Harmonized cross-species cell atlases of trigeminal and dorsal root ganglia.

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.

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

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

The salamander blastema within the broader context of metazoan regeneration.

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.

Harmonized cross-species cell atlases of trigeminal and dorsal root ganglia.

Peripheral sensory neurons in the dorsal root ganglion (DRG) and trigeminal ganglion (TG) are specialized to detect and transduce diverse environmental stimuli including touch, temperature, and pain to the central nervous system. Recent advances in single-cell RNA-sequencing (scRNA-seq) have provided new insights into the diversity of sensory ganglia cell types in rodents, non-human primates, and humans, but it remains difficult to compare transcriptomically defined cell types across studies and species. Here, we built cross-species harmonized atlases of DRG and TG cell types that describe 18 neuronal and 11 non-neuronal cell types across 6 species and 19 studies. We then demonstrate the utility of this harmonized reference atlas by using it to annotate newly profiled DRG nuclei/cells from both human and the highly regenerative axolotl. We observe that the transcriptomic profiles of sensory neuron subtypes are broadly similar across vertebrates, but the expression of functionally important neuropeptides and channels can vary notably. The new resources and data presented here can guide future studies in comparative transcriptomics, simplify cell type nomenclature differences across studies, and help prioritize targets for future pain therapy development.

Limb blastema formation: How much do we know at a genetic and epigenetic level?

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.

A Practical Guide for CRISPR-Cas9-Induced Mutations in Axolotls.

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.

Finding Solutions for Fibrosis: Understanding the Innate Mechanisms Used by Super-Regenerator Vertebrates to Combat Scarring.

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.

A cross-species analysis of systemic mediators of repair and complex tissue regeneration.

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.

Precise control of ion channel and gap junction expression is required for patterning of the regenerating axolotl limb.

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.

Eya2 promotes cell cycle progression by regulating DNA damage response during vertebrate limb regeneration.

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.

von Willebrand factor D and EGF domains is an evolutionarily conserved and required feature of blastemas capable of multitissue appendage regeneration.

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.

Discovery of several thousand highly diverse circular DNA viruses.

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.

Parallels between wound healing, epimorphic regeneration and solid tumors.

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.

Treatment with Human Amniotic Suspension Allograft Improves Tendon Healing in a Rat Model of Collagenase-Induced Tendinopathy.

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.

Bioelectrical controls of morphogenesis: from ancient mechanisms of cell coordination to biomedical opportunities.

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.

Development: How Tadpoles ROC Tail Regeneration.

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.

Common themes in tetrapod appendage regeneration: a cellular perspective.

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.