At Boston College, we would like to thank Dr. Eric Folker for use of his lab&#39;s camera and stereoscope microscope setup for imaging and Bret Judson at the Boston College Imaging Core for infrastructure and support. We would also like to thank the fly community resources: Bloomington Drosophila Stock Center (NIH P40OD018537), Vienna Drosophila Resource Center, and TRiP Center at Harvard Medical School (NIH/NIGMS R01-GM084947) for providing transgenic stocks used in this study. The mouse FasIII antibody were obtained from Developmental Studies Hybridoma Bank supported by NICHD of the NIH and maintained at The University of Iowa, Department of Biology, Iowa City, IA. Research reported in this publication was supported by the National Institute of General Medical Sciences of the National Institutes of Health under Award Number R35GM124691. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

1. Staging and wounding of adult fruit flies
<ol>
	<li>Select D. melanogaster strain of choice (i.e., epi-Gal4/ UAS strain, see Table of Materials). 
	NOTE: Here, the Gal4/UAS system is used to enable epithelial specific gene expression (epi-Gal4) of a gene or RNAi encoded downstream of UAS. This study uses fluorescent membrane protein (UAS-Cd8.mRFP), mitotic inducer (UAS-fzrRNAi, UAS-stg), and WIP inhibitor (UAS-E2F1RNAi; UAS-RacDN).</li>
	<li>Collect two vials of 10-15 newly enclosed female fruit flies each and age on fresh food vials at 25 &#176;C until 3-5 days old. One vial will serve as the uninjured control and the other vial will be wounded as described below. The female flies should be maintained with males (~5/vial).</li>
	<li>To wound the flies, assemble several pin holders each with a single 0.10 mm stainless steel pin. 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.</li>
	<li>Anesthetize aged female fruit flies on a CO2-fly pad under a stereomicroscope and align them into a row using a paint brush. Wearing safety glasses, holding the pin holder in one hand and forceps in the other, use forceps to position a fly with its ventral abdomen facing up.</li>
	<li>Puncture the adult female flies within the epithelial pleurite region of tergite A4 on either side of the ventral midline sternites (Figure 1A). Puncturing this ventral region provides optimal space away from the dissection sites where tissue edges will be torn by mechanical processing.</li>
	<li>Return wounded flies to the food vial and age to desired day post injury (dpi). Epithelial wound healing starts at 1 dpi and ends by 3 dpi. Endoreplication peaks at 2 dpi, which is ideal for the EdU assay (section 4, Figure 2).</li>
</ol>
2. Fly abdominal dissection
NOTE: During this step, it is important to avoid touching the ventral abdominal tissue with the dissection tools because it will compromise the integrity of the epithelium.
<ol>
	<li>Obtain all required materials for dissection: Grace&#39;s solution, forceps, Vanna&#39;s Spring Scissors, 0.10 mm pins, dissecting plates, 9 well glass dissection dish, fixative solution (4% paraformaldehyde in 1x PBS), 1x PBS, wipes, pipettes and tips for 30 &#181;m, and gloves (see Table of Materials).</li>
	<li>Confirm that flies were successfully injured by anesthetizing wounded flies on a CO2-fly pad under a stereomicroscope and checking for the presence of the wound scar (i.e., a melanin spot on abdomen, see Figure 1B). Discard any flies from the experimental group that were not successfully wounded.</li>
	<li>To start dissection, fill one well of a 9 well glass dissection dish with Grace&#39;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&#39;s solution.</li>
	<li>Using forceps in the opposite hand without releasing the 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 the forceps to squeeze out the remaining organs and discard into an empty well.</li>
	<li>Snap off the full abdomen at the thorax junction above tergite A2 using the forceps and transfer the abdomen to an empty well containing ~100 &#181;L of Grace&#39;s solution.</li>
	<li>Repeat steps 2.3-2.5 until all fly abdomens are dissected.</li>
	<li>Reduce the volume of Grace&#39;s solution to 30 &#181;L in the well containing pooled, dissected abdomens.</li>
	<li>Fillet the abdomens open by positioning the abdomen on the dorsal side with the forceps in one hand and then inserting the bottom blade of Vanna&#39;s spring scissors into the abdominal cavity with the other hand. Cut along the dorsal midline until the abdomen is fully opened, which can require up to three cuts (Figure 1C, 1D).</li>
	<li>Set up a dry dissecting plate with four 0.10 mm pins per abdominal mounting area. Each 35 mm dissecting plate can fit up to seven mounting areas. Pipette 30 &#181;L of Grace&#39;s solution on each mounting area and transfer one filleted abdomen to each droplet.</li>
	<li>Pin the filleted abdomens to the dish on the four dorsal corners (Figure 1E). Ensure that the tissue lies flat without tearing or overstretching the abdominal tissue.</li>
	<li>To fix the tissue, pipette off the Grace&#39;s solution and add 30 &#181;L of the fix solution to the pinned abdomen. 
	CAUTION: Wear gloves while handling the fix solution, as paraformaldehyde is toxic.</li>
	<li>Repeat steps 2.10-2.11 until all filleted abdomens are pinned onto the dissecting plate.</li>
	<li>Place a tape label on the bottom of each dish to mark each control and experimental group. Fix samples for 30-60 min at room temperature (RT).</li>
	<li>Wash off fix solution by pipetting on 1.5 mL of 1x PBS to each plate. Dispose of fix solution and plastics in appropriate liquid or dry chemical waste containers according to institutional guidelines.</li>
	<li>Wash plates 2x with 1.5 mL of 1x PBS and store fixed tissue covered in 1.5 mL of 1x 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 &#176;C until ready to immunostain within 1 week of dissection.</li>
</ol>
3. Immunofluorescence
<ol>
	<li>Freshly prepare reagents (see Table of Materials): wash buffer solution (0.3% Triton X 100, 0.3% BSA in 1x PBS). Leftover wash buffer can be saved at 4 &#176;C and used for the duration of the 2 day staining protocol. Prepare enough primary antibody solution per assay (Figure 2) using 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 (1:1,000 rabbit anti-RFP). Primary antibody solutions can be saved at 4 &#176;C and reused multiple times until signal is significantly reduced.</li>
	<li>Permeabilize tissue by pipetting off 1x PBS, adding 1.5 mL of wash buffer, and incubating for at least 30 min on an orbital shaker (80 rpm) at RT.</li>
	<li>Remove wash buffer and stain tissue overnight with 1.5 mL of primary antibody solution, incubating on an orbital shaker (80 rpm) at 4 &#176;C. Collect the primary antibody solution and save in a tube at 4 &#176;C for future experiments.</li>
	<li>First rinse the sample quickly with 1x PBS and then wash 3x with 1.5 mL of wash buffer. For each wash, incubate samples at RT on an orbital shaker for at least 30 min.</li>
	<li>During the final wash, prepare the secondary antibody solution: 1:1,000 donkey anti-rabbit Alexa 488 or 568 and 1:1,000 goat anti-mouse Alexa 488 or 568 (or fluorophores of choice) in wash buffer.</li>
	<li>Remove wash buffer and stain tissues with 1.5 mL of secondary antibody solution. Cover samples with aluminum foil and incubate on an orbital shaker at RT for 3 h. Alternatively, samples can be incubated overnight at 4 &#176;C on an orbital shaker.</li>
