Making a new limb out of old cells: exploring endogenous cell reprogramming and its role during limb regeneration.

Cellular reprogramming is characterized by the induced dedifferentiation of mature cells into a more plastic and potent state. This process can occur through artificial reprogramming manipulations in the laboratory such as nuclear reprogramming and induced pluripotent stem cell (iPSC) generation, and endogenously in vivo during amphibian limb regeneration. In amphibians such as the Mexican axolotl, a regeneration permissive environment is formed by nerve-dependent signaling in the wounded limb tissue. When exposed to these signals, limb connective tissue cells dedifferentiate into a limb progenitor-like state. This state allows the cells to acquire new pattern information, a property called positional plasticity. Here, we review our current understanding of endogenous reprogramming and why it is important for successful regeneration. We will also explore how naturally induced dedifferentiation and plasticity were leveraged to study how the missing pattern is established in the regenerating limb tissue.

Broad host susceptibility of North American amphibian species to Batrachochytrium salamandrivorans suggests high invasion potential and biodiversity risk.

Batrachochytrium salamandrivorans (Bsal) is a fungal pathogen of amphibians that is emerging in Europe and could be introduced to North America through international trade or other pathways. To evaluate the risk of Bsal invasion to amphibian biodiversity, we performed dose-response experiments on 35 North American species from 10 families, including larvae from five species. We discovered that Bsal caused infection in 74% and mortality in 35% of species tested. Both salamanders and frogs became infected and developed Bsal chytridiomycosis. Based on our host susceptibility results, environmental suitability conditions for Bsal, and geographic ranges of salamanders in the United States, predicted biodiversity loss is expected to be greatest in the Appalachian Region and along the West Coast. Indices of infection and disease susceptibility suggest that North American amphibian species span a spectrum of vulnerability to Bsal chytridiomycosis and most amphibian communities will include an assemblage of resistant, carrier, and amplification species. Predicted salamander losses could exceed 80 species in the United States and 140 species in North America.

Integration failure of regenerated limb tissue is associated with incongruencies in positional information in the Mexican axolotl.

Introduction: Little is known about how the newly regenerated limb tissues in the Mexican axolotl seamlessly integrate with the remaining stump tissues to form a functional structure, and why this doesn't occur in some regenerative scenarios. In this study, we evaluate the phenomenological and transcriptional characteristics associated with integration failure in ectopic limb structures generated by treating anterior-located ectopic blastemas with Retinoic Acid (RA) and focusing on the "bulbus mass" tissue that forms between the ectopic limb and the host site. We additionally test the hypothesis that the posterior portion of the limb base contains anterior positional identities. Methods: The positional identity of the bulbus mass was evaluated by assaying regenerative competency, the ability to induce new pattern in the Accessory Limb Model (ALM) assay, and by using qRTPCR to quantify the relative expression of patterning genes as the bulbus mass deintegrates from the host site. We additionally use the ALM and qRTPCR to analyze the distribution of anterior and posterior positional identities along the proximal/distal limb axis of uninjured and regenerating limbs. Results: The bulbus mass regenerates limb structures with decreased complexity when amputated and is able to induce complex ectopic limb structure only when grafted into posterior-located ALMs. Expressional analysis shows significant differences in FGF8, BMP2, TBX5, Chrdl1, HoxA9, and HoxA11 expression between the bulbus mass and the host site when deintegration is occuring. Grafts of posterior skin from the distal limb regions into posterior ALMs at the base of the limb induce ectopic limb structures. Proximally-located blastemas express significantly less HoxA13 and Ptch1, and significantly more Alx4 and Grem1 than distally located blastemas. Discussion: These findings show that the bulbus mass has an anterior-limb identity and that the expression of limb patterning genes is mismatched between the bulbus mass and the host limb. Our findings additionally show that anterior positional information is more abundant at the limb base, and that anterior patterning genes are more abundantly expressed in proximally located blastemas compared to blastemas in the more distal regions of the limb. These experiments provide valuable insight into the underlying causes of integration failure and further map the distribution of positional identities in the mature limb.

The Accessory Limb Model Regenerative Assay and Its Derivatives.

When the Accessory Limb Model (ALM) regenerative assay was first published by Endo, Bryant, and Gardiner in 2004, it provided a robust system for testing the cellular and molecular contributions during each of the basic steps of regeneration: the formation of the wound epithelium, neural induction of the apical epithelial cap, and the formation of a positional disparity between blastema cells. The basic ALM procedure was developed in the axolotl and involves deviating a limb nerve into a lateral wound and grafting skin from the opposing side of the limb axis into the site of injury. In this chapter, we will review the studies that lead to the conception of the ALM, as well as the studies that have followed the development of this assay. We will additionally describe in detail the standard ALM surgery and how to perform this surgery on different limb positions.

