Model systems for regeneration: the spiny mouse, Acomys cahirinus.

The spiny mouse, Acomys spp., is a recently described model organism for regeneration studies. For a mammal, it displays surprising powers of regeneration because it does not fibrose (i.e. scar) in response to tissue injury as most other mammals, including humans, do. In this Primer article, we review these regenerative abilities, highlighting the phylogenetic position of the spiny mouse relative to other rodents. We also briefly describe the Acomys tissues that have been used for regeneration studies and the common features of their regeneration compared with the typical mammalian response. Finally, we discuss the contribution that Acomys has made in understanding the general principles of regeneration and elaborate hypotheses as to why this mammal is successful at regenerating.

A profusion of neural stem cells in the brain of the spiny mouse, Acomys cahirinus.

The vast majority of neural stem cell studies have been conducted on the brains of mice and rats, the classical model rodent. Non-model organisms may, however, give us some important insights into how to increase neural stem cell numbers for regenerative purposes and with this in mind we have characterized these cells in the brain of the spiny mouse, Acomys cahirinus. This unique mammal is highly regenerative and damaged tissue does not scar or fibrose. We find that there are more than three times as many stem cells in the SVZ and more than 3 times as many proliferating cells compared to the CD-1 outbred strain of lab mouse. These additional cells create thick stem cell regions in the wall of the SVZ and very obvious columns of cells moving into the rostral migratory stream. In the dentate gyrus, there are more than 10 times as many cells proliferating in the sub-granular layer and twice the number of doublecortin expressing neuroblasts. A preliminary analysis of some stem cell niche genes has identified Sox2, Notch1, Shh, and Noggin as up-regulated in the SVZ of Acomys and Bmp2 as being down-regulated. The highly increased neural stem cell numbers in Acomys may endow this animal with increased regenerative properties in the brain or improved physiological performance important for its survival.

RA Signaling in Limb Development and Regeneration in Different Species.

This chapter brings together data on the role of retinoic acid (RA) in the embryonic development of fins in zebrafish , limbs in amphibians , chicks , and mice, and regeneration of the amphibian limb . The intention is to determine whether there is a common set of principles by which we can understand the mode of action of RA in both embryos and adults. What emerges from this synthesis is that there are indeed commonalities in the involvement of RA in processes that ventralize, posteriorize, and proximalize the developing and regenerating limb . Different axes of the limb have historically been studied independently; as for example, the embryonic development of the anteroposterior (AP) axis of the chick limb bud versus the regeneration of the limb bud proximodistal (PD) axis . But when we take a broader view, a unifying principle emerges that explains why RA administration to embryos and regenerating limbs results in the development of multiple limbs in both cases. As might be expected, different molecular pathways govern the development of different systems and model organisms, but despite these differences, the pathways involve similar RA signaling genes, such as tbx5, meis, shh, fgfs and hox genes. Studies of developing and regenerating systems have highlighted that RA acts by being synthesized in one embryonic location while acting in another one, exactly as embryonic morphogens do, although there is no evidence for the presence of an RA gradient within the limb . What also emerges is that there is a paucity of information on the involvement of RA in development of the dorsoventral (DV) axis . A molecular explanation as to how RA establishes and alters positional information in all three axes is the most important area of study for the future.

Retinoic acid receptor regulation of epimorphic and homeostatic regeneration in the axolotl.

Salamanders are capable of regenerating amputated limbs by generating a mass of lineage-restricted cells called a blastema. Blastemas only generate structures distal to their origin unless treated with retinoic acid (RA), which results in proximodistal (PD) limb duplications. Little is known about the transcriptional network that regulates PD duplication. In this study, we target specific retinoic acid receptors (RARs) to either PD duplicate (RA treatment or RARγ agonist) or truncate (RARß antagonist) regenerating limbs. RARE-EGFP reporter axolotls showed divergent reporter activity in limbs undergoing PD duplication versus truncation, suggesting differences in patterning and skeletal regeneration. Transcriptomics identified expression patterns that explain PD duplication, including upregulation of proximal homeobox gene expression and silencing of distal-associated genes, whereas limb truncation was associated with disrupted skeletal differentiation. RARß antagonism in uninjured limbs induced a loss of skeletal integrity leading to long bone regression and loss of skeletal turnover. Overall, mechanisms were identified that regulate the multifaceted roles of RARs in the salamander limb including regulation of skeletal patterning during epimorphic regeneration, skeletal tissue differentiation during regeneration, and homeostatic regeneration of intact limbs.

