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Previous studies have implicated epithelial-mesenchymal transition (EMT) in salamander limb regeneration. In this review, we describe putative roles for EMT during each stage of limb regeneration in axolotls and other salamanders. We hypothesize that EMT and EMT-like gene expression programs may regulate three main cellular processes during limb regeneration: (1) keratinocyte migration during wound closure; (2) transient invasion of the stump by epithelial cells undergoing EMT; and (3) use of EMT-like programs by non-epithelial blastemal progenitor cells to escape the confines of their niches. Finally, we propose nontraditional roles for EMT during limb regeneration that warrant further investigation, including alternative EMT regulators, stem cell activation, and fibrosis induced by aberrant EMT., (© 2024 The New York Academy of Sciences.)

Previous studies have reported that amputation invokes body-wide responses in regenerative organisms, but most have not examined the implications of these changes beyond the region of tissue regrowth. Specifically, long-range epidermal responses to amputation are largely uncharacterized, with research on amputation-induced epidermal responses in regenerative organisms traditionally being restricted to the wound site. Here, we investigate the effect of amputation on long-range epidermal permeability in two evolutionarily distant, regenerative organisms: axolotls and planarians. We find that amputation triggers a long-range increase in epidermal permeability in axolotls, accompanied by a long-range epidermal downregulation in MAPK signaling. Additionally, we provide functional evidence that pharmacologically inhibiting MAPK signaling in regenerating planarians increases long-range epidermal permeability. These findings advance our knowledge of body-wide changes due to amputation in regenerative organisms and warrant further study on whether epidermal permeability dysregulation in the context of amputation may lead to pathology in both regenerative and non-regenerative organisms., Competing Interests: CONFLICT OF INTEREST J.L.W. is a co-founder of Matice Biosciences. Other authors declare no conflicts of interest.

Sensory neurons in the dorsal root ganglion (DRG) and trigeminal ganglion (TG) are specialized to detect and transduce diverse environmental stimuli to the central nervous system. Single-cell RNA sequencing has provided insights into the diversity of sensory ganglia cell types in rodents, nonhuman primates, and humans, but it remains difficult to compare cell types across studies and species. We thus constructed harmonized atlases of the DRG and TG that describe and facilitate comparison of 18 neuronal and 11 non-neuronal cell types across six species and 31 datasets. We then performed single-cell/nucleus RNA sequencing of DRG from both human and the highly regenerative axolotl and found that the harmonized atlas also improves cell type annotation, particularly of sparse neuronal subtypes. We observed that the transcriptomes of sensory neuron subtypes are broadly similar across vertebrates, but the expression of functionally important neuropeptides and channels can vary notably. The resources presented here can guide future studies in comparative transcriptomics, simplify cell-type nomenclature differences across studies, and help prioritize targets for future analgesic development.

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 β-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.

Competing Interests: Competing interests The authors declare no competing or financial interests.

Throughout the animal kingdom regenerative ability varies greatly from species to species, and even tissue to tissue within the same organism. The sheer diversity of structures and mechanisms renders a thorough comparison of molecular processes truly daunting. Are "blastemas" found in organisms as distantly related as planarians and axolotls derived from the same ancestral process, or did they arise convergently and independently? Is a mouse digit tip blastema orthologous to a salamander limb blastema? In other fields, the thorough characterization of a reference model has greatly facilitated these comparisons. For example, the amphibian Spemann-Mangold organizer has served as an amazingly useful comparative template within the field of developmental biology, allowing researchers to draw analogies between distantly related species, and developmental processes which are superficially quite different. The salamander limb blastema may serve as the best starting point for a comparative analysis of regeneration, as it has been characterized by over 200 years of research and is supported by a growing arsenal of molecular tools. The anatomical and evolutionary closeness of the salamander and human limb also add value from a translational and therapeutic standpoint. Tracing the evolutionary origins of the salamander blastema, and its relatedness to other regenerative processes throughout the animal kingdom, will both enhance our basic biological understanding of regeneration and inform our selection of regenerative model systems., Competing Interests: Author JW was co-founder of Matice Biosciences. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Tajer, Savage and Whited.)

