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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C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The development of Drosophila melanogaster. , (1993).</li><li>Riechmann, V., Ephrussi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axis+formation+during+Drosophila+oogenesis.">Axis formation during Drosophila oogenesis.</a> Curr Opin Genet Dev. 11 (4), 374-383 (2001).</li><li>Lin, H., Yue, L., Spradling, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Drosophila+fusome,+a+germline-specific+organelle,+contains+membrane+skeletal+proteins+and+functions+in+cyst+formation.">The Drosophila fusome, a germline-specific organelle, contains membrane skeletal proteins and functions in cyst formation.</a> Development. 120 (4), 947-956 (1994).</li><li>Cox, R. T., Spradling, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Balbiani+body+and+the+fusome+mediate+mitochondrial+inheritance+during+Drosophila+oogenesis.">A Balbiani body and the fusome mediate mitochondrial inheritance during Drosophila oogenesis.</a> Development. 130 (8), 1579-1590 (2003).</li><li>Davis, A. F., Clayton, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+situ+localization+of+mitochondrial+DNA+replication+in+intact+mammalian+cells.">In situ localization of mitochondrial DNA replication in intact mammalian cells.</a> J Cell Biol. 135 (4), 883-893 (1996).</li><li>Iborra, F. J., Kimura, H., Cook, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+functional+organization+of+mitochondrial+genomes+in+human+cells.">The functional organization of mitochondrial genomes in human cells.</a> BMC Biol. 2, (2004).</li><li>Rakic, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurogenesis+in+adult+primate+neocortex:+an+evaluation+of+the+evidence.">Neurogenesis in adult primate neocortex: an evaluation of the evidence.</a> Nat Rev Neurosci. 3 (1), 65-71 (2002).</li><li>Salic, A., Mitchison, T. 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I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+DNA+(mtDNA)+Biogenesis:+Visualization+and+Duel+Incorporation+of+BrdU+and+EdU+Into+Newly+Synthesized+mtDNA+In+Vitro.">Mitochondrial DNA (mtDNA) Biogenesis: Visualization and Duel Incorporation of BrdU and EdU Into Newly Synthesized mtDNA In Vitro.</a> Journal of Histochemistry &amp; Cytochemistry. 58 (2), 207-218 (2010).</li><li>Tornoe, C. W., Christensen, C., Meldal, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peptidotriazoles+on+solid+phase:+[1,2,3]-triazoles+by+regiospecific+copper(i)-catalyzed+1,3-dipolar+cycloadditions+of+terminal+alkynes+to+azides.">Peptidotriazoles on solid phase: [1,2,3]-triazoles by regiospecific copper(i)-catalyzed 1,3-dipolar cycloadditions of terminal alkynes to azides.</a> J Org Chem. 67 (9), 3057-3064 (2002).</li><li>Horwitz, H. B., Holt, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specific+inhibition+by+ethidium+bromide+of+mitochondrial+DNA+synthesis+in+physarum+polycephalum.">Specific inhibition by ethidium bromide of mitochondrial DNA synthesis in physarum polycephalum.</a> J. Cell Biol. 49, 546-553 (1971).</li><li>Huberman, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+views+of+the+biochemistry+of+eucaryotic+DNA+replication+revealed+by+aphidicolin,+an+unusual+inhibitor+of+DNA+polymerase+alpha.">New views of the biochemistry of eucaryotic DNA replication revealed by aphidicolin, an unusual inhibitor of DNA polymerase alpha.</a> Cell. 23 (3), 647-648 (1981).</li><li>McClung, C., Hirsh, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stereotypic+behavioral+responses+to+free-base+cocaine+and+the+development+of+behavioral+sensitization+in+Drosophila.">Stereotypic behavioral responses to free-base cocaine and the development of behavioral sensitization in Drosophila.</a> Curr Biol. 8 (2), 109-112 (1998).</li><li>Moore, M. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ethanol+intoxication+in+Drosophila:+Genetic+and+pharmacological+evidence+for+regulation+by+the+cAMP+signaling+pathway.">Ethanol intoxication in Drosophila: Genetic and pharmacological evidence for regulation by the cAMP signaling pathway.</a> Cell. 93 (6), 997-1007 (1998).</li><li>Dzitoyeva, S., Dimitrijevic, N., Manev, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gamma-aminobutyric+acid+B+receptor+1+mediates+behavior-impairing+actions+of+alcohol+in+Drosophila:+adult+RNA+interference+and+pharmacological+evidence.">Gamma-aminobutyric acid B receptor 1 mediates behavior-impairing actions of alcohol in Drosophila: adult RNA interference and pharmacological evidence.</a> Proc Natl Acad Sci U S A. 100 (9), 5485-5490 (2003).</li><li>Koch, E. A., Spitzer, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+effects+of+colchicine+on+oogenesis+in+Drosophila:+induced+sterility+and+switch+of+potential+oocyte+to+nurse-cell+developmental+pathway.">Multiple effects of colchicine on oogenesis in Drosophila: induced sterility and switch of potential oocyte to nurse-cell developmental pathway.</a> Cell Tissue Res. 228 (1), 21-32 (1983).</li></ol>