A constitutively expressed fluorescent ubiquitination-based cell-cycle indicator (FUCCI) in axolotls for studying tissue regeneration.

Regulation of cell cycle progression is essential for cell proliferation during regeneration following injury. After appendage amputation, the axolotl (Ambystoma mexicanum) regenerates missing structures through an accumulation of proliferating cells known as the blastema. To study cell division during blastema growth, we generated a transgenic line of axolotls that ubiquitously expresses a bicistronic version of the fluorescent ubiquitination-based cell-cycle indicator (FUCCI). We demonstrate near-ubiquitous FUCCI expression in developing and adult tissues, and validate these expression patterns with DNA synthesis and mitosis phase markers. We demonstrate the utility of FUCCI for live and whole-mount imaging, showing the predominantly local contribution of cells during limb and tail regeneration. We also show that spinal cord amputation results in increased proliferation at least 5 mm from the site of injury. Finally, we use multimodal staining to provide cell type information for cycling cells by combining fluorescence in situ hybridization, EdU click-chemistry and immunohistochemistry on a single FUCCI tissue section. This new line of animals will be useful for studying cell cycle dynamics using in situ endpoint assays and in vivo imaging in developing and regenerating animals.

Neural control of growth and size in the axolotl limb regenerate.

The mechanisms that regulate growth and size of the regenerating limb in tetrapods such as the Mexican axolotl are unknown. Upon the completion of the developmental stages of regeneration, when the regenerative organ known as the blastema completes patterning and differentiation, the limb regenerate is proportionally small in size. It then undergoes a phase of regeneration that we have called the 'tiny-limb' stage, which is defined by rapid growth until the regenerate reaches the proportionally appropriate size. In the current study we have characterized this growth and have found that signaling from the limb nerves is required for its maintenance. Using the regenerative assay known as the accessory limb model (ALM), we have found that growth and size of the limb positively correlates with nerve abundance. We have additionally developed a new regenerative assay called the neural modified-ALM (NM-ALM), which decouples the source of the nerves from the regenerating host environment. Using the NM-ALM we discovered that non-neural extrinsic factors from differently sized host animals do not play a prominent role in determining the size of the regenerating limb. We have also discovered that the regulation of limb size is not autonomously regulated by the limb nerves. Together, these observations show that the limb nerves provide essential cues to regulate ontogenetic allometric growth and the final size of the regenerating limb.

Humans' ability to regrow lost or damaged body parts is relatively limited, but some animals, such as the axolotl (a Mexican salamander), can regenerate complex body parts, like legs, many times over their lives. Studying regeneration in these animals could help researchers enhance humans' abilities to heal. One way to do this is using the Accessory Limb Model (ALM), where scientists wound an axolotl's leg, and study the additional leg that grows from the wound. The first stage of limb regeneration creates a new leg that has the right structure and shape. The new leg is very small so the next phase involves growing the leg until its size matches the rest of the animal. This phase must be controlled so that the limb stops growing when it reaches the right size, but how this regulation works is unclear. Previous research suggests that the number of nerves in the new leg could be important. Wells et al. used a ALM to study how the size of regenerating limbs is controlled. They found that changing the number of nerves connected to the new leg altered its size, with more nerves leading to a larger leg. Next, Wells et al. created a system that used transplanted nerve bundles of different sizes to grow new legs in different sized axolotls. This showed that the size of the resulting leg is controlled by the number of nerves connecting it to the CNS. Wells et al. also showed that nerves can only control regeneration if they remain connected to the central nervous system. These results explain how size is controlled during limb regeneration in axolotls, highlighting the fact that regrowth is directly controlled by the number of nerves connected to a regenerating leg. Much more work is needed to reveal the details of this process and the signals nerves use to control growth. It will also be important to determine whether this control system is exclusive to axolotls, or whether other animals also use it.

ECM-mediated positional cues are able to induce pattern, but not new positional information, during axolotl limb regeneration.