Regeneration of missing body parts is an incredible ability which is present in a wide number of species. However, this regenerative capability varies among different organisms. Urodeles (salamanders) are able to completely regenerate limbs after amputation through the essential process of blastema formation. The blastema is a collection of relatively undifferentiated progenitor cells that proliferate and repattern to form the internal tissues of a regenerated limb. Understanding blastema formation in salamanders may enable comparative studies with other animals, including mammals, with more limited regenerative abilities and may inspire future therapeutic approaches in humans. This review focuses on the current state of knowledge about how limb blastemas form in salamanders, highlighting both the possible roles of epigenetic controls in this process as well as limitations to scientific understanding that present opportunities for research., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Clustered regularly interspaced short palindromic repeats (CRISPR) is a powerful tool that enables editing of the axolotl genome. In this chapter, we will cover how to retrieve gene sequences, confirm annotation, design CRISPR targets, analyze indels, and screen for Crispant axolotls. This is a comprehensive guide on how to use CRISPR on your favorite gene and gain insights into its function., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Soft tissue fibrosis and cutaneous scarring represent massive clinical burdens to millions of patients per year and the therapeutic options available are currently quite limited. Despite what is known about the process of fibrosis in mammals, novel approaches for combating fibrosis and scarring are necessary. It is hypothesized that scarring has evolved as a solution to maximize healing speed to reduce fluid loss and infection. This hypothesis, however, is complicated by regenerative animals, which have arguably the most remarkable healing abilities and are capable of scar-free healing. This review explores the differences observed between adult mammalian healing that typically results in fibrosis versus healing in regenerative animals that heal scarlessly. Each stage of wound healing is surveyed in depth from the perspective of many regenerative and fibrotic healers so as to identify the most important molecular and physiological variances along the way to disparate injury repair outcomes. Understanding how these powerful model systems accomplish the feat of scar-free healing may provide critical therapeutic approaches to the treatment or prevention of fibrosis., (© 2021 The Authors. Advanced Science published by Wiley-VCH GmbH.)

Regeneration is an elegant and complex process informed by both local and long-range signals. Many current studies on regeneration are largely limited to investigations of local modulators within a canonical cohort of model organisms. Enhanced genetic tools increasingly enable precise temporal and spatial perturbations within these model regenerators, and these have primarily been applied to cells within the local injury site. Meanwhile, many aspects of broader spatial regulators of regeneration have not yet been examined with the same level of scrutiny. Recent studies have shed important insight into the significant effects of environmental cues and circulating factors on the regenerative process. These observations highlight that consideration of more systemic and possibly more broadly acting cues will also be critical to fully understand complex tissue regeneration. In this review, we explore the ways in which systemic cues and circulating factors affect the initiation of regeneration, the regenerative process, and its outcome. As this is a broad topic, we conceptually divide the factors based on their initial input as either external cues (for example, starvation and light/dark cycle) or internal cues (for example, hormones); however, all of these inputs ultimately lead to internal responses. We consider studies performed in a diverse set of organisms, including vertebrates and invertebrates. Through analysis of systemic mediators of regeneration, we argue that increased investigation of these "systemic factors" could reveal novel insights that may pave the way for a diverse set of therapeutic avenues.

Regenerative ability varies tremendously across species. A common feature of regeneration of appendages such as limbs, fins, antlers, and tails is the formation of a blastema-a transient structure that houses a pool of progenitor cells that can regenerate the missing tissue. We have identified the expression of von Willebrand factor D and EGF domains (vwde) as a common feature of blastemas capable of regenerating limbs and fins in a variety of highly regenerative species, including axolotl (Ambystoma mexicanum), lungfish (Lepidosiren paradoxa), and Polpyterus (Polypterus senegalus). Further, vwde expression is tightly linked to the ability to regenerate appendages in Xenopus laevis. Functional experiments demonstrate a requirement for vwde in regeneration and indicate that Vwde is a potent growth factor in the blastema. These data identify a key role for vwde in regenerating blastemas and underscore the power of an evolutionarily informed approach for identifying conserved genetic components of regeneration., (© 2020 The Authors. Evolution & Development published by Wiley Periodicals, Inc.)

