zero population growth: Biological Overview | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References
Gene name - zero population growth
Synonyms - inx4
Cytological map position - 65B5
Function - channel
Symbol - zpg
FlyBase ID: FBgn0024177
Genetic map position - 3L
Classification - innexin
Cellular location - transmembrane
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).
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 germline-soma communication. This study demonstrates that gap junctions may also mediate direct, bi-directional signalling between the soma and germline. When gap junctions between the soma and germline 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 germline and the soma side, respectively. Overall, the results show that bi-directional gap junction-mediated signalling is essential to coordinate the soma and germline to ensure proper spermatogenesis in Drosophila. Moreover, this study shows that stem cell maintenance and differentiation in the testis are directed by gap junction-derived cues (Smendziuk, 2015).
The work presented in this study demonstrates that gap junctions between the soma and germline are essential for fly spermatogenesis. Previous work showing an essential role for Zpg in the fly gonads raised the possibility that signals either from the soma or from other germ cells travel through gap junctions to regulate germline survival and differentiation. Subsequent work in the fly ovaries showed that Zpg was also required for GSC maintenance. This analysis supports and extends these conclusions by finding a cell-autonomous requirement for Zpg in GSC maintenance in the fly testis and demonstrates a role for Inx2 in the soma. Furthermore, it was found that gap junction-mediated signals from the germline also play unique and essential roles in the soma during spermatogenesis, independent of general germline defects. In particular, gap junctions are required to control the proliferation of CySCs and promote the differentiation of their daughters. This work illustrates that the main type of gap junction between the soma and the germline in the fly testis is a heterotypic channel coupling Inx2 in the soma and Zpg in the germline. Importantly, disrupting gap junctions in the soma by knocking down Inx2 phenocopies the zpg mutant phenotype in both the germline and soma. Therefore gap junction-mediated soma-germline regulation in the testis is bi-directional (Smendziuk, 2015).
Gap junctions contribute to stem cell regulation in the testis Recent work has highlighted the importance of gap junctions in stem cell regulation in a number of systems. In line with results from other stem cell models, the data illustrates a specific role for gap junctions in both GSCs and CySCs. The role of gap junctions in stem cell regulation in the testes was illustrated by the requirement for Zpg in the germline and Inx2 in the soma for GSC maintenance. Moreover, loss of Zpg or somatic knockdown of Inx2 also affected CySC proliferation. Furthermore, ultrastructural analysis revealed the presence of gap junctions between GSCs and CySCs. These results, as a whole, suggest a requirement for gap junction-mediated soma-germline communication in both stem cell populations and at the earliest stages of sperm differentiation (Smendziuk, 2015).
Gap junctions facilitate signalling between the soma and germline Following the stem cell stage, strong expression and co-localization of Zpg and Inx2 was consistently detected starting at the 4-cell cyst stage. Expression of Zpg and Inx2 began to diminish after the early spermatocyte stages and was not detected past meiotic stages. The timing at which Inx2 and Zpg expression were most prominent corresponds to a period during which niche signals such as BMP are lost. Loss of these signals causes the germline to undergo rapid differentiation and specialization. It has been shown that as somatic cells move away from the niche and begin differentiating, the soma forms a permeability barrier around the germline, isolating the germline from the outside environment. This transition corresponds with a switch occurs whereby soma-germline communication shifts from predominantly exocrine to juxtacrine signalling. Thus, as the germline becomes increasingly isolated, it becomes more dependent on differentiation signals that arrive via gap junctions from the soma. Once the germline becomes isolated, gap junctions may also play an important nutritive role and permit the movement of essential small metabolites between the germline and soma. Similarly, the soma requires gap junction-mediated signals to allow it to accommodate the increasingly expanded, differentiated, and specialized germline (Smendziuk, 2015).
The observations that gap junctions regulate germline differentiation and soma proliferation are in line with studies from both vertebrate and invertebrate models. In C. elegans, it was recently shown that gap junction-mediated signals are required to maintain GSCs in the niche and for germline differentiation (Starich, 2014). Similarly, work in vertebrates has shown that loss of gap junction-mediated signalling in the soma increased proliferation in post-mitotic Sertoli cells. It is therefore likely that an early role for gap junctions in coordinating soma-germline differentiation is an evolutionarily-conserved mechanism. One recurring feature of germline-soma gap junctions is the expression of different gap junction proteins, resulting in heterotypic gap junctions, exemplified by the Inx2-Zpg gap junctions observed in flies. A key problem in understanding the role of gap junctions in mediating soma-germline communication is identifying the transported signalling cargos. Some possible signals are cAMP, Ca2+, and cGMP, which have been implicated in regulating meiosis in the germline. Attempts to study cAMP and Ca2+ in the testis have proven inconclusive. However, recent work in Drosophila ovaries has suggested that somatic gap junctions may play roles in regulating pH, membrane potential, and ion transport. Overall, multiple signals are likely exchanged between the soma and germline through gap junctions and elucidating their respective functions is a complex task that should be further studied (Smendziuk, 2015).