Insights into the regeneration of skin from Acomys, the spiny mouse.

Members of the Acomys genus, known as spiny mice, are unique among mammals in being perfectly capable of regenerating large areas of skin that have been removed. During this regenerative process hairs, sebaceous glands, erector pili muscles, adipocytes and the panniculus carnosus all regenerate and the dermis does not scar. We review here the processes that the epidermis and the individual components of the dermis undergo during spiny mouse regeneration as well as the molecules that have been identified as potentially important in regeneration. We then relate this to what has been proposed as playing a role in studies from the laboratory mouse, Mus musculus. Differences in the immune systems of spiny mice and laboratory mice are also highlighted as this is suggested to play a part not only in the perfect wound healing that embryos display but also in regeneration in lower vertebrates.

Skin shedding and tissue regeneration in African spiny mice (Acomys).

Evolutionary modification has produced a spectrum of animal defence traits to escape predation, including the ability to autotomize body parts to elude capture. After autotomy, the missing part is either replaced through regeneration (for example, in urodeles, lizards, arthropods and crustaceans) or permanently lost (such as in mammals). Although most autotomy involves the loss of appendages (legs, chelipeds, antennae or tails, for example), skin autotomy can occur in certain taxa of scincid and gekkonid lizards. Here we report the first demonstration of skin autotomy in Mammalia (African spiny mice, Acomys). Mechanical testing showed a propensity for skin to tear under very low tension and the absence of a fracture plane. After skin loss, rapid wound contraction was followed by hair follicle regeneration in dorsal skin wounds. Notably, we found that regenerative capacity in Acomys was extended to ear holes, where the mice exhibited complete regeneration of hair follicles, sebaceous glands, dermis and cartilage. Salamanders capable of limb regeneration form a blastema (a mass of lineage-restricted progenitor cells) after limb loss, and our findings suggest that ear tissue regeneration in Acomys may proceed through the assembly of a similar structure. This study underscores the importance of investigating regenerative phenomena outside of conventional model organisms, and suggests that mammals may retain a higher capacity for regeneration than was previously believed. As re-emergent interest in regenerative medicine seeks to isolate molecular pathways controlling tissue regeneration in mammals, Acomys may prove useful in identifying mechanisms to promote regeneration in lieu of fibrosis and scarring.

Mapping hematopoiesis in a fully regenerative vertebrate: the axolotl.

Hematopoietic 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.

Cellular events during scar-free skin regeneration in the spiny mouse, Acomys.

In contrast to the lab mouse, Mus musculus, several species of spiny mouse, Acomys, can regenerate epidermis, dermis, hairs, sebaceous glands with smooth muscle erector pili muscles and skeletal muscle of the panniculus carnonsus after full thickness skin wounding. Here, we have compared the responses of these scarring and nonscarring organisms concentrating on the immune cells and wound cytokines, cell proliferation, and the collagenous components of the wound bed and scar. The blood of Acomys is very neutropenic but there are greater numbers of mast cells in the Acomys wound than the Mus wound. Most importantly there are no F4/80 macrophages in the Acomys wound and many proinflammatory cytokines are either absent or in very low levels which we suggest may be primarily responsible for the excellent regenerative properties of the skin of this species. There is little difference in cell proliferation in the two species either in the epidermis or mesenchymal tissues but the cell density and matrix composition of the wound is very different. In Mus there are 8 collagens which are up-regulated at least 5-fold in the wound creating a strongly trichrome-positive matrix whereas in Acomys there are very few collagens present and the matrix shows only light trichrome staining. The major component of the Mus matrix is collagen XII which is up-regulated between 10 and 30-fold after wounding. These results suggest that in the Acomys wound the absence of many cytokines resulting in the lack of macrophages is responsible for the failure to up-regulate fibrotic collagens, a situation which permits a regenerative response within the skin rather than the generation of a scar.

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.

Cellular plasticity during vertebrate appendage regeneration.

