In this second volume of Developmental Dynamics' two-part series featuring salamander research, contributions ranged from nomenclature conventions to tool building to developmental and regenerative biology research. The use of salamanders in biological research has grown significantly in recent years, which was on full display during the Annual Salamander Models in Cross-disciplinary Biological Research held virtually in August 2021. In this issue, Masselink et al1 provided an informative overview of this meeting, attended by over 200 people across the globe.
Two articles aimed to bring consistency in gene and transgenic nomenclature across the salamander research community. Nowoshilow et al2 presented guidelines for gene nomenclature in the axolotl, Ambystoma mexicanum, to be consistent with other organisms used in research. In addition, they presented a strategy that identifies the origin of a transgenic axolotl line and distinguishes between random insertion, knockout, or knockin transgenic lines. Yun et al3 proposed adopting the same nomenclature in the Iberian ribbed newt, Pleurodeles waltl. Together, these two articles should increase consistency across studies using salamanders. As an example of the need for transgenic animal naming guidelines, Tilley et al4 presented an overview of all current axolotl lines. They also described the advantages and challenges of generating transgenic axolotl lines.
A group of articles in this special issue focus on the popular theme of regeneration in salamanders. The review by Sader et al5 explores the roles of members of the TGF-b superfamily and downstream targets during wound healing, scarring (in mammals), and salamander limb regeneration. The authors explain how TGF-b signaling plays a key role in the preparation stage of limb regeneration, while the role of BMP signaling is during the redevelopment stage and propose a new model to explain the shift from TGF-b to BMP signaling in the regenerating limb tissue. One of the key activities of TGF-b signaling in the regenerate is to promote cell proliferation, and the study by Carbonell et al6 also focused on the signals that underlie this crucial cellular process. The requirement for reactive oxygen species (ROS) in cell proliferation and outgrowth of regenerating axolotl tails has been recently shown by Al Haj Baddar et al.7 In this issue, Carbonell et al6 build on these previous studies by focusing on a key ROS, H2O2, and its role in tail regeneration during different life stages of the axolotl. H2O2 promotes redox signaling, and its activities have been tied to multiple cellular activities such as proliferation and apoptosis during embryonic and regenerative processes. Using a series of in vivo assays the authors discovered that the inhibition of NOX-dependent ROS production inhibited tail regeneration, and this effect was rescued by treatment with exogenous H2O2. H2O2 treatment not only promoted cell proliferation, but also the recruitment of immune cells, which have been previously shown to play an essential role in blastema formation.8 These activities appear to be linked to the impact of H2O2 on the thioredoxin nAG/AGR2 and Yap1 pathways.
Given the extremely large size of the axolotl genome and that the blastema is a highly proliferative population of cells, limb regeneration relies on mechanisms that combat for replicative stress. The article by Garcia-Lepe et al9 in the current issue pursued the mechanisms by which regenerating cells overcome DNA damage and repair, a major component of replicative stress. Recent studies have highlighted the importance of DNA damage and repair in cell cycle progression during limb regeneration.10 Garcia-Lepe et al build on these previous studies by focusing on components of the homologous recombination repair pathway, Rad51 and MRE11. Using biochemical assays, they validated that both Rad51 and Mre11 bind directly to DNA and that Mre11 presents nuclease activity, as the human homologs do. They also found that Rad51 and Mre11 transcripts are both expressed during the earlier stages of blastema development and that chemical inhibition of both Rad51 and Mre11 at these early stages results in increased DNA damage and senescent cells and a delay in regenerative progression. These studies exhibit the outcome that occurs when the regenerating tissue is unable to overcome the challenges associated with replicative stress.
As we continue to understand the basic biology of salamander limb regeneration, we inch closer to understanding the differences in the mammalian responses to injury. The article by Debuque et al11 in this issue focused on these differences in the immune responses that follow different types of injuries in salamanders and mammals. While immune cells are required for the induction of the regenerative limb blastema,8 the immune response is also a key player in fibrosis and scarring. Debuque et al11 focused on understanding the molecular basis of the immune response in regeneration-permissive injuries in sterile and nonsterile environments. Toll-like receptor signaling is essential for sensing and regulating the immune responses to both injury and infection. The authors discovered that invading macrophages are a major (TLR) bearing cell subset, which responds to both sterile and nonsterile injuries in the axolotl. Delving into the molecular biology of this response, the authors found multiple nonsterile injury-dependent signaling responses through TLR that are also conserved in mammals. The authors then utilized a novel in vitro assay to compare signal transduction in mammalian and axolotl monocytes, which revealed differential in vitro responses to ligands derived from tissue injury in the presence of pathogen antigens. This study highlights an important difference between the axolotl and mammalian responses to injury and opens the door for understanding how TLR signaling pathways regulate the inflammatory response that is unique to injured axolotl tissues.
