The authors wish to thank T. Noguchi for helpful advice on the initial establishment of the culture technique in our laboratory. Research in the lab of R. R.-P. was funded by the DFG within the international collaborative research center TRR81.

Ethics statement: 
The experimental procedures described in this protocol involve exclusively work with Drosophila melanogaster and are not subject to animal welfare laws in Germany.
1. Preparation of Media, Tools, and Flies
<ol>
	<li>Prepare modified commercially available powdered M3 growth medium without potassium bicarbonate17. CAUTION: Irritating to eye and skin. Supplement the medium with 10% fetal bovine serum, 100&#160;U/ml penicillin, and 100&#160;&#181;g/ml streptomycin. Use heat-inactivated and insect-culture-tested fetal bovine serum for best results. Filter the complete medium (referred to hereafter as medium) through a 0.2&#160;&#181;m bottle-top filter unit and store in aliquots at -20&#160;&#176;C. Before use, filter (0.2&#160;&#181;m syringe filter tip) the medium again to remove precipitates.</li>
	<li>Prepare inhibitor stock solutions for the pharmacological assays. Dissolve the chemicals in the appropriate solvent and store in aliquots. For example, prepare a stock solution of 28.69&#160;mM anacardic acid (CAUTION: Irritating to eye and skin) in dimethylsulfoxide.</li>
	<li>Prepare tools for the disruption of dissected pupal testes and dissection of single cysts. Use two sharp and stiff needles, rather than a pair of forceps. The capillary force that develops between the two arms of a pair of forceps makes the dissection of single cysts in small amounts of medium very difficult. For example, use the tip of a stainless steel insect pin inserted into an inoculation loop holder.</li>
	<li>In order to obtain appropriately aged pupae, seed ca. 10 large culture vials (tubes with 4&#160;cm diameter) with a sufficient number of adult flies (ca. 40&#8211;60 flies). To analyze the H-P switch, use flies expressing H2AvD-RFP and ProtamineB-eGFP in their endogenous pattern12,16,22. Incubate the vials under standard conditions at 25&#160;&#176;C for ca. 4&#8211;5 days until third-instar larvae crawl up and start pupation on the walls of the vials25.</li>
</ol>
2. Marking or Collecting White Pre-pupae to Obtain 24-hr-old Pupae for Dissection
<ol>
	<li>To test the influence of certain chemicals on the H-P switch, dissect pupal testes at ca. 24 hr APF12. To obtain staged pupae, take the prepared fly culture vials with pupating larvae (see section 1.4) and identify as many white pre-pupae as possible. Pre-pupae during the very first step in pupation appear as motionless, non-feeding, rather shortened larvae with everted spiracles and white-colored puparium. The puparium becomes tan-brown within 30 to 60 min, which indicates the next stage of pupal development26.</li>
	<li>Mark the positions of the pre-pupae with a permanent marker on the outside of the vials. Incubate the vials at 25&#160;&#176;C for ca. 24 hr.
	<ol>
		<li>Alternatively, pick the pre-pupae from the side of the vials with a small brush moistened with PBS buffer. Then transfer the pre-pupae to the side of a fresh 50&#160;ml reaction tube and incubate at 25&#160;&#176;C for ca. 24 hr.</li>
	</ol>
	</li>
</ol>
3. Dissection of Pupal Testes at ca. 24 hr after Puparium Formation
<ol>
	<li>Distribute several drops (ca. 80&#160;&#181;l each) of medium on a 10&#160;cm plastic cell culture or Petri dish. Using a broad pair of forceps or a brush moistened with medium, pick the pupae at the 24&#160;hr APF stage from the incubated vials (see section 2). Transfer one pupa to each drop of medium on the dish.</li>
	<li>For the dissection, use a stereo-microscope equipped with a fiber-optic illuminator. Do not heat the testes above RT during the dissection as this might prevent proper development in culture.
	<ol>
		<li>Dissect pupae with two pairs of No. 5 forceps. With one pair, grab the pupa at its most posterior part (the posterior spiracles) and hold it in position.</li>
		<li>Grab the anterior spiracles with the other pair of forceps and open the pupal operculum at its anterior end. Peel off the pupal case until at least part of the thorax is exposed.</li>
		<li>Continue to hold the pupa in position by its most posterior end. To release the internal pressure of the pupa, insert both arms of a pair of forceps into the head region of the pupa.</li>
		<li>Rip the pupa open by opening the forceps and moving the forceps in the anterior direction. Ensure that the opening generated with this movement is as large as possible in the first attempt. Avoid damaging the pupa at its posterior end before the pressure has been released as this might result in the testes being pushed directly through the small opening and most often being disrupted.</li>