Based on the results presented in this study, the following model is proposed: GSCs receive multiple cues that control their behaviour, with gap junctions mostly provide a supporting role, allowing the passage of cues from the soma that facilitate long-term GSC maintenance. After stem cell division germline undergoes rapid differentiation. The germline becomes increasingly isolated from the outside environment, and a permeability barrier is formed by the soma. As outside signals from the niche are lost, the germline relies more heavily on gap junctions to allow the passage of small molecules and metabolites from the soma to promote differentiation and provide nourishment. To ensure coordinated growth and differentiation of the soma and germline, signals pass from the germline through the gap junctions into the soma. Taken together, this work defines gap junction-mediated juxtacrine signalling as an additional signalling mechanism in the fly testis. Furthermore, this study provides a clear illustration of the bi-directional regulatory action of soma-germline gap junctions. As this study demonstratea, disrupting innexins in the soma or germline leads to a specific regulatory effect in the other tissue. Therefore bi-directional gap junction-mediated signalling plays a vital role in ensuring proper coordination of the soma and germline during spermatogenesis (Smendziuk, 2015).
The zpg locus encodes a 1.6 kb transcript detected in poly A+ mRNA from whole adult males and females but not from agametic animals. Consistent with the transcript being germline dependent, in situ hybridization to embryos revealed that zpg mRNA was concentrated in germ plasm and in the pole cells of wild-type embryos, from the syncitial blastoderm stage through gonad formation. Zpg protein was also detected in pole cells and primordial germ cells throughout embryogenesis. In wild-type testes, zpg mRNA was detected in the spermatogonial region near the apical tip. The level of zpg mRNA decreased sharply to background at the transition from spermatogonia to spermatocytes (Tazuke, 2002).
In testes, Zpg protein was expressed in spermatogonia and early spermatocytes, where it appears to be concentrated at the interface between germ cells and somatic cyst cells. The anti-Zpg antibody detected discrete patches of protein on the surface of early spermatogonia during the mitotic amplification stage. A similar pattern was seen in both larval and adult testes. The appearance of Zpg protein on the surface of early spermatogonia correlates with the stage at which early germ cells are lost in zpg mutant males. In later spermatogonial and early spermatocyte cysts, anti-Zpg staining is distributed more evenly over the germ cell surface but is especially concentrated at the outer surface of the germ cell cluster, where the germ cells interface with the enveloping somatic cyst cells. Staining with the anti-Zpg antibody becomes weaker and more diffuse during the subsequent primary spermatocyte stage. The spatiotemporal correlation between the appearance of Zpg protein on the surface of spermatogonia in wild-type testis and the defective differentiation and loss of spermatogonial cells in zpg mutant testes suggests that gap junctional communication between spermatogonia and somatic cyst cells may be required for normal differentiation and survival of spermatogonia (Tazuke, 2002).
In ovaries, Zpg protein is present on the surface of developing germ cells, at least up to stage 10 of oogenesis. In developing egg chambers, anti-Zpg antibody staining was particularly striking at the germ cell/somatic follicle cell interface, where under conditions of lighter staining, Zpg protein appeared to be concentrated on the germ cell surface in a discrete patch under each follicle cell. The distribution of Zpg protein appeared more continuous at the nurse cell/nurse cell interface. In the germarium, Zpg protein is detected on the surface of all germ cells, including stem cells. Zpg protein appears to be concentrated in discrete patches on the surface of dividing cysts, where germ cells are in contact with cytoplasmic extensions from the somatically derived inner germarium sheath cells (Tazuke, 2002).
In female germline stem cells, Zpg protein also appears to localize to a small plaque adjacent to the spectrosome at the interface between female germline stem cells and somatic apical cap cells, under conditions where less overall anti-Zpg staining was detected. In an experiment where wild-type ovaries were stained with anti-anti-Spectrin and anti-Zpg antibodies, this dot was detected in 258 of the 289 stem cells scored from 10 different ovaries (Tazuke, 2002).
The position of the spot of Zpg detected just apical to the spectrosome in female germline stem cells by immunofluorescence suggests the possibility that there are gap junctions between the female germline stem cells and the overlying somatic cap cells. The presence of gap junctions in early female germ cells was confirmed by ultrastructural studies. In two separate sets of serial sections through the spectrosome region of female germline stem cells, gap junctions with the characteristic 2x109 m (20 Å) intermembrane spacing were clearly evident between female germline stem cells and adjacent apical cap cells. It is not known whether these gap junctional structures between female germline stem cells and apical cap cells correspond to the spots of Zpg detected adjacent to the spectrosome by immunofluorescence, although their relative positions are the same. In addition, it was observed that the intercellular space between germline stem cells and apical cap cells directly abutting the spectrosome is large (>200 Å; >2x108 m) and filled with lanthanum when stained with this substance. The components of this distinctive space are not known, although the space was characteristic of the five germaria studied by electron microscopy. Adherens junctions were also seen between germline stem cells and apical cap cells. Gap junctions are also observed at the ultrastructural level between adjacent germline stem cells, between cystoblasts, between cysts, between cystoblasts and inner sheath cells, and between adjacent nurse cells. A cluster of multiple gap junction structures was visible by electron microscopy between follicle cells and underlying nurse cells in developing egg chambers, consistent with the patch of Zpg staining at the base of each follicle cell observed by immunofluorescence light microscopy. It is not know whether these gap junctional structures contain Zpg protein (Tazuke, 2002).
