Salamander Limb Regeneration Research Articles

Putative epithelial-mesenchymal transitions during salamander limb regeneration: Current perspectives and future investigations.

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.

Sall4 regulates downstream patterning genes during limb regeneration

Many salamanders can completely regenerate a fully functional limb. Limb regeneration is a carefully coordinated process involving several defined stages. One key event during the regeneration process is the patterning of the blastema to inform cells of what they must differentiate into. Although it is known that many genes involved in the initial development of the limb are re-used during regeneration, the exact molecular circuitry involved in this process is not fully understood. Several large-scale transcriptional profiling studies of axolotl limb regeneration have identified many transcription factors that are up-regulated after limb amputation. Sall4 is a transcription factor that has been identified to play essential roles in maintaining cells in an undifferentiated state during development and also plays a unique role in limb development. Inactivation of Sall4 during limb bud development results in defects in anterior-posterior patterning of the limb. Sall4 has been found to be up-regulated during limb regeneration in both Xenopus and salamanders, but to date it function has been untested. We confirmed that Sall4 is up-regulated during limb regeneration in the axolotl using qRT-PCR and identified that it is present in the skin cells and also in cells within the blastema. Using CRISPR technology we microinjected gRNAs specific for Sall4 complexed with cas9 protein into the blastema to specifically knockout Sall4 in blastema cells only. This resulted in limb regenerate defects, including missing digits, fusion of digit elements, and defects in the radius and ulna. This suggests that during regeneration Sall4 may play a similar role in regulating the specification of anterior-proximal skeletal elements.

David L. Stocum (1939-2023).

David L. Stocum, a scientist whose contributions to and impact on the field of regeneration and developmental biology are legendary, and likely more pervasive than many know, passed away on 21 April 2023. His illustrious career, exploring and characterizing the fundamentals of limb regeneration in salamanders, spanned nearly 60 years. Much of his work dissecting the tissue-level logic of regeneration established the framework for the molecular investigation of regeneration taking place today. His generous spirit as mentor and colleague, encyclopedic understanding of the literature, and enthusiasm for each new discovery and its place within the larger picture of scientific understanding distinguishes him as a giant in the history of regenerative biology. David's career path, the transformative role his teachers and mentors played along the way, and his own role in inspiring the next generation of researchers speaks strongly to the importance and power of basic education to society.

Plant and Salamander Inspired Network Attack Detection and Data Recovery Model

The number of users of the Internet has been continuously rising, with an estimated 5.1 billion users in 2023, which comprises around 64.7% of the total world population. This indicates the rise of more connected devices to the network. On average, 30,000 websites are hacked daily, and nearly 64% of companies worldwide experience at least one type of cyberattack. As per IDC’s 2022 Ransomware study, two-thirds of global organizations were hit by a ransomware attack that year. This creates the desire for a more robust and evolutionary attack detection and recovery model. One aspect of the study is the bio-inspiration models. This is because of the natural ability of living organisms to withstand various odd circumstances and overcome them with an optimization strategy. In contrast to the limitations of machine learning models with the need for quality datasets and computational availability, bio-inspired models can perform in low computational environments, and their performances are designed to evolve naturally with time. This study concentrates on exploring the evolutionary defence mechanism in plants and understanding how plants react to any known external attacks and how the response mechanism changes to unknown attacks. This study also explores how regenerative models, such as salamander limb regeneration, could build a network recovery system where services could be automatically activated after a network attack, and data could be recovered automatically by the network after a ransomware-like attack. The performance of the proposed model is compared to open-source IDS Snort and data recovery systems such as Burp and Casandra.

A small noncoding RNA links ribosome recovery and translation control to dedifferentiation during salamander limb regeneration.

