Upright Imaging of Drosophila Egg Chambers

Summary

The upright imaging method described in this protocol allows for the detailed visualization of the poles of a developing Drosophila melanogaster egg. This end-on view provides a new perspective into the arrangements and morphologies of multiple cell types in the follicular epithelium.

Abstract

Drosophila melanogaster oogenesis provides an ideal context for studying varied developmental processes since the ovary is relatively simple in architecture, is well-characterized, and is amenable to genetic analysis. Each egg chamber consists of germ-line cells surrounded by a single epithelial layer of somatic follicle cells. Subsets of follicle cells undergo differentiation during specific stages to become several different cell types. Standard techniques primarily allow for a lateral view of egg chambers, and therefore a limited view of follicle cell organization and identity. The upright imaging protocol describes a mounting technique that enables a novel, vertical view of egg chambers with a standard confocal microscope. Samples are first mounted between two layers of glycerin jelly in a lateral (horizontal) position on a glass microscope slide. The jelly with encased egg chambers is then cut into blocks, transferred to a coverslip, and flipped to position egg chambers upright. Mounted egg chambers can be imaged on either an upright or an inverted confocal microscope. This technique enables the study of follicle cell specification, organization, molecular markers, and egg development with new detail and from a new perspective.

Introduction

Study of Drosophila melanogaster has provided great insights into the genetic regulation of a wide range of phenomena. In particular, there has been extensive research on egg development, because oogenesis provides a tractable way to investigate many different developmental processes, including tissue patterning, cell polarity changes, cell cycle switching, and translational regulation^1,2,3,4. One important morphogenic event during oogenesis is the specification, acquisition of motility, and migration of a set of cells called border cells (reviewed in ⁵). Since cell migration is a key feature of animal morphogenesis, and because the genetic regulation of this process is well-conserved, mechanisms determined in flies are likely to be important in other contexts. Thus, we are investigating the molecular control of border cell migration. For our studies, we have developed a new method to observe the anterior poles of developing eggs, where border cells arise, to examine how they develop in detail.

Within the ovary, egg chambers at various developmental stages exist along chains called ovarioles, which are encased in a thin sheath (Figure 1A). Each egg chamber will go on to form one egg. During oogenesis, several different cell types must develop in a coordinated manner. The D. melanogaster egg chamber consists of 16 germ-line cells, including one oocyte, surrounded by a single-layer follicular epithelium⁶. A small number of specialized cells, called polar cells, arise at the anterior and posterior poles of the epithelium. The 6-8 border cells originate in the anterior epithelium of the egg chamber, induced by the polar cells^5,7. In mid-oogenesis (stage 9), the border cells detach from their neighbors and migrate between the nurse cells to reach the oocyte at the posterior of the egg chamber^5,7. This movement must be accomplished while the border cells remain in a cluster surrounding two non-motile polar cells, making this a type of collective cell migration. Successful migration of the border cell cluster ensures proper development of the micropyle of the egg shell, which is necessary for fertilization.

The anterior polar cells instruct border cell fate by activating a signal transduction cascade. Polar cells secrete a cytokine, Unpaired (UPD), which binds to a transmembrane receptor, Domeless (DOME), on neighboring follicle cells during stage 8 of oocyte development^8,9. The binding of UPD causes Janus tyrosine kinase (JAK) to phosphorylate the Signal Transducer and Activator of Transcription (STAT)^8,10,11. STAT then moves to the nucleus to activate transcription. Slow Border Cells (SLBO) is a transcription factor that is a direct transcriptional target of STAT and is also required for border cell migration¹². Lateral views of egg chambers indicate that STAT activity is regulated in a gradient across the anterior epithelium^8,11,13. Follicle cells closest to the polar cells have the highest levels of activated STAT, thus they become border cells and invade the adjacent germ-line tissue.