		<li>Observe that once the pupa is opened, the released internal pressure of the pupa presses out agglomerates of fat body cells and tissue through the large opening in the head region. Check whether the pupal testes have already been pushed out of the pupa with this material; this occurs in some cases. The testes are not connected to any other tissue at 24 hr APF and therefore might be expelled easily from the pupa23.</li>
		<li>Avoid squeezing the pupa with the forceps as this might damage the testes if they remain in the pupa. Increase the size of the opening using one pair of forceps.</li>
		<li>Then hold the pupa at its most posterior end. Close the other pair of forceps and gently streak out the contents of the pupa from posterior to anterior to release the testes from the pupa.</li>
	</ol>
	</li>
	<li>Collect the testes by pulling them into a pre-cut 200&#160;&#181;l pipette tip. Try to transfer only a very small amount of medium to reduce the number of fat body cells transferred with the testes. Place the testes into medium in a fresh culture dish. Wash the testes several times with medium until only few fat body cells remain.</li>
	<li>For live imaging, transfer testes to a glass-bottom culture dish with medium and use an inverted imaging microscope. For pharmacological assays, continue with step 5.</li>
</ol>
4. Dissection of Male Germ-line Cysts and Live Imaging
<ol>
	<li>Transfer one or more pupal testis to a glass-bottom culture dish.</li>
	<li>Use a stereo-microscope with fiber-optic illumination and two stainless steel needles (see section 1.3) for the dissection of male germ-line cysts.
	<ol>
		<li>With one needle, carefully try to pin down one testis at its anterior or posterior end. Hold it in position. Insert the other needle into the testis and disrupt the testis sheath by moving the needle sideways. Many of the cysts will be released from the testis by this movement.</li>
		<li>Continue to hold the testis in position with one needle. Gently streak out the remaining cysts from the testis with the other needle.</li>
		<li>Separate aggregated cysts either by gently moving a needle sideways through the cluster of cysts or by transferring the cysts to a fresh culture dish with medium using a 200&#160;&#181;l pipette tip.</li>
	</ol>
	</li>
	<li>Move the culture dish to an inverted imaging microscope for live imaging of single cysts. Disperse cysts again with a needle if necessary.
	<ol>
		<li>Identify the stage of the cysts by using phase-contrast and fluorescence microscopy.</li>
		<li>Focus on a cyst of the desired stage. Confirm focus and position of the cyst frequently during longer imaging experiments, as the cysts do not adhere to the bottom of the culture dish and therefore tend to float out of focus. 
		NOTE: Coating the dish with poly-L-lysine for immobilization is detrimental to the development of early germ-line cysts. Instead, individual cysts can be isolated. With some practice, it is possible to single out a particular cyst by very gently pushing it with the needle. This will allow the transfer of single cysts to separate culture dishes with a glass Pasteur pipette with a drawn-out tip that fits into the field of view of the microscope. The isolated cysts then can be easily relocated in the culture dishes even after long-term incubation.</li>
	</ol>
	</li>
</ol>
5. Pharmacological Assay with Intact Pupal Testes
<ol>
	<li>Fill each well of a 24-well cell culture plate with 500&#160;&#181;l of medium for one experiment of 12 replicates. Transfer one pupal testis to each well, using a pre-cut 200&#160;&#181;l pipette tip.
	<ol>
		<li>Check for the presence of protamines in the isolated testes using an inverted fluorescence microscope. The testes should be free of ProtamineB-eGFP signal, which indicates that the H-P switch has not taken place in any of the cysts. Replace testes that already show ProtamineB-eGFP-positive cysts.</li>
		<li>To the first 12 wells, add 500&#160;&#181;l of medium containing diluted inhibitor compound (anacardic acid) to reach the desired final concentration (150&#160;&#181;M) in 1&#160;ml of medium per well. Use the remaining wells as untreated controls; add 500&#160;&#181;l of medium with the same amount of solvent used as with the inhibitor compounds. Note: If many different compounds and solvents are used, it might be more efficient to use medium alone as control and to test the influence of increasing solvent concentrations in separate experiments.</li>
	</ol>
	</li>
	<li>Incubate the plate at 25&#160;&#176;C for 24 hr in the dark.</li>
	<li>Check again for the presence of protamines in the incubated testes as described above. The control testes should now show one or more ProtamineB-eGFP-positive cysts, which indicates transition through the H-P switch.</li>
	<li>Using a pipette with a 200&#160;&#181;l tip, transfer the testes to poly-L-lysine-coated slides, make squash preparations and stain with appropriate fluorescent antibodies as described elsewhere12,27,28.</li>
</ol>