Many vertebrates have the amazing ability to regenerate all or portions of appendages including limbs, tails, fins, and digits. Unfortunately, our understanding of the cellular and molecular basis of appendage regeneration is severely lacking. However, recent technological advances that facilitate the tracking of cell lineages in vivo through space and time are allowing us to address the unknowns of regeneration, such as characterizing the cells that contribute to regeneration and identifying the tissues these cells differentiate into during regeneration. Here, we describe the experiments and the surprisingly uniform results that have emerged across diverse vertebrate species when specific cell lineages have been tracked during vertebrate appendage regeneration. These investigations show that vertebrates, from zebrafish to salamanders to mammals, utilize a limited amount of cellular plasticity to regenerate missing appendages. The universal approach to appendage regeneration is not to generate pluripotent cells that then differentiate into the new organ, but instead to generate lineage-restricted cells that are propagated in a progenitor-like state. Lessons learned from these natural cases of complex tissue regeneration might inform regenerative medicine on the best approach for re-growing complex tissues.

Identification and Analysis of Axolotl Homologs for Proteins Implicated in Human Neurodegenerative Proteinopathies.

Neurodegenerative proteinopathies such as Alzheimer's Disease are characterized by abnormal protein aggregation and neurodegeneration. Neuroresilience or regenerative strategies to prevent neurodegeneration, preserve function, or restore lost neurons may have the potential to combat human proteinopathies; however, the adult human brain possesses a limited capacity to replace lost neurons. In contrast, axolotls (Ambystoma mexicanum) show robust brain regeneration. To determine whether axolotls may help identify potential neuroresilience or regenerative strategies in humans, we first interrogated whether axolotls express putative proteins homologous to human proteins associated with neurodegenerative diseases. We compared the homology between human and axolotl proteins implicated in human proteinopathies and found that axolotls encode proteins highly similar to human microtubule-binding protein tau (tau), amyloid precursor protein (APP), and ß-secretase 1 (BACE1), which are critically involved in human proteinopathies like Alzheimer's Disease. We then tested monoclonal Tau and BACE1 antibodies previously used in human and rodent neurodegenerative disease studies using immunohistochemistry and western blotting to validate the homology for these proteins. These studies suggest that axolotls may prove useful in studying the role of these proteins in disease within the context of neuroresilience and repair.

Visualization of retinoic acid signaling in transgenic axolotls during limb development and regeneration.

Retinoic acid (RA) plays a necessary role in limb development and regeneration, but the precise mechanism by which it acts during these processes is unclear. The role of RA in limb regeneration was first highlighted by the remarkable effect that it has on respecifying the proximodistal axis of the regenerating limb so that serially repeated limbs are produced. To facilitate the study of RA signaling during development and then during regeneration of the same structure we have turned to the axolotl, the master of vertebrate regeneration, and generated transgenic animals that fluorescently report RA signaling in vivo. Characterization of these animals identified an anterior segment of the developing embryo where RA signaling occurs revealing conserved features of the early vertebrate embryo. During limb development RA signaling was present in the developing forelimb bud mesenchyme, but was not detected during hindlimb development. During limb regeneration, RA signaling was surprisingly almost exclusively observed in the apical epithelium suggesting a different role of RA during limb regeneration. After the addition of supplemental RA to regenerating limbs that leads to pattern duplications, the fibroblast stem cells of the blastema responded showing that they are capable of transcriptionally responding to RA. These findings are significant because it means that RA signaling may play a multifunctional role during forelimb development and regeneration and that the fibroblast stem cells that regulate proximodistal limb patterning during regeneration are targets of RA signaling.

Salamanders as Key Models for Development and Regeneration Research.

For 70 years from the very beginning of developmental biology, the salamander embryo was the pre-eminent model for these studies. Here I review the major discoveries that were made using salamander embryos including regionalization of the mesoderm; patterning of the neural plate; limb development, with the pinnacle being Spemann's Nobel Prize for the discovery of the organizer; and the phenomenon of induction. Salamanders have also been the major organism for elucidating discoveries in organ regeneration, and these are described here too beginning with Spallanzani's experiments in 1768. These include the neurotrophic hypothesis of regeneration, studies of aneurogenic limbs, the concept of dedifferentiation and transdifferentiation, and advances in understanding pattern formation via molecules located on the cell surface. Also described is the prodigious power of brain and spinal cord regeneration and discoveries from lens regeneration, all of which reveal how important salamanders have been as research models.

