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Identification of 14 stages of Drosophila egg chamber. Characteristics of different egg chamber stages.
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The Drosophila egg chamber, whose development is divided into 14 stages, is a well-established model for developmental biology. However, visual stage determination can be a tedious, subjective and time-consuming task prone to errors. Our study presents an objective, reliable and repeatable automated method for quantifying cell features and classify...
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... Drosophila have two ovaries, each containing around 16 ovarioles. The ovariole is a string of 6 or 7 sequentially developing egg chambers. Each Drosophila egg chamber is a basic developmental unit of oogenesis. Furthermore, the egg chamber comprises 16 germ-line cells, one oocyte and 15 nurse cells, which are surrounded by a thin layer of somatic follicle cells 1 . Research on the egg chamber contributes significantly to scientific knowledge. The egg chamber follicle cell epithelium has been a well-established model system to study cell cycle regulation, cell differentiation 2–16 , cell polarity 17–20 , endocytosis 21–23 , exocytosis 24 , morphogenesis 19,25–28 , and cancer metastasis 29,30 . Germline-soma interactions provide developmental cues, especially for anterior-posterior and dorsal-ventral patterning 31–36 . More interestingly, egg chambers have distinctively separated germline and soma, which provides a unique and reliable system to study germline-soma ligand-receptor interaction 1,2,31,37 . The development of the egg chamber during Drosophila oogenesis is divided into 14 stages 1 . Visual identifica- tion of different stages requires significant training, including a thorough understanding of egg chamber growth progression. Even skilled scientists need to cautiously determine the stages based on general features ( Fig. 1) (Fig. 1) 1 and frequently refer to neighboring egg chambers to doubly confirm the determination of stages. However, in a single confocal image of egg chambers, there might be no neighboring egg chambers to help confirm stages. Moreover, even an expert sometimes has difficulty assigning correct stages to inter-stage transitional egg chambers. To address these problems, and to reduce the bias caused by human perceptual variation, an objective, reliable and standardized egg chamber stage identification method is greatly needed. DAPI (4 ′ ,6-diamidino-2-phenylindole) is widely used as a cell nucleus staining dye. In this study, we developed a toolbox that applies understanding of egg chamber morphology 1 extracted automatically from DAPI staining to determine egg chamber stages. We collected 172 confocal microscopy images from different stages to train and evaluate our automatic stage identification method. Egg chamber areas of DAPI images proved to be a stable feature for stage differentiation, while oocyte size played a critical role for late stage differentiation. Integrated with machine learning, our approach provides potential to discover hidden developmental mechanisms, and brings the Drosophila community tools for standardizing stage identification and objectively comparing results obtained from different labs. In this paper, we applied our method to successfully confirm the occasional appearance of Broad expression as early as stage 5, and also unambiguously demonstrated that egg chambers with germline Delta mutation entered midoogenesis, even though the follicle cells did not show the appropriate biomarker, Br 5 . Furthermore, we provided support for the previous understanding that follicle cell mitosis ceases at stage 6 1,38 . Our results clearly showed mitosis occurs in follicle cells of stage-5 egg chambers, while not in those of stage-6 egg chambers. Our findings clarified our understanding that stage 6 should be considered as an entering stage of endocycle, and stage 7 completes endocycle entry 2–6,9,10 . The collected DAPI images should be single cross sections in the middle plane, and represents the largest area size. Before image processing, particular stages were initially assigned to egg chambers by skilled biologists in our team based on standard descriptive guidance 1 (Fig. 1). (Fig. 1). The morphological features, including egg chamber size, ratio, orientation, oocyte size, follicle cell dis- tribution, blob-like chromosomes in polytene nuclei at stage 4, centripetal cell migration at stage 10B, were then extracted to train our machine learning method. Egg chamber size. The development of an organism is often accompanied by increase in size, and this is also the case in Drosophila egg chamber stage development. Since egg chamber size plays a critical role in stage determination, we developed an algorithm for automated egg chamber size measurement, whose workflow is described as follows: Image thresholding: Original images (Fig. 2A) (Fig. 2A) were read into Matlab, and the measurement ( μ m) of each pixel was recorded. Otsu’s method 39 was used to convert the original image into a binary image and initialize the seg- mentation