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.

Bi-directional gap junction-mediated soma-germline communication is essential for spermatogenesis.

Soma-germline interactions play conserved essential roles in regulating cell proliferation, differentiation, patterning and homeostasis in the gonad. In the Drosophila testis, secreted signalling molecules of the JAK-STAT, Hedgehog, BMP and EGF pathways are used to mediate soma-germline communication. Here, we demonstrate that gap junctions may also mediate direct, bi-directional signalling between the soma and germ line. When gap junctions between the soma and germ line are disrupted, germline differentiation is blocked and germline stem cells are not maintained. In the soma, gap junctions are required to regulate proliferation and differentiation. Localization and RNAi-mediated knockdown studies reveal that gap junctions in the fly testis are heterotypic channels containing Zpg (Inx4) and Inx2 on the germ line and the soma side, respectively. Overall, our results show that bi-directional gap junction-mediated signalling is essential to coordinate the soma and germ line to ensure proper spermatogenesis in Drosophila. Moreover, we show that stem cell maintenance and differentiation in the testis are directed by gap junction-derived cues.

A somatic permeability barrier around the germline is essential for Drosophila spermatogenesis.

Interactions between the soma and germline are essential for gametogenesis. In the Drosophila testis, differentiating germ cells are encapsulated by two somatic cells that surround the germline throughout spermatogenesis. chickadee (chic), the fly ortholog of Profilin, mediates soma-germline interactions. Knockdown of Chic in the soma results in sterility and severely disrupted spermatogenesis due to defective encapsulation. To study this defect further, we developed a permeability assay to analyze whether the germline is isolated from the surrounding environment by the soma. We find that germline encapsulation by the soma is, by itself, insufficient for the formation of a permeability barrier, but that such a barrier gradually develops during early spermatogenesis. Thus, germline stem cells, gonialblasts and early spermatogonia are not isolated from the outside environment. By late spermatocyte stages, however, a permeability barrier is formed by the soma. Furthermore, we find that, concomitant with formation of the permeability barrier, septate junction markers are expressed in the soma and localize to junctional sites connecting the two somatic cells that surround the germline. Importantly, knockdown of septate junction components also disrupts the permeability barrier. Finally, we show that germline differentiation is delayed when the permeability barrier is compromised. We propose that the permeability barrier around the germline serves an important regulatory function during spermatogenesis by shaping the signaling events that take place between the soma and the germline.