Fifteen of the 21 innexin (Inx) genes (Hve-inx) found in the genome of the medicinal leech, Hirudo verbana, are expressed in the CNS. Two are expressed pan-neuronally, while the others are restricted in their expression to small numbers of cells, in some cases reflecting the membership of known networks of electrically coupled and dye-coupled neurons or glial cells. When Hve-inx genes characteristic of discrete coupled networks were expressed ectopically in neurons known not to express them, the experimental cells were found to become dye coupled with the other cells in that network. Hve-inx6 is normally expressed by only three neurons in each ganglion, which form strongly dye-coupled electrical connections with each other [Shortening-Coupling interneuron (S-CI) network]. But when Hve-inx6 was ectopically expressed in a variety of central embryonic neurons, those cells became dye coupled with the S-CI network. Similarly, Hve-inx2 is normally uniquely expressed by the ganglion's large glial cells, but when it was ectopically expressed in different central neurons, they became dye coupled to the glial cells. In contrast, overexpression of the pan-neuronal Inx genes Hve-inx1 and Hve-inx14 did not yield any novel instances of dye coupling to pre-existent neuronal networks. These results reveal that expression of certain innexins is sufficient to couple individual neurons to pre-existing networks in the CNS. It is proposed that a primary determinant of selective neuronal connectivity and circuit formation in the leech is the surface expression of unique subsets of gap junctional proteins (Firme, 2013).
Wild-type function of the gene zero population growth is required for early steps in gamete differentiation in both sexes. Although animals carrying a null mutation in zpg were fully viable, they are sterile and have tiny gonads (Tazuke, 2002).
Testes from animals mutant for zpg contained only small numbers of early germ cells up to pre-spermatocyte stage. In wild-type adults, six to nine male germline stem cells lie in a rosette surrounding the cluster of somatic hub cells at the apical tip of the testis. Upon stem cell division, the daughter next to the hub maintains stem cell identity, while the other daughter becomes a gonialblast and initiates four rounds of synchronous mitotic division with incomplete cytokinesis to produce a cyst of 16 interconnected spermatogonial cells, which then differentiate into spermatocytes. Wild-type male germline stem cells and gonialblasts both have a spherical spectrin-rich subcellular structure, the spectrosome. By contrast, interconnected spermatogonia and spermatocytes have a linear and branching spectrin rich fusome. The tiny testes from newly eclosed (0-2 day old) zpg mutant males have only a small number of germ cells, based on immunostaining with germ cell-specific markers. The germ cells usually appear as single or small clusters of cells near the apical tip. Immunostaining revealed that these germ cells contained spherical spectrin rich structures, suggesting stem cell or gonialblast identity. Germ cells in clusters reminiscent of spermatogonia have round or slightly tapered spectrin-rich structures, rather than fusomes, which appear larger than the spectrosomes in stem cells. This suggested that the zpg null mutant spermatogonia attempt, but are unable to complete, differentiation (Tazuke, 2002).
Somatic support cells normally associated with early male germ cells are present in zpg mutant testes, although their morphological arrangement appears abnormal. In wild type, two types of somatic cells, the hub and cyst cells, are in intimate contact with the germ cells. The area and number of the hub cells at the apical tip often appears expanded in zpg-null males compared with wild type. Such abnormalities in the hub may be secondary to a defect in germ cells in zpg mutant testes, since similar abnormalities in hub morphology and cell number are present in testes lacking germ cells altogether. In wild type, a pair of somatic cyst progenitor cells enclose each germline stem cell. Their progeny, the somatic cyst cells, enclose the developing germ cells. Cyst progenitor and cyst cells are present in zpg-null testes, based on the appearance of GFP expressed in these cells under the control of a ptc-GAL4 driver. However, as only a few germ cells were present in the zpg mutant testes, many of the cyst cells do not appear to enclose germ cells, and so do not have the lacy appearance characteristic of cyst cells in wild-type testes (Tazuke, 2002).
Wild-type function of zpg was also required for differentiation of early germ cells in females. The tiny ovaries from newly eclosed zpg mutant females lacked the strings of developing egg chambers characteristic of wild type. Instead, germaria from freshly eclosed females commonly contain only a few germ cells, which appear as single cells at the apical tip of the germarium, located where female germline stem cells and cystoblasts reside. As in the male, female germ line stem cells and cystoblasts can be identified by spherical spectrin rich structures, spectrosomes, while the mitotically amplifying interconnected cysts contain branching fusomes. The germ cells remaining in zpg null mutant germaria have spherical spectrosomes rather than branched fusomes, suggesting stem cell or cystoblast identity. Occasionally, in freshly eclosed females, structures resembling egg chambers were observed (one per 2.9 ovaries, n=58 ovaries) further down the ovary, that contain abnormal number of germ cells that appear to be degenerating (Tazuke, 2002).
