Name: in situ labeling of mitochondrial dna replication in drosophila adult ovaries by edu staining
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Description: Watch this Scientific Journal Video about In Situ Labeling of Mitochondrial DNA Replication in Drosophila Adult Ovaries by EdU Staining at JoVE.com

We thank K. Delaney for comments on the manuscript. This work was supported by the National, Heart, Lung, and Blood Institute (NHLBI) Intramural Program.

1. Tissue Collection and Dissection
<ol>
	<li>In each vial containing dry yeast, culture 10 adult female flies with 10 males for 2-3 days. 
	Note: Maintaining well-fed female flies will improve the overall quality and yield of ovary production and facilitate dissection.</li>
	<li>Anesthetize the female flies on a carbon dioxide (CO2) fly pad.</li>
	<li>Under the stereoscope, place several drops of RT medium (Schneider&#39;s Drosophila medium supplemented with 10% fetal bovine serum (FBS)) on a dissecting pad. Conduct all the dissection procedures in the medium to keep the tissues alive and healthy.</li>
	<li>Grab a fattened female fly with sharp fine-nosed forceps at its lower thorax. Use another set of forceps to tug gently at the extreme posterior of the fly until the tissues in the abdomen are exposed. Detach the two ovaries from other tissues (e.g. guts). 
	Note: Each ovary displays an opaque structure that is composed of 16-20 attached ovarioles.</li>
	<li>To enhance the penetration of reagents, open the ovaries by pulling them apart and passing the tips of the forceps in between each ovariole a couple of times. Keep the ovarioles connected within an ovary to minimize loss of tissues during the following procedures.</li>
	<li>Transfer the ovaries immediately to an 1.5 ml microcentrifuge tube containing 500 &#181;l of Schneider&#39;s Drosophila medium with 10% FBS.</li>
	<li>Repeat the dissection and collect 10-15 ovaries in each microcentrifuge tube.</li>
</ol>
2. EdU Labeling
<ol>
	<li>Aspirate the medium in each tube, and replace with 500 &#181;l of Schneider&#39;s Drosophila medium with 10% FBS containing 7 &#181;M aphidicolin. 
	Note: Aphidicolin is used to block nuclear DNA synthesis by inhibiting DNA polymerase &#945; without affecting mtDNA replication 7,12. It is possible to increase the aphidicolin concentration to up to 70 &#181;M to achieve better inhibition. The stock solution of 3-30 mM aphidicolin in DMSO can be stored in the dark at -20 &#176;C for up to 6 weeks.</li>
	<li>To keep the ovaries healthy, ensure that they are immersed under solutions while changing the medium. Use Schneider&#39;s Drosophila medium with 10% FBS through step 2.6.</li>
	<li>Incubate ovaries for 3 hr at RT on a bench-top rocker with gentle rotation.
	<ol>
		<li>For drug treatment, e.g.,&#160;mitochondrial uncoupler carbonyl cyanide 4-trifluoromethoxy phenylhydrazone (FCCP), add the appropriate concentration of drug (e.g.,&#160;10 &#181;M FCCP) into the medium after 2 hr of aphidicolin treatment. Continue incubating for another 1 hr.</li>
	</ol>
	</li>
	<li>Remove the medium containing aphidicolin with or without drug. Briefly rinse the ovaries with medium (aphidicolin is not necessary) twice.</li>
	<li>Add 1 ml medium containing 10 &#181;M EdU and 7 &#181;M aphidicolin and continue incubating at RT for 2 hr. Store the 10 mM EdU stock solution (in DMSO) at -20 &#176;C.</li>
	<li>Remove the medium containing EdU and aphidicolin. Wash with medium (without aphidicolin) twice for 3 min each.</li>
</ol>
3. Tissue Fixation and Permeabilization
<ol>
	<li>Prepare a 4% paraformaldehyde solution in phosphate buffered saline (PBS, pH 7.4). 
	Note: We used commercial available paraformaldehyde stored in pre-scored ampoules. Open a new ampoule and dilute to 4% before use. 
	CAUTION: Formaldehyde is toxic; it should be handled in a fume hood with skin and eye protection.</li>
	<li>Fix ovaries with 4% paraformaldehyde for 20 min at RT with gentle rotation.</li>
	<li>Remove the fixative and wash ovaries twice in 1 ml of 3% BSA in PBS for 5 min each time.</li>
	<li>To permeabilize the tissues, remove the wash solution and add 1 ml of 0.5% Triton X-100 in PBS. Incubate at RT for 20 min.</li>
	<li>Remove the permeabilization solution and wash twice in 1 ml of 3% BSA in PBS.</li>
</ol>
4. EdU Detection
Note: EdU detection is based on &#34;click&#34; chemistry, a Cu(I)-catalyzed [3+2] cycloaddition 13, which adds fluorescent azides to the terminal alkyne group of EdU, and the fluorescent molecule is subjected to subsequent detection. Since Cu(I), which is easily oxidized to the non-catalytic Cu(II) species, is required to catalyze the reaction, it is recommended to reduce the Cu(II) sulfate in situ to obtain Cu(I). That is, Cu(II) sulfate is used in the presence of a reductant such as ascorbic acid (herein the &#34;buffer additive&#34;) to generate copper (I).
<ol>
	<li>Before the experiment, prepare the working solution of the Alexa Fluor azide (Alexa Fluor 488 azide, Alexa Fluor 555 azide or others, depending on the preferred fluorophore, which are named as &#34;dye azide&#34; hereafter), EdU reaction buffer and EdU buffer additive according to manufacturer&#39;s instructions.
	<ol>
		<li>Reconstitute the dye azide in 70 &#181;l DMSO. Store the working solution at -20 &#176;C for up to 1 year.</li>
		<li>Prepare fresh EdU reaction buffer by diluting the 10x EdU reaction buffer with deionized water. 
		Note: After use, store any remaining 1x solution at 4 &#176;C. The 1x solution is stable for up to 6 months.</li>
		<li>Make a 10x stock solution of the EdU buffer additive by fully dissolving the powder in 2 ml deionized water. 
		Note: This stock solution is stable for up to 1 year at -20 &#176;C. If the solution develops a brown color, it has degraded and should be discarded.</li>
	</ol>
	</li>
	<li>Prepare the EdU reaction cocktail fresh each time, and use within 15 min of preparation. To achieve reproducible results, make sure the reaction components are maintained at the same ratios.
	<ol>
		<li>Make fresh 1x EdU buffer additive solution by diluting the 10x stock solution (prepared in step 4.1.3) 1:10 in deionized water. Use this solution on the same day.</li>
		<li>Prepare 1 ml of EdU reaction cocktail by combining the following ingredients in order: 860 &#181;l 1x EdU reaction buffer, 40 &#181;l CuSO4, 2.5 &#181;l dye azide, 100 &#181;l 1x EdU buffer additive. 
		Note: It is important that the ingredients are added in order to ensure optimal performance.</li>
	</ol>
	</li>
	<li>Remove the PBS with 3% BSA and add 0.5 ml of the EdU reaction cocktail to each tube. Incubate at RT for 30 min with gentle rotation. Keep the samples protected from light.</li>
	<li>Remove the EdU reaction cocktail and wash once with 1 ml of 3% BSA in PBS.</li>
	<li>Wash the samples once with 1 ml of PBS.</li>
</ol>
5. Antibody Labeling
Note: Perform antibody labeling after EdU staining. If no additional staining is desired, one can proceed to mounting and imaging directly. It is important that the samples be protected from light in all the following procedures.
<ol>
	<li>Remove the wash solution. Block the ovaries in 1 ml of blocking solution containing 0.2% BSA and 0.1% Triton X-100 in PBS for 30 min.</li>
	<li>Remove the blocking solution and replace with primary antibody diluted in blocking solution (e.g., mouse ATP synthase subunit &#945; antibody, 1:1,000 dilution). Incubate in the dark at 4 &#176;C O/N.</li>
	<li>Wash the samples with 1 ml of blocking solution 3 times, 10 min each time. To minimize the background, wash two more times for 30 min each. Remove the blocking solution.</li>
	<li>Incubate with secondary antibody diluted in blocking solution (e.g., goat anti-mouse Alexa Fluor 568 secondary antibody, 1:200 dilution) for 2 hr at RT. 
	Note: Use a different color than the one coupled to EdU.</li>
	<li>Repeat the wash steps performed in step 5.3.</li>
	<li>Rinse with 1 ml of PBS once to remove the detergent.</li>
</ol>
6. Mounting and Imaging
<ol>
	<li>Carefully remove all the PBS and immediately cover ovaries with 50 &#181;l mounting medium (with or without DAPI). 
	Note: Use a mounting medium that does not harden while the ovarioles are being separated from each other. Ovaries can be stored in mounting medium at 4 &#176;C for up to a week.</li>
	<li>Cut the end of a pipette tip and use it to transfer ovaries carefully onto a microscope slide.</li>
	<li>Completely separate each ovariole with sharp fine-nosed forceps under the stereomicroscope. Remove the connecting tissues at the posterior end and stage 14 or mature egg chambers. The young egg chambers are at the anterior end and transparent.</li>
	<li>Align the egg chambers with forceps so that they do not overlap with each other.</li>
	<li>Slowly lower a # 1.5 glass coverslip on top of the samples. Allow the mounting medium to polymerize for several hours at RT. Seal with transparent nail polish.</li>
	<li>Image slides the next day, or store at 4 &#176;C prior to imaging.</li>
	<li>Visualize under a confocal microscope with a 63X oil-immersion objective. Capture three-dimensional z-stacks images. 
	Note: There is strong EdU signal in later-stage egg chambers and background fluorescence in the epithelial sheath. When examining EdU labeling in a particular germarium, one should avoid germariums that are overlapped by or close to other tissues or later-stage egg chambers.</li>
</ol>