How salamanders accomplish progenitor cell proliferation while faithfully maintaining genomic integrity and regenerative potential remains elusive. Here we found an innate DNA damage response mechanism that is evident during blastema proliferation (early- to late-bud) and studied its role during tissue regeneration by ablating the function of one of its components, Eyes absent 2. In eya2 mutant axolotls, we found that DNA damage signaling through the H2AX histone variant was deregulated, especially within the proliferating progenitors during limb regeneration. Ultimately, cell cycle progression was impaired at the G1/S and G2/M transitions and regeneration rate was reduced. Similar data were acquired using acute pharmacological inhibition of the Eya2 phosphatase activity and the DNA damage checkpoint kinases Chk1 and Chk2 in wild-type axolotls. Together, our data indicate that highly-regenerative animals employ a robust DNA damage response pathway which involves regulation of H2AX phosphorylation via Eya2 to facilitate proper cell cycle progression upon injury., Competing Interests: KS, DB, JM, SE, ST, GG, JH, KC, ML, JW No competing interests declared, (© 2020, Sousounis et al.)

Although millions of distinct virus species likely exist, only approximately 9000 are catalogued in GenBank's RefSeq database. We selectively enriched for the genomes of circular DNA viruses in over 70 animal samples, ranging from nematodes to human tissue specimens. A bioinformatics pipeline, Cenote-Taker, was developed to automatically annotate over 2500 complete genomes in a GenBank-compliant format. The new genomes belong to dozens of established and emerging viral families. Some appear to be the result of previously undescribed recombination events between ssDNA and ssRNA viruses. In addition, hundreds of circular DNA elements that do not encode any discernable similarities to previously characterized sequences were identified. To characterize these 'dark matter' sequences, we used an artificial neural network to identify candidate viral capsid proteins, several of which formed virus-like particles when expressed in culture. These data further the understanding of viral sequence diversity and allow for high throughput documentation of the virosphere., Competing Interests: MT, DP, NW, BS, AP, GS, YP, SK, PP, DM, PM, JW, BM, JB, SR, BR, JD, BT, OP, JT, SR, BB, MS, AV, AS, CB No competing interests declared

Striking similarities between wound healing, epimorphic regeneration and the progression of solid tumors have been uncovered by recent studies. In this Review, we discuss systemic effects of tumorigenesis that are now being appreciated in epimorphic regeneration, including genetic, cellular and metabolic heterogeneity, changes in circulating factors, and the complex roles of immune cells and immune modulation at systemic and local levels. We suggest that certain mechanisms enabling regeneration may be co-opted by cancer to promote growth at primary and metastatic sites. Finally, we advocate that working with a unified approach could complement research in both fields., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2020. Published by The Company of Biologists Ltd.)

Axolotls and other salamanders have the capacity to regenerate lost tissue after an amputation or injury. Growth and morphogenesis are coordinated within cell groups in many contexts by the interplay of transcriptional networks and biophysical properties such as ion flows and voltage gradients. It is not, however, known whether regulators of a cell's ionic state are involved in limb patterning at later stages of regeneration. Here we manipulated expression and activities of ion channels and gap junctions in vivo, in axolotl limb blastema cells. Limb amputations followed by retroviral infections were performed to drive expression of a human gap junction protein Connexin 26 (Cx26), potassium (Kir2.1-Y242F and Kv1.5) and sodium (NeoNav1.5) ion channel proteins along with EGFP control. Skeletal preparation revealed that overexpressing Cx26 caused syndactyly, while overexpression of ion channel proteins resulted in digit loss and structural abnormalities compared to EGFP expressing control limbs. Additionally, we showed that exposing limbs to the gap junction inhibitor lindane during the regeneration process caused digit loss. Our data reveal that manipulating native ion channel and gap junction function in blastema cells results in patterning defects involving the number and structure of the regenerated digits. Gap junctions and ion channels have been shown to mediate ion flows that control the endogenous voltage gradients which are tightly associated with the regulation of gene expression, cell cycle progression, migration, and other cellular behaviors. Therefore, we postulate that mis-expression of these channels may have disturbed this regulation causing uncoordinated cell behavior which results in morphological defects.