The phenotypic analysis of freshly eclosed zpg-null mutant ovaries suggests that Zpg function is not required for the initial placement of germ cells in the stem cell niche during ovary morphogenesis, although the possibility cannot be ruled out that a minute undetectable amount of maternally derived Zpg product perdures during the larval stages. Analysis of older zpg-null mutant females revealed that the number of germaria with early germ cells in the stem cell niche at the apical tip decreases with age. In three separate experiments, 70%-93% of germaria from newly eclosed zpg/Df females had at least one and usually two or more early germ cells in the stem cell position at the apical tip. By contrast, in 3-week-old zpg-null mutant females of the same genotype, only 17%-24% of germaria had even one Vasa-positive cell at the apical tip, when compared with 100% of wild-type control germaria. In some ovaries, ovarioles that lacked germ cells at the tip of the germarium had one or a few differentiating egg chambers further down the ovariole, as if germline stem cells were not maintained but instead initiated differentiation in the absence of zpg function in older females. The differentiating egg chambers in aged zpg females commonly appeared abnormal (Tazuke, 2002).
Most ovarioles from newly eclosed female flies that carry a strong mutation in the gene zpg have only a few germ cells, located at the anterior tip of the ovariole; the others contain no germ cells. zpg ovarioles that were occupied by germ line harbored 3.8 germ cells on average, as determined by anti-Vasa antibody labeling to mark the germ line. In comparison, wild-type germaria are filled with dividing cysts, the products of cystoblast divisions. The stem cell positions (close to cap cells) were occupied by zpg cells, but some single germ cells or clusters of germ cells were also located away from the tip. The single cells in the zpg mutant contained a round spectrosome, and could therefore be a mixed population of GSCs, cystoblasts or an intermediate between the two. To determine the developmental state of zpg germ cells, wild-type and zpg ovaries were double-labelled with anti-Vasa antibody and an antibody against cytoplasmic Bam protein (BamC), which stains cystoblasts and early cysts, but not GSCs. Of the 169 zpg ovarioles that had germ cells, only 2 contained a single cell that was BamC-positive. By contrast, 13 out of 50 wild-type ovarioles had a single cell stained with anti-BamC (26%). It is concluded that most zpg ovarioles lack cystoblasts (Gilboa, 2003).
In some zpg ovarioles clusters of 2-3 cells were observed that stained positive with anti-BamC antibodies. These may be the developing cysts that give rise to the rare egg chambers observed in these mutants (Tazuke, 2002). As in wild type, the rare developing zpg cysts are interconnected by ring canals. However, the fusome, which normally spreads through the dividing cyst, is either aberrant or absent. The rare egg chambers were almost always composed of less than 16 germ line cells, and DAPI staining indicated degeneration of the chamber. Since the fusome controls the divisions of the cyst, the abnormal number of germ cells in the egg chamber is likely to be the result of a severed fusome in the mutants (Gilboa, 2003).
Using an antibody raised against the cytoplasmic tail of Zpg (Tazuke, 2002), staining was observed in germ cells of every stage of development from GSCs to budding cysts of wild-type germaria. The staining appeared to be at the membrane of the cells and was somewhat stronger in region 2. The presence of Zpg staining in GSCs and dividing cysts correlates with the early defect in germ cell differentiation in zpg mutants and suggests that Zpg acts in the germ line (Gilboa, 2003).
The low number of cystoblasts in zpg ovarioles could be the result of defects in GSC division or in survival of the GSC daughter cell destined to differentiate. To distinguish between these two possibilities, zpg or wild-type germ cells were stained for the mitotic marker phospho-Histone H3. The percentage of marked zpg cells at the stem cell position per ovariole was not statistically different from that of wild-type GSCs. Similarly, the number of wild-type GSCs per ovariole in S phase (detected by BrdU labeling) was comparable to that of zpg cells. Taken together, these data show that zpg and wild-type cells spend a similar proportion of time in M and S phase. Although the possibility that zpg cells have an overall slower cell cycle, as compared to wild-type GSCs, cannot be ruled out, the data show that zpg cells divide. The lack of differentiating cells in zpg ovarioles may therefore indicate that zpg germ cells die when they commence differentiation (Gilboa, 2003).
To test this hypothesis, zpg germ cells were forced to differentiate by inducing expression of Bam protein in these cells, using a heat shock construct. In wild type, overexpression of Bam in GSCs induces the cystoblast differentiation program, resulting in a chain of egg chambers connected to a germarium depleted of germ line. When Bam was induced in zpg flies, no germ cells could be observed with anti-Vasa staining. Heat shock itself was not the cause of germ cell death, since ovaries of heat-shocked zpg animals, which did not carry the hs-bam transgene, still possessed germ cells. Thus, zpg germ cells die when forced to differentiate. This indicates that Zpg is required for the survival of differentiating germ cells. Taken together, these data indicate that zpg cells divide, and that the daughter cells that are destined to differentiate die (Gilboa, 2003).
