β-Thymosin is an essential regulator of stem cell proliferation and neuron regeneration in planarian (Dugesia japonica)

β-Thymosin is a multifunctional peptide ubiquitously expressed in vertebrates and invertebrates. Many studies have found β-thymosin is critical for wound healing, angiogenesis, cardiac repair, hair regrowth, and anti-fibrosis in vertebrates, and plays an important role in antimicrobial immunity in invertebrates. However, whether β-thymosin participates in the regeneration of organisms is still poorly understood. In this study, we identified a β-thymosin gene in Dugesia japonica which played an important role in stem cell proliferation and neuron regeneration during the tissue repair process in D. japonica. Sequencing analysis showed that β-thymosin contained two conserved β-thymosin domains and two actin-binding motifs, and had a high similarity with other β-thymosins of invertebrates. In situ or fluorescence in situ hybridization analysis revealed that Djβ-thymosin was co-localized with DjPiWi in the neoblast cells of intact adult planarians and the blastema of regenerating planarians, suggesting Djβ-thymosin has a potential function of regeneration. Disruption Djβ-thymosin by RNA interference results in a slightly curled up head of planarian and stem cell proliferation defects. Additionally, we found that, upon amputation, Djβ-thymosin RNAi-treated animals had impaired regeneration ability, including impaired blastema formation, delayed eyespot formation, decreased brain area, and disrupted central CNS formation, implying Djβ-thymosin is an essential regulator of stem cell proliferation and neuron regeneration.