Treatment of tendon injuries is challenging, with neither conservative nor surgical approaches providing full recovery. Placental-derived tissues represent a promising tool for the treatment of tendon injuries. In this study, human amniotic suspension allograft (ASA) was investigated in a pre-clinical model of Achilles tendinopathy. Collagenase type I was injected in the right hind limb of Sprague Dawley rats to induce disease. Contralateral tendons were either left untreated or injected with saline as controls. Seven days following induction, tendons were injected with saline, ASA, or left untreated. Rats were sacrificed 14 and 28 days post-treatment. Histological and biomechanical analysis of tendons was completed. Fourteen days after ASA injection, improved fiber alignment and reduced cell density demonstrated improvement in degenerated tendons. Twenty-eight days post-treatment, tendons in all treatment groups showed fewer signs of degeneration, which is consistent with normal tendon healing. No statistically significant differences in histological or biomechanical analyses were observed between treatment groups at 28 days independent of the treatment they received. In this study, ASA treatment was safe, well-tolerated, and resulted in a widespread improvement of the tissue. The results of this study provide preliminary insights regarding the potential use of ASA for the treatment of Achilles tendinopathy.

Specialized epidermal cells are essential for the complex tissue regeneration required to replace tails and limbs, but their exact identities and molecular roles remain murky. Recent work in Xenopus has identified an epidermal cell population critical for tail regeneration, providing intriguing new directions for the field., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Cell-to-cell communication is a cornerstone of multicellular existence. The ancient mechanism of sharing information between cells using the conductance of ions across cell membranes and the propagation of electrical signals through tissue space is a powerful means of efficiently controlling cell decisions and behaviors. Our understanding of how cells use changes in 'bioelectrical' signals to elicit systems-level responses has dramatically improved in recent years. We are now in a position to not just describe these changes, but to also predictively alter them to learn more about their importance for developmental biology and regenerative medicine. Recent work is helping researchers construct a more integrative view of how these simple controls can orchestrate downstream changes in protein signaling pathways and gene regulatory networks. In this review, we highlight experiments and analyses that have led to new insights in bioelectrical controls, specifically as key modulators of complex pattern formation and tissue regeneration. We also discuss opportunities for the development of new therapeutic approaches in regenerative medicine applications by exploiting this fundamental biological phenomenon., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Complete and perfect regeneration of appendages is a process that has fascinated and perplexed biologists for centuries. Some tetrapods possess amazing regenerative abilities, but the regenerative abilities of others are exceedingly limited. The reasons underlying these differences have largely remained mysterious. A great deal has been learned about the morphological events that accompany successful appendage regeneration, and a handful of experimental manipulations can be reliably applied to block the process. However, only in the last decade has the goal of attaining a thorough molecular and cellular biological understanding of appendage regeneration in tetrapods become within reach. Advances in molecular and genetic tools for interrogating these remarkable events are now allowing for unprecedented access to the fundamental biology at work in appendage regeneration in a variety of species. This information will be critical for integrating the large body of detailed observations from previous centuries with a modern understanding of how cells sense and respond to severe injury and loss of body parts. Understanding commonalities between regenerative modes across diverse species is likely to illuminate the most important aspects of complex tissue regeneration., Competing Interests: The authors declare that they have no competing interests.

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.

Background: Defining conserved molecular pathways in animal models of successful cardiac regeneration could yield insight into why adult mammals have inadequate cardiac regeneration after injury. Insight into the transcriptomic landscape of early cardiac regeneration from model organisms will shed light on evolutionarily conserved pathways in successful cardiac regeneration., Methods: Here we describe a cross-species transcriptomic screen in 3 model organisms for cardiac regeneration: axolotl, neonatal mice, and zebrafish. Apical resection to remove ≈10% to 20% of ventricular mass was carried out in these model organisms. RNA-sequencing analysis was performed on the hearts harvested at 3 time points: 12, 24, and 48 hours after resection. Sham surgery was used as internal control., Results: Genes associated with inflammatory processes were found to be upregulated in a conserved manner. Complement receptors (activated by complement components, part of the innate immune system) were found to be highly upregulated in all 3 species. This approach revealed induction of gene expression for complement 5a receptor 1 in the regenerating hearts of zebrafish, axolotls, and mice. Inhibition of complement 5a receptor 1 significantly attenuated the cardiomyocyte proliferative response to heart injury in all 3 species. Furthermore, after left ventricular apical resection, the cardiomyocyte proliferative response was diminished in mice with genetic deletion of complement 5a receptor 1., Conclusions: These data reveal that activation of complement 5a receptor 1 mediates an evolutionarily conserved response that promotes cardiomyocyte proliferation after cardiac injury and identify complement pathway activation as a common pathway of successful heart regeneration., (© 2018 American Heart Association, Inc.)

