Egg Chamber Development Research Articles

Lineage-based scaling of germline intercellular bridges during oogenesis.

Using the fruit fly egg chamber as a model, this study demonstrates that the size and scaling of germline intercellular bridges vary based on lineage.

Singed and vinculin play redundant roles in cell migration by regulating F-actin.

Drosophila Singed (mammalian Fascin) is an actin-binding protein that is known mainly for bundling parallel actin filaments. Among many functions of Singed, it is required for cell motility for both Drosophila and mammalian systems. Increased Fascin-1 levels positively correlate with greater metastasis and poor prognosis in human cancer. Border cell cluster, forms and migrates during Drosophila egg chamber development, shows higher expression of Singed compared with other follicle cells. Interestingly, loss of singed in border cells does not lead to any effect other than delay. In this work, we have screened many actin-binding proteins in search of functional redundancy with Singed for border cell migration. We have found that Vinculin works with Singed to regulate border cell migration, albeit mildly. Although Vinculin is known for anchoring F-actin to the membrane, knockdown of both singed and vinculin leads to a reduced level of F-actin and changes in protrusion characteristics in border cells. We have also observed that they may act together to control microvilli length of brush border membrane vesicles and the shape of egg chambers in Drosophila. We may conclude that singed and vinculin work together to control F-actin and these interactions are consistent across multiple platforms.

Tracking Follicle Cell Development.

Somatic follicle cells are critical support cells for Drosophila oogenesis, as they provide signals and molecules needed to produce a mature egg. Throughout this process, the follicle cells differentiate into multiple subpopulations and transition between three different cell cycle programs to support nurse cell and oocyte development. The follicle cells are mitotic in early egg chamber development, as they cover the germline cyst. In mid-oogenesis, follicle cells switch from mitosis to endocycling, increasing their ploidy from 2C to 16C. Finally, in late oogenesis, cells transition from endocycling to gene amplification, increasing the copy number of a small subset of genes, including the genes encoding proteins required for egg maturation. In order to explore the genetic regulation of these cell cycle switches and follicle cell development and specification, clonal analysis and the GAL4/UAS system are used frequently to reduce or increase expression of genes of interest. These genetic approaches combined with immunohistochemistry and in situ hybridization are powerful tools for characterizing the mechanisms regulating follicle cell development and the mitosis/endocycle and endocycle/gene amplification transitions. This chapter describes the genetic tools available to manipulate gene expression in follicle cells, as well as the methods and reagents that can be utilized to explore gene expression throughout follicle cell development.

Live Imaging of Nurse Cell Behavior in Late Stages of Drosophila Oogenesis.

Drosophila oogenesis is a powerful and tractable model for studies of cell and developmental biology due to the multitude of well-characterized events in both germline and somatic cells, the ease of genetic manipulation in fruit flies, and the large number of egg chambers produced by each fly. Recent improvements in live imaging and ex vivo culturing protocols have enabled researchers to conduct more detailed, longer-term studies of egg chamber development, enabling insights into fundamental biological processes. Here, we present a protocol for dissection, culturing, and imaging of late-stage egg chambers to study intercellular and directional cytoplasmic flow during "nurse cell dumping." This critical developmental process towards the latter stages of oogenesis (stages 10b/11) results in rapid growth of the oocyte and shrinkage of the nurse cells and is accompanied by dynamic changes in cell shape. We also describe a procedure to record high-time-resolution movies of the flow of unlabeled cytoplasmic contents within nurse cells and through cytoplasmic bridges in the nurse cell cluster using reflection microscopy, and we describe two ways to analyze data from nurse cell dumping.

