Eggs over easy: cell death in the Drosophila ovary

Programmed cell death is the most common fate of female germ cells in Drosophila and many animals. In Drosophila, oocytes form in individual egg chambers that are supported by germline nurse cells and surrounded by somatic follicle cells. As oogenesis proceeds, 15 nurse cells die for every oocyte that is produced. In addition to this developmentally regulated cell death, groups of germ cells or entire egg chambers may be induced to undergo apoptosis in response to starvation or other insults. Recent findings suggest that these different types of cell death involve distinct genetic pathways. This review focuses on progress towards elucidating the molecular mechanisms acting during programmed cell death in Drosophila oogenesis.     

Eyes absent,a key repressor of polar cell fate during Drosophila oogenesis

Throughout Drosophila oogenesis, specialized somatic follicle cells perform crucial functions in egg chamber formation and in signaling between somatic and germline cells. In the ovary, at least three types of somatic follicle cells, polar cells, stalk cells and main body epithelial follicle cells, can be distinguished when egg chambers bud from the germarium. Although specification of these three somatic cell types is important for normal oogenesis and subsequent embryogenesis, the molecular basis for establishment of their cell fates is not completely understood. Our studies reveal the gene eyes absent (eya) to be a key repressor of polar cell fate. EYA is a nuclear protein that is normally excluded from polar and stalk cells, and the absence of EYA is sufficient to cause epithelial follicle cells to develop as polar cells. Furthermore, ectopic expression of EYA is capable of suppressing normal polar cell fate and compromising the normal functions of polar cells, such as promotion of border cell migration. Finally, we show that ectopic Hedgehog signaling, which is known to cause ectopic polar cell formation, does so by repressing eya expression in epithelial follicle cells.     

Innexin2 gap junctions in somatic support cells are required for cyst formation and for egg chamber formation in Drosophila

Germ cells require intimate associations with surrounding somatic cells during gametogenesis. During oogenesis, gap junctions mediate communication between germ cells and somatic support cells. However, the molecular mechanisms by which gap junctions regulate the developmental processes during oogenesis are poorly understood. We have identified a female sterile allele of innexin2 (inx2), which encodes a gap junction protein in Drosophila. In females bearing this inx2 allele, cyst formation and egg chamber formation are impaired. In wild-type germaria, Inx2 is strongly expressed in escort cells and follicle cells, both of which make close contact with germline cells. We show that inx2 function in germarial somatic cells is required for the survival of early germ cells and promotes cyst formation, probably downstream of EGFR pathway, and that inx2 function in follicle cells promotes egg chamber formation through the regulation of DE-cadherin and Bazooka (Baz) at the boundary between germ cells and follicle cells. Furthermore, genetic experiments demonstrate that inx2 interacts with the zero population growth (zpg) gene, which encodes a germline-specific gap junction protein. These results indicate a multifunctional role for Inx2 gap junctions in somatic support cells in the regulation of early germ cell survival, cyst formation and egg chamber formation. Inx2 gap junctions may mediate the transfer of nutrients and signal molecules between germ cells and somatic support cells, as well as play a role in the regulation of cell adhesion.     

Stage-specific localization of the small heat shock protein Hsp27 during oogenesis inDrosophila melanogaster

The developmental and heat shock-induced expression of the small heat shock protein Hsp27 was investigated by confocal microscopy of whole-mount immunostained preparations of ovarioles during oogenesis inDrosophila melanogaster. In unstressed flies, Hsp27 was mainly associated with germline nurse cells throughout egg development. A small group of somatic follicle cells also expressed Hsp27 specifically at stages 8 to 10 of oogenesis. Interestingly, this Hsp showed a different intracellular localization depending on the stages of egg chamber development. Thus Hsp27 was localized in the nucleus of nurse cells during the first stages of oogenesis (from germarium to stage 6) whereas it showed a perinuclear and cytoplasmic localization from stage 8. After a heat shock, Hsp27 accumulated in somatic follicle cells surrounding the egg chamber whereas the expression of this small Hsp did not seem to be enhanced in nurse cells. The stage-dependent pattern of intracellular localization of Hsp27 observed in nurse cells of unstressed flies was also observed following heat shock. At late stages of oogenesis, Hsp27 also showed a perinuclear distribution in follicle and nurse cells after heat stress. These observations suggest that different factors may modulate the expression and intracellular distribution of Hsp27. This modulation may be associated with the specific activities occurring in each particular cell type throughout oogenesis during both normal development and under heat shock conditions. Edited by: E.R. Schmidt     