process. Briefly, Otsu’s method applies a threshold that optimizes the separation of image foreground and background. This algorithm has been widely used in image segmentation pre-processing steps 40 . Conditions must be met for Otsu’s method to be effective:1) minimum variability in gray levels of foreground pixels and background pixels, respectively; 2) maximum gray level contrast between background and foreground pixels. In our study, the DAPI-stained images satisfy both conditions, as foreground pixels had much lighter color than the background pixels. We set the threshold at 20% of Otsu’s threshold to include all desired foreground objects. This percentage value was learned through experimentation with data. The Matlab function, graythresh, was used to compute Otsu’s threshold. Another Matlab function, im2bw, applied our threshold and converted the grayscale image to a binary image. The output binary image replaced all pixel values in the input image with luminance greater than the threshold with the value 1 (white, foreground, Fig. 2B) Fig. 2B) and the remaining pixels with the value 0 (black, background, Fig. 2B). Fig. 2B). Largest component extraction: Digital images are prone to noise that may originate from different parts of the image acquisition process. “Noise” is always a relative term and means “unwanted signals”. In our analysis of the binary image obtained in the previous step, we considered any foreground image element other than the egg chamber of interest as noise, including portions of other egg chambers and scattered spotty pixels (Fig. 2A). (Fig. 2A). To detect these noisy pixels and mitigate their negative effects on our analyses, we applied an adjusted average filter, which smoothed the image by replacing each pixel value with the ...
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Citations
... Dissections of the adult flies for D. melanogaster were performed starting at age 0 h and every 6 h for 30 h, while for D. pseudoobscura, dissections were made at age 0 days and daily for 7 days, due to the difference in time of sexual maturation between both species. The description of oocyte developmental stages by Jia et al. (2016) was used to define the time points, the early time point was defined as consisting of only oocytes in stages 1-10, and the late time point was defined as having all stages of oogenesis present. For D. melanogaster at 25℃, the selected time points were 1 and 4 days for control temperature, and for high temperature, 0.25 and 2 days. ...
... Image Analysis. Using the Oocyte developmental stages description by Jia et al. (2016), and the extension Fiji of the software ImageJ (Schindelin et al., 2012), the stage of development of all the visible egg chambers per individual was identified and corrected with size. The identified samples were then pooled into four developmental time points, stages 1 -7, 8 -10, 11, and 12 -14 (Fig. 2). ...
... Region 3 (stage 1) oocytes leave the germarium and enter the vitellarium. Stages 1 to 13 last 5 days, during which meiosis remains in prophase while the oocyte grows and matures [44,45]. At the posterior end of the ovariole (stage [13][14], the oocyte enters prometaphase. ...
... In contrast, surprising patterns were observed in stage 14 oocytes when CID or CAL1 were regulated by MVD1 (Fig 1B). In cid-GFP/MVD1 oocytes, CID was present as centromeric foci in about 50% of oocytes [44,45]. Region 1 contains the stem cell niche as well as the pre-meiotic cyst cells undergoing mitosis. ...
The centromere is an epigenetic mark that is a loading site for the kinetochore during meiosis and mitosis. This mark is characterized by the H3 variant CENP-A, known as CID in Drosophila . In Drosophila , CENP-C is critical for maintaining CID at the centromeres and directly recruits outer kinetochore proteins after nuclear envelope break down. These two functions, however, happen at different times in the cell cycle. Furthermore, in Drosophila and many other metazoan oocytes, centromere maintenance and kinetochore assembly are separated by an extended prophase. We have investigated the dynamics of function of CENP-C during the extended meiotic prophase of Drosophila oocytes and found that maintaining high levels of CENP-C for metaphase I requires expression during prophase. In contrast, CID is relatively stable and does not need to be expressed during prophase to remain at high levels in metaphase I of meiosis. Expression of CID during prophase can even be deleterious, causing ectopic localization to non-centromeric chromatin, abnormal meiosis and sterility. CENP-C prophase loading is required for multiple meiotic functions. In early meiotic prophase, CENP-C loading is required for sister centromere cohesion and centromere clustering. In late meiotic prophase, CENP-C loading is required to recruit kinetochore proteins. CENP-C is one of the few proteins identified in which expression during prophase is required for meiotic chromosome segregation. An implication of these results is that the failure to maintain recruitment of CENP-C during the extended prophase in oocytes would result in chromosome segregation errors in oocytes.