Rotenone mediated developmental toxicity in Drosophila melanogaster

Rotenone (ROT) is a widely used natural pesticide, and its effect on growth and developmental toxicity remain unclear. In the present study, the effects of ROT exposure on the reproductive structure and function of the female Drosophila melanogaster and third instar larvae were investigated. ROT exposure on female Drosophila melanogaster resulted in developmental inhibition and ovarian abnormality, which were evident from the disruptive growth of border cells as well as morphological changes in the orientation of nurse cells during the 9th-10th stage of developing egg chamber of in the Drosophila ovary. Other abnormalities, such as, altered developmental gene expression (Osk, Grk, Nos, Bic-d), inhibition in the kinesin motor protein level (KIF-5B), increased caspases activities (Caspase 3, 8, & 9) and apoptosis were also observed. Subsequently, ROT treated larvae exhibited behavioral deficits and delay in developmental time. The above findings demonstrate that the exposure of ROT causes developmental toxicity in Drosophila melanogaster.

Distinct contributions of ECM proteins to basement membrane mechanical properties in Drosophila.

The basement membrane is a specialized extracellular matrix (ECM) that is crucial for the development of epithelial tissues and organs. In Drosophila, the mechanical properties of the basement membrane play an important role in the proper elongation of the developing egg chamber; however, the molecular mechanisms contributing to basement membrane mechanical properties are not fully understood. Here, we systematically analyze the contributions of individual ECM components towards the molecular composition and mechanical properties of the basement membrane underlying the follicle epithelium of Drosophila egg chambers. We find that the Laminin and Collagen IV networks largely persist in the absence of the other components. Moreover, we show that Perlecan and Collagen IV, but not Laminin or Nidogen, contribute greatly towards egg chamber elongation. Similarly, Perlecan and Collagen, but not Laminin or Nidogen, contribute towards the resistance of egg chambers against osmotic stress. Finally, using atomic force microscopy we show that basement membrane stiffness mainly depends on Collagen IV. Our analysis reveals how single ECM components contribute to the mechanical properties of the basement membrane controlling tissue and organ shape.

PiRNA-guided intron removal from pre-mRNAs regulates density-dependent reproductive strategy.

Animal density-dependent experiences have profound effects on reproductive strategies with marked fecundity differences. Migratory locust adopts distinct population density-dependent reproductive strategies to cope with their respective life cycles, but the mechanisms remain poorly understood. Here, we report that Piwi-interacting RNAs (piRNAs) in the locust germline play key roles in this process. We find that the locust Piwi protein Liwi1 and piRNAs are highly expressed in early developing egg chambers in solitarious locusts, which have higher fecundity than gregarious locusts. Approximately 40% of solitarious locust-associated piRNAs map to protein-coding genes. We find that Liwi1/piRNAs facilitate pre-mRNA splicing of oocyte development-related genes, such as oo18 RNA-binding protein (Orb), in the germline by recruiting the splicing factor U2AF35 to piRNA-targeted introns, thereby increasing fecundity. Such piRNA-guided pre-mRNA splicing is also functional in Drosophila and mouse germ cells. We uncover a piRNA-guidedsplicing mechanism for processing reproduction-related mRNAs and determining animal reproductive strategies.

DaXi\u2014high-resolution, large imaging volume and multi-view single-objective light-sheet microscopy

The promise of single-objective light-sheet microscopy is to combine the convenience of standard single-objective microscopes with the speed, coverage, resolution and gentleness of light-sheet microscopes. We present DaXi, a single-objective light-sheet microscope design based on oblique plane illumination that achieves: (1) a wider field of view and high-resolution imaging via a custom remote focusing objective; (2) fast volumetric imaging over larger volumes without compromising image quality or necessitating tiled acquisition; (3) fuller image coverage for large samples via multi-view imaging and (4) higher throughput multi-well imaging via remote coverslip placement. Our instrument achieves a resolution of 450 nm laterally and 2 μm axially over an imaging volume of 3,000 × 800 × 300 μm. We demonstrate the speed, field of view, resolution and versatility of our instrument by imaging various systems, including Drosophila egg chamber development, zebrafish whole-brain activity and zebrafish embryonic development – up to nine embryos at a time.

