Regeneration: sooner rather than later.

The explosive growth of information from genetics and genomics has led to an appreciation of the conservation of gene regulatory networks between organisms, and between development and regeneration. With ever increasing knowledge, it will be possible to intervene therapeutically to regulate these networks, which will lead to new therapies to induce regeneration. The question then becomes how to do this, rather then when to try. Our thesis is that the time is now, and that this feat can be achieved by combining the insights provided by developmental biologists with the technologies being developed by biomaterial engineers, to achieve the goal of engineering regeneration. We thus envision regenerative engineering as the next step toward achieving the goal of human regeneration. Among the most important discoveries about regeneration from studies of salamanders that regenerate exceptionally well, is that both pattern-following and pattern-forming cells are required. Much progress is being made toward understanding the former cells, but little is known about the cells that control positional information and pattern formation. Within the near future, it will become possible to provide the information needed for regeneration exogenously in the form of an engineered extracellular matrix that is a biomimetic of the endogenous information. Since growth factors (morphogens) can control pattern formation, an engineered grid could be based on spatially organized patterns of sulfation of glycosaminoglycans that control the behavior of cells by modulating morphogen activity. Progress in engineering the positional information grid for regeneration will necessitate learning the sulfation codes associated with successful regeneration in animals such as salamanders.

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

PURPOSE OF REVIEW: 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? RECENT FINDINGS: 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. SUMMARY: 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.

Regulation of regeneration by Heparan Sulfate Proteoglycans in the Extracellular Matrix.

Just as the building of a house requires a blueprint, the rebuilding of lost or damaged body parts through regeneration requires a set of instructions for the assembly of the various tissues into the right places. Much progress has been made in understanding how to control the differentiation of different cell types to provide the building blocks for regeneration, such as bone, muscle, blood vessels and nerves/Schwann cells. These are the cells that follow the blueprint (the pattern-following cells) and end up in the right places relative to each other in order to restore the lost function. Much less is known about the cells that are specialized to generate and regenerate the blueprint (the pattern-forming cells) in order to instruct the pattern-following cells as to how and where to rebuild the structures. Recent studies provide evidence that the pattern-forming cells synthesize an information-rich extracellular matrix (ECM) that controls the behavior of pattern-following cells leading to the regeneration of limb structures. The ability of the ECM to do this is associated with glycosaminoglycans that have specific spatial and temporal modifications of sulfation patterns. This mechanism for controlling pattern formation appears to be conserved between salamanders and mammals, and thus the next challenge for inducing human regeneration is to identify and understand the biology of these pattern-forming cells and the ECM that they synthesize.

The relationship between growth and pattern formation.

Successful development depends on the creation of spatial gradients of transcription factors within developing fields, and images of graded distributions of gene products populate the pages of developmental biology journals. Therefore the challenge is to understand how the graded levels of intracellular transcription factors are generated across fields of cells. We propose that transcription factor gradients are generated as a result of an underlying gradient of cell cycle lengths. Very long cell cycles will permit accumulation of a high level of a gene product encoded by a large transcription unit, whereas shorter cell cycles will permit progressively fewer transcripts to be completed due to gating of transcription by the cell cycle. We also propose that the gradients of cell cycle lengths are generated by gradients of extracellular morphogens/growth factors. The model of cell cycle gated transcriptional regulation brings focus back to the functional role of morphogens as cell cycle regulators, and proposes a specific and testable mechanism by which morphogens, in their roles as growth factors (how they were originally discovered), also determine cell fate.

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.

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.

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.

The accessory limb model: an alternative experimental system of limb regeneration.

Accessory limb model (ALM) was developed as an experimental model and functional assay for limb regeneration. The ALM provides several ways to identify pathways and test for signaling molecules that regulate limb regeneration. Here, we summarize the history of the ALM and describe the specific details involved in inducing ectopic blastemas and limbs from a skin wound on the side of the arm.

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.

The axolotl limb blastema: cellular and molecular mechanisms driving blastema formation and limb regeneration in tetrapods.

