The Secreted Protein Disulfide Isomerase Ag1 Lost by Ancestors of Poorly Regenerating Vertebrates Is Required for Xenopus laevis Tail Regeneration

Abstract

Warm-blooded vertebrates regenerate lost limbs and their parts in general much worse than fishes and amphibians. We previously hypothesized that this reduction in regenerative capability could be explained in part by the loss of some genes important for the regeneration in ancestors of warm-blooded vertebrates. One of such genes could be ag1, which encodes secreted protein disulfide isomerase of the Agr family. Ag1 is activated during limb and tail regeneration in the frog Xenopus laevis tadpoles and is absent in warm-blooded animals. The essential role of another agr family gene, agr2, in limb regeneration was demonstrated previously in newts. However, agr2, as well as the third member of agr family, agr3, are present in all vertebrates. Therefore, it is important to verify if the activity of ag1 lost by warm-blooded vertebrates is also essential for regeneration in amphibians, which could be a further argument in favor of our hypothesis. Here, we show that in the Xenopus laevis tadpoles in which the expression of ag1 or agr2 was artificially suppressed, regeneration of amputated tail tips was also significantly reduced. Importantly, overexpression of any of these agrs or treatment of tadpoles with any of their recombinant proteins resulted in the restoration of tail regeneration in the refractory period when these processes are severely inhibited in normal development. These findings demonstrate the critical roles of ag1 and agr2 in regeneration in frogs and present indirect evidence that the loss of ag1 in evolution could be one of the prerequisites for the reduction of regenerative ability in warm-blooded vertebrates.

Post-amputation reactive oxygen species production is necessary for axolotls limb regeneration

“…Of great relevance, our results show that blocking ROS production and exogenous H 2 O 2 affect Agr2 expression levels, which has been previously detected in Schwann cells during limb regeneration in axolotls (Kumar et al, 2010) and overexpression of this gene promotes blastema formation and denervated limb regeneration in newts (Kumar et al, 2007). Notably, limb denervation in Xenopus reduces the ROS production required for the regeneration of this structure and Agr2 inhibition reduces the regenerated tail area in this same animal model (Zhang et al, 2018;Ivanova et al, 2021). Additionally, during tail regeneration in salamanders, blocking ROS production reduces Agr2 expression levels and the final size of the regenerate (Carbonell M et al, 2021).…”

Potential Role of AGR2 for Mammalian Skin Wound Healing

“…Indeed, if Agr genes were blocked, the tadpole's tail regeneration was inhibited accompanied by mitotic activity and signaling pathway inhibition. Additionally, in contrast, if these genes were overexpressed in a temporary regeneration incompetent stage (refractory period), regeneration reactivation was observed [17,18]. These data point to the importance of AGR secretory PDI for regeneration stimulation and appear to be promising objects for a detailed study of their role in the skin of amniotes in normal conditions and during wound healing.…”

“…AGR proteins belong to the large evolutionary conserved family of proteins with disulfide isomerase activity (PDI) [15]. Initially found and characterized in the early development of Xenopus laevis, AGR genes were shown to be involved in the specification of dorsoanterior ectodermal fate, i.e., in the formation of the cement gland and induction of the forebrain fate of Xenopus [16,17]. According to the refined phylogeny, proposed in 2013, there are three subfamilies: AG1, AGR2 and AGR3 [18].…”

“…In the process of studying wound healing in amphibians, a weak immune response after injury was shown [1]. The EGF pathway and the expression of MMP9 are activated after damage in the blastema area [3,17]. In experiments with Xenopus laevis, a high level of expression of FGF8, 10 in the late blastema was shown [13].…”