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Stages of egg chamber development.Nomarski optics images of unstained dissected egg chambers, all of which are oriented with anterior to the left. (a) An ovariole including the anterior tip (the germarium, bracket), four early-stage egg chambers, a stage 8 egg chamber and a stage 10B egg chamber. The oocyte of a stage 8 egg chamber occupies less than one-third the volume of the egg chamber but contains yolk, which is obvious based on its refractile properties and autofluoresence. For more detailed staging criteria, see King23 or Spradling24. (b) Another ovariole, containing the germarium through to early stage 9, which is defined based on the criteria that the oocyte occupies approximately one-third the total volume of the egg chamber, the outer follicle cells are in the process of rearranging such that most of them will stack up and assume a columnar morphology and contact the oocyte in the posterior half of the egg chamber. The anterior follicle cells (other than the border cells) will spread out into a thin, flat layer to cover the nurse cells and become difficult to see because they are thin. (c) Part of an ovariole showing a stage 10 egg chamber in which the oocyte occupies approximately one-half the volume of the egg chamber, and both the epithelial follicle cell rearrangement and border cell migration are complete. (d) A stage 12 egg chamber. (e) A stage 13, nearly mature egg chamber. The oocyte (o) is outlined in stages 8–10 and the position of the border cells is indicated (arrowhead) where possible. Approximate duration of stages at room temperature: stage 8 (6 h); stage 9 (6 h); stage 10 (10 h).
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This protocol describes a method for the dissection of egg chambers from intact Drosophila females and culture conditions that permit live imaging of them, with a particular emphasis on stage 9. This stage of development is characterized by oocyte growth and patterning, outer follicle cell rearrangement and migration of border cells. Although in vi...
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... step can be repeated a few times for each ovary to obtain a number of ovarioles from each. Figure 2 8| If some late-stage egg chambers (stage 11 or older) are still attached to the stage 8-9 egg chambers, carefully remove them without touching the egg chamber of interest. Hold one end of the largest egg chamber with one pair of forceps and, with the other, sever the connection between the stage 10 egg chamber and the older one, never touching the desired egg chamber. ...
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... One-to three-day-old females were fattened on yeast with males for 1-2 days before dissection. Drosophila egg chambers were dissected and mounted in live-imaging medium (Invitrogen Schneider's insect medium with 20% FBS and with a final pH adjusted to 6.9), using a similar version of the protocol described in 52 . In contrast to the normal mounting conditions, egg chambers were slightly compressed to overcome the endogenous curvature. ...
Actomyosin networks constrict cell area and junctions to alter cell and tissue shape. However, during cell expansion under mechanical stress, actomyosin networks are strengthened and polarized to relax stress. Thus, cells face a conflicting situation between the enhanced actomyosin contractile properties and the expansion behaviour of the cell or tissue. To address this paradoxical situation, we study late Drosophila oogenesis and reveal an unusual epithelial expansion wave behaviour. Mechanistically, Rac1 and Rho1 integrate basal pulsatile actomyosin networks with ruffles and focal adhesions to increase and then stabilize basal area of epithelial cells allowing their flattening and elongation. This epithelial expansion behaviour bridges cell changes to oocyte growth and extension, while oocyte growth in turn deforms the epithelium to drive cell spreading. Basal pulsatile actomyosin networks exhibit non-contractile mechanics, non-linear structures and F-actin/Myosin-II spatiotemporal signal separation, implicating unreported expanding properties. Biophysical modelling incorporating these expanding properties well simulates epithelial cell expansion waves. Our work thus highlights actomyosin expanding properties as a key mechanism driving tissue morphogenesis.
... Drosophila egg chamber live imaging. We followed the protocol of Prasad et al. 52 . Briefly, Drosophila egg chambers (stage 9 to stage 10A) were dissected from adult females and mounted in Schneider's insect medium supplemented with 20% FBS and adjusted to a pH of 6.9. ...
The ultimate aim of fluorescence microscopy is to achieve high-resolution imaging of increasingly larger biological samples. Extended depth of field presents a potential solution to accelerate imaging of large samples when compression of information along the optical axis is not detrimental to the interpretation of images.
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... Imaging chambers were fabricated as described previously [10,45]. Ovaries from flies with C587-GAL4, UAS-FUCCI genotypes described above and incubated at 29C for 3d were dissected into imaging medium formulated as in [65] (20% FBS in Schneiders insect medium, 0.2 mg/mL insulin, penicillin and streptomycin). After separating ovaries into individual ovarioles in imaging medium, 135 μL medium containing ovarioles was mixed with 15 μL Matrigel (Corning), added to the imaging chamber and left covered for 15 min to gel. ...
