Regulation of stem cell identity by miR-200a during spinal cord regeneration.

Axolotls are an important model organism for multiple types of regeneration, including functional spinal cord regeneration. Remarkably, axolotls can repair their spinal cord after a small lesion injury and can also regenerate their entire tail following amputation. Several classical signaling pathways that are used during development are reactivated during regeneration, but how this is regulated remains a mystery. We have previously identified miR-200a as a key factor that promotes successful spinal cord regeneration. Here, using RNA-seq analysis, we discovered that the inhibition of miR-200a results in an upregulation of the classical mesodermal marker brachyury in spinal cord cells after injury. However, these cells still express the neural stem cell marker sox2. In vivo cell tracking allowed us to determine that these cells can give rise to cells of both the neural and mesoderm lineage. Additionally, we found that miR-200a can directly regulate brachyury via a seed sequence in the 3'UTR of the gene. Our data indicate that miR-200a represses mesodermal cell fate after a small lesion injury in the spinal cord when only glial cells and neurons need to be replaced.

Wound healing across the animal kingdom: Crosstalk between the immune system and the extracellular matrix.

Tissue regeneration is widespread in the animal kingdom. To date, key roles for different molecular and cellular programs in regeneration have been described, but the ultimate blueprint for this talent remains elusive. In animals capable of tissue regeneration, one of the most crucial stages is wound healing, whose main goal is to close the wound and prevent infection. In this stage, it is necessary to avoid scar formation to facilitate the activation of the immune system and remodeling of the extracellular matrix, key factors in promoting tissue regeneration. In this review, we will discuss the current state of knowledge regarding the role of the immune system and the interplay with the extracellular matrix to trigger a regenerative response.

Learning from regeneration research organisms: The circuitous road to scar free wound healing.

The skin is the largest organ in the body and plays multiple essential roles ranging from regulating temperature, preventing infection and ultimately defining who we are physically. It is a highly dynamic organ that constantly replaces the outermost cells throughout life. However, when faced with a major injury, human skin cannot restore a significant lesion to its original functionality, instead a reparative scar is formed. In contrast to this, many other species have the unique ability to regenerate full thickness skin without formation of scar tissue. Here we review recent advances in the field that shed light on how the skin cells in regenerative species react to injury to prevent scar formation versus scar forming humans.

Dynamic membrane depolarization is an early regulator of ependymoglial cell response to spinal cord injury in axolotl.

Salamanders, such as the Mexican axolotl, are some of the few vertebrates fortunate in their ability to regenerate diverse structures after injury. Unlike mammals they are able to regenerate a fully functional spinal cord after injury. However, the molecular circuitry required to initiate a pro-regenerative response after spinal cord injury is not well understood. To address this question we developed a spinal cord injury model in axolotls and used in vivo imaging of labeled ependymoglial cells to characterize the response of these cells to injury. Using in vivo imaging of ion sensitive dyes we identified that spinal cord injury induces a rapid and dynamic change in the resting membrane potential of ependymoglial cells. Prolonged depolarization of ependymoglial cells after injury inhibits ependymoglial cell proliferation and subsequent axon regeneration. Using transcriptional profiling we identified c-Fos as a key voltage sensitive early response gene that is expressed specifically in the ependymoglial cells after injury. This data establishes that dynamic changes in the membrane potential after injury are essential for regulating the specific spatiotemporal expression of c-Fos that is critical for promoting faithful spinal cord regeneration in axolotl.

Identification of Conserved and Novel MicroRNAs during Tail Regeneration in the Mexican Axolotl.

