Germ cells require intimate associations and signals from the surrounding somatic cells throughout gametogenesis. The zero population growth (zpg) locus of Drosophila encodes a germline-specific gap junction protein, Innexin 4, that is required for survival of differentiating early germ cells during gametogenesis in both sexes. Animals with a null mutation in zpg are viable but sterile and have tiny gonads. Adult zpg-null gonads contain small numbers of early germ cells, resembling stem cells or early spermatogonia or oogonia, but lack later stages of germ cell differentiation. In the male, Zpg protein localizes to the surface of spermatogonia, primarily on the sides adjacent to the somatic cyst cells. In the female, Zpg protein localizes to germ cell surfaces, both those adjacent to surrounding somatic cells and those adjacent to other germ cells. It is proposed that Zpg-containing gap junctional hemichannels in the germ cell plasma membrane may connect with hemichannels made of other innexin isoforms on adjacent somatic cells. Gap junctional intercellular communication via these channels may mediate passage of crucial small molecules or signals between germline and somatic support cells required for survival and differentiation of early germ cells in both sexes (Tazuke, 2002).

Zpg is required during oogenesis for the survival of the germ line stem cell daughter as it moves away from the niche and begins to differentiate. Germ-line stem cells (GSCs) lacking Zpg can divide, but the daughter cell destined to differentiate dies. These results suggest that zpg may be necessary for the differentiation process itself, as well as for survival of differentiated germ cells, and that zpg probably acts in parallel to bam and bgcn. The differentiation of the GSC to a cystoblast is gradual, and it is suggested many of the germ cells in 'stem cell tumors' caused either by strong mutations in bam or by overexpression of Dpp may be at an intermediate state between GSCs and cystoblasts. These findings suggest that germ line stem cells differentiate upon losing contact with their niche, that gap junction mediated cell-cell interactions are required for germ cell differentiation, and that in Drosophila germ line stem cell differentiation to a cystoblast is gradual. (Gilboa, 2003).

Gap junctions are intercellular channels assembled from connexin (vertebrate) or innexin (invertebrate) subunits, six of which oligomerize to form a cylindrical hemichannel in the plasma membrane (Bruzzone, 1996; White, 1996; Phelan, 1998; Curtin, 1999). Hemichannels on two adjacent cell surfaces dock end-to-end to form gap junctions, which are commonly voltage gated and permit passage of ions and small molecules, such as nucleotides between the coupled cells. Vertebrates and invertebrates both have several gap junction protein isoforms, which can combine to form gap junctions with different permeability properties and regulation (Bruzzone, 1996; White, 1996; Phelan, 1998; Curtin, 1999). The zpg protein has been shown to form functional, voltage-gated, heterotypic gap junctions in the paired Xenopus oocytes system, with one oocyte expressing zpg and the partner oocyte expressing a different Drosophila gap junction protein, Inx2 (J. Davies, personal communication to Tazuke, 2002). Strikingly, functional channels did not form when both oocytes expressed the Zpg protein (J. Davies, personal communication to Tazuke, 2002), suggesting that Zpg forms heterotypic but not homotypic gap junctions (Tazuke, 2002).

In both sexes, the Zpg protein was detected on the surface of germ cells where they interface with adjacent somatic cells. Gap junctions have been observed at the ultrastructural level between germ cells and associated somatic cells in both sexes in insects including Drosophila (Szöllösi, 1980; Huebner, 1981; Adler, 2000). It is proposed that hemichannels made of Zpg on the surface of germ cells dock with hemichannels made of other innexin isoforms on the surface of somatic cells to form functional gap junctions. Of the eight innexins in the Drosophila genome (Curtin, 1999; Phelan, 2001), ogre, inx2, and inx3 have been found to be expressed in follicle cells (Stebbings, 2002). inx2 message is expressed at the apical tip of the testis and follicle cells of egg chambers. Furthermore, ESTs matching inx2, inx5 and ogre transcripts are found in adult testis cDNA library, suggesting that, in both sexes, other innexins are expressed in the Drosophila gonad, in addition to zpg. Heterotypic gap junctions between germline and soma, that are required for gametogenesis, are reminiscent of connexin-derived gap junctions in the mammalian gonad. The mammalian connexin Cx37 (Gja4), which is expressed on the mouse oocyte surface, is thought to form a heterotypic channel with a gap junction hemichannel containing Cx43 (Gja1), which is expressed on the surrounding somatic cumulus cells (Sutovsky, 1993; Juneja, 1999). Mice with targeted disruption of Cx37 have defects in follicular growth with premature granulosa cell luteinization, resulting in infertility (Simon, 1997). Zpg protein was also detected on the surfaces between adjacent germ cells, where it may form a hemichannel together with other innexin isoforms possibly expressed in germ cells in small amounts to give rise to functional gap junctions between adjacent germ cells. Alternatively, the Zpg protein at the interface between adjacent germ cells may not form functional channels (Tazuke, 2002).

