Salamanders represent some of the first experimental models that were embraced internationally to study the mechanisms of embryonic development. Published works on urodeles date back to the early 1800s, but their popularity skyrocketed in the early 1900s as pioneering biologists such as Spemann embraced these easily-manipulated embryos to answer pivotal questions on the mechanisms of cell specification and tissue organization during development. Salamanders were also recognized early on for their profound regenerative abilities as adults, an aspect of urodele biology that makes them stand apart from all other tetrapods. Salamanders are a diverse group of animals, exhibiting distinct life cycles and habitats. Thus, they provide a unique platform to study the conserved properties of embryonic and post-embryonic development in a group of animals under the influence of many different selective pressures. In this special issue on Salamander Models, we have curated 11 research and review articles that embrace the diversity of these organisms and their biology.
In modern day, salamanders are by far and large used to study regenerative mechanisms with the conviction that these studies will 1 day lead to the development of regenerative therapies in humans. Regenerative mechanisms are well-represented in this special issue, with articles that explore limb, tail, lung, and liver regeneration. One question that has remained unanswered in the regeneration field is how, and whether, the mechanisms underlying limb regeneration change as the larval salamander develops into an adult. This concept is further explored in a perspective piece by Tribondeau et al that explores the commonalities and differences in salamander larva as they mature into paedomorphic or metamorphic adults.1 In a study of larval newt regeneration, Khan and Crawford used scanning electron microscopy to track larval and adult limb regeneration.2 The authors show the well-known phenomenon that urodeles first develop their digits in a preaxial dominant manner, which is opposite to the postaxial dominance in most tetrapod limbs. Limb regeneration in the larva also follows this mechanism, however, in adults the story changes. Digits emerge at the same time in regenerating adult newt limbs, suggesting they might not be determined in a preaxial manner. This intriguing finding warrants further studies to determine if a similar phenomenon occurs in other species. Subtle differences in limb regeneration have also been reported among the various species of urodeles, highlighting the importance of more diverse sampling from this order. With this in mind, Vieira et al teamed up with the US Fish and Wildlife Service in San Marcos Texas to characterize limb and tail regeneration in the Texas blind salamander (Eurycea rathbuni), a threatened cave-dwelling and aquatic species.3 In addition to charactering blastema formation and regeneration in this species for the first time, the team documented multiple aspects of this species biology including methods of sexing, mating behavior, and details of animal husbandry to encourage the research community to adopt this species as a regenerative model organism.
Salamander regeneration has been thoroughly investigated for over a century, but most studies have been performed on appendage regeneration such as limbs and tails. In an effort to understand the regeneration capacity of internal organs, Jensen et al, studied the regenerative capacity of the axolotl lung.4 By characterizing cell proliferation in a number of cell types following injury at the distal tip of the lung, the authors found that increased proliferation was equal throughout the injured lung suggesting an organ-wide response to injury. In limb regeneration, while proliferation has also been documented to occur at more distant sites,5 it is more focused at the site of injury compared to in lung regeneration. This difference exemplifies the need to investigate multiple regenerative processes to understand the shared and unique properties of repair in diverse situations. In line with this idea, Ohashi et al sets their focus on studying the process of liver regeneration and have discovered a novel mechanism of regeneration that the authors have named “compensatory congestion.”6 Liver regeneration in mammalian and fish systems occurs through the process known as compensatory growth, led by the activation of the cell cycle of the differentiated cell populations in the liver to replace the missing or damaged tissues. In contrast, compensatory congestion is the regenerative mechanism by which cell density is increased without the activation of liver development genes, or regrowth of the missing tissue. Thus, the means of liver regeneration appears to differ among vertebrates.