Activation of progenitor cells is crucial to promote tissue repair following injury in adult animals. In the context of successful limb regeneration following amputation, progenitor cells residing within the stump must re-enter the cell cycle to promote regrowth of the missing limb. We demonstrate that in axolotls, amputation is sufficient to induce cell-cycle activation in both the amputated limb and the intact, uninjured contralateral limb. Activated cells were found throughout all major tissue populations of the intact contralateral limb, with internal cellular populations (bone and soft tissue) the most affected. Further, activated cells were additionally found within the heart, liver, and spinal cord, suggesting that amputation induces a common global activation signal throughout the body. Among two other injury models, limb crush and skin excisional wound, only limb crush injuries were capable of inducing cellular responses in contralateral uninjured limbs but did not achieve activation levels seen following limb loss. We found this systemic activation response to injury is independent of formation of a wound epidermis over the amputation plane, suggesting that injury-induced signals alone can promote cellular activation. In mammals, mTOR signaling has been shown to promote activation of quiescent cells following injury, and we confirmed a subset of activated contralateral cells is positive for mTOR signaling within axolotl limbs. These findings suggest that conservation of an early systemic response to injury exists between mammals and axolotls, and propose that a distinguishing feature in species capable of full regeneration is converting this initial activation into sustained and productive growth at the site of regeneration., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Appendage regeneration is not a simple task. The animal must harness all of its energy and resources to orchestrate perhaps one of the most complicated events since its development. Balancing the immune response, wound healing, proliferation, patterning and differentiation is an elegant job, and how some animals achieve that still leaves researchers enchanted today. In this work, we review some of the molecular aspects of regeneration, with a focus on the axolotl, the champion of tetrapod limb regeneration, and the mouse, an excellent mammalian model for digit tip regeneration. Advances in molecular and genomic tools have enabled the discovery of exciting fundamental features of limb regeneration. Integrating the data from different animal systems will be crucial to understanding the common requirements of successful appendage regeneration and places for flexibility. The combination of these efforts is paving the way to grasping how good regenerators respond to the loss of body parts, how these mechanisms might compare in modest regenerators, and, ultimately, in developing approaches for improving regenerative outcomes in humans.

Axolotl salamanders are powerful models for understanding how regeneration of complex body parts can be achieved, whereas mammals are severely limited in this ability. Factors that promote normal axolotl regeneration can be examined in mammals to determine if they exhibit altered activity in this context. Furthermore, factors prohibiting axolotl regeneration can offer key insight into the mechanisms present in regeneration-incompetent species. We sought to determine if we could experimentally compromise the axolotl's ability to regenerate limbs and, if so, discover the molecular changes that might underlie their inability to regenerate. We found that repeated limb amputation severely compromised axolotls' ability to initiate limb regeneration. Using RNA-seq, we observed that a majority of differentially expressed transcripts were hyperactivated in limbs compromised by repeated amputation, suggesting that mis-regulation of these genes antagonizes regeneration. To confirm our findings, we additionally assayed the role of amphiregulin , an EGF-like ligand, which is aberrantly upregulated in compromised animals. During normal limb regeneration, amphiregulin is expressed by the early wound epidermis, and mis-expressing this factor lead to thickened wound epithelium, delayed initiation of regeneration, and severe regenerative defects. Collectively, our results suggest that repeatedly amputated limbs may undergo a persistent wound healing response, which interferes with their ability to initiate the regenerative program. These findings have important implications for human regenerative medicine., Competing Interests: The authors declare no competing financial interests.