The Mexican Axolotl is able to regenerate missing limb structures in any position along the limb axis throughout its life and serves as an excellent model to understand the basic mechanisms of endogenous regeneration. How the new pattern of the regenerating axolotl limb is established has not been completely resolved. An accumulating body of evidence indicates that pattern formation occurs in a hierarchical fashion, which consists of two different types of positional communications. The first type (Type 1) of communication occurs between connective tissue cells, which retain memory of their original pattern information and use this memory to generate the pattern of the regenerate. The second type (Type 2) of communication occurs from connective tissue cells to other cell types in the regenerate, which don't retain positional memory themselves and arrange themselves according to these positional cues. Previous studies suggest that molecules within the extracellular matrix (ECM) participate in pattern formation in developing and regenerating limbs. However, it is unclear whether these molecules play a role in Type 1 or Type 2 positional communications. Utilizing the Accessory Limb Model, a regenerative assay, and transcriptomic analyses in regenerates that have been reprogrammed by treatment with Retinoic Acid, our data indicates that the ECM likely facilities Type-2 positional communications during limb regeneration.

The Regulation of Growth in Developing, Homeostatic, and Regenerating Tetrapod Limbs: A Minireview.

The size and shape of the tetrapod limb play central roles in their functionality and the overall physiology of the organism. In this minireview we will discuss observations on mutant animal models and humans, which show that the growth and final size of the limb is most impacted by factors that regulate either limb bud patterning or the elongation of the long bones. We will also apply the lessons that have been learned from embryos to how growth could be regulated in regenerating limb structures and outline the challenges that are unique to regenerating animals.

Characterizing the regenerative capacity and growth patterns of the Texas blind salamander (Eurycea rathbuni).

BACKGROUND: Regeneration of complex patterned structures is well described among, although limited to a small sampling of, amphibians. This limitation impedes our understanding of the full range of regenerative competencies within this class of vertebrates, according to phylogeny, developmental life stage, and age. To broaden the phylogenetic breath of this research, we characterized the regenerative capacity of the Texas blind salamander (Eurycea rathbuni), a protected salamander native to the Edwards Aquifer of San Marcos, Texas and colonized by the San Marcos Aquatic Resource Center. As field observations suggested regenerative abilities in this population, the forelimb stump of a live captured female was amputated in the hopes of restoring the structure, and thus locomotion in the animal. Tails were clipped from two males to additionally document tail regeneration. RESULTS: We show that the Texas blind salamander exhibits robust limb and tail regeneration, like all other studied Plethodontidae. Regeneration in this species is associated with wound epithelium formation, blastema formation, and subsequent patterning and differentiation of the regenerate. CONCLUSIONS: The study has shown that the Texas blind salamander is a valuable model to study regenerative processes, and that therapeutic surgeries offer a valuable means to help maintain and conserve this vulnerable species.

Advancements to the Axolotl Model for Regeneration and Aging.

Loss of regenerative capacity is a normal part of aging. However, some organisms, such as the Mexican axolotl, retain striking regenerative capacity throughout their lives. Moreover, the development of age-related diseases is rare in this organism. In this review, we will explore how axolotls are used as a model system to study regenerative processes, the exciting new technological advancements now available for this model, and how we can apply the lessons we learn from studying regeneration in the axolotl to understand, and potentially treat, age-related decline in humans.

Hierarchical pattern formation during amphibian limb regeneration.

In 1901 T.H. Morgan proposed in "Regeneration" that pattern formation in amphibian limb regeneration is a stepwise process. Since, biologist have continued to piece together the molecular components of this process to better understand the "patterning code" responsible for regenerate formation. Within this context, several different models have been proposed; however, all are based on one of two underlying hypotheses. The first is the "morphogen hypothesis" that dictates that pattern emerges from localized expression of signaling molecules, which produce differing position-specific cellular responses in receptive cells depending on the intensity of the signal. The second hypothesis is that cells in the remaining tissues retain memory of their patterning information, and use this information to generate new cells with the missing positional identities. A growing body of evidence supports the possibility that these two mechanisms are not mutually exclusive. Here, we propose our theory of hierarchical pattern formation, which consists of 4 basic steps. The first is the existence of cells with positional memory. The second is the communication of positional information through cell-cell interactions in a regeneration-permissive environment. The third step is the induction of molecular signaling centers. And the last step is the interpretation of these signals by specialized cell types to ultimately restore the limb in its entirety. Biological codes are intertwined throughout this model, and we will discuss their multiple roles and mechanisms.

FGF, BMP, and RA signaling are sufficient for the induction of complete limb regeneration from non-regenerating wounds on Ambystoma mexicanum limbs.