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The authors have no competing interests.

The presented protocol describes two different applications based both on the ability to dissect Drosophila testes and single germ-line cysts at a given developmental stage and on the ex vivo development of these cells and tissues in a culture dish. One application is the pharmacological manipulation of vital processes during sperm development. Importantly, this allows access to post-meiotic spermatogenesis that is not easily gained using functional analyses based on fly genetics. The other application is the microscopic observation of living and developing germ cells and testes. Especially this application benefits from the ability of Drosophila cells and organs in culture to survive and develop at RT and under normal atmosphere. Hence, an inverted imaging microscope equipped with a heated stage (heating to standard breeding temperature of 25&#160;&#176;C) is sufficient to obtain meaningful results in live-imaging experiments. Furthermore, many transgenic fly strains expressing fluorescent protein-tagged proteins for live imaging exist or can be easily established.
To obtain pupal testes or germ-line cysts of a specific developmental stage, it is crucial that one is familiar with the timing of Drosophila development. The exact timing for each stage might vary among laboratories, culture conditions, and fly strains and must be established empirically. As outlined above, this is especially important for pharmacological assays that, for example, target the H-P switch. For such experiments, it is advisable to always check for spermatids with a ProtamineB-eGFP signal before the actual inhibitor test because the timing can vary slightly even within one population.
Following the protocol above should enable the experimenter to dissect testes from early pupal stages as intact organs free of attached fat body tissue. These testes and, even more so, isolated germ-line cysts are very fragile. Hence, it is crucial to develop the manual dexterity and experience necessary for handling and transferring testes and cysts using forceps, needles, and pipettes. Especially studies targeting meiotic and early post-meiotic germ-cell development would benefit from this. While it is comparatively easy to dissect cysts of late spermatids with long flagella from adult testes, it is difficult to obtain earlier stages. Dissecting these cysts from early pupal testes following this protocol is much more efficient. Keep in mind that in a usual fly strain, only ca. 50% of the pupae will be male. Therefore, prepare to dissect at least twice as many pupae as the number of testes required. Alternatively, it is possible to identify male L3 larvae using a stereo-microscope, as the testes can be identified as translucent round organs embedded in the lateral fat body tissues of the intact larva23,34, and to collect them in fresh culture vials that can be used to pick pre-pupae for staging and dissections. It is also possible to identify male pre-pupae35.
We noticed that both the isolated germ-line cysts and the intact pupal testes in culture are sensitive to light, as has also been reported previously18. This light sensitivity might also hold for some chemical compounds used in pharmacological assays. Therefore, it is best to keep the culture dishes of an assay in the dark during incubation. Likewise, when planning time-lapse imaging experiments with developing testes and, especially, isolated cysts, keep in mind that too-long exposure times and/or too-high sampling rates may inhibit ex vivo development. The appropriate sampling rate should be determined experimentally.
Bacterial and/or fungal infection and over-growth in the culture dishes poses a severe problem. Infected culture dishes with too many bacteria do not support ex vivo development of the testes and cysts. Therefore, the described culture medium contains penicillin and streptomycin (see step 1.1), but during long-term incubation, these antibiotics might be degraded and their activity might diminish. This could be one of the reasons for the limited time of survival (max. 72&#160;hr) of cultured cysts observed when using the protocol above. Yet this time frame could not be extended by regular replacement of the culture medium in the dishes. We conclude that other factors might influence the survival in culture, such as a lack of growth signals from other tissues of the genital tract. On the other hand, post-meiotic development is quicker in Drosophila than in mammals. In Drosophila, spermiogenesis takes approximately 90 hr, and the H-P switch takes place between 50 and 60 hr after meiosis9,12. Thus, the usual survival time of around 48 hr achieved with this protocol is well suited to follow pivotal developmental processes in spermatogenesis, such as meiotic divisions or the H-P switch. Although bacterial or fungal contamination can be avoided by using clean tools and media, the presented protocol does not make use of strictly sterile work conditions because flies and fly testes already contain bacteria when raised under standard conditions. Nevertheless, some applications of this culture technique might benefit from flies dissected or even raised under aseptic conditions (for protocols, see&#160;17,36,37).
Pharmacological assays depend on the use of chemical compounds acting as inhibitors or activators of various enzymes. The compounds commonly used in such experiments often differ widely in their target specificity. As an advantage, this offers the potential to target whole enzyme classes, for example, with anacardic acid inhibiting p300/CBP, PCAF, and MYST family members of HATs38. The downside of this could be potential off-target or cytotoxic effects induced by such compounds. Therefore, each compound used in an assay with pupal testes or cysts should be tested for its cytotoxicity and specific effect at different concentrations. Furthermore, slight variations in the culture conditions, compound concentration or activity, and variations in the cell permeability might lead to variability of the induced effects. Therefore, it is necessary to monitor the success of the treatment in each experiment. For example, when we used anacardic acid at 150 &#181;M, we observed slight variability in the strength of the chromatin condensation phenotype between and sometimes within some testes, while acetylation of histone H4 was consistently decreased.
Pharmacological assays with Drosophila testes in culture have been used to analyze pre- and post-meiotic mechanisms of spermatogenesis4,12,14,21. Since it is difficult to access post-meiotic spermatogenesis with genetic tools to target players with essential pre-meiotic and meiotic functions, this ex vivo approach constitutes an alternative. Furthermore, in principle, the method could be adapted to pulse-chase experiments to follow the development of treated germ cells over time. However, we have not yet tested this possibility. Making use of early pupal testes is an advantage in pharmacological assays. First, the thin outer sheath of the young pupal testes (around 24 hr APF) could allow improved permeability of chemical compounds. Second, when studying the post-meiotic H-P switch, the pupal testes dissected at 24 hr APF from the Protamine-GFP-expressing fly line enable a direct readout of whether the H-P switch was affected or not.
To date, several protocols for ex vivo cultivation of Drosophila organs and tissues including testes and germ-line cysts have been developed (e.g.,&#160;see&#160;4,14,17,20,39,40). The cultivation medium and the general conditions used in the protocol presented here were established in 197917. The culture system was later adapted to in vivo imaging of the individualization mechanism in late post-meiotic cysts isolated from adult testes4. Previously, we have reported that these conditions also support survival and differentiation of meiotic and early post-meiotic germ cells in their cysts for around 48 hr12. In contrast to other culture systems19 the observed developmental timing in these cultures was approximately consistent with the timing determined from fixed samples (for details, see12). H2AvD-RFP and Protamine-GFP fluorescence signals can be used to visualize the nuclear morphology and chromatin composition of the germ cells in culture. We found that for clear identification of developmental stages in culture, this is advantageous over other criteria, such as coiling of cysts. Importantly, the presented protocol does not involve addition of fly extract or other growth factors other than fetal bovine serum. Furthermore, we show here that the conditions are compatible with fluorescence microscopy imaging of meiotic divisions ex vivo in culture. Images can be obtained with reasonable resolution in an easy-to-use medium that supports long-term development. This is in contrast to protocols enabling short-term (ca. 3 hr) observations in Voltalef oil41,42.
The procedures described above for cultivation and pharmacological treatment of pupal testes and single male germ-line cysts are easily adaptable to the many genetic, epigenetic, cell biological, or developmental questions that can be researched in Drosophila spermatogenesis. This includes especially research focusing on germ-line stem cells. It has been shown that stem cell development can be followed by live imaging of cultured adult testes20,43. However, the peristaltic movement of the mature testis sheath interferes with image acquisition. Therefore, either imaging is done using fragmented testes43 or the number of imaging experiments performed has to be increased to account for lost datasets20. Early pupal testes (24 hr APF) are not yet enclosed with muscle cells. Even slightly older testes (36&#8211;45 hr APF) appear to show few peristaltic movements (compare Figure 3 and Supplemental video 1). Therefore, cultivation of young pupal testes as intact organs could serve as an alternative.
Furthermore, once mastered, the ability to dissect pupal testes and eventually isolated germ-line cysts will allow further applications using single cysts as a source of a homogeneous, albeit small, cell population. For example, it is thus possible to perform RT-qPCR to analyze the transcriptional regulation of specific genes during defined stages of spermatogenesis, as has already been demonstrated15.