The Mexican axolotl salamander (Ambystoma mexicanum) is one member of a select group of vertebrate animals that have retained the amazing ability to regenerate multiple body parts. In addition to being an important model system for regeneration, the axolotl has also contributed extensively to studies of basic development. While many genes known to play key roles during development have now been implicated in various forms of regeneration, much of the regulatory apparatus controlling the underlying molecular circuitry remains unknown. In recent years, microRNAs have been identified as key regulators of gene expression during development, in many diseases and also, increasingly, in regeneration. Here, we have used deep sequencing combined with qRT-PCR to undertake a comprehensive identification of microRNAs involved in regulating regeneration in the axolotl. Specifically, among the microRNAs that we have found to be expressed in axolotl tissues, we have identified 4564 microRNA families known to be widely conserved among vertebrates, as well as 59,811 reads of putative novel microRNAs. These findings support the hypothesis that microRNAs play key roles in managing the precise spatial and temporal patterns of gene expression that ensures the correct regeneration of missing tissues.

Spinal cord regeneration: where fish, frogs and salamanders lead the way, can we follow?

Major trauma to the mammalian spinal cord often results in irreversible loss of function, i.e. paralysis, and current therapies ranging from drugs, implantations of stem cells and/or biomaterials, and electrically stimulated nerve regrowth, have so far offered very limited success in improving quality-of-life. However, in marked contrast with this basic shortcoming of ours, certain vertebrate species, including fish and salamanders, display the amazing ability to faithfully regenerate various complex body structures after injury or ablation, restoring full functionality, even in the case of the spinal cord. Despite the inherently strong and obvious translational potential for improving treatment strategies for human patients, our in-depth molecular-level understanding of these decidedly more advanced repair systems remains in its infancy. In the present review, we will discuss the current state of this field, focusing on recent progress in such molecular analyses using various regenerative species, and how these so far relate to the mammalian situation.

A Reproducible Spinal Cord Crush Injury in the Regeneration-Permissive Axolotl.

Following injury, axolotls are able to functionally regenerate their spinal cord, regaining both motor and sensory control. In contrast, humans respond to severe spinal cord injury by forming a glial scar, which prevents further damage but also inhibits any regenerative growth, resulting in loss of function caudal to the injury site. The axolotl has become a popular system to elucidate the underlying cellular and molecular events that contribute to successful CNS regeneration. However, the experimental injuries (tail amputation and transection) that are utilized in axolotls do not mimic the blunt trauma that is often sustained in humans. Here, we report a more clinically relevant model for spinal cord injuries in the axolotl using a weight-drop technique. This reproducible model allows precise control over the severity of the injury by regulating the drop height, weight, compression, and position of the injury.

LRRK2 kinase activity is necessary for development and regeneration in Nematostella vectensis.

Background: The starlet sea anemone, Nematostella vectensis, is an emerging model organism with a high regenerative capacity, which was recently found to possess an orthologue to the human LRRK2 gene (nvLRRK2). The leucine rich repeat kinase 2 (LRRK2) gene, when mutated, is the most common cause of inherited Parkinson's Disease (PD). Its protein product (LRRK2) has implications in a variety of cellular processes, however, the full function of LRRK2 is not well established. Current research is focusing on understanding the function of LRRK2, including both its physiological role as well as its pathobiological underpinnings. Methods: We used bioinformatics to determine the cross-species conservation of LRRK2, then applied drugs targeting the kinase activity of LRRK2 to examine its function in development, homeostasis and regeneration in Nematostella vectensis. Results: An in-silico characterization and phylogenetic analysis of nvLRRK2 comparing it to human LRRK2 highlighted key conserved motifs and residues. In vivo analyses inhibiting the kinase function of this enzyme demonstrated a role of nvLRRK2 in development and regeneration of N. vectensis. These findings implicate a developmental role of LRRK2 in Nematostella, adding to the expanding knowledge of its physiological function. Conclusions: Our work introduces a new model organism with which to study LRRK biology. We show a necessity for LRRK2 in development and regeneration. Given the short generation time, genetic trackability and in vivo imaging capabilities, this work introduces Nematostella vectensis as a new model in which to study genes linked to neurodegenerative diseases such as Parkinson's.

