Development of a ZRS Reporter System for the Newt (Cynops pyrrhogaster) during Terrestrial Limb Regeneration

Newts, a type of urodele amphibian, offer remarkable insights into regenerative medicine due to their extraordinary tissue regeneration capabilities—a challenging feat in humans. During limb regeneration of adult newts, fascinating cellular and molecular processes are revealed, including scarless healing, de-differentiation of mature cells, and regeneration of limbs and digits. Sonic hedgehog (Shh), crucial for vertebrate limb development, is regulated by the zone of polarizing activity regulatory sequence (ZRS) in the limb bud zone of polarizing activity (ZPA). The metamorphosed (terrestrial) newt can reactivate Shh during regeneration, facilitating proper limb patterning. Cell types capable of regulating the ZRS in metamorphosed newts remain unknown. The identification of such cell types provides invaluable insight to novel regenerative mechanisms. Therefore, in this study, we developed the first newt ZRS reporter. First, we isolated and characterized the newt ZRS enhancer (nZRS), identifying conserved DNA binding sites. Several binding sites with medical relevance were conserved in the newt ZRS. In a functional analysis, we developed a system composed of a transgenic nZRS reporter newt and a new newt anti-Shh antibody, together allowing Shh monitoring during limb regeneration. This system provides a valuable in vivo approach for future genetic studies of patterning during limb regeneration.