Humans and other mammals are limited in their natural abilities to regenerate lost body parts. By contrast, many salamanders are highly regenerative and can spontaneously replace lost limbs even as adults. Because salamander limbs are anatomically similar to human limbs, knowing how they regenerate should provide important clues for regenerative medicine. Although interest in understanding the mechanics of this process has never wavered, until recently researchers have been vexed by seemingly impenetrable logistics of working with these creatures at a molecular level. Chief among the problems has been the very large size of salamander genomes, and not a single salamander genome has been fully sequenced to date. Recently the enormous gap in sequence information has been bridged by approaches that leverage mRNA as the starting point. Together with functional experimentation, these data are rapidly enabling researchers to finally uncover the molecular mechanisms underpinning the astonishing biological process of limb regeneration., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Perfusion techniques have been used for centuries to visualize the circulation of tissues. Axolotl (Ambystoma mexicanum) is a species of salamander that has emerged as an essential model for regeneration studies. Little is known about how revascularization occurs in the context of regeneration in these animals. Here we report a simple method for visualization of the vasculature in axolotl via perfusion of 1,1'-Dioctadecy-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI). DiI is a lipophilic carbocyanine dye that inserts into the plasma membrane of endothelial cells instantaneously. Perfusion is done using a peristaltic pump such that DiI enters the circulation through the aorta. During perfusion, dye flows through the axolotl's blood vessels and incorporates into the lipid bilayer of vascular endothelial cells upon contact. The perfusion procedure takes approximately one hour for an eight-inch axolotl. Immediately after perfusion with DiI, the axolotl can be visualized with a confocal fluorescent microscope. The DiI emits light in the red-orange range when excited with a green fluorescent filter. This DiI perfusion procedure can be used to visualize the vascular structure of axolotls or to demonstrate patterns of revascularization in regenerating tissues.

Matching appendage size to body size is fundamental to animal function. Generating an appropriately-sized appendage is a robust process executed during development which is also critical for regeneration. When challenged, larger animals are programmed to regenerate larger limbs than smaller animals within a single species. Understanding this process has important implications for regenerative medicine. To approach this complex question, models with altered appendage size:body size ratios are required. We hypothesized that repeatedly challenging axolotls to regrow limb buds would affect their developmental program resulting in altered target morphology. We discovered that after 10 months following this experimental procedure, limbs that developed were permanently miniaturized. This altered target morphology was preserved upon amputation and regeneration. Future experiments using this platform should provide critical information about how target limb size is encoded within limb progenitors., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Mammals have extremely limited regenerative capabilities; however, axolotls are profoundly regenerative and can replace entire limbs. The mechanisms underlying limb regeneration remain poorly understood, partly because the enormous and incompletely sequenced genomes of axolotls have hindered the study of genes facilitating regeneration. We assembled and annotated a de novo transcriptome using RNA-sequencing profiles for a broad spectrum of tissues that is estimated to have near-complete sequence information for 88% of axolotl genes. We devised expression analyses that identified the axolotl orthologs of cirbp and kazald1 as highly expressed and enriched in blastemas. Using morpholino anti-sense oligonucleotides, we find evidence that cirbp plays a cytoprotective role during limb regeneration whereas manipulation of kazald1 expression disrupts regeneration. Our transcriptome and annotation resources greatly complement previous transcriptomic studies and will be a valuable resource for future research in regenerative biology., (Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.)

The Mexican axolotl (Ambystoma mexicanum) is capable of fully regenerating amputated limbs, but denervation of the limb inhibits the formation of the post-injury proliferative mass called the blastema. The molecular basis behind this phenomenon remains poorly understood, but previous studies have suggested that nerves support regeneration via the secretion of essential growth-promoting factors. An essential nerve-derived factor must be found in the blastema, capable of rescuing regeneration in denervated limbs, and its inhibition must prevent regeneration. Here, we show that the neuronally secreted protein Neuregulin-1 (NRG1) fulfills all these criteria in the axolotl. Immunohistochemistry and in situ hybridization of NRG1 and its active receptor ErbB2 revealed that they are expressed in regenerating blastemas but lost upon denervation. NRG1 was localized to the wound epithelium prior to blastema formation and was later strongly expressed in proliferating blastemal cells. Supplementation by implantation of NRG1-soaked beads rescued regeneration to digits in denervated limbs, and pharmacological inhibition of NRG1 signaling reduced cell proliferation, blocked blastema formation and induced aberrant collagen deposition in fully innervated limbs. Taken together, our results show that nerve-dependent NRG1/ErbB2 signaling promotes blastemal proliferation in the regenerating limb and may play an essential role in blastema formation, thus providing insight into the longstanding question of why nerves are required for axolotl limb regeneration., (© 2016. Published by The Company of Biologists Ltd.)