	<li>Wash samples by first discarding secondary antibody solution and then rinse sample quickly with 1x PBS followed by three washes with 1.5 mL of wash buffer. For each wash, incubate samples at RT on an orbital shaker for at least 30 min.</li>
	<li>Prepare DAPI solution by diluting DAPI to 10 &#181;g/mL in wash buffer. After the final wash, stain samples with 1.5 mL DAPI solution incubating at RT for 30 min.</li>
	<li>Discard DAPI solution and rinse samples 2x in 1.5 mL of 1x PBS. Store stained tissue in 1.5 mL of 1x PBS in the dark, covered with aluminum foil, at 4 &#176;C until ready to mount on a glass slide with coverslip. The mounting step should be performed within 1 week.</li>
</ol>
4. Cell cycle activity (EdU Assay)
<ol>
	<li>Make up a 10 mM EdU stock solution from Click-iT kit (see Table of Materials) by dissolving EdU powder in dH20 and mixing for ~15 min until completely dissolved. The stock solution can be aliquoted (250 &#181;L per tube) and stored at -80 &#176;C.</li>
	<li>Feed flies EdU by first diluting EdU stock to 5 mM in dH2O. Add dry yeast until the solution is cloudy and briefly vortex to mix. Cut off a 0.5 mL tube cap and place it at the bottom of the fly food vial. Push the cap into the food, so it is stable.</li>
	<li>Anesthetize the flies and transfer 3-5 day old flies into the vial. Tap the flies to one edge so none are stuck in the cap.</li>
	<li>Pipette 75 &#181;L of yeast-EdU solution into the cap. Flies should be fed fresh yeast-EdU solution every day and transferred to a fresh food vial with a cap every other day to ensure that the flies do not get stuck in the bottom of the vial.</li>
	<li>To transfer flies, flip to a new vial with a cap, put flies to sleep, tap flies to one side, and add fresh yeast-EdU solution.</li>
	<li>On the third day, injure the flies and continue to feed yeast-EdU until dissection at 2 dpi (Figure 4A). See protocol section 2 for dissection and fixation methods.</li>
	<li>Prepare EdU staining reagents: wash buffer (0.3% Triton X 100, 0.3% BSA in 1x PBS), permeabilization buffer (0.5% Triton X 100 in 1x PBS), blocking buffer (3% BSA in 1x PBS), and prepare reagents from the EdU assay kit (see Table of Materials), including 1x reaction buffer and 1x reaction buffer additive as directed by the manufacturer.</li>
	<li>Wash samples for 1 h, mixing at RT in 1.5 mL of wash buffer.</li>
	<li>Add 1.5 mL of permeabilization buffer and incubate samples for 20 min. 
	NOTE: Thaw and prepare reaction cocktail solution using a volume of 500 &#181;L/plate.</li>
	<li>Wash samples 1x quickly with 1x PBS and then 3x quickly with 1 mL of blocking buffer.</li>
	<li>Pipette off all remaining blocking buffer and add 500 &#181;L of reaction cocktail solution per plate. Swirl plates to ensure tissues are completely covered. Incubate in a drawer in the dark for 1 h at RT.</li>
	<li>Wash samples 1x quickly with 1.5 mL of blocking buffer.</li>
	<li>Stain with 1.5 mL of DAPI solution at 1:5,000 in wash buffer for 30 min.</li>
	<li>Wash 2x with 1x PBS quickly, wrap in foil, and store in the dark at 4 &#176;C until ready to mount samples within 3 days.</li>
</ol>
5. Mount stained tissue
<ol>
	<li>Obtain all required materials for mounting: glass slides, glass coverslips, clear nail polish, mounting media, a pair of forceps, and wipes.</li>
	<li>To mount stained fly tissue, unpin abdomens from the dissecting plate using forceps under the stereomicroscope. Transfer tissue to ~30 &#181;L of mounting media on a glass coverslip by gently grabbing the tissue with forceps by its dorsal flanks, taking care to avoid touching the ventral area with forceps.</li>
	<li>Under the stereomicroscope orient the abdominal tissue so that the inside is facing down toward the coverslip (i.e., the external cuticle/bristles are facing up). Pull the oriented abdomens to the edge of the 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.</li>
	<li>Label 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.</li>
	<li>Seal the edges of the coverslip with clear nail polish and repeat for all remaining experimental groups. Store slides in a slide box at 4 &#176;C until ready to image.</li>
</ol>
6. Imaging and processing
<ol>
	<li>Image the fly abdominal wound area by first locating the melanin scar with a confocal microscope (Figure 1B), either a point scanner or a structured illumination (ApoTome) with a 40x oil or dry objective.</li>
	<li>Check the exposure on each channel, ensuring that the signal is below saturation. The imaging settings should be based on the 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.</li>
	<li>Take a full z-stack image in all three channels with an optimal distance of at least 0.50 &#181;m between slices. Save captured images and open the file in the image analysis program Fiji (also known as ImageJ).</li>
	<li>For each image, create a z-stack projection using the sum of slices option for all channels.</li>
	<li>Rotate the images as necessary to ensure the nuclei are lined up horizontally across images (Figure 3A and Figure 4E).</li>
	<li>Crop all the images to a rectangular selection of 300 &#181;m x 300 &#181;m centered around the wound site or center of uninjured control. Identify the area by drawing a rectangle and selecting Edit | Selection | Specify. Check that scaled units are in microns to ensure the same sized box is used for all images to be analyzed.</li>
</ol>
7. Endoreplication (ploidy) analysis
<ol>
	<li>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).</li>
	<li>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 1x when finished to generate the final mask.</li>
	<li>Generate a region of interest (ROI) map by using the Analyze Particles function: set size to 5 &#181;m-60 &#181;m in order to capture most of the nuclei without including the background.</li>
	<li>Manually adjust the 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).</li>
	<li>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.</li>
	<li>Delete any selections where the epithelial nuclei outline overlaps with nonepithelial nuclei (e.g., nuclei from muscle or fat) from the ROI map. 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.</li>
	<li>Measure the area and integrated density of each epithelial nuclei in the ROI map using the analysis tools in Fiji. Export the values into a spreadsheet program.</li>
	<li>Measure the average image background using the circular selection tool. Draw three circles that do not overlap with any nuclei in different areas of the DAPI image. Add the area and integrated density of each of the circles to a spreadsheet program to establish the background image brightness.</li>
	<li>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 three integrated density per area measurements for the image in order to obtain the average background per unit area.</li>
	<li>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.</li>
	<li>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 a reference for calculating ploidy in the epithelial nuclei from the experimental conditions8.</li>
	<li>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): 
	(Nuclear Integrated Density - Background Nuclear Integrated Density)/Average Nuclear Integrated Density (uninjured epithelial nuclei = 2C) x 2 = epithelial nuclear ploidy (C)</li>
	<li>Graph nuclei with ploidy values as a 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 &#62;32C [&#62;25.0C] (Figure 3F).</li>
</ol>