The salvador-warts-hippo pathway is required for epithelial proliferation and axis specification in Drosophila

In Drosophila, the body axes are specified during oogenesis through interactions between the germline and the overlying somatic follicle cells [1-5]. A Gurken/TGF-alpha signal from the oocyte to the adjacent follicle cells assigns them a posterior identity [6, 7]. These posterior cells then signal back to the oocyte, thereby inducing the repolarization of the microtubule cytoskeleton, the migration of the oocyte nucleus, and the localization of the axis specifying mRNAs [8-10]. However, little is known about the signaling pathways within or from the follicle cells responsible for these patterning events. We show that the Salvador Warts Hippo (SWH) tumor-suppressor pathway is required in the follicle cells in order to induce their Gurken- and Notch-dependent differentiation and to limit their proliferation. The SWH pathway is also required in the follicle cells to induce axis specification in the oocyte, by inducing the migration of the oocyte nucleus, the reorganization of the cytoskeleton, and the localization of the mRNAs that specify the anterior-posterior and dorsal-ventral axes of the embryo. This work highlights a novel connection between cell proliferation, cell growth, and axis specification in egg chambers.     

PAR-1 is required for the maintenance of oocyte fate in Drosophila

The PAR-1 kinase is required for the posterior localisation of the germline determinants in C. elegans and Drosophila, and localises to the posterior of the zygote and the oocyte in each case. We show that Drosophila PAR-1 is also required much earlier in oogenesis for the selection of one cell in a germline cyst to become the oocyte. Although the initial steps in oocyte determination are delayed, three markers for oocyte identity, the synaptonemal complex, the centrosomes and Orb protein, still become restricted to one cell in mutant clones. However, the centrosomes and Orb protein fail to translocate from the anterior to the posterior cortex of the presumptive oocyte in region 3 of the germarium, and the cell exits meiosis and becomes a nurse cell. Furthermore, markers for the minus ends of the microtubules also fail to move from the anterior to the posterior of the oocyte in mutant clones. Thus, PAR-1 is required for the maintenance of oocyte identity, and plays a role in microtubule-dependent localisation within the oocyte at two stages of oogenesis. Finally, we show that PAR-1 localises on the fusome, and provides a link between the asymmetry of the fusome and the selection of the oocyte.     

Drosophila Argonaute 1 and its miRNA biogenesis partners are required for oocyte formation and germline cell division

Argonaute 1 (Ago1) is a member of the Argonaute/PIWI protein family involved in small RNA-mediated gene regulation. In Drosophila, Ago1 plays a specific role in microRNA (miRNA) biogenesis and function. Previous studies have demonstrated that Ago1 regulates the fate of germline stem cells. However, the function of Ago1 in other aspects of oogenesis is still elusive. Here we report the function of Ago1 in developing egg chambers. We find that Ago1 protein is enriched in the oocytes and is also highly expressed in the cytoplasm of follicle cells. Clonal analysis of multiple ago1 mutant alleles shows that many mutant egg chambers contain only 8 nurse cells without an oocyte which is phenocopied in dicer-1, pasha and drosha mutants. Our results suggest that Ago1 and its miRNA biogenesis partners play a role in oocyte determination and germline cell division in Drosophila.     

Cct1, a phosphatidylcholine biosynthesis enzyme, is required for Drosophila oogenesis and ovarian morphogenesis

Patterning of the Drosophila egg requires cooperation between the germline cells and surrounding somatic follicle cells. In order to identify genes involved in follicle cell patterning, we analyzed enhancer trap lines expressed in specific subsets of follicle cells. Through this analysis, we have identified tandem Drosophila genes homologous to CTP: phosphocholine cytidylyltransferase (CCT), the second of three enzymes in the CDP-choline pathway, which is used to synthesize phosphatidylcholine. Drosophila Cct1 is expressed at high levels in three specific subsets of follicle cells, and this expression is regulated, at least in part, by the TGF-beta and Egfr signaling pathways. Mutations in Cct1 result in a number of defects, including a loss of germline stem cell maintenance, mispositioning of the oocyte, and a shortened operculum, suggesting that Cct1 plays multiple roles during oogenesis. In addition, Cct1 mutants display a novel branched ovariole phenotype, demonstrating a requirement for this gene during ovarian morphogenesis. These data provide the first evidence for a specific role for CCT, and thus for phosphatidylcholine, in patterning during development.     