... Z stacks of triple color images of the ovarioles stained with DAPI, anti-Orb antibody, and rhodamine-conjugated phalloidin were acquired on a Nikon A1plus scanning confocal microscope with a GaAsP detector and a 20× 0.75 N.A. lens using Galvano scanning, at every 1 µm/ step. Egg chamber stages' characterization was previously described (50,87). Nurse cell area and oocyte area were specified (at the largest cross-section) and measured by manual polygon selection (area size) in FIJI. ...
... Z stack images of the ovarioles stained with DAPI, rhodamine-conjugated phalloidin, and Cy5-labeled FLAP-X smiFISH probe (some samples were also labeled with YFP) were acquired on a Nikon W1 spinning disk confocal microscope (Yokogawa CSU with pinhole size 50 µm) with a Hamamatsu ORCA-Fusion Digital CMOS Camera, and a 40× 1.25 N.A. silicone oil lens, at every 0.5 µm/step. Egg chamber stages' characterization was previously described (50,87). A sum Z-projection of a total 2.5 µm z-stack image (six z-slices in total) of each egg chamber was created by FIJI (Image>Stacks>Z projection>Sum slices), and cell area and fluorescence intensity were measured by manual polygon selection in FIJI. ...
... Z stacks of triple color images of the ovarioles stained with DAPI, rhodamineconjugated phalloidin, and FITC-conjugated β-tubulin or EMTB-3XGFP labeling were acquired on a Nikon W1 spinning disk confocal microscope (Yokogawa CSU with pinhole size 50 µm) with a Hamamatsu ORCA-Fusion Digital CMOS Camera, with a 100× 1.35 N.A. silicone oil lens, at every 0.5 µm/step. Egg chamber stages' characterization was previously described (50,87). A sum Z-projection of a total 2.5 µm z-stack image (six z-slices in total) of a stage 6 to 7 egg chamber was created by FIJI (Image>Stacks>Z projection>Sum slices), and cell area and fluorescence intensity were measured by manual polygon selection in FIJI. ...
In many species, only one oocyte is specified among a group of interconnected germline sister cells. In Drosophila melanogaster , 16 interconnected cells form a germline cyst, where one cell differentiates into an oocyte, while the rest become nurse cells that supply the oocyte with mRNAs, proteins, and organelles through intercellular cytoplasmic bridges named ring canals via microtubule-based transport. In this study, we find that a microtubule polymerase Mini spindles (Msps), the Drosophila homolog of XMAP215, is essential for maintenance of the oocyte specification. mRNA encoding Msps is transported and concentrated in the oocyte by dynein-dependent transport along microtubules. Translated Msps stimulates microtubule polymerization in the oocyte, causing more microtubule plus ends to grow from the oocyte through the ring canals into nurse cells, further enhancing nurse cell-to-oocyte transport by dynein. Knockdown of msps blocks the oocyte growth and causes gradual loss of oocyte determinants. Thus, the Msps-dynein duo creates a positive feedback loop, ensuring oocyte fate maintenance by promoting high microtubule polymerization activity in the oocyte, and enhancing dynein-dependent nurse cell-to-oocyte transport.
... To identify the different follicular stages, the detailed description of the stagespecific characteristics according to Jia et al. served as guidance. 63 ...