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The authors have nothing to disclose.

EdU labeling is a novel and efficient method to detect DNA synthesis in proliferating cells, which is based on the incorporation and staining of nucleoside analogues in newly synthesized DNA. This method is superior to the BrdU labeling method in that it is faster and highly sensitive. More importantly, it allows for good structural preservation and efficient EdU-dye penetration of whole-mount tissues 9, 10. Historically, as a superior alternative to BrdU labeling, EdU labeling was used for studying nuclear DNA replication during the S-phase of the cell cycle. Aphidicolin is an inhibitor of DNA polymerase &#945;, which is the main polymerase for nuclear DNA replication at S-phase 1215. mtDNA replication is carried out by DNA polymerase &#947;, which is insensitive to aphidicolin treatment. Hence, treatment with aphidicolin prior to and during the EdU incubation significantly inhibited EdU incorporation into nuclear DNA. It should be noted that aphidicolin may be stable for several weeks if appropriately stored, and the efficacy of aphidicolin in inhibiting nuclear DNA replication was variable in our hands. The ovarioles or egg chambers with strong nuclear DNA labeling should be excluded from further data analyses.
mtDNA replication can be readily visualized as puncta in the cytoplasm, which also affords a straightforward way to quantify the level of mtDNA replication by counting the number of EdU puncta, normalized to the total volume of cytoplasm. Imaging software can be applied to automatically identify EdU puncta in microscopic images, which is especially useful for computational analyses of large data sets. However, precautions should be taken, because the incomplete inhibition of nuclear DNA replication can lead to the EdU incorporation in distinct loci on the chromosome and display as puncta inside the nucleus. Also in other cases, the intensity of EdU incorporation into replicating mtDNA might be weak, while the background and noise could be high. Therefore, the individual parameters for automatic image analyses should be carefully defined. It is also recommended that images should be examined with trained eyes to make sure that proper EdU puncta are identified.
Visualization of newly synthesized mtDNA during Drosophila oogenesis provides an opportunity to investigate how mtDNA replication is regulated under physiological or pathological conditions, by carrying out the experiment in flies subjected to a variety of pharmacological or genetic perturbations. In a previous study, EdU incorporation assay was performed in a mtDNA mutant Drosophila mt:CoIT300I11. Furthermore, to disrupt the mitochondrial membrane potential, ovaries were treated with various concentrations of mitochondrial uncoupler FCCP or 2,4-dinitrophenol (DNP) prior to EdU incorporation. Depending on the experimental purposes and drug characteristics, different methods might be adopted for effective delivery. For adult flies, drugs can be presented as a vapor (e.g., ethanol and cocaine) 16,17 or drugs can be injected into the abdomen, where it quickly diffuses throughout the body 18. The most common practice is that drugs are added to the fly food or a sucrose/drug-saturated filter paper. For example, the inhibitor of microtubule assembly, colchicine, was fed to flies for 2-3 days before ovary dissection 19. Hence, it is important to evaluate the drug delivery method and choose appropriate concentrations.
To ensure the successful imaging of mtDNA replication in Drosophila ovaries, several critical steps must be carefully executed. Foremost, preserving the viability and health of the ovaries during dissection and EdU incorporation is essential (steps 1 and 2). The Schneider&#39;s Drosophila medium with FBS needs to be warmed to RT before use. One should minimize direct contact between the egg chambers and dissecting tools or pipette tips. The ovaries should be immersed under solutions all times to avoid dehydration. Mishandling the tissues can readily lead to faint or no fluorescent signals. During the tissue mounting step, ovarioles should be separated from each other and spread out on the microscope slide. Make sure the egg chambers are not stacked on top of each other. We noticed that the egg chambers beyond stage 14 displayed little EdU incorporation. Additionally, because of the large size, the late stage egg chambers often cause the neighboring younger egg chambers to be out of focus. Thus it is recommended that the late stage egg chambers be discarded.
Here we provide a detailed protocol for labeling replicating mtDNA in Drosophila adult ovaries. This method allows for simple quantification of mtDNA replication under various genetic and pharmacological perturbations, and will be useful for dissecting mechanisms underlying developmental mitochondrial biogenesis and mtDNA inheritance.

