displays a lower molecular weight isoform in non-regenerating cord. In the Axolotl, embryos and juveniles maintain Msi-1 expression in the intact cord. In the adult Axolotl, Msi-1 is absent, but upregulates after injury. Msi-2 levels are more variable among Axolotl life stages: rising between late tailbud embryos and juveniles and decreasing in adult cord. Cultures of regeneration-competent Xenopus tadpole cord and injury-responsive adult Axolotl cord ependymal cells showed an identical growth factor response. Epidermal growth factor (EGF) maintains mesenchymal outgrowth in vitro, the cells are proliferative and maintain msi-1 expression. Non-regeneration competent Xenopus ependymal cells, NF 62+, failed to attach or grow well in EGF+ medium. Ependymal Msi-1 expression in vivo and in vitro is a strong indicator of regeneration competence in the amphibian spinal cord.

Regulation of Injury-Induced Ovarian Regeneration by Activation of Oogonial Stem Cells.

Some animals have the ability to generate large numbers of oocytes throughout life. This raises the question whether persistent adult germline stem cell populations drive continuous oogenesis and whether they are capable of mounting a regenerative response after injury. Here we demonstrate the presence of adult oogonial stem cells (OSCs) in the adult axolotl salamander ovary and show that ovarian injury induces OSC activation and functional regeneration of the ovaries to reproductive capability. Cells that have morphological similarities to germ cells were identified in the developing and adult ovaries via histological analysis. Genes involved in germ cell maintenance including Vasa, Oct4, Sox2, Nanog, Bmp15, Piwil1, Piwil2, Dazl, and Lhx8 were expressed in the presumptive OSCs. Colocalization of Vasa protein with H3 mitotic marker showed that both oogonial and spermatogonial adult stem cells were mitotically active. Providing evidence of stemness and viability of adult OSCs, enhanced green fluorescent protein (EGFP) adult OSCs grafted into white juvenile host gonads gave rise to EGFP OSCs, and oocytes. Last, the axolotl ovaries completely regenerated after partial ovariectomy injury. During regeneration, OSC activation resulted in rapid differentiation into new oocytes, which was demonstrated by Vasa+ /BrdU+ coexpression. Furthermore, follicle cell proliferation promoted follicle maturation during ovarian regeneration. Overall, these results show that adult oogenesis occurs via proliferation of endogenous OSCs in a tetrapod and mediates ovarian regeneration. This study lays the foundations to elucidate mechanisms of ovarian regeneration that will assist regenerative medicine in treating premature ovarian failure and reduced fertility. Stem Cells 2017;35:236-247.

Adult axolotls can regenerate original neuronal diversity in response to brain injury

The axolotl can regenerate multiple organs, including the brain. It remains, however, unclear whether neuronal diversity, intricate tissue architecture, and axonal connectivity can be regenerated; yet, this is critical for recovery of function and a central aim of cell replacement strategies in the mammalian central nervous system. Here, we demonstrate that, upon mechanical injury to the adult pallium, axolotls can regenerate several of the populations of neurons present before injury. Notably, regenerated neurons acquire functional electrophysiological traits and respond appropriately to afferent inputs. Despite the ability to regenerate specific, molecularly-defined neuronal subtypes, we also uncovered previously unappreciated limitations by showing that newborn neurons organize within altered tissue architecture and fail to re-establish the long-distance axonal tracts and circuit physiology present before injury. The data provide a direct demonstration that diverse, electrophysiologically functional neurons can be regenerated in axolotls, but challenge prior assumptions of functional brain repair in regenerative species.

Evaluation of the anesthetic effects of MS222 in the adult Mexican axolotl (Ambystoma mexicanum).

