The Utility of Stage-specific Mid-to-late Drosophila Follicle Isolation

Summary

Stage-specific isolation of mid-to-late Drosophila follicles is useful for a variety of purposes. Such follicles develop in culture, which allows for genetic and/or pharmacologic manipulations to be coupled with in vitro development assays and live imaging. Additionally, follicles can be used for molecular studies, such as isolating mRNA and protein.

Abstract

Drosophila oogenesis or follicle development has been widely used to advance the understanding of complex developmental and cell biologic processes. This methods paper describes how to isolate mid-to-late stage follicles (Stage 10B-14) and utilize them to provide new insights into the molecular and morphologic events occurring during tight windows of developmental time. Isolated follicles can be used for a variety of experimental techniques, including in vitro development assays, live imaging, mRNA expression analysis and western blot analysis of proteins. Follicles at Stage 10B (S10B) or later will complete development in culture; this allows one to combine genetic or pharmacologic perturbations with in vitro development to define the effects of such manipulations on the processes occurring during specific periods of development. Additionally, because these follicles develop in culture, they are ideally suited for live imaging studies, which often reveal new mechanisms that mediate morphological events. Isolated follicles can also be used for molecular analyses. For example, changes in gene expression that result from genetic perturbations can be defined for specific developmental windows. Additionally, protein level, stability, and/or posttranslational modification state during a particular stage of follicle development can be examined through western blot analyses. Thus, stage-specific isolation of Drosophila follicles provides a rich source of information into widely conserved processes of development and morphogenesis.

Introduction

Each Drosophila ovary is composed of ~16 ovarioles, or chains of sequentially maturing egg chambers or follicles. Each follicle is composed of a single oocyte, 15 germ line derived nurse or support cells, and ~650 somatic cells termed follicle cells (Figure 1A). Drosophila oogenesis is divided into 14 morphologically defined stages of development¹. Each stage of follicle development is observed many times within a single fly, making it relatively easy to isolate a substantial number of stage-specific follicles.

The mid-to-late stages of oogenesis (Stages 10B-14) are particularly well suited for stage isolation (Figure 1). At Stage 10B (S10B), the follicle is fully elongated (i.e. its length is equal to that of a Stage 14 (S14) follicle, see Figure 1 and Figure 2H) and half the length of the follicle is composed of nurse cells while the other half is the oocyte (Figure 1C). At this stage the nurse cells undergo dramatic actin remodeling, strengthening the cortical actin and generating parallel bundles of actin filaments². At the same time, a population of follicle cells, termed centripetal cells, migrate in between the nurse cells and the oocyte, and two dorsal groups of follicle cells become specified to undergo migration to form the dorsal appendages, tubular respiratory apparatuses for the embryo³. The nurse cells then contract (S11), squeezing their cytoplasmic contents into the oocyte in a process called nurse cell dumping, which provides the oocyte with the factors necessary for it to complete embryogenesis (Figure 1D). The nurse cells then undergo cell death (S12-S13)⁴, and the follicle cells secrete and pattern the eggshell⁵ (Figures 1E-G). Thus, the end of oogenesis is rich with important developmental and morphogenetic processes.

Isolated mid-to-late stage follicles (S10B-S14) can be used for a variety of purposes, including molecular analyses. For example, mRNA from staged follicles can be isolated for RT-PCR, microarray, or RNA-seq analyses. This allows one to look at gene expression within a short developmental window, with only a few cell types present, and determine how gene expression is changed by either pharmacologic or genetic perturbations. Stage isolation can also be used to look at proteins by western blotting. Such analysis is important because it allows one to quantify the level of protein expression in wild-type versus mutants at specific stages. While one could use immunofluorescent analyses to achieve similar results, quantification of fluorescence is less robust due to the strict requirements that all of the pixels be within the linear range of detection⁶. Additionally, western blot analysis may provide other information, such as if the protein is posttranslationally modified or is expressed from a specific splice isoform. Isolated stages can also be used for further protein purification, including subcellular fractionation or coimmunoprecipitation.