The development of male germ cells of many species is a sequential process. It is characterized by a series of crucial events that culminates in the formation of morphologically and epigenetically highly specialized spermatozoa. In Drosophila melanogaster, germ-line stem cells at the tip of the testis divide asymmetrically to give rise to a new stem cell and the spermatogonium, i.e.,&#160;the daughter cell that is committed to differentiation (see Figure 1 for an overview)1,2. The spermatogonium undergoes four rounds of mitotic divisions and, with the onset of meiosis, a phase of impressive growth and transcriptional activity called the spermatocyte stage. The cells originating from one spermatogonium remain connected to each other and develop as a synchronized bundle wrapped by two somatic cells&#8212;a structure referred to as a cyst. After completion of meiosis, the morphology of the male germ cells is completely reorganized (schematically shown in Figure 1). Initially, round spermatids elongate to form the hydrodynamic head structure, and the flagellum develops. Eventually, the sperm cells are separated from each other by a complex, apoptosis-related mechanism called individualization3&#8211;5.
In many species, including Drosophila melanogaster and mammalian species, the morphological changes of the germ cells are accompanied by a remarkable epigenetic rearrangement of the haploid male germ-line chromatin, called the histone-to-protamine switch (H-P switch)6&#8211;9. Possibly nearly all canonical histone and many histone-variant molecules are stripped from the DNA and are replaced by small basic proteins, the protamines, resulting in the highly compact nucleoprotamine structure specific to the chromatin of mature sperm. General characteristics of the H-P switch are the replacement of canonical histones first by histone variants, then by transitional proteins, and finally by protamines. All this is accompanied by several changes to epigenetic marks, such as a surge in acetylation levels of histone H4 just prior to histone removal7,10-12.
Most, if not all, of the above-mentioned processes are essential for the development of mature, fully fertile sperm. This is true not only in Drosophila but also in humans, as mammalian spermatogenesis shares a considerable amount of similarity with the invertebrate system1,8,9. Studying male germ-cell development is greatly facilitated in the Drosophila model system. Flies are genetically highly accessible. The generation of mutants as well as the establishment of fly strains expressing fusion genes or RNA interference constructs takes little effort. However, in the study of post-meiotic spermatogenesis, the use of mutants and simple genetic tools could reach a limit. In Drosophila spermatogenesis, transcription ceases almost completely with entry into meiotic divisions. Thus, post-meiotic spermatogenesis is mainly based on translationally repressed and stored mRNAs synthesized in an extended meiotic prophase13-16. Therefore, tools like RNA interference or the use of non-conditional mutants are not suitable for studying specifically the post-meiotic roles of gene products that also fulfill essential functions in pre-meiotic or meiotic germ cell development.
One advantage of Drosophila that can be exploited in such cases is the ability of dissected intact testes and even cysts of germ cells to develop ex vivo in culture medium4,12,17&#8211;20. The dissection and cultivation of testes or germ-line cysts allows not only microscopic observation of germ-cell development in living cells, but also the use of pharmacological assays with, e.g.,&#160;inhibitors of enzyme complexes such as histone acetyltransferases (HATs), histone deacetylases (HDACs), topoisomerases, or the proteasome. Thus, it is possible to manipulate enzymatic functions during post-meiotic spermatogenesis and to monitor the developmental outcomes regardless of embryonic, pre-meiotic, or meiotic functions4,12.
In pharmacological assays, we previously analyzed the acetylation-dependent localization of a bromodomain protein during meiotic prophase as well as the relevance of histone acetylation levels for the epigenetic H-P switch in post-meiotic spermatogenesis by using specific inhibitors targeting HATs and HDACs12,21. Targeting the H-P switch in these assays was made possible by using a fly strain that expresses the fusion genes histone2AvD-RFP and protamineB-eGFP12,22 in the endogenous patterns, and the observation that the first spermatids in pupal testes pass through the H-P switch between 24 and 48 hr APF12.
The protocol presented describes the dissection of testes and cysts from Drosophila pupae, the culture conditions, and a pharmacological assay with intact pupal testes. For this purpose, pupal testes at around 24 hr after puparium formation (APF) are dissected (see Figures 1 and 2). At this time, the testes have not made connections to other tissues and are surrounded by only a thin outer sheath, which allows better penetration by the inhibitor compounds23,24. The testes are bean- or pear-shaped organs that can be easily taken into culture. These testes are dissected, cultured, and treated with specific inhibitors. Subsequently, the effects of the inhibitors are monitored using immunofluorescence analyses and fluorescence microscopy of fusion protein expression, and by comparison of the nuclear morphology of the treated germ cells to that of untreated germ cells. The conditions presented in this protocol also allow live imaging of developing testes and germ-line cysts in culture.

