Drosophila as a model to study wound-induced polyploidization

Abstract

Polyploidy is a frequently occurring phenomenon whose impact on organismal health and disease is still poorly understood. A cell is defined as polyploid if it contains more than the diploid copy of its chromosomes, which occurs by endoreplication or cell fusion. In tissue repair, wound-induced polyploidization (WIP) has been found to be a conserved healing strategy from fruit flies to vertebrates. WIP has several advantages over cell proliferation including resistance to oncogenic growth and genotoxic stress. The challenge has been to faithfully identify whether polyploid cells arise and how these unique cells function. Here we provide a detailed protocol to study WIP in the adult fruit fly epithelium where polyploid cells are generated within two days after a puncture wound. Taking advantage of D. melanogaster’s extensive genetic tool kit, the genes required to initiate and regulate WIP, including Myc, have begun to be identified. Continued studies using this method with reveal how other genetic and physiological cues including sex, diet, and age regulate and influence WIP’s function.

SUMMARY:

Wound-induced polyploidization is a conserved tissue repair strategy where cells grow in size instead of dividing to compensate for cell loss. Here we provide a detailed protocol on how to use the fruit fly as a model to measure ploidy and its genetic regulation in epithelial wound repair.

INTRODUCTION:

Drosophila melanogaster is an attractive model system to study the cellular and molecular mechanisms of epithelial wound repair. Like in mammals, the tissue repair mechanisms used depends on both the tissue and its developmental stage. Scarless wound healing occurs in the fruit fly embryo where an actomyosin purse string forms at the epithelial leading edge enabling the wound to seamlessly close^1,2. Post-embryonic wound healing in larval, pupa, and adult fruit fly results in extracellular matrix remodeling, melanin scar formation, and epithelial cell growth^3-6. The epithelial cells enlarge in size by cell fusion and the endocycle, an incomplete cell cycle that bypasses mitosis^3,4,7,8. As result, cell loss is compensated by polyploid cell growth instead of cell division. The adult fly hindgut, midgut, and follicular epithelium also rely on polyploid cell growth to compensate for cell loss after tissue damage^9-11.

Polyploidy is a well-known aspect of organismal development in plants and insects, but in the last few years, it has become more apparent that polyploidy is a conserved tissue repair strategy in vertebrates¹². The zebrafish, which has the capacity to regenerate its heart, relies on polyploid cell growth to heal damaged epicardium¹³. Polyploidy also contributes to mammalian liver regeneration and kidney tubule epithelium repair after acute injury^14,15. In these examples, polyploid cells are generated by endoreplication either endocycle or endomitosis, which results in a binucleated cell due to a block in cytokinesis ¹². The enigma is why polyploid cells arise during wound repair and how polyploidy affects tissue function. Recent studies have provided new insight into the question of whether polyploidy offers a healing advantage or disadvantage. In zebrafish epicardium, polyploidy enhanced the speed of wound healing¹³. In D. melanogaster hindgut and mammalian liver, polyploidy was found to be protective against oncogenic growth^11,14. In adult fly epithelium, we recently found that polyploidy enables wound repair in the presence of genotoxic stress¹⁶. Endoreplication is resistant to DNA damage, allowing wound healing when cell proliferation would otherwise be compromised¹⁷. For cardiomyocytes in mouse and zebrafish hearts, however, polyploidy slows healing, resulting in enhanced scar formation^18,19. Therefore, depending on the organ and/ or cell type, polyploidy can be a beneficial or detrimental tissue repair strategy. The accessibility of D. melanogaster genetics coupled with analysis of the wound-induced polyploidization (WIP) response is key for elucidating the molecular and cellular mechanisms that guide this alternative wound healing strategy.

Here, we present a protocol for analyzing WIP in the adult D. melanogaster epithelium. We include instructions for fruit fly injury, dissection, immunostaining, mounting, imaging, and analysis of re-epithelialization, cell fusion, and endoreplication (ploidy). The imaging and ploidy analysis can also be adapted to other models to test whether WIP occurs. It should be noted that with an increase in nuclear DNA content there is often a corresponding increase in nuclear size. However, there are many examples in biology where nuclear size does not result in a corresponding change in ploidy²⁰. Even more caution should be taken when interpreting nuclear size in the context of a wound environment where cells will often spread or stretch to cover the wound site. Therefore, the only definitive proof of change in ploidy is to measure DNA content by this method or others, such as whole genome sequencing²¹. Finally, we envision that this method will increase the accessibility of the adult D. melanogaster abdominal epithelium as a model to study the role and regulation of polyploidy in wound repair.

PROTOCOL:

1. Staging and wounding of adult fruit flies

1.1 Select D. melanogaster strain of choice (i.e. epi-Gal4/ UAS strain, see Table of Materials).

Table of Materials.