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W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphological+analysis+of+Drosophila+larval+peripheral+sensory+neuron+dendrites+and+axons+using+genetic+mosaics.">Morphological analysis of Drosophila larval peripheral sensory neuron dendrites and axons using genetic mosaics.</a> Journal of Visualized Experiments. (57), e3111 (2011).</li><li>Dai, W., Montell, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+Imaging+of+Border+Cell+Migration+in+Drosophila.">Live Imaging of Border Cell Migration in Drosophila.</a> Methods in Molecular Biology. 1407, 153-168 (2016).</li></ol>

Presented is 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, the proto-oncogene Myc was recently identified as a key regulator of WIP. Myc is required for epithelial cells to endoreplicate post injury and is sufficient for quiescent epithelial cells to endocycle both in adult fly epithelium and accessory glands16,22. It was also found that switching epithelial cells to a mitotic cell cycle by expression of stg, fzrRNAi is detrimental to wound repair. Continued studies using this method will identify other genes 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 the easy induction of polyploidy with a mechanical puncture and the fact that polyploid cells are generated within days4. The tissue dissection and preparation protocols are based on larval dissection techniques23, but the 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&#39;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 opaque 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 the future, this model will be ideal to study cell-to-cell crosstalk and the contribution of other cell types to WIP by regulating gene expression with the 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 variables influence WIP, including sex, diet, infection, age, and environmental stressors. While the protocol uses the adult female fly due to its larger 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 heart12. The fruit fly model will enable researchers to study polyploidization in physiological and disease contexts&#160;because human disease-related genes are highly conserved.