Stage-specific follicle isolation can also be used for in vitro development assays⁷ and live-imaging⁸. Isolated S10B-S13 follicles will continue to develop to S14 in simple culture media (see below). It is important to note that S10A follicles will not progress through nurse cell dumping using the culture conditions discussed in this manuscript. We have used S10B in vitro development assays to define the role of prostaglandins, both pharmacologically and genetically, in regulating actin remodeling by using nurse cell dumping and development as read-outs^7,9. Similarly, the later stages of development can also be isolated to determine the effects of pharmacologic treatments or genetic manipulations on particular processes such as centripetal cell migration, dorsal appendage migration/formation¹⁰, and nurse cell death. Such assays can be used to perform dominant interaction screens or assays; for example, while heterozygosity for mutations in pxt or fascin alone have no effect on S10B in vitro development, follicles from double heterozygotes exhibit nurse cell dumping defects and a block in development⁹.

Additionally, because S10B-13 can develop in culture, all of the processes that occur during this time can be observed by live-imaging. Such imaging can be performed simply using transmitted light (if one is only interested in gross changes in morphology) or with confocal microscopy using transgenic flies expressing fluorescent probes or follicles stained with live imaging dyes. Live imaging is being used to substantially advance our understanding of developmental processes. Indeed, live imaging of late stage follicles has expanded the knowledge of dorsal appendage migration, an example of tubulogenesis¹⁰. We expect that live imaging of additional late stage processes, including actin dynamics during nurse cell dumping, will provide novel insights into these developmental events. It is important to note that while S10A and early stages of follicle development will not continue to develop into a S14 in culture, live-imaging of events occurring during those stages of development is possible using alternative culture conditions^11-14 (see Discussion for more information).

Here we provide detailed protocols for isolating late stage follicles for either in vitro development and live-imaging, or molecular analyses (mRNA and protein isolation).

Protocol

1. Preparing Drosophila Prior to Stage Isolation

1. Make wet yeast paste by combining 50 g of active dry yeast with 90 ml of distilled water. Mix with a spatula to combine. Let the mixture stand for ~30 min before assessing consistency. The consistency should be just thick enough to adhere to the side of a fly vial but not run down the side. To achieve the desired consistency it may be necessary to add up to 10 ml of additional water or a small amount of dry yeast. Store in a covered container at 4 °C. The stored yeast can be used for ~1-2 weeks.
2. Collect <24 hr old adult flies (both males and females) of the desired genotypes and put them into a vial with fly food plus wet yeast paste smeared on the side of the vial. The temperature at which the flies are maintained will vary depending upon the experiment. For routine experiments, flies are maintained at room temperature. Whereas experiments in which a gene is being overexpressed using the UAS/GAL4 system¹⁵ may require maintaining the flies at 25 °C or higher, as appropriate.
3. Provide the flies with a fresh smear of yeast paste daily for 2 days. NOTE: As follicle development is tightly regulated by the nutritional status of the female fly, it is essential to provide the fly with the nutrient rich yeast paste for at least 36 hr prior to dissection.

2. Isolating Mid-to-late Staged Drosophila Follicles

1. Allow the media to come to room temperature (~30 min). If the staged follicles are going to be used for in vitro development assays, freshly prepare IVEM media (Grace's insect media, 10% heat inactivated fetal bovine serum, and 1x penicillin/streptomycin). If the staged follicles are going to be used for mRNA or protein isolation use either Grace's insect or IVEM media. IMPORTANT: Media temperature is critical. If the media is cold it will alter both the actin and microtubule cytoskeletons and block development.
2. Anesthetize the flies in the yeasted vial by injecting CO₂ gas and place 3-5 female flies onto a fly pad under CO₂. IMPORTANT: Do not leave more females than can be dissected in a 10 min time period on the fly pad as extended exposure to CO₂ can cause sterility and death.
3. Fill one well of a 9-spot plate with media and place it on top of a dark surface (a piece of black plexiglass works well). Focus the dissecting scope on the bottom of the well.
4. Using #5 Dumont forceps pick up a single female with one hand (this is best done using the dominant hand). It is often easiest to pick up the female by the wings, legs, or thorax to abdomen transition. Submerge the female in the media filled well. Adjust the focus of the microscope as necessary.
5. Using a second pair of forceps held in the nondominant hand, grab the female at the anterior end of the abdomen so that the posterior end is positioned towards the dominant hand (Figure 2A). Make sure to keep the fly submerged in the media.
6. With the dominant hand, use the forceps to grab the cuticle at the 2nd most posterior pigmented segment and rip it away from the rest of the abdomen (Figures 2B-C).
7. Using the forceps in the dominant hand, gently squeeze the anterior end of the abdomen until the ovaries emerge (Figures 2D-E). If necessary, place the forceps in between the two ovaries and pull them out of the carcass.
8. Either move the ovaries to a new well with fresh media (Figure 2F) or remove the carcass to a Kimwipe or paper towel. This is particularly important if follicles are being isolated for in vitro development.
9. Repeat until 3-5 pairs of ovaries have been isolated. NOTE: Each ovary is composed of ~16 ovarioles or chains of sequentially maturing follicles. Each ovariole is contained within a muscle sheath.
10. Using pin vises and the needles supplied with them, gently pull apart the ovary by running the needles between the ovarioles (Figure 2G). This will sometimes release the individual follicles as well.
11. Individual ovarioles and the easily distinguishable morphological stages of follicle development should now be visible (Figure 2G, teased apart ovary). Refer to Figures 1 and 2H for morphological differences that can be used to distinguish the different stages. To isolate follicles that are within intact ovarioles and still contained within a muscle sheath, use a needle to cut across the ovariole at the anterior of the preceding follicle and at the posterior of the follicle immediately following the follicle of interest. This will break the muscle sheath and the follicle of interest away can now be cut away from the neighboring follicles without damaging it.
12. As individual stages are separated, move the follicles of interest, using a glass pipette, to a new well with fresh media. This is particularly important when isolating follicles for in vitro development. NOTE: It is important to squeeze the pipette bulb before entering the media to prevent the air bubbles from redistributing the follicles of interest throughout the well. Additionally, if the follicles are sticking to the glass pipette, the pipette can be precoated with 3% bovine serum albumin (BSA) by pipetting the BSA solution up and down several times and rinsing with dissecting media.
13. Once enough follicles have been isolated, proceed with subsequent experiments.