List of all reagents and equipment required for this study.

Name	Company	Catalog Number	Notes
Drosophila husbandry
Fly food	Archon Scientific	N/A	Corn Syrup/Soy food
Dissection, wounding, staining, and mounting
Stereomicroscope	Olympus	SZ51	For dissecting and mounting fly tissue
Flystuff Flypad	Genesee Scientific	59-114	For anethesizing D. melanogaster strains
Blowgun mini	Genesee Scientific	54-104M	For anethesizing D. melanogaster strains
Drosophila tubing and fittings	Genesee Scientific	59-124C, 59-123, 59-140	For anethesizing D. melanogaster strains
Carbon dioxide tank	various distributors	N/A	For anethesizing D. melanogaster strains
Glass dissecting dish	Fisher Scientific	13-748B	For performing dissections in
35mm Petri dishes	Fisher Scientific	FB0875713	For creating plates to dissect in
Dissecting Plates (use Sylgard 184 Sil Elastic Kit)	Ellsworth Adhesives	184SIL	For creating plates to dissect in. Mix epoxy as directed, let dry overnight
Insect pins	Fine Science Tools	26002-10	For wounding and pinning fly abdomens flat
Dumont #5 Forceps	Fine Science Tools	11252-20	For dissecting
Vannas Spring Scissors	Fine Science Tools	15000-00	For dissecting
Pin holders	Fine Science Tools	91606-07	For wounding
Grace's Insect Medium, unsupplemented	Thermofisher	11595030	For dissecting in
Phosphate Buffered Saline, PH 7.4	Sigma	P3813-10PAK	For staining
Rabbit anti-Grainyhead Primary Antibody	N/A	N/A	For immunostaining epithelial nuclei. Protocol to make antibody can be found (Ref. #4 and 8)
Rabbit anti-RFP Primary Antibody	MBL	PM005	For immunostaining mCD8-RFP fly epithelium
Mouse anti-Fasciclin III (Drosophila) Primary Antibody	Developmental Studies Hybridoma Bank	7G10	For immunostaining epithelial cell-cell junctions
Goat anti-Mouse IgG (H+L) Secondary Antibody, Alexa Fluor 488 conjugate	Thermofisher	A11001	For secondary immuostaining
Goat anti-Mouse IgG (H+L) Secondary Antibody, Alexa Fluor 568 conjugate	Thermofisher	A11031	For secondary immuostaining
Donkey anti-Rabbit IgG (H+L) Secondary Antibody, Alexa Fluor 488 conjugate	Thermofisher	A21206	For secondary immuostaining
Donkey anti-Rabbit IgG (H+L) Secondary Antibody, Alexa Fluor 568 conjugate	Thermofisher	A10042	For secondary immuostaining
Triton X-100	Sigma	10789704001	For immuostaining
Bovine Serum Albumin, 30%	Sigma	A7284-500ML	For immuostaining
Click-iT EdU 594 Kit	Thermofisher	C10339	For EdU assay
DAPI	Sigma	D9542-10MG	For immuostaining
Glass slides	Fisher Scientific	12-518-104C	For mounting
Coverslips	Thermofisher	3406	For mounting
Mouting media	Vector Laboratories	H-1000	Anti-fade mounting media to prevent photo bleaching during imaging
Nail polish	Electron Microscopy Sciences	72180	For sealing slides
50mL Conical Centrifuge Tubes	Fisher Scientific	14-432-22	For preparing staining reagents in
Ortibal shaker	Fisher Scientific	02-217-988	For immuostaining
Imaging and Analysis
AxioImager M2 with Apotome	Zeiss	NA	For imaging samples
Fiji/ ImageJ (image analysis software)	NIH	https://imagej.nih.gov/ij	For image analysis
Excel	Microsoft		For performing ploidy calculations
D. melanogaster strains
epi-Gal4	Bloomington Drosophila Stock Center (b)	b38793	Losick et al. Current Biology, 2013
epi-Gal4, UAS-mCD8.RFP	Bloomington Drosophila Stock Center (b)	b38793, b27392	Losick et al. Current Biology, 2013
UAS-myc RNAi	Bloomington Drosophila Stock Center (b)	b36123	Grendler et al. Development, 2019
UAS-Myc	Bloomington Drosophila Stock Center (b)	b9674	Grendler et al. Development, 2019
UAS-fzr RNAi, UAS-Stg	VDRC (v) and Bloomington Drosophila Stock Center (b)	v25550, b56562	Grendler et al. Development, 2019
UAS-E2F RNAi, UAS-RacDN	VDRC (v) and Bloomington Drosophila Stock Center (b)	v108837, b6292	Losick et al. Current Biology, 2013

Here, the Gal4/ UAS system is used to enable epithelial specific gene expression (epi-Gal4) of the a gene or RNAi encoded downstream of UAS. We used fluorescent membrane protein (UAS-Cd8.mRFP), mitotic inducer (UAS-fzr^RNAi, UAS-stg) and WIP inhibitor (UAS-E2F1^RNAi; UAS-Rac^DN).