Drosophila ovarian Follicle Stem Cells (FSCs) present a favorable paradigm for understanding how stem cell division and differentiation are balanced in communities where those activities are independent. FSCs also allow exploration of how this balance is integrated with spatial stem cell heterogeneity. Posterior FSCs become proliferative Follicle Cells (FCs), while anterior FSCs become quiescent Escort Cells (ECs) at about one fourth the frequency. A single stem cell can nevertheless produce both FCs and ECs because it can move between anterior and posterior locations. Studies based on EdU incorporation to approximate division rates suggested that posterior FSCs divide faster than anterior FSCs. However, direct measures of cell cycle times are required to ascertain whether FC output requires a net flow of FSCs from anterior to posterior. Here, by using live imaging and FUCCI cell-cycle reporters, we measured absolute division rates. We found that posterior FSCs cycle more than three times faster than anterior FSCs and produced sufficient new cells to match FC production. H2B-RFP dilution studies supported different cycling rates according to A/P location and facilitated live imaging, showing A/P exchange of FSCs in both directions, consistent with the dynamic equilibrium inferred from division rate measurements. Inversely graded Wnt and JAK-STAT pathway signals regulate FSC differentiation to ECs and FCs. JAK-STAT promotes both differentiation to FCs and FSC cycling, affording some coordination of these activities. When JAK-STAT signaling was manipulated to be spatially uniform, the ratio of posterior to anterior division rates was reduced but remained substantial, showing that graded JAK-STAT signaling only partly explains the graded cycling of FSCs. By using FUCCI markers, we found a prominent G2/M cycling restriction of posterior FSCs together with an A/P graded G1/S restriction, that JAK-STAT signaling promotes both G1/S and G2/M transitions, and that PI3 kinase signaling principally stimulates the G2/M transition.
... ; https://doi.org/10. 1101 Vasa binds to cluster transcripts and localizes to nuage granules that are biased toward regions of the nuclear envelope opposite clusters (Zhang et al. 2012 Leptomycin B and assayed localization (Prasad et al. 2007;Findley et al. 2003). After 2 hours of incubation in 10 µM Leptomycin B, GFP-Vasa localized to the nucleus and nuage ( Figure 4B). ...
... Treatment with Leptomycin B was carried out in the media mix in Prasad et al. 2007 at 25 o C for the indicated time in a rocking incubator. ...
Environmental stress activates transposons and is proposed to generate genetic diversity that facilitates adaptive evolution. piRNAs guide germline transposon silencing, but the impact of stress on the piRNA pathway is not well understood. In Drosophila, the Rhino-Deadlock-Cuff complex (RDC) drives transcription of clusters composed of nested transposon fragments, generating precursors that are processed into mature piRNAs in the cytoplasm. We show that acute heat shock triggers rapid, reversable, loss of RDC localization and cluster transcript expression with coordinate changes in the cytoplasmic processing machinery. Maternal piRNAs bound to Piwi are proposed to guide Rhino localization to clusters during early embryogenesis. However, RDC re-localization after heat shock is accelerated in piwi mutants and delayed in thoc7 mutants, which disrupt piRNA precursor binding to THO complex, and we show that maternally deposited piRNAs are dispensable for RDC localization to the major 42AB cluster. Cluster specification is reconsidered in light of these findings.
... Our ex vivo culture protocol was adapted from previous work (Cetera et al., 2016;Prasad et al., 2007). Briefly, egg chambers were dissected from ovaries in a culture ...
During Drosophila oogenesis, somatic follicle cells differentiate to secrete components of the eggshell. Prior to secretion, the epithelium reorganizes to shape eggshell specializations, including border FC collective cell migration and later dorsal formation. These FC movements provide valuable insights into collective cell migration. However, little is known about centripetal migration, which encloses the oocyte after secretion has begun. Centripetal migration begins with apical extension of a few follicle cells (FCs) that move away from the basement membrane to invade between germ cells. We define a timeline of reproducible milestones, using time-lapse imaging of egg chamber explants. Inward migration occurs in two phases. First, leading centripetal FCs ingress, extending apically over the anterior oocyte, and constricting basally. Second, following FCs move collectively toward the anterior, then around the corner to move inward with minimal change in aspect ratio. E-Cadherin was required in leading centripetal FCs for their normal ingression, assessed with homozygous shotgun mutant or RNAi knockdown clones; ingression was influenced non-autonomously by mutant following FCs. This work establishes centripetal migration as an accessible model for biphasic, E-Cadherin-adhesion mediated, collective migration.
... Individual egg chambers were dissected in live imaging media (LIM) [77]. After dissection, the egg chambers were incubated in LIM supplemented with mouse anti-Delta (1:20 dilution) for 1 hour at 25˚C. ...