Some organisms, such as the Mexican axolotl, have the capacity to regenerate complicated biological structures throughout their lives. Which molecular pathways are sufficient to induce a complete endogenous regenerative response in injured tissue is an important question that remains unanswered. Using a gain-of-function regeneration assay, known as the Accessory Limb Model (ALM), we and others have begun to identify the molecular underpinnings of the three essential requirements for limb regeneration; wounding, neurotrophic signaling, and the induction of pattern from cells that retain positional memory. We have previously shown that treatment of Mexican axolotls with exogenous retinoic acid (RA) is sufficient to induce the formation of complete limb structures from blastemas that were generated by deviating a nerve bundle into an anterior-located wound site on the limb. Here we show that these ectopic structures are capable of regenerating and inducing new pattern to form when grafted into new anterior-located wounds. We additionally found that the expression of Alx4 decreases, and Shh expression increases in these anterior located blastemas, but not in the mature anterior tissues, supporting the hypothesis that RA treatment posteriorizes blastema tissue. Based on these and previous observations, we used the ALM assay to test the hypothesis that a complete regenerative response can be generated by treating anterior-located superficial limb wounds with a specific combination of growth factors at defined developmental stages. Our data shows that limb wounds that are first treated with a combination of FGF-2, FGF-8, and BMP-2, followed by RA treatment of the resultant mid-bud stage blastema, will result in the generation of limbs with complete proximal/distal and anterior/posterior limb axes. Thus, the minimal signaling requirements from the nerve and a positional disparity are achieved with the application of this specific combination of signaling molecules.

Regenerative Models for the Integration and Regeneration of Head Skeletal Tissues.

Disease of, or trauma to, the human jaw account for thousands of reconstructive surgeries performed every year. One of the most popular and successful treatment options in this context involves the transplantation of bone tissue from a different anatomical region into the affected jaw. Although, this method has been largely successful, the integration of the new bone into the existing bone is often imperfect, and the integration of the host soft tissues with the transplanted bone can be inconsistent, resulting in impaired function. Unlike humans, several vertebrate species, including fish and amphibians, demonstrate remarkable regenerative capabilities in response to jaw injury. Therefore, with the objective of identifying biological targets to promote and engineer improved outcomes in the context of jaw reconstructive surgery, we explore, compare and contrast the natural mechanisms of endogenous jaw and limb repair and regeneration in regenerative model organisms. We focus on the role of different cell types as they contribute to the regenerating structure; how mature cells acquire plasticity in vivo; the role of positional information in pattern formation and tissue integration, and limitations to endogenous regenerative and repair mechanisms.

Exogenous Vitamin D signaling alters skeletal patterning, differentiation, and tissue integration during limb regeneration in the axolotl.

Urodele amphibians such as the axolotl regenerate complete limbs as adults, and understanding how the "blueprint", or pattern, of the regenerate is established and manipulated are areas of intense interest. Nutrient signaling plays an important role in pattern formation during regeneration. Retinoic acid signaling is the most characterized pathway during this process. Exogenous retinoic acid (RA) reprograms the pattern information in regenerating cells to a more posterior, ventral, and proximal identity. Vitamin D signaling shares several molecular similarities with RA and has been shown to alter pattern formation during zebrafish pectoral fin regeneration. To determine if exogenous Vitamin D signaling is capable of reprograming pattern in the axolotl limb blastema, we treated regenerating limbs with a potent Vitamin D agonist. Under the studied conditions, exogenous Vitamin D did not act in a manner similar to RA and failed to proximalize the pattern of the resulting regenerates. The Vitamin D treatment did result in several skeletal defects during regeneration, including carpal fusions along the A/P axis; failure to integrate the newly regenerated tissue with the existing tissue, formation of ectopic nodules of cartilage at the site of amputation, and altered bone morphology in uninjured skeletal tissue.

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.

Histological image data of limb skeletal tissue from larval and adult Ambystoma mexicanum.

The data presented in this article are related to the article entitled "Cartilage and bone cells do not participate in skeletal regeneration in Ambystoma mexicanum limbs" [1]. Here we present image data of the post-embryonic development of the forelimb skeletal tissue of Ambystoma Mexicanum. Histological staining was performed on sections from the intact limbs of young (6.5 cm) and old (25 cm) animals, and on dissected skeletal tissues (cartilage, bone, and periosteum) from these animals.

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.

Positional plasticity in regenerating Amybstoma mexicanum limbs is associated with cell proliferation and pathways of cellular differentiation.