1.2 Collect 2 vials of 10 to 15 newly enclosed female fruit flies each and age on fresh food vial at 25°C until 3-5 days old. One vial will serve as the uninjured control and other vial will be wounded as described below. The female flies should be maintained with males (~5/ vial).

1.3 To wound the flies assemble several pin holders each with a single 0.10 mm stainless steel pin per holder wearing safety glasses. Ensure the sharp end of the pin is facing out. Pins can easily bend or chip after puncturing the fly and hooked or damaged pins should be discarded.

1.4 Anesthetize aged fruit flies on a CO₂-fly pad under stereomicroscope and align flies into a row using a paint brush. Holding the pin holder in one hand and forceps in the other, use forceps to position the fly with its ventral abdomen facing up .

1.5 Puncture adult female flies within the epithelial pleurite region of tergite A4 on either side ventral midline sternites (Figure 1A). Puncturing in this ventral region provides optimal space away from the dissection sites where tissue edges will be torn by mechanical processing.

Figure 1. Adult fruit fly abdominal wounding, dissecting, and tissue mounting.

(A) Schematic of adult abdominal wounding assay. Flies should be injured on either side of the abdomen at tergite 4 (T4). (B) Adult female fruit fly 3 dpi with melanin scab formed from wound healing (white box). Scale bar, 50μm. (C) Dissected adult abdomen, dorsal view, with tergites labeled. Abdomens should be filleted down the midline of the dorsal side (white dashed line). Scale bar, 50μm. (D) Dissected and filleted adult abdomens, prior to pinning. Scale bar, 50μm. (E) Pinned adult abdomen on a dissecting plate. A pin should be placed in each of the four corners of the abdomen on the dorsal side. The tissue should be gently opened, but not stretched to tear. (F) Adult abdomens mounted and placed on a glass coverslip with the inside of the abdomen facing down towards the coverslip and cuticle oriented toward the glass slide. (G) FasIII staining of intact wound area with no processing perturbation and a central syncytium (dashed yellow line). Scale bar, 50μm. (H) Image of a scratched wound area (*) with an unstained FasIII region that disrupts the syncytium.

1.6 Return wounded flies to food vial and age to desired day post injury (dpi). Epithelial wound healing initiates at 1 dpi and completes by 3 dpi, whereas endoreplication peaks at 2 dpi (ideal for EdU assay, Figure 2).

Figure 2. WIP analysis workflow.

The flowchart depicts the 3 assays described in this study and overlapping and distinct steps to detect and measure the WIP response. The EdU assay measures cell cycle activity (blue boxes), ploidy and re-epithelialization are detected by Grh/FasIII immunostaining (green boxes), and expression of membrane RFP allows the extent of epithelial wound closure to be measured (pink boxes). Common steps are in gray boxes and D. melanogaster strain genotypes are listed above.

2. Fly abdominal dissection

NOTE: During this step, it is important to avoid touching the ventral abdominal tissue with dissections tools as it will compromise the integrity of the epithelium.

2.1 Obtain all required materials for dissection: Grace’s solution, a pair of forceps, Vanna’s Spring Scissors, 0.10 mm pins, dissecting plates, 9-well glass dissection dish, fixative solution (4% paraformaldehyde in 1 X PBS), 1 X PBS, wipes, pipettes and tips for 30μm, and gloves (see Table of Materials).

2.2 Confirm flies were successfully injured by anesthetizing wounded flies on a CO₂-fly pad under stereomicroscope and checking for the presence of the wound scar (a melanin spot on abdomen, see Figure 1B). Discard any flies from the experimental group which were not successfully wounded.

2.3 To start dissection, fill one well of a 9-well glass dissection dish with Grace’s solution. Use a pair of forceps to grasp a wounded female fly by the dorsal side of the thorax and submerge the fly in the well containing Grace’s solution.

2.4 Using forceps in the opposite hand without releasing thorax, puncture the dorsal cuticle below tergite A6 and pull the cuticle off the rear end of the fruit fly. The internal organs (ovary and intestine) will usually come out at this step, if not, gently push on the dorsal side of the abdomen with forceps to squeeze out the remaining organs and discard into an empty well.

2.5 Snap off the full abdomen at the thorax junction above tergite A2 using the forceps and transfer the abdomen to an empty well containing a ~100μl of Grace’s solution. Repeat Steps 2.3-2.5 until all fly abdomens are dissected.

