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

Abstract

Background: In the Drosophila ovary, germ-line and soma cells are interconnected via gap junctions. The main gap-junction proteins in invertebrates are members of the innexin family. In order to reveal the role that innexins play in cell-cell communication during oogenesis, we investigated the localization of innexins 1, 2, 3 and 4 using immunohistochemistry, and analyzed follicle development following channel blockade.

Results: We found innexin 1 predominantly localized to the baso-lateral domain of follicle cells, whereas innexin 2 is positioned apico-laterally as well as apically between follicle cells and germ-line cells. Innexin 3 was observed laterally in follicle cells and also in nurse cells, and innexin 4 was detected in the oolemma up to stage 8 and in nurse-cell membranes up to stage 12. In order to test whether innexins form channels suitable for intercellular communication, we microinjected innexin antibodies in combination with a fluorescent tracer into the oocyte of stage-10 follicles. We found that dye-coupling between oocyte and follicle cells was largely reduced by innexin-2 antibodies directed against the intracellular C-terminus as well as against the intracellular loop. Analyzing in vitro, between stages 10 and 14, the developmental capacities of follicles following microinjections of innexin-2 antibodies revealed defects in follicle-cell differentiation, nurse-cell regression, oocyte growth and choriogenesis.

Conclusion: Our results suggest that all analyzed innexins are involved in the formation of gap junctions in the ovary. While innexins 2 and 3 are colocalized between soma cells, innexins 2 and 4 are colocalized between soma and germ-line cells. Innexin 2 is participating in cell-cell communication via hemichannels residing in the oolemma. It is obvious that gap-junctional communication between germ-line and soma cells is essential for several processes during oogenesis.

Figure 1

Inx1 is localized to baso-lateral follicle-cell membranes. A: Drosophila stage-10b follicle, showing different cell types and membrane regions relevant for the present study (blue: nuclei, stained with DAPI; red: F-actin, stained with rhodaminylphalloidin). B: Scheme of the predicted protein structure of innexins (white: intracellular domains (NT, N-terminus; CL, cytopasmic loop; CT, C-terminus), black: transmembrane domains, grey: extracellular domains). Hemichannels made of six molecules can be either homo- or heteromeric, channels made of two hemichannels either homo- or heterotypic. C: On immunoblots of ovary extracts using Inx1-antiserum (AInx1Rb-CT) a weak band at the calculated molecular mass of 46 kDa (asterisk) and two bands at 43 and 41 kDa are recognized. D: Inx1 is detected in a continuous pattern as well as in presumed gap-junction plaques at the lateral follicle-cell membranes (FC, arrows, stage 10a, surface view, WFF). E: Inx1 is restricted to the baso-lateral domain (arrowheads) of the follicle cells, i. e. the apical domain and the oolemma (asterisk) is not stained (stage 10a, optical median section, WFF). a, anterior; aFCM, apical follicle-cell membrane; bFCM, basal follicle-cell membrane; BM, basement membrane; cFC, centripetally migrating follicle cells; NC, nurse cells; Ooc, oocyte; Ool, oolemma.

Figure 2

Inx2 is localized to membrane plaques in follicle cells and germ-line cells and is distributed stage-specifically in nurse cells. On immunoblots of ovary extracts using Inx2-antisera (A: AInx2Rb-CT(KLRH), A': AInx2GP-CL) a band at the calculated molecular mass of 42 kDa (asterisk) and a band at 39 kDa are recognized. B: Inx2 is localized to presumed gap-junction plaques between follicle cells as well as between follicle cells and germ-line cells (stage 10b, AInx2Rb-CT(REM), optical median section, WFF; see also Fig. 4D). Black arrows point to apico-laterally situated plaques between follicle cells, white arrows point to plaques between follicle cells and oocyte, and arrowheads point to plaques between centripetally migrating follicle cells and nurse cells. C: Control follicle (NIS) showing the anterior-dorsal region of the oocyte and the follicular epithelium (comparable to the region shown in B). Different membrane regions are marked with white lines. Apical follicle-cell membranes make contact with the oolemma via microvilli spanning the developing vitelline membrane (VM). D, E: Inx2 is found in lateral and apical, but not in basal follicle-cell membranes (arrowheads, stage 11, Anti-Inx2GP-CL, D: LSM, E: optical tangential section, WFF). F: Inx 2 is located in the oolemma (arrowhead) as well as in nurse-cell membranes (white arrows, stage 8, Anti-Inx2GP-CL, optical median section, WFF). G-I: Stage-specific distribution of Inx2 in the nurse cells (AInx2Rb-CT(REM), WFF): During stage 10a (G), Inx2 accumulates around nurse-cell nuclei (NCN). During stage 10b (H), Inx2 becomes dispersed in the cytoplasm. During nurse-cell regression (stage 11, I), Inx2 is found in cytoplasmic clouds and in particles that become transported into the oocyte. FCN, follicle-cell nucleus (not stained); for further abbreviations, see legend to Fig. 1.