Specification of migratory cell fate from a stationary population is complex and indispensable both for metazoan development as well for the progression of the pathological condition like tumor metastasis. Though this cell fate transformation is widely prevalent, the molecular understanding of this phenomenon remains largely elusive. We have employed the model of border cells (BC) in Drosophila oogenesis and identified germline activity of an RNA binding protein, Cup that limits acquisition of migratory cell fate from the neighbouring follicle epithelial cells. As activation of JAK-STAT in the follicle cells is critical for BC specification, our data suggest that Cup, non-cell autonomously restricts the domain of JAK-STAT by activating Notch in the follicle cells. Employing genetics and Delta endocytosis assay, we demonstrate that Cup regulates Delta recycling in the nurse cells through Rab11GTPase thus facilitating Notch activation in the adjacent follicle cells. Since Notch and JAK-STAT are antagonistic, we propose that germline Cup functions through Notch and JAK-STAT to modulate BC fate specification from their static epithelial progenitors.
... Live imaging of egg chamber culture was as previously described (Law et al., 2013;Prasad et al., 2007) with slight modification. Briefly, media for both dissection and live-imaging, comprised of Schneider media (Gibco), 15% fetal bovine serum, 0.1 mg/ml acidified insulin (Sigma), 9 µM FM4-64 dye (Molecular Probes) and 0.1 mg/ml Penstrep (Gibco) was freshly prepared. ...
Ataxin-7 is a key component of the Spt-Ada-Gcn5-acetyltransferase (SAGA) chromatin-modifying complex that anchors Non-stop/USP22, a deubiquitinase, to the complex, thereby helping to coordinate SAGA's different activities. Recently, we found that non-stop is required in the Drosophila ovary for expression of Hippo signalling pathway components ex and mer, and polarisation of the actin cytoskeleton during collective epithelial cell migration. Here we show that in addition to being required for collective migration, Ataxin-7 plays an essential role in posterior follicle cells (PFCs) to control epithelial maturation and architecture, as well as body axis specification which depends on correct PFC differentiation. Loss of both the deubiquitinase and acetyltransferase modules of SAGA phenocopy loss of Ataxin-7 in PFCs, demonstrating a redundant requirement for SAGA's enzymatic modules. Strikingly, loss of yorkie completely suppressed effects of Ataxin-7 loss-of-function in PFCs, indicating that the only essential function of Ataxin-7 in PFCs is to suppress yorkie function. This may have broad relevance to the roles of SAGA and Ataxin-7 in development and disease.
... Dissection of ovaries and measurements were performed in Schneider medium with supplements (Prasad et al., 2007). Basement membrane stiffness was measured using an atomic force microscope (Nanowizard I, JPK Instruments) mounted on a Zeiss Axiovert 200M wide-field microscope with a 20× objective (Zeiss, Plan Apochromat, NA=0.8) and a CCD camera (DMK 23U445, The Imaging Source). ...
... Egg chambers were dissected in egg chamber culture medium (Prasad et al., 2007) and adhered to glass bottom microwell dishes (MatTek) coated with poly-D-lysine. Egg chambers were imaged every minute for 20 min. ...
... Egg chambers were dissected in Schneider medium with supplements (Prasad et al., 2007). Dissected egg chambers were adhered to glass bottom microwell dishes (MatTek) coated with poly-D-lysine and were either treated with or without (control) 0.05% Collagenase I (Merck). ...
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.
... For Drosophila fly imaging, the egg chambers were dissected and cultured as described previously 51 . D. melanogaster ovaries were removed from females 3 d after eclosion for observation. ...
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.
... Egg chamber culture and time-lapse imaging of live egg chambers Live imaging of egg chamber culture was as previously described (Law et al., 2013;Prasad et al., 2007), with slight modification. Briefly, media for both dissection and live imaging comprising Schneider media (Gibco), 15% FBS, 0.1 mg/ml acidified insulin (Sigma), 9 µM FM4-64 dye (Molecular Probes), and 0.1 mg/ml penicillin-streptomycin (Gibco) were freshly prepared. ...
Polarization of the actin cytoskeleton is vital for the collective migration of cells in vivo. During invasive border cell migration in Drosophila, actin polarization is directly controlled by the Hippo signaling complex, which resides at contacts between border cells in the cluster. Here, we identify, in a genetic screen for deubiquitinating enzymes involved in border cell migration, an essential role for nonstop/USP22 in the expression of Hippo pathway components expanded and merlin. Loss of nonstop function consequently leads to a redistribution of F-actin and the polarity determinant Crumbs, loss of polarized actin protrusions, and tumbling of the border cell cluster. Nonstop is a component of the Spt-Ada-Gcn5-acetyltransferase (SAGA) transcriptional coactivator complex, but SAGA’s histone acetyltransferase module, which does not bind to expanded or merlin, is dispensable for migration. Taken together, our results uncover novel roles for SAGA-independent nonstop/USP22 in collective cell migration, which may help guide studies in other systems where USP22 is necessary for cell motility and invasion.