2.6 Reduce Grace’s solution volume to 30μl in well containing pooled dissected abdomens.

2.7 Fillet the abdomens open by positioning the abdomen on dorsal side with the forceps in one hand and then inserting the bottom blade of Vanna’s spring scissor into abdominal cavity with the other hand. Cut along the dorsal midline until abdomen is fully opened, which can require up to three cuts (Figure 1C and 1D).

2.8 Setup a dry dissecting plate with four 0.10mm pins per abdominal mounting area. Each 35mm dissecting plate can fit up to seven mounting areas. Pipette 30μl of Grace’s solution on each mounting area and transfer one filleted abdomen to each droplet.

2.9 Pin the filleted abdomens to the dish on the four dorsal corners (Figure 1E). Take care to ensure that the tissue lies flat without tearing or overstretching the abdominal tissue.

2.10 To fix tissue, pipette off the Grace’s solution and add 30μl of the fix solution to the pinned abdomen. CAUTION: Wear gloves while handling fix solutions as paraformaldehyde is toxic.

Repeat Steps 2.9-2.10 until all filleted abdomens are pinned onto the dissecting plate.

2.11 Place a tape label on the bottom of each dish to mark each control and experimental group. Fix samples for 30-60 minutes at room temperature.

2.12 Wash off fix solution by pipetting on 1.5 ml of 1 X PBS to each plate. Dispose of fix solution and plastics in appropriate liquid or dry chemical waste containers according to institutional guidelines.

2.13 Wash plates twice with 1.5 ml of 1 X PBS and store fixed tissue covered in 1.5 ml of 1 X PBS in a plastic container with a lid. Add a layer of damp paper towel to the bottom of the container and store samples at 4°C until ready to immunostain within at least one week of dissection.

3. Immunofluorescence

3.1 Freshly prepare reagents (see Table of Materials): wash buffer solution (0.3% Triton-X-100, 0.3% BSA in 1 X PBS). Leftover wash buffer can be saved a 4°C and used for the duration of the 2-day staining protocol. Prepare primary antibody solution per assayed used (Figure 2). Anti-FasIII (1:50 mouse anti-Fasciclin-III) in wash buffer with either anti-Grh (1:300 affinity purified rabbit anti-Grainyhead⁸) or anti-RFP (1:1000 rabbit anti-RFP). Primary antibody solutions can be saved at 4°C and reused multiple times until signal is significantly reduced.

3.2 Permeabilize tissue by pipetting off 1 X PBS and adding 1.5 ml of wash buffer for at least 30 minutes on an orbital shaker (80 rpms) at room temperature.

3.3 Remove wash buffer and stain tissue overnight with 1.5 ml of primary antibody solution on an orbital shaker (80 rpms) at 4°C. Collect the primary antibody solution and save in a tube at 4°C for future experiments.

3.4 Rinse sample first quickly with 1 x PBS and then wash samples 3 x with 1.5 ml of wash buffer. For each wash, rotate samples at room temperature on an orbital shaker for at least 30 minutes.

3.5 During the final wash, prepare secondary antibody solution: 1:1000 Donkey anti-Rabbit Alexa 488 or 568 and 1:1000 Goat anti-Mouse Alexa 488 or 568 (or fluorophores of choice) in wash buffer.

3.6 Remove wash buffer and stain tissues with 1.5 ml of secondary antibody solution. Cover samples with aluminum foil and rotate on an orbital shaker at room temperature for 3 hours. Alternatively, samples can be rotated overnight at 4°C on an orbital shaker.

3.7 Wash samples by first discarding secondary antibody solution and then rinse sample quickly with 1 x PBS followed by 3 washes with 1.5 ml of wash buffer. For each wash, rotate samples at room temperature on an orbital shaker for at least 30 minutes.

3.8 Prepare DAPI solution by diluting DAPI to 10 μg/ml in wash buffer. After final wash, stain samples with 1.5 ml DAPI solution rotating at room temperature for 30 minutes.

3.9 Discard DAPI solution and rinse samples twice in 1.5 ml of 1 X PBS. Store stained tissue in 1.5 ml of 1 X PBS in dark (covered with aluminum foil) at 4°C until ready to mount on coverslip-glass slide. The mounting step should be performed within one week.

4. Cell cycle activity (EdU Assay):

4.1 Make up a 10 mM EdU stock solution from Click-iT kit (see Table of Materials) by dissolving EdU powder in dH₂0 and rotating for ~15 minutes until completely dissolved. Stock solution can be aliquoted (250 μl per tube) and stored at −80°C.

4.2 Feed flies EdU by first diluting EdU stock to 5 mM in dH₂O. Add dry yeast until solution is cloudy and briefly vortex to mix. Cut off a 0.5 ml tube cap and place it at the bottom of fly food vial. Push cap into food, so it is sturdy.