The trace element lithium exerts a versatile bioactivity in humans, to some extend overlapping with in vivo findings in the model organism Drosophila melanogaster . A potentially essential function of lithium in reproduction has been suggested since the 1980s and multiple studies have since been published postulating a regulatory role of lithium in female gametogenesis. However, the impact of lithium on fruit fly egg production has not been at the center of attention to date. In the present study, we report that dietary lithium (0.1–5.0 mM LiCl) substantially improved life time egg production in D. melanogaster w 1118 females, with a maximum increase of plus 45% when supplementing 1.0 mM LiCl. This phenomenon was not observed in the insulin receptor mutant InR E19 , indicating a potential involvement of insulin‐like signaling in the lithium‐mediated fecundity boost. Analysis of the whole‐body and ovarian transcriptome revealed that dietary lithium affects the mRNA levels of genes encoding proteins related to processes of follicular maturation. To the best of our knowledge, this is the first report on dietary lithium acting as an in vivo fecundity stimulant in D. melanogaster , further supporting the suggested benefit of the trace element in female reproduction.
... 44,90 Timing of oocyte stages and embryonic nuclear cycles-Staging of oocytes and embryonic nuclear cycles was done as described previously. 94,95 In short, oocytes and embryos were stained with the DAPI nuclear stain to determine the position and the number of the nuclei in oocytes and embryos, which then enabled us to stage the oocytes and embryos into the correct oogenic stage and nuclear cycle. ...
RNA granules are membraneless condensates that provide functional compartmentalization within cells. The mechanisms by which RNA granules form are under intense investigation. Here, we characterize the role of mRNAs and proteins in the formation of germ granules in Drosophila. Super-resolution microscopy reveals that the number, size, and distribution of germ granules is precisely controlled. Surprisingly, germ granule mRNAs are not required for the nucleation or the persistence of germ granules but instead control their size and composition. Using an RNAi screen, we determine that RNA regulators, helicases, and mitochondrial proteins regulate germ granule number and size, while the proteins of the endoplasmic reticulum, nuclear pore complex, and cytoskeleton control their distribution. Therefore, the protein-driven formation of Drosophila germ granules is mechanistically distinct from the RNA-dependent condensation observed for other RNA granules such as stress granules and P-bodies.
... Subsequently, all females were dissected under the stereomicroscope, mature oocytes at stage 14 (Jia et al., 2016) were counted from both ovaries, and the measurements for body size ( Figure 2) and crop size ( Figure 3) were taken. Each crop was then crushed in 500 μL saline and the entire suspension of 500 μL was used for the spread plating as described by Sanders (2012) on selective yeast medium (Sanders, 2012). ...
Parent-to-offspring transmission of beneficial microorganisms is intimately interwoven with the evolution of social behaviors. Ancestral stages of complex sociality-microbe vectoring interrelationships may be characterized by high costs of intensive parental care and hence only a weak link between the transmission of microbial symbionts and offspring production. We investigate the relationship between yeast symbiont transmission and egg-laying, as well as some general factors thought to drive the "farming" of microscopic fungi by the fruit fly Drosophila melanogaster, an insect with no obvious parental care but which is highly dependent on dietary microbes during offspring development. The process of transmitting microbes involves flies ingesting microbes from their previous environment, storing and vectoring them, and finally depositing them to a new environment. This study revealed that fecal materials of adult flies play a significant role in this process, as they contain viable yeast cells that support larval development. During single patch visits, egg-laying female flies transmitted more yeast cells than non-egg-laying females, suggesting that dietary symbiont transmission is not random, but linked to offspring production. The crop, an extension of the foregut, was identified as an organ capable of storing viable yeast cells during travel between egg-laying sites. However, the amount of yeast in the crop reduced rapidly during periods of starvation. Although females starved for 24 h deposited a smaller amount of yeast than those starved for 6 h, the yeast inoculum produced still promoted the development of larval offspring. The results of these experiments suggest that female Drosophila fruit flies have the ability to store and regulate the transfer of microorganisms beneficial to their offspring via the shedding of fecal material. We argue that our observation may represent an initial evolutionary stage of maternal care through the manipulation of microbial load, from which more specialized feedbacks of sociality and microbe management may evolve.