<table><tbody><tr><td>Anacardic acid</td><td>Merck Millipore</td><td>172050</td><td>brand: Calbiochem (EMD Millipore)</td></tr><tr><td>Anti-acetyl-Histone H4</td><td>Merck Millipore</td><td>06-598</td><td>brand: Upstate</td></tr><tr><td>AxioCam MRm&amp;nbsp;</td><td>Zeiss</td><td></td><td></td></tr><tr><td>AxioObserver.Z1 inverted microscope</td><td>Zeiss</td><td></td><td></td></tr><tr><td>Dimethyl sulfoxide</td><td>Sigma</td><td>D8418</td><td></td></tr><tr><td>Dumont 5-Inox forceps</td><td>Dumont</td><td></td><td>multiple suppliers</td></tr><tr><td>Fetal Bovine Serum, Heat Inactivated</td><td>Sigma</td><td>F4135</td><td></td></tr><tr><td>Fiber optical&amp;nbsp; illuminator</td><td></td><td></td><td>multiple suppliers</td></tr><tr><td>Glass Bottom Microwell Dishes</td><td>MatTek</td><td>P35G-1.5-14-C</td><td></td></tr><tr><td>Goat anti-rabbit IgG (H+L)-Cy5</td><td>Dianova</td><td>711-175-152</td><td></td></tr><tr><td>Hoechst</td><td>Sigma</td><td>B1155</td><td></td></tr><tr><td>Inoculation loop or pin holder</td><td></td><td></td><td>multiple suppliers</td></tr><tr><td>Austerlitz INSECT PINS stainless steel needles, size 0</td><td>Plano GmbH</td><td>N5018</td><td>In our hands both of these pin sizes worked well in the dissections. The minutiens are less rigid but have a smaller tip diameter.</td></tr><tr><td>Austerlitz INSECT PINS stainless steel minutiens, diam. 0.1 mm</td><td>Plano GmbH</td><td>N5014</td><td></td></tr><tr><td>Multiwell plates, 24-wells</td><td>Becton Dickinson</td><td>351147</td><td>brand: Falcon</td></tr><tr><td>Pen Strep</td><td>invitrogen</td><td>15070</td><td>brand: Gibco</td></tr><tr><td>Shields and Sang M3 Insect Medium</td><td>Sigma</td><td>S8398</td><td></td></tr><tr><td>Stemi SV6 binocular</td><td>Zeiss</td><td></td><td></td></tr></tbody></table>

ex vivo culture of drosophila pupal testis and single male germ-line cysts: dissection, imaging, and pharmacological treatment

During spermatogenesis in mammals and in Drosophila melanogaster, male germ cells develop in a series of essential developmental processes. This includes differentiation from a stem cell population, mitotic amplification, and meiosis. In addition, post-meiotic germ cells undergo a dramatic morphological reshaping process as well as a global epigenetic reconfiguration of the germ line chromatin&#8212;the histone-to-protamine switch.
Studying the role of a protein in post-meiotic spermatogenesis using mutagenesis or other genetic tools is often impeded by essential embryonic, pre-meiotic, or meiotic functions of the protein under investigation. The post-meiotic phenotype of a mutant of such a protein could be obscured through an earlier developmental block, or the interpretation of the phenotype could be complicated. The model organism Drosophila melanogaster offers a bypass to this problem: intact testes and even cysts of germ cells dissected from early pupae are able to develop ex vivo in culture medium. Making use of such cultures allows microscopic imaging of living germ cells in testes and of germ-line cysts. Importantly, the cultivated testes and germ cells also become accessible to pharmacological inhibitors, thereby permitting manipulation of enzymatic functions during spermatogenesis, including post-meiotic stages.
The protocol presented describes how to dissect and cultivate pupal testes and germ-line cysts. Information on the development of pupal testes and culture conditions are provided alongside microscope imaging data of live testes and germ-line cysts in culture. We also describe a pharmacological assay to study post-meiotic spermatogenesis, exemplified by an assay targeting the histone-to-protamine switch using the histone acetyltransferase inhibitor anacardic acid. In principle, this cultivation method could be adapted to address many other research questions in pre- and post-meiotic spermatogenesis.

Watch this Scientific Journal Video about Ex vivo Culture of Drosophila Pupal Testis and Single Male Germ-line Cysts: Dissection, Imaging, and Pharmacological Treatment at JoVE.com

Ex vivo Culture of Drosophila Pupal Testis and Single Male Germ-line Cysts: Dissection, Imaging, and Pharmacological Treatment

This protocol describes the dissection and cultivation of intact testes and germ-line cysts from Drosophila melanogaster pupae. This method allows microscopic observation of spermatogenesis ex vivo. Furthermore, we describe a pharmacological assay of the effect of inhibitors on specific stages of germ-cell development in pupal testes.

Ex vivo Culture of Drosophila Pupal Testis and Single Male Germ-line Cysts: Dissection, Imaging, and Pharmacological Treatment

During spermatogenesis in mammals and in Drosophila melanogaster, male germ cells develop in a series of essential developmental processes. This includes ...

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Nanyang Technological University

Research

JoVE Journal

Biology

17.4K Views.  Philipps-Universität Marburg. This protocol describes the dissection and cultivation of intact testes and germ-line cysts from Drosophila melanogaster pupae. This method allows microscopic observation of spermatogenesis ex vivo. Furthermore, we describe a pharmacological assay of the effect of inhibitors on specific stages of germ-cell development in pupal testes.