Target of Rapamycin Drives Unequal Responses to Essential Amino Acid Depletion for Egg Laying in Drosophila Melanogaster.

Nutrition shapes a broad range of life-history traits, ultimately impacting animal fitness. A key fitness-related trait, female fecundity is well known to change as a function of diet. In particular, the availability of dietary protein is one of the main drivers of egg production, and in the absence of essential amino acids egg laying declines. However, it is unclear whether all essential amino acids have the same impact on phenotypes like fecundity. Using a holidic diet, we fed adult female Drosophila melanogaster diets that contained all necessary nutrients except one of the 10 essential amino acids and assessed the effects on egg production. For most essential amino acids, depleting a single amino acid induced as rapid a decline in egg production as when there were no amino acids in the diet. However, when either methionine or histidine were excluded from the diet, egg production declined more slowly. Next, we tested whether GCN2 and TOR mediated this difference in response across amino acids. While mutations in GCN2 did not eliminate the differences in the rates of decline in egg laying among amino acid drop-out diets, we found that inhibiting TOR signalling caused egg laying to decline rapidly for all drop-out diets. TOR signalling does this by regulating the yolk-forming stages of egg chamber development. Our results suggest that amino acids differ in their ability to induce signalling via the TOR pathway. This is important because if phenotypes differ in sensitivity to individual amino acids, this generates the potential for mismatches between the output of a pathway and the animal’s true nutritional status.

The SAGA core module is critical during Drosophila oogenesis and is broadly recruited to promoters.

The Spt/Ada-Gcn5 Acetyltransferase (SAGA) coactivator complex has multiple modules with different enzymatic and non-enzymatic functions. How each module contributes to gene expression is not well understood. During Drosophila oogenesis, the enzymatic functions are not equally required, which may indicate that different genes require different enzymatic functions. An analogy for this phenomenon is the handyman principle: while a handyman has many tools, which tool he uses depends on what requires maintenance. Here we analyzed the role of the non-enzymatic core module during Drosophila oogenesis, which interacts with TBP. We show that depletion of SAGA-specific core subunits blocked egg chamber development at earlier stages than depletion of enzymatic subunits. These results, as well as additional genetic analyses, point to an interaction with TBP and suggest a differential role of SAGA modules at different promoter types. However, SAGA subunits co-occupied all promoter types of active genes in ChIP-seq and ChIP-nexus experiments, and the complex was not specifically associated with distinct promoter types in the ovary. The high-resolution genomic binding profiles were congruent with SAGA recruitment by activators upstream of the start site, and retention on chromatin by interactions with modified histones downstream of the start site. Our data illustrate that a distinct genetic requirement for specific components may conceal the fact that the entire complex is physically present and suggests that the biological context defines which module functions are critical.

The Drosophila ribosome protein S5 paralog RpS5b promotes germ cell and follicle cell differentiation during oogenesis.

Emerging evidence suggests that ribosome heterogeneity may have important functional consequences in the translation of specific mRNAs within different cell types and under various conditions. Ribosome heterogeneity comes in many forms, including post-translational modification of ribosome proteins (RPs), absence of specific RPs and inclusion of different RP paralogs. The Drosophila genome encodes two RpS5 paralogs: RpS5a and RpS5b. While RpS5a is ubiquitously expressed, RpS5b exhibits enriched expression in the reproductive system. Deletion of RpS5b results in female sterility marked by developmental arrest of egg chambers at stages 7-8, disruption of vitellogenesis and posterior follicle cell (PFC) hyperplasia. While transgenic rescue experiments suggest functional redundancy between RpS5a and RpS5b, molecular, biochemical and ribo-seq experiments indicate that RpS5b mutants display increased rRNA transcription and RP production, accompanied by increased protein synthesis. Loss of RpS5b results in microtubule-based defects and in mislocalization of Delta and Mindbomb1, leading to failure of Notch pathway activation in PFCs. Together, our results indicate that germ cell-specific expression of RpS5b promotes proper egg chamber development by ensuring the homeostasis of functional ribosomes.