To further explore the role of Zpg in early germ cell differentiation and survival, zpg alleles were recombined with mutant alleles of the gene pumilio (pum). pum mutant ovaries exhibit a compound phenotype. Many ovarioles lack germ line completely, a defect that may be attributed to preoogenic defects. Ovarioles with germ line have a germ line-depleted germarium connected to a few defective egg chambers. This phenotype suggests that Pum has roles in GSC maintenance. In ovaries from zpg, pum double-mutant females, many ovarioles were empty. This is consistent with the embryonic and larval requirement for pum. Ovarioles occupied by germ line exhibited a phenotype more similar to zpg than to pum mutants: few germ cells at the tip of the ovariole. Thus Zpg function is required for the differentiation of pum mutant germ cells. The apparent difference between the zpg, pum and hs-Bam; zpg phenotypes may reflect the different roles of Pum and Bam in GSC differentiation. Pum, as a translational repressor may permit GSC maintenance by repressing differentiation, which requires Zpg. By contrast, Bam may have a more instructive role in GSC differentiation, such that its overexpression can overrule GSC-maintenance cues emanating from the niche, independent of zpg (Gilboa, 2003).
In addition to defects in GSC maintenance, pum mutants also show defects in cyst development. This function is also evident in the zpg, pum phenotype. Although zpg, pum ovarioles occupied by germ line mostly resemble the zpg phenotype, they contain more germ cells, and have more dividing cysts and differentiating egg chambers, than those of the single zpg mutant. The double mutant had an average of 0.23 egg chambers per ovariole (n=290), compared with 0.02 (n=500) in flies homozygous for zpg and heterozygous for pum. The higher number of single cells and egg chambers in zpg, pum double mutants may indicate that Zpg function is less essential when cyst development is abrogated, as is the case in pum mutants (Gilboa, 2003).
This analysis suggests that Zpg wild-type function is required for the differentiation of GSCs. At least two other genes, bam and bgcn, are required for early germ cell differentiation. However, the phenotype of bam and bgcn mutant ovaries is strikingly different from that of zpg mutants. Ovaries mutant for bam or bgcn are filled with many undifferentiated single germ cells harboring a spherical spectrosome, which have been described as GSC tumors. By contrast, ovaries from zpg flies have only a few germ cells at the tip of the ovariole. To determine the functional relationship between these genes, flies were made doubly mutant for zpg with either bam or bgcn. Ovaries from newly eclosed females were stained to visualize the germ line and the spectrosomes. In the double mutant lacking both zpg and bam, only a few germ cells were detected at the ovariole tip. However, most double-mutant ovarioles had somewhat more germ cells than zpg ovarioles. Similar results were obtained with double mutants of zpg and bgcn (Gilboa, 2003).
Since GSCs can survive in a zpg background, the predicted phenotype of a zpg, bam or bgcn; zpg double mutant would be similar to a bam (or bgcn) phenotype (i.e. a germarium filled with undifferentiated GSCs). By contrast, the double-mutant phenotype more closely resembles the zpg phenotype. To test whether slow division of zpg cells accounts for the lack of tumors in young females, older (1- to 2-week-old) females were analyzed; a similar phenotype to that of young females was found. Thus, wild-type Zpg function is required for the accumulation of bam or bgcn mutant germ cell tumors removed from the niche (Gilboa, 2003).
To investigate further a possible role for Zpg-mediated intercellular communication in the development of germ cell tumors, the genetic interaction between dpp and zpg was analyzed. It has been proposed that an increased Dpp signal induces over-proliferation of GSC-like cells. It was therefore reasoned that an increased Dpp signal could induce zpg cells to over-proliferate. To test this, flies carrying several insertions of a heat-shock dpp transgene (hs-dpp) were crossed into a zpg background. Flies were heat shocked, and then dissected and stained to reveal the germ line and spectrosomes. Control animals heterozygous for zpg, carrying a subset of the hs-dpp transgenes, had more single germ cells with spherical spectrosomes than did wild type. Homozygous zpg animals, which carried at least the same number of hs-dpp transgenes as the control, did not show an increase in germ cell number. To test whether zpg cells could respond to a Dpp signal, ovaries of zpg animals were stained with antibodies against phosphorylated Mad (p-Mad). Mad is a component of the Dpp signal transduction pathway and is phosphorylated upon activation of the pathway. In wild type, p-Mad staining can be detected in GSCs, cystoblasts and dividing cysts in region 1 of the germarium. The highest level of staining is observed in early germ cells; staining then gradually declines towards the posterior. p-Mad is also detected in the single cells that accumulate following Dpp overexpression. Similarly, p-Mad staining is detected in zpg germ cells, suggesting that the mutant cells are able to receive the Dpp signal. There may be several explanations for the failure of zpg cells to proliferate or survive in response to a Dpp signal. (1) The Dpp pathway could be blocked downstream of Mad in zpg cells. (2) zpg cells may not be able to survive when unattached to the tip of the ovary. (3) Proliferating cells in hs-dpp flies, that move away from the niche, are in a more differentiated state than the cells in the niche, and therefore die in a zpg background (Gilboa, 2003).