Mitochondrial development during Drosophila oogenesis: distribution, density and in situ RNA hybridizations.

The changes in distribution and density of mitochondria and the level of mitochondrial RNA during Drosophila oogenesis were studied simultaneously in the 3 cell types ie follicle cells, nurse cells and oocyte, making up the egg chamber. Up to stage 6, mitochondrial density (mitochondrial and cellular areas ratio) was elevated and increased similarly in both follicle and nurse cells. Thereafter the mitochondrial density of follicle cells continued to increase and that of the nurse cells declined markedly while the nurse cell mitochondria assembled in dense groups and decreased in size. This can be related to a transfer of nurse cell cytoplasm, including mitochondria, to the oocyte. In the oocyte from stage 4 to stage 7 we observed a significant decrease of the mitochondrial density due to the absence of mitochondrial biogenesis. Then the cytoplasm transfer caused mitochondrial density to increase up to the level found in the nurse cells at the end of oogenesis. The mature oocyte contains enough mitochondria to supply 15,000 somatic cells. Our results strongly suggest that the variations in size, distribution and density of mitochondria relate to the particular energetic requirements of the different cell types during the first half of oogenesis. Later they relate to the developmental requirements of the nurse cells and the oocyte, in particular the storage of mitochondria in the oocyte. The level of mitochondrial RNA was studied through in situ hybridization. Throughout oogenesis the follicle and nurse cell RNA evolved similarly. Up to stage 9, there was no change in RNA densities in these cells, suggesting a correlation with the cell volume and/or the nuclear DNA content. Thereafter the cellular RNA concentration declined rapidly. In the oocyte the RNA concentration evolved differently especially from stage 10 to the end, the RNA density being stabilized. This can be related to the injection of nurse cell mitochondria, followed by their assignment to reserve status. Our results suggest that the mt RNA density is under extramitochondrial control mechanisms.     

Drosophila par-1 is required for oocyte differentiation and microtubule organization

BACKGROUND: Drosophila oocyte determination involves a complex process by which a single cell within an interconnected cyst of 16 germline cells differentiates into an oocyte. This process requires the asymmetric accumulation of both specific messenger RNAs and proteins within the future oocyte as well as the proper organization of the microtubule cytoskeleton, which together with the fusome provides polarity within the developing germline cyst. RESULTS: In addition to its previously described late oogenic role in the establishment of anterior-posterior polarity and subsequent embryonic axis formation, the Drosophila par-1 gene is required very early in the germline for establishing cyst polarity and for oocyte specification. Germline clonal analyses, for which we used a protein null mutation, reveal that Drosophila par-1 (par-1) is required for the asymmetric accumulation of oocyte-specific factors as well as the proper organization of the microtubule cytoskeleton. Similarly, somatic clonal analyses indicate that par-1 is required for microtubule stabilization in follicle cells. The PAR-1 protein is localized to the fusome and ring canals within the developing germline cyst in direct contact with microtubules. Likewise, in the follicular epithelium, PAR-1 colocalizes with microtubules along the basolateral membrane. However, in either case PAR-1 localization is independent of microtubules. CONCLUSIONS: The Drosophila par-1 gene plays at least two essential roles during oogenesis; it is required early in the germline for organization of the microtubule cytoskeleton and subsequent oocyte determination, and it has a second, previously described role late in oogenesis in axis formation. In both cases, par-1 appears to exert its effects through the regulation of microtubule dynamics and/or stability, and this finding is consistent with the defined role of the mammalian PAR-1 homologs.     

The Hippo pathway controls polar cell fate through Notch signaling during Drosophila oogenesis

During Drosophila oogenesis, the somatic follicle cells form an epithelial layer surrounding the germline cells to form egg chambers. In this process, follicle cell precursors are specified into polar cells, stalk cells, and main-body follicle cells. Proper specification of these three cell types ensures correct egg chamber formation and polarization of the anterior–posterior axis of the germline cells. Multiple signaling cascades coordinate to control the follicle cell fate determination, including Notch, JAK/STAT, and Hedgehog signaling pathways. Here, we show that the Hippo pathway also participates in polar cell specification. Over-activation of yorkie (yki) leads to egg chamber fusion, possibly through attenuation of polar cell specification. Loss-of-function experiments using RNAi knockdown or generation of mutant clones by mitotic recombination demonstrates that reduction of yki expression promotes polar cell formation in a cell-autonomous manner. Consistently, polar cells mutant for hippo (hpo) or warts (wts) are not properly specified, leading to egg chamber fusion. Furthermore, Notch activity is increased in yki mutant cells and reduction of Notch activity suppresses polar cell formation in yki mutant clones. These results demonstrate that yki represses polar cell fate through Notch signaling. Collectively, our data reveal that the Hippo pathway controls polar cell specification. Through repressing Notch activity, Yki serves as a key repressor in specifying polar cells during Drosophila oogenesis.     