Comparison of Blastema Formation after Injury in Two Cephalopod Species.

Regeneration is the ability to functionally replace significant amounts of lost tissue or whole appendages like arms, limbs or tentacles. The amount of tissue that can be regenerated varies among species, but regeneration is found in both invertebrate and vertebrate animals. Cephalopods have been broadly reported in the literature to regenerate their arms. There are over 800 species of Cephalopod; however, regeneration has only been documented in the literature in a few species (1). Here we compare arm regeneration in two species of cephalopod, the Octopus bimaculoides and the hummingbird bobtail squid Euprymna berryi.

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.

Zebrafishing for enhancers of hearing regeneration.

The discovery of regeneration-specific enhancer elements has added an exciting player to the field of regeneration biology. In this issue of Cell Genomics, Jimenez et al.1 demonstrate the power of combining single-cell genomics with the genetically tractable zebrafish to identify modulators of adult hair cell regeneration.

Spinal cord regeneration - the origins of progenitor cells for functional rebuilding.

The spinal cord is one of the most important structures for all vertebrate animals as it connects almost all parts of the body to the brain. Injury to the mammalian spinal cord has devastating consequences, resulting in paralysis with little to no hope of recovery. In contrast, other vertebrate animals have been known for centuries to be capable of functionally regenerating large lesions in the spinal cord. Here, we will review the current knowledge of spinal cord regeneration and recent work in different proregenerative animals that has begun to shed light on the cellular and molecular mechanisms these animals use to direct cells to rebuild a complex, functional spinal cord.

The Axolotl's journey to the modern molecular era.

The salamander Ambystoma mexicanum, commonly called "the axolotl" has a long, illustrious history as a model organism, perhaps with one of the longest track records as a laboratory-bred vertebrate, yet it also holds a prominent place among the emerging model organisms. Or rather it is by now an "emerged" model organism, boasting a full cohort molecular genetic tools that allows an expanding community of researchers in the field to explore the remarkable traits of this animal including regeneration, at cellular and molecular precision-which had been a dream for researchers over the years. This chapter describes the journey to this status, that could be helpful for those developing their respective animal or plant models.

Salamanders: The molecular basis of tissue regeneration and its relevance to human disease.

Salamanders are recognized for their ability to regenerate a broad range of tissues. They have also have been used for hundreds of years for classical developmental biology studies because of their large accessible embryos. The range of tissues these animals can regenerate is fascinating, from full limbs to parts of the brain or heart, a potential that is missing in humans. Many promising research efforts are working to decipher the molecular blueprints shared across the organisms that naturally have the capacity to regenerate different tissues and organs. Salamanders are an excellent example of a vertebrate that can functionally regenerate a wide range of tissue types. In this review, we outline some of the significant insights that have been made that are aiding in understanding the cellular and molecular mechanisms of tissue regeneration in salamanders and discuss why salamanders are a worthy model in which to study regenerative biology and how this may benefit research fields like regenerative medicine to develop therapies for humans in the future.

miR-196 is an essential early-stage regulator of tail regeneration, upstream of key spinal cord patterning events.

Salamanders have the remarkable ability to regenerate many body parts following catastrophic injuries, including a fully functional spinal cord following a tail amputation. The molecular basis for how this process is so exquisitely well-regulated, assuring a faithful replication of missing structures every time, remains poorly understood. Therefore a study of microRNA expression and function during regeneration in the axolotl, Ambystoma mexicanum, was undertaken. Using microarray-based profiling, it was found that 78 highly conserved microRNAs display significant changes in expression levels during the early stages of tail regeneration, as compared to mature tissue. The role of miR-196, which was highly upregulated in the early tail blastema and spinal cord, was then further analyzed. Inhibition of miR-196 expression in this context resulted in a defect in regeneration, yielding abnormally shortened tails with spinal cord defects in formation of the terminal vesicle. A more detailed characterization of this phenotype revealed downstream components of the miR-196 pathway to include key effectors/regulators of tissue patterning within the spinal cord, including BMP4 and Pax7. As such, our dataset establishes miR-196 as an essential regulator of tail regeneration, acting upstream of key BMP4 and Pax7-based patterning events within the spinal cord.