bam tumor cells and germ cells proliferating after Dpp overexpression (hs-dpp tumor cells) are considered to be GSCs because of their round spectrosomes and lack of BamC staining. Yet, these cells do not accumulate in a zpg background. One possible explanation for this observation is that bam and hs-dpp cells, as they move away from the niche, are at an intermediate state (pre-cystoblast) between a stem cell and a cystoblast, and that cystoblast development and survival requires Zpg. To determine whether an intermediate state between GSCs and cystoblasts exists in wild type, ovarioles were triple-labeled with anti-Vasa, 1B1 monoclonal antibody and anti-BamC, to mark the germ line, spectrosomes and cystoblasts, respectively. BamC antibody stains cysts of 4 or 8 cells strongly. Two-cell cysts had notably weaker staining. Only rarely were cystoblasts, i.e., single cells, stained with anti-BamC. In many ovarioles, single cells with a spherical spectrosome were observed that were removed from the stem cell position yet did not stain for the cystoblast marker BamC. The number of cells were counted that carried a spherical spectrosome and did not stain with anti-BamC. These cells would comprise the GSC population plus the presumptive intermediate population. Of 100 ovarioles scored, most had between 3 and 5 single cells that did not stain with anti-BamC. The average number of these cells was 3.9. This is a greater number than the average number of GSCs that populate an ovariole (between 2 and 3), as determined by cell-lineage analysis and electron microscopy. These data support the hypothesis that an intermediate state between a stem cell and a cystoblast exists in wild type (Gilboa, 2003).
An intermediate cell population between slowly dividing stem cells and differentiating cells, described as transit amplifying cells, is a common feature in stem cell systems, including those giving rise to blood cells, skin and the gut epithelium. These are the products of stem cell division and have limited potential to divide prior to differentiation. In principle, the existence of a transit amplifying cell population in the Drosophila germ line could be suggested if the GSC division rate is too low to account for the number of cysts/egg chambers produced (including dying ones) in a set period of time. Acquiring this information, especially in region 1 of the germarium has proved to be difficult. Therefore the products of possible division of cells were directly examined at the transition state. If a pre-cystoblast cell amplifies, it should give rise to more than one egg chamber. By contrast, a cystoblast divides incompletely, giving rise to a two-cell cyst and, eventually, to one egg chamber. To distinguish between 'intermediate-state' divisions and cystoblast divisions, mosaic analysis was conducted in wild-type germaria using the FLP-FRT marker system, in which cells that are produced by mitotic recombination are marked as twin-spots by the copy number of the gene (lacZ), encoding the marker ß-Galactosidase (0 or 2 copies). Recombination in GSCs would result in a marked GSC, a string of marked cysts arising from its subsequent division, and one cyst that is the twin-spot of the original recombination event. A recombination event in the cystoblast would not be observed under the experimental conditions because marked cells within a cyst share the diffusable ß-Gal. It was reasoned that, if a cell at the transition state divides, twin-spot cysts would be observed even in germaria where GSCs are not marked. Such a situation could also arise if a marked GSC left the niche and differentiated. However, GSC loss is unlikely to affect this analysis, since the half-life of wild-type GSCs is 4.6 weeks, whereas the females were dissected 2-3 days after induction of Flipase. Of 177 ovarioles scored, 104 contained no marked GSCs and no marked cysts (~60%). 73 ovarioles contained marked cysts and a marked GSC (~40%). Ovarioles were observed that contained only marked cysts and marked GSCs. These results led to the conclusion that cells at the transition state in wild type do not divide at an appreciable rate but proceed to differentiate to a cystoblast. Thus, they do not constitute the equivalent of a transient amplifying population. By contrast, tumor cells in bam mutants, or when Dpp is overexpressed, continue to divide, since their differentiation is blocked (Gilboa, 2003).
Thus, zpg ovarioles contain single germ cells at the anterior tip. Most of these cells do not reach the cystoblast stage. Since zpg cells are not arrested at a particular stage of their cell cycle, and can divide, it is concluded that GSCs that lack zpg divide to give another stem cell and a daughter cell that dies at early stages of differentiation. Accordingly, overexpression of bam, which is necessary and sufficient to promote germ cell differentiation, kills zpg cells (Gilboa, 2003).
zpg encodes innexin 4, one of eight innexins in Drosophila (Phelan, 2001; Stebbings, 2002; Tazuke, 2002). Innexins are the functional homologs of the vertebrate connexins, or gap junction proteins (Phelan, 2001). In mammalian oogenesis, gap junctions have been implicated in cell-cell signaling. Early luteinization of granulosa cells is observed either when the oocyte is physically removed from immature wild-type follicles, or in mice lacking connexin 37 (Simon, 1997), suggesting a gap junction-mediated signaling mechanism between the oocyte and granulosa cells (Gilboa, 2003).