3. In vitro Development of S10B Follicles

1. Isolate S10B follicles using the stage isolation protocol (step 2) in IVEM media. Make sure that the follicles are rapidly moved away from debris. As these follicles will continue to mature, it is important that S10Bs are collected for 30-60 min and then placed into the maturation media of choice. NOTE: S10B need to be carefully distinguished from S10A follicles (Figure 1C compared to 1B), as S10A follicles will not mature in the IVEM culture media. In both S10A and S10B follicles, half of the length is composed of nurse cells and the other half is the oocyte, but in S10B the length of the follicle is equal to that of the S14 follicles.
2. Using a glass pipette, move ~30 S10B follicles into a well of a 24-well tissue culture plate. NOTE: Make sure to verify that all of the follicles being transferred are S10B and have not matured to S11.
3. Prepare 1 μl of maturation media per well, i.e. IVEM media plus pharmacological reagents.
4. Using a pulled glass pipette*, remove as much media from the well (step 3.2) as possible, and quickly add maturation media of choice. NOTE: It is essential to use a pulled glass pipette as staged follicles can be easily taken up with either glass pipettes or pipette tips with a Pipetman.
   *To make pulled pipettes: 1) heat the thin portion of long, glass pipettes in the flame of a Bunsen burner just until the glass begins to soften; 2) immediately move the pipette out of the flame and pull horizontally to draw the pipette into a finer tube; 3) break to generate a fine point. CAUTION: Use eye protection as fragments of glass may fly off.
5. Repeat steps 3.1-3.4 for as many wells as the experiment requires.
6. Allow follicles to develop for >10 hr and score the developmental progression under a dissecting scope. NOTE: Experiments are usually scored the next day to allow sufficient time for the follicles to develop. S10B is ~5 hr, S11 is ~30 min, S12 is ~2 hr, and S13 is ~1 hr in duration at 25 °C¹ and development at room temperature will take slightly longer. Similar experiments to that described for S10B follicles can be performed using S11-S13 follicles.

4. Stage Isolation for Live Imaging

1. Isolate late stage follicles (S10B-14) expressing the fluorescent marker of choice using the stage isolation protocol (step 2) in IVEM media, quickly moving follicles away from debris.
2. Move follicles of interest into new media and then transfer by glass pipette to a coverslip bottom Petri plate. Take care to add media so that it bubbles up in the coverslip bottom area but does not spill over.
3. For longer time-lapse movies, it is sometimes necessary to maintain humidity within the Petri plate by adding a rolled up Kimwipe, moistened with water, to the inside edge of the Petri plate. Place the lid on top.
4. Image on an inverted microscope. Detailed resolution will require confocal microscopy. It is necessary to balance frequency of imaging, strength of illumination, and length of imaging; this will need to be independently worked out for each labeling tool and developmental process.

