Cell Tracing by a Multicolor Reporter Transgenic Iberian Ribbed Newt Pleurodeles waltl

Abstract

Living organisms exhibit varying regenerative abilities depending on the species. Among them, urodele amphibians have been widely used in regeneration biology due to their remarkable regenerative capacity. Iberian ribbed newts, in particular, have been established as a prominent model for regeneration research, offering advantages such as a large number of eggs spawned, a short period of sexual maturation, and the development of genetic manipulation techniques. Cell tracing is an essential method for deciphering cellular processes during organ regeneration. The multicolor reporter Brainbow, which stochastically manifests multiple fluorescent proteins based on the Cre/lox recombination system, has been utilized for clonal analysis in regenerative animal models. In this study, we aimed to utilize this valuable multicolor reporter in Iberian ribbed newts, which are gaining increasing importance as a regenerative animal model. We generated transgenic Iberian ribbed newts carrying the Brainbow3.0 reporter cassette under the control of the CAG (cytomegalovirus early enhancer/chicken beta-actin promoter/rabbit beta-globin splice acceptor) promoter. Cre recombinase induction via electroporation led to recombinant reporter expression in the brain, spinal cord, and muscle. Recombinant reporter-expressing cells could be traced in regenerating tail muscle, midbrain, and spinal cord. Additionally, we applied laser ablation to reporter-positive epithelial cells of Brainbow3.0 newts, enabling clonal analyses at the cellular level. We expect that this long-lasting multicolor reporter will prove versatile for a broad range of research fields.