Two aspects that appear to apply to almost all regenerating systems is the essential role of epigenetic modifications and the inflammatory response. In a follow-up to their published work demonstrating histone deacetylase (HDAC) inhibition blocks axolotl tail regeneration, Baddar et al asked if co-treatment with Cobalt chloride, which can induce hypoxia and stress, can rescue this outcome.7 The authors discovered that while an extremely brief CoCl2 treatment post-amputation can mitigate the similarly brief treatment with an HDAC inhibitor, longer treatments led to deleterious consequences for regeneration. Using microarray analyses, they implicated 28 genes as being differentially regulated between tails treated with HDAC inhibitor alone vs in conjunction with CoCl2. Among these genes are many transcription factors, as well as genes implicated in stress and inflammatory responses, whose expression is normally induced in the very early stages of successful tail and limb regeneration. Immune system inputs and controls on regenerative processes are becoming increasingly appreciated in a variety of model systems, salamanders included. A thorough review, and insightful consideration of future directions, on the topic of how the immune system regulates regenerative responses in salamanders is presented by Bolanos-Castro et al.8
One fascinating aspect of salamander biology is the massive size of their genomes which can range anywhere between 14 and 120 Gb. This feature provides challenges to regenerative processes, but also may play a mechanistic role itself. For example, the question of how genomic stability is maintained in the highly proliferative blastema cell population during amphibian limb regeneration has become an area if intense interest. Garcia-Lepe et al have written a comprehensive review that lays out the exceptional challenge of repairing DNA-damage during regeneration, how regenerating cells respond to DNA damage, and the methods that cells use to cope with genomic instability when this challenge has not been met.9 Sessions and Wake present an intriguing hypothesis that genome size is linked to regenerative ability.10 The authors first pose that internal organ regeneration may be fundamentally different than blastema-based appendage regeneration, which was observed by Jensen et al and Ohashi et al in this special issue, but all utilize some reactivation of developmental programs. They pose that transposable element genome expansion in some species correlates with regeneration. Specifically, the large intron sizes in some salamanders leads to “intron delay” that slows the cell cycle, morphogenesis, and cellular responses. This intronic delay maintains many cells in an incomplete state of differentiation, potentially making them more capable of accessing developmental programs.
Related to the salamander genome, a new high-resolution study of the genetic composition of two axolotls in the Ambystoma Genetic Stock Center at the University of Kentucky, performed by Timoshevskaya et al provides the community with >700 million polymorphisms.11 They also compared the identified loci to orthologous sites in a wild-caught tiger salamander (Ambystoma tigrinum) sample and present evidence that many of the polymorphisms now present in laboratory axolotls may have arisen from crosses between axolotls and tiger salamanders in the stock center when the albino mutant line was established.
While salamanders are exceptional models to study regeneration, as we have previously stated, they have also been historically wonderful tools to understand the underpinnings of embryonic development. In the current issue, Davis et al focused on the eyeless mutant axolotl, which was originally discovered in the 1960s by Humphry.12 These mutants exhibit an arrest of eye morphogenesis at the optic-vesicle stage in addition to altered development of the forebrain. Only a handful of mutant axolotl strains have been discovered or generated to date, yet the recent sequencing of the axolotl genome has made it easier to identify the genetic causes for these mutant phenotypes. The authors discovered that the eyeless phenotype is caused by a frameshift mutation in the retinal and anterior neural fold homeobox (rax) gene.
Last, but not least, this special issue also features two articles that provide tools to better visualize diverse developmental processes in salamanders. Masselink and Tanaka have focused on improving our ability to understand the critical spatial aspects of regeneration at a cellular and molecular level, which is limited by imaging technologies, both the hardware and the methods used for preparing samples.13 They outlined some of the imaging work that has been done to date in salamander models. They also predicted new approaches for reducing bottlenecks in workflows for imaging whole tissues and even entire appendages. A new innovation in tissue clearing of axolotls is also presented in this issue by Adrados et al using a method called Salamander-Eci.14 This method builds upon previous clearing approaches,15, 16 but modified parameters to reduce tissue shrinkage that often occurs with previous Eci clearing approaches. They also show that Salamander-Eci is compatible with immunohistochemistry to stain specific cell types and click chemistry to image EdU-based cell proliferation. It is of significance that the samples can be stored long term in Salamander-Eci with little to no loss of fluorescence intensity, and that this method is much improved in is its speed, low cost, non-toxic solutions, and compatibility with staining for proteins and cell proliferation.
The recent advancements in technological and genomic tools that can be applied to salamander systems have allowed researchers to tackle many long-standing questions in development and regeneration, as well as identify new conceptual holes in our understating of their biology. The articles in this Special Issue showcase how developmental biology research in salamanders is rapidly advancing and represents the diverse expertise within this field. This will be the first part of a two-part series on Salamander models in developmental biology, evolution, and regeneration.