The axolotl is one of the few tetrapods that are capable of regenerating complicated biological structures, such as complete limbs, throughout adulthood. Upon injury the axolotl generates a population of regeneration-competent limb progenitor cells known as the blastema, which will grow, establish pattern, and differentiate into the missing limb structures. In this review we focus on the crucial early events that occur during wound healing, the neural-epithelial interactions that drive the formation of the early blastema, and how these mechanisms differ from those of other species that have restricted regenerative potential, such as humans. We also discuss how the presence of cells from the different axes of the limb is required for the continued growth and establishment of pattern in the blastema as described in the polar coordinate model, and how this positional information is reprogrammed in blastema cells during regeneration. Multiple cell types from the mature limb stump contribute to the blastema at different stages of regeneration, and we discuss the contribution of these types to the regenerate with reference to whether they are "pattern-forming" or "pattern-following" cells. Lastly, we explain how an engineering approach will help resolve unanswered questions in limb regeneration, with the goal of translating these concepts to developing better human regenerative therapies.

Positional information in axolotl and mouse limb extracellular matrix is mediated via heparan sulfate and fibroblast growth factor during limb regeneration in the axolotl (Ambystoma mexicanum).

Urodele amphibians are unique among adult vertebrates in their ability to regenerate complex body structures after traumatic injury. In salamander regeneration, the cells maintain a memory of their original position and use this positional information to recreate the missing pattern. We used an in vivo gain-of-function assay to determine whether components of the extracellular matrix (ECM) have positional information required to induce formation of new limb pattern during regeneration. We discovered that salamander limb ECM has a position-specific ability to either inhibit regeneration or induce de novo limb structure, and that this difference is dependent on heparan sulfates that are associated with differential expression of heparan sulfate sulfotransferases. We also discovered that an artificial ECM containing only heparan sulfate was sufficient to induce de novo limb pattern in salamander limb regeneration. Finally, ECM from mouse limbs is capable of inducing limb pattern in axolotl blastemas in a position-specific, developmental-stage-specific, and heparan sulfate-dependent manner. This study demonstrates a mechanism for positional information in regeneration and establishes a crucial functional link between salamander regeneration and 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.

Characterization of in vitro transcriptional responses of dorsal root ganglia cultured in the presence and absence of blastema cells from regenerating salamander limbs.

During salamander limb regeneration, nerves provide signals that induce the formation of a mass of proliferative cells called the blastema. To better understand these signals, we developed a blastema-dorsal root ganglia (DRG) co-culture model system to test the hypothesis that nerves differentially express genes in response to cues provided by the blastema. DRG with proximal and distal nerve trunks were isolated from axolotls (Ambystoma mexicanum), cultured for five days, and subjected to microarray analysis. Relative to freshly isolated DRG, 1,541 Affymetrix probe sets were identified as differentially expressed and many of the predicted genes are known to function in injury and neurodevelopmental responses observed for mammalian DRG. We then cultured 5-day DRG explants for an additional five days with or without co-cultured blastema cells. On Day 10, we identified 27 genes whose expression in cultured DRG was significantly affected by the presence or absence of blastema cells. Overall, our study established a DRG-blastema in vitro culture system and identified candidate genes for future investigations of axon regrowth, nerve-blastema signaling, and neural regulation of limb regeneration.

Positional information is reprogrammed in blastema cells of the regenerating limb of the axolotl (Ambystoma mexicanum).

The regenerating region of an amputated salamander limb, known as the blastema, has the amazing capacity to replace exactly the missing structures. By grafting cells from different stages and regions of blastemas induced to form on donor animals expressing Green Fluorescent Protein (GFP), to non-GFP host animals, we have determined that the cells from early stage blastemas, as well as cells at the tip of late stage blastemas are developmentally labile such that their positional identity is reprogrammed by interactions with more proximal cells with stable positional information. In contrast, cells from the adjacent, more proximal stump tissues as well as the basal region of late bud blastemas are positionally stable, and thus form ectopic limb structures when grafted. Finally, we have found that a nerve is required to maintain the blastema cells in a positionally labile state, thus indicating a role for reprogramming cues in the blastema microenvironment.