Figure 3

Inx3 is colocalized with Inx2 to membrane plaques in follicle cells and nurse cells. On immunoblots of ovary extracts using Inx3-antisera (A: AInx3GP-CT, A': AInx3Rb-CL) a weak band at the calculated molecular mass of 45 kDa (asterisk) and two bands at 35 and 30 kDa are recognized. B, C: Inx3 is localized to presumed gap-junction plaques between follicle cells (stage 10a). It is expressed in a continuous as well as punctate pattern at the lateral membranes (arrowheads, B: AInx3Rb-CL, WFF, C: AInx3GP-CT, LSM), but is missing at the apical membranes and at the oolemma (arrows). D, E: Inx2 (red, AInx2Rb-CT(REM)) and Inx3 (green, AInx3GP-CT) are colocalized in lateral follicle-cell membranes (yellow, arrows in D, stage 10a, LSM) as well as in nurse-cell membranes (yellow, arrow in E, stage 10b, LSM). F: In young follicles, Inx3 is located in plaques between nurse cells and follicle cells (arrows) and around nurse-cell nuclei (stage 7, AInx3Rb-CL, WFF). For further abbreviations, see legend to Fig. 1.

Figure 4

Inx4 is localized to membrane plaques in germ-line cells and seems to interact with Inx2 in follicle cells. A, B: Inx4-plaques (arrows in A (tangential section) and B (median section), stage 6, LSM) were detected between each follicle cell and the underlying oocyte and nurse cells, respectively. Continuous staining was found at the nurse-cell membranes (arrowhead in B). C: Inx4-plaques (black arrowheads, stage 10b, WFF) are located between nurse cells and the centripetally migrating follicle cells. D: Inx2 in follicle-cell membranes (green, AInx2GP-CL) and Inx4 in nurse-cell membranes (red, AInx4Rb-CT) are in close vicinity (arrowheads, stage 8, LSM) and seem to interact. Beginning with stage 8, only Inx2 is present between oocyte and follicle cells (arrow). For further abbreviations, see legend to Fig. 1.

Figure 5

Microinjected Inx2-antisera inhibit communication between oocyte and follicle cells. In 85% (H₂O dest.) and 48% (NIS), respectively, of the control follicles (stage 10), Lucifer Yellow CH was found to move from the microinjected oocyte (Ooc) into the nurse cells (NC) as well as into the surrounding follicle cells (arrowhead in inset A). In contrast, in 83% (AInx2Rb-CT) and 70% (AInx2GP-CL), respectively, of the follicles microinjected with Inx2-antisera, gap-junctional communication between oocyte and follicle cells was blocked (arrowhead in inset B). For further details, see Table 1.

Figure 6

Communication via Inx2-containing channels is essential for normal oogenesis in vitro. Of the microinjected control follicles, 70% (H₂O dest.) and 55% (NIS), respectively, developed in R-14 medium from stage 10b (0 h) to stages 12–14 (8 h, inset shows example). In contrast, in 76% (AInx2Rb-CT) and 73% (AInx2GP-CL), respectively, of the follicles microinjected with Inx2-antisera, oogenesis in vitro up to stage 12 was either incomplete or arrested in stage 10b (8 h, inset shows example). For further details, see Table 2.