Name: preparation of drosophila larval and pupal testes for analysis of cell division in live, intact tissue
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Description: Watch this Scientific Journal Video about Preparation of Drosophila Larval and Pupal Testes for Analysis of Cell Division in Live, Intact Tissue at JoVE.com

This work was supported by Department of Defense start-up funds to J.T.S.

1. Prepare Animals, Tools, and Media for Dissection
<ol>
	<li>Cross male and female flies to obtain progeny of the desired genotype. Useful transgenic markers for identification and staging of meiotic spermatocytes include GFP-tubulin to label the meiotic spindle and RFP-histone 2A to label chromsomes8. Use five to ten females per cross and maintain crosses on standard fly food in vials in a 25 &#176;C incubator until third instar larvae begin crawling up the sides of the vial (4&#8211;5 days). ...

<ol>
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T., Bate, M., Martinez-Arias, A. <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. , 71-147 (1993).</li><li>Dunleavy, E. M., 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=The+Cell+Cycle+Timing+of+Centromeric+Chromatin+Assembly+in+Drosophila+Meiosis+Is+Distinct+from+Mitosis+Yet+Requires+CAL1+and+CENP-C.">The Cell Cycle Timing of Centromeric Chromatin Assembly in Drosophila Meiosis Is Distinct from Mitosis Yet Requires CAL1 and CENP-C.</a> PLOS Biology. 10 (12), 1001460 (2012).</li><li>Fabian, L., Brill, J. 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D., Ma, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+Drosophila+melanogaster+testes.">Isolation of Drosophila melanogaster testes.</a> Journal of Visualized Experiments. (51), (2011).</li><li>Lerit, D. A., Plevock, K. M., Rusan, N. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+imaging+of+Drosophila+larval+neuroblasts.">Live imaging of Drosophila larval neuroblasts.</a> Journal of Visualized Experiments. 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We have described a protocol for the preparation of larval or early pupal Drosophila testes, optimized for long-term, live imaging of spermatocyte cell division. This is a powerful method for analysis of cell division in the physiological context of intact tissue. The power of this method is further expanded when combined with Drosophila genetic tools, such as specific gene mutations, tissue-specific RNAi-mediated suppression, and fluorescently labeled protein and organelle markers. In addition, the sma...