... /2023 fluoxetine After two days flies were transferred to fresh food -control or conditioned with fluoxetine -and maintained an additional two days On Day 5 ovaries were dissected in ice-cold PBST (PBS with 0.5% Tween 20), individual ovarioles separated, fixed for 20 min with 4% paraformaldehyde, rinsed three times with PBS, incubated for 30 min with 0.3% BSA (blocking solution) in PBS at room temperature. Stages of egg chambers were determined as described (Jia et al., 2016). Follicle cells were counted in images that showed sagittal sections of each egg chamber. ...
A bstract
Declining germline quality is a major cause of reproductive senescence. Potential remedies could be found by studying regulatory pathways that promote germline quality. Several lines of evidence, including a C. elegans male pheromone ascr#10 that counteracts the effects of germline aging in hermaphrodites, suggest that the nervous system plays an important role in regulating germline quality. Inspired by the fact that serotonin mediates ascr#10 signaling, here we show that serotonin reuptake inhibitors recapitulate the effects of ascr#10 on the germline and promote healthy oocyte aging in C. elegans . Surprisingly, we found that pharmacological increase of serotonin signaling stimulates several developmental processes in D. melanogaster , including improved oocyte quality, although underlying mechanisms appear to be different between worms and flies. Our results reveal a plausibly conserved role for serotonin in maintaining germline quality and identify a class of therapeutic interventions using available compounds that could efficiently forestall reproductive aging.
... Z stacks of triple color images of the ovarioles stained with DAPI, anti-Orb antibody, and rhodamine conjugated-phalloidin staining were acquired on a Nikon A1plus scanning confocal microscope with a GaAsP detector and a 20X 0.75 N.A. lens using Galvano scanning. Egg chamber stages characterization was as previously described (50,83). ...
... Z stacks of triple color images of the ovarioles stained with DAPI, rhodamine conjugated-phalloidin, and Cy5-labeled FLAP-X smiFISH probe staining were acquired on a Nikon W1 spinning disk confocal microscope (Yokogawa CSU with pinhole size 50 µm) with a Hamamatsu ORCA-Fusion Digital CMOS Camera, and a 40X 1.25 N.A. silicone oil lens, at every 0.5 µm/step. Egg chamber stages characterization was as previously described (50,83). A sum Z-projection of a total 2.5 µm z-stack image (6 z slices in total) of each egg chamber was created by FIJI (Image>Stacks>Z projection>Sum slices), and cell area and fluorescence intensity were measured by manual polygon selection in FIJI. ...
... ; https://doi.org/10.1101/2023.03.09.531953 doi: bioRxiv preprint 100X 1.35 N.A. silicone oil lens, at every 0.5 µm/step. Egg chamber stages characterization was as previously described (50,83). A sum Z-projection of a total 2.5 µm z-stack image (6 z slices in total) of a stage 6-7 egg chamber was created by FIJI (Image>Stacks>Z projection>Sum slices), and cell area and fluorescence intensity were measured by manual polygon selection in FIJI. ...
In many species, only one oocyte is specified among a group of interconnected germline sister cells. In Drosophila melanogaster , 16-cell interconnected cells form a germline cyst, where one cell differentiates into an oocyte, while the rest become nurse cells that supply the oocyte with mRNAs, proteins, and organelles through intercellular cytoplasmic bridges named ring canals via microtubule-based transport. In this study, we find that a microtubule polymerase Mini spindles (Msps), the Drosophila homolog of XMAP215, is essential for the oocyte fate determination. mRNA encoding Msps is concentrated in the oocyte by dynein-dependent transport along microtubules. Translated Msps stimulates microtubule polymerization in the oocyte, causing more microtubule plus ends to grow from the oocyte through the ring canals into nurse cells, further enhancing nurse cell-to-oocyte transport by dynein. Knockdown of msps blocks the oocyte growth and causes gradual loss of oocyte determinants. Thus, the Msps-dynein duo creates a positive feedback loop, enhancing dynein-dependent nurse cell-to-oocyte transport and transforming a small stochastic difference in microtubule polarity among sister cells into a clear oocyte fate determination.