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Schneider&#8217;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.&#160;</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, CuSO4, Alexa Fluor 488 azide, EdU reaction buffer and Edu buffer additive are included</td>
		</tr>
		<tr>
			<td>Mouse ATP synthase subunit &#945; antibody, (15H4C4)</td>
			<td>MitoSciences</td>
			<td>Ab14748</td>
			<td>1:1000 dilution</td>
		</tr>
		<tr>
			<td>Mouse Hts antibody (clone RC)</td>
			<td>Developmental Studies Hybridoma Bank (DSHB)</td>
			<td>hts RC</td>
			<td>1:1000 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 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 ...

Targeted in Situ Mutagenesis of Histone Genes in Budding Yeast

We describe a PCR- and homologous recombination-based system for generating targeted mutations in histone genes in budding yeast cells. The resulting mutant alleles reside at their endogenous genomic sites and no exogenous DNA sequences are left in the genome following the procedure. Since in haploid yeast cells each of the four core histone proteins is encoded by two non-allelic genes with highly homologous open reading frames (ORFs), targeting mutagenesis specifically to one of two genes encoding a particular histone protein can be problematic. The strategy we describe here bypasses this problem by utilizing sequences outside, rather than within, the ORF of the target genes for the homologous recombination step. Another feature of this system is that the regions of DNA driving the homologous recombination steps can be made to be very extensive, thus increasing the likelihood of successful integration events. These features make this strategy particularly well-suited for histone gene mutagenesis, but can also be adapted for mutagenesis of other genes in the yeast genome.

Targeted in Situ Mutagenesis of Histone Genes in Budding Yeast

A strategy for generating mutations in histone genes at their endogenous location in Saccharomyces cerevisiae is presented.

We describe a PCR- and homologous recombination-based system for generating targeted mutations in histone genes in budding yeast cells. The resulting ...

Kinetics of Lagging-strand DNA Synthesis In Vitro by the Bacteriophage T7 Replication Proteins

Here we provide protocols for the kinetic examination of lagging-strand DNA synthesis in vitro by the replication proteins of bacteriophage T7. The T7 replisome is one of the simplest replication systems known, composed of only four proteins, which is an attractive feature for biochemical experiments. Special emphasis is placed on the synthesis of ribonucleotide primers by the T7 primase-helicase, which are used by DNA polymerase to initiate DNA synthesis. Because the mechanisms of DNA replication are conserved across evolution, these protocols should be applicable, or useful as a conceptual springboard, to investigators using other model systems. The protocols described here are highly sensitive and an experienced investigator can perform these experiments and obtain data for analysis in about a day. The only specialized piece of equipment required is a rapid-quench flow instrument, but this piece of equipment is relatively common and available from various commercial sources. The major drawbacks of these assays, however, include the use of radioactivity and the relative low throughput.

Kinetics of Lagging-strand DNA Synthesis In Vitro by the Bacteriophage T7 Replication Proteins

We describe sensitive, gel-based discontinuous assays to examine the kinetics of lagging-strand initiation using the replication proteins of bacteriophage T7.

Here we provide protocols for the kinetic examination of lagging-strand DNA synthesis in vitro by the replication proteins of bacteriophage T7. The T7 ...

High Resolution Fluorescent In Situ Hybridization in Drosophila Embryos and Tissues Using Tyramide Signal Amplification

In our efforts to determine the patterns of expression and subcellular localization of Drosophila RNAs on a genome-wide basis, and in a variety of tissues, we have developed numerous modifications and improvements to our original fluorescent in situ hybridization (FISH) protocol. To facilitate throughput and cost effectiveness, all steps, from probe generation to signal detection, are performed using exon 96-well microtiter plates. Digoxygenin (DIG)-labelled antisense RNA probes are produced using either cDNA clones or genomic DNA as templates. After tissue fixation and permeabilization, probes are hybridized to transcripts of interest and then detected using a succession of anti-DIG antibody conjugated to biotin, streptavidin conjugated to horseradish peroxidase (HRP) and fluorescently conjugated tyramide, which in the presence of HRP, produces a highly reactive intermediate that binds to electron dense regions of immediately adjacent proteins. These amplification and localization steps produce a robust and highly localized signal that facilitates both cellular and subcellular transcript localization. The protocols provided have been optimized to produce highly specific signals in a variety of tissues and developmental stages. References are also provided for additional variations that allow the simultaneous detection of multiple transcripts, or transcripts and proteins, at the same time.