Regulation of cell proliferation and patterning in Drosophila oogenesis by Hedgehog signaling

The localized expression of Hedgehog (Hh) at the extreme anterior of Drosophila ovarioles suggests that it might provide an asymmetric cue that patterns developing egg chambers along the anteroposterior axis. Ectopic or excessive Hh signaling disrupts egg chamber patterning dramatically through primary effects at two developmental stages. First, excess Hh signaling in somatic stem cells stimulates somatic cell over-proliferation. This likely disrupts the earliest interactions between somatic and germline cells and may account for the frequent mis-positioning of oocytes within egg chambers. Second, the initiation of the developmental programs of follicle cell lineages appears to be delayed by ectopic Hh signaling. This may account for the formation of ectopic polar cells, the extended proliferation of follicle cells and the defective differentiation of posterior follicle cells, which, in turn, disrupts polarity within the oocyte. Somatic cells in the ovary cannot proliferate normally in the absence of Hh or Smoothened activity. Loss of protein kinase A activity restores the proliferation of somatic cells in the absence of Hh activity and allows the formation of normally patterned ovarioles. Hence, localized Hh is not essential to direct egg chamber patterning.     

Organizer activity of the polar cells during Drosophila oogenesis

Patterning of the Drosophila egg requires the establishment of several distinct types of somatic follicle cells, as well as interactions between these follicle cells and the oocyte. The polar cells occupy the termini of the follicle and are specified by the activation of Notch. We have investigated their role in follicle patterning by creating clones of cells mutant for the Notch modulator fringe. This genetic ablation of polar cells results in cell fate defects within surrounding follicle cells. At the anterior, the border cells, the immediately adjacent follicle cell fate, are absent, as are the more distant stretched and centripetal follicle cells. Conversely, increasing the number of polar cells by expressing an activated form of the Notch receptor increases the number of border cells. At the posterior, elimination of polar cells results in abnormal oocyte localization. Moreover, when polar cells are mislocalized laterally, the surrounding follicle cells adopt a posterior fate, the oocyte is located adjacent to them, and the anteroposterior axis of the oocyte is re-oriented with respect to the ectopic polar cells. Our observations demonstrate that the polar cells act as an organizer that patterns surrounding follicle cells and establishes the anteroposterior axis of the oocyte. The origin of asymmetry during Drosophila development can thus be traced back to the specification of the polar cells during early oogenesis.     

Bicaudal-D is essential for egg chamber formation and cytoskeletal organization in drosophila oogenesis

Bicaudal-D (Bic-D) is required for the transport of determinant mRNAs and proteins to the presumptive oocyte, an essential step in the differentiation of the oocyte. Bic-D protein contains four well-defined heptad repeat domains characteristic of intermediate filament proteins. We characterized the ovarian phenotypes of females expressing mutant Bic-D proteins (Bic-D(H)) deleted for each of the heptad repeat domains. The altered migration of follicle cells we observe in mutant ovaries suggests that Bic-D functions in the germline and directs the inward migration of somatic follicle cells. In the germarium Bic-D is required for the organization of the egg chamber and the structural integrity of the oocyte and nurse cells. Examination of the polarized microtubule network in Bic-D(H) ovaries shows that Bic-D function is required for both the establishment of the polarized microtubule network and its maintenance throughout oogenesis. To explain the multiple functions suggested by the pleiotropic Bic-D phenotype, we propose that Bic-D protein could form itself a filamentous structure and represent an integral, essential part of the cytoskeleton.     