Drosophila melanogaster is an attractive model system to study the cellular and molecular mechanisms of epithelial wound repair. As in mammals, the tissue repair mechanisms used depend&#160;on both the tissue and its developmental stage. Scarless wound healing occurs in the fruit fly embryo where an actomyosin &#34;purse string&#34; forms at the epithelial leading edge enabling the wound to seamlessly close1,2. Post-embryonic wound healing in larvae, pupae, and adult fruit flies results in extracellular matrix remodeling, melanin scar formation, and epithelial cell growth3,4,5,6. The epithelial cells increase in size by cell fusion and the endocycle, an incomplete cell cycle that bypasses mitosis3,4,7,8. 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 damage9,10,11.
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 injury14,15. In these examples, polyploid cells are generated by endoreplication via either endocycle or endomitosis, which results in a binucleated cell due to a block in cytokinesis12. 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 growth11,14. In adult fly epithelium, it was 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 formation18,19. 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 make it an ideal model system for elucidating the molecular and cellular mechanisms that guide this wound healing strategy.
Here, we present a protocol for analyzing WIP in the adult D. melanogaster epithelium. Included are 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 reflect 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 sequencing)21. This method increases the suitability of the adult D. melanogaster abdominal epithelium as a model to study the role and regulation of polyploidy in wound repair.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>35 mm Petri dishes</td>
			<td>Fisher Scientific</td>
			<td>FB0875713</td>
			<td>For creating plates to dissect in</td>
		</tr>
		<tr>
			<td>50 mL Conical Centrifuge Tubes</td>
			<td>Fisher Scientific</td>
			<td>14-432-22</td>
			<td>For preparing staining reagents in</td>
		</tr>
		<tr>
			<td>AxioImager M2 with Apotome</td>
			<td>Zeiss</td>
			<td>NA</td>
			<td>For imaging samples</td>
		</tr>
		<tr>
			<td>Blowgun mini</td>
			<td>Genesee Scientific</td>
			<td>54-104M</td>
			<td>For anethesizing D. melanogaster strains</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin, 30%</td>
			<td>Sigma</td>
			<td>A7284-500ML</td>
			<td>For immuostaining</td>
		</tr>
		<tr>
			<td>Carbon dioxide tank</td>
			<td>various distributors</td>
			<td>N/A</td>
			<td>For anethesizing D. melanogaster strains</td>
		</tr>
		<tr>
			<td>Click-iT EdU 594 Kit</td>
			<td>Thermofisher</td>
			<td>C10339</td>
			<td>For EdU assay</td>
		</tr>
		<tr>
			<td>Coverslips</td>
			<td>Thermofisher</td>
			<td>3406</td>
			<td>For mounting</td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Sigma</td>
			<td>D9542-10MG</td>
			<td>For immuostaining</td>
		</tr>
		<tr>
			<td>Dissecting Plates (use Sylgard 184 Sil Elastic Kit)</td>
			<td>Ellsworth Adhesives</td>
			<td>184SIL</td>
			<td>For creating plates to dissect in. Mix epoxy as directed, let dry overnight</td>
		</tr>
		<tr>
			<td>Donkey anti-Rabbit IgG (H+L) Secondary Antibody, Alexa Fluor 488 conjugate</td>
			<td>Thermofisher</td>
			<td>A21206</td>
			<td>For secondary immuostaining</td>
		</tr>
		<tr>
			<td>Donkey anti-Rabbit IgG (H+L) Secondary Antibody, Alexa Fluor 568 conjugate</td>
			<td>Thermofisher</td>
			<td>A10042</td>
			<td>For secondary immuostaining</td>
		</tr>
		<tr>
			<td>Drosophila tubing and fittings</td>
			<td>Genesee Scientific</td>
			<td>59-124C, 59-123, 59-140</td>
			<td>For anethesizing D. melanogaster strains</td>
		</tr>
		<tr>
			<td>Dumont #5 Forceps</td>
			<td>Fine Science Tools</td>
			<td>11252-20</td>
			<td>For dissecting</td>
		</tr>
		<tr>
			<td>epi-Gal4</td>
			<td>Bloomington Drosophila Stock Center (b)</td>
			<td>b38793</td>
			<td>Losick et al. Current Biology, 2013</td>
		</tr>
		<tr>
			<td>epi-Gal4, UAS-mCD8.RFP</td>
			<td>Bloomington Drosophila Stock Center (b)</td>
			<td>b38793, b27392</td>
			<td>Losick et al. Current Biology, 2013</td>
		</tr>
		<tr>
			<td>Excel</td>
			<td>Microsoft</td>
			<td></td>
			<td>For performing ploidy calculations</td>
		</tr>
		<tr>
			<td>Fiji/ImageJ (image analysis software)</td>
			<td>NIH</td>
			<td><a href="https://imagej.nih.gov/ij" target="_blank">https://imagej.nih.gov/ij</a></td>
			<td>For image analysis</td>
		</tr>
		<tr>
			<td>Fly food</td>
			<td>Archon Scientific</td>
			<td>N/A</td>
			<td>Corn Syrup/Soy food</td>
		</tr>
		<tr>
			<td>Flystuff Flypad</td>
			<td>Genesee Scientific</td>
			<td>59-114</td>
			<td>For anethesizing D. melanogaster strains</td>
		</tr>
		<tr>
			<td>Glass dissecting dish</td>
			<td>Fisher Scientific</td>
			<td>13-748B</td>
			<td>For performing dissections in</td>
		</tr>
		<tr>
			<td>Glass slides</td>
			<td>Fisher Scientific</td>
			<td>12-518-104C</td>
			<td>For mounting</td>
		</tr>
		<tr>
			<td>Goat anti-Mouse IgG (H+L) Secondary Antibody, Alexa Fluor 488 conjugate</td>
			<td>Thermofisher</td>
			<td>A11001</td>
			<td>For secondary immuostaining</td>
		</tr>
		<tr>
			<td>Goat anti-Mouse IgG (H+L) Secondary Antibody, Alexa Fluor 568 conjugate</td>
			<td>Thermofisher</td>
			<td>A11031</td>
			<td>For secondary immuostaining</td>
		</tr>
		<tr>
			<td>Grace&#39;s Insect Medium, unsupplemented</td>
			<td>Thermofisher</td>
			<td>11595030</td>
			<td>For dissecting in</td>
		</tr>
		<tr>
			<td>Insect pins</td>
			<td>Fine Science Tools</td>
			<td>26002-10</td>
			<td>For wounding and pinning fly abdomens flat</td>
		</tr>
		<tr>
			<td>Mouse anti-Fasciclin III (Drosophila) Primary Antibody</td>
			<td>Developmental Studies Hybridoma Bank</td>
			<td>7G10</td>
			<td>For immunostaining epithelial cell-cell junctions</td>
		</tr>
		<tr>
			<td>Mouting media</td>
			<td>Vector Laboratories</td>
			<td>H-1000</td>
			<td>Anti-fade mounting media to prevent photo bleaching during imaging</td>
		</tr>
		<tr>
			<td>Nail polish</td>
			<td>Electron Microscopy Sciences</td>
			<td>72180</td>
			<td>For sealing slides</td>
		</tr>
		<tr>
			<td>Ortibal shaker</td>
			<td>Fisher Scientific</td>
			<td>02-217-988</td>
			<td>For immuostaining</td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline, PH 7.4</td>
			<td>Sigma</td>
			<td>P3813-10PAK</td>
			<td>For staining</td>
		</tr>
		<tr>
			<td>Pin holders</td>
			<td>Fine Science Tools</td>
			<td>91606-07</td>
			<td>For wounding</td>
		</tr>
		<tr>
			<td>Rabbit anti-Grainyhead Primary Antibody</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>For immunostaining epithelial nuclei. Protocol to make antibody can be found (Ref. #4 and 8)</td>
		</tr>
		<tr>
			<td>Rabbit anti-RFP Primary Antibody</td>
			<td>MBL</td>
			<td>PM005</td>
			<td>For immunostaining mCD8-RFP fly epithelium</td>
		</tr>
		<tr>
			<td>Stereomicroscope</td>
			<td>Olympus</td>
			<td>SZ51</td>
			<td>For dissecting and mounting fly tissue</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma</td>
			<td>10789704001</td>
			<td>For immuostaining</td>
		</tr>
		<tr>
			<td>UAS-E2F RNAi, UAS-RacDN</td>
			<td>VDRC (v) and Bloomington Drosophila Stock Center (b)</td>
			<td>v108837, b6292</td>
			<td>Losick et al. Current Biology, 2013</td>
		</tr>
		<tr>
			<td>UAS-fzr RNAi, UAS-Stg</td>
			<td>VDRC (v) and Bloomington Drosophila Stock Center (b)</td>
			<td>v25550, b56562</td>
			<td>Grendler et al. Development, 2019</td>
		</tr>
		<tr>
			<td>UAS-Myc</td>
			<td>Bloomington Drosophila Stock Center (b)</td>
			<td>b9674</td>
			<td>Grendler et al. Development, 2019</td>
		</tr>
		<tr>
			<td>UAS-myc RNAi</td>
			<td>Bloomington Drosophila Stock Center (b)</td>
			<td>b36123</td>
			<td>Grendler et al. Development, 2019</td>
		</tr>
		<tr>
			<td>Vannas Spring Scissors</td>
			<td>Fine Science Tools</td>
			<td>15000-00</td>
			<td>For dissecting</td>
		</tr>
	</tbody>
</table>

a drosophila model to study wound-induced polyploidization

Polyploidy is a frequent 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 is a result of 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 identify why polyploid cells arise and how these unique cells function. Provided is a detailed protocol to study WIP in the adult fruit fly epithelium where polyploid cells are generated within 2 days after a puncture wound. Taking advantage of D. melanogaster&#39;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 can reveal how other genetic and physiological variables including sex, diet, and age regulate and influence WIP&#39;s function.