Cell division is often studied using cell lines grown in culture1. While we have gained a wealth of invaluable insight and understanding of fundamental mechanisms from these studies2, cells grown in culture cannot fully recapitulate the physiology of cell division as it occurs in intact, living tissue. For example, in intact tissues and organs, cells must divide at the right place and at the right time so that progeny cells are properly situated within the tissue, so that they can undergo appropriate differentiation or functional programs, and so that cell proliferation is properly coordinated with tissue growth or homeostasis3. For cells grown in culture on the other hand, cell division is generally regulated by growth factors in the culture medium4, and thus we cannot learn from these cells how in vivo environmental factors such as tissue architecture or developmental signaling influence the division process. It is also important to note that many of the cell lines used to study cell division, such as HeLa and U2OS cells, were derived from metastatic tumors5. Therefore, many aspects of the basic physiology of these cancer cells, such as cell cycle regulatory mechanisms and chromosomal stability, have likely been altered compared to healthy cells. Complete understanding of cell division physiology, therefore, depends on our ability to study dividing cells in their native, in vivo environments that preserve physiological regulatory mechanisms and tissue architecture.
Advances in understanding how cell division operates within intact tissues and organs are hampered by difficulties inherent to in vivo or ex vivo analysis. First, it can be difficult or impossible to access dividing cells for microscopic analysis within large organs or thick tissues. Second, it is often difficult to predict when individual cells will divide in vivo. Third, tissue physiology may rapidly deteriorate during ex vivo culture. In this protocol, we describe readily accessible methods for live and fixed analysis of Drosophila melanogaster spermatocytes as they undergo meiotic cell divisions within fully intact testes. These cells are ideal for live, ex vivo analysis because they are readily accessible with standard optical methods such as confocal microscopy, they divide at predictable times and locations, and intact testes can be maintained in ex vivo culture for up to about 24 h. In addition, Drosophila spermatocytes are large round cells (approximately 20&#8211;30 &#181;m in diameter) that do not change shape when they divide, making them ideal for high resolution, time-lapse imaging of cellular components such as the spindle apparatus and cytoplasmic organelles. Although these cells undergo meiosis as opposed to mitosis, many of the essential cell division processes are very similar between these two cell division mechanisms6. These advantages, combined with powerful Drosophila genetic tools, have made Drosophila spermatocytes an extensively used model for ex vivo analysis of essential cell division processes including spindle formation and regulation, cytokinesis, and organelle remodeling and partitioning7,8,9,10,11.
Spermatocytes are cells that are at the meiotic stage of spermatogenesis. In Drosophila, spermatogenesis begins with a group of germline stem cells that are located in a small region or &#8220;hub&#8221; at one pole of the testis12,13. These cells divide by an asymmetric mitosis, giving rise to a single differentiated spermatogonium. Spermatogonia then undergo four synchronous mitoses to produce a group of 16 cells that remain closely associated within a single cyst. At this stage, the cells transition from a mitotic to a meiotic cell cycle and are referred to as spermatocytes. Spermatocytes spend about two to three days in an extended G2 stage of the cell cycle, during which they grow dramatically and undergo cytological changes in preparation for the two meiotic divisions and subsequent spermiogenesis13,14. The entire group of 16 spermatocytes within a single cyst then enter the first meiotic division at the same time. Thus, several meiotic spermatocytes can be imaged simultaneously as they proceed through cell division. The first meiotic division proceeds for approximately 1.5 h and is followed almost immediately by the second meiotic division, yielding 64 total spermatids that go on to differentiate into mature spermatozoa.
A unique advantage of using spermatocytes to study cell division in live, intact tissue is that groups or cysts of cells constantly progress through the different stages of spermatogenesis, and cells at all stages of spermatogenesis can usually be identified in any given testis (see Figure 3A). Therefore, it is relatively easy to find meiotic cells in whole testes. We generally focus our analyses on the first as opposed to second meiosis because these cells are much larger and more amenable to high resolution imaging, but the entire process encompassing both meiotic divisions can be successfully imaged. It should also be noted that the general protocol for testis preparation and culture can be used to analyze other processes of spermatogenesis as well, such as the earlier mitotic stem cell or spermatogonial divisions or the cytological changes that occur as spermatids mature into spermatozoa15. Many of these aspects of spermatogenesis are highly conserved between Drosophila and humans16.
Drosophila spermatocytes begin to reach the meiotic phase of spermatogenesis during the third instar larval stage of Drosophila development13. Therefore, testes isolated from lifecycle stages beginning with third instar larvae and including pupae and adults can be used for analysis of dividing spermatocytes. Several excellent protocols are available for extraction of testes from adult male flies for live and fixed analysis of spermatogenesis17,18,19. These protocols may be preferable for studying late stages of spermatogenesis or if genetic markers that are only visible in adults must be used. The protocol focuses instead on testis preparation from larvae and early pupae, because testes at these stages have several advantages that are specifically pertinent to cell division analysis by high resolution, time-lapse microscopy. First, testes from larvae and pupae are smaller than those from adults, and the cells within the organ are less crowded. Because of this, dividing spermatocytes can often be imaged near the outer surface of larval testes, without having to penetrate through multiple layers of light scattering tissue. Second, adult testes move rhythmically due to contractions of the attached accessory organs, and these movements make time-lapse imaging of individual cells challenging. And third, larval testes are advantageous when studying gene mutations that cause pupal or adult lethality. Our methods are optimized for long-term culture of testes on the microscope stage, allowing for imaging of multiple rounds of cell division or the progression of individual cells through multiple stages of spermatogenesis in the same preparation. We also describe a protocol for fixation and immunostaining of whole testes. Overall, our protocols are particularly useful for those interested in studying cell division in intact tissue, and the ability to combine spermatocyte analysis with highly tractable Drosophila genetic tools makes this an especially powerful approach.