Retinoic Acid-Induced Limb Duplications.

Retinoic acid (RA) and the family of molecules based on vitamin A known as retinoids have remarkable effects on limb regeneration in salamanders and newts and cause whole limb duplications in a concentration-dependent manner. They respecify all three axes of the limb-the proximodistal, the anteroposterior, and the dorsoventral axis. As a result, complete limbs can be induced to regenerate from distal amputation planes producing two limbs in tandem. Here, we describe the basic methods for undertaking these experiments as well as the use of new synthetic retinoids which have retinoic acid receptor-selective actions. These will be valuable tools in future studies on the molecular basis of limb duplications and thus our understanding of the nature of positional information in the regenerating salamander limb.

African Spiny Mice (Acomys) Exhibit Mild Osteoarthritis Following Meniscal Injury.

OBJECTIVE: Hyaline cartilage has limited innate healing abilities and hyaline cartilage loss is a hallmark of osteoarthritis (OA). Animal models can provide important insights into cartilage regeneration potential. One such animal model, the African spiny mouse (Acomys), is capable of regenerating skin, skeletal muscle, and elastic cartilage. This study aims to evaluate whether these regenerative abilities protect Acomys with meniscal injury from OA-related joint damage and behaviors indicative of joint pain and dysfunction. DESIGN: Acomys received destabilization of the medial meniscus (DMM) surgery (n = 11) or a skin incision (n = 10). Gait testing occurred at 4, 6, 8, 10, and 12 weeks after surgery. At endpoint, joints were processed for histology to assess cartilage damage. RESULTS: Following joint injury, Acomys with DMM surgery altered their walking patterns by increasing the percent stance time on the contralateral limb relative to the operated limb, thereby reducing the amount of time the injured limb must bear weight on its own throughout the gait cycle. Histological grading indicated evidence of OA-related joint damage in Acomys with DMM surgery; these changes were primarily driven by loss of structural integrity in the hyaline cartilage. CONCLUSIONS: Acomys developed gait compensations, and the hyaline cartilage in Acomys is not fully protected from OA-related joint damage following meniscal injury, although this damage was less severe than that historically found in C57BL/6 mice with an identical injury. Thus, Acomys do not appear to be completely protected from OA-related changes, despite the ability to regenerate other wounded tissues.

Osteoderms in a mammal the spiny mouse Acomys and the independent evolution of dermal armor.

Osteoderms are bony plates found in the skin of vertebrates, mostly commonly in reptiles where they have evolved independently multiple times, suggesting the presence of a gene regulatory network that is readily activated and inactivated. They are absent in birds and mammals except for the armadillo. However, we have discovered that in one subfamily of rodents, the Deomyinae, there are osteoderms in the skin of their tails. Osteoderm development begins in the proximal tail skin and is complete 6 weeks after birth. RNA sequencing has identified the gene networks involved in their differentiation. There is a widespread down-regulation of keratin genes and an up-regulation of osteoblast genes and a finely balanced expression of signaling pathways as the osteoderms differentiate. Future comparisons with reptilian osteoderms may allow us to understand how these structures have evolved and why they are so rare in mammals.

Bite Wounds and Dominance Structures in Male and Female African Spiny Mice (Acomys cahirinus): Implications for Animal Welfare and the Generalizability of Experimental Results.

Bite wounds due to aggression in male laboratory mice (Mus musculus) are a major welfare concern, often leading to attrition, chronic activation of the innate immune system, and significant impacts on the experimental results derived from the use of these animals as models. Bite wounding within the home-cage of spiny mice (Acomys cahirinus)-a valuable research model for wound healing and menstruation-is poorly characterized. While we have anecdotally observed frequent bite wounding in Acomys, the frequency of aggression within the home-cage, the severity of the bite wounds, and the types of dominance structures remain unstudied. Here, we report that 46% of Acomys cages in our colony had at least one bite wound over the course of a year and that same-sex pairs fought in the home-cage 10% of the time during their dark/active phase. Both sexes inflicted wounds and frequently engaged in agonistic behaviors, even with stable dominance structures. We found that females inflicted less severe bite wounds in same-sex housing. Also, aged females in same-sex pairs were never observed fighting, and no bite wounds were observed in aged Acomys. These results suggest that we should consider whether bite wounding negatively impacts our experimental results since physical trauma is known to alter menstrual cycling and healing.