A detailed protocol is provided 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 was easily induced by a mechanical puncture with an insect pin and polyploid cells were generated within a short period of time (2-3 dpi) (Figure 1A, 1B)4. The major challenge is the dissection of intact abdominal tissue without any perturbations to the epithelium. The D. melanogaster epithelium is easily accidentally bumped or scratched with the sharp dissection tools. Therefore, the steps of this protocol should be practiced prior to use and analysis.
First, the injury was restricted to the ventral female abdomen, which provides a large, flat opaque tissue area ideal for imaging. The puncture wounds were inflicted in the pleurite epithelium, which lies on either side of the ventral midline sternites and targeted between tergite (T) segments T4-T5 (Figure 1A-C). This wound placement provides a large visible area that is not disrupted by the dissection. Challenging steps include the abdominal spring scissor cut and pinning steps (Figure 1D, 1E). The spring cutting step worked best when the abdomens were cut in a reduced volume of Grace&#39;s solution (~30 &#181;L) to decrease tissue movement. A well-centered cut along the dorsal midline was necessary to provide sufficient area on the abdominal dorsal flaps to pin open on the dissection plate (Figure 1C). The abdomen must 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 must be discarded. Once the abdominal tissue was fixed, it remained on the dissection plate until immunofluorescence staining was complete and the abdomens were 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 fusion4,16. The septate junction protein FasIII, which labels cell-cell junctions, provided an indicator for whether any processing perturbations occurred during preparation (Figure 1G, 1H). Abdomens with large scratches (unstained area) that perturb the wound area must be discarded and were not used for further analysis (Figure 1H).
The next step was to analyze intact samples for any defects in WIP. This protocol includes distinct assays to detect different aspects of the WIP response (Figure 2). Wound repair was complete when a central, large, multinucleated cell covered the wound scab (Figure 3A). Here cell fusion was detected by staining for FasIII/Grh and quantifying the number of Grh+ epithelial nuclei encompassed in the FasIII outlined area4. Defects in wound closure or re-epithelialization were detected when gaps of &#62;10 &#181;m in the epithelial sheet were observed (Figure 3B, red arrow). This was the case, for example, when WIP was inhibited by the activation of mitotic cycle via expression of stg, fzrRNAi, as recently reported16. In this genetic condition, 52% of the wounds were not able to form a continuous epithelial sheet over the wound scab (Figure 3B, 3C).
Another method to measure wound repair in this model was by visualizing the epithelial membrane with epi-Gal4 expression of UAS-mCD8-ChRFP4 (Figure 2, Figure 3D). In the control, 91% of epithelial wounds closed completely by 3 dpi, but inhibiting WIP by blocking endoreplication (E2f1RNAi) and cell fusion (RacDN) simultaneously, as previously reported, caused 92% of epithelial wounds to remain completely open (Figure 3D, 3E)8,16. The activation of mitotic cell cycle by expression of stg, fzrRNAi also resulted in an epithelial wound closure defect. However, by visualizing the epithelial cell membrane, the extent of re-epithelialization defect could be determined. The WIP mutant (E2f1RNAi, RacDN) fly wounds were more open than the stg, fzrRNAi wounds (Figure 3D, dashed red outline)16. This membrane wound healing assay provided more information on the extent of the wound repair defect. As a result, re-epithelialization defects could be grouped as either completely open, partially closed (i.e., &#62;10 &#181;m gaps), or completely closed (Figure 3D, 3E).
In addition to cell fusion, epithelial cells grow in size by endoreplication, an incomplete cell cycle that doubles the nuclear DNA content. Endoreplication was assayed by both cell cycle activity and direct nuclear DNA ploidy measurements (Figure 2 and Figure 4). Here, cell cycle activity was detected by incorporation of the thymidine analog, EdU (Figure 4A, 4B). D. melanogaster epithelial cells were found to enter the endocycle, an incomplete cell cycle that oscillates between the S and G phases without an intervening M phase4,12. The adult D. melanogaster diet was supplemented with EdU+ food prior to injury and the flies were maintained on an EdU+ diet until dissection at 2 dpi (Figure 4A). The EdU was then detected using the manufacturer&#39;s Click-iT protocol. This EdU assay was used to determine where, when, and how many nuclei were triggered to enter S phase in response to a wound. Using the Gal4/ UAS system, it was recently found that epithelial specific expression of myc can either block (mycRNAi) or exacerbate (Myc overexpression) the competence of epithelial cells to enter S phase. As result, it has been shown that Myc is sufficient to induce endoreplication in postmitotic cells, even without injury16,22.
Next, epithelial ploidy was determined by directly measuring nuclear DNA content. Epithelial nuclei were identified by immunofluorescence staining for the epithelial specific marker, Grh (Figure 4D). In the Fiji imaging software, epithelial nuclei were systemically identified and then thresholded using Grh nuclear stain. Nuclei were then separated and the ROIs overlaid on to the SUM of stacks DAPI image (Figure 4D, green arrow). Any overlapping nuclei were manually deleted before the Integrated Density of the selected nuclei was measured (Figure 4D, red arrows). This semiautomated method allows one to quantify the distribution and ploidy of most nuclei throughout the uninjured and repaired fly abdominal epithelium8. As recently reported, the epithelial nuclei surrounding the wound were composed of 44% polyploid nuclei with DNA content more than 3C at 3 dpi (Figure 4E, 4F)16. As expected from the EdU results, knockdown of myc led to a significant block in endoreplication, as only 9% of epithelial nuclei were 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, because factors such as cell stretching can also influence nuclear size without affecting nuclear DNA content20.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61252/61252fig1.jpg" /> 
Figure 1: Adult fruit fly abdominal wounding, dissecting, and tissue mounting. (A) Diagram of the 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 = 50 &#181;m. (C) Dissected adult abdomen, dorsal view, with tergites labeled. Abdomens were filleted down the midline of the dorsal side (white dashed line). Scale bar = 50 &#181;m. (D) Dissected and filleted adult abdomens prior to pinning. Scale bar = 50 &#181;m. (E) Pinned adult abdomen on a dissecting plate. A pin was placed in each of the four corners of the abdomen on the dorsal side. The tissue was gently opened but not stretched, to avoid tearing. (F) Adult abdomens were 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 = 50 &#181;m. (H) Image of a scratched wound area (*) with an unstained FasIII region that disrupts the syncytium. <a href="https://www.jove.com/files/ftp_upload/61252/61252fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61252/61252fig2.jpg" /> 
Figure 2: WIP analysis workflow. The flowchart depicts the three 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 measurement of the extent of epithelial wound closure (pink boxes). Common steps are in gray boxes and D. melanogaster strain genotypes are listed above. <a href="https://www.jove.com/files/ftp_upload/61252/61252fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61252/61252fig3.jpg" /> 
Figure 3: Methods to detect re-epithelialization during WIP. Re-epithelialization was perturbed when WIP was genetically inhibited. Immunofluorescent images of control (A) and stg, fzrRNAi (B) at 3 dpi. Epithelial nuclei and septate junctions were stained with Grh (green) and FasIII (magenta), respectively. (A&#39; and B&#39;) FasIII staining alone showed that re-epithelialization was impaired (red arrow) in stg, fzrRNAi epithelium. Scale bar = 50 &#181;m. (C) Quantification of re-epithelialization defects (%) at 3 dpi (gray): control (n = 8), stg, fzrRNAi (n = 6). Error bars indicate standard error; statistical significance was measured via Student&#39;s T-test, **P &#60; 0.01. Re-epithelialization during wound repair could also be detected by expression of a membrane linked RFP using epi-Gal4, UAS-mCD8-RFP. (D) Immunofluorescent images of control, E2F1RNAi, RacDN, and stg, fzrRNAi at 3 dpi. Scale bar = 20 &#181;m. Wound scab (yellow outline) and open epithelial wound area (red outline). (E) Quantification of wound closure in ctrl (n = 11), E2F1RNAi, RacDN (n = 13), and stg, fzrRNAi (n = 13). Adapted from Grendler et al.16. <a href="https://www.jove.com/files/ftp_upload/61252/61252fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/61252/61252fig4.jpg" /> 
Figure 4: Methods to detect endoreplication during WIP. (A) Timeline of EdU assay: adult Drosophila were fed 75 &#181;L of 5 mM yeast-EdU every day 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 = 50 &#181;m. (C) Average number of EdU+ epithelial nuclei per fly at 2 dpi: ctrl (n = 37), mycRNAi#1 (n = 10), and Myc (n = 8). Error bars indicate standard error; statistical significance was measured via Student&#39;s T-test, *P &#60; 0.05, **P &#60; 0.01. (D) Schematic of detection and measurement of epithelial nuclear ploidy. Epithelial nuclei were identified and thresholded by the anti-Grh stain in Fiji. Overlapping epithelial nuclei were separated (green arrowheads) or removed (red arrowheads) if overlaid by nonepithelial nuclei. The integrated density and nuclear area of corresponding DAPI stained nuclei image were measured. (E) Epithelial nuclear size (Grh) was altered by myc expression at 3 dpi. (F) Epithelial nuclear ploidy (%) at 3 dpi: ctrl (n = 4), mycRNAi#1 (n = 6), and Myc (n = 3). Adapted from Grendler et al.16. <a href="https://www.jove.com/files/ftp_upload/61252/61252fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about A Drosophila Model to Study Wound-induced Polyploidization at JoVE.com