The requirement for zpg function appears to be different in germ cells occupying the stem cell niche than in dividing cyst cells or spermatogonia, since stem cells were initially present in newly eclosed zpg-null animals. The striking loss of early germ cells at the onset of gamete differentiation in zpg-null animals raises the possibility that gap junctions may mediate passage of small molecule nutrients or signals from the surrounding somatic cells that are required for germ cell differentiation or survival. Gap junctional intercellular communication could be required for early stages of gamete differentiation, with germ cells undergoing cell death if unable to follow the normal differentiation program properly. The observation that spectrin-rich structures remain spherical and never reach the branched fusome stage, even in clustered germ cells resembling mitotic spermatogonia or cyst cells, suggests that the earliest stages of gamete differentiation are defective in zpg-null gonads. The spectrin-rich structures in the clustered zpg-null spermatogonia are larger than the usual spherical spectrosomes and often have abnormal morphology, suggesting that the differentiation program may initiate but fails to complete. Although zpg germ cells do not accumulate, no striking increase in Acridine Orange staining is detected in zpg gonads, suggesting that zpg germ cells may be rapidly lost after the onset of differentiation. Furthermore, the small number of germ cells present in a zpg mutant gonad is not due to failure in mitosis; germline stem cells appear to divide at the same frequency in newly eclosed zpg null mutant females as in wild type (Tazuke, 2002).

Interactions between early germ cells and somatic cells are known to play an essential role in early germ cell differentiation in both sexes. In males, for example, normal differentiation of spermatogonia from male germline stem cells requires a functional EGFR signaling pathway in the surrounding somatic cells. Later, after mitotic amplification of spermatogonial cells, activation in somatic cyst cells of a receptor in the TGFß signaling pathway is essential for germ cells to transition from the mitotic amplification program to spermatocyte growth, meiosis and spermiogenesis. In neither case have the crucial signals from somatic support cells to the germ cells they enclose been identified. The data on the mutant phenotype and the molecular identity of zpg gene product raise the possibility that crucial small molecule nutrients or signals regulating Drosophila germ cell differentiation and survival may be transmitted via gap junctions. Intriguingly, in mammals, gap junction permeability is regulated by EGFR pathway signaling via phosphorylation of the cytoplasmic tails of connexins by MAPK (Warn-Cramer, 1998). Activation of the EGFR in somatic cyst cells could signal to germ cells by changing the permeability of gap junctions for small molecule second messengers between germline and soma (Tazuke, 2002 and references therein).

Gap junctions in the Drosophila gonad may also mediate transfer of small molecule nutrients between germline and soma. Mammalian follicle cells have been shown to take up and phosphorylate labeled nucleotides from the culture medium, then release them to the oocyte (Heller, 1980), possibly via gap junctional intercellular channels. In developing egg chambers, Zpg protein was especially concentrated at the interface between each follicle cell and the underlying germ cell, consistent with the observation of gap junctions between germ cells and follicle cells of other insects by electron microscopy. Because Zpg function is required during the earlier steps of oogenesis, the precise function of Zpg-derived gap junctions in egg chambers could not be determined. However, electrical coupling and permeability to Lucifer Yellow dye, both characteristics of gap junctions, have been observed between germ cells and follicle cells in Drosophila and other insects (Woodruff, 1979; Huebner, 1981; Adler, 2000). Thus, it is possible that insect follicle cells also function to contribute to the growth of the oocyte by the uptake, metabolic conversion and intercellular transfer of small molecules via gap junctions (Tazuke, 2002).

Gap junctional communication between female germline stem cells and somatic apical cap cells may play a role in long term stem cell maintenance at the tip of the ovariole. Under specific staining conditions, zpg protein in female germline stem cells localized to a distinct dot adjacent to the spectrosome at the side where the germline stem cells abut the somatic apical cap cells. The terminal filament and cap cells at the apical tip of the germarium regulate germline stem cell behavior, in part through a signaling pathway involving the TGFß homolog, decapentaplegic. The loss of female germline stem cells with age in zpg mutants raises the possibility that gap junctional communication dependent on zpg might also be required to mediate signaling from apical cap cells for stem cell maintenance. Alternatively, gap junctions containing zpg may help maintain female germline stem cells in their niche by contributing to mechanical adhesion between stem cells and the apical cap cells, perhaps in conjunction with the adherens junctions observed adjacent to gap junctions between germline stem cells and adjoining cap cells (Tazuke, 2002 and references therein).