Video: Ex vivo Culture of Drosophila Pupal Testis and Single Male Germ-line Cysts: Dissection, Imaging, and Pharmacological Treatment

Ex vivo Culturing of Whole, Developing Drosophila Brains

We describe a method for ex vivo culturing of whole Drosophila brains. This can be used as a counterpoint to chronic genetic manipulations for investigating the cell biology and development of central brain structures by allowing acute pharmacological interventions and live imaging of cellular processes. As an example of the technique, prior work from our lab1 has shown that a previously unrecognized subcellular compartment lies between the axonal and somatodendritic compartments of axons of the Drosophila central brain. The development of this compartment, referred to as the axon initial segment (AIS)2, was shown genetically to depend on the neuron-specific cyclin-dependent kinase, Cdk5. We show here that ex vivo treatment of wild-type Drosophila larval brains with the Cdk5-specific pharmacological inhibitors roscovitine and olomoucine3 causes acute changes in actin organization, and in localization of the cell-surface protein Fasciclin 2, that mimic the changes seen in mutants that lack Cdk5 activity genetically.
A second example of the ex vivo culture technique is provided for remodeling of the connections of embryonic mushroom body (MB) gamma neurons during metamorphosis from larva to adult. The mushroom body is the center of olfactory learning and memory in the fly4, and these gamma neurons prune their axonal and dendritic branches during pupal development and then re-extend branches at a later timepoint to establish the adult innervation pattern5. Pruning of these neurons of the MB has been shown to occur via local degeneration of neurite branches6, by a mechanism that is triggered by ecdysone, a steroid hormone, acting at the ecdysone receptor B17, and that is dependent on the activity of the ubiquitin-proteasome system6. Our method of ex vivo culturing can be used to interrogate further the mechanism of developmental remodeling. We found that in the ex vivo culture setting, gamma neurons of the MB recapitulated the process of developmental pruning with a time course similar to that in vivo. It was essential, however, to wait until 1.5 hours after puparium formation before explanting the tissue in order for the cells to commit irreversibly to metamorphosis; dissection of animals at the onset of pupariation led to little or no metamorphosis in culture. Thus, with appropriate modification, the ex vivo culture approach can be applied to study dynamic as well as steady state aspects of central brain biology.

Ex vivo Culturing of Whole, Developing Drosophila Brains

This article describes a method by which one can mimic in vivo development of the Drosophila mushroom body in an ex vivo culture system.

We describe a method for ex vivo culturing of whole Drosophila brains. This can be used as a counterpoint to chronic genetic manipulations for ...

Neuroscience

A Protocol for Genetic Induction and Visualization of Benign and Invasive Tumors in Cephalic Complexes of Drosophila melanogaster

Drosophila has illuminated our understanding of the genetic basis of normal development and disease for the past several decades and today it continues to contribute immensely to our understanding of complex diseases 1-7. Progression of tumors from a benign to a metastatic state is a complex event 8 and has been modeled in Drosophila to help us better understand the genetic basis of this disease 9. Here I present a simple protocol to genetically induce, observe and then analyze the progression of tumors in Drosophila larvae. The tumor induction technique is based on the MARCM system 10 and exploits the cooperation between an activated oncogene, RasV12 and loss of cell polarity genes (scribbled, discs large and lethal giant larvae) to generate invasive tumors 9. I demonstrate how these tumors can be visualized in the intact larvae and then how these can be dissected out for further analysis. The simplified protocol presented here should make it possible for this technique to be utilized by investigators interested in understanding the role of a gene in tumor invasion.

A Protocol for Genetic Induction and Visualization of Benign and Invasive Tumors in Cephalic Complexes of Drosophila melanogaster

Cooperation between an activated oncogene like RASV12 and mutations in cell polarity genes like scribbled, result in tumor growth in Drosophila where tumor cells also display invasive behaviors. Here a simple protocol for the induction and observation of the benign and invasive tumors is presented.

Drosophila has illuminated our  understanding of the genetic basis of normal development and disease for the  past several decades and today it continues ...

Medicine

Imaging and Analysis of Tissue Orientation and Growth Dynamics in the Developing Drosophila Epithelia During Pupal Stages

Within multicellular organisms, mature tissues and organs display high degrees of order in the spatial arrangements of their constituent cells. A remarkable example is given by sensory epithelia, where cells of the same or distinct identities are brought together via cell-cell adhesion showing highly organized planar patterns. Cells align to one another in the same direction and display equivalent polarity over large distances. This organization of the mature epithelia is established over the course of morphogenesis. To understand how the planar arrangement of the mature epithelia is achieved, it is crucial to track cell orientation and growth dynamics with high spatiotemporal fidelity during development in vivo. Robust analytical tools are also essential to identify and characterize local-to-global transitions. The Drosophila pupa is an ideal system to evaluate oriented cell shape changes underlying epithelial morphogenesis. The pupal developing epithelium constitutes the external surface of the immobile body, allowing long-term imaging of intact animals. The protocol described here is designed to image and analyze cell behaviors at both global and local levels in the pupal abdominal epidermis as it grows. The methodology described can be easily adapted to the imaging of cell behaviors at other developmental stages, tissues, subcellular structures, or model organisms.

Imaging and Analysis of Tissue Orientation and Growth Dynamics in the Developing Drosophila Epithelia During Pupal Stages

This protocol is designed for the imaging and analysis of the dynamics of cell orientation and tissue growth in the Drosophila abdominal epithelia as the fruit fly undergoes metamorphosis. The methodology described here can be applied to the study of different developmental stages, tissues, and subcellular structures in Drosophila or other model organisms.

