Gap junctions mediate discrete regulatory steps during fly spermatogenesis.

Gametogenesis requires coordinated signaling between germ cells and somatic cells. We previously showed that Gap junction (GJ)-mediated soma-germline communication is essential for fly spermatogenesis. Specifically, the GJ protein Innexin4/Zero population growth (Zpg) is necessary for somatic and germline stem cell maintenance and differentiation. It remains unknown how GJ-mediated signals regulate spermatogenesis or whether the function of these signals is restricted to the earliest stages of spermatogenesis. Here we carried out comprehensive structure/function analysis of Zpg using insights obtained from the protein structure of innexins to design mutations aimed at selectively perturbing different regulatory regions as well as the channel pore of Zpg. We identify the roles of various regulatory sites in Zpg in the assembly and maintenance of GJs at the plasma membrane. Moreover, mutations designed to selectively disrupt, based on size and charge, the passage of cargos through the Zpg channel pore, blocked different stages of spermatogenesis. Mutations were identified that progressed through early germline and soma development, but exhibited defects in entry to meiosis or sperm individualisation, resulting in reduced fertility or sterility. Our work shows that specific signals that pass through GJs regulate the transition between different stages of gametogenesis. Author summary: Gap-junctions allow neighboring cells to communicate by connecting their cytoplasm. Gap-junctions play an essential role during sperm development by facilitating communication between the two cell types found in the testes, the germline which produces sperm, and the soma, which provides an essential supportive environment to the germline. We sought to better understand the ways in which gap-junctions help germline and somatic cells to communicate. We introduced nearly twenty different mutations into a gap-junction gene that connects the soma and germline in the fly testes. These mutations were chosen based on bioinformatics and analysis of the predicted structure of the gap-junction protein. We replaced the normal version of the gap-junction with the mutated versions in flies, and analysed how sperm development was affected. Based on this analysis we identified key parts of the protein that were required for the assembly and maintenance of the gap-junctions. Moreover, mutations designed to selectively disrupt the passage of specific materials through the gap-junction blocked different stages of sperm development. Mutations were identified that progressed through early sperm development, but exhibited defects in later stages, resulting in sterility. Our work shows that specific signals that pass-through gap-junctions regulate the transition between different stages of sperm development. [ABSTRACT FROM AUTHOR]