Inx4 (mutants of which are named zero population growth, zpg) is the only germ cell specific innexin and is expressed in germ line cells of both males and females (Tazuke et al, 2002). Putative loss of function zpg mutant adults have grossly reduced reproductive organs, produce no viable gametes, and are sterile (Tazuke et al, 2002). Therefore, Zpg performs an essential role and its loss cannot be compensated for by the other innexins expressed in the same tissue. One of the requirements of Zpg is for the maintenance of germ cells as they divide, move away from the germ line stem cell niche and initiate differentiation (Gilboa et al, 2003), however mutant phenotypes are also observed in associated somatic cells, possibly due to secondary effects. The distribution of Zpg and some of the other innexins (Ogre, Inx2 and Inx3) also expressed in the reproductive organs have been examined using immunocytochemistry (Bohrmann and Zimmermann, 2008), the results of which are summarized in the image (Inx1,2,3,4 distribution in the follicle). This study confirmed observations already made of Zpg, that it is localised to the plasma membrane of the oocyte (the oolemma) and in the cell membranes of nurse cells and is particularly prominent between germ cells and supporting somatic follicle cells (Tazuke et al, 2002, Gilboa et al, 2003). Importantly, the Bohrmann and Zimmermann immunocytological study places Zpg in the context of the other innexins present. So we can see that Zpg and Inx2 might interact in the oolemma and Zpg, Inx2 and Inx3 may interact in the plasma membranes of nurse cells. Ogre and Zpg probably do not interact given that Ogre protein was restricted to the baso-lateral end of follicle cells, the wrong end of the cell to interact with the Zpg containing oocyte and nurse cells. As far as communication between the oocyte and follicle cells goes, plaques consisting of Zpg and Inx2 exist in the oolemma (oocyte cell membrane) and potentially form complete gap junction channels by docking with hemichannels consisting solely of Inx2 (…or possibly Inx2+Inx3) at the apical end of follicle cells. Whether the oolemma hemichannels are homomeric, with each channel consisting solely of Zpg, or solely of Inx2, or are heteromeric consisting of mixed Zpg/Inx2 subunits, is not known. A "personal communication" note in Tazuke et al, 2002 reports that in the Xenopus oocyte system gap junction coupling occurs when Zpg is expressed in one oocyte and Inx2 in the neighbour (…going off on a tangent…this suggests that the presence of two cysteines in the extracellular loops of Inx2 and three cysteines in the extracellular loops of Zpg/Inx4 has no consequence regarding the docking of hemichannels to form heterotypic holochannels). Inx2 can also form functional holochannels on its own (Stebbings et al, 2000). Despite the obvious importance of Zpg during gametogenesis, Bohrmann and Zimmermann report that, as far gap junction mediated intercellular transfer of dye and embryonic development go, perturbation of gap junctions by injecting antibodies specific for Inx2 into the oocyte had far greater consequences than the injection of antibodies specific for Zpg or Inx3. Genetic experiments have shown previously that embryos produced from homozygous mutant inx2kropf(P16) germ line clones (Bauer et al, 2004) exhibit structural deformities. Bohrmann and Zimmermann note that the pattern of innexin distribution within the developing follicle is dynamic. Inx4 is found in the oolemma up to stage 8 (the start of vitellogenesis) but not afterwards and Inx2 is observed as cytoplasmic clouds in nurse cells after stage 10 but is transferred to the oocyte thereafter, as the nurse cells regress. This is comparable to the dynamic nature of Inx2 protein distribution in other cell types. For instance where the subcellular localisation differs between embryonic and pupal salivary gland cells and expression levels change between larval and pupal salivary gland cells. Little is known concerning the nature of the signals that pass through gap junction channels to mediate the diverse biological roles associated with them. The case of Zpg and egg development is no different. It is argued that the developmental defects observed in zpg mutants and Inx2-antibody-challenged oocytes are unlikely to arise due to a failure of nutrient transfer via gap junctions (Bohrmann and Zimmermann). It is possible that mutant gap junctions interfere with some, or all, of the many ligand/receptor signalling pathways (Lechner et al, 2007) that must function normally to produce viable eggs, although zpg mutant germaria can definitely receive a Decapentaplegic (dpp) mediated signal resulting in the phosphorylation of Mothers against decapentaplegic (mad) (Gilboa et al, 2003). Just because a signalling pathway is activated normally doesn't mean that it continues normally or will produce the correct output, however. There are a number of possible ways for mutant gap junctions to interfere with ligand/receptor pathways. Dominant mutants of inx2, Inx2TA181 and Inx2UA104 have been found to interact with alleles of the Na+ / K+ ATPase α and β subunits (the sodium pump) in genetic screens and the interaction generates phenotypes similar to weak Notch pathway mutants. The underlying mechanism is unknown but potentially involves disregulated ionic homeostasis or perturbed cell polarisation which alters ligand/receptor signalling. Interestingly, the zpgz-0918 allele has a point mutation near a highly conserved proline in the 2nd extracellular domain close to the substitution sites that generate the Inx2TA181 and Inx2UA104 alleles (Inx2/Inx4(Zpg) protein seq comparison). There has been no report of a dominant phenotype for zpgz-0918 (Tazuke et al, 2002), but that is not unexpected as the phenotypes associated with the dominant Inx2 alleles are relatively weak also. It would be interesting to know if zpgz-0918 genetically interacts with sodium pump components, not only to confirm the Inx2 observations but also potentially providing a good system (compact cells, self-contained system, known Innexin protein localisation, etc.) in which to elucidate the underlying interaction mechanism.
Drosophila innexins - zpg / inx4
(Publications prior to 2009 are based on data extracted from the textpresso text mining facility.)
Gap junctions in the ovary of Drosophila melanogaster: localization of innexins 1, 2, 3 and 4 and evidence for intercellular communication via innexin-2 containing channels., BMC Dev Biol., Bohrmann J, Zimmermann J., 2008, Nov 27;8:111.
Intercellular communication: the Drosophila innexin multiprotein family of gap junction proteins., Chem Biol., Bauer R, Loer B, Ostrowski K, Martini J, Weimbs A, Lechner H, Hoch M., 2005, 12(5):515-26
Germ line stem cell differentiation in Drosophila requires gap junctions and proceeds via an intermediate state., Development, Gilboa L, Forbes A, Tazuke SI, Fuller MT, Lehmann R., 2003, 130(26):6625-34
Gap junctions in Drosophila: developmental expression of the entire innexin gene family., Mech Dev., Stebbings LA, Todman MG, Phillips R, Greer CE, Tam J, Phelan P, Jacobs K, Bacon JP, Davies JA., 2002, 113(2):197-205
A germline-specific gap junction protein required for survival of differentiating early germ cells., Development, Tazuke SI, Schulz C, Gilboa L, Fogarty M, Mahowald AP, Guichet A, Ephrussi A, Wood CG, Lehmann R, Fuller MT., 2002, 129(10):2529-39
Innexins get into the gap., Bioessays, Phelan P, Starich TA., 2001, 23(5):388-96