Zpg is required for the survival of the germ line stem cell daughter as it moves away from the niche and begins to differentiate. From its expression pattern, and the specificity of its function, it is clear that Zpg acts in a germ line autonomous way. However, it is not yet known whether Zpg facilitates communication between germ cells, or between germ line and soma. The germarium is a compact structure where early germ cells contact each other, the somatic cap cells contact GSCs, and inner-sheath cells contact GSCs and their daughters. Further study is needed to clarify which cells communicate with germ cells through Zpg-gap junctions. Likewise, the nature of the requirement for zpg in GSC differentiation is still uncertain. Gap junctions may be used to supply the GSC daughter cell with nutrients, or with a survival factor required for its subsequent growth. Alternatively, gap junctions may be used to deposit a factor that is required for the differentiation process itself, rather than for survival, while an accessory mechanism eliminates cells that begin differentiating without that factor. The major argument in favor of a role for Zpg in differentiation at the stem cell stage comes from the phenotypic analysis of zpg, pum double mutants in which, unlike in pum single mutants, GSCs do not differentiate. Although Pum-mediated repression is removed in the double mutants, GSCs cannot differentiate as they may lack a differentiation signal provided by Zpg. It is harder to imagine how nutritional deficiency per se could block differentiation of the double-mutant cells because single-mutant zpg cells do begin to differentiate (and then die) (Gilboa, 2003).
Recent studies suggest that the niche promotes stem cell fate through Dpp signaling. This may be achieved through repression of bam. GSCs are also tightly tethered to the niche via adherens junctions. Other, as-yet unidentified mechanisms may be used by the niche to protect GSCs (Gilboa, 2003).
Once germ cells leave the niche they activate the differentiation pathway. It is proposed that differentiation of GCSs to cystoblasts is not direct but proceeds via an intermediate state. Most wild-type cells, which by their position within the germarium were judged to be cystoblasts, are not stained with BamC antibody. This finding concurs with Ohlstein (1997), who observed cytoplasmic Bam just before the cystoblasts divide to form a two-cell cyst and proposed the existence of an intermediate/pre-cystoblast state between GSCs and cystoblasts. In other stem cell systems, the intermediate cell population has a biological function, namely increasing the progeny of a single stem cell division. The results indicate that in Drosophila females, cells at the intermediate state do not constitute a transit-amplifying cell population. However, the 'pre-cystoblast state' may have a different biological significance. A vacant niche can be filled by a neighboring GSC that divides 'sideways' instead of along the anteroposterior axis. An alternative for filling a vacant GSC position might be for a cell at the intermediate state to reoccupy the niche (Gilboa, 2003).
The suggested model, adding a transitory state between the stem cell and the cystoblast, raises an interesting question. Is the differentiation of a GSC to a cystoblast a continuous process or a discrete one? It is notable that none of the markers that are currently used is specific to the stem cell, the cystoblast or the intermediate. Bam is present (in its fusomal form) already in the stem cell, and BamC gradually accumulates in the cystoblast. Pumilio protein and phosphorylated Mad are also detected from GSCs to cystoblasts and early cysts. In other stem cell systems, including mammalian hematopoiesis, many intermediate cell types are known, and can be isolated by specific marker combinations. Due to the relative lack of markers, the isolation of these 'cell types' from Drosophila ovaries is currently impossible. The overlap of expression patterns of the proteins that are known to have a role in stem cell maintenance and differentiation may indicate that differentiation is gradual, and possibly reversible (Gilboa, 2003).
It has been suggested that in bam or bgcn mutant ovaries, or when Dpp is overexpressed, the germaria are filled with GSC tumor cells. The findings of an intermediate cell population in wild type raises the possibility that GSC tumor cells share some properties with precystoblasts. Both these populations are single, do not stain for BamC, but exist outside of the niche. Some support to the analogy between pre-cystoblasts and 'GSC tumors' is evident in the fact that the latter do not survive past the niche in a zpg background. Thus, the zpg double mutants allow two cell populations in 'GSC tumors' to be distinguish - cells that are inside or outside of the niche. It is suggest that when Dpp is overexpressed, or in bam/bgcn mutants, cells outside the niche cannot fully activate the differentiation program and are at an intermediate state between a GSC and a cystoblast. These cells die in a zpg background, whereas the tumor cells in contact with the niche survive. The results thus suggest that beyond Zpg gap junctions and Dpp signaling, there must be additional signaling between GSCs and the niche, which helps maintain GSCs. Additional markers are needed to determine unequivocally whether bam tumors are similar to Dpp tumors, and whether they share properties with wild-type precystoblasts (Gilboa, 2003).
Differentiation of the stem cell daughter requires gap junctions. It is assumed that zpg is acting in parallel to pum, dpp, bam and bgcn because the double mutants showed incomplete epistasis of zpg over each of these genes. Although a role for gap junction proteins has been established in mammalian oogenesis (Ackert, 2001; Carabatsos, 2000; Juneja, 1999; Simon, 1997), this is thought to be the first instance where a gap junction protein is shown to control stem cell differentiation. What passes through these gap junctions, and which cells are connected to GSCs through Zpg channels, is still unknown. One intriguing option is that Zpg forms part of the channels that connect GSCs to the surrounding somatic niche cells. If so, that would suggest that the niche in the Drosophila germarium is necessary, not only for stem cell maintenance, but also for stem cell differentiation (Gilboa, 2003).