4.3 Anesthetize flies and transfer 3-5 days old flies into the vial. Tap the flies to one edge so none are stuck in the cap.

4.4 Pipette 75 μl of yeast-EdU solution into cap. Flies should be fed fresh yeast-EdU solution every day and transferred to a fresh food vial with cap every other day to ensure that the flies do not get stuck in bottom of the vial.

4.5 To transfer flies: flip to new vial with cap then put flies to sleep, tap flies to one side, and add fresh yeast-EdU solution.

4.6 On the third day, injure the flies and continue to feed yeast-EdU until dissection at 2 dpi (Figure 4A). See protocol part 2 for dissection and fixation methods.

Figure 4. Methods to detect endoreplication during WIP.

(A) Timeline of EdU assay: adult Drosophila were fed every day 75 μl of 5 mM yeast-EdU diet 2 days prior to injury and continued until 2 dpi. (B) Immunofluorescent images of EdU label in fly strains expressed with epi-Gal4/ UAS system at 2 dpi. Wound scab (W). Scale bar, 50μm. (C) Average number of EdU+ epithelial nuclei per fly at 2 dpi: ctrl (n=37), myc^RNAi#1 (n=10), and Myc (n=8). Standard error and statistical significance were measured via Student’s T-test, *p<0.05, **p<0.01. (D) Schematic of detection and measurement of epithelial nuclear ploidy. Epithelial nuclei are identified and thresholded by the anti-Grh stain in Fiji. Overlapping epithelial nuclei are separated (green arrowheads) or removed (red arrowheads) if overlaid by non-epithelial nuclei. The Integrated Density and nuclear area of corresponding DAPI stained nuclei image is measured. (E) Epithelial nuclear size (Grh) is altered by myc expression at 3 dpi. (F) Epithelial nuclear ploidy (%) at 3 dpi: ctrl (n=4), myc^RNAi#1 (n=6), and Myc (n=3). Adapted from Grendler et al., 2019.

4.7 Prepare EdU Staining Reagents: wash buffer (0.3% Triton X-100, 0.3% BSA in 1 X PBS), permeabilization buffer (0.5% Triton X-100 in 1 x PBS), blocking buffer (3% BSA in 1 x PBS), and prepare reagents from Click-iT Kit, including 1x Click-iT reaction buffer and 1 x reaction buffer additive as directed by manufacturer.

4.8 Wash samples for 1 hr rotating at room temperature in 1.5 ml of wash buffer.

4.9 Add 1.5 ml of permeabilization buffer and rotate samples for 20 min. NOTE: Thaw and prepare Click-IT reaction cocktail solution = number of samples (500 μl/ plate).

4.10 Wash samples once quickly with 1 x PBS and then 3 x quickly with 1 ml of blocking buffer.

4.11 Pipette off all remaining blocking buffer and add 500 μl Click-iT cocktail. Swirl plates to ensure tissues are completely covered. Incubate in the dark (drawer) for 1 hr at room temperature.

4.12 Wash samples 1x quickly with 1.5 ml of blocking buffer.

4.13 Stain with 1.5 ml of DAPI solution at 1:5000 in wash buffer for 30 minutes.

4.14 Wash twice with 1 x PBS quickly and store in dark (wrap in foil) at 4°C until ready to mount samples within 3 days.

5. Mount Stained Tissue

5.1 Obtain all required materials for mounting: glass slides, glass coverslips, clear nail polish, mounting media, a pair of forceps, and wipes.

5.2 To mount stained fly tissue, unpin abdomens from the dissecting plate using forceps under the stereomicroscope. Transfer tissue to a drop (~30μl) of mounting media on glass coverslip by gently grabbing tissue with forceps on its dorsal flanks, taking care to avoid touching the ventral area with forceps.

5.3 Under stereomicroscope orient the abdominal tissue so that the inside is facing down toward the coverslip (i.e. external cuticle/bristles are facing up). Pull the oriented abdomens to edge of media droplet using the forceps; surface tension will help keep the tissue flat (Figure 1F). NOTE: It is helpful for imaging to organize the abdomens in a column or row at this stage.

5.4 Labeled a glass slide (i.e. control or experimental) and pick up the coverslip by slowly bringing the slide closer to the coverslip. Flip slide over and gently blot with a wipe to remove excess mounting media.

5.5 Seal edges of the coverslip with clear nail polish and repeat for all remaining experimental groups. Store slides in a slide box at 4°C until ready to image.

6. Imaging and Processing

6.1 Image the fly abdominal wound area by first locating the melanin scar on confocal microscope (Figure 1B), either a point scanner or a structured illumination (ApoTome) with a 40x oil or dry objective.