Building a blastema from the stump is a key step of salamander limb regeneration. Stump-derived cells temporarily suspend their identity as they contribute to the blastema by a process generally referred to as dedifferentiation. Here, we provide evidence for a mechanism that involves an active inhibition of protein synthesis during blastema formation and growth. Relieving this inhibition results in a higher number of cycling cells and enhances the pace of limb regeneration. By small RNA profiling and fate mapping of skeletal muscle progeny as a cellular model for dedifferentiation, we find that the downregulation of miR-10b-5p is critical for rebooting the translation machinery. miR-10b-5p targets ribosomal mRNAs, and its artificial upregulation causes decreased blastema cell proliferation, reduction in transcripts that encode ribosomal subunits, diminished nascent protein synthesis, and retardation of limb regeneration. Taken together, our data identify a link between miRNA regulation, ribosome biogenesis, and protein synthesis during newt limb regeneration.

Altered developmental programs and oriented cell divisions lead to bulky bones during salamander limb regeneration

There are major differences in duration and scale at which limb development and regeneration proceed, raising the question to what extent regeneration is a recapitulation of development. We address this by analyzing skeletal elements using a combination of micro-CT imaging, molecular profiling and clonal cell tracing. We find that, in contrast to development, regenerative skeletal growth is accomplished based entirely on cartilage expansion prior to ossification, not limiting the transversal cartilage expansion and resulting in bulkier skeletal parts. The oriented extension of salamander cartilage and bone appear similar to the development of basicranial synchondroses in mammals, as we found no evidence for cartilage stem cell niches or growth plate-like structures during neither development nor regeneration. Both regenerative and developmental ossification in salamanders start from the cortical bone and proceeds inwards, showing the diversity of schemes for the synchrony of cortical and endochondral ossification among vertebrates.

Retinoic Acid-Induced Limb Duplications.

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

Wnt Signaling Coordinates the Expression of Limb Patterning Genes During Axolotl Forelimb Development and Regeneration.

After amputation, axolotl salamanders can regenerate their limbs, but the degree to which limb regeneration recapitulates limb development remains unclear. One limitation in answering this question is our lack of knowledge about salamander limb development. Here, we address this question by studying expression patterns of genes important for limb patterning during axolotl salamander limb development and regeneration. We focus on the Wnt signaling pathway because it regulates multiple functions during tetrapod limb development, including limb bud initiation, outgrowth, patterning, and skeletal differentiation. We use fluorescence in situ hybridization to show the expression of Wnt ligands, Wnt receptors, and limb patterning genes in developing and regenerating limbs. Inhibition of Wnt ligand secretion permanently blocks limb bud outgrowth when treated early in limb development. Inhibiting Wnt signaling during limb outgrowth decreases the expression of critical signaling genes, including Fgf10, Fgf8, and Shh, leading to the reduced outgrowth of the limb. Patterns of gene expression are similar between developing and regenerating limbs. Inhibition of Wnt signaling during regeneration impacted patterning gene expression similarly. Overall, our findings suggest that limb development and regeneration utilize Wnt signaling similarly. It also provides new insights into the interaction of Wnt signaling with other signaling pathways during salamander limb development and regeneration.

Rebuilding limbs, one cell at a time.

Key Findings New techniques for visualizing and interrogating single cells hold the key to unlocking the underlying mechanisms of salamander limb regeneration.

Tig1 regulates proximo-distal identity during salamander limb regeneration

Salamander limb regeneration is an accurate process which gives rise exclusively to the missing structures, irrespective of the amputation level. This suggests that cells in the stump have an awareness of their spatial location, a property termed positional identity. Little is known about how positional identity is encoded, in salamanders or other biological systems. Through single-cell RNAseq analysis, we identified Tig1/Rarres1 as a potential determinant of proximal identity. Tig1 encodes a conserved cell surface molecule, is regulated by retinoic acid and exhibits a graded expression along the proximo-distal axis of the limb. Its overexpression leads to regeneration defects in the distal elements and elicits proximal displacement of blastema cells, while its neutralisation blocks proximo-distal cell surface interactions. Critically, Tig1 reprogrammes distal cells to a proximal identity, upregulating Prod1 and inhibiting Hoxa13 and distal transcriptional networks. Thus, Tig1 is a central cell surface determinant of proximal identity in the salamander limb.