C. elegans body-wall muscle cells are electrically coupled through gap junctions. Previous studies suggest that UNC-9 is an important, but not the only, innexin mediating the electrical coupling. This study analyzed junctional current (Ij) for mutants of additional innexins to identify the remaining innexin(s) important to the coupling. The results suggest that a total of six innexins contribute to the coupling, including UNC-9, INX-1, INX-10, INX-11, INX-16, and INX-18. The Ij deficiency in each mutant was rescued completely by expressing the corresponding wild-type innexin specifically in muscle, suggesting that the innexins function cell-autonomously. Comparisons of Ij between various single, double, and triple mutants suggest that the six innexins probably form two distinct populations of gap junctions with one population consisting of UNC-9 and INX-18 and the other consisting of the remaining four innexins. Consistent with their roles in muscle electrical coupling, five of the six innexins showed punctate localization at muscle intercellular junctions when expressed as GFP- or epitope-tagged proteins, and muscle expression was detected for four of them when assessed by expressing GFP under the control of innexin promoters. The results may serve as a solid foundation for further explorations of structural and functional properties of gap junctions in C. elegans body-wall muscle (Liu, 2013).
Neurons and glia of the medicinal leech CNS express different subsets of the 21 innexin genes encoded in its genome. The punctal distributions of fluorescently tagged innexin transgenes varies in a stereotypical pattern depending on the innexin expressed. Furthermore, whereas certain innexins colocalize extensively (INX1 and INX14), others do not (e.g., INX1 and INX2 or INX6). It was then demonstrated that the mutation of a highly conserved proline residue in the second transmembrane domain of innexins creates a gap junction protein with dominant negative properties. Coexpressing the mutated INX1 gene with its wild type blocks the formation of fluorescent puncta and decouples the expressing neuron from its normal gap junction-coupled network of cells. Similarly, expression of an INX2 mutant transgene (a glial cell innexin), blocks endogenous INX2 puncta and wild-type transgene puncta, and decouples the glial cell from the other glial cells in the ganglion. In cell culture dye-uptake and plasma membrane labeling experiments it was shown that the mutant innexin transgene is not expressed on the cell membrane but instead appears to accumulate in the cell's perinuclear region. Lastly, these mutant innexin transgenes were used to show that the INX1 mutant transgene blocks not only INX1 puncta formation, but also puncta of INX14, with which INX1 usually colocalizes. By contrast, the formation of INX6 puncta was unaffected by the INX1 mutant. Together, these experiments suggest that leech innexins can selectively interact with one another to form gap junction plaques, which are heterogeneously located in cellular arbors (Yazdani, 2013).
Notch (N) is a transmembrane receptor that mediates cell-cell interactions to determine many cell-fate decisions. N contains EGF-like repeats, many of which have an O-fucose glycan modification that regulates N-ligand binding. This modification requires GDP-L-fucose as a donor of fucose. The GDP-L-fucose biosynthetic pathways are well understood, including the de novo pathway, which depends on GDP-mannose 4,6 dehydratase (Gmd) and GDP-4-keto-6-deoxy-D-mannose 3,5-epimerase/4-reductase (Gmer). However, the potential for intercellularly supplied GDP-L-fucose and the molecular basis of such transportation have not been explored in depth. To address these points, the genetic effects of mutating Gmd and Gmer on fucose modifications were studied in Drosophila. These mutants were found to function cell-nonautonomously, and GDP-L-fucose was found to be supplied intercellularly through gap junctions composed of Innexin-2. GDP-L-fucose was not supplied through body fluids from different isolated organs, indicating that the intercellular distribution of GDP-L-fucose is restricted within a given organ. Moreover, the gap junction-mediated supply of GDP-L-fucose was sufficient to support the fucosylation of N-glycans and the O-fucosylation of the N EGF-like repeats. These results indicate that intercellular delivery is a metabolic pathway for nucleotide sugars in live animals under certain circumstances (Ayukawa, 2012).
Search PubMed for articles about Drosophila zero population growth
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Ayukawa, T., Matsumoto, K., Ishikawa, H. O., Ishio, A., Yamakawa, T., Aoyama, N., Suzuki, T. and Matsuno, K. (2012). Rescue of Notch signaling in cells incapable of GDP-L-fucose synthesis by gap junction transfer of GDP-L-fucose in Drosophila. Proc Natl Acad Sci U S A 109: 15318-15323. PubMed ID: 22949680
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Liu, P., Chen, B., Altun, Z. F., Gross, M. J., Shan, A., Schuman, B., Hall, D. H. and Wang, Z. W. (2013). Six innexins contribute to electrical coupling of C. elegans body-wall muscle. PLoS One 8: e76877. PubMed ID: 24130800
Ohlstein, B., Lavoie, C. A., Vef, O., Gateff, E. and McKearin, D. M. (2000). The Drosophila cystoblast differentiation factor, benign gonial cell neoplasm, is related to DExH-box proteins and interacts genetically with bag-of-marbles. Genetics 155: 1809-1819. 10924476
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date revised: 30 August 2015
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