A Drosophila Model to Study Wound-induced Polyploidization

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

A Drosophila Model to Study Wound-induced Polyploidization

Polyploidy is a frequent phenomenon whose impact on organismal health and disease is still poorly understood. A cell is defined as polyploid if it ...

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Research

JoVE Journal

Developmental Biology

5.2K Views.  Boston College. Wound-induced polyploidization is a conserved tissue repair strategy where cells grow in size instead of dividing to compensate for cell loss. Here is a detailed protocol on how to use the fruit fly as a model to measure ploidy and its genetic regulation in epithelial wound repair.

Video: A Drosophila Model to Study Wound-induced Polyploidization

Generating CRISPR/Cas9 Mediated Monoallelic Deletions to Study Enhancer Function in Mouse Embryonic Stem Cells

Enhancers control cell identity by regulating tissue-specific gene expression in a position and orientation independent manner. These enhancers are often located distally from the regulated gene in intergenic regions or even within the body of another gene. The position independent nature of enhancer activity makes it difficult to match enhancers with the genes they regulate. Deletion of an enhancer region provides direct evidence for enhancer activity and is the gold standard to reveal an enhancer's role in endogenous gene transcription. Conventional homologous recombination based deletion methods have been surpassed by recent advances in genome editing technology which enable rapid and precisely located changes to the genomes of numerous model organisms. CRISPR/Cas9 mediated genome editing can be used to manipulate the genome in many cell types and organisms rapidly and cost effectively, due to the ease with which Cas9 can be targeted to the genome by a guide RNA from a bespoke expression plasmid. Homozygous deletion of essential gene regulatory elements might lead to lethality or alter cellular phenotype whereas monoallelic deletion of transcriptional enhancers allows for the study of cis-regulation of gene expression without this confounding issue. Presented here is a protocol for CRISPR/Cas9 mediated deletion in F1 mouse embryonic stem (ES) cells (Mus musculus129 x Mus castaneus). Monoallelic deletion, screening and expression analysis is facilitated by single nucleotide polymorphisms (SNP) between the two alleles which occur on average every 125 bp in these cells.

Experimental validation of enhancer activity is best approached by loss-of-function analysis. Presented here is an efficient protocol that uses CRISPR/Cas9 mediated deletion to study allele-specific regulation of gene transcription in F1 ES cells which contain a hybrid genome (Mus musculus129 x Mus castaneus).

Enhancers control cell identity by regulating tissue-specific gene expression in a position and orientation independent manner. These enhancers are often ...