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

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

Vincenzo Colucci’s 1886 memoir, Intorno alla rigenerazione degli arti e della coda nei tritoni, annotated and translated into English as: Concerning regeneration of the limbs and tail in salamanders

Vincenzo Colucci taught and did research in several Italian veterinary schools during the latter part of the nineteenth century. Although he usually dealt with normal and pathological histology of domestic animals, he was also interested in regeneration. His 1886 description of limb and tail regeneration in salamanders was one of the earliest studies of events at the cellular level during body part regeneration in any animal. Although he was sometimes incorrect (for example, he believed that white blood cells gave rise to a large part of the regenerating tissues), his work represents a striking advance over the gross anatomical studies of the subject published by Spallanzani in 1768. Unfortunately, Colucci’s memoir is in an issue of a journal that had become almost unobtainable by the turn of the twentieth century. Recently, however, a digitized version of his work has become available online. The chief purpose of the present contribution is to supply an English translation of the original Italian text and figure captions. In addition, the translation is annotated to help modern readers understand some of Colucci’s now outdated assumptions. A discussion of his sometimes disconcerting notions affords an interesting glimpse of an era when some biological ideas now taken for granted were still only dimly understood. For example, in those times: there were several different (and rapidly changing) concepts of the blastema; not everyone accepted all the tenets of cell theory; the possibility of cell division without mitosis was seriously entertained; and some rather baroque ideas about vasculogenesis and angiogenesis were in play. An English translation will make Colucci’s 1886 memoir more widely accessible and, one hopes, help rescue him from his long and undeserved oblivion.

Salamander‐Eci: An optical clearing protocol for the three‐dimensional exploration of regeneration

Salamander limb regeneration is a complex biological process that entails the orchestration of multiple cellular and molecular mechanisms in a three-dimensional space. Hence, a comprehensive understanding of this process requires whole-structure level explorations. Recent advances in imaging and optical clearing methods have transformed the study of regenerative phenomena, allowing the three-dimensional visualization of structures and entire organisms. Here we introduce Salamander-Eci, a rapid and robust optical clearing protocol optimized for the widely used axolotl model, which allows simultaneous immunohistochemistry and Click-chemistry detection with minimal volume disruption. We provide examples of its application, from whole larva to adult limbs and organs, and complement it with an image analysis pipeline for volumetric cell quantification. Further, we offer a detailed 3D quantitation of cell proliferation throughout axolotl limb regeneration. Salamander-Eci enables the comprehensive volumetric analysis of regenerative phenomena at both local and systemic levels.

Limb regeneration in salamanders: the plethodontid tale.

Salamanders are the only vertebrates that can regenerate limbs as adults. This makes them ideal models to investigate the cellular and molecular mechanisms of tissue regeneration. Ambystoma mexicanum and Nothopthalmus viridescens have long served as primary salamander models of limb regeneration, and the recent sequencing of the axolotl genome now provides a blueprint to mine regeneration insights from other salamander species. In particular, there is a need to study South American plethodontid salamanders that present different patterns of limb development and regeneration. A broader sampling of species using next-generation sequencing approaches is needed to reveal shared and unique mechanisms of regeneration, and more generally, the evolutionary history of salamander limb regeneration.

Remembering where we are: Positional information in salamander limb regeneration.

Fifty years ago, Lewis Wolpert defined an important question in developmental biology: how are cell fates determined by the positions of cells within a system? He proposed that cells retain positional values as if they lie within a coordinate system and that the interpretation of these values produces patterns in development. He referred to this concept as positional information. Though initially controversial, this concept of positional information has proven to be profoundly influential in developmental biology. One area in which the influence of Wolpert's theoretical work can be clearly demonstrated is the study of limb regeneration in salamanders. Here, we review the work in limb regeneration leading up to Wolpert defining the concept of positional information and how his theory has guided regeneration research over the subsequent 50 years.