Besides the nuclear genome, each eukaryotic cell also contains thousands of copies of small circular DNA in the mitochondrial matrix. While mitochondrial DNA (mtDNA) encodes essential subunits of electron transport chain, the majority of the mitochondrial proteome, including all factors for replication and transcription of mtDNA are encoded by the nuclear genome. In contrast to the nuclear genome that follows Mendelian laws of inheritance, the animal mitochondrial genome is inherited exclusively through the maternal lineage. Therefore, understanding how mtDNA is proliferated and transmitted from one generation to the next through the female oocyte is fundamentally important. However, there is continuing debate over how mtDNA replication is regulated in the female germline. Additionally, the widely accepted mtDNA bottleneck theory for mtDNA transmission suggests that the population of mtDNA in primordial germ cells is subsampled to a relatively small number during development 12. It also implies that mtDNA replication is temporally and spatially regulated during oogenesis. Hence, the in situ detection of mtDNA replication during germline development will facilitate the understanding of the mechanism of mtDNA inheritance.
Drosophila oogenesis provides a genetically tractable system to study mtDNA replication and transmission. In each of the two Drosophila ovaries, there are 16-20 independent strings of egg chambers called ovarioles 3, which are the functional units of egg production (see Figure 1A). Each ovariole contains a progressive linear organization of oogenesis, where the anterior tip is composed of a structure called germarium. The germarium is further divided into four regions that contain germ cells at distinct developmental stages. In region 1, germline stem cells go through asymmetric division to produce daughter cells known as cystoblasts. Region 2a contains the cystoblasts that have completed their final division. Cystoblasts undergo four rounds of division producing 16-cell groups. The 16 cells remain connected to each other by cytoplasmic bridges called ring canals. Only one of the cells commits to differentiation as the oocyte, while the other 15 develop as polyploid nurse cells. An essential cytoplasmic structure, known as fusome, is proposed to facilitate with the formation of ring canals, determine the cyst polarity and the nurse cell-oocyte interactions 4,5. As the cysts move towards region 2b, the cyst structures span the entire width of the germarium, and become more associated with follicle cells. The germarium structures end with region 3 containing the first budding egg chamber. Subsequently, the egg chambers are assembled at the posterior end of the germarium, which progress through the ovariole in 14 morphologically distinct stages. The growth of oocyte depends on the nurse cells, which transport proteins, mRNA and endomembrane structures (e.g., Golgi) via the ring canals into the oocyte. During Drosophila oogenesis, it was found that a fraction of the mitochondria within each 16-cell cyst were associated with the fusome, moved through the ring canals and were delivered into the single oocyte in a large mass called Balbiani body 6. This phenomenon was proposed to contribute to the quality control of mitochondrial inheritance through female ovaries.
Detection of mtDNA synthesis relies on the incorporation of labeled DNA precursors into cellular DNA. Traditionally, a nucleoside analog of thymidine 5-bromo-2&#39;-deoxyuridine (BrdU) was used to label the mtDNA replication in tissue culture cells 7,8. However, the antibody-based BrdU labeling exhibits several limitations, especially for whole-mount tissue staining. One major disadvantage of BrdU labeling is that it requires DNA denaturation to expose the BrdU epitope, so that it can be detected by the anti-BrdU antibody. The specimen has to be treated under harsh denaturing conditions such as chemicals (e.g., hydrochloric acid or mixtures of methanol and acetic acid), heat, or digestion with DNase, which could disrupt the structure of the specimen and complicate the following staining procedure 9,10.
Here, an alternative thymidine analog 5-ethynyl-2&#180;-deoxyuridine (EdU) is used to label the replicating mitochondrial DNA in Drosophila adult ovaries. This method is faster and highly sensitive. EdU is readily incorporated into cellular DNA during DNA replication. The following detection is based on a &#34;click&#34; reaction, a Cu (I) -catalyzed covalent reaction between the terminal alkyne group and a fluorescent azide 10. Because the reaction does not require the denaturation of the specimen, it allows good structural preservation. Furthermore, the size of dye azide is only 1/500 of that of an antibody molecule 10, which enables fast and efficient penetration of whole-mount tissues. We have used this method to detect mtDNA replication during Drosophila oogenesis and found a striking spatial pattern in the germarium region of Drosophila ovary 11, which lead us to propose a replication-dependent mtDNA selective inheritance mechanism. We present here a detailed protocol on EdU labeling of mtDNA replication in Drosophila ovaries. To demonstrate the application of the protocol, we also tested the mtDNA replication in a mtDNA mutant Drosophila (mt:CoIT300I) 11, as well as with diverse treatment of mitochondrial uncouplers, which dissipate the mitochondrial membrane potential and potentially disrupt mtDNA replication.