Retinoic acid in the development, regeneration and maintenance of the nervous system.

Retinoic acid (RA) is involved in the induction of neural differentiation, motor axon outgrowth and neural patterning. Like other developmental molecules, RA continues to play a role after development has been completed. Elevated RA signalling in the adult triggers axon outgrowth and, consequently, nerve regeneration. RA is also involved in the maintenance of the differentiated state of adult neurons, and disruption of RA signalling in the adult leads to the degeneration of motor neurons (motor neuron disease), the development of Alzheimer's disease and, possibly, the development of Parkinson's disease. The data described here strongly suggest that RA could be used as a therapeutic molecule for the induction of axon regeneration and the treatment of neurodegeneration.

Tumor suppressors inhibit reprogramming of African spiny mouse ( Acomys) fibroblasts to induced pluripotent stem cells.

Background: The African spiny mouse ( Acomys) is an emerging mammalian model for scar-free regeneration, and further study of Acomys could advance the field of regenerative medicine. Isolation of pluripotent stem cells from Acomys would allow for development of transgenic or chimeric animals and in vitro study of regeneration; however, the reproductive biology of Acomys is not well characterized, complicating efforts to derive embryonic stem cells. Thus, we sought to generate Acomys induced pluripotent stem cells (iPSCs) by reprogramming somatic cells back to pluripotency. Methods: To generate Acomys iPSCs, we attempted to adapt established protocols developed in Mus. We utilized a PiggyBac transposon system to genetically modify Acomys fibroblasts to overexpress the Yamanaka reprogramming factors as well as mOrange fluorescent protein under the control of a doxycycline-inducible TetON operon system. Results: Reprogramming factor overexpression caused Acomys fibroblasts to undergo apoptosis or senescence. When SV40 Large T antigen (SV40 LT) was added to the reprogramming cocktail, Acomys cells were able to dedifferentiate into pre-iPSCs. Although use of 2iL culture conditions induced formation of colonies resembling Mus PSCs, these Acomys iPS-like cells lacked pluripotency marker expression and failed to form embryoid bodies. An EOS-GiP system was unsuccessful in selecting for bona fide Acomys iPSCs; however, inclusion of Nanog in the reprogramming cocktail along with 5-azacytidine in the culture medium allowed for generation of Acomys iPSC-like cells with increased expression of several naïve pluripotency markers. Conclusions: There are significant roadblocks to reprogramming Acomys cells, necessitating future studies to determine Acomys-specific reprogramming factor and/or culture condition requirements. The requirement for SV40 LT during Acomys dedifferentiation may suggest that tumor suppressor pathways play an important role in Acomys regeneration and that Acomys may possess unreported cancer resistance.

Retinoic Acid Receptors and the Control of Positional Information in the Regenerating Axolotl Limb.

We know little about the control of positional information (PI) during axolotl limb regeneration, which ensures that the limb regenerates exactly what was amputated, and the work reported here investigates this phenomenon. Retinoic acid administration changes the PI in a proximal direction so that a complete limb can be regenerated from a hand. Rather than identifying all the genes altered by RA treatment of the limb, we have eliminated many off-target effects by using retinoic acid receptor selective agonists. We firstly identify the receptor involved in this respecification process as RARα and secondly, identify the genes involved by RNA sequencing of the RARα-treated blastemal mesenchyme. We find 1177 upregulated genes and 1403 downregulated genes, which could be identified using the axolotl genome. These include several genes known to be involved in retinoic acid metabolism and in patterning. Since positional information is thought to be a property of the cell surface of blastemal cells when we examine our dataset with an emphasis on this aspect, we find the top canonical pathway is integrin signaling. In the extracellular matrix compartment, we find a MMP and several collagens are upregulated; several cell membrane genes and secretory factors are also upregulated. This provides data for future testing of the function of these candidates in the control of PI during limb regeneration.