Midkine is a dual regulator of wound epidermis development and inflammation during the initiation of limb regeneration.

Formation of a specialized wound epidermis is required to initiate salamander limb regeneration. Yet little is known about the roles of the early wound epidermis during the initiation of regeneration and the mechanisms governing its development into the apical epithelial cap (AEC), a signaling structure necessary for outgrowth and patterning of the regenerate. Here, we elucidate the functions of the early wound epidermis, and further reveal midkine (mk) as a dual regulator of both AEC development and inflammation during the initiation of axolotl limb regeneration. Through loss- and gain-of-function experiments, we demonstrate that mk acts as both a critical survival signal to control the expansion and function of the early wound epidermis and an anti-inflammatory cytokine to resolve early injury-induced inflammation. Altogether, these findings unveil one of the first identified regulators of AEC development and provide fundamental insights into early wound epidermis function, development, and the initiation of limb regeneration.

Comparative transcriptomics of limb regeneration: Identification of conserved expression changes among three species of Ambystoma

Transcriptome studies are revealing the complex gene expression basis of limb regeneration in the primary salamander model – Ambystoma mexicanum (axolotl). To better understand this complexity, there is need to extend analyses to additional salamander species. Using microarray and RNA-Seq, we performed a comparative transcriptomic study using A. mexicanum and two other ambystomatid salamanders: A. andersoni, and A. maculatum. Salamanders were administered forelimb amputations and RNA was isolated and analyzed to identify 405 non-redundant genes that were commonly, differentially expressed 24 h post amputation. Many of the upregulated genes are predicted to function in wound healing and developmental processes, while many of the downregulated genes are typically expressed in muscle. The conserved transcriptional changes identified in this study provide a high-confidence dataset for identifying factors that simultaneous orchestrate wound healing and regeneration processes in response to injury, and more generally for identifying genes that are essential for salamander limb regeneration.

An integrative framework for salamander and mouse limb regeneration.

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.

ITRAQ-based quantitative proteomic analysis of Cynops orientalis limb regeneration

BackgroundSalamanders regenerate their limbs after amputation. However, the molecular mechanism of this unique regeneration remains unclear. In this study, isobaric tags for relative and absolute quantification (iTRAQ) coupled with liquid chromatography tandem mass spectrometry (LC-MS/MS) was employed to quantitatively identify differentially expressed proteins in regenerating limbs 3, 7, 14, 30 and 42 days post amputation (dpa).ResultsOf 2636 proteins detected in total, 253 proteins were differentially expressed during different regeneration stages. Among these proteins, Asporin, Cadherin-13, Keratin, Collagen alpha-1(XI) and Titin were down-regulated. CAPG, Coronin-1A, AnnexinA1, Cathepsin B were up-regulated compared with the control. The identified proteins were further analyzed to obtain information about their expression patterns and functions in limb regeneration. Functional analysis indicated that the differentially expressed proteins were associated with wound healing, immune response, cellular process, metabolism and binding.ConclusionsThis work indicated that significant proteome alternations occurred during salamander limb regeneration. The results may provide fundamental knowledge to understand the mechanism of limb regeneration.

Regeneration and Regrowth Potentials of Digit Tips in Amphibians and Mammals.

Tissue regeneration and repair have received much attention in the medical field over the years. The study of amphibians, such as newts and salamanders, has uncovered many of the processes that occur in these animals during full-limb/digit regeneration, a process that is highly limited in mammals. Understanding these processes in amphibians could shed light on how to develop and improve this process in mammals. Amputation injuries in mammals usually result in the formation of scar tissue with limited regrowth of the limb/digit; however, it has been observed that the very tips of digits (fingers and toes) can partially regrow in humans and mice under certain conditions. This review will summarize and compare the processes involved in salamander limb regeneration, mammalian wound healing, and digit regeneration in mice and humans.