6.2 Check the exposure on each channel ensuring that signal is below saturation. The imaging settings should be based on ‘brightest’ sample group. This is particularly important for ploidy analysis as the DAPI channel needs to stay in the linear range to accurately measure DNA content.

6.3 Take a full Z-stack image in all three channels with optimal distance of at least 0.50μm between slices. Save captured images and open the file in the image analysis program Fiji (also known as ImageJ).

6.4 For each image, create a Z-stack projection using the sum of slices option for all channels.

6.5 Rotate images as necessary to ensure the nuclei are lined up horizontally across image (Figure 3A and Figure 4E).

Figure 3. Methods to detect re-epithelialization during WIP.

Re-epithelialization is perturbed when WIP is genetically inhibited. Immunofluorescent images of control (A) and stg, fzr^RNAi (B) at 3 dpi. Epithelial nuclei and septate junctions are stained with Grh (green) and FasIII (magenta), respectively. (A’ and B’) FasIII staining alone shows that re-epithelialization is impaired (red arrow) in stg, fzr^RNAi epithelium. Scale bar, 50μm. (C) Quantification of re-epithelialization defects (%) at 3 dpi (gray): control (n=8), stg, fzr^RNAi (n=6). Standard error and statistical significance was measured via Student’s T-test, **p<0.01. Re-epithelialization during wound repair can also be detected by expression of a membrane linked RFP using epi-Gal4, UAS-mCD8-RFP. (D) Immunofluorescent images of control, E2F1^RNAi, RacDN, and stg, fzr^RNAi at 3 dpi. Scale bar, 20μm. Wound scab (yellow outline) and open epithelial wound area (red outline). (E) Quantification of wound closure in ctrl (n=11), E2F1^RNAi, RacDN (n=13), and stg, fzr^RNAi (n=13). Adapted from Grendler et al., 2019.

6.6 Crop all the images to a rectangular selection of 300μm x 300μm centered around the wound site or center of uninjured control. Identify the area by drawing a rectangle and selecting Edit→ Selection → Specify, check scaled units are in microns to ensure the same sized box is used for all images to be analyzed.

7. Endoreplication (Ploidy) Analysis

7.1 Using Fiji, select the Grh channel window and duplicate the image. Then use the threshold tool to create a mask. Manually adjust the threshold by sliding the top bar to minimize background without causing the nuclei to shrink drastically (Figure 4D).

7.2 If any nuclei in the Grh channel are touching in the threshold image, use the paintbrush tool (2 pixel width) in the same color as the background, to draw a line between the nuclei. Click to apply one time when finished to generate the final mask.

7.2 Generate an ROI map by using the Analyze Particles function: set size to 5-60μm in order to capture most of the nuclei without including the background.

7.3 Manually adjust ROI map as needed in the ROI manager. Delete any selections that are not nuclei and add any nuclei to the list that were not identified by outlining the nucleus with the freehand selection tool and adding it to the ROI manager (Figure 4D).

7.4 Select the DAPI channel then click show all in the ROI manager to apply the generated ROI map in the Grh channel onto the DAPI channel.

7.5 Delete from the ROI map any selections where the epithelial nuclei outline overlaps with non-epithelial nuclei (e.g. nuclei from muscle or fat body). Grainyhead only stains epithelial nuclei, whereas DAPI stains all nuclei. Check that each outlined selection contains only one nucleus and delete or edit any selections with more than one nucleus. Save edited ROI list.

7.6 Measure the area and integrated density of each epithelial nuclei in the ROI map using the analysis tools in Fiji. Export the values into an excel spreadsheet.

7.7 Measure the average image background by using the circular selection tool. Draw 3 circles on the DAPI image in different areas of the image that do not overlap with any nuclei. Add the area and integrated density of each of the circles to the excel spreadsheet to establish the background image brightness.

7.8 Start by calculating the average background per unit area for each image by dividing each background integrated density value by its corresponding area. Then average the 3 integrated density per area measurements for the image in order to obtain the average background per unit area.

7.9 Next, calculate the total background of each DAPI nucleus by multiplying the area of the nucleus by the average background per unit area. The normalized DAPI intensity for each nucleus measured can then be calculated by subtracting the total background of each nucleus from its measured integrated density.

7.10 Average all the normalized DAPI intensity values from the uninjured epithelial control. The uninjured epithelial nuclei were previously calculated to have a ploidy value of 2C and can serve as reference for calculating ploidy in the epithelial nuclei from the experimental conditions⁸.

7.11 Calculate the ploidy of each nucleus by dividing the normalized DAPI intensity of each nucleus by the normalized value from reference uninjured epithelial control (2C) then multiply the value by 2 to equal the normalized ploidy (C-value).