Mitochondrial Dynamics in the Drosophila Ovary Regulates Germ Stem Cell Number, Cell Fate, and Female Fertility.

The fate and proliferative capacity of stem cells have been shown to strongly depend on their metabolic state. Mitochondria are the powerhouses of the cell being responsible for energy production via oxidative phosphorylation (OxPhos) as well as for several other metabolic pathways. Mitochondrial activity strongly depends on their structural organization, with their size and shape being regulated by mitochondrial fusion and fission, a process known as mitochondrial dynamics. However, the significance of mitochondrial dynamics in the regulation of stem cell metabolism and fate remains elusive. Here, we characterize the role of mitochondria morphology in female germ stem cells (GSCs) and in their more differentiated lineage. Mitochondria are particularly important in the female GSC lineage. Not only do they provide these cells with their energy requirements to generate the oocyte but they are also the only mitochondria pool to be inherited by the offspring. We show that the undifferentiated GSCs predominantly have fissed mitochondria, whereas more differentiated germ cells have more fused mitochondria. By reducing the levels of mitochondrial dynamics regulators, we show that both fused and fissed mitochondria are required for the maintenance of a stable GSC pool. Surprisingly, we found that disrupting mitochondrial dynamics in the germline also strongly affects nurse cells morphology, impairing egg chamber development and female fertility. Interestingly, reducing the levels of key enzymes in the Tricarboxylic Acid Cycle (TCA), known to cause OxPhos reduction, also affects GSC number. This defect in GSC self-renewal capacity indicates that at least basal levels of TCA/OxPhos are required in GSCs. Our findings show that mitochondrial dynamics is essential for female GSC maintenance and female fertility, and that mitochondria fusion and fission events are dynamically regulated during GSC differentiation, possibly to modulate their metabolic profile.

Mutations in the insulator protein Suppressor of Hairy wing induce genome instability.

Insulator proteins orchestrate the three-dimensional organization of the genome. Insulators function by facilitating communications between regulatory sequences and gene promoters, allowing accurate gene transcription regulation during embryo development and cell differentiation. However, the role of insulator proteins beyond genome organization and transcription regulation remains unclear. Suppressor of Hairy wing [Su(Hw)] is a Drosophila insulator protein that plays an important function in female oogenesis. Here we find that su(Hw) has an unsuspected role in genome stability during cell differentiation. We show that su(Hw) mutant developing egg chambers have poorly formed microtubule organization centers (MTOCs) in the germarium and display mislocalization of the anterior/posterior axis specification factor gurken in later oogenesis stages. Additionally, eggshells from partially rescued su(Hw) mutant female germline exhibit dorsoventral patterning defects. These phenotypes are very similar to phenotypes found in the important class of spindle mutants or in piRNA pathway mutants in Drosophila, in which defects generally result from the failure of germ cells to repair DNA damage. Similarities between mutations in su(Hw) and spindle and piRNA mutants are further supported by an excess of DNA damage in nurse cells, and because Gurken localization defects are partially rescued by mutations in the ATR (mei-41) and Chk1 (grapes) DNA damage response genes. Finally, we also show that su(Hw) mutants produce an elevated number of chromosome breaks in dividing neuroblasts from larval brains. Together, these findings suggest that Su(Hw) is necessary for the maintenance of genome integrity during Drosophila development, in both germline and dividing somatic cells.