Dlx5 and Msx2 regulate mouse anterior neural tube closure through ephrinA5-EphA7.

Homeodomain-containing transcription factors Dlx5 and Msx2 are able to form a heterodimer, and together can regulate embryonic development including skeletogenesis. Dlx5 functions as a transcriptional activator and Msx2 a transcriptional repressor, and they share common target genes. During mouse digit development, the expression domains of Dlx5 and Msx2 overlap at the distal region of the developing terminal phalange, although digit formation and regeneration are not altered in the Dlx5 and Msx2 null mutant embryos. Interestingly, we observed a high rate of defects in neural tube formation in Dlx5 and Msx2 double null mutants. In the absence of both Dlx5 and Msx2, a high occurrence of exencephaly and severe defects in craniofacial morphology are observed. Additionally, Dlx5 and Msx2 expression domain analysis showed overlap of the genes at the apex of the neural folds just prior to neural fold fusion. The expression patterns of ephrinA5 and two isoforms of EphA7 were tested as downstream targets of Dlx5 and Msx2. Results show that EphrinA5 and the truncated isoform of EphA7 are regulated by Dlx5 and Msx2 together, although the full length isoform of EphA7 expression is not altered. Overall, these data show that Dlx5 and Msx2 play a critical role in controlling cranial neural tube morphogenesis by regulating cell adhesion via the ephrinA5 and EphA7 pathway.

Gene expression patterns specific to the regenerating limb of the Mexican axolotl.

Salamander limb regeneration is dependent upon tissue interactions that are local to the amputation site. Communication among limb epidermis, peripheral nerves, and mesenchyme coordinate cell migration, cell proliferation, and tissue patterning to generate a blastema, which will form missing limb structures. An outstanding question is how cross-talk between these tissues gives rise to the regeneration blastema. To identify genes associated with epidermis-nerve-mesenchymal interactions during limb regeneration, we examined histological and transcriptional changes during the first week following injury in the wound epidermis and subjacent cells between three injury types; 1) a flank wound on the side of the animal that will not regenerate a limb, 2) a denervated limb that will not regenerate a limb, and 3) an innervated limb that will regenerate a limb. Early, histological and transcriptional changes were similar between the injury types, presumably because a common wound-healing program is employed across anatomical locations. However, some transcripts were enriched in limbs compared to the flank and are associated with vertebrate limb development. Many of these genes were activated before blastema outgrowth and expressed in specific tissue types including the epidermis, peripheral nerve, and mesenchyme. We also identified a relatively small group of transcripts that were more highly expressed in innervated limbs versus denervated limbs. These transcripts encode for proteins involved in myelination of peripheral nerves, epidermal cell function, and proliferation of mesenchymal cells. Overall, our study identifies limb-specific and nerve-dependent genes that are upstream of regenerative growth, and thus promising candidates for the regulation of blastema formation.

Regeneration of limb joints in the axolotl (Ambystoma mexicanum).

In spite of numerous investigations of regenerating salamander limbs, little attention has been paid to the details of how joints are reformed. An understanding of the process and mechanisms of joint regeneration in this model system for tetrapod limb regeneration would provide insights into developing novel therapies for inducing joint regeneration in humans. To this end, we have used the axolotl (Mexican Salamander) model of limb regeneration to describe the morphology and the expression patterns of marker genes during joint regeneration in response to limb amputation. These data are consistent with the hypothesis that the mechanisms of joint formation whether it be development or regeneration are conserved. We also have determined that defects in the epiphyseal region of both forelimbs and hind limbs in the axolotl are regenerated only when the defect is small. As is the case with defects in the diaphysis, there is a critical size above which the endogenous regenerative response is not sufficient to regenerate the joint. This non-regenerative response in an animal that has the ability to regenerate perfectly provides the opportunity to screen for the signaling pathways to induce regeneration of articular cartilage and joints.