AP-1cFos/JunB/miR-200a regulate the pro-regenerative glial cell response during axolotl spinal cord regeneration.

Salamanders have the remarkable ability to functionally regenerate after spinal cord transection. In response to injury, GFAP+ glial cells in the axolotl spinal cord proliferate and migrate to replace the missing neural tube and create a permissive environment for axon regeneration. Molecular pathways that regulate the pro-regenerative axolotl glial cell response are poorly understood. Here we show axolotl glial cells up-regulate AP-1cFos/JunB after injury, which promotes a pro-regenerative glial cell response. Injury induced upregulation of miR-200a in glial cells supresses c-Jun expression in these cells. Inhibition of miR-200a during regeneration causes defects in axonal regrowth and transcriptomic analysis revealed that miR-200a inhibition leads to differential regulation of genes involved with reactive gliosis, the glial scar, extracellular matrix remodeling and axon guidance. This work identifies a unique role for miR-200a in inhibiting reactive gliosis in axolotl glial cells during spinal cord regeneration.

Development of a 3D matrix for modeling mammalian spinal cord injury in vitro.

Spinal cord injury affects millions of people around the world, however, limited therapies are available to improve the quality of life of these patients. Spinal cord injury is usually modeled in rats and mice using contusion or complete transection models and this has led to a deeper understanding of the molecular and cellular complexities of the injury. However, it has not to date led to development of successful novel therapies, this is in part due to the complexity of the injury and the difficulty of deciphering the exact roles and interactions of different cells within this complex environment. Here we developed a collagen matrix that can be molded into the 3D tubular shape with a lumen and can hence support cell interactions in a similar architecture to a spinal cord. We show that astrocytes can be successfully grown on this matrix in vitro and when injured, the cells respond as they do in vivo and undergo reactive gliosis, one of the steps that lead to formation of a glial scar, the main barrier to spinal cord regeneration. In the future, this system can be used to quickly assess the effect of drugs on glial scar protein activity or to perform live imaging of labeled cells after exposure to drugs.

Genome-wide analysis reveals conserved transcriptional responses downstream of resting potential change in Xenopus embryos, axolotl regeneration, and human mesenchymal cell differentiation.

Endogenous bioelectric signaling via changes in cellular resting potential (V mem) is a key regulator of patterning during regeneration and embryogenesis in numerous model systems. Depolarization of V mem has been functionally implicated in dedifferentiation, tumorigenesis, anatomical re-specification, and appendage regeneration. However, no unbiased analyses have been performed to understand genome-wide transcriptional responses to V mem change in vivo. Moreover, it is unknown which genes or gene networks represent conserved targets of bioelectrical signaling across different patterning contexts and species. Here, we use microarray analysis to comparatively analyze transcriptional responses to V mem depolarization. We compare the response of the transcriptome during embryogenesis (Xenopus development), regeneration (axolotl regeneration), and stem cell differentiation (human mesenchymal stem cells in culture) to identify common networks across model species that are associated with depolarization. Both subnetwork enrichment and PANTHER analyses identified a number of key genetic modules as targets of V mem change, and also revealed important (well-conserved) commonalities in bioelectric signal transduction, despite highly diverse experimental contexts and species. Depolarization regulates specific transcriptional networks across all three germ layers (ectoderm, mesoderm, and endoderm) such as cell differentiation and apoptosis, and this information will be used for developing mechanistic models of bioelectric regulation of patterning. Moreover, our analysis reveals that V mem change regulates transcripts related to important disease pathways such as cancer and neurodegeneration, which may represent novel targets for emerging electroceutical therapies.