<table><tbody><tr><td>Schneider&amp;rsquo;s Drosophila medium</td><td>Invitrogen</td><td>21720-024</td><td></td></tr><tr><td>FBS</td><td>Invitrogen</td><td>10100-147</td><td></td></tr><tr><td>Pair of Dumont #5 Forceps</td><td>Fine Science Tools</td><td>11252-20</td><td></td></tr><tr><td>Aphidicolin</td><td>Sigma</td><td>A0781</td><td>Aliquot after dissolving in DMSO. Avoid repetitive thawing and freezing. Protect from light. May be used within 6 weeks after dissolving.&amp;nbsp;</td></tr><tr><td>FCCP</td><td>Sigma</td><td>C2920</td><td></td></tr><tr><td>DMSO</td><td>Sigma</td><td>D2650</td><td></td></tr><tr><td>Paraformaldehyde, 16% EM grade</td><td>Electron Microscopy Sciences</td><td>15710</td><td>Formaldehyde is toxic; it should be handled in a fume hood with skin and eye protection.</td></tr><tr><td>PBS</td><td>KD Medical</td><td>RGF-3190</td><td></td></tr><tr><td>BSA</td><td>Sigma</td><td>A7030</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma</td><td>T9284</td><td></td></tr><tr><td>Click-iT EdU Alexa Fluor 488 Imaging Kit</td><td>Invitrogen</td><td>C10337</td><td>EdU, CuSO&lt;sub&gt;4&lt;/sub&gt;, Alexa Fluor 488 azide, EdU reaction buffer and Edu buffer additive are included</td></tr><tr><td>Mouse ATP synthase subunit &amp;alpha; antibody, (15H4C4)</td><td>MitoSciences</td><td>Ab14748</td><td>1:1,000 dilution</td></tr><tr><td>Mouse Hts antibody (clone RC)</td><td>Developmental Studies Hybridoma Bank (DSHB)</td><td>hts RC</td><td>1:1,000 dilution</td></tr><tr><td>Goat anti-mouse Alexa Fluor 568 secondary antibody</td><td>Invitrogen</td><td>A-11004</td><td>1:200 dilution</td></tr><tr><td>Vectashield mounting medium with DAPI</td><td>Vector Laboratories</td><td>H-1500</td><td></td></tr><tr><td>Glass coverslips, #1.5 22 mm x 22 mm</td><td>Fisher Scientific</td><td>12-541-B</td><td></td></tr><tr><td>Microscope slide</td><td>Fisher Scientific</td><td>22-038-103</td><td></td></tr><tr><td>Nail polish</td><td>Elf</td><td></td><td>Many of the pigments used in nail polishes are fluorescent and leach into specimens. Only clear nail polish should be used.</td></tr></tbody></table>

in situ labeling of mitochondrial dna replication in drosophila adult ovaries by edu staining

The mitochondrial genome is inherited exclusively through the maternal line. Understanding of how the mitochondrion and its genome are proliferated and transmitted from one generation to the next through the female oocyte is of fundamental importance. Because of the genetic tractability, and the elegant, ordered simplicity by which oocyte development proceeds, Drosophila oogenesis has become an invaluable system for mitochondrial study. An EdU (5-ethynyl-2&#180;-deoxyuridine) labeling method was utilized to detect mitochondrial DNA (mtDNA) replication in Drosophila ovaries. This method is superior to the BrdU (5-bromo-2'-deoxyuridine) labeling method in that it allows for good structural preservation and efficient fluorescent dye penetration of whole-mount tissues.
Here we describe a detailed protocol for labeling replicating mitochondrial DNA in Drosophila adult ovaries with EdU. Some technical solutions are offered to improve the viability of the ovaries, maintain their health during preparation, and ensure high-quality imaging. Visualization of newly synthesized mtDNA in the ovaries not only reveals the striking temporal and spatial pattern of mtDNA replication through oogenesis, but also allows for simple quantification of mtDNA replication under various genetic and pharmacological perturbations.