Methods to Examine the Lymph Gland and Hemocytes in Drosophila Larvae

Many parallels exist between the Drosophila and mammalian hematopoietic systems, even though Drosophila lack the lymphoid lineage that characterize mammalian adaptive immunity. Drosophila and mammalian hematopoiesis occur in spatially and temporally distinct phases to produce several blood cell lineages. Both systems maintain reservoirs of blood cell progenitors with which to expand or replace mature lineages. The hematopoietic system allows Drosophila and mammals to respond to and to adapt to immune challenges. Importantly, the transcriptional regulators and signaling pathways that control the generation, maintenance, and function of the hematopoietic system are conserved from flies to mammals. These similarities allow Drosophila to be used to genetically model hematopoietic development and disease.
Here we detail assays to examine the hematopoietic system of Drosophila larvae. In particular, we outline methods to measure blood cell numbers and concentration, visualize a specific mature lineage in vivo, and perform immunohistochemistry on blood cells in circulation and in the hematopoietic organ. These assays can reveal changes in gene expression and cellular processes including signaling, survival, proliferation, and differentiation and can be used to investigate a variety of questions concerning hematopoiesis. Combined with the genetic tools available in Drosophila, these assays can be used to evaluate the hematopoietic system upon defined genetic alterations. While not specifically outlined here, these assays can also be used to examine the effect of environmental alterations, such as infection or diet, on the hematopoietic system.

Methods to Examine the Lymph Gland and Hemocytes in Drosophila Larvae

Drosophila and mammalian hematopoietic systems share many common features, making Drosophila an attractive genetic model to study hematopoiesis. Here we demonstrate dissection and mounting of the major larval hematopoietic organ for immunohistochemistry. We also describe methods to assay various larval hematopoietic compartments including circulating hemocytes and sessile crystal cells.

Many parallels exist between the Drosophila and mammalian hematopoietic systems, even though Drosophila lack the lymphoid lineage that characterize ...

Isolation and Characterization of Primary Rat Valve Interstitial Cells: A New Model to Study Aortic Valve Calcification

Calcific aortic valve disease (CAVD) is characterized by the progressive thickening of the aortic valve leaflets. It is a condition frequently found in the elderly and end-stage renal disease (ESRD) patients, who commonly suffer from hyperphosphatemia and hypercalcemia. At present, there are no medication therapies that can stop its progression. The mechanisms that underlie this pathological process remain unclear. The aortic valve leaflet is composed of a thin layer of valve endothelial cells (VECs) on the outer surfaces of the aortic cusps, with valve interstitial cells (VICs) sandwiched between the VECs. The use of a rat model enables the in vitro study of ectopic calcification based on the in vivo physiopathological serum phosphate (Pi) and calcium (Ca) levels of patients who suffer from hyperphosphatemia and hypercalcemia. The described protocol details the isolation of a pure rat VIC population as shown by the expression of VIC markers: alpha-smooth muscle actin (&#945;-SMA) vimentin and tissue growth factor beta (TGF&#946;) 1 and 2, and the absence of cluster of differentiation (CD) 31, a VEC marker. By expanding these VICs, biochemical, genetic, and imaging studies can be performed to study and unravel the key mediators underpinning CAVD.

This protocol describes the isolation, culture, and calcification of rat-derived valve interstitial cells, a highly physiological in vitro model of calcific aortic valve disease (CAVD). Exploitation of this rat model facilitates CAVD research in exploring the cell and molecular mechanisms that underlie this complex pathological process.

Calcific aortic valve disease (CAVD) is characterized by the progressive thickening of the aortic valve leaflets. It is a condition frequently found in ...

Application of Chronic Stimulation to Study Contractile Activity-induced Rat Skeletal Muscle Phenotypic Adaptations

Skeletal muscle is a highly adaptable tissue, as its biochemical and physiological properties are greatly altered in response to chronic exercise. To investigate the underlying mechanisms that bring about various muscle adaptations, a number of exercise protocols such as treadmill, wheel running, and swimming exercise have been used in the animal studies. However, these exercise models require a long period of time to achieve muscle adaptations, which may be also regulated by humoral or neurological factors, thus limiting their applications in studying the muscle-specific contraction-induced adaptations. Indirect low frequency stimulation (10 Hz) to induce chronic contractile activity (CCA) has been used as an alternative model for exercise training, as it can successfully lead to muscle mitochondrial adaptations within 7 days, independent of systemic factors. This paper details the surgical techniques required to apply the treatment of CCA to the skeletal muscle of rats, for widespread application in future studies.

This protocol describes the use of the chronic contractile activity model of exercise to observe stimulation-induced skeletal muscle adaptations in the rat hindlimb.

Skeletal muscle is a highly adaptable tissue, as its biochemical and physiological properties are greatly altered in response to chronic exercise. To ...

Whole-mount Confocal Microscopy for Adult Ear Skin: A Model System to Study Neuro-vascular Branching Morphogenesis and Immune Cell Distribution

Here, we present a protocol of a whole-mount adult ear skin imaging technique to study comprehensive three-dimensional neuro-vascular branching morphogenesis and patterning, as well as immune cell distribution at a cellular level. The analysis of peripheral nerve and blood vessel anatomical structures in adult tissues provides some insights into the understanding of functional neuro-vascular wiring and neuro-vascular degeneration in pathological conditions such as wound healing. As a highly informative model system, we have focused our studies on adult ear skin, which is readily accessible for dissection. Our simple and reproducible protocol provides an accurate depiction of the cellular components in the entire skin, such as peripheral nerves (sensory axons, sympathetic axons, and Schwann cells), blood vessels (endothelial cells and vascular smooth muscle cells), and inflammatory cells. We believe this protocol will pave the way to investigate morphological abnormalities in peripheral nerves and blood vessels as well as the inflammation in the adult ear skin under different pathological conditions.

Here, we describe a high resolution whole-mount imaging method in the entire adult mouse ear skin, which enables us to visualize branching morphogenesis and patterning of peripheral nerves and blood vessels, as well as immune cell distribution.

Here, we present a protocol of a whole-mount adult ear skin imaging technique to study comprehensive three-dimensional neuro-vascular branching ...

Organotypic Culture Method to Study the Development Of Embryonic Chicken Tissues

The embryonic chicken is commonly used as a reliable model organism for vertebrate development. Its accessibility and short incubation period makes it ideal for experimentation. Currently, the study of these developmental pathways in the chicken embryo is conducted by applying inhibitors and drugs at localized sites and at low concentrations using a variety of methods. In vitro tissue&#160;culturing is a technique that enables the study of tissues separated from the host organism, while simultaneously bypassing many of the physical limitations present when working with whole embryos, such as the susceptibility of embryos to high doses of potentially lethal chemicals. Here, we present an organotypic culturing protocol for culturing the embryonic chicken half head in vitro, which presents new opportunities for the examination of developmental processes beyond the currently established methods.

Here, we present an organotypic culturing protocol to grow embryonic chicken organs in vitro. Using this method, the development of embryonic chicken tissue can be studied, while maintaining a high degree of control over the culture environment.