Within multicellular organisms, mature tissues and organs display high degrees of order in the spatial arrangements of their constituent cells. A ...

Developmental Biology

Preparation of Drosophila Larval and Pupal Testes for Analysis of Cell Division in Live, Intact Tissue

Experimental analysis of cells dividing in living, intact tissues and organs is essential to our understanding of how cell division integrates with development, tissue homeostasis, and disease processes. Drosophila spermatocytes undergoing meiosis are ideal for this analysis because (1) whole Drosophila testes containing spermatocytes are relatively easy to prepare for microscopy, (2) the spermatocytes&#8217; large size makes them well suited for high resolution imaging, and (3) powerful Drosophila genetic tools can be integrated with in vivo analysis. Here, we present a readily accessible protocol for the preparation of whole testes from Drosophila third instar larvae and early pupae. We describe how to identify meiotic spermatocytes in prepared whole testes and how to image them live by time-lapse microscopy. Protocols for fixation and immunostaining whole testes are also provided. The use of larval testes has several advantages over available protocols that use adult testes for spermatocyte analysis. Most importantly, larval testes are smaller and less crowded with cells than adult testes, and this greatly facilitates high resolution imaging of spermatocytes. To demonstrate these advantages and the applications of the protocols, we present results showing the redistribution of the endoplasmic reticulum with respect to spindle microtubules during cell division in a single spermatocyte imaged by time-lapse confocal microscopy. The protocols can be combined with expression of any number of fluorescently tagged proteins or organelle markers, as well as gene mutations and other genetic tools, making this approach especially powerful for analysis of cell division mechanisms in the physiological context of whole tissues and organs.

Preparation of Drosophila Larval and Pupal Testes for Analysis of Cell Division in Live, Intact Tissue

The goal of this protocol is to analyze cell division in intact tissue by live and fixed cell microscopy using Drosophila meiotic spermatocytes. The protocol demonstrates how to isolate whole, intact testes from Drosophila larvae and early pupae, and how to process and mount them for microscopy.

Experimental analysis of cells dividing in living, intact tissues and organs is essential to our understanding of how cell division integrates with ...

Dissection and Live-Imaging of the Late Embryonic Drosophila Gonad

The Drosophila melanogaster male embryonic gonad is an advantageous model to study various aspects of developmental biology including, but not limited to, germ cell development, piRNA biology, and niche formation. Here, we present a dissection technique to live-image the gonad ex vivo during a period when in vivo live-imaging is highly ineffective. This protocol outlines how to transfer embryos to an imaging dish, choose appropriately-staged male embryos, and dissect the gonad from its surrounding tissue while still maintaining its structural integrity. Following dissection, gonads can be imaged using a confocal microscope to visualize dynamic cellular processes. The dissection procedure requires precise timing and dexterity, but we provide insight on how to prevent common mistakes and how to overcome these challenges. To our knowledge this is the first dissection protocol for the Drosophila embryonic gonad, and will permit live-imaging during an otherwise inaccessible window of time. This technique can be combined with pharmacological or cell-type specific transgenic manipulations to study any dynamic processes occurring within or between the&#160;cells in their natural gonadal environment.

Dissection and Live-Imaging of the Late Embryonic Drosophila Gonad

Here, we provide a dissection protocol required to live-image the late embryonic Drosophila male gonad. This protocol will permit observation of dynamic cellular processes under normal conditions or after transgenic or pharmacological manipulation.

The Drosophila melanogaster male embryonic gonad is an advantageous model to study various aspects of developmental biology including, but not limited to, ...

Isolation of Drosophila melanogaster Testes

The testes of Drosophila melanogaster provide an important model for the study of stem cell maintenance and differentiation, meiosis, and soma-germline interactions. Testes are typically isolated from adult males 0-3 days after eclosion from the pupal case. The testes of wild-type flies are easily distinguished from other tissues because they are yellow, but the testes of white mutant flies, a common genetic background for laboratory experiments are similar in both shape and color to the fly gut. Performing dissection on a glass microscope slide with a black background makes identifying the testes considerably easier. Testes are removed from the flies using dissecting needles. Compared to protocols that use forceps for testes dissection, our method is far quicker, allowing a well-practiced individual to dissect testes from 200-300 wild-type flies per hour, yielding 400-600 testes. Testes from white flies or from mutants that reduce testes size are harder to dissect and typically yield 200-400 testes per hour.

Isolation of Drosophila melanogaster Testes

Drosophila melanogaster testes can be rapidly and efficiently isolated from adult males using dissecting needles. With practice, one can readily isolate in one or two days an amount of testes sufficient for the analysis of DNA or RNA by high throughput sequencing or more traditional molecular biology methods or of protein for antibody- or enzyme-based assays.

The testes of Drosophila melanogaster provide an important model for the study of stem cell maintenance and differentiation, meiosis, and soma-germline ...

Imaging Centrosomes in Fly Testes

Centrosomes are conserved microtubule-based organelles whose structure and function change dramatically throughout the cell cycle and cell differentiation. Centrosomes are essential to determine the cell division axis during mitosis and to nucleate cilia during interphase. The identity of the proteins that mediate these dynamic changes remains only partially known, and the function of many of the proteins that have been implicated in these processes is still rudimentary. Recent work has shown that Drosophila spermatogenesis provides a powerful system to identify new proteins critical for centrosome function and formation as well as to gain insight into the particular function of known players in centrosome-related processes. Drosophila is an established genetic model organism where mutants in centrosomal genes can be readily obtained and easily analyzed. Furthermore, recent advances in the sensitivity and resolution of light microscopy and the development of robust genetically tagged centrosomal markers have transformed the ability to use Drosophila testes as a simple and accessible model system to study centrosomes. This paper describes the use of genetically-tagged centrosomal markers to perform genetic screens for new centrosomal mutants and to gain insight into the specific function of newly identified genes.