High Resolution Fluorescent In Situ Hybridization in Drosophila Embryos and Tissues Using Tyramide Signal Amplification

The described RNA in situ hybridization protocol allows the detection of RNA in whole Drosophila embryos or dissected tissues. Using 96-well microtiter plates and tyramide signal amplification, transcripts can be detected at high resolution, sensitivity, and throughput, and at a relatively low cost.

In our efforts to determine the patterns of expression and subcellular localization of Drosophila RNAs on a genome-wide basis, and in a variety of ...

Profiling DNA Replication Timing Using Zebrafish as an In Vivo Model System

DNA replication timing is an important cellular characteristic, exhibiting significant relationships with chromatin structure, transcription, and DNA mutation rates. Changes in replication timing occur during development and in cancer, but the role replication timing plays in development and disease is not known. Zebrafish were recently established as an in vivo model system to study replication timing. Here is detailed the protocols for using the zebrafish to determine DNA replication timing. After sorting cells from embryos and adult zebrafish, high-resolution genome-wide DNA replication timing patterns can be constructed by determining changes in DNA copy number through analysis of next generation sequencing data. The zebrafish model system allows for evaluation of the replication timing changes that occur in vivo throughout development, and can also be used to assess changes in individual cell types, disease models, or mutant lines. These methods will enable studies investigating the mechanisms and determinants of replication timing establishment and maintenance during development, the role replication timing plays in mutations and tumorigenesis, and the effects of perturbing replication timing on development and disease.

Profiling DNA Replication Timing Using Zebrafish as an In Vivo Model System

Zebrafish were recently used as an in vivo model system to study DNA replication timing during development. Here is detailed the protocols for using zebrafish embryos to profile replication timing. This protocol can be easily adapted to study replication timing in mutants, individual cell types, disease models, and other species.

DNA replication timing is an important cellular characteristic, exhibiting significant relationships with chromatin structure, transcription, and DNA ...

Genome-wide Surveillance of Transcription Errors in Eukaryotic Organisms

Accurate transcription is required for the faithful expression of genetic information. Surprisingly though, little is known about the mechanisms that control the fidelity of transcription. To fill this gap in scientific knowledge, we recently optimized the circle-sequencing assay to detect transcription errors throughout the transcriptome of Saccharomyces cerevisiae, Drosophila melanogaster, and Caenorhabditis elegans. This protocol will provide researchers with a powerful new tool to map the landscape of transcription errors in eukaryotic cells so that the mechanisms that control the fidelity of transcription can be elucidated in unprecedented detail.

This protocol provides researchers with a new tool to monitor the fidelity of transcription in multiple model organisms.

Accurate transcription is required for the faithful expression of genetic information. Surprisingly though, little is known about the mechanisms that ...

Visualization of Microbiota in Tick Guts by Whole-mount In Situ Hybridization

Infectious diseases transmitted by arthropod vectors continue to pose a significant threat to human health worldwide. The pathogens causing these diseases, do not exist in isolation when they colonize the vector; rather, they likely engage in interactions with resident microorganisms in the gut lumen. The vector microbiota has been demonstrated to play an important role in pathogen transmission for several vector-borne diseases. Whether resident bacteria in the gut of the Ixodes scapularis tick, the vector of several human pathogens including Borrelia burgdorferi, influence tick transmission of pathogens is not determined. We require methods for characterizing the composition of the bacteria associated with the tick gut to facilitate a better understanding of potential interspecies interactions in the tick gut. Using whole-mount in situ hybridization to visualize RNA transcripts associated with particular bacterial species allows for the collection of qualitative data regarding the abundance and distribution of the microbiota in intact tissue. This technique can be used to examine changes in the gut microbiota milieu over the course of tick feeding and can also be applied to analyze expression of tick genes. Staining of whole tick guts yield information about the gross spatial distribution of target RNA in the tissue without the need for three-dimensional reconstruction and is less affected by environmental contamination, which often confounds the sequencing-based methods frequently used to study complex microbial communities. Overall, this technique is a valuable tool that can be used to better understand vector-pathogen-microbiota interactions and their role in disease transmission.

Visualization of Microbiota in Tick Guts by Whole-mount In Situ Hybridization

Here, we present a protocol to spatially and temporally assess the presence of viable microbiota in tick guts using a modified whole-mount in situ hybridization approach.

Infectious diseases transmitted by arthropod vectors continue to pose a significant threat to human health worldwide. The pathogens causing these ...