The Mexican axolotl (Ambystoma mexicanum) is a unique research model in several fields of medicine, where surgical and invasive procedures may be required. As yet, little is known about the efficacy of MS222 (tricaine methanesulfonate), which is the most commonly used anesthetic agent in amphibians. The main objectives of this study were to evaluate the anesthetic effects and physiological changes in adult axolotls following a 20-minute immersion bath, containing progressive MS222 concentrations starting at 0.1%. Depth of anesthesia and physiological changes were evaluated every 15 minutes post-MS222 exposure with the following parameters: righting behavior, withdrawal reflex, acetic acid test response, heart rate, and blood oxygen saturation, as well as cloacal and body surface temperatures. A 20-minute exposure in a 0.1% MS222 immersion bath (n=6 animals) had no anesthetic effects on adult axolotls after 20 minutes of exposure. With a 0.2% MS222 solution, all axolotls (n=9) were deeply anesthetized at 15 minutes, and 80% were still unresponsive at 30 minutes postexposure. Blood oxygen saturation and heart rate were slightly, but significantly, increased when compared with the baseline value and remained stable up to recovery. There was no significant increase in surface and cloaca temperatures, compared with baseline. With the 0.4% MS222 solution, the duration of anesthesia lasted for 90 minutes to at least 120 minutes (n=3 animals) and this concentration was deemed too high. In conclusion, a 20-minute immersion bath with 0.2% MS222 may be used for short procedures (15–30 minutes) requiring anesthesia of adult axolotls.

Thyroxine-induced metamorphosis in the axolotl (Ambystoma mexicanum).

The axolotl (Ambystoma mexicanum) has remained an important model for regeneration and developmental biology for over a century. Although axolotls in captive-bred colonies usually exist in an aquatic form, they retain the ability to undergo metamorphosis following exposure to thyroid hormone. Here we present a robust method for inducing metamorphosis in adult axolotls that results in high survivability and produces terrestrial animals that can be maintained in long-term captivity.

Mapping hematopoiesis in a fully regenerative vertebrate: the axolotl

AbstractHematopoietic stem cell (HSC)-derived cells are involved in wound healing responses throughout the body. Unfortunately for mammals, wound repair typically results in scarring and nonfunctional reparation. Among vertebrates, none display such an extensive ability for adult regeneration as urodele amphibians, including 1 of the more popular models: the axolotl. However, a lack of knowledge of axolotl hematopoiesis hinders the use of this animal for the study of hematopoietic cells in scar-free wound healing and tissue regeneration. We used white and cytomegalovirus:green fluorescent protein+ transgenic white axolotl strains to map sites of hematopoiesis and develop hematopoietic cell transplant methodology. We also established a fluorescence-activated cell sorter enrichment technique for major blood lineages and colony-forming unit assays for hematopoietic progenitors. The liver and spleen are both active sites of hematopoiesis in adult axolotls and contain transplantable HSCs capable of long-term multilineage blood reconstitution. As in zebrafish, use of the white axolotl mutant allows direct visualization of homing, engraftment, and hematopoiesis in real time. Donor-derived hematopoiesis occurred for >2 years in recipients generating stable hematopoietic chimeras. Organ segregation, made possible by embryonic microsurgeries wherein halves of 2 differently colored embryos were joined, indicate that the spleen is the definitive site of adult hematopoiesis.

Probability of Regenerating a Normal Limb After Bite Injury in the Mexican Axolotl (Ambystoma mexicanum).

Multiple factors are thought to cause limb abnormalities in amphibian populations by altering processes of limb development and regeneration. We examined adult and juvenile axolotls (Ambystoma mexicanum) in the Ambystoma Genetic Stock Center (AGSC) for limb and digit abnormalities to investigate the probability of normal regeneration after bite injury. We observed that 80% of larval salamanders show evidence of bite injury at the time of transition from group housing to solitary housing. Among 717 adult axolotls that were surveyed, which included solitary‐housed males and group‐housed females, approximately half presented abnormalities, including examples of extra or missing digits and limbs, fused digits, and digits growing from atypical anatomical positions. Bite injury probably explains these limb defects, and not abnormal development, because limbs with normal anatomy regenerated after performing rostral amputations. We infer that only 43% of AGSC larvae will present four anatomically normal looking adult limbs after incurring a bite injury. Our results show regeneration of normal limb anatomy to be less than perfect after bite injury.

Experimentally induced metamorphosis in axolotls reduces regenerative rate and fidelity.