Significance statement
Oocyte determination in Drosophila melanogaster provides a valuable model for studying cell fate specification. We describe the crucial role of the duo of microtubule polymerase Mini spindles (Msps) and cytoplasmic dynein in this process. We show that Msps is essential for oocyte fate determination. Msps concentration in the oocyte is achieved through dynein-dependent transport of msps mRNA along microtubules. Translated Msps stimulates microtubule polymerization in the oocyte, further enhancing nurse cell-to-oocyte transport by dynein. This creates a positive feedback loop that transforms a small stochastic difference in microtubule polarity among sister cells into a clear oocyte fate determination. Our findings provide important insights into the mechanisms of oocyte specification and have implications for understanding the development of multicellular organisms.
... While loss of Atf4 in the somatic gonad did not result in increased follicle death, we observed a delay in border cell migration in these follicles. We quantified the extent of border cell migration by measuring follicle size and percent border cell migration across stage 9-10 follicles (Fig. S1E) 22 . The average area of follicles with border cells in the first, second, or final third of migration was then plotted for each genotype, similar to previously published characterizations of border cell migration 23 . ...
Reproduction is an energy-intensive process requiring systemic coordination. However, the inter-organ signaling mechanisms that relay nutrient status to modulate reproductive output are poorly understood. Here, we use Drosophila melanogaster as a model to establish the Integrated Stress Response (ISR) transcription factor, Atf4, as a fat tissue metabolic sensor which instructs oogenesis. We demonstrate that Atf4 regulates the lipase Brummer to mediate yolk lipoprotein synthesis in the fat body. Depletion of Atf4 in the fat body also blunts oogenesis recovery after amino acid deprivation and re-feeding, suggestive of a nutrient sensing role for Atf4. We also discovered that Atf4 promotes secretion of a fat body-derived neuropeptide, CNMamide, which modulates neural circuits that promote egg-laying behavior (ovulation). Thus, we posit that ISR signaling in fat tissue acts as a "metabolic sensor" that instructs female reproduction: directly, by impacting yolk lipoprotein production and follicle maturation, and systemically, by regulating ovulation.
... These cells were sampled from egg chambers present at progressive stages of development, since follicle cells normally increase their nuclear area following the onset of midoogenesis. 21 A positive correlation was indeed detected between the distribution of RFP intensities and the progressively increasing developmental stages of follicle cells (Figures 1D and S1). When RFP intensities were measured and plotted against the per-quartile distribution of nuclear area for both Lgl-KD (n = 57) and Lgl-KD + N ICD (n = 363) multilayered cells, a similar correlation was detected ( Figures 1E and 1F). ...
In proliferating neoplasms, microenvironment-derived selective pressures promote tumor heterogeneity by imparting diverse capacities for growth, differentiation, and invasion. However, what makes a tumor cell respond to signaling cues differently from a normal cell is not well understood. In the Drosophila ovarian follicle cells, apicobasal-polarity loss induces heterogeneous epithelial multilayering. When exacerbated by oncogenic-Notch expression, this multilayer displays an increased consistency in the occurrence of morphologically distinguishable cells adjacent to the polar follicle cells. Polar cells release the Jak/STAT ligand Unpaired (Upd), in response to which neighboring polarity-deficient cells exhibit a precursor-like transcriptomic state. Among the several regulons active in these cells, we could detect and further validate the expression of Snail family transcription factor Escargot (Esg). We also ascertain a similar relationship between Upd and Esg in normally developing ovaries, where establishment of polarity determines early follicular differentiation. Overall, our results indicate that epithelial-cell polarity acts as a gatekeeper against microenvironmental selective pressures that drive heterogeneity.