Epithelial nuclear ploidy calculation:

(Nuclear Integrated Density – Background Nuclear Integrated Density)/ Average Nuclear Integrated Density (uninjured epithelial nuclei=2C) x 2= epithelial nuclear ploidy (C)

7.12 Nuclei with ploidy values can be graph as dot plot, histogram, or grouped into a bar graph accordingly (i.e. 2C (0.6-2.9C), 4C (3.0-5.9C), 8C (6.0-12.9C), 16C (13.0-24.9C), and >32C (>25.0C) (Figure 3F).

REPRESENTATIVE RESULTS:

Herein we provide a detailed protocol on how to use D. melanogaster as a model to study wound-induced polyploidization (WIP). This wound healing model provides many advantages over mammalian and other fly models of WIP. Polyploidy is easily induced by a mechanical puncture with an insect pin and polyploid cells are generated within a short period of time (2 to 3 dpi) (Figure 1A and 1B)⁴. The challenge lies more in the dissection of intact abdominal tissue without any perturbations to epithelium. The D. melanogaster epithelium is easily disrupted by an accidental bump or scratch with the sharp dissection tools. Therefore, the steps of this protocol should be practiced prior to use and analysis.

This protocol provides additional notes on where issues can arise. First, the injury is restricted to the ventral female abdomen, which provides a large, flat opaque tissue area ideal for imaging. The puncture wounds are inflicted in the pleurite epithelium which lies on either side of the ventral midline sternites and targeted between tergite (T) segments T4 to T5 (Figure 1A-C). This wound placement provides a large area to view that is not disrupted by the dissection. The other challenging steps include the abdominal spring scissor cut and pinning steps (Figure 1D and 1E). The spring cutting step works best when the abdomens are cut in a reduced volume of Grace’s solution (~30 μl) to decrease tissue movement. A well centered cut (along the dorsal midline) is necessary to provide sufficient area on the abdominal dorsal flaps to pin open on the dissection plate (Figure 1C). The abdomen should be gently pinned on the four corners without excessive force (Figure 1E). A pin push that is too hard will distort the abdominal tissue and could even push the tissue into the dissection plate. If this happens, the tissue should be discarded. Once the abdominal tissue is fixed, it remains on the dissection plate until immunofluorescence staining is complete and the abdomens are mounted on a glass coverslip for imaging (Figure 1F).

Wound healing requires a continuous epithelial sheet to form, which is dependent on endoreplication and cell fusion^4,16. The septate junction protein, FasIII, which labels cell-cell junctions provides an indicator for whether any processing perturbations have occurred during the preparation (Figure 1G and 1H). Abdomens with large scratches (unstained area) that perturb the wound area should be discarded and not used for further analysis (Figure 1H). The next step is to analyze intact samples for any defects in WIP. Within this protocol are distinct assays, which are used to detect different aspects of the WIP response (Figure 2). Wound repair is complete when a central large multinucleated cell covers the wound scab (Figure 3A). Here cell fusion is detected by staining for FasIII/ Grh and quantifying the number Grh⁺ epithelial nuclei encompassed in the FasIII outlined area⁴. Defects in wound closure or re-epithelialization are detected when gaps of >10 μm in the epithelial sheet are observed (Figure 3B, red arrow). This is the case, for example, when WIP is inhibited by activation of mitotic cycle via expression of stg, fzr^RNAi as we recently reported¹⁶. In this genetic condition, 52% of the wounds are not able to form a continuous epithelial sheet over the wound scab (Figure 3B and 3C).

Another method to measure wound repair in this model is by visualizing the epithelial membrane with epi-Gal4 expression of UAS-mCD8-ChRFP⁴ (Figure 2 and 3D). In the control, 91% of epithelial wounds close completely by 3 dpi, but inhibiting WIP by blocking endoreplication (E2f1^RNAi) and cell fusion (RacDN) simultaneously, as we previously reported, causes 92% of epithelial wounds to remain completely open (Figure 3D and 3E)^8,16. The activation of mitotic cell cycle by expression of stg, fzr^RNAi, also resulted in an epithelial wound closure defect. However, by visualizing epithelial cell membrane, the extent of re-epithelialization defect can be determined. The WIP mutant (E2f1^RNAi, RacDN) fly wound were more open than the stg, fzr^RNAi wounds (Figure 3D, dashed red outline)¹⁶. This membrane wound healing assays provides more resolution on the extent of the wound repair defect. As a result, re-epithelialization defects can be grouped as either completely open, partial (>10 μm gaps), or completely closed (Figure 3D and 3E).