Methodology for Accurate Detection of Mitochondrial DNA Methylation

Quantification of DNA methylation can be achieved using bisulfite sequencing, which takes advantage of the property of sodium bisulfite to convert unmethylated cytosine into uracil, in a single-stranded DNA context. Bisulfite sequencing can be targeted (using PCR) or performed on the whole genome and provides absolute quantification of cytosine methylation at the single base-resolution. Given the distinct nature of nuclear- and mitochondrial DNA, notably in the secondary structure, adaptions of bisulfite sequencing methods for investigating cytosine methylation in mtDNA should be made. Secondary and tertiary structure of mtDNA can indeed lead to bisulfite sequencing artifacts leading to false-positives due to incomplete denaturation poor access of bisulfite to single-stranded DNA. Here, we describe a protocol using an enzymatic digestion of DNA with BamHI coupled with bioinformatic analysis pipeline to allow accurate quantification of cytosine methylation levels in mtDNA. In addition, we provide guidelines for designing the bisulfite sequencing primers specific to mtDNA, in order to avoid targeting undesirable NUclear MiTochondrial segments (NUMTs) inserted into the nuclear genome.

Here, we present a protocol to allow accurate quantification of mitochondrial DNA (mtDNA) methylation. In this protocol, we describe an enzymatic digestion of DNA with BamHI coupled with a bioinformatic analysis pipeline which can be used to avoid overestimation of mtDNA methylation levels caused by the secondary structure of mtDNA.

Quantification of DNA methylation can be achieved using bisulfite sequencing, which takes advantage of the property of sodium bisulfite to convert ...

Characterizing Histone Post-translational Modification Alterations in Yeast Neurodegenerative Proteinopathy Models

Neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS) and Parkinson&#39;s disease (PD), cause the loss of hundreds of thousands of lives each year. Effective treatment options able to halt disease progression are lacking. Despite the extensive sequencing efforts in large patient populations, the majority of ALS and PD cases remain unexplained by genetic mutations alone. Epigenetics mechanisms, such as the post-translational modification of histone proteins, may be involved in neurodegenerative disease etiology and progression and lead to new targets for pharmaceutical intervention. Mammalian in vivo and in vitro models of ALS and PD are costly and often require prolonged and laborious experimental protocols. Here, we outline a practical, fast, and cost-effective approach to determining genome-wide alterations in histone modification levels using Saccharomyces cerevisiae as a model system. This protocol allows for comprehensive investigations into epigenetic changes connected to neurodegenerative proteinopathies that corroborate previous findings in different model systems while significantly expanding our knowledge of the neurodegenerative disease epigenome.

This protocol outlines experimental procedures to characterize genome-wide changes in the levels of histone post-translational modifications (PTM) occurring in connection with the overexpression of proteins associated with ALS and Parkinson&#39;s disease in Saccharomyces cerevisiae models. After SDS-PAGE separation, individual histone PTM levels are detected with modification-specific antibodies via Western blotting.

Neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS) and Parkinson&#39;s disease (PD), cause the loss of hundreds of thousands of lives ...

In Vivo Functional Study of Disease-associated Rare Human Variants Using Drosophila

Advances in sequencing technology have made whole-genome and whole-exome datasets more accessible for both clinical diagnosis and cutting-edge human genetics research. Although a number of in silico algorithms have been developed to predict the pathogenicity of variants identified in these datasets, functional studies are critical to determining how specific genomic variants affect protein function, especially for missense variants. In the Undiagnosed Diseases Network (UDN) and other rare disease research consortia, model organisms (MO) including Drosophila, C. elegans, zebrafish, and mice are actively used to assess the function of putative human disease-causing variants. This protocol describes a method for the functional assessment of rare human variants used in the Model Organisms Screening Center Drosophila Core of the UDN. The workflow begins with gathering human and MO information from multiple public databases, using the MARRVEL web resource to assess whether the variant is likely to contribute to a patient&#39;s condition as well as design effective experiments based on available knowledge and resources. Next, genetic tools (e.g., T2A-GAL4 and UAS-human cDNA lines) are generated to assess the functions of variants of interest in Drosophila. Upon development of these reagents, two-pronged functional assays based on rescue and overexpression experiments can be performed to assess variant function. In the rescue branch, the endogenous fly genes are &#34;humanized&#34;&#160;by replacing the orthologous Drosophila gene with reference or variant human transgenes. In the overexpression branch, the reference and variant human proteins are exogenously driven in a variety of tissues. In both cases, any scorable phenotype (e.g., lethality, eye morphology, electrophysiology) can be used as a read-out, irrespective of the disease of interest. Differences observed between reference and variant alleles suggest a variant-specific effect, and thus likely pathogenicity. This protocol allows rapid, in vivo assessments of putative human disease-causing variants of genes with known and unknown functions.

In Vivo Functional Study of Disease-associated Rare Human Variants Using Drosophila

The goal of this protocol is to outline the design and performance of in vivo experiments in Drosophila melanogaster to assess the functional consequences of rare gene variants associated with human diseases.

Advances in sequencing technology have made whole-genome and whole-exome datasets more accessible for both clinical diagnosis and cutting-edge human ...