Imaging of centrosomal proteins during Drosophila spermatogenesis is a powerful method to identify new proteins critical for centrosome biology as well as to elucidate the particular function of known players in this process.

Centrosomes are conserved microtubule-based organelles whose structure and function change dramatically throughout the cell cycle and cell ...

Cytological Analysis of Spermatogenesis: Live and Fixed Preparations of Drosophila Testes

Drosophila melanogaster is a powerful model system that has been widely used to elucidate a variety of biological processes. For example, studies of both the female and male germ lines of Drosophila have contributed greatly to the current understanding of meiosis as well as stem cell biology. Excellent protocols are available in the literature for the isolation and imaging of Drosophila ovaries&#160;and testes3-12. Herein, methods for the dissection and preparation of Drosophila testes for microscopic analysis are described with an accompanying video demonstration. A protocol for isolating testes from the abdomen of adult males and preparing slides of live tissue for analysis by phase-contrast microscopy as well as a protocol for fixing and immunostaining testes for analysis by fluorescence microscopy are presented. These techniques can be applied in the characterization of Drosophila mutants that exhibit defects in spermatogenesis as well as in the visualization of subcellular localizations of proteins.

Cytological Analysis of Spermatogenesis: Live and Fixed Preparations of Drosophila Testes

Methods for isolating and preparing Drosophila testes samples (live and fixed) for imaging by phase-contrast and fluorescence microscopy are described herein.&#160;

Drosophila melanogaster is a powerful model system that has been widely used to elucidate a variety of biological processes. For example, studies of both ...

Sonication-facilitated Immunofluorescence Staining of Late-stage Embryonic and Larval Drosophila Tissues In Situ

Studies performed in Drosophila melanogaster embryos and larvae provide crucial insight into developmental processes such as cell fate specification and organogenesis. Immunostaining allows for the visualization of developing tissues and organs. However, a protective cuticle that forms at the end of embryogenesis prevents permeation of antibodies into late-stage embryos and larvae. While dissection prior to immunostaining is regularly used to analyze Drosophila larval tissues, it proves inefficient for some analyses because small tissues may be difficult to locate and isolate. Sonication provides an alternative to dissection in larval Drosophila immunostaining protocols. It allows for quick, simultaneous processing of large numbers of late-stage embryos and larvae and maintains in situ morphology. After fixation in formaldehyde, a sample is sonicated. Sample is then subjected to immunostaining with antigen-specific primary antibodies and fluorescently labeled secondary antibodies to visualize target cell types and specific proteins via fluorescence microscopy. During the process of sonication, proper placement of a sonicating probe above the sample, as well as the duration and intensity of sonication, is critical. Additonal minor modifications to standard immunostaining protocols may be required for high quality stains. For antibodies with low signal to noise ratio, longer incubation times are typically necessary. As a proof of concept for this sonication-facilitated protocol, we show immunostains of three tissue types (testes, ovaries, and neural tissues) at a range of developmental stages.

Sonication-facilitated Immunofluorescence Staining of Late-stage Embryonic and Larval Drosophila Tissues In Situ

Immunostaining is an effective technique for visualizing specific cell types and proteins within tissues. By utilizing sonication, the protocol described here alleviates the need to dissect Drosophila melanogaster tissues from late-stage embryos and larvae before immunostaining. We provide an efficient methodology for the immunostaining of formaldehyde-fixed whole mount larvae.

Studies performed in Drosophila melanogaster embryos and larvae provide crucial insight into developmental processes such as cell fate specification and ...

Immunostaining of Whole-Mount Drosophila Testes for 3D Confocal Analysis of Large Spermatocytes

Drosophila testes are a powerful model system for studying biological processes including stem cell biology, nuclear architecture, meiosis and sperm development. However, immunolabeling of the whole Drosophila testis is often associated with significant non-uniformity of staining due to antibody penetration. Squashed preparations only partially overcome the problem since it decreases the 3D quality of the analyses. Herein, we describe a whole-mount protocol using NP40 and heptane during fixation together with immunolabeling in liquid media. It preserves the volume suitable for confocal microscopy together with reproducible and reliable labeling. We show different examples of 3D reconstitution of spermatocyte nuclei from confocal sections. The intra- and inter-testes reproducibility allows 3D quantification and comparison of fluorescence between single cells from different genotypes. We used different components of the intranuclear MINT structure (Mad1-containing Intra Nuclear Territory) as well as two components associated with the nuclear pore complex to illustrate this protocol and its applications on the largest cells of the testis, the S4-S5 spermatocytes.

Immunostaining of Whole-Mount Drosophila Testes for 3D Confocal Analysis of Large Spermatocytes

We present here an improved protocol for the whole-mount preparation and immunostaining of Drosophila testes, suitable for confocal microscopy and also allowing reproducible and reliable labeling. We illustrate this protocol by 3D representation and quantification of colocalization experiments on the large S4-S5 spermatocytes.

Drosophila testes are a powerful model system for studying biological processes including stem cell biology, nuclear architecture, meiosis and sperm ...

Ex vivo Culture of Drosophila Pupal Testis and Single Male Germ-line Cysts: Dissection, Imaging, and Pharmacological Treatment

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