Analysis of the Temporal Patterning of Notch Downstream Targets during Drosophila melanogaster Egg Chamber Development

Living organisms require complex signaling interactions and proper regulation of these interactions to influence biological processes. Of these complex networks, one of the most distinguished is the Notch pathway. Dysregulation of this pathway often results in defects during organismal development and can be a causative mechanism for initiation and progression of cancer. Despite previous research entailing the importance of this signaling pathway and the organismal processes that it is involved in, less is known concerning the major Notch downstream targets, especially the onset and sequence in which they are modulated during normal development. As timing of regulation may be linked to many biological processes, we investigated and established a model of temporal patterning of major Notch downstream targets including broad, cut, and hindsight during Drosophila melanogaster egg chamber development. We confirmed the sequential order of Broad upregulation, Hindsight upregulation, and Cut downregulation. In addition, we showed that Notch signaling could be activated at stage 4, one stage earlier than the stage 5, a previously long-held belief. However, our further mitotic marker analysis re-stated that mitotic cycle continues until stage 5. Through our study, we once again validated the effectiveness and reliability of our MATLAB toolbox designed to systematically identify egg chamber stages based on area size, ratio, and additional morphological characteristics.

The Arp2/3 complex and the formin, Diaphanous, are both required to regulate the size of germline ring canals in the developing egg chamber

Intercellular bridges are an essential structural feature found in both germline and somatic cells throughout the animal kingdom. Because of their large size, the germline intercellular bridges, or ring canals, in the developing fruit fly egg chamber are an excellent model to study the formation, stabilization, and growth of these structures. Within the egg chamber, the germline ring canals connect 15 supporting nurse cells to the developing oocyte, facilitating the transfer of materials required for successful oogenesis. The ring canals are derived from a stalled actomyosin contractile ring; once formed, additional actin and actin-binding proteins are recruited to the ring to support the 20-fold growth that accompanies oogenesis. These behaviors provide a unique model system to study the actin regulators that control incomplete cytokinesis, intercellular bridge formation, and growth. By temporally controlling their expression in the germline, we have demonstrated that the Arp2/3 complex and the formin, Diaphanous (Dia), coordinately regulate ring canal size and growth throughout oogenesis. Dia is required for successful incomplete cytokinesis and the initial stabilization of the germline ring canals. Once ring canals have formed, the Arp2/3 complex and Dia cooperate to determine ring canal size and maintain stability. Our data suggest that nurse cells must maintain a precise balance between the activity of these two nucleators during oogenesis.

A putative stem-loop structure in Drosophila crumbs is required for mRNA localisation in epithelia and germline cells.

Crumbs (Crb) is an evolutionarily conserved transmembrane protein localised to the apical membrane of epithelial cells. Loss or mislocalisation of Crb is often associated with disruption of apicobasal cell polarity. crb mRNA is also apically enriched in epithelial cells, and, as shown here, accumulates in the oocyte of developing egg chambers. We narrowed down the localisation element (LE) of crb mRNA to 47 nucleotides, which form a putative stem-loop structure that may be recognised by Egalitarian (Egl). Mutations in conserved nucleotides abrogate apical transport. crb mRNA enrichment in the oocyte is affected in egl mutant egg chambers. A CRISPR-based genomic deletion of the crb locus that includes the LE disrupts asymmetric crb mRNA localisation in epithelia and prevents its accumulation in the oocyte during early stages of oogenesis, but does not affect Crb protein localisation in embryonic and follicular epithelia. However, flies lacking the LE show ectopic Crb protein expression in the nurse cells. These data suggest an additional role for the Drosophila 3'-UTR in regulating translation in a tissue-specific manner.This article has an associated First Person interview with the first author of the paper.

Mercury chloride exposure induces DNA damage, reduces fertility, and alters somatic and germline cells in Drosophila melanogaster ovaries.