5. Stage Isolation for mRNA Preparation

1. Isolate individual stage follicles using the stage isolation protocol (step 2) with either Grace's or IVEM media.
2. Using a glass pipette, move the individual stages of interest into new wells with media; it is possible to collect multiple stages at once.
3. Keep collection times under 1 hr. Move the follicles, using a glass pipette, to 1.5 ml microfuge tubes.
4. Spin the tube briefly in a mini-microcentrifuge to pellet all of the follicles. Using a pulled glass pipette (see step 3.4) carefully remove all of the media. NOTE: If using a full size microcentrifuge, spin down the follicles at low speed.
5. Add 100 μl of Trizol and grind by hand for ~20 sec using a plastic pestle. Spin down at full-speed in a microcentrifuge and move Trizol to a new 1.5 ml microfuge tube being careful not to disturb any pelleted debris. Store at -80 °C.
6. Repeat steps 5.1-5.5 until enough follicles have been obtained for the experiment. Routinely, ~75 S10B, ~75 S12, and ~100 S14 follicles yield ~10 μg of RNA.
7. Thaw the samples on ice. Combine samples, as appropriate, into one microfuge tube and bring the Trizol volume up to 800 μl. Proceed with RNA isolation as directed by the manufacturer. IMPORTANT: Remember to DNase treat the isolated RNA with RNase free DNase.

6. Stage Isolation for Western Blotting

1. Preheat a heat block to 100 °C.
2. Isolate individual stage follicles using the stage isolation protocol (step 2) with either Grace's or IVEM media. NOTE: It is necessary to empirically determine how many of a particular stage follicle are needed to observe each specific protein. For highly expressed proteins 1-3 S10B follicles/well is enough to see a strong signal on a western blot. However, it is important to make a representative sample, taking follicles from multiple females. Thus, 15-20 follicles of a particular stage are usually collected.
3. Move follicles, using a glass pipette, to a 1.5 ml microfuge tube. Spin briefly in a mini-microcentrifuge to pellet the follicles (see step 5.4). Carefully remove all of the media using a pulled glass pipette (see step 3.4) and add 50 μl of 1x PBS and 50 μl of 2x Laemmli buffer.
4. Grind by hand for ~20 sec with a plastic pestle. NOTE: Plastic pestles can be reused for grinding western samples by washing and autoclaving them.
5. Boil the samples for 10 min in the heat block.
6. Chill briefly on ice and spin at full-speed for 15 sec in a microcentrifuge. Either immediately load onto a SDS-PAGE gel or store at -20 °C. NOTE: If the samples are stored, remember to reboil the samples before loading onto the gel.
7. Perform western blot analysis following standard protocols.

Results

When isolating specific stages of Drosophila follicle development it is essential to be able to accurately distinguish the different morphological stages. This is somewhat challenging for S10A and S10B, as the nurse cells and the oocyte each take up half the length of the follicle at these stages (Figure 1B compared to 1C). However, S10A follicles are shorter in length than S10B follicles, as the S10B follicles are fully elongated and thus equal in length to a S14 follicle (

Discussion

The Drosophila follicle is composed of only a small number of cell types, making it ideal for both morphologic and molecular analyses. Furthermore, due to the structure of the ovary, it is relatively easy to obtain large numbers of specific stages of follicle development with a common dissecting scope and minimal training. As each stage represents a short temporal window, stage isolation can provide significant molecular insights into the developmental processes occurring during that stage. For example, we have ...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Active Dry Yeast	Genesee Scientific	62-103	Any source of Active Dry Yeast is fine
Grace’s Insect Media	Lonza	04-457F
Heat Inactivated Fetal Bovine Serum	Atlanta Biologicals	S11050H	Any Heat Inactivated FBS should work
10x Pen/Strep	Gibco/Invitrogen	15140-122
Pin Vises and Needles	Ted Pella, Inc.	13561-10
Spot Plate, Nine Well	Corning	7220-85
#5 Dumont forceps	Fine Science Tools	11252-20
24 multi-well plates	Becton Dickinson	35 3226	Any 24-well tissue culture dish should work
Coverslip Bottom Dishes (35mm)	MatTek Corporation	P35G-1.0-14-C	Coverslip thickness will depend on the microscope/objective being used
Glass pipettes	Corning	7095B-5x (for transferring follicles)
Glass pipettes - long	Corning	7095B-9 (for producing pulled pipettes)
Sample pestle (1.5 μl; RNase/DNase free)	Research Products International	199228	Any plastic pestle that fits 1.5 μl microfuge tubes can be used
Trizol	Invitrogen	15596-018