Mitochondrial development during Drosophila oogenesis: distribution,density and in situ RNA hybridizations

The changes in distribution and density of mitochondria and the level of mitochondrial RNA during Drosophila oogenesis were studied simultaneously in the 3 cell types ie follicle cells, nurse cells and oocyte, making up the egg chamber. Up to stage 6, mitochondrial density (mitochondrial and cellular areas ratio) was elevated and increased similarly in both follicle and nurse cells. Thereafter the mitochondrial density of follicle cells continued to increase and that of the nurse cells declined markedly while the nurse cell mitochondria assembled in dense groups and decreased in size. This can be related to a transfer of nurse cell cytoplasm, including mitochondria, to the oocyte. In the oocyte from stage 4 to stage 7 we observed a significant decrease of the mitochondrial density due to the absence of mitochondrial biogenesis. Then the cytoplasm transfer caused mitochondrial density to increase up to the level found in the nurse cells at the end of oogenesis. The mature oocyte contains enough mitochondria to supply 15 000 somatic cells. Our results strongly suggest that the variations in size, distribution and density of mitochondria relate to the particular energetic requirements of the different cell types during the first half of oogenesis. Later they relate to the developmental requirements of the nurse cells and the oocyte, in particular the storage of mitochondria in the oocyte. The level of mitochondrial RNA was studied through in situ hybridization. Throughout oogenesis the follicle and nurse cell RNA evolved similarly. Up to stage 9, there was no change in RNA densities in these cells, suggesting a correlation with the cell volume and/or the nuclear DNA content. Thereafter the cellular RNA concentration declined rapidly. In the oocyte the RNA concentration evolved differently especially from stage 10 to the end, the RNA density being stabilized. This can be related to the injection of nurse cell mitochondria, followed by their assignment to reserve status. Our results suggest that the mt RNA density is under extramitochondrial control mechanisms.     

Oogenesis in four species of Piscicola (Hirudinea, Rhynchobdellida)

Piscicola has a pair of elongated sac-shaped ovaries. Inside the ovaries are numerous small somatic cells and regularly spherical egg follicles. Each follicle is composed of three types of cells: many (average 30) germ cells (cystocytes) interconnected by intercellular bridges in clones (cysts), one intermediate cell, and three to five outer follicle cells (envelope cells). Each germ cell in a clone has one intercellular bridge connecting it to the central anucleate cytoplasmic mass, the cytophore. Each cluster of germ cells is completely embedded inside a single huge somatic follicle cell, the intermediate (interstitial) cell. The most spectacular feature of the intermediate cell is its development of a system of intracytoplasmic canals apparently formed of invaginations of its cell membrane. Initially the complex of germ cell cluster + intermediate cell is enclosed within an envelope composed of squamous cells. As oogenesis progresses the envelope cells gradually degenerate. All the germ cells that have terminated their mitotic divisions are of similar size and enter meiotic prophase, but one of the cystocytes promptly starts to grow faster and differentiates into the oocyte, whereas the remaining cystocytes withdraw from meiosis and become nurse cells (trophocytes). Numerous mitochondria, ER, and a vast amount of ribosomes are transferred from the trophocytes via the cytophore toward the oocyte. Eventually the oocyte ingests all the content of the cytophore, and the trophocytes degenerate. Little vitellogenesis takes place; the oocyte gathers nutrients in the form of small lipid droplets. At the end of oogenesis, an electron-dense fibrous vitelline envelope appears around the oocyte, among short microvilli. At the same time, electron-dense cortical granules occur in the oocyte cortical cytoplasm; at the end of oogenesis they are numerous, but after fertilization they disappear from the ooplasm. In the present article we point out many differences in the course of oogenesis in two related families of rhynchobdellids: piscicolids and glossiphoniids.     

The role of brinker in eggshell patterning

Drosophila oogenesis provides a useful system to study signal transduction pathways and their interactions. Through clonal analysis, we found that brinker (brk), a repressor of Dpp signaling, plays an important role in the Drosophila ovary, where its function is essential for dorsal appendage formation. In the absence of brk, operculum fates are specified at the expense of dorsal appendage fates. Brk is expressed by most of the oocyte associated follicle cells, starting from stage 8 of oogenesis. Transforming Growth Factor beta (TGFbeta) signaling represses brk expression in both the early stage egg chambers and in the anterior follicle cells. In brk mutant follicle cell clones at the dorsal anterior region, Broad Complex (BR-C) expression is down-regulated in a larger domain than in wild type. We show that BR-C is required for dorsal appendage development. In large anterior BR-C mutant clones, dorsal appendages are absent, and instead, the eggshell has an enlarged operculum like region at the anterior. In addition, we show that the Epidermal Growth Factor (EGF) receptor signaling represses the TGFbeta signaling in oogenesis by up-regulating brk expression. From our results and previously published data, it appears that anterior follicle cells integrate the levels of EGF receptor activation and TGFbeta receptor activation. Operculum fate results when the sum of the level of activation of both pathways reaches a threshold level, and reduction of activity of one pathway can be compensated to some extent by increase in the other pathway.