Wnt6 is required for germline stem cell self-renewal and differentiation via regulating adhesion junction and ERK signaling in Drosophila testis

Objective Spermatogenesis is a highly conserved and widespread process in eukaryotes, and Drosophila testes serve as an effective model system for its study. Many well- characterized signaling pathways are crucial for germline differentiation. Wnt proteins play important roles in regulating stem and progenitor cell proliferation and differentiation. In this study, we used the Drosophila model to investigate the function of Wnt6 in germline development. Methods Drosophila strains were maintained under specific conditions. Transgenic RNA interference (RNAi) lines were obtained from relevant sources, and fly crosses were carried out. Immunostaining was performed using specific primary and secondary antibodies, confocal images were acquired and analyzed, and statistical analysis was conducted. Results Knockdown of Wnt6 in early germline cells led to partial loss of germline stem cells (GSCs) and impaired GSC differentiation. Wnt6 regulated the adhesion junctions in the stem cell niche of Drosophila testes, as its knockdown reduced the expression of Arm and Rho1. Wnt6 deficiency in spermatogonia caused Arm- and Rho1- dependent adhesion junction impairment. Rac1 and Cdc42 were essential for GSC maintenance and differentiation, and Wnt6 regulated Rac1- and Cdc42- mediated adhesion junction signals. The loss of Wnt6, Rac1, and Cdc42 in Drosophila testes led to abnormal inactivation of dpERK. Conclusion Wnt6 plays a critical role in regulating GSC differentiation in the stem cell niche of Drosophila testes. Wnt6 regulates GSC self-renewal and differentiation through Rac1- and Cdc42- mediated adhesion junctions, highlighting the crosstalk between Wnt signaling and adhesion junctions. The regulation of germline stem cell self-renewal and differentiation by Wnt6 is closely associated with dpERK signaling. We hypothesize that Wnt6 regulates germ cell differentiation by influencing dpERK signaling through the adhesion junction function mediated by Rac1 and Cdc42.