Loss-of-function genetics provides strong evidence for a gene's function in a wild-type context. In many model systems, this approach has been invaluable for discovering the function of genes in diverse biological processes. Axolotls are urodele amphibians (salamanders) with astonishing regenerative abilities, capable of regenerating entire limbs, portions of the tail (including spinal cord), heart, and brain into adulthood. With their relatively short generation time among salamanders, they offer an outstanding opportunity to interrogate natural mechanisms for appendage and organ regeneration provided that the tools are developed to address these long-standing questions. Here we demonstrate targeted modification of the thrombospondin-1 (tsp-1) locus using transcription-activator-like effector nucleases (TALENs) and identify a role of tsp-1 in recruitment of myeloid cells during limb regeneration. We find that while tsp-1-edited mosaic animals still regenerate limbs, they exhibit a reduced subepidermal collagen layer in limbs and an increased number of myeloid cells within blastemas. This work presents a protocol for generating and genotyping mosaic axolotls with TALEN-mediated gene edits.

The ability to introduce DNA elements into host cells and analyze the effects has revolutionized modern biology. Here we describe a protocol to generate Moloney murine leukemia virus (MMLV)-based, replication-incompetent pseudotyped retrovirus capable of infecting axolotls and incorporating genetic information into their genome. When pseudotyped with vesicular stomatitis virus (VSV)-G glycoprotein, the retroviruses can infect a broad range of proliferative axolotl cell types. However, if the retrovirus is pseudotyped with an avian sarcoma leukosis virus (ASLV)-A envelope protein, only axolotl cells experimentally manipulated to express the cognate tumor virus A (TVA) receptor can be targeted by infections. These strategies enable robust transgene expression over many cell divisions, cell lineage tracing, and cell subtype targeting for gene expression.

The actin-Capping Protein heterodimer, composed of the α and β subunits, is a master F-actin regulator. In addition to its role in many cellular processes, Capping Protein acts as a main tumor suppressor module in Drosophila and in humans, in part, by restricting the activity of Yorkie/YAP/TAZ oncogenes. We aimed in this report to understand how both subunits regulate each other in vivo. We show that the levels and capping activities of both subunits must be tightly regulated to control F-actin levels and consequently growth of the Drosophila wing. Overexpressing capping protein α and β decreases both F-actin levels and tissue growth, while expressing forms of Capping Protein that have dominant negative effects on F-actin promote tissue growth. Both subunits regulate each other's protein levels. In addition, overexpressing one of the subunit in tissues knocked-down for the other increases the mRNA and protein levels of the subunit knocked-down and compensates for its loss. We propose that the ability of the α and β subunits to control each other's levels assures that a pool of functional heterodimer is produced in sufficient quantities to restrict the development of tumor but not in excess to sustain normal tissue growth.

Axolotls are poised to become the premiere model system for studying vertebrate appendage regeneration. However, very few molecular tools exist for studying crucial cell lineage relationships over regeneration or for robust and sustained misexpression of genetic elements to test their function. Furthermore, targeting specific cell types will be necessary to understand how regeneration of the diverse tissues within the limb is accomplished. We report that pseudotyped, replication-incompetent retroviruses can be used in axolotls to permanently express markers or genetic elements for functional study. These viruses, when modified by changing their coat protein, can infect axolotl cells only when they have been experimentally manipulated to express the receptor for that coat protein, thus allowing for the possibility of targeting specific cell types. Using viral vectors, we have found that progenitor populations for many different cell types within the blastema are present at all stages of limb regeneration, although their relative proportions change with time.