Several articles and reviews also built on scientific understanding of development in salamander models as it relates to animal age, size, and life cycle transitions. A wonderful review article from Adamson et al12 brings us through the past, present, and future of biological research on the axolotl model. Additionally, two studies using this model leveraged the response of axolotls to exogenous l-thyroxine stimulation, whereby the typically aquatic axolotls transition to a metamorphic form. Metamorphosed axolotls exhibit anatomical changes such as lost gills and reshaped tails, and they adopt a terrestrial lifestyle. Exploring the anatomical consequences of induced metamorphosis in axolotls, Olejnickova et al13 focused on the heart. While some physiological features such as heart rate and ventricular activation time remained constant between pedomorphs (premetamorphosis) and metamorphs (postmetamorphosis), other attributes changed substantially. For example, the atrioventricular canal was shown to be substantially thicker in metamorphs versus pedomorphs. Additionally, ventricular trabeculae exhibited a thick, sheet-like architecture in metamorphs, whereas this tissue appeared to have a more random and “spongy” architecture in pedomorphs. Physiologically, metamorphs displayed faster conduction speed compared to the pedomorphic counterparts. More research on how metamorphosis impacts the anatomy, physiology, and regenerative responses of salamanders may provide essential clues for how regenerative prowess can be impacted by factors such as life cycle and maturation.
Expanding on this theme, Riquelme-Guzman et al14 analyzed morphological characteristics of the appendicular skeleton in axolotls during normal maturation and aging and postinduced metamorphosis. Their research indicates that their growth rates, as measured by two different metrics of body length, generally follow a “two-line” model whereby rapid growth occurs until around 10 months of age and slow growth occurs thereafter. Intriguingly, they also show that axolotls may continue to grow in length as late as 20 years of age, supporting the anecdotal claim that axolotls may be indeterminant growers since they did not detect evidence of growth plateau even in the oldest animals they sampled. This work also showed that long bones in the axolotl zeugopod undergo progressive ossification, with the earliest onset of detectable changes starting at 4 cm (snout-tail length) and overt ossification signs continuing through 20 cm. Bone volume was also analyzed using microCT and showed parallel kinetics. The same work also provided images of adipocyte-filled marrow cavities and vascular architecture within axolotl bones. Using transgenic knockin lines, they also reported that while Col1a2 marks all skeletal cells, Sox9 is specific to chondrogenic cells in axolotl. Fate-mapping revealed the Col1a2-expressing cells serve as progenitors for both marrow cells and bone cells in the fully developed axolotl skeleton. They also documented that forced metamorphosis of axolotls leads to accelerated changes in the appendicular skeleton, with metamorphosed animals exhibiting increased bone volume in both the radius and ulna compared to age-matched pedomorphs.
If life cycle transitions such as the ones described in Olejnickova et al13 and Riquel-Guzman et al14 intrigue you, you will also enjoy two articles by Bonnet et al15 and Bonnet and Ledbetter16 in the current issue. Bonnet et al review explored the various causes of life cycle transitions in salamander species and the link between these transitions with body size, genome size, and skeletal evolution. The perspective by Bonnet and Ledbetter explored the differences between pedomorphic amphibians and those that complete their transformation into a terrestrial form and the links between these differences and life cycle evolution.
Finally, in a rare and inspiring example of a single-author article, Solovieva17 made the most of the time at home many researchers experienced due to Covid-19 lab restrictions. She analyzed the life cycle of the newt Lissotriton vulgaris, commonly known as the smooth newt, with widespread distribution across Europe. This work is beautifully executed and provides a modern, thorough, baseline/fundamental description of this species, complete with a photographic embryology series. It also features developmental rate plots in aggregate and for individual examples. Solovieva also reported descriptive features of the habitat and measurements of key environmental factors in their habitat, such as water and air temperature. Her article is likely to remind many readers of how they were inspired to become biologists from their own backyard explorations, and it may revive interest in experimentation with other salamander species beyond those now in common laboratory use.