The above protocol allows visualization of the punctate structures associated with mitochondria (Figure 1B-C), which indicate mitochondrial DNA replication during Drosophila oogenesis. The EdU puncta localized with mitochondria marked by staining for ATP synthase alpha subunit (Figure 2). The observed signals were absent in ovaries treated with ethidium bromide 11, an inhibitor for mtDNA replication 14, validating that these puncta indeed label replicating mtDNA.Aphidicolin was used to inhibit nuclear DNA staining without affecting mtDNA replication (Figure 2). Without aphidicolin treatment, intense EdU signals label the nuclei, and mtDNA puncta were barely detected (Figure 3). However, in the presence of aphidicolin, nuclear incorporation was dramatically reduced and many puncta associated with mitochondria were observed.
There is a high level of mtDNA replication in post-germarium egg chambers (Figure 1B). However, notably, mtDNA replication displayed a spatial pattern in the germarium. As indicated by the number of EdU puncta, there is a moderate level of mtDNA replication in region 1 of the germarium, but almost no EdU incorporation in region 2A (Figure 1C). As the cyst moves down to region 2B in the germarium, mtDNA replication resumed and the number of EdU puncta in the posterior cyst of region 2B was much higher than that in region 2A (Figure 1C). Specifically, intensive EdU incorporation was concentrated around the ring canals and the fusome structures, as stained by the hu li tai shao (Hts) protein. mtDNA kept replicating at a high level in region 3 of the germarium (Figure 1C)
To demonstrate the association of specific genes or treatment with mtDNA proliferation, Drosophila ovaries could be subjected to gene manipulation or drug treatment. We treated ovaries with different concentrations of FCCP, a classic mitochondrial protonophore, which dissipates mitochondrial membrane potential. As a control, DMSO had no effect on mtDNA replication (Figure 4A). High doses of FCCP (10 &#181;M) almost depleted mtDNA replication entirely throughout the germarium (Figure 4D). Nonetheless, lower concentration of FCCP (2 or 5 &#181;M) had a minor impact on mtDNA replication in region 1 but inhibited replication in regions 2B and 3 (Figure 4B-C), suggesting regions 2B and 3 are more sensitive to mitochondrial disruption, or they maintain relative slower replication kinetics. The above results indicated that mtDNA replication is associated with mitochondrial activity. Particularly, different regions of the germarium responded differently to mitochondrial impairment.
<img alt="Figure 1" src="/files/ftp_upload/54516/54516fig1.jpg" /> 
Figure 1. mtDNA replication during Drosophila oogenesis. (A) Diagram of a Drosophila ovariole and a magnified view of the germarium. The ovariole illustrates from left to right, anterior to posterior, successive developmental stages of egg chambers. In the germarium, the fusome (red), germline stem cells (blue), mitochondria (green), future oocyte (broken line, recognized by positioning, fusome structure and mitochondrial clusters), developing cysts (peach) and four developmental regions are shown. (B) Representative z-stack projection of a wild-type ovariole labeled by EdU and antibody against Hts-RC, a marker for ring canals and fusome. In the presence of aphidicolin, the EdU was incorporated into mtDNA (arrowheads) and nuclei (arrows). Scale bar, 10 &#181;m. (C) Magnified view of germaria outlined in the boxed region in B. The four developmental regions are indicated. Scale bar, 5 &#181;m 11 . <a href="https://www.jove.com/files/ftp_upload/54516/54516fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/54516/54516fig2.jpg" /> 
Figure 2. mtDNA replication in the Drosophila germarium visualized by EdU incorporation with aphidicolin treatment. (A)-(D) Representative confocal section of a wild type germarium showing EdU incorporation (green, b), mitochondria, marked by ATP Synthase alpha subunit staining (red, C), and nuclei, labeled with DAPI staining (blue, D) with pre-incubation with DNA polymerase-&#945; inhibitor aphidicolin. (A&#39;-D&#39;) A magnified image of the boxed area in (A) showing EdU incorporation (B&#39;), mitochondria (C&#39;) and nuclei (D&#39;). In the presence of aphidicolin, nuclear incorporation (arrows) is reduced and many puncta were localized within mitochondria (arrowheads). Scale bars, 10 &#181;m. The figure has been modified from 11. <a href="https://www.jove.com/files/ftp_upload/54516/54516fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/54516/54516fig3.jpg" /> 
Figure 3. mtDNA replication is barely detected in the Drosophila germarium without aphidicolin treatment. (A)-(D) Representative confocal section of a wild type germarium showing EdU incorporation (green), mitochondria, marked by ATP Synthase alpha subunit staining (red), and nuclei, labeled with DAPI staining (blue) in the absence of the DNA polymerase-&#945; inhibitor aphidicolin. (A&#39;-D&#39;) A magnified image of the boxed area in (A) showing EdU incorporation (B&#39;), mitochondria (C&#39;) and nuclei (D&#39;). Without aphidicolin, intense EdU signals label nuclei (arrows), and mtDNA puncta were barely detected. Scale bars, 10 &#181;m. The figure has been modified from 11. <a href="https://www.jove.com/files/ftp_upload/54516/54516fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/54516/54516fig4.jpg" /> 
Figure 4. Mitochondrial uncoupler impairs the mtDNA replication. Representative z-stack projects showing wild-type germarium treated with DMSO (A) or the mitochondrial uncoupler FCCP at concentrations of 2 &#181;M (B), 5 &#181;M (C), 10 &#181;M (D) during EdU incorporation. Note the impaired EdU labeling in region 2B treated with 2 &#181;M FCCP (B), and in both region 2B and 3 treated with 5 &#181;M FCCP (outlined in boxes). Four developmental regions are shown. Arrows, nuclear DNA; arrowheads, mtDNA. Scale bar, 10 &#181;m. The figure has been modified from 11. <a href="https://www.jove.com/files/ftp_upload/54516/54516fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about In Situ Labeling of Mitochondrial DNA Replication in Drosophila Adult Ovaries by EdU Staining at JoVE.com

In Situ Labeling of Mitochondrial DNA Replication in Drosophila Adult Ovaries by EdU Staining

Drosophila oogenesis continues to be exceptionally useful in the study of mitochondrial proliferation and inheritance. This manuscript describes a detailed protocol used to label the replicating mitochondrial DNA (mtDNA) in Drosophila adult ovaries with 5-ethynyl-2&#180;-deoxyuridine (EdU), which facilitates uncovering mechanisms associated with mitochondrial inheritance that were previously debatable.

In Situ Labeling of Mitochondrial DNA Replication in Drosophila Adult Ovaries by EdU Staining

The mitochondrial genome is inherited exclusively through the maternal line. Understanding of how the mitochondrion and its genome are proliferated and ...

in-situ-labeling-mitochondrial-dna-replication-drosophila-adult

Research

JoVE Journal

Genetics

9.0K Views.  National Institutes of Health. Drosophila oogenesis continues to be exceptionally useful in the study of mitochondrial proliferation and inheritance. This manuscript describes a detailed protocol used to label the replicating mitochondrial DNA (mtDNA) in Drosophila adult ovaries with 5-ethynyl-2´-deoxyuridine (EdU), which facilitates uncovering mechanisms associated with mitochondrial inheritance that were previously debatable.

Video: In Situ Labeling of Mitochondrial DNA Replication in Drosophila Adult Ovaries by EdU Staining

Genome-wide Determination of Mammalian Replication Timing by DNA Content Measurement

Replication of the genome occurs during S phase of the cell cycle in a highly regulated process that ensures the fidelity of DNA duplication. Each genomic region is replicated at a distinct time during S phase through the simultaneous activation of multiple origins of replication. Time of replication (ToR) correlates with many genomic and epigenetic features and is linked to mutation rates and cancer. Comprehending the full genomic view of the replication program, in health and disease is a major future goal and challenge.
This article describes in detail the &#34;Copy Number Ratio of S/G1 for mapping genomic Time of Replication&#34; method (herein called: CNR-ToR), a simple approach to map the genome wide ToR of mammalian cells. The method is based on the copy number differences between S phase cells and G1 phase cells. The CNR-ToR method is performed in 6 steps: 1. Preparation of cells and staining with propidium iodide (PI); 2. Sorting G1 and S phase cells using fluorescence-activated cell sorting (FACS); 3. DNA purification; 4. Sonication; 5. Library preparation and sequencing; and 6. Bioinformatic analysis. The CNR-ToR method is a fast and easy approach that results in detailed replication maps.