Mercury exposure has been shown to affect the reproductive system in many organisms, although the molecular mechanisms are still elusive. In the present study, we exposed Drosophila melanogaster Canton-S adult females to concentrations of 0 mM, 0.1 mM, 0.3 mM, 3 mM, and 30 mM of mercury chloride (HgCl2) for 24 h, 48 h, or 72 h to determine how mercury could affect fertility. Alkaline assays performed on dissected ovaries showed that mercury induced DNA damage that is not only dose-dependent but also time-dependent. All ovaries treated for 72 h have incorporated mercury and exhibit size reduction. Females treated with 30 mM HgCl2, the highest dose, had atrophied ovaries and exhibited a drastic 7-fold reduction in egg laying. Confocal microscopy analysis revealed that exposure to HgCl2 disrupts germinal and somatic cell organization in the germarium and leads to the aberrant expression of a germline-specific gene in somatic follicle cells in developing egg chambers. Together, these results highlight the potential long-term impact of mercury on germline and ovarian cells that might involve gene deregulation.

The dPix-Git complex is essential to coordinate epithelial morphogenesis and regulate myosin during Drosophila egg chamber development.

How biochemical and mechanical information are integrated during tissue development is a central question in morphogenesis. In many biological systems, the PIX-GIT complex localises to focal adhesions and integrates both physical and chemical information. We used Drosophila melanogaster egg chamber formation to study the function of PIX and GIT orthologues (dPix and Git, respectively), and discovered a central role for this complex in controlling myosin activity and epithelial monolayering. We found that Git’s focal adhesion targeting domain mediates basal localisation of this complex to filament structures and the leading edge of migrating cells. In the absence of dpix and git, tissue disruption is driven by contractile forces, as reduction of myosin activators restores egg production and morphology. Further, dpix and git mutant eggs closely phenocopy defects previously reported in pak mutant epithelia. Together, these results indicate that the dPix-Git complex controls egg chamber morphogenesis by controlling myosin contractility and Pak kinase downstream of focal adhesions.

The Exocyst Component Sec3 Controls Egg Chamber Development Through Notch During Drosophila Oogenesis.

The exocyst complex plays multiple roles via tethering secretory or recycling vesicles to the plasma membrane. Previous studies have demonstrated that the exocyst contains eight components, which possibly have some redundant but distinct functions. It is therefore interesting to investigate the biological function of each component. Here, we found that Sec3, one component of exocyst complex, is involved in Drosophila egg chamber development. Loss of sec3 results in egg chamber fusion through the abolishment of cell differentiation. In addition, loss of sec3 increases cell numbers but decreases cell size. These defects phenocopy Notch pathway inactivation. In line with this, loss of sec3 indeed leads to Notch protein accumulation, suggesting that the loss of Sec3 inhibits the delivery of Notch onto the plasma membrane and accumulates inactive Notch in the cytoplasm. Loss of sec3 also leads to the ectopic expression of two Notch pathway target genes, Cut and FasciclinIII, which should normally be downregulated by Notch. Altogether, our study revealed that Sec3 governs egg chamber development through the regulation of Notch, and provides fresh insights into the regulation of oogenesis.

IMP regulates Kuzbanian to control the timing of Notch signalling in Drosophila follicle cells.

ABSTRACTThe timing of Drosophila egg chamber development is controlled by a germline Delta signal that activates Notch in the follicle cells to induce them to cease proliferation and differentiate. Here, we report that follicle cells lacking the RNA-binding protein IMP go through one extra division owing to a delay in the Delta-dependent S2 cleavage of Notch. The timing of Notch activation has previously been shown to be controlled by cis-inhibition by Delta in the follicle cells, which is relieved when the miRNA pathway represses Delta expression. imp mutants are epistatic to Delta mutants and give an additive phenotype with belle and Dicer-1 mutants, indicating that IMP functions independently of both cis-inhibition and the miRNA pathway. We find that the imp phenotype is rescued by overexpression of Kuzbanian, the metalloprotease that mediates the Notch S2 cleavage. Furthermore, Kuzbanian is not enriched at the apical membrane in imp mutants, accumulating instead in late endosomes. Thus, IMP regulates Notch signalling by controlling the localisation of Kuzbanian to the apical domain, where Notch cleavage occurs, revealing a novel regulatory step in the Notch pathway.