The embryonic chicken is commonly used as a reliable model organism for vertebrate development. Its accessibility and short incubation period makes it ...

A Hyperandrogenic Mouse Model to Study Polycystic Ovary Syndrome

Hyperandrogenemia plays a critical role in reproductive and metabolic function in females and is the hallmark of polycystic ovary syndrome. Developing a lean PCOS-like mouse model that mimics women with PCOS is clinically meaningful. In this protocol, we describe such a model. By inserting a 4 mm length of DHT (dihydrotestosterone) crystal powder pellet (total length of pellet is 8 mm), and replacing it monthly, we are able to produce a PCOS-like mouse model with serum DHT levels 2 fold higher than mice not implanted with DHT (no-DHT). We observed reproductive and metabolic dysfunction without changing body weight and body composition. While exhibiting a high degree of infertility, a small subset of these PCOS-like female mice can get pregnant and their offspring show delayed puberty and increased testosterone as adults. This PCOS-like lean mouse model is a useful tool to study the pathophysiology of PCOS and the offspring from these PCOS-like dams.

We describe the development of a lean PCOS-like mouse model with&#160;dihydrotestosterone pellet to study the pathophysiology of PCOS and the offspring from these PCOS-like dams.

Hyperandrogenemia plays a critical role in reproductive and metabolic function in females and is the hallmark of polycystic ovary syndrome. Developing a ...

An In Vitro Model of a Parallel-Plate Perfusion System to Study Bacterial Adherence to Graft Tissues

Various valved conduits and stent-mounted valves are used for right ventricular outflow tract (RVOT) valve replacement in patients with congenital heart disease. When using prosthetic materials however, these grafts are susceptible to bacterial infections and various host responses.
Identification of bacterial and host factors that play a vital role in endovascular adherence of microorganisms is of importance to better understand the pathophysiology of the onset of infections such as infective endocarditis (IE) and to develop preventive strategies. Therefore, the development of competent models to investigate bacterial adhesion under physiological shear conditions is necessary. Here, we describe the use of a newly designed in vitro perfusion chamber based on parallel plates that allows the study of bacterial adherence to different components of graft tissues such as exposed extracellular matrix, endothelial cells and inert areas. This method combined with colony-forming unit (CFU) counting is adequate to evaluate the propensity of graft materials towards bacterial adhesion under flow. Further on, the flow chamber system might be used to investigate the role of blood components in bacterial adhesion under shear conditions. We demonstrated that the source of tissue, their surface morphology and bacterial species specificity are not the major determining factors in bacterial adherence to graft tissues by using our in-house designed in vitro perfusion model.

An In Vitro Model of a Parallel-Plate Perfusion System to Study Bacterial Adherence to Graft Tissues

We describe an in-house designed in vitro flow chamber model, which allows the investigation of bacterial adherence to graft tissues.

Various valved conduits and stent-mounted valves are used for right ventricular outflow tract (RVOT) valve replacement in patients with congenital heart ...

Thawing, Culturing, and Cryopreserving Drosophila Cell Lines

There are currently over 160 distinct Drosophila cell lines distributed by the Drosophila Genomics Resource Center (DGRC). With genome engineering, the number of novel cell lines is expected to increase. The DGRC aims to familiarize researchers with using Drosophila cell lines as an experimental tool to complement and drive their research agenda. Procedures for working with a variety of Drosophila cell lines with distinct characteristics are provided, including protocols for thawing, culturing, and cryopreserving cell lines. Importantly, this publication demonstrates the best practices required to work with Drosophila cell lines to minimize the risk of contaminations from adventitious microorganisms or from other cell lines. Researchers who become familiar with these procedures will be able to delve into the many applications that use Drosophila cultured cells including biochemistry, cell biology and functional genomics.

Thawing, Culturing, and Cryopreserving Drosophila Cell Lines

Drosophila cell lines are important reagents for both fundamental and biomedical research. This article provides protocols for thawing, subculturing, and the cryopreservation of commonly used Drosophila cell lines to assist researchers in incorporating the use of these reagents in their research.

There are currently over 160 distinct Drosophila cell lines distributed by the Drosophila Genomics Resource Center (DGRC). With genome engineering, the ...

Methods to Test Endocrine Disruption in Drosophila melanogaster

In recent years there has been growing evidence that all organisms and the environment are exposed to hormone-like chemicals, known as endocrine disruptor chemicals (EDCs). These chemicals may alter the normal balance of endocrine systems and lead to adverse effects, as well as an increasing number of hormonal disorders in the human population or disturbed growth and reduced reproduction in the wildlife species. For some EDCs, there are documented health effects and restrictions on their use. However, for most of them, there is still no scientific evidence in this sense. In order to verify potential endocrine effects of a chemical in the full organism, we need to test it in appropriate model systems, as well as in the fruit fly, Drosophila melanogaster. Here we report detailed in vivo protocols to study endocrine disruption in Drosophila, addressing EDC effects on the fecundity/fertility, developmental timing, and lifespan of the fly. In the last few years, we used these Drosophila life traits to investigate the effects of exposure to 17-&#945;-ethinylestradiol (EE2), bisphenol A (BPA), and bisphenol AF (BPA F). Altogether, these assays covered all Drosophila life stages and made it possible to evaluate endocrine disruption in all hormone-mediated processes. Fecundity/fertility and developmental timing assays were useful to measure the EDC impact on the fly reproductive performance and on developmental stages, respectively. Finally, the lifespan assay involved chronic EDC exposures to adults and measured their survivorship. However, these life traits can also be influenced by several experimental factors that had to be carefully controlled. So, in this work, we suggest a series of procedures we have optimized for the right outcome of these assays. These methods allow scientists to establish endocrine disruption for any EDC or for a mixture of different EDCs in Drosophila, although to identify the endocrine mechanism responsible for the effect, further essays could be needed.

Methods to Test Endocrine Disruption in Drosophila melanogaster

Endocrine disruptor chemicals (EDCs) represent a serious problem for organisms and for natural environments. Drosophila melanogaster represents an ideal model to study EDC effects in vivo. Here, we present methods to investigate endocrine disruption in Drosophila, addressing EDC effects on fecundity, fertility, developmental timing, and lifespan of the fly.

In recent years there has been growing evidence that all organisms and the environment are exposed to hormone-like chemicals, known as endocrine disruptor ...