Limb regeneration in a direct-developing terrestrial salamander, Bolitoglossa ramosi (Caudata: Plethodontidae): Limb regeneration in plethodontid salamanders.

Appendage regeneration is one of the most compelling phenomena in regenerative biology and is extensively studied in axolotls and newts. However, the regenerative capacity in other families of salamanders remains poorly described. Here we characterize the limb regeneration process in Bolitoglossa ramosi, a direct-developing terrestrial salamander of the plethodontid family. We (1) describe the major morphological features at different stages of limb regeneration, (2) show that appendage regeneration in a terrestrial salamander varies from other amphibians and (3) show that limb regeneration in this species is considerably slower than in axolotls and newts (95 days post-amputation for complete regeneration) despite having a significantly smaller genome size than axolotls or newts.

Distinctive adaptive response to repeated exposure to hydrogen peroxide associated with upregulation of DNA repair genes and cell cycle arrest.

Many environmental and physiological stresses are chronic. Thus, cells are constantly exposed to diverse types of genotoxic insults that challenge genome stability, including those that induce oxidative DNA damage. However, most in vitro studies that model cellular response to oxidative stressors employ short exposures and/or acute stress models. In this study, we tested the hypothesis that chronic and repeated exposure to a micromolar concentration of hydrogen peroxide (H2O2) could activate DNA damage responses, resulting in cellular adaptations. For this purpose, we developed an in vitro model in which we incubated mouse myoblast cells with a steady concentration of ~50µM H2O2 for one hour daily for seven days, followed by a final challenge of a 10 or 20X higher dose of H2O2 (0.5 or 1mM). We report that intermittent long-term exposure to this oxidative stimulus nearly eliminated cell toxicity and significantly decreased genotoxicity (in particular, a >5-fold decreased in double-strand breaks) resulting from subsequent acute exposure to oxidative stress. This protection was associated with cell cycle arrest in G2/M and induction of expression of nine DNA repair genes. Together, this evidence supports an adaptive response to chronic, low-level oxidative stress that results in genomic protection and up-regulated maintenance of cellular homeostasis.

Maintaining plethodontid salamanders in the laboratory for regeneration studies.

Limb regeneration studies have been extensively carried out in species of Ambystomatidae and Salamandridae families. So far limited research has been conducted in species belonging to the Plethodontidae family, where some of the species differs from other salamander families due to their direct development, thus absence of a larval life. Here, we describe a protocol to maintain the plethodontid salamanders of genus Bolitoglossa species under laboratory conditions to perform regeneration studies.