Transgenesis promises a powerful means for assessing gene function during amphibian limb regeneration. This approach is complicated, however, by the need for embryonic appendage development to proceed unimpeded despite the genetic alterations one wishes to test later in the context of regeneration. Achieving conditional gene regulation in this amphibian has not proved to be as straightforward as in many other systems. In this report we describe a unique method for obtaining temporal control over exogenous gene expression in the axolotl. Based on technology derived from the Escherichia coli Lac operon, uninduced transgenes are kept in a repressed state by the binding of constitutively expressed Lac repressor protein (LacI) to operator sequences within the expression construct. Addition of a lactose analog, IPTG, to the swimming water of the axolotl is sufficient for the sugar to be taken up by cells, where it binds the LacI protein, thereby inducing expression of the repressed gene. We use this system to demonstrate an in vivo role for thrombospondin-4 in limb regeneration. This inducible system will allow for systematic analysis of phenotypes at defined developmental or regenerative time points. The tight regulation and robustness of gene induction combined with the simplicity of this strategy will prove invaluable for studying many aspects of axolotl biology.

The molecular processes underlying regeneration remain largely unknown. Several potential factors have been elucidated by focusing on the regenerative function of genes originally identified in a developmental context. A complementary approach is to consider the roles of factors involved in wound healing. Here we focus on the Thrombospondins, a family of secreted extracellular matrix proteins that have been implicated in skin wound healing in mammals. We show that a subset of Thrombospondins are expressed at distinct times and in particular cell types during axolotl limb regeneration. Our studies have revealed the axolotl orthologs of thrombospondin-1 (tsp-1) and thrombospondin-4 (tsp-4) are highly upregulated during limb regeneration in patterns both distinct and similar to larval limb development. Our data suggest that thrombospondins may be key regulators of limb regeneration in axolotl, while their activation appears to be relegated solely to wound healing in vertebrates that have lost the ability to regenerate limbs., (Copyright © 2011 Wiley-Liss, Inc.)

There have been notable advances in the scientific understanding of regeneration within the past year alone, including two recently published in BMC Biology. Increasingly, progress in the regeneration field is being inspired by comparisons with stem cell biology and enabled by newly developed techniques that allow simultaneous examination of thousands of genes and proteins.

The investigation of vertebrate limb regeneration, a favorite topic of early developmental biologists, is enjoying a renaissance thanks to recently developed molecular and genetic tools, as indicated in recent papers in BMC Biology and BMC Developmental Biology. Classical experiments provide a rich context for interpreting modern functional studies.

Ptpmeg is a cytoplasmic tyrosine phosphatase containing FERM and PDZ domains. Drosophila Ptpmeg and its vertebrate homologs PTPN3 and PTPN4 are expressed in the nervous system, but their developmental functions have been unknown. We found that ptpmeg is involved in neuronal circuit formation in the Drosophila central brain, regulating both the establishment and the stabilization of axonal projection patterns. In ptpmeg mutants, mushroom body (MB) axon branches are elaborated normally, but the projection patterns in many hemispheres become progressively abnormal as the animals reach adulthood. The two branches of MB alpha/beta neurons are affected by ptpmeg in different ways; ptpmeg activity inhibits alpha lobe branch retraction while preventing beta lobe branch overextension. The phosphatase activity of Ptpmeg is essential for both alpha and beta lobe formation, but the FERM domain is required only for preventing alpha lobe retraction, suggesting that Ptpmeg has distinct roles in regulating the formation of alpha and beta lobes. ptpmeg is also important for the formation of the ellipsoid body (EB), where it influences the pathfinding of EB axons. ptpmeg function in neurons is sufficient to support normal wiring of both the EB and MB. However, ptpmeg does not act in either MB or EB neurons, implicating ptpmeg in the regulation of cell-cell signaling events that control the behavior of these axons.

How a nucleus is positioned within a highly polarized postmitotic animal cell is not well understood. In this work, we demonstrate that the Dynactin complex (a regulator of the microtubule motor protein Dynein) is required to maintain the position of the nucleus within post-mitotic Drosophila melanogaster photoreceptor neurons. We show that multiple independent disruptions of Dynactin function cause a relocation of the photoreceptor nucleus toward the brain, and that inhibiting Dynactin causes the photoreceptor to acquire a bipolar appearance with long leading and trailing processes. We find that while the minus-end directed motor Dynein cooperates with Dynactin in positioning the photoreceptor nucleus, the plus-end directed microtubule motor Kinesin acts antagonistically to Dynactin. These data suggest that the maintenance of photoreceptor nuclear position depends on a balance of plus-end and minus-end directed microtubule motor function.