While most tetrapods are unable to regenerate severed body parts, amphibians display a remarkable ability to regenerate an array of structures. Frogs can regenerate appendages as larva, but they lose this ability around metamorphosis. In contrast, salamanders regenerate appendages as larva, juveniles, and adults. However, the extent to which fundamental traits (e.g., metamorphosis, body size, aging, etc.) restrict regenerative ability remains contentious. Here we utilize the ability of normally paedomorphic adult axolotls (Ambystoma mexicanum) to undergo induced metamorphosis by thyroxine exposure to test how metamorphosis and body size affects regeneration in age‐matched paedomorphic and metamorphic individuals. We show that body size does not affect regeneration in adult axolotls, but metamorphosis causes a twofold reduction in regeneration rate, and lead to carpal and digit malformations. Furthermore, we find evidence that metamorphic blastemal cells may take longer to traverse the cell cycle and display a lower proliferative rate. This study identifies the axolotl as a powerful system to study how metamorphosis restricts regeneration independently of developmental stage, body size, and age; and more broadly how metamorphosis affects tissue‐specific changes.

Proliferation zones in the axolotl brain and regeneration of the telencephalon

BackgroundAlthough the brains of lower vertebrates are known to exhibit somewhat limited regeneration after incisional or stab wounds, the Urodele brain exhibits extensive regeneration after massive tissue removal. Discovering whether and how neural progenitor cells that reside in the ventricular zones of Urodeles proliferate to mediate tissue repair in response to injury may produce novel leads for regenerative strategies. Here we show that endogenous neural progenitor cells resident to the ventricular zone of Urodeles spontaneously proliferate, producing progeny that migrate throughout the telencephalon before terminally differentiating into neurons. These progenitor cells appear to be responsible for telencephalon regeneration after tissue removal and their activity may be up-regulated by injury through an olfactory cue.ResultsThere is extensive proliferation of endogenous neural progenitor cells throughout the ventricular zone of the adult axolotl brain. The highest levels are observed in the telencephalon, especially the dorsolateral aspect, and cerebellum. Lower levels are observed in the mesencephalon and rhombencephalon. New cells produced in the ventricular zone migrate laterally, dorsally and ventrally into the surrounding neuronal layer. After migrating from the ventricular zone, the new cells primarily express markers of neuronal differentiative fates. Large-scale telencephalic tissue removal stimulates progenitor cell proliferation in the ventricular zone of the damaged region, followed by proliferation in the tissue that surrounds the healing edges of the wound until the telencephalon has completed regeneration. The proliferative stimulus appears to reside in the olfactory system, because telencephalic regeneration does not occur in the brains of olfactory bulbectomized animals in which the damaged neural tissue simply heals over.ConclusionThere is a continual generation of neuronal cells from neural progenitor cells located within the ventricular zone of the axolotl brain. Variable rates of proliferation were detected across brain regions. These neural progenitor cells appear to mediate telencephalic tissue regeneration through an injury-induced olfactory cue. Identification of this cue is our future goal.

Skin Regeneration in Adult Axolotls: A Blueprint for Scar-Free Healing in Vertebrates

While considerable progress has been made towards understanding the complex processes and pathways that regulate human wound healing, regenerative medicine has been unable to develop therapies that coax the natural wound environment to heal scar-free. The inability to induce perfect skin regeneration stems partly from our limited understanding of how scar-free healing occurs in a natural setting. Here we have investigated the wound repair process in adult axolotls and demonstrate that they are capable of perfectly repairing full thickness excisional wounds made on the flank. In the context of mammalian wound repair, our findings reveal a substantial reduction in hemostasis, reduced neutrophil infiltration and a relatively long delay in production of new extracellular matrix (ECM) during scar-free healing. Additionally, we test the hypothesis that metamorphosis leads to scarring and instead show that terrestrial axolotls also heal scar-free, albeit at a slower rate. Analysis of newly forming dermal ECM suggests that low levels of fibronectin and high levels of tenascin-C promote regeneration in lieu of scarring. Lastly, a genetic analysis during wound healing comparing epidermis between aquatic and terrestrial axolotls suggests that matrix metalloproteinases may regulate the fibrotic response. Our findings outline a blueprint to understand the cellular and molecular mechanisms coordinating scar-free healing that will be useful towards elucidating new regenerative therapies targeting fibrosis and wound repair.