We describe here a relatively fast and simple approach for mapping genome-wide mammalian replication timing, from cell isolation to the basic analysis of the sequencing results. A genomic map of a representative replication program will be provided following the protocol.

Replication of the genome occurs during S phase of the cell cycle in a highly regulated process that ensures the fidelity of DNA duplication. Each genomic ...

Visualization of Mitochondrial DNA Replication in Individual Cells by EdU Signal Amplification

Mitochondria are key regulators of cellular energy and mitochondrial biogenesis is an essential component of regulating mitochondria numbers in healthy cells1-3. One approach for monitoring mitochondrial biogenesis is to measure the rate of mitochondrial DNA (mtDNA) replication4. We developed a sensitive technique to label newly synthesized mtDNA in individual cells in order to study mtDNA biogenesis. The technique combines the incorporation of 5-ethynyl-2'-deoxyuridine (EdU)5-7 with a tyramide signal amplification (TSA)8 protocol to visualize mtDNA replication within subcellular compartments of neurons. EdU is superior to other thymidine analogs, such as 5-bromo-2-deoxyuridine (BrdU), because the initial click reaction to label EdU5-7 does not require the harsh acid treatments or enzyme digests that are required for exposing the BrdU epitope. The milder labeling of EdU allows for direct comparison of its incorporation with other cellular markers9-10. The ability to visualize and quantify mtDNA biogenesis provides an essential tool for investigating the mechanisms used to regulate mitochondrial biogenesis and would provide insight into the pathogenesis associated with drug toxicity, aging, cancer and neurodegenerative diseases. Our technique is applicable to sensory neurons as well as other cell types. The use of this technique to measure mtDNA biogenesis has significant implications in furthering the understanding of both normal cellular physiology as well as impaired disease states.

We developed a sensitive technique to label newly synthesized mitochondrial DNA (mtDNA) in individual cells in order to study mtDNA biogenesis. The technique combines the incorporation of EdU together with a tyramide signal amplification (TSA) protocol to visualize mtDNA replication within subcellular compartments of neurons.

Mitochondria are key regulators of cellular energy and mitochondrial biogenesis is an essential component of regulating mitochondria numbers in healthy ...

Neuroscience

Cell Cycle Analysis in the C. elegans Germline with the Thymidine Analog EdU

Cell cycle analysis in eukaryotes frequently utilizes chromosome morphology, expression and/or localization of gene products required for various phases of the cell cycle, or the incorporation of nucleoside analogs. During S-phase, DNA polymerases incorporate thymidine analogs such as EdU or BrdU into chromosomal DNA, marking the cells for analysis. For C. elegans, the nucleoside analog EdU is fed to the worms during regular culture and is compatible with immunofluorescent techniques. The germline of C. elegans is a powerful model system for the studies of signaling pathways, stem cells, meiosis, and cell cycle because it is transparent, genetically facile, and meiotic prophase and cellular differentiation/gametogenesis occur in a linear assembly-like fashion. These features make EdU a great tool to study dynamic aspects of mitotically cycling cells and germline development. This protocol describes how to successfully prepare EdU bacteria, feed them to wild-type C. elegans hermaphrodites, dissect the hermaphrodite gonad, stain for EdU incorporation into DNA, stain with antibodies to detect various cell cycle and developmental markers, image the gonad and analyze the results. The protocol describes the variations in the method and analysis for the measurement of S-phase index, M-phase index, G2 duration, cell cycle duration, rate of meiotic entry, and rate of meiotic prophase progression. This method can be adapted to study the cell cycle or cell history in other tissues, stages, genetic backgrounds, and physiological conditions.

Cell Cycle Analysis in the C. elegans Germline with the Thymidine Analog EdU

An imaging-based method is described that can be used to identify S-phase and analyze cell cycle dynamics in the C. elegans hermaphrodite germline using the thymidine analog EdU. This method requires no transgenes and is compatible with immunofluorescent staining.

Cell cycle analysis in eukaryotes frequently utilizes chromosome morphology, expression and/or localization of gene products required for various phases ...

Developmental Biology

High-Throughput Quantification of the Synthesis and Distribution of mtDNA

High-Throughput Image-Based Quantification of Mitochondrial DNA Synthesis and Distribution

The vast majority of cellular processes require a continuous supply of energy, the most common carrier of which is the ATP molecule. Eukaryotic cells produce most of their ATP in the mitochondria by oxidative phosphorylation. Mitochondria are unique organelles because they have their own genome that is replicated and passed on to the next generation of cells. In contrast to the nuclear genome, there are multiple copies of the mitochondrial genome in the cell. The detailed study of the mechanisms responsible for the replication, repair, and maintenance of the mitochondrial genome is essential for understanding the proper functioning of mitochondria and whole cells under both normal and disease conditions. Here, a method that allows the high-throughput quantification of the synthesis and distribution of mitochondrial DNA (mtDNA) in human cells cultured in vitro is presented. This approach is based on the immunofluorescence detection of actively synthesized DNA molecules labeled by 5-bromo-2'-deoxyuridine (BrdU) incorporation and the concurrent detection of all the mtDNA molecules with anti-DNA antibodies. Additionally, the mitochondria are visualized with specific dyes or antibodies. The culturing of cells in a multi-well format and the utilization of an automated fluorescence microscope make it easier to study the dynamics of mtDNA and the morphology of mitochondria under a variety of experimental conditions in a relatively short time.

Author Spotlight: High-Throughput Image-Based Quantification of Mitochondrial DNA Synthesis and Distribution  

A procedure for studying the dynamics of mitochondrial DNA (mtDNA) metabolism in cells using a multi-well plate format and automated immunofluorescence imaging to detect and quantify mtDNA synthesis and distribution is described. This can be further used to investigate the effects of various inhibitors, cellular stresses, and gene silencing on mtDNA metabolism.

The vast majority of cellular processes require a continuous supply of energy, the most common carrier of which is the ATP molecule. Eukaryotic cells ...