To understand how the border cells are specified within and detach from the epithelium, we need to observe how the tissue is organized. If we view egg chambers from an anterior-on perspective, we would expect radial symmetry of STAT activity in the follicle cells surrounding the polar cells. An end-on view would also more accurately show differences of membrane proteins and cell-cell interfaces prior to and during detachment than comparing cells in different focal planes. Because egg chambers are oblong and attached to each other by stalk cells, they settle onto slides laterally, making it difficult to observe the anterior architecture. Thus, much information about the cells at the poles of the egg chamber has been inferred from lateral views. Although some information can be obtained through algorithmic 3-D reconstructions of optical sections, light scattering, photobleaching, and poorer limits of resolution in the Z-axis make this information less detailed and reliable in the absence of expensive techniques like super-resolution microscopy¹⁴. Other kinds of section-based imaging (for example, electron microscopy or microtome sectioning) require extensive manipulation of tissues, including dehydration, increasing the likelihood for artifacts. Thus, we developed a new method to image D. melanogaster egg chambers while upright. This method has already proven useful in elucidating how motile cells are fated (see Representative results and Manning et al, under review), and is likely to be more broadly valuable in studies of other aspects of oogenesis.

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Protocol

1. Dissection of Ovarioles

1. Transfer about fifteen 2-4-day-old female flies and a few males to a fresh fly food vial with added active dry yeast. Use cotton as a plug for the vials, and add a few drops of water to the cotton to keep the humidity high.
2. Place vial in a 25 °C incubator for 14-16 hr to maximize the number of stage 8-10 egg chambers.
   NOTE: Incubation time varies depending on temperature and desired stage.
3. Prepare dissection media, 0.1M KPO₄ and NP40 solutions as needed for the next day (see Materials).
4. Anesthetize the flies using CO₂, and place under a dissection microscope. Pipet several drops of dissection media into each cavity in the glass depression slide.
5. Hold forceps in each hand at an approximately 30° angle to the table top and orient one female fly with the wings down. With your non-dominant hand, pick up the female using forceps, grasping her at the anterior of the abdomen, near the thorax, and submerge her into the dissection media in the depression slide (Figure 1A inset).
6. With the other pair of forceps, grasp the exoskeleton at the ventral posterior of the abdomen and pierce through. Grab the ovaries at the posterior end, then pull them out of the abdomen and place into the fresh media on the other side of the depression slide. (Figure 1A inset). Avoid pulling apart the gut. Discard the carcass.
7. Repeat with the other female flies until all ovaries are dissected. Discard the males.
8. With one pair of forceps, hold an ovary at the posterior end (near the largest egg chambers), and with the other hand use a pair of forceps to pinch one of the most anterior egg chambers (germarium). (Figure 1A).
9. Slowly and steadily, pull an ovariole chain out of the ovary sheath. Continue to dissect out ovarioles individually until all desired egg chambers are removed.
10. Repeat steps 1.7-1.8 for all dissected ovaries.
11. Once complete, transfer ovarioles to a 0.6ml tube using a plastic transfer pipette.
12. Let ovarioles settle to the bottom of tube, then proceed to fixing and staining steps.

2. Immunofluorescent (IF) Staining of Ovarioles

1. Carefully remove dissection media from ovarioles with a pipette. Be sure not to remove any ovarioles or egg chambers. Leave approximately 50 μl of ovarioles and dissection media.
2. Add 50 μl of 16% paraformaldehyde to 150 μl of 0.1 M KPO₄ Buffer in a tube, and add solution to egg chambers to fix them. Incubate while rocking for 10 min. CAUTION: paraformaldehyde is toxic.
3. Remove fixative and rinse twice with 0.5 ml NP40 wash buffer.
4. Wash two times for 15 min each at RT.
5. Refer to standard IF protocol¹⁵ for subsequent steps.
   1. Briefly, after fixation, add primary antibodies at the appropriate dilutions and incubate O/N at 4 °C. Wash in NP40 several times, then add secondary antibodies (1:200 dilutions).
   2. Wash several times in NP40, then add DAPI (1:1,000) for 10 min, then repeat washes. Once IF protocol is complete, add 200 μl of 50% glycerol in PBS to egg chambers and let sit at RT for 2 hr.
   3. Remove solution, replace it with 70% glycerol in PBS, and cover with foil. Place sample at 4 ᵒC until ready for mounting. Meanwhile, continue with step 3.