BACKGROUND: The endogenous ability to dedifferentiate, re-pattern, and re-differentiate adult cells to repair or replace damaged or missing structures is exclusive to only a few tetrapod species. The Mexican axolotl is one example of these species, having the capacity to regenerate multiple adult structures including their limbs by generating a group of progenitor cells, known as the blastema, which acquire pattern and differentiate into the missing tissues. The formation of a limb regenerate is dependent on cells in the connective tissues that retain memory of their original position in the limb, and use this information to generate the pattern of the missing structure. Observations from recent and historic studies suggest that blastema cells vary in their potential to pattern distal structures during the regeneration process; some cells are plastic and can be reprogrammed to obtain new positional information while others are stable. Our previous studies showed that positional information has temporal and spatial components of variation; early bud (EB) and apical late bud (LB) blastema cells are plastic while basal-LB cells are stable. To identify the potential cellular and molecular basis of this variation, we compared these three cell populations using histological and transcriptional approaches. RESULTS: Histologically, the basal-LB sample showed greater tissue organization than the EB and apical-LB samples. We also observed that cell proliferation was more abundant in EB and apical-LB tissue when compared to basal-LB and mature stump tissue. Lastly, we found that genes associated with cellular differentiation were expressed more highly in the basal-LB samples. CONCLUSIONS: Our results characterize histological and transcriptional differences between EB and apical-LB tissue compared to basal-LB tissue. Combined with our results from a previous study, we hypothesize that the stability of positional information is associated with tissue organization, cell proliferation, and pathways of cellular differentiation.

Principles and mechanisms of regeneration in the mouse model for wound-induced hair follicle neogenesis.

Wound induced hair follicle neogenesis (WIHN) describes a regenerative phenomenon in adult mammalian skin, wherein fully functional hair follicles regenerate de novo in the center of large excisional wounds. Originally described in rats, rabbits, sheep, and humans in 1940-60, the WIHN phenomenon was reinvestigated in mice only recently. The process of de novo hair regeneration largely duplicates the morphological and signaling features of normal embryonic hair development. Similar to hair development, WIHN critically depends on the activation of canonical WNT signaling. However, unlike hair development, WNT activation in WIHN is dependent on Fgf9 signaling generated by the immune system's gamma delta (γδ) T cells. The cellular bases of WIHN remain to be fully characterized, however, the available evidence leaves open the possibility for a blastema-like mechanism, wherein epidermal and/or dermal wound cells undergo epigenetic reprogramming toward a more plastic, embryonic-like state. De novo hair follicles do not regenerate from preexisting hair-fated bulge stem cells. This suggests that hair neogenesis is not driven by preexisting lineage-restricted progenitors, as is the case for amputation-induced mouse digit tip regeneration, but rather may require a blastema-like mechanism. The WIHN model is characterized by several intriguing features, which await further explanation. These include: (i) minimum wound size requirement for activating neogenesis, (ii) restriction of hair neogenesis to the wound's center, (iii) imperfect patterning outcomes, both in terms of neogenic hair positioning within the wound and in terms of their orientation. Future inquires into the WIHN process, made possible by a wide array of the available skin-specific genetic tools, will undoubtedly expand our understanding of the regeneration mechanisms in adult mammals.

Understanding positional cues in salamander limb regeneration: implications for optimizing cell-based regenerative therapies.

Regenerative medicine has reached the point where we are performing clinical trials with stem-cell-derived cell populations in an effort to treat numerous human pathologies. However, many of these efforts have been challenged by the inability of the engrafted populations to properly integrate into the host environment to make a functional biological unit. It is apparent that we must understand the basic biology of tissue integration in order to apply these principles to the development of regenerative therapies in humans. Studying tissue integration in model organisms, where the process of integration between the newly regenerated tissues and the 'old' existing structures can be observed and manipulated, can provide valuable insights. Embryonic and adult cells have a memory of their original position, and this positional information can modify surrounding tissues and drive the formation of new structures. In this Review, we discuss the positional interactions that control the ability of grafted cells to integrate into existing tissues during the process of salamander limb regeneration, and discuss how these insights could explain the integration defects observed in current cell-based regenerative therapies. Additionally, we describe potential molecular tools that can be used to manipulate the positional information in grafted cell populations, and to promote the communication of positional cues in the host environment to facilitate the integration of engrafted cells. Lastly, we explain how studying positional information in current cell-based therapies and in regenerating limbs could provide key insights to improve the integration of cell-based regenerative therapies in the future.