Biochemistry

Stimulating and Analyzing Adult Neurogenesis in the Drosophila Central Brain

The molecular and cellular mechanisms underlying neurogenesis in response to disease or injury are not well understood. However, understanding these mechanisms is crucial for developing neural regenerative therapies. Drosophila melanogaster is a leading model for studies of neural development but historically has not been exploited to investigate adult brain regeneration. This is primarily because the adult brain exhibits very low mitotic activity. Nonetheless, penetrating traumatic brain injury (PTBI) to the adult Drosophila central brain triggers the generation of new neurons and new glia. The powerful genetic tools available in Drosophila combined with the simple but rigorous injury protocol described here now make adult Drosophila brain a robust model for neural regeneration research. Provided here are detailed instructions for (1) penetrating injuries to the adult central brain and (2) dissection, immunohistochemistry, and imaging post-injury. These protocols yield highly reproducible results and will facilitate additional studies to dissect mechanisms underlying neural regeneration.

Stimulating and Analyzing Adult Neurogenesis in the Drosophila Central Brain

This article provides detailed protocols for inflicting Penetrating Traumatic Brain Injury (PTBI) to adult Drosophila and examining the resulting neurogenesis.

The molecular and cellular mechanisms underlying neurogenesis in response to disease or injury are not well understood. However, understanding these ...

Experimental Approaches to Study Mitochondrial Localization and Function of a Nuclear Cell Cycle Kinase, Cdk1

Although mitochondria possess their own transcriptional machinery, merely 1% of mitochondrial proteins are synthesized inside the organelle. The nuclear-encoded proteins are transported into mitochondria guided by their mitochondria targeting sequences (MTS); however, a majority of mitochondrial localized proteins lack an identifiable MTS. Nevertheless, the fact that MTS can instruct proteins to go into the mitochondria provides a valuable tool for studying mitochondrial functions of normally nuclear and/or cytoplasmic proteins. We have recently identified the cell cycle kinase CyclinB1/Cdk1 complex in the mitochondria. To specifically study the mitochondrial functions of this complex, mitochondrial overexpression and knock-down of this complex without interfering with its nuclear or cytoplasmic functions were essential. By tagging CyclinB1/Cdk1 with MTS, we were able to achieve mitochondrial overexpression of this complex to study its mitochondrial targets as well as functions. Via tagging dominant-negative Cdk1 with MTS, inhibition of Cdk1 activity was accomplished particularly in the mitochondria. Potential mitochondrial targets of CyclinB1/Cdk1 complex were identified using a gel-based proteomics approach. Unlike traditional 2D gel analysis, we employed 2-dimensional difference gel electrophoresis (2D-DIGE) technology followed by phosphoprotein staining to fluorescently label differentially phosphorylated proteins in mitochondrial Cdk1 expressing cells. Identification of phosphoprotein spots that were altered in wild type versus dominant negative Cdk1 bearing mitochondria revealed the identity of mitochondrial targets of Cdk1. Finally, to determine the effect of CyclinB1/Cdk1 mitochondrial localization in cell cycle progression, a cell proliferation assay using a synthetic thymidine analogue EdU (5-ethynyl-2&#8242;-deoxyuridine) was used to monitor the cells as they go through the cell cycle and replicate their DNA. Altogether, we demonstrated a variety of approaches available to study mitochondrial localization and activity of a cell cycle kinase. These are advanced, yet easy to follow methods that will be beneficial to many cell biology researchers.

Here, we outline how to study mitochondrial localization of a (cell cycle) kinase, and how to determine its sub-mitochondrial location as well as potential mitochondrial substrates/targets. Forced expression of proteins into the mitochondria provides a useful tool for studying the functional consequences of mitochondrial localization of a protein of interest.

Although mitochondria possess their own transcriptional machinery, merely 1% of mitochondrial proteins are synthesized inside the organelle. The ...

Biology

Studying Mitochondrial Structure and Function in Drosophila Ovaries

Analysis of the mitochondrial structure-function relationship is required for a thorough understanding of the regulatory mechanisms of mitochondrial functionality. Fluorescence microscopy is an indispensable tool for the direct assessment of mitochondrial structure and function in live cells and for studying the mitochondrial structure-function relationship, which is primarily modulated by the molecules governing fission and fusion events between mitochondria. This paper describes and demonstrates specific methods for studying mitochondrial structure and function in live as well as in fixed tissue in the model organism Drosophila melanogaster. The tissue of choice here is the Drosophila ovary, which can be isolated and made amenable for ex vivo live confocal microscopy. Furthermore, the paper describes how to genetically manipulate the mitochondrial fission protein, Drp1, in Drosophila ovaries to study the involvement of Drp1-driven mitochondrial fission in modulating the mitochondrial structure-function relationship. The broad use of such methods is demonstrated in already-published as well as in novel data. The described methods can be further extended towards understanding the direct impact of nutrients and/or growth factors on the mitochondrial properties ex vivo. Given that mitochondrial dysregulation underlies the etiology of various diseases, the described innovative methods developed in a genetically tractable model organism, Drosophila, are anticipated to contribute significantly to the understanding of the mechanistic details of the mitochondrial structure-function relationship and to the development of mitochondria-directed therapeutic strategies.

Studying Mitochondrial Structure and Function in Drosophila Ovaries

Analysis of the mitochondrial structure-function relationship is required for a thorough understanding of the regulatory mechanisms of mitochondrial functionality. Specific methods for studying mitochondrial structure and function in live and fixed Drosophila ovaries are described and demonstrated in this paper.

Analysis of the mitochondrial structure-function relationship is required for a thorough understanding of the regulatory mechanisms of mitochondrial ...