3. Advance Preparation of Glycerin Jelly Slides

1. Measure at least 4 ml of glycerin jelly using a spatula and fill a glass cell culture tube to the half way mark. Melt glycerin jelly in a water bath at 55 °C for 30 min.
2. Pour a small drop of glycerol, approximately 300 μl, from the stock solution onto two microscope slides. Using a tissue, spread glycerol in a thin layer across each of the slides. This provides a base and allows for easy removal of glycerin jelly at step 5.6.
3. Cut the tip off of a filtered 1,000 μl pipette tip. Use cut pipette tip to transfer 1,000-1,500 μl of warm glycerin jelly slowly to each microscope slide. Take care to prevent bubbles.
4. Let glycerin jelly slides sit for 5 min at RT to set.
5. Place each glycerin jelly slide in 60 mm × 15 mm petri dish and cover with plastic wrap. Place at 4 °C O/N. Optionally, store glycerin jelly slides for up to 5 days at 4 °C.

4. Mounting Egg Chambers

1. Using samples from step 2.5, pipette several 3 μl drops of egg chambers in 70% glycerol across one glycerin jelly slide. Continue pipetting 3 μl drops until all egg chambers are on this slide. Make sure each drop contains at least ten egg chambers. Meanwhile, heat a culture tube of glycerin jelly in a 55 °C water bath.
2. Place the glycerin jelly slide with egg chambers under a dissection microscope. Adjust magnification so that only one drop of egg chambers is in the field of view (Figure 1B).
3. Using a 30 gauge needle as a spoon, pick up the desired stage egg chamber. When necessary, separate desired egg chambers from an ovariole chain using the needle as a knife.
4. To avoid debris that may interfere with imaging, transfer the egg chamber to the second glycerin jelly slide, with the anterior facing left (Figure 1E-1). Repeat until there are at least seven egg chambers lined up in a column (Figure 1C).
5. Form new columns of seven until all desired egg chambers are transferred to the second glycerin jelly slide.
6. Tear off a small piece of tissue and twist. Use this to absorb the excess PBS-Glycerol from the egg chambers by lightly touching each one. Take care to not remove egg chambers.
7. Cut off the tip of a filtered 200 μl pipette tip. Use this to transfer 150 μl of glycerin jelly to cover all the columns of egg chambers.
   NOTE: The amount of glycerin jelly needed to cover the columns is based on egg chamber stages and number of columns. Amount may be adjusted.
8. Let mounted egg chambers sit for 5 min at RT until the top layer of glycerin jelly is completely solidified (Figure 1D).
9. Place slide of mounted egg chambers in a 60 mm × 15 mm petri dish and cover with plastic wrap. Store at 4 °C O/N to allow the glycerin jelly layers to solidify together (place in the dark if there is concern about photobleaching).

5. Preparation of Egg Chambers for Imaging

1. Place slide of mounted egg chambers under a dissection microscope. Adjust magnification so that only one column of egg chambers is in the field of view.
2. Using a 45° angle miniature scalpel, cut a straight line along the right side of the first column of egg chambers (close to the posterior side of the egg chambers). (Figure 1D-E)
3. Next, cut above the first egg chamber at the top and below the last egg chamber in the column. At this point the column of egg chambers should be cut on three sides.
4. Using a microknife, slice along the left side of the column, as close as possible to the anterior side of the egg chambers (Figure 1E-3).
5. Pipette 100 μl of cold 1X PBT over the cut column.
6. Use a round edged spatula to remove the excess glycerin jelly around three sides of the cut column of egg chambers and discard, leaving the rest on the slide.
7. Insert the spatula in the cut made at the anterior side of egg chambers and separate the column from the primary glycerin jelly block.
8. Prepare samples for either inverted (5.8.1) or upright (5.8.2) microscopy.
   1. For Inverted Microscope: Transfer the column of egg chambers to a coverslip with anterior side of egg chambers facing down. Repeat steps 5.2-5.8 until all columns of glycerin jelly have been removed and mounted. Add a couple of drops of 50% PBS-Glycerol onto each block of egg chambers to prevent them from drying out. Image egg chambers directly on coverslip.
   2. For Upright Microscope: Transfer the column of egg chambers to a microscope slide with the anterior side facing up. Repeat steps 5.2-5.9 until all columns of glycerin jelly have been removed and mounted on microscope slides. Add a couple of drops of 50% PBS-Glycerol onto each block of egg chambers and cover blocks with a #1 ½ coverslip.