<table>
	<tbody>
		<tr>
			<td>5&#34; Dissecting Probe</td>
			<td>Fisher</td>
			<td>08-965-A</td>
			<td></td>
		</tr>
		<tr>
			<td>5.75&#34; Glass Pasteur Pipet</td>
			<td>Fisher</td>
			<td>13-678-20A</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin (BSA)</td>
			<td>Fisher</td>
			<td>BP9703-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont #5 Forceps</td>
			<td>Fine Science Tools</td>
			<td>11252-20</td>
			<td>Straight forcepts with fine tips</td>
		</tr>
		<tr>
			<td>Frosted Microscope Slides</td>
			<td>Fisher</td>
			<td>12-544-2</td>
			<td>Slides for mounting fixed tissue, with frosted writing surface</td>
		</tr>
		<tr>
			<td>Halocarbon oil 700</td>
			<td>Sigma</td>
			<td>H8898-100 mL</td>
			<td></td>
		</tr>
		<tr>
			<td>Lumox Dish 50</td>
			<td>Sarstedt</td>
			<td>SAR946077410</td>
			<td>Gas-permeable tissue culture dish</td>
		</tr>
		<tr>
			<td>Microscope Cover Glass</td>
			<td>Fisher</td>
			<td>12-541-B</td>
			<td>22x22 mm, #1.5 glass coverslip</td>
		</tr>
		<tr>
			<td>Minutien Pins</td>
			<td>Fine Science Tools</td>
			<td>26002-15</td>
			<td>Insect pins used to make scalpel tool</td>
		</tr>
		<tr>
			<td>Nickel Plated Pin Holder</td>
			<td>Fine Science Tools</td>
			<td>26018-17</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde 32% Solution, EM Grade</td>
			<td>Electron Microsocopy Sciences</td>
			<td>15714</td>
			<td></td>
		</tr>
		<tr>
			<td>Plan Beveled Edge Microscope Slides</td>
			<td>Fisher</td>
			<td>12-549-5</td>
			<td>Slides used for dissections</td>
		</tr>
		<tr>
			<td>PYREX Spot Plates</td>
			<td>Fisher</td>
			<td>13-748B</td>
			<td>9-well glass dissecting dish</td>
		</tr>
		<tr>
			<td>Schneider&#39;s Drosophila medium</td>
			<td>Fisher</td>
			<td>21720-024</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe Filter, 0.22 &#181;m</td>
			<td>EMD Millipore</td>
			<td>SLGS033SB</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Fisher</td>
			<td>BP151-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Vectashield</td>
			<td>Vector Labs</td>
			<td>H-1000</td>
			<td>Microscopy mounting medium</td>
		</tr>
	</tbody>
</table>

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.

When this protocol is successfully executed, testes will remain fully intact for imaging by confocal microscopy or other fluorescence microscopy methods. As seen in Figure 3A, the cellular organization of the testes is preserved, and the progression of cell differentiation from one end of the testis to the other including spermatogonia, spermatocytes, and haploid spermatids is visible. GFP-tubulin is a useful marker for identifying dividing spermatocytes and for live imaging of the progressi...

Watch this Scientific Journal Video about Preparation of Drosophila Larval and Pupal Testes for Analysis of Cell Division in Live, Intact Tissue at JoVE.com

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.

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 ...

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Dissection, Immunohistochemistry and Mounting of Larval and Adult Drosophila Brains for Optic Lobe Visualization

Dissection, Immunohistochemistry and Mounting of Larval and Adult Drosophila Brains for Optic Lobe Visualization

The Drosophila optic lobe, comprised of four neuropils: the lamina, medulla, lobula and lobula plate, is an excellent model system for exploring the ...