Visualizing and Tracking Endogenous mRNAs in Live Drosophila melanogaster Egg Chambers

Fluorescence-based imaging techniques, in combination with developments in light microscopy, have revolutionized how cell biologists conduct live cell imaging studies. Methods for detecting RNAs have expanded greatly since seminal studies linked site-specific mRNA localization to gene expression regulation. Dynamic mRNA processes can now be visualized via approaches that detect mRNAs, coupled with microscopy set-ups that are fast enough to capture the dynamic range of molecular behavior. The molecular beacon technology is a hybridization-based approach capable of direct detection of endogenous transcripts in living cells. Molecular beacons are hairpin-shaped, internally quenched, single-nucleotide discriminating nucleic acid probes, which fluoresce only upon hybridization to a unique target sequence. When coupled with advanced fluorescence microscopy and high-resolution imaging, they enable one to perform spatial and temporal tracking of intracellular movement of mRNAs. Although this technology is the only method capable of detecting endogenous transcripts, cell biologists have not yet fully embraced this technology due to difficulties in designing such probes for live cell imaging. A new software application, PinMol, allows for enhanced and rapid design of probes best suited to efficiently hybridize to mRNA target regions within a living cell. In addition, high-resolution, real-time image acquisition and current, open source image analysis software allow for a refined data output, leading to a finer evaluation of the complexity underlying the dynamic processes involved in the mRNA&#39;s life cycle.
Here we present a comprehensive protocol for designing and delivering molecular beacons into Drosophila melanogaster egg chambers. Direct and highly specific detection and visualization of endogenous maternal mRNAs is performed via spinning disc confocal microscopy. Imaging data is processed and analyzed using object detection and tracking in Icy software to obtain details about the dynamic movement of mRNAs, which are transported and localized to specialized regions within the oocyte.

Visualizing and Tracking Endogenous mRNAs in Live Drosophila melanogaster Egg Chambers

Here, we present a protocol for the visualization, detection, analysis and tracking of endogenous mRNA trafficking in live Drosophila melanogaster egg chamber using molecular beacons, spinning disc confocal microscopy, and open-source analysis software.

Fluorescence-based imaging techniques, in combination with developments in light microscopy, have revolutionized how cell biologists conduct live cell ...

Specific Labeling of Mitochondrial Nucleoids for Time-lapse Structured Illumination Microscopy

Mitochondrial nucleoids are compact particles formed by mitochondrial DNA molecules coated with proteins. Mitochondrial DNA encodes tRNAs, rRNAs, and several essential mitochondrial polypeptides. Mitochondrial nucleoids divide and distribute within the dynamic mitochondrial network that undergoes fission/fusion and other morphological changes. High resolution live fluorescence microscopy is a straightforward technique to characterize a nucleoid&#39;s position and motion. For this technique, nucleoids are commonly labeled through fluorescent tags of their protein components, namely transcription factor a (TFAM). However, this strategy needs overexpression of a fluorescent protein-tagged construct, which may cause artifacts (reported for TFAM), and is not feasible in many cases. Organic DNA-binding dyes do not have these disadvantages. However, they always show staining of both nuclear and mitochondrial DNAs, thus lacking specificity to mitochondrial nucleoids. By taking into account the physico-chemical properties of such dyes, we selected a nucleic acid gel stain (SYBR Gold) and achieved preferential labeling of mitochondrial nucleoids in live cells. Properties of the dye, particularly its high brightness upon binding to DNA, permit subsequent quantification of mitochondrial nucleoid motion using time series of super-resolution structured illumination images.

The protocol describes specific labeling of mitochondrial nucleoids with a commercially available DNA gel stain, acquisition of time lapse series of live labeled cells by super-resolution structured illumination microscopy (SR-SIM), and automatic tracking of nucleoid motion.

Mitochondrial nucleoids are compact particles formed by mitochondrial DNA molecules coated with proteins. Mitochondrial DNA encodes tRNAs, rRNAs, and ...

5-Ethynyl-2'-Deoxyuridine/Phospho-Histone H3 Dual-Labeling Protocol for Cell Cycle Progression Analysis in Drosophila Neural Stem Cells

In vivo cell cycle progression analysis is routinely performed in studies on genes regulating mitosis and DNA replication. 5-Ethynyl-2'-deoxyuridine (EdU) has been utilized to investigate replicative/S-phase progression, whereas antibodies against phospho-histone H3 have been utilized to mark mitotic nuclei and cells. A combination of both labels would enable the classification of G0/G1 (Gap phase), S (replicative), and M (mitotic) phases and serve as an important tool to evaluate the effects of mitotic gene knockdowns or null mutants on cell cycle progression. However, the reagents used to mark EdU-labelled cells are incompatible with several secondary antibody-fluorescent tags. This complicates immunostaining, where primary and tagged secondary antibodies are used to mark pH3-positive mitotic cells. This paper describes a step-by-step protocol for the dual-labeling of EdU and pH3 in Drosophila larval neural stem cells, a system utilized extensively to study mitotic factors. Additionally, a protocol is provided for image analysis and quantification to allocate labeled cells in 3 distinct categories, G0/G1, S, S&gt;G2/M (progression from S to G2/M), and M phases.

5-Ethynyl-2'-Deoxyuridine/Phospho-Histone H3 Dual-Labeling Protocol for Cell Cycle Progression Analysis in Drosophila Neural Stem Cells

Cell cycle analysis with 5-ethynyl-2'-deoxyuridine (EdU) and phospho-histone H3 (pH3) labeling is a multi-step procedure that may require extensive optimization. Here, we present a detailed protocol that describes all steps for this procedure including image analysis and quantification to distinguish cells in different cell cycle phases.

In vivo cell cycle progression analysis is routinely performed in studies on genes regulating mitosis and DNA replication. 5-Ethynyl-2'-deoxyuridine (EdU) ...

In Situ Labeling of Mitochondrial DNA Replication in Drosophila Adult Ovaries by EdU Staining

Summary

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Chapters in this video

Genome-wide Determination of Mammalian Replication Timing by DNA Content Measurement

Visualization of Mitochondrial DNA Replication in Individual Cells by EdU Signal Amplification

Cell Cycle Analysis in the C. elegans Germline with the Thymidine Analog EdU

High-Throughput Image-Based Quantification of Mitochondrial DNA Synthesis and Distribution

Stimulating and Analyzing Adult Neurogenesis in the Drosophila Central Brain

Experimental Approaches to Study Mitochondrial Localization and Function of a Nuclear Cell Cycle Kinase, Cdk1

Studying Mitochondrial Structure and Function in Drosophila Ovaries

Visualizing and Tracking Endogenous mRNAs in Live Drosophila melanogaster Egg Chambers

Specific Labeling of Mitochondrial Nucleoids for Time-lapse Structured Illumination Microscopy

5-Ethynyl-2'-Deoxyuridine/Phospho-Histone H3 Dual-Labeling Protocol for Cell Cycle Progression Analysis in Drosophila Neural Stem Cells