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Results

The upright imaging method allowed us to see directly the organization of cells in the anterior follicular epithelium at stage 8. A general marker for follicle cell fate, the Eyes Absent (EYA) protein, as well as the nuclear DNA marker DAPI, showed even expression across this field of cells, and demonstrated that all cells could be seen with similar staining intensities (Figure 2B”). Proteins regulated in response to the cytokine UPD, however, showed variable patterns and expression levels. Polar c...

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Discussion

Here we describe a method to mount and image small, developing egg chambers from a end-on perspective. Common techniques for imaging egg chambers are optimized for lateral views and primarily allow precise visualization of medio-lateral follicle cells when stained with fluorescent antibodies. The use of Z-stacks or 3-D reconstructions aids in viewing multiple focal planes, but is still inadequate for sub-cellular resolution of the poles of elliptical egg chambers (Figure 2D). While this can be overcome p...

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Materials

Name	Company	Catalog Number	Comments
0.1 M Potassium phosphate Buffer (KPO4 Buffer)			Add 3.1 g of NaH2PO4•H2O and 10.9 g of Na2HPO4 (anhydrous) to distilled H2O to make a volume of 1 L. The pH of the final solution will be 7.4. This buffer can be stored for up to 1 month at 4°C.
16% Paraformaldehyde aqueous solution, EM grade	Electron Microscopy Sciences	15710	methanol-free to preserve GFP fluorescence
30G x 1/2 inch needle	VWR	BD305106	Regular bevel
Active Dry Yeast	Genesee Scientific	62-103	To fatten female flies
Bovine Serum Albumin	PAA-cell culture company	A15-701	Used in NP40 Wash Buffer
Dumont #5 Forceps	Fine Science Tools	11295-10	Dumostar alloy, biologie tip; sharp tips are essential for ovariole dissection
DAPI (4′,6-Diamidino-2-phenylindole dihydrochloride)	Sigma Aldrich	D9542	5 mg/ml stock solution
Fetal Bovine Serum (FBS)	Life Technologies	16140-071	Added to supplement dissection medium (10%)
Glass culture tube	VWR	47729-576	14 ml
Glass Depression Slides	VWR	470019-020	1.2 mm Thick, Double Cavity
Glycerin Jelly	Electron Microsocpy Sciences	17998-10	Mounting media
Glycerol	IBI Scientific	IB15760	70% in PBS
IGEPAL CA-630	Sigma Aldrich	I3021-500ML	(interchangable for Nonidet P-40)  Used in NP40 Wash Buffer
Leica Fluorescent Stereoscope	Leica Microsystems
Leica SP5 Confocal Microscope	Leica Microsystems		40x/0.55NA dry objective
Micro spatula	VWR	82027-518	Stainless steel
Microscope Slide	VWR	16004-368	75x25x1 mm
NP40 Wash Buffer			50 mM TRIS-HCl, 150 mM NaCl, 0.5% Ipegal, 1 mg/ml BSA, and 0.02% sodium azide
Penicillin-Streptomycin-Glutamine	Life Technologies	10378-016	Added to supplement dissection medium (0.6X)
Petridish- Polysterine, sterile	VWR	82050-548	60 W x 15 H mm
Phosphate Buffer Saline	Sigma Aldrich	P3813-10PAK	10 packs of Powder
Potassium phosphate dibasic	Sigma Aldrich	P3786-500G	Used in 0.1 M KPO4 Buffer
Potassium phosphate monobasic	Sigma Aldrich	P9791-500G	Used in 0.1 M KPO4 Buffer
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Schneider’s Insect Medium	Life Technologies	11720018	With L-glutamine and sodium bicarbonate
Sodium azide	Sigma Aldrich	S2002-25G	Used in fluorescent antibody staining
Sodium chloride	Sigma Aldrich	S3014-500G	Used in NP40 Wash Buffer
TRIS-HCl	IBI Scientific	IB70144	1 M TRIS-HCl, pH 7.4
Volocity 3D Image Analysis Software	PerkinElmer		For processing confocal Z-stacks