In addition to cell fusion, epithelial cells grow in size by endoreplication, an incomplete cell cycle that doubles the nuclear DNA content. Endoreplication is assayed by both cell cycle activity and direct nuclear DNA ploidy measurements (Figure 2 and 4). Here cell cycle activity is detected by incorporation of the thymidine analog, EdU (Figure 4A and 4B). D. melanogaster epithelial cells were found to enter the endocycle, an incomplete cell cycle that oscillates between S and G phases without an intervening M phase^4,12. The adult D. melanogaster diet is supplemented with EdU⁺ food prior to injury and the flies are maintained on EdU⁺ diet until dissection at 2 dpi (Figure 4A). The EdU is then detected using the manufacturer’s Click-iT protocol. This EdU assay is used to determine where, when, and how many nuclei are triggered to enter S phase in response to a wound. Using the Gal4/ UAS system, we recently found that epithelial specific expression of myc can either block (myc^RNAi) or exacerbate (Myc overexpression) the competence of epithelial cells to enter S phase. As result, we and others have now shown that Myc is sufficient even without injury to induce endoreplication in postmitotic cells^16,22.

Next, epithelial ploidy is determined by directly measuring nuclear DNA content. Epithelial nuclei are identified by immunofluorescence staining for the epithelial specific marker, Grh (Figure 4D). In Fiji imaging software, epithelial nuclei are systemically identified and then thresholded using Grh nuclear stain. Nuclei are then separated and the ROIs overlaid on to the SUM of stacks DAPI image (Figure 4D, green arrow). Any overlapping nuclei are manually deleted before the Integrated Density of the selected nuclei are measured (Figure 4D, red arrows). This semi-automated method allows one to quantify the distribution and ploidy of most nuclei throughout the uninjured and repaired fly abdominal epithelium⁸. As we recently reported, the epithelial nuclei surrounding the wound are composed of 44% polyploid nuclei with DNA content more than 3C at 3 dpi (Figure 4E and 4F)¹⁶. Knockdown of myc as expected from the EdU results led to a significant block in endoreplication as only 9% of epithelial nuclei are polyploid, whereas overexpression of Myc resulted in 100% polyploid epithelial nuclei around the wound site (Figure 4F)¹⁶. Epithelial nuclear size was also visibly affected by myc expression with either reduced or enlarged nuclei present. However nuclear area is not an accurate measure of ploidy and physiological effects, such as cell stretching can also influence nuclear size without effecting nuclear DNA content²⁰.

DISCUSSION:

Here we provide a detailed protocol on how to dissect and use the adult D. melanogaster abdominal epithelium to study how genes regulate WIP by altering re-epithelialization and endoreplication during wound repair¹⁶. Using this method, we recently identified proto-oncogene Myc as a key regulator of WIP. Myc is required for the epithelial cells to endoreplicate post injury and is sufficient for quiescent epithelial cells to endocycle both in adult fly epithelium and accessory gland^16,22. We also found that switching epithelial cells to a mitotic cell cycle by expression of stg, fzr^RNAi is detrimental to wound repair. Continued studies using this method will identify other genes that are required to regulate re-epithelialization and endoreplication during WIP revealing both similarities and differences to how polyploidy is regulated and functions in a variety of tissues.

This model and method offer unique advantages, including that polyploidy is easily induced with a mechanical puncture and polyploid cells are generated within days⁴. The tissue dissection and preparation is based on larval dissection technique²³, but adult fly abdomen is more rigid and therefore easily perturbed. As a result, this protocol requires practice and precision to isolate an intact tissue to study WIP. Once dissected, however, the epithelium is clearly visible and easily imaged, yielding a snapshot of the wound healing process. This method provides a wealth of information on the adult fly’s epithelial organization, cell and syncytium size, and the ploidy of cells and individual nuclei. While live imaging is not yet possible within the intact fruit fly due to its non-transparent cuticle. This protocol could be adapted to include currently available ex vivo culture conditions used in D. melanogaster to perform short-term live imaging studies²⁴.

In future, this model would be ideal to study cell-to-cell crosstalk and the contribution of other cell-types to WIP by regulating gene expression with Gal4/ UAS system in other cell-types of interest. Similar questions can also be answered using a variety of genetic and mutant backgrounds. The dissected adult fly abdomen contains a variety of cell-types that can be easily visualized using this method, including fat body and oenocytes, lateral muscle fibers, sensory neurons, trachea, and macrophage-like hemocytes. In addition, this model will allow researchers to investigate how physiological effects influence WIP including sex, diet, infection, age, and environmental stressors. While the protocol uses the adult female fly due to its enlarged size, WIP also occurs in the male fruit fly (Gjelsvik and Losick, unpublished). Polyploid cells have been found to arise during aging and age-associated disease in the mammalian liver, brain, eye, and heart ¹². The fruit fly model will enable researchers to study polyploidization in these physiological and disease contexts as human disease-related genes are highly conserved.